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Effects of Parental Dietary Exposure to GM Rice TT51 on the Male Reproductive System of Rat Offspring

OBJECTIVE: To evaluate the health effects of parental dietary exposure to GM rice TT51 on the male reproductive system of rat offspring. METHODS: Rice-based diets, containing 60% ordinary grocery rice, MingHui63, or TT51 by weight, were given to parental rats (15 males/30 females each group) for 70 days prior mating and throughout pregnancy and lactation. After weaning, eight male offspring rats were randomly selected at each group and fed with diets correspondent to their parents’ for 70 days. The effects of exposure to TT51 on male reproductive system of offspring rats were assessed through sperm parameters, testicular function enzyme activities, serum hormones (FSH, LH, and testosterone levels), testis histopathological examination, and the relative expression levels of selected genes along the hypothalamic-pituitary- testicular (HPT) axis. RESULTS: No significant differences were observed in body weight, food intake, organ/body weights, serum hormone, sperm parameters, testis function enzyme ACP, LDH, and SDH activities, testis histopathological changes, and relative mRNA expression levels of GnRH-R, FSH-R, LH-R, and AR along the HPT axis. CONCLUSION: The results of this study demonstrate that parental dietary exposure to TT51 reveals no significant differences on the reproductive system of male offspring rats compared with MingHui63 and control.

Detection of respiratory syncytial virus infection in nasal aspirate samples by flow cytometry

Hypotheses: (i) Flow cytometry has the potential for rapid detection of respiratory viral antigens. (ii) This technique can be applied to viral diagnosis in clinical samples. Objectives and study design: (i) To study the identification of six common respiratory viral pathogens by flow cytometry, in virus infected and uninfected cultured cells, as models of positive and negative clinical samples. (ii) To compare flow cytometry with the established techniques of viral isolation and immunofluorescent microscopy in the diagnosis of respiratory syncytial virus infection in 68 naso-pharyngeal aspirates taken from children and sent to the virology laboratory for routine virological diagnosis. Results: (i) For each virus analysed, populations of infected and non-infected cells were clearly discernable, confirming potential for this method in rapid viral diagnosis in clinical samples. (ii) Two definitions were employed for a sample to be positive by flow cytometry, these were compared with the combined established techniques. The sensitivity, specificity, positive and negative predictive values of flow cytometry were 41%, 98%, 92% and 71% for the first definition and 74%, 88%, 80% and 84% for the second definition respectively. Conclusions: As tested in this study, flow cytometry is less sensitive than established techniques as well as recently developed rapid diagnostic techniques for the diagnosis of respiratory syncytial virus infection. Further evaluation of the potential of flow cytometry in rapid viral diagnosis is warranted.

The use of macrolides in respiratory tract infections

Macrolides have enjoyed continued use for over 40 years, being increasingly usedfor the treatment of respiratory tract infections. Newer macrolides have been introduced that show improved absorption after oral administration, better gastrointestinal tolerance, and delivery of increased amounts of drug to the infection site. Macrolides are commonly used in community-acquired pneumonia, as well as in atypical pneumonia and legionellosis. The newer macrolides, in comparative studies, have been shown to be as effective as the conventional therapies for treating acute otitis media, acute sinusitis and acute pharyngitis, with a low incidence of side-effects. However, dosing can be simplified because of their unique pharmacokinetic properties. Limitations in the use of macrolides for respiratory infections include rather marginal activity in the most severe cases of Haemophilus influenzae infections, lack of activity against Klebsiella and other coliforms, which precludes their use as single agents in the therapy of pneumonia in patients with significant underlying disease or in the elderly, and development of resistance in streptococci and staphylococci.

Rapid diagnosis of respiratory virus infections in patients with acute respiratory disease

Viral respiratory infections represent a significant segment of the total respiratory disease spectrum; however, until recently the laboratory diagnosis of viral respiratory infections was relatively inefficient. Development of new and improved immunologic assay systems has paved the way for accurate and reliable rapid diagnostic tests that detect viral antigens in clinical specimens. We conducted a careful and elaborate study in which radioimmunoassay for antigen detection was compared with a battery of tissue culture systems for viral isolation and identification. Using a fine plastic catheter, a specimen of mucus was aspirated from the nasopharynx of patients with clinical signs and symptoms of acute viral upper respiratory tract infections. Each specimen was divided into two portions; one was used to inoculate a variety of tissue culture cell lines and the other was used for radioimmunoassay tests for influenza A and B, adenovirus, parainfluenza 1, 2, and 3, and respiratory syncytial virus. Radioimmunoassay results compared very favorably with the tissue culture data with only one exception—adenovirus. Essentially this degree of accuracy and reproducibility was obtained with an enzyme-linked immunosorbent assay test, which has replaced radioimmunoassay. Tissue cultures are still used for backup, but with a rapid antigen detection system in place, coupled with a modern computer program to facilitate the laboratory data to the clinician, considerable strides have been made, and will continue to be made, in the diagnosis and therapy of viral respiratory tract infections.

Pathogens in livestock waste, their potential for movement through soil and environmental pollution

Livestock wastes contain many pathogenic microorganisms including bacteria, viruses and protozoa. Following the application of these wastes to land the potential exists for environmental contamination. Plants, soil and ultimately water courses which may subsequently be used as catchments for public water supplies may all be affected. Research attention is now being focused on this possibility, especially in the case of protozoan pathogens which may be the most important as they are often resistant to current methods used in public water treatment. In this review we highlight some of the many factors that are likely to influence the degree of pollution by their effect on both the vertical and horizontal transport of microorganisms through soil. Soil pH, temperature, the presence of plants, microbial surface properties, type of waste, soil type and soil water content and flow may all affect the rate and extent of vertical transport, with the latter two generally considered to be the most important. Lateral movement is a particular problem in soils with impermeable substrata or in waterlogged conditions and in these cases the major factors affecting movement include rainfall rate, topography of the land and the rate at which microorganisms partition into the runoff.

The virulence of mouse hepatitis virus 3, as evidenced by permissivity of cultured hepatic cells toward escape mutants

Deux mutants d'échappement à la neutralisation du MHV3 (MHV 51.6 et MHV Cl12) montrent, en comparaison au MHV3 sauvage (wt), un ralentissement de leur multiplication chez des souris sensibles BALB/c et une atténuation du pouvoir pathogène provoquant un retard considérable de la mortalité. Le MHV3 wt se réplique à des titres élevés dans toutes les cellules hépatiques en culture. Le MHV3 51.6, de virulence intermédiaire, se multiplie dans toutes les cellules hépatiques excepté dans les cellules endothéliales. Le mutant qui tue le plus tardivement les souris (MHV Cl12) se multiplie uniquement dans les cellules de Ito, les autres étant non permissives. Ces résultats montrent une corrélation directe entre la virulence du MHV3 et son aptitude à se répliquer de manière efficace dans les cellules hépatiques in vitro.

Calf Pneumonia

Infectious calf pneumonia is a high-morbidity pneumonia of housed dairy-type calves. Viruses are important etiologic agents and infection with bovine respiratory syncytial virus (RSV) and parainfluenza type 3 virus (PI-3 virus) may result in extensive, and sometimes fatal, lung damage. Respiratory viral infections are frequently followed by mycoplasmal and secondary bacterial invasion of the lower respiratory tract, which increases the extent and severity oflung damage. Bad housing, particularly bad ventilation, will increase the severity of pneumonia outbreaks. Although the source of respiratory viral infections is not always obvious, it is likely that a proportion of calves acquire infection from their dams early in life. The possibility of cross-infections from other domestic animals and from humans must also be considered. Diagnosis of respiratory virus infections necessitates submission of suitable respiratory tract specimens that are taken at an early stage in the outbreak together with paired sera. Various therapeutic and prophylactic regimens for the control of calf pneumonia are described.

Intravenous Fluid Therapy in Calves

Effective intravenous fluid therapy requires determination of the volume required and the composition of the appropriate parenteral solution. Specific abnormalities in major electrolytes, acid-base balance, and energy balance should be considered in fluid selection. Parenteral solutions should be delivered in a sterile manner, with attention to potential complications.

Diagnosis of Neonatal Pig Diarrhea

General intestinal protection mechanisms and pathogenic mechanisms of diarrhea are provided as a means to explain selection of sites for diagnostic sampling. Five common enteric pathogens of neonatal pigs are covered in depth, including colibacillosis, transmissible gastroenteritis, rotavirus, Clostridium perfringens, and coccidiosis. Tables are provided for clinical differential diagnosis, as well as diagnostic sampling. Additionally, general treatment, vaccination, and management protocols are presented.

Controversies in Clostridium difficile testing

Recent reports of two nosocomial outbreaks of Clostridium difficile-associated disease caused by toxin A-deficient strains emphasize that these strains can cause disease. Laboratories using an assay that detects only toxin A as their primary diagnostic test risk misdiagnosis of cases or outbreaks in the institutions they serve. Repeat testing can account for a significant portion of a laboratory's C. difficile testing workload. Published data are available to support laboratory rules for rejection of repeat stool specimens within 7 days of an initial specimen. There are also substantial published data to support laboratory rejection of formed stools sent to the laboratory for C. difficile testing.

Intravenous Fluid Therapy of Calves

Dehydration in neonatal calves with diarrhea is a common cause of death. Severely dehydrated calves that are unable to suckle need intravenous fluids for effective resuscitation. This article gives an overview of the principles of intravenous fluid therapy for dehydrated calves including the types of fluids commonly used and methods of fluid administration. Practical on-farm options for simple clinical assessment and treatment of dehydration and acidosis in calves are also discussed in this article.

IDENTIFICATION OF A T CELL LYMPHOMA CATEGORY DERIVED FROM INTESTINAL-MUCOSA-ASSOCIATED T CELLS

2 cases of precursor T cell lymphoma and 37 cases of peripheral T cell lymphoma were investigated for their reactivity with the monoclonal antibody (mAb) HML-1, which recognises human intestinal T lymphocytes but not lymph-node T cells. In all but one of the lymphomas studied, the tumour cells were unreactive with the mAb HML-1. The HML-1(+) lymphoma was the only tumour that was primarily localised in the epithelium and lamina propria of the small intestine, and was associated with ulcerative jejunitis and coeliac disease. This result suggests that the HML-1(+) lymphoma was derived from intestinal mucosa T lymphocytes and differs from precursor T cell lymphoblastic lymphomas and nodal and cutaneous peripheral T cell lymphomas.

CALICIVIRUS GASTROENTERITIS IN NORTH WEST LONDON

During a thirty-month study of gastroenteritis in North West London, 592 cases were found to be associated with excretion of viruses. 39 (6·6%) of these patients, most of whom were admitted to hospital because of gastroenteritis, were shedding caliciviruses. The cases occurred throughout the year with a peak incidence in the winter. The 39 patients ranged in age from 6 weeks to 13 years, the peak incidence beig among infants aged 1-6 months. The clinical features of calcivirus infection are not distinguishable from those of rotavirus infection.

Expression of nitric oxide synthase type II in the spinal cord under conditions producing thermal hyperalgesia

There is evidence supporting spinal cord nitric oxide (NO) production in the mechanisms underlying hyperalgesia, presumed to arise from the activity of neuronal nitric oxide synthase type I (NOS I). Intrathecal administration of interleukin-1β and interferon-γ to rats results in a thermal hyperalgesia which peaks at 2 h post-injection but which is undetectable 8 h post-injection. Expression of mRNA for nitric oxide synthase type II (NOS II) was detected by reverse transcription-polymerase chain reaction followed by Southern hybridization utilizing specific oligonucleotides in spinal cord tissue from animals 4 h and 8 h after cytokine injection, but not at longer time points. NOS II protein was detected in soluble fractions of spinal cords from animals 4 h and 8 h after cytokine injection. In situ hybridization for NOS II mRNA revealed positive cells bilaterally in the spinal cord 4 h after cytokine injection in a perivascular distribution and scattered throughout the gray and white matter. Immunohistochemistry for NOS II showed a similar distribution which could only be partially accounted for by macrophages/microglia. These results provide evidence for induction of NOS II expression under conditions producing thermal hyperalgesia and suggest a possible role in this behavior for the production of NO by a variety of cell types in the CNS.

Viral Infections of the Newborn

Viral infections of the newborn result in significant morbidity and mortality each year. The fetus and newborn are particularly wlnerable to viral infection. The range of expression may vary from no clinical disease to devastating illness and infection occurring before, during, or after birth. Nursing management is determined by the specific viral infection, the severity of the illness, and the unique conditions of the newborn and his/her family. Promising new therapies are on the horizon that may lessen the severity of viral disease. Until such time, the major thrusts of management of neonatal viral disease are prevention of infection and supportive care for the acutely ill newborn.

Golgi retention signals: do membranes hold the key?

The diverse forms and functions of cellular organelles are, presumably, a consequence of their particular molecular compositions. The generation and maintenance of this diversity is achieved by the targeting of newly synthesized proteins to specific locations and their subsequent retention there. Sequences that retain proteins in the endoplasmic reticulum (ER) have been identified at the C-termini of resident ER proteins, where they are readily accessible to potential receptors. By contrast, recent results have demonstrated that retention of proteins in the Golgi complex involves sequences located within transmembrane domains. This suggests the novel possibility that the membrane composition of the Golgi complex plays a role in retention of resident Golgi proteins.

CHRONIC ENCEPHALOMYELITIS WITH SPECIFIC INCREASE IN INTRATHECAL MUMPS ANTIBODIES

Symptoms of severe encephalomyelitis developed in a 31-year-old man in 1967. He had a high serum antibody titre to mumps virus associated with a polymorphic cell reaction and an increased protein concentration in cerebrospinal fluid (CSF). He recovered considerably within a year and was able to resume work. In 1975 his condition deteriorated again; it improved during the following few years, but a further deterioration then occurred. In March, 1981, the complement-fixing antibody titre to mumps virus was 1/32 in the serum and 1/4 in the CSF. In November, 1981, the CSF IgG index was increased and the altered serum/CSF antibody ratio persisted. The specificity of the altered antibody ratio was confirmed by the single radial haemolysis test and an immunoassay specific for mumps virus. Antibodies against the mumps virus envelope glycoprotein, M-protein, and nucleoprotein could be demonstrated by immunoprecipitation and the antibody patterns in serum and CSF were similar. Antibodies against other microorganisms were not detected in the patient's CSF, and mumps antibodies were not found in the CSF specimens of 57 control patients. This case may be an example of a new disease—chronic mumps virus infection in the central nervous system.

The where, what and how of ribosomal frameshifting in retroviral protein synthesis

The gag and pol genes of most retroviruses occur in different reading frames and their translation as a single polypeptide is carried out by ribosomal frameshifting in the −1 direction. The alignment of the different reading frames occurs by overlapping reading in response to at least two signals within the RNA: one is a heptanucleotide stretch at the frameshift site and the other is a stem-loop structure which occurs just downstream of the first signal.

Rotavirus excretion by kids in a naturally infected goat herd

A cross-sectional study was carried out in a dairy goat herd, investigating the presence of rotavirus by means of ELISA, polyacrylamide gel electrophoresis (PAGE) and two latex agglutination tests in feces of 63 goat kids younger than 1 month, with and without diarrhea, and in feces of 19 adult goats during the first few days after parturition. All animals belonged to a herd located in the mountains of the León province (NW Spain). Rotaviruses were found in 18 out of 63 goat kid fecal samples but no significant association between shedding of rotavirus and presence of diarrhea could be established. Rotaviruses were found in kids aged 6 to 21 days, and more frequently between 6 and 10 days. No shedding of virus was detected in any of the adults. Considering ELISA as the reference test, PAGE and both latex agglutination tests were less sensitive. One of the latex tests was also highly non-specific. All PAGE-positive samples showed the typical electropherotype of group A rotavirus. Feces were also screened for other pathogens including Escherichia coli, Clostridium perfringens and Cryptosporidium parvum. C. parvum oocysts were detected in the feces of six out of 45 goat kids tested, all six suffering from diarrhea. This paper represents the first description of rotavirus infections in goats in Spain. The possible mechanisms of viral diffusion within the herd and its role as pathogen in goats are discussed.

Expression vectors for quatitating in vivo translational ambiguity: Their potential use to analyse frameshifting at the HIV gag-pol junction

Translational errors are necessary so as to allow gene expression in various organisms. In retroviruses, synthesis of pol gene products necessitates either readthrough of a stop codon or frameshifting. Here we present an experimental system that permits quantification of translational errors in vivo. It consists of a family of expression vectors carrying different mutated versions of the luc gene as reporter. Mutations include both an in-frame stop codon and 1-base-pair deletions that require readthough or frameshift, respectively, to give rise to an active product. This system is sensitive enough to detect background errors in mammalian cells. In addition, one of the vectors contains two unique cloning sites that make it possible to insert any sequence of interest. This latter vector was used to analyse the effect of a DNA fragment, proposed to be the target of high level slippage at the gag-pol junction of HIV. The effect of paromomycin and kasugamycin, two antibiotics known to influence translational ambiguity, was also tested in cultured cells. The results indicate that paromomycin diversely affects readthrough and frameshifting, while kasugamycin had no effect. This family of vectors can be used to analyse the influence of structural and external factors on translational ambiguity in both mammalian cells and bacteria.

The asymmetric distribution of phosphatidylcholine in rat brain synaptic plasma membranes

The distribution of phosphatidylcholine between inner and outer monolayers of rat brain synaptic plasma membrane was investigated by means of a phosphatidylcholine specific exchange protein. About 70% of the total membranal phosphatidylcholine was in the outer leaflet, 33% of which was exposed and readily exchanged in intact synaptosomes while the remainder was exchangeable following osmotic shock. Permeabilization of the synaptic plasma membranes by overnight incubation in buffer or by saponin (<0.08%) exposed an additional 30% of phosphatidylcholine to exchange, presumably from the inner cytoplasmic leaflet. Phosphatidylcholine is therefore asymmetrically distributed in the synaptosomal plasma membrane, as it is in other plasma membranes.

Synthesis of long cDNA from viral RNA template()

Methods to make long and reliable cDNA from viral RNA template have been optimized. The conditions of the denaturation of the viral RNA template were most critical. For synthesis of the first DNA strand, the concentration of the primer and the presence of an RNase inhibitor were important. During the synthesis of the second strand, the incubation temperature was found to have effect on the length of the transcripts. Application of our optimized conditions on coronaviral genomic RNA as template resulted in cDNA libraries with inserts in the range of 0.5–5 kb without a separate cDNA size selection. Furthermore, a convenient variant of the alcohol precipitation and the analysis of single-stranded DNA on neutral agarose gels are described.

Advances in the diagnosis of respiratory virus infections

Background: Advances have been made in selecting sensitive cell lines for isolation, in early detection of respiratory virus growth in cells by rapid culture assays, in production of monoclonal antibodies to improve many tests such as immunofluorescence detection of virus antigens in nasopharyngeal aspirates, in highly sensitive antigen detections by time-resolved fluoroimmunoassays (TR-FIAs) and biotin-enzyme immunoassays (BIOTH-E), and, finally, in the polymerase chain reaction (PCR) detection of respiratory virus DNA or RNA in clinical specimens. All of these advances have contributed to new or improved possibilities for the diagnosis of respiratory virus infections. Objectives and study design: This review summarizes our experiences during the last 15 years in the development of diagnostic tests for respiratory virus infections, and in use of these tests in daily diagnostic work and in epidemiological studies. Results: Immunofluorescence tests based on monoclonal antibodies, all-monoclonal TR-FIAs, and biotin-enzyme immunoassays (EIAs) have about the same sensitivities and specificities. They compare well with the sensitivity of virus culture. PCR followed by liquid-phase hybridization is a sensitive method for detecting adenovirus DNA and enterovirus and rhinovirus RNA in clinical specimens. IgG EIA on paired acute and convalescent phase sera is the most sensitive serological test for respiratory virus infections and is a valuable reference method when evaluating the sensitivity of new diagnostic tests. The IgG avidity test can distinguish primary infections from re-infections at least in respiratory syncytial virus (RSV) infections. IgM antibody assays, on the other hand, had low sensitivities in our studies. Conclusions: The choice of diagnostic methods for respiratory virus infections depends on the type and location of the laboratory, the number of specimens tested, and the previous experience of the laboratory. Virus culture, whenever possible, should be the basic diagnostic method; the results, including identification of the virus, should be available no more than 24 h later than the results of rapid diagnostic tests. In small laboratories, especially in hospitals where specimen transportation is well organized, immunofluorescence may be the best choice for antigen detection with the provision that an experienced microscopist and a good UV microscope are available. If the laboratory receives a large number of specimens and has previous experience with EIAs, then biotin-EIAs or TR-FIAs may be the most practical techniques. Their advantages include the stability of the antigens in clinical samples since intact, exfoliated epithelial cells are not required, treatment of specimens is practical, testing of large numbers of specimens is possible, and reading the printed test result is less subjective than reading fluorescence microscopy. The larger role of PCR in the diagnosis of respiratory virus infections depends on future developments such as practical methods to extract DNA or RNA and to purify the extracts from nonspecific inhibitors, plus further improvements to minimize cross-contamination. Group-specific detection of enteroviruses and rhinoviruses is an example of the potential for PCR technology. In experienced laboratories. EIA IgG antibody tests should be available. Recombinant antigens may be a useful part of such assays.

Immunohistochemical and behaviour pharmacological analysis of rats inoculated intranasally with vesicular stomatitis virus

A temperature-sensitive mutant of vesicular stomatitis virus was inoculated intranasally into infant Sprague Dawley rats aged 9 to 17 days. Rats receiving the virus at 9 days of age had an extensive spread of infection throughout the brain and the animals died after a few days. Rats inoculated at day 11 postnatally survived and the infection was limited to the olfactory pathways, hypothalamus, diagonal bands and the anterior raphe nuclei. Stereological measurements showed that the volume of infected neurons constituted 67 ± 10% of the total neuronal volume in the dorsal raphe nucleus. Double-labelling experiments revealed that both 5-hydroxytryptamine- and substance P-immunoreactive neurons contained the virus antigen. The motor stimulant effect of amphetamine was studied at 3 months post infection. The increase in amphetamine-induced frequency and duration of rearing was significantly attenuated in infected rats and the amphetamine-induced locomotion was slightly reduced.

Sequential variation in genomic RNA patterns of human rotaviruses isolated from infantile gastroenteritis

The incidence and RNA electrophoretypes of rotaviral isolates obtained from infants and young children with acute gastroenteritis were studied from October, 1985 through April, 1986. Analysis of the viral RNA was carried out by Polyacrylamide gel electrophoresis followed by silver staining. Fourteen electrophoretypes were identified. A single dominant electrophoretype was found during the first months of the rotavirus seasonal outbreak. In contrast, a large variety of RNA patterns were observed during the latter portion of the study period. Subgrouping of rotavirus isolates by a double-sandwich enzyme-linked immunosorbent assay using monoclonal-detecting antibodies showed that all strains belonged to subgroup II. Mixed rotavirus electrophoretypes appeared in 4 cases (8.16 %).

RAISED URINARY FIBRIN-DEGRADATION PRODUCTS, COMPLEMENT, AND IgG DURING AN INFLUENZA-LIKE ILLNESS

Urine from eight normal controls in whom an influenza-like illness developed contained high concentrations of fibrin-degradation products (F.D.P.), IgG, and C(3). The study was carried out when influenza A was prevalent in the community. However, a wide range of serological investigations revealed no evidence for influenza A or other viruses. The infection may have been caused by other viruses which produce upper-respiratory-tract infections and which are not readily diagnosed by serology. Urinary fibrin-degradation products are a well-known marker of glomerulonephritic activity and viral antigens may have induced an immune-complex glomerulonephritis in the 8 controls in whom an influenza-like disease developed. A larger normal population should be investigated during a virus epidemic.

Differential effects of orally versus parenterally administered qinghaosu derivative artemether in dogs

Artemether (AM) is an antimalarial drug derived from artemisinin (Qinghaosu), an extract of the herb Artemisia annua L., sweet wormwood. Its antiparasitic effect is that of a schizontocide and is explained by rapid uptake by parasitized erythrocytes and interaction with a component of hemoglobin degradation resulting in formation of free radicals. It has been shown to exhibit a high clinical cure rate. Previous animal safety studies with Qinghaosu derivatives revealed dose-dependent neurotoxicity with movement disturbances and neuropathic changes in the hindbrain of intramuscularly treated dogs, rats and monkeys. Such effects have not been seen in man. The objective of our present studies was to compare the effects of high levels of AM administered to dogs p.o. versus i.m. In a pilot study 20 mg/kg/day of AM was given i.m. to groups of 3 male Beagle dogs for 5 and 30 days, respectively. Clinical signs of neurotoxicity were noted in some individual dogs from test day 23 on. One dog had to be sacrificed pre-term. Hematologic findings indicated a hypochromic, microcytic anemia. Microscopic examination demonstrated neuropathic changes only at 30 days, but not at 5 days. The animals had neuronal and secondary axonal damage, most prominent in the cerebellar roof, pontine and vestibular nuclei, and in the raphe/paralemniscal region. The affected neurons showed loss of Nissl substance, cytoplasmic eosinophilia, shrinkage of the nucleus and in advanced stages scavenging by microglia. In a subsequent experiment, AM was administered to groups of 4 male and 4 female dogs, respectively, at 8 daily doses of 0, 20, 40 and 80 mg/kg i.m., or 0, 50, 150 and 600 mg/kg p.o. Neurologic signs were seen at high i.m. doses only. In most animals they were inconspicuous and consisted of reduced activity with convulsions seen in single dogs shortly before death. Neuronal damage occurred in all animals at 40 and 80 mg/kg following i.m. treatment. At 20 mg/kg minimal effects occurred in 5/8 dogs only, indicating that this level was close to tolerated exposure. No comparable lesions were observed after oral administration. Both i.m. and p.o. exposure at high dose levels was associated with a prolongation of mean QT interval of ECG, suggesting slowing of repolarization of the myocardium. Individual data indicated that in 1 of 4 females at 80 mg/kg i.m. this prolongation was above the 25 % level considered as threshold for concern. After intramuscular administration pharmacokinetics indicated peak plasma levels of AM at 2 to 4 hours post-dose, slow elimination and a tendency to accumulate after repeated administration. Only low levels of the major metabolite, dihydroartemisinin (DHA), were found. AM levels in the cerebrospinal fluid (CSF) were < 10 % of plasma levels. After oral administration AM concentrations were considerably lower than after i.m. administration. The concentration of DHA was high on day 1 but almost nil on day 7 indicating its fast inactivation in dogs. Two hours after the 8(th) oral administration neither AM nor DHA was detected in CSF which may explain the absence of neurotoxicity in dogs after oral administration of AM.

Growth of canine distemper virus in cultured astrocytes: relationship to in vivo persistence and disease

Canine distemper virus (CDV) causes an encephalomyelitis in dogs which varies with the viral strain. The CDV Cornell A75-17 strain produces a delayed, subacute to chronic, demyelinating CNS disease. In contrast, the Snyder Hill (CDV-SH) strain-associated neurological disease is more acute in onset, is usually non-demyelinating and primarily produces lesions in the gray matter. In these studies we describe the effects of these two virulent and one avirulent CDV strain, Rockborn (CDV-RO), on astrocytes in dissociated canine brain cell cultures. In multiple replicate experiments, astrocytes were infected most rapidly by CDV-RO [100% of astrocytes were infected by 14 days post-inoculation (pi.)]. This strain caused severe cytopathic effect (CPE) and cytolysis. CDV-SH similarly produced a rapid infection of the astrocytes. In contrast, CDV A75-17 infected less than 25% of the astrocyte population during the first 28 days p.i. (± 7 days); after 28 days p.i., a rapid rise in astrocyte infection occurred. Both virulent viruses caused astrocytic syncytial formation but did not cause cytolysis of the astrocyte population as was observed with the attenuated virus. Titers of infectious virus, released into the supernatant fluid, reflected the degree of astrocyte infection. Virus released by the cultures late in CDV A75-17 infection showed enhanced ability to infect newly derived astrocytes; in contrast, brain cell passaged CDV-SH did not show increased growth in these cells. These results show that (1) there is a difference in growth rate, CPE and capacity for adaptation of three different CDV strains in astrocytes in vitro, and (2) some aspects of the disease (such as persistence in white matter) produced by the virulent strains in vivo may be related to the course of astrocyte infection observed in vitro.

Comparison of detection methods for adenovirus from enteric clinical specimens

Fecal samples submitted for virus examination over July 1990 to June 1991 from children <3 years of age were examined by electron microscopy (EM), virus culture (VC), and enzyme immunoassay [EIA, group-reactive and adenovirus (Ad) 40/41 specific; Cambridge BioScience] to compare the detection rate of adenovirus from pediatric fecal specimens. Ad isolates of serotypes 1–7 grown in HEp-2 or primary rhesus monkey kidney cells were identified by neutralization. Graham 293 cell cultures were used only when specimens were found to be positive for Ad by EM, type-specific Ad40/41 EIA, and for isolates not identified by neutralization. Ads grown in 293 cells were identified by DNA restriction endonuclease analysis. Of the 1187 specimens examined, 105 (9%) were found to be positive for Ad. VC detected 93, while 12 additional positives were detected by EM or EIA. The relative sensitivity of VC, EIA, and EM for the 105 specimens was 89% (93), 45% (47), and 35% (37), respectively. Among the 105 positive specimens, enteric Ad, nonenteric Ad, and untypeable Ad were 28% (29), 65% (68), and 7% (8), respectively. Of 37 EM positives, 62% (23) were enteric Ad; 27% (10) were nonenteric including serotypes 2, 3, 4, 5, 12, and 31, with 4, 1, 1, 2, 1, and 1 isolates of each type positive, respectively; and 11% (4) were detectable only by EM. Five isolates were identified as variant of Ad 2(3), Ad 3(1) and Ad 31(1). Over a 1-year period, a single Ad41 variant strain was the most frequently detected enteric Ad in Winnipeg, Manitoba, Canada. For maximum detection rates of Ad viruses in pediatric fecal specimens, a combination of EM, VC, and EIA is required, but group-reactive EIA, or EM followed by Ad40/41-specific EIA of initial positives, are the most direct and efficient methods for enteric Ad detection.

RESPIRATORY SYNCYTIAL AND OTHER VIRUSES ASSOCIATED WITH RESPIRATORY DISEASE IN INFANTS

Diagnosis by virus isolation and serology was attempted in 377 cases of respiratory-tract infection in infants under one year of age admitted to hospital during two winters. A diagnosis of infection with respiratory syncytial (R.S.) virus was made in 40%, rhinovirus in 6·1%, adenovirus in 3·7%, parainfluenza in 2·1%, enterovirus in 1·9%, and influenza in 1·3%. R.S.-virus infections were more severe than others and occurred mostly in the first five months of life, with a peak at two months. Rhinovirus infections occurred at all ages, and often involved the lower respiratory tract. Of the 12 deaths, only 1 (due to R.S. virus) was not associated with a contributory cause. Maternal antibody to R.S. virus did not notably affect the incidence or severity of R.S.-virus infections.

Lewis rats of the inbred strain LEW/Han: Life expectancy, spectrum and incidence of spontaneous neoplasms

Although Lewis rats are frequently used in biomedical research, little is known about their life-data and spontaneous pathology. Therefore, it was the aim of this study to determine the life expectancy, spectrum and incidence of spontaneous neoplasms of the inbred rat strain LEW/Han. A total of 629 LEW/Han rats (305 females and 324 males) from a specified pathogen-free breeding colony were kept from weaning up to their natural death under defined environmental conditions. A complete histological examination was performed on all organs and macroscopically altered tissues of all animals which died during the first three years of the study. These were 296 female (98%) and 213 male (66%) rats. The mean lifespan of the females (27.7±5.1 months) was significantly shorter than that of the males (32.5±6.6 months). In both sexes, the lifespan was mainly determined by the occurrence of neoplasms. Of the large spectrum of 52 histologically different tumour types, the highest incidences were observed for adenomas of the pituitary gland and adenomas/adenocarcinomas of the adrenal cortex in both sexes, mammary gland tumours and endometrial carcinomas in females, and C-cell adenomas/adenocarcinomas of the thyroid gland and tumours of the haemopoietic system in males. Of these, the high incidences of tumours of the haemopoietic system in males (27.7%) and of endometrial carcinomas in females (45.2%) should be considered as characteristic features of the strain.

Epitope spreading

Epitope (determinant) spreading is the development of immune responses to endogenous epitopes secondary to the release of self antigens during a chronic autoimmune or inflammatory response. The past year has seen considerable advances in our understanding of the contribution of epitope spreading to the chronic pathogenesis of experimental T-cell-mediated and antibody-mediated autoimmune diseases. Most significantly, conclusive functional evidence for a major role for epitope spreading in the chronic pathogenesis of murine relapsing-remitting experimental autoimmune encephalomyelitis, a CD4(+) T-cell-mediated model of multiple sclerosis, was forthcoming.

4 Haematological disorders associated with feline retrovirus infections

Feline oncornavirus and lentivirus infections have provided useful models to characterize the virus and host cell factors involved in a variety of marrow suppressive disorders and haematological malignancies. Exciting recent progress has been made in the characterization of the viral genotypic features involved in FeLV-associated diseases. Molecular studies have clearly defined the causal role of variant FeLV env gene determinants in two disorders: the T-lymphocyte cytopathicity and the clinical acute immuno-suppression induced by the FeLV-FAIDS variant and the pure red cell aplasia induced by FeLV-C/Sarma. Variant or enFeLV env sequences also appear to play a role in FeLV-associated lymphomas. Additional studies are required to determine the host cell processes that are perturbed by these variant env gene products. In the case of the FeLV-FAIDS variant, the aberrant env gene products appear to impair superinfection interference, resulting in accumulation of unintegrated viral DNA and cell death. In other cases it is likely that the viral env proteins interact with host products that are important in cell viability and/or proliferation. Understanding of these mechanisms will therefore provide insights to factors involved in normal The haematological consequences of FIV are quite similar to those associated with HIV. As with HIV, FIV does not appear to directly infect myeloid or erythroid precursors, and the mechanisms of marrow suppression likely involve virus, viral antigen, and/or infected accessory cells in the marrow microenvironment. Studies using in vitro experimental models are required to define the effects of each of these microenvironmental elements on haematopoietic progenitors. As little is known about the molecular mechanisms of FIV pathogenesis, additional studies of disease-inducing FIV strains are needed to identify the genotypic features that correlate with virulent phenotypic features. Finally, experimental FIV infection in cats provides the opportunity to correlate in vivo virological and haematological changes with in vitro observations in a large animal model that closely mimics HIV infection in man.

CEA adhesion molecules: multifunctional proteins with signal-regulatory properties

The carcinoembryonic antigen family comprises a large number of complex molecules, several of which possess cell adhesion activities. The primordial adhesion molecules of this family are the cell—cell adhesion molecules (C-CAMs), which have been found to be multifunctional, signal-regulatory proteins. C-CAMs inhibit tumor growth, interact with calmodulin, protein tyrosine kinases and protein tyrosine phosphatases, and are subject to specific dimerization reactions. These new insights indicate that C-CAMs are important regulators of cellular functions.

Mouse hepatitis virus A59 increases steady-state levels of MHC mRNAs in primary glial cell cultures and in the murine central nervous system()

Infection of mixed glial cell cultures with mouse hepatitis virus (MHV)-A59 results in an approximately six-fold increase in the level of major histocompatibility complex (MHC) class I mRNA. In situ hybridization of glial cell cultures infected with MHV-A59 again showed enhanced MHC mRNA expression, both in infected and uninfected cells. These results extend our earlier finding that MHC surface antigens are enhanced on astrocytes and oligodendrocytes after MHV-A59 infection and suggest that this enhancement is a result of an increase in the steady-state level of MHC mRNA. We further demonstrate that increases in MHC mRNA occur in the murine central nervous system (CNS) following infection in vivo. Northern blot analysis of RNA from the brains of infected animals showed transient expression of both MHC class I and class II mRNA over the first 14 days of infection. Expression coincided with viral replication and clearance. In situ hybridization of brain sections from infected animals showed that class I and class II expression was widespread throughout all portions of the brain and in uninfected as well as infected cells. Viral RNA, in contrast, was observed in small foci of cells and mostly within the limbic system. Thus enhancement of MHC mRNA was not restricted either to areas of infection or inflammation. The spatial relationship between viral and MHC expression supports our hypothesis that a soluble mediator is involved in the mechanism of the increase in MHC levels. The fact that MHC induction occurs in vivo as well as in vitro suggests MHC may be important in the mechanism of MHV-induced disease.

Pulmonary cystic keratinizing squamous cell lesions of rats after inhalation/instillation of different particles

Cystic keratinizing squamous cell lesions from three inhalation studies (Study A, B, C) and one intratracheal instillation study (Study D) in rats were reclassified and a certain number of lesions examined immunohistochemically for PCNA (proliferating cell nuclear antigen) as a marker of cellular proliferation. The following classification was used: squamous cell metaplasia with marked keratinization, keratinizing cyst, cystic keratinizing epithelioma, cystic keratinizing squamous cell carcinoma, keratinizing squamous cell carcinoma and non-keratinizing squamous cell carcinoma. In study A (inhalation of coal oven exhaust and subcutaneous injection of a high dose of DB (ah)A) 49.3 % of rats developed cystic keratinizing squamous cell carcinomas. Inhalation of coal oven exhaust gas together with intratracheal instillation of crocidolite or subcutaneous injection of a low dose DB(ah)A (dibenz(ah)anthracene) resulted in cystic keratinizing squamous cell carcinomas in 23 % to 24 % of the rats. High incidences of cystic squamous cell carcinomas in the range of 31.9 % to 76.4 % were observed in rats of Study B1 after a 10-months exposure to tar/pitch condensation aerosol (different B(a)P (benzo(a)pyrene) concentrations) with added carbon black in some groups. After a 20-months exposure period to the same inhalation atmospheres (Study B2) the incidence of squamous cell carcinomas was increased up to 95.8 %. Exposure of rats to various concentrations of unfiltered diesel exhaust (Study C) resulted in incidences of cystic keratinizing epitheliomas ranging from 2.5 % (2.5 mg/m(3)) to 10.7 % (7.5 mg/m(3)). Epitheliomas were also observed in 16.2 % of carbon black and 16.0 % of titanium dioxide exposed rats. Only a few cystic keratinizing squamous cell carcinomas occurred. In the intratrachel instillation study (Study D) increased incidences of cystic keratinizing epitheliomas occurred in rats exposed to native diesel exhaust particles (16.7 %), high dose of extracted diesel exhaust particles (14.6 %), extracted printex 90-carbon black particles (18.8 %), and extracted printex 90-carbon black particles + B(a)P (18.8 %). High indicences of cystic keratinizing squamous cell carcinomas were noted in rats that received 15 mg B(a)P (14.6 %) or 30 mg B(a)P (72.7 %) intratracheally. Immunohistochemical labeling of nuclei with PCNA demonstrated proliferative activity in one or two (and focally more than two) peripheral cell layers of cystic keratinizing epitheliomas and in more than three peripheral cell layers of cystic keratinizing squamous cell carcinomas and keratinizing squamous cell carcinomas. The wall of keratinizing cysts showed no or a weak reaction.

Mapping neuroinvasiveness of the herpes simplex virus type 1 encephalitis-inducing strain 2762 by the use of monoclonal antibodies

Monoclonal antibodies (MAbs) directed against herpes simplex virus (HSV)-coded glycoproteins gB, gC, gD and gE were employed in an in vitro model of neuroinvasiveness using sensory neurons from rat dorsal root ganglion (DRG) cells. The neurons were cultured in a two-chamber system allowing infection via the neuritic extensions exclusively. The effects of 30 MAbs on viral replication of the encephalitis-derived HSV-1 strain 2762 and its less neuroinvasive variant 2762p11 were assayed in this model. One MAb reactive with gD gave a nine-fold reduction of the virus yields of both strains. One MAb directed against gB gave an enhanced virus yield of strain 2762, but not of the 2762p11 variant. Another gB-reactive MAb decreased the virus yield of strain 2762p11, but not of 2762 after neuritic infection. The findings indicate that an alteration of gB has occurred during the passage of the strain 2762. Mutants of the same strain were derived by infecting hybridomas producing MAb reactive with gB, gC, gD and gE, respectively. The gB hybridoma mutant showed a significantly lower neuroinvasiveness in the DRG model, and was non-virulent after snout infection of mice. We suggest that the structure of gB of the strain 2762 is of importance for the neuroinvasiveness of this strain.

SJL/J resistance to mouse hepatitis virus-JHM-induced neurologic disease can be partially overcome by viral variants of S and host immunosuppression()

The basis of the resistance of SJL/J mice to various strains of mouse hepatitis virus (MHV) has been the subject of some debate, especially as it relates to the number and nature of the determinants involved. Our previous work demonstrated that resistance by primary SJL/J glial cultures may involve events subsequent to viral gene expression, possibly due to a defect in cell-to-cell spread of the infection. Since S, the virion's major spike glycoprotein, is known to facilitate the spread of infection due to its syncytiogenic properties, we decided to investigate the role of this viral structural protein in resistance by primary SJL/J glial cells. Variants possessing deletions within the S coding region were able to grow in SJL/J glial cells 10–100 times easier and fuse five-times more efficiently than wt virus. Induction of neurologic disease in SJL/J mice following intracranial inoculation with either wt JHMV or the S deletion variant, AT11f cord, was age-dependent, occurring only in animals inoculated under 4 weeks of age. Resistance in older animals to wt and variant viruses could be abrogated by immunosuppression with cyclosporin A. However, both disease incidence and viral brain titers were higher in animals receiving the JHM variant AT11f cord virus, suggesting that SJL/J resistance to neurologic disease may manifest itself through interactions between inefficient cell-to-cell spread of the infection and protective aspects of the immune response.

Apoptosis in brain-specific autoimmune disease

Recent neuropathological studies of experimental autoimmune encephalomyelitis have focused attention on the high number of cells in the lesions that show typical morphological features of apoptosis. Surprisingly, it has turned out that the vast majority of apoptotic cells are T lymphocytes and that they actually represent the antigen-specific T-cell population responsible for the induction of the disease. Taken together, these data suggest that clearance of autoimmune inflammation in the nervous system is accomplished by the destruction of the antigen-specific T-cell population within the lesions. This may explain the low level of central nervous system specific T-cell memory formation, as well as previously unexplained phenomena of ‘epitope spreading’, in autoimmune inflammation of the nervous system

The astrocyte is a target cell in mice persistently infected with mouse hepatitis virus, strain JHM()

Mouse hepatitis virus, strain JHM (MHV-JHM), causes a late onset, clinically apparent, demyelinating encephalomyelitis in 40% of suckling C57BL/6 mice born to immunized dams. Suckling mice born to unimmunized dams rapidly succumb to an acute encephalomyelitis. MHV-JHM can be isolated from the brains and spinal cords of maternal antibody-protected mice when the late onset disease becomes clinically apparent, showing that the virus must be present in these mice when they are still asymptomatic. To determine which cells of the central nervous system (CNS) were potential reservoirs for the virus during the asymptomatic period, tissue sections were assayed simultaneously by immunoperoxidase and immunofluorescence staining for the presence of viral antigen and for glial fibrillary acidic protein (GFAP), a marker for astrocytes.(2) The results indicate that 20% (range 0–52%) of the MHV-JHM infected cells in asymptomatic mice were astrocytes. In mice symptomatic with late onset hindlimb paralysis, a higher percentage of infected cells were astrocytes. These results indicate that astrocytes are a target cell in both symptomatic and asymptomatic mice persistently infected with MHV-JHM, and suggest that the astrocyte is a potential cellular reservoir for MHV-JHM in asymptomatic mice.

Neuron-specific expression of a hamster prion protein minigene in transgenic mice induces susceptibility to hamster scrapie agent

To study the effect of cell type-restricted hamster PrP expression on susceptibility to the hamster scrapie agent, we generated transgenic mice using a 1 kb hamster cDNA clone containing the 0.76 kb HPrP open reading frame under control of the neuron-specific enolase promoter. In these mice, expression of HPrP was detected only in brain tissue, with highest levels found in neurons of the cerebellu, hippocampus, thalamus, and cerebral cortex. These transgenic mice were susceptible to infection by the 263K strain of hamster scrapie with an average incubation period of 93 days, compared to 72 days in normal hamsters. In contrast, nontransgenic mice were not susceptible to this agent. These results indicate that neuron-specific expression of the 1 kb HPrP minigene including the HPrP open-reading frame is sufficient to mediate susceptibility to hamster scraple, and that HPrP expression in nonneuronal brain cells is not necessary to overcome the TSE species barrier.

Rapidly decreased HBV RNA predicts responses of pegylated interferons in HBeAg-positive patients: a longitudinal cohort study

BACKGROUND: As an important anti-HBV drug, pegylated interferon α (PegIFNα) offers promising clinical efficacy, but biomarkers that accurately forecast treatment responses are yet to be elucidated. Here, we evaluated whether HBV RNA could act as an early monitor of pegylated interferon responses. METHODS: We analyzed a phase 3, multicenter, randomized cohort of 727 HBeAg-positive non-cirrhotic patients receiving a 48-week treatment of PegIFNα-2a or PegIFNα-2b and a 24-week treatment-free follow-up. Serum levels of HBV RNA, HBV DNA, HBeAg, and HBsAg were measured at weeks 0, 12, 24, 48, and 72. RESULTS: HBeAg seroconversion and HBsAg loss at week 72 were observed in 217 (29.8%) and 21 (2.9%) patients, respectively. During the 48-week treatment, HBV RNA decreased more rapidly than HBV DNA and HBsAg, but HBV RNA and HBeAg shared similar dynamics with positive correlations. Multivariate regression analyses consistently revealed the significance of HBV RNA at weeks 0, 12, 24, and 48 to monitor HBeAg seroconversion but not HBsAg loss. Although baseline HBV RNA only showed a modest AUC performance, HBV RNA with a significant increase of AUC at week 12 outperformed other HBV biomarkers to forecast HBeAg seroconversion (p value < 0.05). HBV RNA ≤ 1000 copies/mL was an optimized cutoff at week 12 that offered better prediction than other HBV biomarkers. This optimized cutoff plus patient age, HBV genotype B, and HBeAg offered a strong estimation of HBeAg seroconversion (accuracy 95.2%, true negative rate 99.8%). CONCLUSION: HBV RNA at week 12 is an effective monitor of HBeAg seroconversion in HBeAg-positive patients treated with pegylated interferons. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12072-020-10015-3) contains supplementary material, which is available to authorized users.

Opportunistic infections in immunosuppressed patients with juvenile idiopathic arthritis: analysis by the Pharmachild Safety Adjudication Committee

BACKGROUND: To derive a list of opportunistic infections (OI) through the analysis of the juvenile idiopathic arthritis (JIA) patients in the Pharmachild registry by an independent Safety Adjudication Committee (SAC). METHODS: The SAC (3 pediatric rheumatologists and 2 pediatric infectious disease specialists) elaborated and approved by consensus a provisional list of OI for use in JIA. Through a 5 step-procedure, all the severe and serious infections, classified as per MedDRA dictionary and retrieved in the Pharmachild registry, were evaluated by the SAC by answering six questions and adjudicated with the agreement of 3/5 specialists. A final evidence-based list of OI resulted by matching the adjudicated infections with the provisional list of OI. RESULTS: A total of 772 infectious events in 572 eligible patients, of which 335 serious/severe/very severe non-OI and 437 OI (any intensity/severity), according to the provisional list, were retrieved. Six hundred eighty-two of 772 (88.3%) were adjudicated as infections, of them 603/682 (88.4%) as common and 119/682 (17.4%) as OI by the SAC. Matching these 119 opportunistic events with the provisional list, 106 were confirmed by the SAC as OI, and among them infections by herpes viruses were the most frequent (68%), followed by tuberculosis (27.4%). The remaining events were divided in the groups of non-OI and possible/patient and/or pathogen-related OI. CONCLUSIONS: We found a significant number of OI in JIA patients on immunosuppressive therapy. The proposed list of OI, created by consensus and validated in the Pharmachild cohort, could facilitate comparison among future pharmacovigilance studies. TRIAL REGISTRATION: Clinicaltrials.gov NCT 01399281; ENCePP seal: awarded on 25 November 2011.

Serum sphingosine-1-phosphate levels and Sphingosine-1-Phosphate gene polymorphisms in acute respiratory distress syndrome: a multicenter prospective study

BACKGROUND: Sphingosine-1-phosphate (S1P) is a signaling phospholipid involved in pathophysiologic progression of acute respiratory distress syndrome (ARDS) through its roles in endothelial barrier function and immune modulation. We hypothesized that decreased serum S1P level is associated with the clinical outcomes of ARDS and polymorphisms in the S1P gene are associated with serum S1P levels. METHODS: This multicenter prospective study includes ARDS patients and healthy blood donors as controls. Serum S1P levels were quantified using enzyme-linked immunosorbent assays. Eight tag single nucleotide polymorphisms (SNPs) in the S1P gene were detected, and their associations with S1P levels were evaluated. RESULTS: A total of 121 ARDS patients and 100 healthy individuals were enrolled. Serum S1P levels were lower in ARDS patients than in controls (P < 0.001). Decreased S1P levels correlated with more organ dysfunction and higher Acute Physiology and Chronic Health Evaluation II scores. Changes in S1P levels in ARDS patients were associated with the clinical outcomes. The recessive model for SNP rs3743631 suggests that GG homozygote is associate with a higher risk for ARDS. The dominant model for SNP rs907045 suggests that AA or TA genotype might increase the risk for ARDS. In ARDS patients, the rs3743631 GG genotype showed lower S1P levels than those harboring AG and AA genotypes. The serum S1P levels of rs907045 AA or TA genotype patients were lower than those of TT genotype. CONCLUSIONS: Serum S1P levels are dramatically decreased in ARDS patients. Reduced S1P levels are associated with worse clinical outcomes. There is a significant association between S1P rs3743631, rs907045 polymorphisms and susceptibility of ARDS.

Plasmodium vivax HAP2/GCS1 gene exhibits limited genetic diversity among parasite isolates from the Greater Mekong Subregion

BACKGROUND: Antigens expressed in sexual stages of the malaria parasites are targets of transmission-blocking vaccines (TBVs). HAP2/GCS1, a TBV candidate, is critical for fertilization in Plasmodium. Here, the genetic diversity of PvHAP2 was studied in Plasmodium vivax parasite populations from the Greater Mekong Subregion (GMS). METHODS: Plasmodium vivax clinical isolates were collected in clinics from the China-Myanmar border region (135 samples), western Thailand (41 samples) and western Myanmar (51 samples). Near full-length Pvhap2 (nucleotides 13–2574) was amplified and sequenced from these isolates. Molecular evolution studies were conducted to evaluate the genetic diversity, selection and population differentiation. RESULTS: Sequencing of the pvhap2 gene for a total of 227 samples from the three P. vivax populations revealed limited genetic diversity of this gene in the GMS (π = 0.00036 ± 0.00003), with the highest π value observed in Myanmar (0.00053 ± 0.00009). Y133S was the dominant mutation in the China-Myanmar border (99.26%), Myanmar (100%) and Thailand (95.12%). Results of all neutrality tests were negative for all the three populations, suggesting the possible action of purifying selection. Codon-based tests identified specific codons which are under purifying or positive selections. Wright’s fixation index showed low to moderate genetic differentiation of P. vivax populations in the GMS, with F(ST) ranging from 0.04077 to 0.24833, whereas high levels of genetic differentiation were detected between the China-Myanmar border and Iran populations (F(ST) = 0.60266), and between Thailand and Iran populations (F(ST) = 0.44161). A total of 20 haplotypes were identified, with H2 being the abundant haplotype in China-Myanmar border, Myanmar and Thailand populations. Epitope mapping prediction of Pvhap2 antigen showed that high-score B-cell epitopes are located in the S307-G324, L429-P453 and V623-D637 regions. The E317K and D637N mutations located within S307-G324 and V623-D637 epitopes slightly reduced the predicted score for potential epitopes. CONCLUSIONS: The present study showed a very low level of genetic diversity of pvhap2 gene among P. vivax populations in the Greater Mekong Subregion. The relative conservation of pvhap2 supports further evaluation of a Pvhap2-based TBV. [Image: see text]

Cytokine response in human leptospirosis with different clinical outcomes: a systematic review

BACKGROUND: Leptospirosis is a neglected zoonotic disease which is a major challenge for clinicians and public health professionals in tropical countries. The cytokine storm during the second (immune) phase is thought to be a major contributory factor for the leptospirosis disease severity. We aim to summarize evidence for cytokine response in leptospirosis at different clinical outcomes. METHODS: A systematic review was carried out to examine the cytokine response in leptospirosis patients using relevant scientific databases. Reference lists of the selected articles were also screened. Quality of the selected studies was assessed by using the National Institutes of Health Quality Assessment Tool for Observational Cohort and Cross-Sectional Studies. RESULTS: Of the 239 articles retrieved in the initial search, 18 studies fulfilled the selection criteria. India and Thailand have produced the highest number of studies (17% each, n = 3). The majority were comparative cross-sectional studies (72%, n = 13). Overall the quality of the selected studies was fair regardless of few drawbacks such as reporting of sample size and the lack of adjustment for confounders. Microscopic agglutination test (67% - 12/18) and enzyme-linked immunosorbent assay (50% - 9/18) were commonly used for the confirmation of leptospirosis and the measurement of cytokines respectively. IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-α levels were found to be significantly higher in severe than in mild leptospirosis. There were equivocal findings on the association between IL-1β, TNF-α and IL-10/TNF-α ratio and disease severity. CONCLUSIONS: Leptospirosis had a wide-range of elevated cytokines. However, prospective studies in-relation to the onset of the symptom are required to better understand the pathophysiology of cytokine response in leptospirosis.

Acute respiratory distress syndrome subphenotypes and therapy responsive traits among preclinical models: protocol for a systematic review and meta-analysis

BACKGROUND: Subphenotypes were recently reported within clinical acute respiratory distress syndrome (ARDS), with distinct outcomes and therapeutic responses. Experimental models have long been used to mimic features of ARDS pathophysiology, but the presence of distinct subphenotypes among preclinical ARDS remains unknown. This review will investigate whether: 1) subphenotypes can be identified among preclinical ARDS models; 2) such subphenotypes can identify some responsive traits. METHODS: We will include comparative preclinical (in vivo and ex vivo) ARDS studies published between 2009 and 2019 in which pre-specified therapies were assessed (interleukin (IL)-10, IL-2, stem cells, beta-agonists, corticosteroids, fibroblast growth factors, modulators of the receptor for advanced glycation end-products pathway, anticoagulants, and halogenated agents) and outcomes compared to a control condition. The primary outcome will be a composite of the four key features of preclinical ARDS as per the American Thoracic Society consensus conference (histologic evidence of lung injury, altered alveolar-capillary barrier, lung inflammatory response, and physiological dysfunction). Secondary outcomes will include the single components of the primary composite outcome, net alveolar fluid clearance, and death. MEDLINE, Embase, and Cochrane databases will be searched electronically and data from eligible studies will be extracted, pooled, and analyzed using random-effects models. Individual study reporting will be assessed according to the Animal Research: Reporting of In Vivo Experiments guidelines. Meta-regressions will be performed to identify subphenotypes prior to comparing outcomes across subphenotypes and treatment effects. DISCUSSION: This study will inform on the presence and underlying pathophysiological features of subphenotypes among preclinical models of ARDS and should help to determine whether sufficient evidence exists to perform preclinical trials of subphenotype-targeted therapies, prior to potential clinical translation. SYSTEMATIC REVIEW REGISTRATION: PROSPERO (ID: CRD42019157236).

Diagnosis of severe scrub typhus infection by next-generation sequencing:a case report

BACKGROUND: Scrub typhus is an acute febrile illness, which was caused by Orientia tsutsugamushi and transmitted through the bite of chiggers. The diagnosis of scrub typhus could be missed diagnosis due to the absence of the pathognomonic eschar. CASE PRESENTATION: A 76-year-old man was hospitalized with fever and kidney injury and was diagnosed of hemorrhagic fever with renal syndrome first. However, the situation of the illness deteriorated into refractory septic shock and multiple organ dysfunction rapidly,although the treatment of anti-sepsis was used in 3rd-5th day. Orientia tsutsugamushi was determined to be the causative pathogen by Next-generation sequencing of his plasma sample in 6th day. Then, the patient was treated with doxycycline and azithromycin and recovered quickly. CONCLUSIONS: Next-generation sequencing was a new diagnostic technology and could identify scrub typhus in accurately and fast without the pathognomonic eschar.

Krebspatienten in der operativen Intensivmedizin

Cancer is one of the leading causes of death worldwide. New targeted and individualized therapies and drugs provide a survival benefit for an increasing number of patients, but can also cause severe side effects. An increasing number of oncology patients are admitted to intensive care units (ICU) because of cancer-related complications or treatment-associated side effects. Postoperative care, respiratory distress and sepsis are the leading causes for admission. Tumor mass syndromes and tumor lysis may require urgent treatment. Traditional anticancer chemotherapy is associated with infections and immunosuppression. Newer agents are generally well-tolerated and side effects are mild or moderate, but overwhelming inflammation and autoimmunity can also occur. Cellular treatment, such as with chimeric antigen receptor modified T‑cells, monoclonal and bispecific antibodies targeting immune effectors and tumor cells are associated with cytokine release syndrome (CRS) with hypotension, skin reactions and fever. It is related to excessively high levels of inflammatory cytokines. Immune checkpoint inhibitors can lead to immune-related adverse events (IRAEs), such as colitis and endocrine disorders. Noninfectious respiratory complications, such as pneumonitis can also occur. Recent studies revealed that short-term and medium-term survival of cancer patients is better than previously expected. In this review article we summarize diagnostic and treatment strategies for common life-threatening complications and emergencies requiring ICU admission. Furthermore, strategies for rational admission policies are presented.

Hämato-onkologische Intensivpatienten: Gefordert: Spezifisches Wissen der Intensivmediziner und Zusammenarbeit mit Onkologen

Die Lebenserwartung und Prävalenz von Krebserkrankungen steigt stetig an, was unweigerlich zu einer Zunahme an kritisch erkrankten Krebspatienten führt. Dieser Artikel erläutert, warum es in den letzten Jahrzehnten zu einer deutlichen Verbesserung der Prognose von intensivmedizinisch behandelten Krebspatienten kam, welche Gründe am häufigsten zur Aufnahme führen, und welche Risikofaktoren sich auf die Mortalität auswirken. Ferner wird die Wichtigkeit einer adäquaten Patientenselektion besprochen, sowie auf weitere Spezifika eingegangen. So bringt zum Beispiel das akute respiratorische Versagen als weitaus häufigste Organdysfunktion in dieser Patientengruppe sowohl prognostisch, diagnostisch, als auch therapeutisch etliche wichtige Besonderheiten mit sich. Die erfolgreiche Versorgung von Krebspatienten auf einer Intensivstation (ICU) setzt ein spezifisches Wissen der Intensivmediziner und eine gute Zusammenarbeit mit den behandelnden Hämatologen und Onkologen voraus.

Akutes Leberversagen: Zeitgerechte Diagnose und Therapie ist entscheidend für die Prognose

Acute liver failure (ALF) is a rare condition with fatal outcome. Characteristic is rapid onset of liver damage without preexisting liver diseases, including hepatic encephalopathy and coagulopathy. Early and correct diagnosis is essential for further management of patients, since diagnosis impacts therapy choice. Survival of patients with ALF has improved dramatically due to advances in critical care medicine and the use of liver transplantation.

Thrombopenie auf der Intensivstation: Diagnose, Differenzialdiagnose und Therapie

Thrombocytopenia is a frequent phenomenon in intensive care medicine. A variety of conditions can be responsible for low platelet counts. As platelets are part of the primary hemostatic system, bleeding is the most important complication of thrombocytopenia. Proper workup of the differential diagnosis to identify the cause of thrombocytopenia is essential because the various underlying disorders require different diagnostic and therapeutic management strategies. A low platelet count is a strong predictor of outcome in critically ill patients. This article summarizes the differential diagnosis and diagnostic workup of thrombocytopenia in the critically ill, describes the most important conditions, and gives an overview on therapeutic options and strategies.

Infectiepreventie vereist kennis van zaken

Besmetting op het werk kan een reëel gevaar voor de volksgezondheid betekenen. Tijdens de SARS- en kippeninfluenza-epidemie van vorig jaar, maar ook bij de voorjaarsinfluenza en de tbc-uitbraak in een supermarkt te Zeist van dit jaar, kwam duidelijk naar voren hoezeer volksgezondheid en werknemersgezondheid samenhangen. Bij SARS waren de eerste patiënten voornamelijk werknemers in de Chinese gezondheidszorg.

Efficient functional screening of a cellular cDNA library to identify severe fever with thrombocytopenia syndrome virus entry factors

The identification of host cell factors for virus entry is useful for the molecular explanation of viral tropisms and often leads to a more profound understanding of virus-induced diseases. Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus. No countermeasures against the disease exist. In this report, we show an efficient method using virus-like particles for the functional screening of a cellular cDNA library to identify SFTS virus entry factors. Two variants encoding dendritic cell-specific ICAM-3 grabbing non-integrin related (DC-SIGNR), a calcium-dependent lectin known to enhance SFTS virus infection, were successfully identified from a human liver cDNA library. We will discuss applications for yet unidentified factor(s) for SFTS virus entry and for entry factor(s) for other viruses related to SFTS virus.

Occurrence of pendelluft under pressure support ventilation in patients who failed a spontaneous breathing trial: an observational study

BACKGROUND: Pendelluft, the movement of gas within different lung regions, is present in animal models of assisted mechanical ventilation and associated with lung overstretching. Due to rebreathing of CO(2) as compared to fresh gas, pendelluft might reduce ventilatory efficiency possibly exacerbating patient’s respiratory workload during weaning. Our aim was to measure pendelluft by electrical impedance tomography (EIT) in patients who failed a spontaneous breathing trial (SBT). METHODS: This is an observational study conducted in a general intensive care unit of a tertiary-level teaching hospital. EIT signal was recorded in 20 patients while pressure support (PS) ventilation was progressively reduced from clinical level (baseline) to 2 cmH(2)O, as in an SBT; four ventral-to-dorsal lung regions of interest were identified for pendelluft measurement. A regional gas movement (> 6 mL) occurring in a direction opposite to the global EIT signal was considered diagnostic for high pendelluft. RESULTS: Eight patients out of 20 (40%) were classified as high-pendelluft; baseline clinical characteristics did not differ between high- and low-pendelluft patients. At PS reduction, pendelluft and EtCO(2) increased more in the high-pendelluft group (p < .001 and .011, respectively). The volume of gas subject to pendelluft moved almost completely from the ventral towards the dorsal lung regions, while the opposite movement was minimal (16.3 [10:32.8] vs. 0 [0:1.8] mL, p = .001). In a subgroup of patients, increased pendelluft volumes positively correlated with markers of respiratory distress such as increased respiratory rate, p0.1, and EtCO(2). CONCLUSIONS: Occult pendelluft can be measured by EIT, and is frequently present in patients failing an SBT. When present, pendelluft increases with the reduction of ventilator support and is associated with increased EtCO(2), suggesting a reduction of the ability to eliminate CO(2).

Thiopurine Derivative-Induced Fpg/Nei DNA Glycosylase Inhibition: Structural, Dynamic and Functional Insights

DNA glycosylases are emerging as relevant pharmacological targets in inflammation, cancer and neurodegenerative diseases. Consequently, the search for inhibitors of these enzymes has become a very active research field. As a continuation of previous work that showed that 2-thioxanthine (2TX) is an irreversible inhibitor of zinc finger (ZnF)-containing Fpg/Nei DNA glycosylases, we designed and synthesized a mini-library of 2TX-derivatives (TXn) and evaluated their ability to inhibit Fpg/Nei enzymes. Among forty compounds, four TXn were better inhibitors than 2TX for Fpg. Unexpectedly, but very interestingly, two dithiolated derivatives more selectively and efficiently inhibit the zincless finger (ZnLF)-containing enzymes (human and mimivirus Neil1 DNA glycosylases hNeil1 and MvNei1, respectively). By combining chemistry, biochemistry, mass spectrometry, blind and flexible docking and X-ray structure analysis, we localized new TXn binding sites on Fpg/Nei enzymes. This endeavor allowed us to decipher at the atomic level the mode of action for the best TXn inhibitors on the ZnF-containing enzymes. We discovered an original inhibition mechanism for the ZnLF-containing Fpg/Nei DNA glycosylases by disulfide cyclic trimeric forms of dithiopurines. This work paves the way for the design and synthesis of a new structural class of inhibitors for selective pharmacological targeting of hNeil1 in cancer and neurodegenerative diseases.

The Controversial Roles of ADP-Ribosyl Hydrolases MACROD1, MACROD2 and TARG1 in Carcinogenesis

Post-translational modifications (PTM) of proteins are crucial for fine-tuning a cell’s response to both intracellular and extracellular cues. ADP-ribosylation is a PTM, which occurs in two flavours: modification of a target with multiple ADP-ribose moieties (poly(ADP-ribosyl)ation or PARylation) or with only one unit (MARylation), which are added by the different enzymes of the PARP family (also known as the ARTD family). PARylation has been relatively well-studied, particularly in the DNA damage response. This has resulted in the development of PARP inhibitors such as olaparib, which are increasingly employed in cancer chemotherapeutic approaches. Despite the fact that the majority of PARP enzymes catalyse MARylation, MARylation is not as well understood as PARylation. MARylation is a dynamic process: the enzymes reversing intracellular MARylation of acidic amino acids (MACROD1, MACROD2, and TARG1) were discovered in 2013. Since then, however, little information has been published about their physiological function. MACROD1, MACROD2, and TARG1 have a ‘macrodomain’ harbouring the catalytic site, but no other domains have been identified. Despite the lack of information regarding their cellular roles, there are a number of studies linking them to cancer. However, some of these publications oppose each other, some rely on poorly-characterised antibodies, or on aberrant localisation of overexpressed rather than native protein. In this review, we critically assess the available literature on a role for the hydrolases in cancer and find that, currently, there is limited evidence for a role for MACROD1, MACROD2, or TARG1 in tumorigenesis.

Hepatitis C Virus Entry: An Intriguingly Complex and Highly Regulated Process

Hepatitis C virus (HCV) is a major cause of chronic hepatitis and liver disease worldwide. Its tissue and species tropism are largely defined by the viral entry process that is required for subsequent productive viral infection and establishment of chronic infection. This review provides an overview of the viral and host factors involved in HCV entry into hepatocytes, summarizes our understanding of the molecular mechanisms governing this process and highlights the therapeutic potential of host-targeting entry inhibitors.

Virus-induced pathogenesis, vaccine development, and diagnosis of novel H7N9 avian influenza A virus in humans: a systemic literature review

H7N9 avian influenza virus (AIV) caused human infections in 2013 in China. Phylogenetic analyses indicate that H7N9 AIV is a novel reassortant strain with pandemic potential. We conducted a systemic review regarding virus-induced pathogenesis, vaccine development, and diagnosis of H7N9 AIV infection in humans. We followed PRISMA guidelines and searched PubMed, Web of Science, and Google Scholar to identify relevant articles published between January 2013 and December 2018. Pathogenesis data indicated that H7N9 AIV belongs to low pathogenic avian influenza, which is mostly asymptomatic in avian species; however, H7N9 induces high mortality in humans. Sporadic human infections have recently been reported, caused by highly pathogenic avian influenza viruses detected in poultry. H7N9 AIVs resistant to adamantine and oseltamivir cause severe human infection by rapidly inducing progressive acute community-acquired pneumonia, multiorgan dysfunction, and cytokine dysregulation; however, mechanisms via which the virus induces severe syndromes remain unclear. An H7N9 AIV vaccine is lacking; designs under evaluation include synthesized peptide, baculovirus-insect system, and virus-like particle vaccines. Molecular diagnosis of H7N9 AIVs is suggested over conventional assays, for biosafety reasons. Several advanced or modified diagnostic assays are under investigation and development. We summarized virus-induced pathogenesis, vaccine development, and current diagnostic assays in H7N9 AIVs.

Hirnstammenzephalitis eines 8-jährigen Mädchens: Seltene Todesursache

Encephalitis is often caused by viral infections and can affect various brain regions. The clinical symptoms are highly variable. Oligosymptomatic cerebral infections may remain undetected but there are also cases with very severe symptoms (e.g. paralysis, convulsions). This article presents the case of an 8-year-old girl who suffered from headaches and vomiting for several days following a harmless fall with impact on the back of the head. The health condition decreased rapidly and 7 days after the fall the girl was presented to a children’s hospital. After a further foudroyant course of the disease the girl was hospitalized 2 days later. Resuscitation became necessary due to convulsions and pulmonary obstruction. Cranial computed tomography (cCT) revealed temporal hypodense areas and local edema. Cardiac echocardiography revealed a decreased left ventricular pump function so that encephalitis and myocarditis were suspected. Despite antiviral and antibiotic therapy the girl died 4 days after admission due to intravital brain death and multiorgan failure. This cause of death was confirmed by forensic autopsy. The fall had not led to any intracranial injury and predisposing diseases were not found. Histological examination revealed necrotizing brainstem encephalitis.

Local orders in international organisations: the World Health Organization's global programme on AIDS

In 1990, the World Health Organization (WHO) started to downsize its renowned Global Programme on AIDS, despite continued donor and member state support. This turnaround has decisively contributed to WHO's loss of leadership in HIV/AIDS politics. From the viewpoint of both rationalist and constructivist theories of international organisation (IO) agency, an IO engaging in ‘mission shrink’ is a striking irregularity. In order to account for such apparently self-defeating behaviour, this article adopts an open systems view of IOs and identifies trans-organisational coalitions as important agents of IO change. I argue that subunit dynamics rather than systemic conditions drive IO behaviour, in particular where member states’ material power and their formal control of organisational veto positions do not coincide. This approach will be used to retrace the changes in subunit coalitions that drove WHO's erratic HIV/AIDS programme and thus to solve this puzzle of ‘mission shrink’. On the basis of insights from the WHO case, the article concludes by offering a heuristic of trans-organisational coalitions and the types of IO change associated with them.

Is universal access to antiretroviral drugs an emerging international norm?

The international community appears to have embraced a new norm — that of universal access to antiretroviral drugs. The process by which this norm has found acceptance raises interesting questions about how norm entrepreneurs frame their arguments, the role of non-state actors in realizing a norm, and the importance of existent complementary norms. To understand the success of the norm of universal antiretroviral access, I examine the failure of an earlier health-related norm — that of universal primary health care. The campaign for universal antiretroviral access points to a need for a more nuanced understanding of norm evolution within the international community and a more holistic vision of which actors can facilitate the realization of a norm.

Diagnose akuter Transfusionsreaktionen

The erroneous transfusion of AB0-incompatible red cells may result in hemolysis and complement-mediated shock (acute hemolytic transfusion reaction, AHTR), leading to death in less than 10%. The mistake will be detected by re-checking the patient’s and the blood product’s identity. Evidence of the incompatible transfusion is supplied by serology and, in cases of a fatal outcome, by immunohistochemistry. Differential diagnoses to be distinguished from AHTR are other immunologically mediated events like the most important transfusion-related acute lung injury (TRALI), febrile non-hemolytic transfusion reaction (FNHTR), allergic transfusion reactions, along with a variety of nonimmunologic incidents like transfusion-transmitted bacterial infections, hypervolemia, and other rare events such as citrate reaction, air embolism, and foreign body embolism. If the outcome is lethal, the question of causality has to be answered by a comprehensive evaluation including the clinical data as well as serological, microbiological, autoptic, and histological findings.

Diagnostik granulomatöser Erkrankungen mit Lungenbefall

Granulome als Zeichen der spezifischen Entzündung im Lungengewebe treten bei vielen Erkrankungen auf. Das radiologische Standardverfahren bei pulmonalen Granulomatosen ist meistens die Dünnschichtcomputertomografie. Bei klinischem Verdacht und entsprechenden Hinweisen in der Thoraxübersicht sind eine Dünnschicht-CT und die interdisziplinäre Diskussion der Befunde zu empfehlen.

Effect of initial infusion rates of fluid resuscitation on outcomes in patients with septic shock: a historical cohort study

BACKGROUND: Fluid resuscitation has become the cornerstone of early septic shock management, but the optimal fluid rate is still not well studied. The goal of this investigation is to examine the relationship between fluid resuscitation rate and septic shock resolution. METHOD: We retrospectively studied adult (≥ 18 years) patients with septic shock, defined based on sepsis III definition, from January 1, 2006, through May 31, 2018, in the medical intensive care unit (MICU) of Mayo Clinic Rochester. The fluid resuscitation time was defined as the time required to infuse the initial fluid bolus of 30 ml/kg, based on the recommendations of the 2016 surviving sepsis campaign. The cohort was divided into four groups based on the average fluid rate (group 1 ≥ 0.5, group 2 0.25–0.49, group 3 0.17–0.24, and group 4 < 0.17 ml/kg/min). The primary outcome was the time to shock reversal. Multivariable regression analyses were conducted to account for potential confounders. RESULT: A total of 1052 patients met eligibility criteria and were included in the analysis. The time-to-shock reversal was significantly different among the groups (P < .001). Patients in group 1 who received fluid resuscitation at a faster rate had a shorter time to shock reversal (HR = 0.78; 95% CI 0.66–0.91; P = .01) when compared with group 4 with a median (IQR) time-to-shock reversal of 1.7 (1.5, 2.0) vs. 2.8 (2.6, 3.3) days, respectively. Using 0.25 ml/kg/min as cutoff, the higher fluid infusion rate was associated with a shorter time to shock reversal (HR = 1.22; 95% CI 1.06–1.41; P = .004) and with decreased odds of 28-day mortality (HR = 0.71; 95% CI 0.60–0.85; P < .001). CONCLUSION: In septic shock patients, initial fluid resuscitation rate of 0.25–0.50 ml/kg/min (i.e., completion of the initial 30 ml/kg IV fluid resuscitation within the first 2 h), may be associated with early shock reversal and lower 28-day mortality compared with slower rates of infusion.

Tumor microenvironment complexity and therapeutic implications at a glance

The dynamic interactions of cancer cells with their microenvironment consisting of stromal cells (cellular part) and extracellular matrix (ECM) components (non-cellular) is essential to stimulate the heterogeneity of cancer cell, clonal evolution and to increase the multidrug resistance ending in cancer cell progression and metastasis. The reciprocal cell-cell/ECM interaction and tumor cell hijacking of non-malignant cells force stromal cells to lose their function and acquire new phenotypes that promote development and invasion of tumor cells. Understanding the underlying cellular and molecular mechanisms governing these interactions can be used as a novel strategy to indirectly disrupt cancer cell interplay and contribute to the development of efficient and safe therapeutic strategies to fight cancer. Furthermore, the tumor-derived circulating materials can also be used as cancer diagnostic tools to precisely predict and monitor the outcome of therapy. This review evaluates such potentials in various advanced cancer models, with a focus on 3D systems as well as lab-on-chip devices.

Interferon-β Stimulation Elicited by the Influenza Virus Is Regulated by the Histone Methylase Dot1L through the RIG-I-TRIM25 Signaling Axis

Influenza virus infection increases the methylation of lysine 79 of histone 3 catalyzed by the Dot1L enzyme. The role of Dot1L against infections was highlighted by an increase of influenza A and vesicular stomatitis virus replication in Dot1L-inhibited cells mediated by a decreased antiviral response. Interferon-beta (IFN-β) reporter assays indicate that Dot1L is involved in the control of retinoic acid-inducible gene-I protein (RIG-I) signaling. Accordingly, Dot1L inhibition decreases the IFN-β promoter stimulation and RIG-I- mitochondria-associated viral sensor (RIG-I-MAVS) association upon viral infection. Replication of an influenza A virus lacking NS1 (delNS1), incapable of counteracting the antiviral response, is not affected by Dot1L inhibition. Consequently, RIG-I-MAVS association and nuclear factor–κB (NF-κB) nuclear translocation, are not affected by the Dot1L inhibition in delNS1 infected cells. Restoration of NS1 expression in trans also reinstated Dot1L as a regulator of the RIG-I-dependent signaling in delNS1 infections. Interferon-inducible E3 ligase tripartite motif-containing protein 25 (TRIM25) expression increases in influenza virus infected cells, but Dot1L inhibition reduces both the TRIM25 expression and TRIM25 protein levels. TRIM25 overexpression reverses the defective innate response mediated by Dot1L inhibition elicited upon virus infection or by overexpression of RIG-I signaling intermediates. Thus, TRIM25 is a control point of the RIG-I recognition pathway controlled by Dot1L and may have a general role in RNA viruses recognized by the RIG-I sensor.

Burden of Community-Acquired Pneumonia and Unmet Clinical Needs

Community-acquired pneumonia (CAP) is the leading cause of death among infectious diseases and an important health problem, having considerable implications for healthcare systems worldwide. Despite important advances in prevention through vaccines, new rapid diagnostic tests and antibiotics, CAP management still has significant drawbacks. Mortality remains very high in severely ill patients presenting with respiratory failure or shock but is also high in the elderly. Even after a CAP episode, higher risk of death remains during a long period, a risk mainly driven by inflammation and patient-related co-morbidities. CAP microbiology has been altered by new molecular diagnostic tests that have turned viruses into the most identified pathogens, notwithstanding uncertainties about the specific role of each virus in CAP pathogenesis. Pneumococcal vaccines also impacted CAP etiology and thus had changed Streptococcus pneumoniae circulating serotypes. Pathogens from specific regions should also be kept in mind when treating CAP. New antibiotics for CAP treatment were not tested in severely ill patients and focused on multidrug-resistant pathogens that are unrelated to CAP, limiting their general use and indications for intensive care unit (ICU) patients. Similarly, CAP management could be personalized through the use of adjunctive therapies that showed outcome improvements in particular patient groups. Although pneumococcal vaccination was only convincingly shown to reduce invasive pneumococcal disease, with a less significant effect in pneumococcal CAP, it remains the best therapeutic intervention to prevent bacterial CAP. Further research in CAP is needed to reduce its population impact and improve individual outcomes.

Post-transplantation Outcomes in Patients with PA or MMA: A Review of the Literature

INTRODUCTION: Liver transplantation is recognised as a treatment option for patients with propionic acidemia (PA) and those with methylmalonic acidemia (MMA) without renal impairment. In patients with MMA and moderate-to-severe renal impairment, combined liver–kidney transplantation is indicated. However, clinical experience of these transplantation options in patients with PA and MMA remains limited and fragmented. We undertook an overview of post-transplantation outcomes in patients with PA and MMA using the current available evidence. METHODS: A literature search identified publications on the use of transplantation in patients with PA and MMA. Publications were considered if they presented adequate demographic and outcome data from patients with PA or MMA. Publications that did not report any specific outcomes for patients or provided insufficient data were excluded. RESULTS: Seventy publications were identified of which 38 were full papers. A total of 373 patients underwent liver/kidney/combined liver–kidney transplantation for PA or MMA. The most typical reason for transplantation was recurrent metabolic decompensation. A total of 27 post-transplant deaths were reported in patients with PA [14.0% (27/194)]. For patients with MMA, 18 post-transplant deaths were reported [11% (18/167)]. A total of 62 complications were reported in 115 patients with PA (54%) with cardiomyopathy (n = 12), hepatic arterial thrombosis (HAT; n = 14) and viral infections (n = 12) being the most commonly reported. A total of 52 complications were reported in 106 patients with MMA (49%) with viral infections (n = 14) and renal failure/impairment (n = 10) being the most commonly reported. CONCLUSIONS: Liver transplantation and combined liver–kidney transplantation appears to benefit some patients with PA or MMA, respectively, but this approach does not provide complete correction of the metabolic defect and some patients remain at risk from disease-related and transplantation-related complications, including death. Thus, all treatment avenues should be exhausted before consideration of organ transplantation and the benefits of this approach must be weighed against the risk of perioperative complications on an individual basis.

Molecular Mechanisms for the Mechanical Modulation of Airway Responsiveness

The smooth muscle of the airways is exposed to continuously changing mechanical forces during normal breathing. The mechanical oscillations that occur during breathing have profound effects on airway tone and airway responsiveness both in experimental animals and humans in vivo and in isolated airway tissues in vitro. Experimental evidence suggests that alterations in the contractile and mechanical properties of airway smooth muscle tissues caused by mechanical perturbations result from adaptive changes in the organization of the cytoskeletal architecture of the smooth muscle cell. The cytoskeleton is a dynamic structure that undergoes rapid reorganization in response to external mechanical and pharmacologic stimuli. Contractile stimulation initiates the assembly of cytoskeletal/extracellular matrix adhesion complex proteins into large macromolecular signaling complexes (adhesomes) that undergo activation to mediate the polymerization and reorganization of a submembranous network of actin filaments at the cortex of the cell. Cortical actin polymerization is catalyzed by Neuronal-Wiskott–Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, which are activated by pathways regulated by paxillin and the small GTPase, cdc42. These processes create a strong and rigid cytoskeletal framework that may serve to strengthen the membrane for the transmission of force generated by the contractile apparatus to the extracellular matrix, and to enable the adaptation of smooth muscle cells to mechanical stresses. This model for the regulation of airway smooth muscle function can provide novel perspectives to explain the normal physiologic behavior of the airways and pathophysiologic properties of the airways in asthma.

High serum procalcitonin concentrations in patients with sepsis and infection

High concentrations of calcitonin-like immunoreactivity have been found in the blood of patients with various extrathyroid diseases. By means of a monoclonal immunoradiometric assay for calcitonin precursors, we have measured serum concentrations of procalcitonin in patients with various bacterial and viral infections. 79 children (newborn to age 12 years) in hospital with suspected infections were investigated prospectively. 19 patients with severe bacterial infections had very high serum concentrations of procalcitonin at diagnosis (range 6-53 ng/mL) in comparison with 21 children found to have no signs of infection (baseline concentrations <0·1 ng/mL). Serum procalcitonin values decreased rapidly during antibiotic therapy. 11 patients with peripheral bacterial colonisation or local infections without invasive sepsis and 18 (86%) of 21 patients with viral infections had concentrations within or slightly above the normal range (0·1-1·5 ng/mL). Among 9 severely burned patients studied in an intensive care unit, the post-traumatic course of procalcitonin concentrations (range 0·1-120 ng/mL) was closely related to infectious complications and acute septic episodes. Concentrations of mature calcitonin were normal in all subjects, whatever procalcitonin concentrations were found. Concentrations of a substance immunologically identical to procalcitonin are raised during septic conditions. Serum concentrations seem to be correlated with the severity of microbial invasion.

Route of inoculation and mosquito vector exposure modulate dengue virus replication kinetics and immune responses in rhesus macaques

Dengue virus (DENV) is transmitted by infectious mosquitoes during blood-feeding via saliva containing biologically-active proteins. Here, we examined the effect of varying DENV infection modality in rhesus macaques in order to improve the DENV nonhuman primate (NHP) challenge model. NHPs were exposed to DENV-1 via subcutaneous or intradermal inoculation of virus only, intradermal inoculation of virus and salivary gland extract, or infectious mosquito feeding. The infectious mosquito feeding group exhibited delayed onset of viremia, greater viral loads, and altered clinical and immune responses compared to other groups. After 15 months, NHPs in the subcutaneous and infectious mosquito feeding groups were re-exposed to either DENV-1 or DENV-2. Viral replication and neutralizing antibody following homologous challenge were suggestive of sterilizing immunity, whereas heterologous challenge resulted in productive, yet reduced, DENV-2 replication and boosted neutralizing antibody. These results show that a more transmission-relevant exposure modality resulted in viral replication closer to that observed in humans.

Polymorphisms and drug resistance analysis of HIV-1 isolates from patients on first line antiretroviral therapy (ART) in South-eastern Nigeria

Acquisition of resistance mutations by HIV-1 isolates causes treatment failure among infected patients receiving antiretroviral therapy (ART). This study determined patterns of drug-resistance mutations (DRMs) among HIV-1 isolates from patients receiving first-line ART in South-eastern Nigeria. Blood samples were collected from HIV-1 infected patients accessing antiretroviral treatment centers at General Hospital Awo-Omamma, Imo state, State Hospital Asaba, Delta state and St Joseph’s Catholic Hospital Adazi, Anambra state and used for HIV-1 DNA sequencing and phylogenetic analysis. DRMs were scored using combination of Stanford algorithm and the 2015 International Antiviral Society-USA list while drug susceptibility was predicted using Stanford algorithm. Twenty eight of the HIV-1 isolates were sequenced and identified as subtypes G (35.7%), CRF02_AG (57.1%) and unclassifiable, U(G) (7.1%). Major PI resistance-associated mutations were identified at two sites including M46L (16.7% of subtype G/U(G)) and V82L (6.3% of CRF02_AG). Minor PI resistance-associated mutations identified among subtype G/U(G) are L10V/I (8.3%) and K20I (100%) while L10V/I (50%), K20I (100%), L33F (6.3%) and N88D (6.3%) were identified among CRF02_AG. Other polymorphisms found include; I13V/A, E35Q, M36I/L, N37D/S/E/H, R57K/G, L63T/P/S/Q, C67E/S, H69K/R, K70R, V82I and L89M in the range of 28.6% to 100% among the different subtypes. Interpretation based on Stanford algorithm showed that Darunavir/ritonavir is the only regimen whose potency was not compromised by the circulating mutations. Identification of major and minor PI resistance mutations in this study underscores the need for drug resistance testing prior to initiation of second line antiretroviral therapy in Nigeria.

Digital Image Analysis of Heterogeneous Tuberculosis Pulmonary Pathology in Non-Clinical Animal Models using Deep Convolutional Neural Networks

Efforts to develop effective and safe drugs for treatment of tuberculosis require preclinical evaluation in animal models. Alongside efficacy testing of novel therapies, effects on pulmonary pathology and disease progression are monitored by using histopathology images from these infected animals. To compare the severity of disease across treatment cohorts, pathologists have historically assigned a semi-quantitative histopathology score that may be subjective in terms of their training, experience, and personal bias. Manual histopathology therefore has limitations regarding reproducibility between studies and pathologists, potentially masking successful treatments. This report describes a pathologist-assistive software tool that reduces these user limitations, while providing a rapid, quantitative scoring system for digital histopathology image analysis. The software, called ‘Lesion Image Recognition and Analysis’ (LIRA), employs convolutional neural networks to classify seven different pathology features, including three different lesion types from pulmonary tissues of the C3HeB/FeJ tuberculosis mouse model. LIRA was developed to improve the efficiency of histopathology analysis for mouse tuberculosis infection models, this approach has also broader applications to other disease models and tissues. The full source code and documentation is available from https://Github.com/TB-imaging/LIRA.

Lipid moieties of Mycoplasma pneumoniae lipoproteins are the causative factor of vaccine-enhanced disease

Vaccine-enhanced disease (VED) occurs as a result of vaccination followed by infection with virulent Mycoplasma pneumoniae. To date VED has prevented development of an efficacious vaccine against this significant human respiratory pathogen. Herein we report that vaccination of BALB/c mice with M. pneumoniae lipid-associated membrane proteins (LAMPs) induces lung lesions consistent with exacerbated disease following challenge, without reducing bacterial loads. Removal of lipid moieties from LAMPs prior to vaccination eliminates VED and reduces bacterial loads after infection. Collectively, these data indicate that lipid moieties of lipoproteins are the causative factors of M. pneumoniae VED.

Non-Anticoagulant Heparins as Heparanase Inhibitors

The chapter will review early and more recent seminal contributions to the discovery and characterization of heparanase and non-anticoagulant heparins inhibiting its peculiar enzymatic activity. Indeed, heparanase displays a unique versatility in degrading heparan sulfate chains of several proteoglycans expressed in all mammalian cells. This endo-β-D-glucuronidase is overexpressed in cancer, inflammation, diabetes, atherosclerosis, nephropathies and other pathologies. Starting from known low- or non-anticoagulant heparins, the search for heparanase inhibitors evolved focusing on structure-activity relationship studies and taking advantage of new chemical-physical analytical methods which have allowed characterization and sequencing of polysaccharide chains. New methods to screen heparanase inhibitors and to evaluate their mechanism of action and in vivo activity in experimental models prompted their development. New non-anticoagulant heparin derivatives endowed with anti-heparanase activity are reported. Some leads are under clinical evaluation in the oncology field (e.g., acute myeloid leukemia, multiple myeloma, pancreatic carcinoma) and in other pathological conditions (e.g., sickle cell disease, malaria, labor arrest).

Two Weeks

These 2 weeks of attending are ones that I will never forget, but I worry about what the next 2 weeks will bring.

Comparative neutralizing potencies of antibodies suggest conservation as well as mechanistic differences in human cytomegalovirus entry into epithelial and endothelial cells

Antibody neutralization of cytomegalovirus (CMV) entry into diverse cell types is a key consideration for development of vaccines and immunotherapeutics. CMV entry into fibroblasts differs significantly from entry into epithelial or endothelial cells: fibroblast entry is mediated by gB and gH/gL/gO, whereas both epithelial and endothelial cell entry require an additional pentameric complex (PC) comprised of gH/gL/UL128/UL130/UL131A. Because PC-specific antibodies in CMV-seropositive human sera do not affect fibroblast entry but potently block entry into epithelial or endothelial cells, substantially higher neutralizing potencies for CMV-positive sera are observed when assayed using epithelial cells as targets than when using fibroblasts. That certain sera exhibit similar discordances between neutralizing potencies measured using epithelial vs. endothelial cells (Gerna G. et al.J Gen Virol, 89:853–865, 2008) suggested that additional mechanistic differences may also exist between epithelial and endothelial cell entry. To further explore this issue, neutralizing potencies using epithelial and endothelial cells were simultaneously determined for eight CMV-positive human sera, CMV-hyperimmune globulin, and a panel of monoclonal or anti-peptide antibodies targeting specific epitopes in gB, gH, gH/gL, or the PC. No significant differences were observed between epithelial and endothelial neutralizing potencies of epitope-specific antibodies, CMV-hyperimmune globulin, or seven of the eight human sera. However, one human serum exhibited a six-fold higher potency for neutralizing entry into epithelial cells vs. endothelial cells. These results suggest that epitopes exist that are important for epithelial entry but are less critical, or perhaps dispensable, for endothelial cell entry. Their existence should be considered when developing monoclonal antibody therapies or subunit vaccines representing limited epitopes.

Cytokine Polymorphisms and Susceptibility to Severe Infectious Diseases

Cytokines are key regulators of the host response to infection, controlling the inflammatory reaction which is an essential component of the defense mechanisms. The major importance of these proteins in the pathogenesis and outcome of infectious diseases has been clearly demonstrated. In humans, there is increasing evidence that the host's cytokine response is genetically determined and that the genetic variability of cytokines underlies the complexity of interindividual differences in the immune response to micro organism invasions. We review the influence of host cytokine gene on the susceptibility to and the severity of parasitic, bacterial and viral infections. Proinflammatory cytokine polymorphisms are discussed in detail because of their importance in the course of severe infections such as meningococcal disease, cerebral malaria and septic shock. Genetic variants of the IL-10 gene, an antiinflammatory cytokine appear to be responsible for an uncontrolled and intense CARS and may have also dramatic consequences as an overwhelming inflammatory response. Our greater understanding of the genetic factors that influence mortality and morbidity of infectious diseases will permit identification of genomic markers which may be required for risk stratification of patients targeted for novel immunomodulatory treatments helping clinicians to select the most appropriate treatment options for their patients.

Treatment of Argentine hemorrhagic fever

Argentine hemorrhagic fever (AHF) is a rodent-borne illness caused by the arenavirus Junin that is endemic to the humid pampas of Argentina. AHF has had significant morbidity since its emergence in the 1950s, with a case-fatality rate of the illness without treatment between 15% and 30%. The use of a live attenuated vaccine has markedly reduced the incidence of AHF. Present specific therapy involves the transfusion of immune plasma in defined doses of neutralizing antibodies during the prodromal phase of illness. However, alternative forms of treatment are called for due to current difficulties in early detection of AHF, related to its decrease in incidence, troubles in maintaining adequate stocks of immune plasma, and the absence of effective therapies for severely ill patients that progress to a neurologic–hemorrhagic phase. Ribavirin might be a substitute for immune plasma, provided that the supply is guaranteed. Immune immunoglobulin or monoclonal antibodies should also be considered. New therapeutic options such as those being developed for systemic inflammatory syndromes should also be valuated in severe forms of AHF.

Real-time kinetics and high-resolution melt curves in single-molecule digital LAMP to differentiate and study specific and non-specific amplification

Isothermal amplification assays, such as loop-mediated isothermal amplification (LAMP), show great utility for the development of rapid diagnostics for infectious diseases because they have high sensitivity, pathogen-specificity and potential for implementation at the point of care. However, elimination of non-specific amplification remains a key challenge for the optimization of LAMP assays. Here, using chlamydia DNA as a clinically relevant target and high-throughput sequencing as an analytical tool, we investigate a potential mechanism of non-specific amplification. We then develop a real-time digital LAMP (dLAMP) with high-resolution melting temperature (HRM) analysis and use this single-molecule approach to analyze approximately 1.2 million amplification events. We show that single-molecule HRM provides insight into specific and non-specific amplification in LAMP that are difficult to deduce from bulk measurements. We use real-time dLAMP with HRM to evaluate differences between polymerase enzymes, the impact of assay parameters (e.g. time, rate or florescence intensity), and the effect background human DNA. By differentiating true and false positives, HRM enables determination of the optimal assay and analysis parameters that leads to the lowest limit of detection (LOD) in a digital isothermal amplification assay.

New Cysteine Protease Inhibitors: Electrophilic (Het)arenes and Unexpected Prodrug Identification for the Trypanosoma Protease Rhodesain

Electrophilic (het)arenes can undergo reactions with nucleophiles yielding π- or Meisenheimer (σ-) complexes or the products of the S(N)Ar addition/elimination reactions. Such building blocks have only rarely been employed for the design of enzyme inhibitors. Herein, we demonstrate the combination of a peptidic recognition sequence with such electrophilic (het)arenes to generate highly active inhibitors of disease-relevant proteases. We further elucidate an unexpected mode of action for the trypanosomal protease rhodesain using NMR spectroscopy and mass spectrometry, enzyme kinetics and various types of simulations. After hydrolysis of an ester function in the recognition sequence of a weakly active prodrug inhibitor, the liberated carboxylic acid represents a highly potent inhibitor of rhodesain (K(i) = 4.0 nM). The simulations indicate that, after the cleavage of the ester, the carboxylic acid leaves the active site and re-binds to the enzyme in an orientation that allows the formation of a very stable π-complex between the catalytic dyad (Cys-25/His-162) of rhodesain and the electrophilic aromatic moiety. The reversible inhibition mode results because the S(N)Ar reaction, which is found in an alkaline solvent containing a low molecular weight thiol, is hindered within the enzyme due to the presence of the positively charged imidazolium ring of His-162. Comparisons between measured and calculated NMR shifts support this interpretation.

Genetic responses of inbred chicken lines illustrate importance of eIF2 family and immune-related genes in resistance to Newcastle disease virus

Newcastle disease virus (NDV) replication depends on the translation machinery of the host cell; therefore, the eukaryotic translation initiation factor 2 (eIF2) gene family is a likely candidate for control of viral replication. We hypothesized that differential expression of host genes related to translation and innate immune response could contribute to differential resistance to NDV in inbred Fayoumi and Leghorn lines. The expression of twenty-one genes related to the interferon signaling pathway and the eIF2 family was evaluated at two- and six-days post infection (dpi) in the spleen from both lines, either challenged by NDV or nonchallenged. Higher expression of OASL in NDV challenged versus nonchallenged spleen was observed in Leghorns at 2 dpi. Lower expression of EIF2B5 was found in NDV challenged than nonchallenged Fayoumis and Leghorns at 2 dpi. At 2 dpi, NDV challenged Fayoumis had lower expression of EIF2B5 and EIF2S3 than NDV challenged Leghorns. At 6 dpi, NDV challenged Fayoumis had lower expression of EIF2S3 and EIF2B4 than NDV challenged Leghorns. The genetic line differences in expression of eIF2-related genes may contribute to their differential resistance to NDV and also to understanding the interaction between protein synthesis shut-off and virus control in chickens.

Cancer neoantigen prioritization through sensitive and reliable proteogenomics analysis

Genomics-based neoantigen discovery can be enhanced by proteomic evidence, but there remains a lack of consensus on the performance of different quality control methods for variant peptide identification in proteogenomics. We propose to use the difference between accurately predicted and observed retention times for each peptide as a metric to evaluate different quality control methods. To this end, we develop AutoRT, a deep learning algorithm with high accuracy in retention time prediction. Analysis of three cancer data sets with a total of 287 tumor samples using different quality control strategies results in substantially different numbers of identified variant peptides and putative neoantigens. Our systematic evaluation, using the proposed retention time metric, provides insights and practical guidance on the selection of quality control strategies. We implement the recommended strategy in a computational workflow named NeoFlow to support proteogenomics-based neoantigen prioritization, enabling more sensitive discovery of putative neoantigens.

Verlauf und prognostische Parameter bei Still-Syndrom des Erwachsenen: Eigene Erfahrungen und Literaturübersicht

□ PATIENTS AND METHODS: Ten patients with adult onset of Still’s disease (AOSD) were examined one to nine years after the established diagnosis. Clinical symptoms, laboratory parameters and the outcome of the cases are presented and compared to international literature and to Yamagushi’s in 1992 proposed diagnostic criteria. Nine patients were reexamined in our out-patient clinic. The chart of one additional patient, who died 10 month after the initial symptoms was also available for data analysis. Retrospectively, it was investigated whether any parameters were predictive for a chronic or severe form of the disease. □ RESULTS: One patient died 10 month after the diagnosis was established due to a secondary haemophagozytic syndrome. One patient developed a chronic form of the disease, whereas 2 patients had a chronic-remitting form. Six patients presented a self-limiting, shorter than 12 month lasting course of AOSD with a restitutio ad integrum. All patients fulfilled the diagnostic criteria of Yamagushi et al. Three of 10 patients developed a chronic form of AOSD, compared to up to 70% of the patients reported by others. The patient who died was significantly older (46 years) than the average age (24,9 years) of all patients. Interestingly, he did not present Still’s rash or lymphadenopathy, but rather developed a secondary hemophagocytic syndrome with an excessive hyperferritinaemia. □ CONCLUSION: Predicting parameters for a chronic course of the disease could not be found. Each patient’s diagnosis retrospectively could be confirmed using the Yamagushi’s diagnostic criteria. Thus, these criteria appear helpful in the difficult diagnostic process of this disease.

Die Transplantation hämatopoetischer Stammzellen: Teil I: Definitionen, prinzipielle Anwendungsmöglichkeiten, Komplikationen

The transplantation of hematopoietic and lymphopoetic stem and progenitor cells has become a standard procedure for the treatment of many malignant diseases. Autologous stem cells are derived from the patient himself, allogeneic cells from an HLA-identical or HLA-compatible family or unrelated donor. Hematopoetic stem cells can be obtained from bone marrow, blood and fetal cord blood. After 3 to 5 days treatment, the granulocyte-colony stimulating factor (G-CSF) mobilizes stem- and progenitor cells from the marrow into the blood. This method is now standard in autologous transplantation and is increasingly preferred in allogeneic transplantation. The time to hematopoietic recovery is shorter with blood stem cells than with bone marrow cells. With myeloablative high dose therapy followed by stem cell transplantation, long term disease free survival is possible in many cases and great proportions of patients can be cured (see part II). Improvements of supportive care have reduced toxicity of treatment substantially, however severe complications still occur at oropharynx, gastrointestinal tract, liver, lung, skin, kidney, urinary tract and nervous system. After allogeneic transplantation immunocompetent donor cells can react with the recipients tissue. In HLA-identical donor and recipients differences in the minor histocompatibility antigens account for this graft-versus-host-reaction (GvH), which is mainly mediated by transplanted T-cells. The GvH-reaction can affect skin, liver, gut and other organs and cause clinically relevant GvH-disease (GvHD). The GvHD is more severe in HLA-mis-matched or unrelated transplantations. Immunodeficiency and organ dysfunction due to GvHD may predispose to life threatening infections and impair the outcome of transplantion. Unrelated cord blood stem cells may have a minor risk of inducing acute GvHD, as stem and T-cells are immature. After allogeneic stem cell transplantation, the relapse rate of leukemia or lymphoma is significantly reduced by immunoreactive cells: graft-versus-tumor (GvT) or graftversus-leukemia effect (GvL).

Total-body irradiation before bone marrow transplantation for acute leukemia in first or second complete remission: Results and prognostic factors in 326 consecutive patients

AIM: In order to assess the influence of total-body irradiation (TBI) on the outcome and incidence of complication after bone marrow transplantation (BMT), we retrospectively analyzed our patients treated for acute leukemia and conditioned with TBI prior to BMT. PATIENTS AND METHODS: Between 1980 and 1993, 326 patients referred to our department with acute non-lymphoblastic leukemia (ANLL, n=182) and acute lymphoblastic leukemia (ALL, n=144) in complete remission underwent TBI either in single dose (190 patients: 10 Gy administered to the midplane, and 8 Gy to the lungs [STBI]) or in 6 fractions (136 patients: 12 Gy on 3 consecutive days, and 9 Gy to the lungs [FTBI]) before BMT. The male-to-female ratio was 204/122 (1.67), and the median age was 30 years (mean: 30 ± 11, range: 3 to 63). The patients were analyzed according to 3 instantaneous dose rate groups: 118 patients in the LOW group (≤0.048 Gy/min), 188 in the MEDIUM group (> 0.048 and ≤ 0.09 Gy/min), and 20 in the HIGH group (> 0.09 cGy/min). Conditioning chemotherapy consisted of cyclophosphamide (CY) alone in 250 patients, CY and other drugs in 54, and 22 patients were conditioned using combinations without CY. Following TBI, allogeneic and autologous BMT were realized respectively in 118 and 208 patients. Median follow-up period was 68 months (mean: 67 ± 29, range: 24 to 130 months). RESULTS: Five-year survival, LFS, RI and TRM rates were 42%, 40%, 47%, and 24%, respectively. Five-year LFS was 36% in the STBI and 45% in the FTBI group (p = 0.17). It was 36% in the LOW group, 42% in the MEDIUM group, and 30% in the HIGH group (p > 0.05). Five-year RI was 50% in STBI, 43% in FTBI, 55% in LOW, 41% in MEDIUM, and 44% in HIGH groups (STBI vs. FTBI, p = 0.48; LOW vs. MEDIUM, p = 0.03: MEDIUM vs. HIGH, p = 0.68). TRM was not influenced significantly by the different TBI techniques. When analyzing separately the influence of fractionation and the instantaneous dose rate either in ANLL or ALL patients, no difference in terms of survival and LFS was observed. Fractionation did not influence the 5-year RI both in ANLL and ALL patients. However, among the patients with ANLL, 5-year RI was significantly higher (58%) in the LOW group than the MEDIUM group (31%, p = 0.001), whereas instantaneous dose rate did not significantly influence the RI in ALL patients. The 5-year TRM rate was significantly higher in allogeneic BMT group both in ANLL (37%) and ALL (37%) patients than those treated by autologous BMT (ANLL: 15%, ALL: 18%: p = 0.002 and 0.02, respectively). The 5-year estimated interstitial pneumonitis (IP) and cataract incidence rates were 22% and 19%, respectively, in all patients. IP incidence seemed to be higher in the HIGH group (46%) than the MEDIUM (19%, p = 0.05) or LOW (25%, p = 0.15) groups. Furthermore, cataract incidence was significantly influenced by fractionation (STBI vs. FTBI, 29% vs. 9%; p = 0.003) and instantaneous dose rate (LOW vs. MEDIUM vs. HIGH, 0% vs. 27% vs. 33%; p < 0.0001). Multivariate analyses revealed that the best factors influencing the survival were 1st CR (p = 0.0007), age ≤ 40 years (p = 0.003), and BMT after 1985 (p = 0.008). The RI was influenced independently only by the remission status (p = 0.0002). On the other hand, the TRM rate was lower in patients who did not experience graft-vs.-host disease (GvHD, p < 0.0001), and in those treated after 1985 (p = 0.0005). GvHD was the only independent factor involved in the development of IP (p = 0.01). When considering the cataract incidence, the only independent factor was the instantaneous dose rate (p = 0.0008). CONCLUSION: The outcome of BMT patients conditioned with TBI for acute leukemia was not significantly influenced by the TBI technique, and TRM seemed to be lower in patients treated after 1985. On the other hand, cataract incidence was significantly influenced by the instantaneous dose rate.

Broussonetia papyrifera Root Bark Extract Exhibits Anti-inflammatory Effects on Adipose Tissue and Improves Insulin Sensitivity Potentially Via AMPK Activation

The chronic low-grade inflammation in adipose tissue plays a causal role in obesity-induced insulin resistance and its associated pathophysiological consequences. In this study, we investigated the effects of extracts of Broussonetia papyrifera root bark (PRE) and its bioactive components on inflammation and insulin sensitivity. PRE inhibited TNF-α-induced NF-κB transcriptional activity in the NF-κB luciferase assay and pro-inflammatory genes’ expression by blocking phosphorylation of IκB and NF-κB in 3T3-L1 adipocytes, which were mediated by activating AMPK. Ten-week-high fat diet (HFD)-fed C57BL6 male mice treated with PRE had improved glucose intolerance and decreased inflammation in adipose tissue, as indicated by reductions in NF-κB phosphorylation and pro-inflammatory genes’ expression. Furthermore, PRE activated AMP-activated protein kinase (AMPK) and reduced lipogenic genes’ expression in both adipose tissue and liver. Finally, we identified broussoflavonol B (BF) and kazinol J (KJ) as bioactive constituents to suppress pro-inflammatory responses via activating AMPK in 3T3-L1 adipocytes. Taken together, these results indicate the therapeutic potential of PRE, especially BF or KJ, in metabolic diseases such as obesity and type 2 diabetes.

Rotavirus outbreak in central Australia

In May 2001, one of the largest outbreaks of Rotavirus in living memory swept through central Australia, resulting in 246 emergency department presentations and the hospitalisation of 137 children in a single month. Hundreds more throughout the region were afflicted. Of the hospitalised cases, 96 per cent were under 4 years of age and over 90 per cent were Aboriginal. There were no reported deaths from the outbreak. The response by health personnel was similar to that experienced during other natural disasters, stretching local resources beyond their normal capacity. This report summarises the pathogenesis, clinical features and treatment of Rotavirus, and describes the management of a major outbreak of this potentially lethal and devastating disease in a unique and isolated context.

Examen virologique des sécrétions nasopharyngées et examen cytobactériologique de l'expectoration

Les sécrétions nasopharyngées et bronchiques se prêtent à différents examens bactériologiques, virologiques et autres. Assez courants à l'hôpital, ils sont beaucoup moins utilisés en pédiatrie ambulatoire. Néanmoins, pour que leurs résultats soient interprétables et utiles au diagnostic, la technique de prélèvement doit être soigneuse. L'intérêt pratique de ces examens mérite également d'être précisé pour que les demandes soient justifiées.

Infectious diseases in child day care facilities

Infections in children in day care are common, but can be limited by education of providers and staff in standards of hygiene, maintenance of basic techniques of infection control, appropriate use of the physical facilities of the day care unit, and maintenance of recommended levels of children and staff.

Chapter 6 Protein Sorting in the Secretory Pathway

This chapter focuses on protein sorting in the secretory pathway. From primary and secondary biosynthetic sites in the cytosol and mitochondrial matrix, respectively, proteins and lipids are distributed to more than 30 final destinations in membranes or membrane-bound spaces, where they carry out their programmed function. Molecular sorting is defined, in its most general sense, as the sum of the mechanisms that determine the distribution of a given molecule from its site of synthesis to its site of function in the cell. The final site of residence of a protein in a eukaryotic cell is determined by a combination of various factors, acting in concert: (1) site of synthesis, (2) sorting signals or zip codes, (3) signal recognition or decoding mechanisms, (4) cotranslational or posttranslational mechanisms for translocation across membranes, (5) specific fusion–fission interactions between intracellular vesicular compartments, and (6) restrictions to the lateral mobility in the plane of the bilayer. Improvements in cell fractionation, protein separation, and immune precipitation procedures in the past decade have made them possible. Very little is known about the mechanisms that mediate the localization and concentration of specific proteins and lipids within organelles. Various experimental model systems have become available for their study. The advent of recombinant DNA technology has shortened the time needed for obtaining the primary structure of proteins to a few months.

Biological and morphological consequences of dsRNA-induced suppression of tetraspanin mRNA in developmental stages of Echinococcus granulosus

BACKGROUND: Cystic echinococcosis, caused by the cestode Echinococcus granulosus, is a neglected tropical disease with remarkable morbidity in humans and a problem of worldwide economic importance in livestock industry. Understanding the molecular basis of the parasite growth and development is essential for the disease diagnosis, management and control. The tetraspanin (TSP) family of proteins are transmembrane proteins with a role in many physiological processes of eukaryotic organisms. TSPs present in the tegumental surface of platyhelminths play pivotal roles in host-parasite interaction. However, little is known about the role of TSPs in growth and development in the Platyhelminthes. To understand the role of TSP1 in the growth and development of E. granulosus we investigated the effect of EgTSP1-specific long dsRNA in different in vitro stages of the parasite. METHODS: Different stages of E. granulosus, protoscoleces and strobilated worms, were cultivated In vitro in di-phasic media. Using long dsRNA and two delivery methods, i.e. electroporation and electro-soaking, EgTSP1 silencing was performed with an EgTSP1-specific dsRNA. The TSP1 expression profile was assessed as well as the biological and ultrastructural properties of the parasites. RESULTS: After three days of dsRNA treatment, EgTSP1 expression was significantly reduced in both stages of E. granulosus as compared to irrelevant/unrelated dsRNA and untreated controls. Silencing expression of EgTSP1 in different stages of E. granulosus resulted in reduced viability and body contractions, inhibition of protoscoleces evagination and distinctive tegumental changes. Ultrastructural morphology of the strobilated worms treated with EgTSP1-specific dsRNA was indicative of the microtriches impairments and vacuolated tegument compared to the control helminths. CONCLUSIONS: Results of the present study suggest that EgTSP1 plays important structural roles in tegument configuration in E. granulosus. EgTSP1 is proved to be a potential target for the development of vaccines and RNAi-based drugs. [Image: see text]

High prevalence of urogenital infection/inflammation in patients with azoospermia does not impede surgical sperm retrieval

Considering infection/inflammation to be an important risk factor in male infertility, the aim of this study was to make a comprehensive evaluation of the prevalence of urogenital tract infection/inflammation and its potential impact on sperm retrieval in azoospermic patients. In this prospective study, 71 patients with azoospermia were subjected to an extensive andrological workup including comprehensive microbiological diagnostics (2‐glass test, semen, testicular swab and testicular tissue analysis) and testicular biopsy/testicular sperm extraction (TESE). Medical history suggested urogenital tract infection/inflammation in 7% of patients, 11% harboured STIs, 14% showed significant bacteriospermia, 15% had seminal inflammation, 17% fulfilled the MAGI definition, and 27% had relevant pathogens. At the testicular level, 1 patient had a swab positive for bacteria, no viruses were detected, tissue specimens never indicated pathogens, whereas histopathology revealed focal immune cell infiltrates in 23% of samples. Testicular sperm retrieval rate was 100% in obstructive and 46% in nonobstructive azoospermia. None of the infection/inflammation‐related variables was associated with the success of sperm retrieval or inflammatory lesions in the testis. The high prevalence of urogenital infection/inflammation among azoospermic men underpins their role as significant aetiologic factors in male infertility. However, this observation does not refer to the chances of sperm retrieval at the time of surgery/TESE.

Type I interferon in HIV treatment: from antiviral drug to therapeutic target

Type I interferons (IFNs) are soluble molecules that exert potent antiviral activity and are currently used for the treatment of a panel of viral infections. In the case of HIV, the use of type I IFN has had limited success, and has almost been abandoned. During the last decade, a series of studies has highlighted how HIV infection may cause overactivation of type I IFN production, which contributes to the exhaustion of the immune system and to disease progression. This review describes the transition from the proposed use of type I IFN as antiviral drugs in HIV infection, to the idea that blocking their activity or production may provide an immunologic benefit of much greater importance than their antiviral activity.

Assessing the implications of positive genomic screening results

AIM: Before population screening of ‘healthy’ individuals is widely adopted, it is important to consider the harms and benefits of receiving positive results and how harms and benefits may differ by age. SUBJECTS & METHODS: Participants in a preventive genomic screening study were screened for 17 genes associated with 11 conditions. We interviewed 11 participants who received positive results. RESULTS: Interviewees expressed little concern about their positive results in light of their older age, the risk condition for which they tested positive, or other pressing health concerns. CONCLUSION: Researchers and clinicians should recognize that returning positive results may not have the impact they presume given the diversity of the conditions screened and those who choose to undergo screening.

Content analysis of systematic reviews on the effectiveness of Traditional Chinese Medicine

OBJECTIVE: To evaluate evidence for the efficacy of Traditional Chinese Medicine (TCM) in systematic reviews. METHODS: Chinese (TCMPeriodical Literature Database, Chinese Biological Medicine database, Chinese Medical Current Contents, China Hospital Knowledge Database journal fulltext database, Virtual Machining and Inspection System, and Wanfang) and English (Cochrane Database of Systematic Reviews, PubMed and Embase) databases were searched. RESULTS: Three thousand, nine hundred and fifty-five articles were initially identified, 606 of which met the inclusion criteria, including 251 in English (83 from the Cochrane Database) and 355 in Chinese. The number of articles published each year increased between 1989 and 2009. Cardiocerebrovascular disease was the most studied target disease. Intervention measures includedTCM preparations (177 articles), acupuncture (133 articles) and combinations of TCM and western medicine (38 articles). Control measures included positive medical (177 articles), basic treatment (100 articles), placebo (219 articles), and blank and mutual (107 articles). All articles included at least one reference; the greatest number was 268. Six of 10 articles with high quality references demonstrated curative effectsagainst target diseasesincludingupper respiratory tract infection, dementia and depression. Interventions that were not recommendedwere tripterygium for rheumatoid arthritis andTCM syndrome differentiation for pediatric nocturia. In 10.4% of the studies, the authors concluded that the intervention had a curative effect. The assessors agreed with the authors' conclusions in 88.32% of cases, but rejected 8.94% (54 articles). CONCLUSION: 1) Training in systematic review methods, including topic selection, study design, methods and technology, should be improved. 2) Upper respiratory tract infection, dementia and depression may become the predominant diseases treatedby TCM, and the corresponding interventions could be developed into practical applications. 3) Use of non-recommended interventions should be controlled, and there should be more research on side effects.

Column liquid chromatography of integral membrane proteins

Biological membranes have as a major function the compartmentation of biological processes in cells and organelles. They consist of a bilayer of phospholipid molecules in which proteins are embedded. These integral membrane proteins, which cross the bilayer once or several times, generally have a higher than average hydrophobicity and tend to aggregate. Detergents are needed to remove integral membrane proteins from the lipid bilayer and they have to be present during further chromatographic purification. Predominantly, four modes of HPLC have been used alone or in combination for the puridication of integral membrane proteins. These are based on differences of proteins in size (size-exclusion chromatography, SEC), electrostatic interaction (ion-exchange chromatography, IEC), bioaffinity (bioaffinity chromatography, BAC) and hydrophobic interaction (reversed-phase chromatography, RPC, and hydrophobic-interaction chromatography, HIC). SEC, IEC, BAC and HIC are used under relatively mild conditions, and buffer systems generally contain a non-ionic detergent. RPC generally has a denaturing effect on the protein and should preferably be used for the purification of integral membrane proteins smaller than 50 kD.

Alteration of intracellular traffic by monensin; mechanism, specificity and relationship to toxicity

Monensin, a monovalent ion-selective ionophore, facilitates the transmembrane exchange of principally sodium ions for protons. The outer surface of the ionophore-ion comples is composed largely of nonpolar hydrocarbon, which imparts a high solubility to the complexes in nonpolar solvents. In biological systems, these complexes are freely soluble in the lipid components of membranes and, presumably, diffuse or shuttle through the membranes from one aqueous membrane interface to the other. The net effect for monensin is a trans-membrane exchange of sodium ions for protons. However, the interaction of an ionophore with biological membranes, and its ionophoric expression, is highly dependent on the biochemical configuration of the membrane itself. One apparent consequence of this exchange is the neutralization of acidic intracellular compartments such as the trans Golgi apparatus cisternae and associated elements, lysosomes, and certain endosomes. This is accompanied by a disruption of trans Golgi apparatus cisternae and of lysosome and acidic endosome function. At the same time, Golgi apparatus cisternae appear to swell, presumably due to osmotic uptake of water resulting from the inward movement of ions. Monensin effects on Golgi apparatus are observed in cells from a wide range of plant and animal species. The action of monensin is most often exerted on the trans half of the stacked cisternae, often near the point of exit of secretory vesicles at the trans face of the stacked cisternae, or, especially at low monensin concentrations or short exposure times, near the middle of the stacked cisternae. The effects of monensin are quite rapid in both animal and plant cells; i.e., changes in Golgi apparatus may be observed after only 2–5 min of exposure. It is implicit in these observations that the uptake of osmotically active cations is accompanied by a concomitant efflux of H(+) and that a net influx of protons would be required to sustain the ionic exchange long enough to account for the swelling of cisternae observed in electron micrographs. In the Golgi apparatus, late processing events such as terminal glycosylation and proteolytic cleavages are most susceptible to inhibition by monensin. Yet, many incompletely processed molecules may still be secreted via yet poorly understood mechanisms that appear to bypass the Golgi apparatus. In endocytosis, monensin does not prevent internalization. However, intracellular degradation of internalized ligands may be prevented. It is becoming clear that endocytosis involves both acidic and non-acidic compartments and that monensin inhibits those processes that normally occur in acidic compartments. Thus, monensin, which is capable of collapsing Na(+) and H(+) gradients, has gained wide-spread acceptance as a tool for studying Golgi apparatus function and for localizing and identifying the molecular pathways of subcellular vesicular traffic involving acid compartments. Among its advantages are the low concentrations at which inhibitions are produced (0.01–1.0 μM), a minimum of troublesome side effects (e.g., little or no change of protein synthesis or ATP levels) and a reversible action. Because the affinity of monensin for Na(+) is ten times that for K(+), its nearest competitor, monensin mediates primarily a Na(+)-H(+) exchange. Monensin has little tendency to bind calcium. Not only is monensin of importance as an experimental tool, it is of great commercial value as a coccidiostat for poultry and to promote more efficient utilization of feed in cattle. The mechanisms by which monensin interact with coccidia and rumen microflora to achieved these benefits are reasonably well documented. However, the interactions between monensin and the tissues of the host animal are not well understood although the severe toxicological manifestations of monensin poisoning are well known. Equine species are particularly susceptible to monensin poisoning, and a common effect of monensin poisoning is vacuolization and/or swelling of mitochondria in striated muscle. Other pathological injuries to striated muscle, spleen, lung, liver and kidney also have been noted. A consistent observation is cardiac myocyte degeneration as well as vacuolization. Differences in cellular response resulting from exposure to monensin (i.e., Golgi apparatus swelling in cultured cells, isolated tissues, and plants vs.mitochondrial swelling in animals fed monensin) suggest that myocardial damage is due either to a monensin metabolite or is a secondary response to some other derivation. However, as pointed out by Bergen and Bates [26], the underlying mode of action of ionophores is on transmembrane ion fluxes which dissipate cation and proton gradients. Consequently, some or all of the observed monensin effects in vivo in animals could be secondary phenomena caused by disruption of normal membrane physiology resulting from altered ion fluxes.

Catalytic site studies on tuna (Thunnus albacares) pyloric caeca aminopeptidase

Tuna pyloric caeca aminopeptidase (tAP) is a glycosylated zinc-metalloenzyme containing apparently two identical subunits. The enzyme is reversibly inhibited in a time-dependent manner by amastatin. Slow development of tAP inhibition by this inhibitor could be demonstrated. Dissociation of the complex of tAP with amastatin is also slow. Two molar equivalents of the inhibitor are bound by the enzyme suggesting the presence of one catalytic site in each subunit. Chemical modification of tAP with 1-cyclohexyl-3-(2-morpholinoethyl)carbonyl-metho-p-toluene sulfonate and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinone revealed the presence of essential acidic amino acid residues probably located at the active site. Compatible with the presence of arginine and tyrosine residues at the catalytic site of most metalloproteinases, tAP is reversibly inhibited by phenylglyoxal and inactivated by tetranitromethane in a time-dependent fashion. The rate of inhibition by these modifiers could be significantly decreased if the enzyme was previously treated with amastatin suggesting that the modified amino acid residues are located at the catalytic site. Diethylpyrocarbonate did not affect the activity of both native and zinc-depleted tAP suggesting that histidine is not involved in the zinc-ligand formation.