bluuebunny/crossref_metadata_2025 huggingface dataset:
Vaccine immune response and side effects with the use of acetaminophen with influenza vaccine
Authors: Gross P A , Levandowski R A , Russo C , Weksler M , Bonelli J , Dran S , Munk G , Deichmiller S , Hilsen R , Panush R F
Published: January 2020

The purpose of this study was to determine whether acetaminophen impairs the immune response to influenza vaccine. Influenza vaccine is an under-utilized preventive measure, partly because of the unfounded perception that fever and myalgias frequently follow vaccination. While acetaminophen may decrease these infrequent side effects, it may also alter the immune response to vaccination. We compare the effect of acetaminophen with placebo on the humoral immune response to the 1991-1992 commercially available influenza vaccine. We studied 60 healthy, elderly subjects from a geriatric clinic and 20 infirm, elderly subjects from a nursing home. The subjects were randomly assigned to receive placebo or acetaminophen (1,000 mg every 6 h) for 2 days. Acetaminophen did not depress or enhance the immune development of serum hemagglutination inhibition antibody to the three vaccine antigens. The systemic side effects of fever and myalgia were uncommon in both groups. The healthy elderly subjects mounted a significantly better immune response to the influenza virus A/Taiwan/1/86 (H1N1) vaccine strain than did the infirm elderly subjects (geometric mean titer, 115 versus 51; P = 0.003). The functional activity score obtained by using the chronic healthy evaluation component of the Acute Physiology and Chronic Health Evaluation system could be used to distinguish the healthy from the infirm elderly (scores of 1.27 versus 3.75, P < 0.001). Acetaminophen neither depressed nor enhanced the serum antibody response to the vaccine in the healthy and infirm elderly subjects studied.
Lack of antibody response to invasin in humans with yersiniosis
Authors: Fortineau N , Beretti J L , Berche P , Simonet M
Published: January 2020

The Yersinia pseudotuberculosis inv gene encodes invasin, a 103-kDa outer membrane protein allowing bacteria to penetrate mammalian cells. This protein is produced in vitro at below 30 degrees C. In this work, we studied the antibody response against invasin in humans suffering from yersiniosis and in mice orally infected with a virulent strain of Y. pseudotuberculosis. Infection with enteropathogenic Yersinia strains did not induce either a systemic or a gut antibody response to invasin. Our results suggest that the inv gene is not expressed in the gut at 37 degrees C and, therefore, that invasin is not present to the immune system when microorganisms multiply in the host tissues.
In vitro cytokine production by normal human peripheral blood mononuclear cells as a measure of immunocompetence or the state of activation
Authors: Friberg D , Bryant J , Shannon W , Whiteside T L
Published: January 2020

Measurements of cytokine levels in serum may not adequately reflect the cytokine-producing potential of immune cells because of the short half-lives of cytokines and the presence of various inhibitors in human sera. In vitro cytokine production by peripheral blood mononuclear cells (PBMCs) can be an important and reliable measure of immunocompetence. Also, spontaneous in vitro release of cytokines by PBMCs may serve as a measure of their activation in vivo. In the present study, normal ranges for the in vitro production by PBMCs of interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha), IL-2, and gamma interferon (IFN-gamma) were established; the feasibility of using cryopreserved PBMCs for assays of in vitro cytokine production was evaluated; and spontaneous (unstimulated) versus induced production of cytokines by fresh and cryopreserved PBMCs from healthy donors was compared. Supernatants obtained from paired fresh and frozen PBMCs were quantitated for IL-1 beta, TNF-alpha, IL-2, and IFN-gamma by using enzyme-linked immunosorbent assay or a radioimmunoassay standardized against World Health Organization cytokine standards. Fresh or cryopreserved PBMCs activated with lipopolysaccharide produced comparable levels of IL-1 beta. However, the mean levels of stimulated production of TNF-alpha, IFN-gamma, and IL-2 were significantly higher in cryopreserved versus fresh PBMCs (P < or = 0.0004). Correlations between the level of production of each cytokine by fresh versus cryopreserved in vitro-stimulated PBMCs were statistically significant, although of moderate magnitude. Spontaneous cytokine release by fresh versus cryopreserved cells was not significantly different.
Antigenic structure of the complete nonstructural (NS) 2 and 5 proteins of hepatitis C virus (HCV): anti-HCV NS2 and NS5 antibody reactivities in relation to HCV serotype, presence of HCV RNA, and acute HCV infection
Authors: Zhang Z X , Chen M , Sönnerborg A , Sällberg M
Published: January 2020

Antigenic regions within the nonstructural (NS) 2 and 5 proteins of hepatitis C virus (HCV) were identified and characterized by the use of 127 overlapping synthetic peptides and a serum panel consisting of 167 human serum samples from persons with antibodies to HCV. Initially, 20 anti-HCV-positive serum samples were used to screen the peptides covering the complete NS2 and NS5 proteins. Among the 27 overlapping peptides spanning the NS2 protein of HCV, only the peptide covering residues 960 to 975 was recognized by human sera. Within the 100 peptides covering the NS5 protein, major linear antigenic regions were located at residues 2284 to 2329 within the putative NS5a and at residues 2584 to 2599 and 2944 to 2959 within the putative NS5b. Additional minor linear antigenic regions were also identified within the NS5. The sequence of the antigenic region of the NS2 protein is, unlike most parts of the NS2 protein, highly conserved among the described types of HCV, whereas the sequence of the major antigenic region of NS5 shows variability among HCV types. The recognition of a peptide corresponding to a part of the major region of NS5 was found to be dependent on HCV type. In 129 anti-HCV-positive serum samples, the prevalence of antibodies to the NS2 protein was found to be 23% among HCV RNA-positive sera and 10% among HCV RNA-negative sera. In the same samples, reactivity to the major linear antigenic regions of HCV NS5 was found in 68% of the HCV RNA-positive sera and 67% of the HCV RNA-negative sera. Of 18 serum samples from five patients with acute HCV infections, and who seroconverted with respect to anti-HCV, 4 were found to be reactive to one or more of the 100 NS5 peptides and in three serum samples the NS5 reactivities were found to shorten the time for serodiagnosis of cross-reactive with a region form residues 2584 to 2599 of NS5, which has 67% homology with a six-residue sequence of NS2. In conclusion, in this study we have identified and evaluated the potential use of synthetic peptides corresponding to linear antigenic regions of the NS2 and NS5 proteins.
Substance P augments tumor necrosis factor release in human monocyte-derived macrophages
Authors: Lee H R , Ho W Z , Douglas S D
Published: January 2020

Substance P (SP) is an undecapeptide that has the amino sequence Arg-Pro-Lys-Pro-Gin-Gln-Phe-Phe-Gly-Leu-Met-NH2 and that belongs to a family of structurally related peptides known as tachykinins, the latter are widely distributed in the central nervous system. SP is involved in the biological activities of cells in the immune system, including the induction of cytokines in immune cells. We have investigated the effects of SP on constitutive and/or lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF) in cultured blood monocyte-derived macrophages (MDM). Cells cultured in vitro for 14 days were treated with SP at various concentrations (10(-10) to 10(-6M) in the presence of LPS before culture supernatants were harvested. TNF bioactivity in culture supernatants was measured with L929 cell line MDM from 10 of 12 donors treated with a SP alone showed increased TNF production. SP and LPS also interacted in a synergistic fashion in upregulating TNF production in MDM from responders. The stimulatory effect of SP was inhibited by two SP antagonists, spantide ([D-Arg-1-D-Trp-7-D-Trp-7-D-Trp-9-leu-11]-SP) and CP-96,345 (a nonpeptide antagonist of the SP receptor). In addition, an anti SP polyclonal antibody blocked the SP effect on TNF production in cultured MDM, further indicating the specificity of these effects. These results demonstrate that SP is an important regulator of monokine production by human monocytes/macrophages.
Inhibitory effect of 3'-azido-3'-deoxythymidine on proliferative responsiveness of CD8+ lymphocytes to interleukin-2
Authors: Mercure L , Lalonde R , Phaneuf D , Brenner B , Wainberg M A
Published: January 2020

Although several studies have shown that 3'-azido-3'-deoxythymidine (AZT) is not toxic for CD4+ lymphocytes, its effect on CD8+ cells has never been studied in a systematic way. We purified CD8+ cells from the peripheral blood mononuclear cells of both human immunodeficiency virus (HIV)-seronegative and HIV-infected individuals by means of magnetic beads that had been coated with monoclonal antibodies. We report that AZT, but not two other nucleosides tested, inhibited the interleukin-2-dependent proliferation of CD8+ lymphocytes in a dose-dependent manner. No such effect was observed with regard to CD4(+)-enriched populations. The AZT-mediated antiproliferative effect did not appear to be related to either the CD4+ count or to prior treatment with this drug in the case of HIV-seropositive subjects.
Humoral response to mycobacterial heat shock proteins in patients with constrictive pericarditis caused by tuberculosis and its implications for pathogenesis
Authors: Ng T T , Strang J I , Wilkins E G
Published: January 2020

Tuberculous pericarditis is one of the commonest causes of cardiac failure in Transkei and the surrounding regions in southeast Africa. About 20% of patients with clinically diagnosed tuberculous pericardial effusion go on to develop pericardial fibrosis (i.e., construction), a complication which is associated with significant mortality and morbidity. The pathological mechanisms underlying this aberrant inflammatory response are poorly understood, and there is a lack of reliable pointers (clinical or laboratory) in predicting the likelihood of development of constriction. We studied the humoral response to mycobacterial heat shock proteins (65 and 71 kDa) in 25 patients with culture-positive tuberculous pericardial effusion and found a significant correlation between high anti-mycobacterial hsp60 antibody titers (before treatment) and subsequent development of fibrosis (P = 0.035 by logistic regression), which is independent of the effect of the use of prednisolone as adjuvant therapy. Possible mechanisms underlying the pathogenesis of pericardial constriction in tuberculosis are postulated.
Rotavirus-specific intestinal immune response in mice assessed by enzyme-linked immunospot assay and intestinal fragment culture
Authors: Khoury C A , Brown K A , Kim J E , Offit P A
Published: January 2020

Primate rotavirus strain RRV and bovine strain WC3 or reassortants made between these animal viruses and human rotaviruses have been administered to infants as candidate vaccines. We compared RRV and WC3 in a murine model of oral infection. We determined the relative capacities of these viruses to induce a virus-specific humoral immune response by intestinal lymphocytes as tested by enzyme-linked immunospot assay, intestinal fragment culture, and enzyme-linked immunosorbent assay of intestinal contents. We found that inoculation of mice with RRV induced higher frequencies of virus-specific immunoglobulin A (IgA)-secreting cells in the lamina propria, greater quantities of virus-specific IgA in intestinal fragment cultures, and greater quantities of virus-specific IgA in intestinal secretions than did inoculation with WC3 or inactivated RRV (iRRV). The induction of an IgA response in serum was predictive of an IgA response among intestinal lymphocytes after inoculation with RRV but not WC3. In addition, large quantities of IgG, IgA, and IgM not specific for rotavirus were produced in fragment cultures from mice inoculated with RRV but not in cultures from mice inoculated with WC3 or iRRV. Possible mechanisms of RRV-induced polyclonal stimulation of intestinal B cells are discussed.
Investigation of cross-reactions against Trichinella spiralis antigens by enzyme-linked immunosorbent assay and enzyme-linked immunoelectrotransfer blot assay in patients with various diseases
Authors: De-la-Rosa J L , Alcantara P , Correa D
Published: January 2020

Data regarding cross-reactions against Trichinella spiralis in humans are scarce and controversial. For this reason, we tested serum samples from patients with typhoid fever, brucellosis, toxoplasmosis, amoebiasis, cysticercosis, trichocephaliasis, ascariasis, and onchocerciasis against an antigenic extract of T. spiralis infective larvae in an enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunoelectrotransfer blot (EITB) assay. All except one serum sample from the group of patients with onchocerciasis were negative in the ELISA; in the EITB assay, only faint bands were observed with the samples from patients with onchocerciasis and ascariasis and negative results were obtained with the samples from patients with other diseases. In conclusion, cross-reactions were found only in the groups of patients with other nematode infections and were of very low magnitude, most of them virtually negative.
Prevalence of antibodies against Borrelia species in patients with unclassified uveitis in regions in which Lyme disease is endemic and nonendemic
Authors: Kimura K , Isogai E , Isogai H , Nishikawa T , Masuzawa T , Yoshikawa K , Kotake S , Ohno S , Fujii N
Published: January 2020

We studied 93 patients with unclassified uveitis from two regions in Japan (Hokkaido and Kanagawa) to assess the contribution of Borrelia species to this condition. The seroprevalence of antibody to Borrelia species was higher in patients from Hokkaido than in those from Kanagawa. The unclassified uveitis of seropositive patients was probably a complication of Lyme borreliosis since (i) the antibody titers were as high as those in clinically diagnosed Lyme disease patients, (ii) healthy controls from Hokkaido showed low seroprevalence in contrast with unclassified uveitis patients from Hokkaido, and (iii) the reaction pattern of antibodies in sera from patients with unclassified uveitis was the same as that in patients with Lyme disease. This is the first report to reveal the high risk of Lyme borreliosis in patients with unclassified uveitis in regions endemic for Lyme disease. In case of unclassified uveitis as well as in cases of inflammatory disease of unknown origin, Lyme disease should be taken into consideration, especially in regions in which Lyme diseases is endemic, even if it is reported only in animals.
Rapid whole-blood flow cytometry assay for diagnosis of chronic granulomatous disease
Authors: O'Gorman M R , Corrochano V
Published: January 2020

Chronic granulomatous disease (CGD) is characterized by defective killing of intracellular microorganisms due to mutations in one of the four known components of the NADPH oxidase system. This system is responsible for the generation of superoxide and related antimicrobial oxidants. Diagnosis of CGD requires the demonstration of an abnormal oxidase system in the leukocytes of affected patients. Recently, several flow cytometry-based procedures which measure various reactive oxygen intermediates generated by the NADPH oxidase system have been developed. Most of the procedures developed to date require time-consuming granulocyte isolation, washing, and counting procedures, or they lack sensitivity. We have modified an existing procedure such that cell labelling and stimulation are performed directly in whole blood. Optimization of this procedure and its use in the diagnosis of patients with CGD or X-linked carriers are presented.
Connective tissue mast cells exhibit time-dependent degranulation heterogeneity
Authors: Kaminer M S , Murphy G F , Zweiman B , Lavker R M
Published: January 2020

Previous studies have identified two ultrastructurally distinct forms of mast cell (MC) degranulation following activation. Immunoglobulin E (IgE)-mediated reactions are characterized by a very rapid swelling and fusion of MC granules and abrupt mediator release. In certain chronic disease states (e.g., bullous pemphigoid), there is "piecemeal" degranulation with a more-gradual mediator release effected by microvesicular transport of "pieces" of granules to the cell surface. It is unclear whether these two degranulation patterns are determined by the different natures of the stimuli, heterogeneity among responding MC granules, or temporal factors. To investigate these issues, we have carried out electron microscopic studies with skin biopsies obtained from ragweed-sensitive subjects 15 and 30 s and 1, 3, 5, and 10 min after intradermal ragweed injection. "Anaphylactic"-type granule changes began by 15 s after ragweed injection and were complete by 5 min; unaffected granules were juxtaposed with granules that were swollen and fused. The remaining granules subsequently underwent changes in appearance similar to those seen in piecemeal degranulation. However, microvesicular transport of granule components to the surface was not observed. These findings indicate that skin MC changes in sites of IgE-mediated reactions include not only the typical very rapid anaphylactic degranulation but also a slower onset of gradual alteration of other granules, frequently within the same MC. These different patterns could reflect MC granule heterogeneity with attendant different responses to IgE-mediated stimuli.
Comparative evaluation of a commercial enzyme immunoassay for the detection of human antibody to Rickettsia typhi
Authors: Kelly D J , Chan C T , Paxton H , Thompson K , Howard R , Dasch G A
Published: January 2020

A commercial enzyme immunoassay kit called the Dip-S-Ticks (DS) for the detection of total immunoglobulin (Ig) G and IgM human antibodies to Rickettsia typhi was evaluated. In tests with 340 serum samples from patients with diagnosed cases of rickettsial diseases, patients suffering from other febrile illnesses, and normal subjects, the DS compared favorably with the standard indirect fluorescent-antibody (IFA) test. At IFA cutoff titers of > or = 1.64 and > or = 1:128, the DS showed sensitivities of 88.2 and 91.4% and specificities of 91.8 and 87.7%, respectively. The DS test correlated significantly with both the IFA IgG (r = 0.84, P < 0.0005) and IgM (r = 0.63, P < 0.0005) titers. Only 80% of IgG and 82% of IgM IFA readings determined by two technicians were within one dilution, while the DS was more reliable, with 100% within one dot. The rapidity, reliability, and simplicity of the DS suggest that it is a suitable test for use in clinical laboratories unable to perform the IFA test.
Decreased T-cell proliferative response to common environmental antigens could be an indicator of early human immunodeficiency virus-mediated lymphocyte lesions
Authors: Tassinari P , Deibis L , Blanca I , Bianco N E , Echeverría de Pérez G
Published: January 2020

To evaluate CD4+/CD29+ cells and their responses to different antigens in polar stages of human immunodeficiency virus (HIV) infection, we studied 26 HIV-seropositive carriers (SPCs) and 15 patients with AIDS simultaneously with 20 healthy volunteers (HVs) and 10 seronegative homosexual and bisexual men (SNH). CD3, CD4, CD29, and CD45RA phenotypes were analyzed by two-color flow cytometry. Significant depletion of CD4+ T cells and both memory (CD4+/CD29+) and naive (CD4+/CD45RA+) T-cell subsets was found among SPCs and AIDS patients compared with the numbers of such cells in the HV and SNH groups. Responses to optimal doses of Candida albicans, streptokinase, and tetanus toxoid were explored in peripheral blood mononuclear cells and CD4(+)- and CD4+/CD29(+)-enriched cell populations. In SPCs, the response to C. albicans in peripheral blood mononuclear cells showed a statistically significant diminution compared with the response of HVs (15,308 versus 35,951 cpm). In addition, a significantly reduced response to streptokinase was evident only when cell preparations were CD4+/CD29+ enriched (3,048 versus 10,367 cpm). Furthermore, the SPC group comprised seven responders to at least one antigen and seven nonresponders to any of the selected specific antigens. Absence of a response in these latter patients was independent of the absolute counts of memory and naive T-cell populations. The response to tetanus toxoid, although diminished in SPCs, was not significantly different from that in controls. Our results suggest that defective responses to common environmental antigens, unrelated to the absolute number of CD4+/CD29+ cells, is probably an early indicator of an HIV-induced lymphocyte lesion.
Anti-human immunodeficiency virus type 1 antibodies of noninfected subjects are not related to autoantibodies occurring in systemic diseases
Authors: Kämmerer R , Bürgisser P , Frei P C
Published: January 2020

Indeterminate Western blot (WB) (immunoblot) patterns for anti-human immunodeficiency virus type 1 (HIV-1) antibodies are often observed when testing serum samples from noninfected individuals. We investigated here the possible involvement of some frequently occurring autoantibodies (anti-SmB/B', U1snRNP [68 kDa, A, and C], Ro/SS-A [60 and 52 kDa], and Jo-1) in the generation of such indeterminate HIV-1 WB. In particular, the role of a reported sequence homology between p24 gag and the SmB/B' autoantigen was investigated. Serum samples were obtained from 50 healthy controls, 51 patients with systemic lupus erythematosus (SLE), 46 with systemic sclerosis, 6 with Sjögren's disease, 3 with mixed connective tissue disease, and 41 healthy subjects with persistent indeterminate HIV-1 WB. Reactivity to HIV-1 p24 gag was slightly but not significantly more frequent in patients with SLE than in controls (25.5% versus 14.0%; P > 0.1), whereas reactivity to HIV-1 p17 gag was significantly more frequent in the former subjects (23.5% versus 8.0%; P = 0.03). Simultaneous reactivity to p17 and p24 was observed in patients with SLE (11.8%; P = 0.014) or systemic sclerosis (8.7%; P = 0.049) but not in controls. There was no association found between the presence of any autoantibody and the occurrence of indeterminate HIV-1 WB nor between the presence of p24-reactive antibodies and anti-SmB/B'; this indicates that most p24-reactive antibodies are directed to epitopes other than the proline-rich sequences shared by p24 gag and SmB/B'.
Multireactive pattern of serum autoantibodies in asymptomatic individuals with immunoglobulin A deficiency
Authors: Barka N , Shen G Q , Shoenfeld Y , Alosachie I J , Gershwin M E , Reyes H , Peter J B
Published: January 2020

Selective immunoglobulin A (IgA) deficiency (sIgAD) is associated with certain autoimmune states. Increased production of autoantibodies and eventual development of overt autoimmune disease are related in part to genetic and environmental factors as well as to the immune deficiency. We surveyed serum specimens from 60 healthy subjects with sIgAD for the presence of 21 different autoantibodies by enzyme-linked immunosorbent assays. The frequencies of 16 autoantibodies were higher in sIgAD patients than in normal healthy controls. Autoantibodies to Jo-1 (28%), cardiolipin (21%), phosphatidylserine (20%), Sm (15%), asialo-GM1 (21%), sulfatide (32%), sulfoglucuronyl paragloboside (11%), and collagen type I (10%) were detected at high frequencies in comparison to those of normal healthy controls. Many of the serum samples were multireactive (i.e., exhibited binding to more than two autoantigens). Forty percent (24 of 60) of sIgAD serum samples reacted against six or more autoantigens; 10% (6 of 60) of sIgAD serum samples were not reactive with any of the 21 autoantigens. Three percent (7 of 209) of consecutive serum samples submitted for autoimmune antibody analysis that were positive for autoantibodies were from patients with IgA deficiency. Our finding of an increased frequency of autoantibodies in sIgAD patients supports the notion of polyclonal stimulation by repeated environmental stimuli as an etiologic mechanism. Alternatively, the increased frequency may be caused by a dysregulation of the immune response in such individuals. The mere detection of autoantibodies cannot predict whether a subject with sIgAD will develop an autoimmune disease or determine which specific disease will emerge.
Levels of interleukin 6 and tumor necrosis factor in serum from humans vaccinated with live, attenuated Francisella tularensis
Authors: Krakauer T
Published: January 2020

The levels of interleukin 6 (IL-6) and tumor necrosis factor alpha in serum did not change significantly during the course of immunization with the live vaccine strain of Francisella tularensis. Higher levels of circulating IL-6 were found in the sera of vaccinees with good antibody responses than in the sera from nonresponders. Preimmunization levels of IL-6 in serum were also higher in responders than in nonresponders.
Bioactive tumor necrosis factor in the sputa of cystic fibrosis patients
Authors: Kelly N , Clee S , Nakielna B
Published: January 2020

Eighty sputum samples from 37 cystic fibrosis patients were assayed for tumor necrosis factor alpha (TNF-alpha) in the L929 bioactivity assay. In addition, the presence of TNF-alpha-specific mRNA in the sputum was determined by using TNF-alpha-specific DNA probes in a PCR amplification step after reverse transcription of the mRNA to cDNA. Bioactive TNF-alpha was detected in 69 (86%) of the 80 sputum samples tested and, on at least one occasion, in 35 (95%) of the 37 cystic fibrosis patients sampled. The highest levels of TNF-alpha were observed in sputa obtained from patients presenting with an exacerbation of their disease.
Antibody responses to the capsular polysaccharide of Neisseria meningitidis serogroup B in patients with meningococcal disease
Authors: Granoff D M , Kelsey S K , Bijlmer H A , Van Alphen L , Dankert J , Mandrell R E , Azmi F H , Scholten R J
Published: January 2020

We measured antibody responses to meningococcal serogroup B (MenB) polysaccharide (PS) by enzyme-linked immunosorbent assay (ELISA) in sera from 94 patients from The Netherlands with disease caused by Neisseria meningitidis group B. The patients ranged in age from 3 to 73 years (mean age, 18.8 years). In initial studies we showed that the binding of a panel of MenB PS-reactive human immunoglobulin M (IgM) paraproteins to biotinylated MenB PS bound to avidin-coated microtiter wells was inhibited > 90% by the addition of soluble MenB PS or encapsulated group B meningococci. In contrast, inhibition of IgM anti-MenB PS antibody-binding activity in many of the patient sera was less than 50% (range, 20 to 94%). These data suggested a high frequency of nonspecific binding in the patient sera. Therefore, all serum samples were assayed in replicate in the presence or absence of soluble MenB PS, and only the inhibitable fraction of the binding signal was used to calculate the anti-MenB PS antibody concentrations. In 17 control patients with meningococcal disease caused by serogroup A or C strains, there was no significant difference in the respective IgM or IgG anti-MenB PS antibody concentrations in paired acute- and convalescent-phase sera. In contrast, in patients with MenB disease, the geometric mean IgM anti-MenB PS antibody concentration increased from 3.9 units/ml in acute-phase serum to 10.5 units/ml in convalescent-phase serum (P < 0.001). The corresponding geometric mean IgG anti-MenB PS antibody titers were 1:27 and 1:36 (P < 0.05). There was only a weak relationship between age and the magnitude of the logarithm of the antibody concentrations in convalescent-phase sera (for IgM, r2 = 0.06 and P < 0.05; for IgG, r2 = 0.08 and P < 0.01). Our data indicate that precautions are needed to avoid nonspecificity in measuring serum antibody responses to MenB PS by ELISA. Furthermore, although this PS is thought to be a poor immunogen, patients as young as 3 years of age recovering from MenB disease demonstrate both ImG and IgG antibody responses in serum.
In vitro detection of apoptotic stimuli by use of the HL-60 myeloid leukemic cell line
Authors: Shellhaas J L , Zuckerman S H
Published: January 2020

The human histiocytic lymphoma line HL-60 has served as a model of myeloid cell differentiation and can be induced to differentiate along the neutrophil or monocytic lineage, depending on the external stimulus. The nondifferentiated cell line retains a premyeloid leukemic phenotype and is capable of anchorage-independent growth and proliferation. The role of apoptosis in the regulation of immunologic and inflammatory events associated with homeostasis and disease has been most intensively studied in lymphocytes. In the present study, nondifferentiated HL-60 has served as a model for studying myeloid cell apoptosis by investigating apoptotic changes induced by camptothecin, a DNA topoisomerase inhibitor, as well as physiologic stimuli, including ceramide analogs and a monoclonal antibody against the Fas antigen. Multiparameter flow cytometry was used to evaluate apoptosis by measuring changes in both side scatter and propidium iodide staining. The appearance of apoptotic cells was confirmed biochemically by measuring DNA endonuclease activity by both enzyme-linked immunosorbent assay quantitation and DNA ladder formation on agarose gels and morphologically with the detection of micronuclei by confocal laser microscopy. These studies demonstrate that HL-60 can serve as an in vitro model for the detection of physiologic and pharmacologic apoptotic stimuli and for understanding the early and late cellular changes associated with induction of the apoptotic program.
The hook protein of Borrelia burgdorferi, encoded by the flgE gene, is serologically recognized in Lyme disease
Authors: Jwang B , Dewing P , Fikrig E , Flavell R A
Published: January 2020

The periplasmic flagellum of Borrelia burgdorferi consists of a unipeptide flagellar filament, a hook, and a basal body. Here, we report the cloning and expression of the hook gene, flgE, of B. burgdorferi N40. The flgE gene is 1,119 nucleotides long and is located on the 950-kb linear chromosome of B. burgdorferi. The primary protein sequence of FlgE shows 73% similarity to the FlgE protein of Treponema phagedenis and approximately 50% similarity to the FlgG proteins of both gram-positive and gram-negative bacteria. The flgE gene was cloned into an Escherichia coli expression plasmid, pMX, to produce FlgE protein. Subsequently, FlgE murine antiserum was prepared by immunizing mice with the partially purified B. burgdorferi FlgE protein. By Western blot (immunoblot) analysis, the antiserum was found to react with a 40-kDa peptide in the whole-cell lysates, confirming the expression of the flgE gene in B. burgdorferi. Additionally, antibodies to FlgE were found in serum specimens from 19 of 42 patients with Lyme disease. Moreover, when other antigens, including 41G (the immunodominant domain of flagellin), OspE, OspF, and p22, were used to test for the development of corresponding antibodies in these patients, 67% of these patients (28 of 42) reacted to at least one of these five antigens, suggesting that a combination of FlgE with other available B. burgdorferi recombinant proteins is a good candidate for substrates in assays to aid in the diagnosis of Lyme disease.
Effects of pretreatment with SDZ MRL 953, a novel immunostimulatory lipid A analog, on endotoxin-induced acute lung injury in guinea pigs
Authors: Nakamura H , Ishizaka A , Urano T , Sayama K , Sakamaki F , Terashima T , Waki Y , Soejima K , Tasaka S , Hasegawa N
Published: January 2020

SDZ MRL 953 (SDZ), a novel immunostimulatory lipid A analog, has been reported to have immunopharmacological activities similar to those of lipopolysaccharide (LPS) but to have little of the toxicity of LPS. We investigated the effects of pretreatment with SDZ on Escherichia coli endotoxin-induced acute lung injury in guinea pigs. Four experimental groups consisted of saline control (n = 16), SDZ (-12 h) plus LPS (2 mg/kg of SDZ per kg of body weight injected intravenously 12 h before intravenous injection of 2 mg of LPS per kg; n = 15), SDZ (-10 min) plus LPS (SDZ injected 10 min before LPS injection; n = 10), and LPS alone (n = 16). The animals were sacrificed, and lung tissue was sampled 4 h after LPS or saline infusion. Lung injury was assessed by measuring the wet weight-to-dry weight ratio and the level of 125I-labeled albumin accumulation in bronchoalveolar lavage fluid relative to that in plasma. In the SDZ (-12 h) plus LPS group, these two parameters of acute lung injury were decreased compared with those in the LPS alone group. However, they were not decreased in the SDZ (-10 min) plus LPS group. We conclude that SDZ attenuates endotoxin-induced acute lung injury when it is administered 12 h before LPS injection. The attenuating effects of SDZ are speculated to be due to down regulation of the response to endotoxin rather than to receptor blocking.
Effect of xanthine derivates and dexamethasone on Streptococcus pneumoniae-stimulated production of tumor necrosis factor alpha, interleukin-1 beta (IL-1 beta), and IL-10 by human leukocytes
Authors: van Furth A M , Seijmonsbergen E M , Langermans J A , van der Meide P H , van Furth R
Published: January 2020

The present study concerns the release of the proinflammatory cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha and of the anti-inflammatory cytokine IL-10 by human leukocytes in whole blood during stimulation with Streptococcus pneumoniae and the effects of various xanthine derivates, i.e., pentoxifylline (PTX), caffeine, and theofylline, and of dexamethasone (DXM). All three xanthine derivates and DXM inhibited the release of tumor necrosis factor alpha, PTX being the most effective. PTX, theofylline, and DXM inhibited the release of IL-1 beta, but caffeine did not affect IL-1 beta release. The release of IL-10 was significantly reduced by PTX at 24 h and by caffeine at 48 h, but DXM increased the release of this cytokine. In sum, the results of this study demonstrate that DXM inhibits only the release of proinflammatory cytokines but not of the anti-inflammatory cytokine IL-10 by human leukocytes, while PTX is the most potent inhibitor of both proinflammatory and anti-inflammatory cytokines.
Identification of outer membrane proteins as target antigens of Pseudomonas aeruginosa Homma serotype M
Authors: Yokota S
Published: January 2020

Pseudomonas aeruginosa is routinely serotyped in Japan by using the Homma scheme. The serotypes (O serotypes) are based on the chemical structure of the O-polysaccharide portion of the lipopolysaccharide (LPS). However, the nature of the Homma serotype M antigen has remained obscure because strains classified as serotype M usually have the rough phenotype. I characterized the target antigen of serotype M. The results of Western blotting (immunoblotting) showed that commercially available typing monoclonal antibody (MAb) against serotype M specifically bound to outer membrane protein (Opr) G and that typing rabbit antiserum specific for serotype M mainly contained antibodies against Oprs F and H2. These Oprs were distributed among all P. aeruginosa strains tested, including the serotype standard, serotype M and nontypeable strains, and a series of LPS-core-defective mutants derived from strain PAC1. However, the rough mutants derived from strain PAC1 agglutinated with the anti-serotype M antibodies, whereas the smooth strains did not. LPS preparations from serotype M strains possessed few or no polysaccharide chains. These strains had higher levels of binding activity with anti-serotype M MAb, as well as with anti-lipid A MAb, which specifically bound to the cell surface of the rough-natured gram-negative bacterial strains with high activity. The anti-serotype M antiserum also contained rough-LPS-specific antibodies, but the epitope was distributed among only a few strains. The results suggested that the Oprs acted as the serotype M antigen and that LPS did not. In conclusion, the rough strains agglutinated with anti-Opr antibodies and were distinguished as serotype M from the smooth strains of other serotypes, because the antibodies were accessible to the cell surface lacking O polysaccharides. I supposed that Homma serotype M is an index of the rough nature of P. aeruginosa strains rather than one of the O serotypes.
Quantitative analysis of phagocytosis and killing of Cryptococcus neoformans by human peripheral blood mononuclear cells by flow cytometry
Authors: Chaka W , Scharringa J , Verheul A F , Verhoef J , Van Strijp A G , Hoepelman I M
Published: January 2020

Monocytes may represent an important defense mechanism in disseminated cryptococcosis. We have developed a flow cytometric method to study the interaction of Cryptococcus neoformans with monocytes. For phagocytosis, C. neoformans was labelled with fluorescein isothiocynate (FITC). Monocytes were identified on the flow cytometer by labelling with anti-CD14-R-phycoerythrin. Discrimination between attached cells (association) and internalized cells (uptake) was made by quenching FITC-labelled C. neoformans with trypan blue. Only internalized cells kept their FITC fluorescence after quenching. For comparison under the microscope, specific staining of the cell wall of C. neoformans with Uvitex was used. Internalized C. neoformans cells were not stained, as Uvitex was occluded from phagocytes. To assay killing, C. neoformans was labelled with 0.2 mM 2'-7(1)-bis(2-carboxyethyl)-5-carboxyfluorescein acetoxymethylester. After phagocytosis of labelled cells by monocytes, blood cells were lysed with 25 mM deoxycholate. Viable yeast cells retained the fluorescence, but nonviable cells lost it. Quantitative counts of viable cells on Sabouraud dextrose agar were performed for comparison. The change in the relative fluorescence of green within the monocyte region was used to quantitate association, uptake, and killing of C. neoformans by monocytes on the flow cytometer. The flow cytometry methods showed that 18% +/- 2%, 35% +/- 14%, 50% +/- 11%, 51% +/- 6% of monocytes had become associated with C. neoformans after 0, 30, 60, and 120 min, respectively. After 2 h of phagocytosis time, 30% of C. neoformans-associated monocytes had taken up the cells, and killing rates of 23% +/- 17%, 22% +/- 9%, and 40% +/- 13% were obtained with effector-to-target cell ratios of 1:1, 10:1, and 50:1, respectively. Results with the flow cytometry methods compared favorably with those by the conventional methods used, but the flow cytometry methods are simpler, rapid, more reproducible, and objective.
Isolation and characterization of human placental trophoblast subpopulations from first-trimester chorionic villi
Authors: Aboagye-Mathiesen G , Laugesen J , Zdravkovic M , Ebbesen P
Published: January 2020

A method for the simultaneous preparation of highly enriched human placental trophoblast populations (villous and extravillous) from first-trimester placental villi (5 to 12 weeks) by using sequential trypsinization, percoll gradient centrifugation, and negative selection with anti-CD9 immunomagnetic separation is described. The purification method resulted in the isolation of four distinct trophoblast populations identified on the basis of morphology and phenotyping: (i) mononuclear villous cytotrophoblast cells which, through differentiation, become committed to syncytium formation; (ii) an extravillous trophoblast population which appeared as a "crazy pavement" and, with subsequent subculturing, differentiated morphologically to mononuclear cells; (iii) an extravillous trophoblast fraction which fused to form multinucleated trophoblast giant cells; and (iv) floating intermediate extravillous trophoblast cells which fused together to form cell clumps and which further differentiated to a mononuclear anchoring intermediate extravillous trophoblast. Short-term cultures of the freshly isolated cell fractions consisted of heterogeneous trophoblasts at different differentiation stages as determined by their varied biochemical and morphological properties. All the isolated trophoblast populations expressed the cytokeratin intermediate filament and the epithelium-specific cell-cell adhesion molecule E-cadherin. The isolated villous trophoblasts in culture expressed integrins alpha 6 and beta 4 and reduced levels of beta 1 subunits, whereas the proliferating extravillous trophoblast cultures expressed alpha 1, alpha 3, and alpha 5 and high levels of beta 1 integrin subunits, vitronectin receptor (alpha V beta 3/beta 5), and major histocompatibility complex class 1 molecules. Furthermore, the isolated trophoblast populations secreted metalloproteases (such as type IV collagenases [mainly 72- and 92-kDa enzymes, i.e., gelatinases A and B]) and urokinase plasminogen activator, as evaluated by substrate gel zymography. This method of isolation should facilitate in vitro studies of trophoblast proliferation, differentiation, invasion, virus interactions, cytokenesis, and immunology.
Regulation of early complement components C3 and C4 in the synovium
Authors: Collins T , Winkelstein J A , Sullivan K E
Published: January 2020

To determine the cytokine inducibility of early complement component (C3 and C4) expression in the synovium, explant tissue was maintained in culture for 7 days. C3 and C4 production was measured by specific enzyme-linked immunosorbent assay, and RNA was evaluated by semiquantitative PCR. The effects of leukemia inhibitory factor (LIF), gamma interferon (IFN-gamma), IFN-alpha, and estrogen on C3 and C4 expression were evaluated. C3 levels were unaffected by 7 days of LIF, IFN-gamma, or IFN-alpha treatment. In contrast, C4 levels were significantly induced in synovial samples treated for 7 days with either IFN-gamma or IFN-alpha. LIF had no effect on C4 levels in this system. Estrogen was found to down-modulate the induction of expression due to IFN-gamma. These data provide evidence for cytokine regulation of C4 expression in the synovium and for estrogen modulation of those effects.
Normal range estimation for repeated immunologic measures
Authors: Taylor J M , Cumberland W G , Meng X , Giorgi J V
Published: January 2020

A method for estimating a normal range from a set of immunologic measurements on control subjects when there may be more than one observation per subject is described. The method is nonparametric, makes efficient use of all observations, and is very simple to apply. It is illustrated on a set of 152 CD38 measurements from 58 healthy men.
Expression of the somatostatin gene in human astrocytoma cell lines
Authors: Mercure L , Tannenbaum G S , Schipper H M , Phaneuf D , Wainberg M A
Published: January 2020

Somatostatin (somatotropin release-inhibiting hormone; SRIH) has been demonstrated in neurons of the central nervous system (CNS) as well as in endocrine cells of the pancreas and gastrointestinal tract and can suppress various immune functions including lymphocyte proliferation, immunoglobulin synthesis, and cytokine production. Since astrocytes possess antigen-presenting activity and can secrete a wide array of immunoregulatory and inflammatory cytokines, we studied SRIH gene expression in both astrocyte cell lines and mitogen-stimulated peripheral blood mononuclear leukocytes from healthy donors. We now report by means of a complementary DNA-based reverse transcription PCR that differential levels of SRIH mRNA were expressed in 9 of 11 human astrocytoma cell lines tested but were undetectable in activated peripheral blood mononuclear leukocytes as well as in a variety of human lymphocyte and monocyte cell lines. The synthesis and secretion of SRIH protein by astrocytoma cells that expressed SRIH transcripts were confirmed by specific radioimmunoassay of cell culture fluids. These findings support the notion that SRIH gene expression occurs in human astrocytoma cells but not in mature lymphoid cells of the immune system.
Enzyme-linked immunosorbent assay with partially purified cytosoluble 28-kilodalton protein for serological differentiation between Brucella melitensis-infected and B. melitensis Rev.1-vaccinated sheep
Authors: Salih-Alj Debbarh H , Cloeckaert A , Bézard G , Dubray G , Zygmunt M S
Published: January 2020

The problem of differentiating sheep infected with Brucella melitensis from those vaccinated or exposed to cross-reaching organisms has not been resolved by conventional serological tests or through the use of the smooth lipopolysaccharide in primary binding assays. We therefore analyzed sera from ewes experimentally infected with B. melitensis H38, from ewes naturally infected with B. melitensis, and from B. melitensis Rev.1-vaccinated ewes by enzyme-linked immunosorbent assay with three antigenic fractions: O polysaccharide, a cytosoluble protein extract (CPE) from the rough strain B. melitensis B115, and a partially purified cytosoluble protein of 28 kDa (CP28) from the CPE. Immunoglobulin G anti-O polysaccharide and anti-CPE responses were detected in all groups of animals tested (Rev.1 vaccinated and B. melitensis infected). However, false-positive reactions with CPE occurred with sera from Brucella-free ewes. The use of partially purified CP28 abolished these false-positive reactions. Furthermore, no immunoglobulin G antibodies against CP28 were detected in sera from vaccinated ewes, whereas 80% (8 of 10) of ewes experimentally infected with B. melitensis H38 and 89% (25 of 28) of naturally infected ewes showed various degrees of anti-CP28 reactivity (absorbance values of between 0.5 and 2.5). The results obtained with CP28 showed the potential usefulness of this antigen to permit the detection of B. melitensis-infected ewes and their differentiation from B. melitensis Rev.1-vaccinated ones.
The DNA-binding protein pUL57 of human cytomegalovirus: comparison of specific immunoglobulin M (IgM) reactivity with IgM reactivity to other major target antigens
Authors: Maine G T , Lazzarotto T , Chovan L E , Flanders R , Landini M P
Published: January 2020

In this work we used PCR to amplify the DNA regions coding for two polypeptides from pUL57 of human cytomegalovirus (amino acids 540 to 601 and 1144 to 1233) and showed that both portions reacted very efficiently with immunoglobulin M in sera of acutely infected subjects. However, pUL57 is not an essential antigen for the replacement of or supplement to a cocktail of recombinant protein antigens containing portions of ppUL32, -44, -83, and -80a in immunoglobulin M serology.
Determination of varicella-zoster virus (VZV) immune status with the VIDAS VZV immunoglobulin G automated immunoassay and the VZVScan latex agglutination assay
Authors: Gleaves C A , Schwarz K A , Campbell M B
Published: January 2020

The VIDAS varicella-zoster virus (VZV) immunoglobulin G immunoassay and the VZVScan latex agglutination (LA) test were compared for the detection of VZV antibody in patient sera. Of 625 samples tested, 554 were positive for VZV antibody by one or both methods. Five hundred thirteen (82.1%) samples were positive by both methods, 71 (11.4%) were negative by both methods, and there were 41 (6.6%) discrepant samples. Statistically significant differences in sensitivity and specificity were not found (P = 0.37); however, several observations were made. All 23 VIDAS repeat equivocal samples were LA positive. Fifty-four samples showed prozone effects with the LA assay, all of which resolved as positive.