bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of europium and chromium release assays: cytotoxicity in healthy individuals and patients with cervical carcinoma
Authors: von Zons P , Crowley-Nowick P , Friberg D , Bell M , Koldovsky U , Whiteside T L
Published: January 2020

Natural killer (NK) and lymphokine-activated killer (LAK) cell activities were measured in peripheral blood obtained from healthy women to compare a standard 51Cr release assay with a nonradioactive europium (Eu3+) release assay based on time-resolved fluorescence. The two types of cytotoxicity assays were first compared in paired determinations performed on 28 samples of peripheral blood mononuclear cells obtained from healthy women who had normal pap smears or no biopsy evidence of cervical squamous intraepithelial lesions (SIL). Target cells (NK-sensitive K562 and NK-resistant Raji cell lines) were labeled with Eu3+ only, 51Cr only, or both labels and compared in cytotoxicity assays using fresh or interleukin 2 (IL-2)-activated effector cells. Spontaneous release in the Eu3+ release assay was comparable to that observed in the 51Cr release assay, but maximum Eu3+ release always exceeded that of 51Cr. In 4-h assays, specific release of Eu3+ from target cells was more rapid than that of 51Cr, consistently resulting in 30 to 40% higher levels of activity. However, a significant linear correlation (P < 0.001) was observed between cytotoxicity levels based on measurements of Eu3+ and 51Cr release in 4-h assays. The Eu3+ release assay was then used to measure NK and LAK activities in the peripheral blood of women with cervical SIL or cervical squamous cell carcinoma (SCC). Mean NK activity of women with advanced SIL (121 lytic units [LU]) or SCC (93 LU) was found to be similar to that of controls (101 LU) or patients with normal cervical biopsies (90 LU), as was the ability to generate IL-2-stimulated NK activity. However, LAK activity during 18 h of incubation in the presence of IL-2 was reduced in patients with cervical SCC (P < 0.05) compared with that in normal controls. Results of 51Cr assays performed in parallel with patient samples gave comparable results. Advantages of EU3+ release assays for routine evaluation of cytotoxicity are discussed.
Inflammatory myopathy, bronchiolitis obliterans/organizing pneumonia, and anti-Jo-1 antibodies--an interesting association
Authors: Kalenian M , Zweiman B
Published: January 2020

We report an interesting association of inflammatory myopathy, characterized pathologically as dermatomyositis, with bronchiolitis obliterans/organizing pneumonia and anti-histidyl-tRNA synthetase (Jo-1) antibody. The relations of different types of pulmonary involvement to inflammatory myopathy and antisynthetase antibodies are discussed.
Detection of HLA class I-specific antibodies by the QuikScreen enzyme-linked immunosorbent assay
Authors: Lucas D P , Paparounis M L , Myers L , Hart J M , Zachary A A
Published: January 2020

The GTI QuikScreen test is an enzyme-linked immunosorbent assay (ELISA) that uses soluble HLA class I antigens as targets. In tests of 5,893 human serum specimens, we evaluated the reliability, sensitivity, and utility of the GTI QuikScreen test for detecting HLA class I-specific antibody. We found that the test could reliably detect HLA-specific antibodies of the immunoglobulin G (IgG) but not the IgM class. The degree of correlation with lymphocytotoxicity testing varied among the different serum sources, with the best correlation achieved with sera from renal transplant candidates (r > 0.7) and the poorest with sera from patients with end-stage liver disease (r = 0.26), possibly because of elevated alkaline phosphatase levels in the liver patients. Test reproducibility was high (96%), and test failure rate was low (1.7%). The test sensitivity is comparable to that of the antiglobulin cytotoxicity and, possibly, even flow cytometric tests. There was a highly significant (P < 0.001) correlation between the optical densities obtained in the ELISA and the percent panel reactive antibody determined by cytotoxicity testing. Therefore, although designed only to determine the presence or absence of HLA-specific antibody, GTI QuikScreen test results also provided an indication of the extent of sensitization. The test is one of the most effective and efficient ways to determine if antibodies producing a positive result in crossmatch tests are specific for HLA class I antigens. As an adjunct to serum screening by cytotoxicity testing, the GTI QuikScreen test can produce a substantial savings of time and effort that reduces the cost to the laboratory and to the patient.
Increased frequency of HLA-DR11 in pediatric human immunodeficiency virus-associated parotid gland enlargement
Authors: Schuval S J , O'Reilly M E , Bonagura V R
Published: January 2020

We sought to determine whether an increased frequency of the HLA-DR11 (formerly DR5) phenotype is found in human immunodeficiency virus (HIV)-infected children with parotid gland enlargement. In HIV-infected adults, parotid gland enlargement may be part of the diffuse infiltrative CD8 lymphocytosis syndrome. An increased frequency of expression of HLA-DR11 has been described in association with diffuse infiltrative CD8 lymphocytosis syndrome. We conducted a case-control study with 26 HIV-infected children, 13 of whom had parotid gland enlargement and 13 of whom did not but who were matched for age, race, and sex with those with parotid gland enlargement. Clinical and laboratory parameters (including HLA-DR11 phenotype) were compared between the two groups. HIV-positive children with parotid gland enlargement showed an increased frequency of HLA-DR11, similar to their adult counterparts with diffuse infiltrative CD8 lymphocytosis syndrome. The HLA-DR11 phenotype may be associated with the development of parotid gland enlargement in HIV-infected children and may be a marker for a more benign outcome of HIV infection.
In vivo semen-associated pH neutralization of cervicovaginal secretions
Authors: Tevi-Bénissan C , Bélec L , Lévy M , Schneider-Fauveau V , Si Mohamed A , Hallouin M C , Matta M , Grésenguet G
Published: January 2020

Physiological cervicovaginal acidity can partly inactivate human immunodeficiency virus (HIV). Basic semen components should be able to partially neutralize in vivo cervicovaginal pH. The goals of the study were to evaluate the relationship between cervicovaginal pH and presence of semen components in sexually active African women and to assess whether vaginal douching with water performed just after sexual intercourse could significantly reduce semen components and restore physiological cervicovaginal pH. Cervicovaginal secretion (CVS) from 56 heterosexual African women (19 to 45 years old), living in Bangui, Central African Republic, were evaluated for pH, semen components (prostatic acid phosphatase [PAP] and prostatic specific antigen [PSA]), cellularity, and hemoglobin at inclusion and after vaginal douching with 100 ml of water by using a bock. Before douching, semen components were found in 46 of 56 CVS (82%). The mean vaginal pH was 5.2 (range, 3.6 to 7.7), and concentrations of both PAP and PSA correlated positively and strongly with cervicovaginal pH (P < 0.001). After douching, semen components were found in 35 of 56 CVS (62%) (P = 0.03). Cervicovaginal PAP and PSA levels were significantly decreased (respectively, P < 0.0001 and P < 0.01; PAP, -72%; PSA, -87%), as was the total cell count (-60%; P < 0.0001). Furthermore, in CVS previously positive for both PAP and PSA, the mean vaginal pH was significantly decreased (6.5 versus 5.3, P < 0.01); no genital bleeding was observed. Frequent persistence of semen in CVS from heterosexually active African women leads to a shift from acidity to neutrality that could favor male to female HIV transmission. Vaginal douching provides significant elimination of semen after sexual intercourse; it should be considered for study as a supplementary means for the prevention of heterosexual HIV transmission.
Immune responses in mothers of term and preterm very-low-birth-weight infants
Authors: Gennaro S , Fehder W P , Cnaan A , York R , Campbell D E , Gallagher P R , Douglas S D
Published: January 2020

Differences in the levels of immune cell subsets present in peripheral blood have been demonstrated based on sociodemographic factors such as age and race. Postpartal women, who are recovering from the immune changes that are concomitant with pregnancy, have lymphocyte and monocyte values that differ from other populations. A subgroup of postpartal women, mothers who deliver preterm very-low-birth-weight (VLBW) (< or = 1,500 g) infants, may have further differences in values of immune cell subsets and in immune functioning either because of hormonal factors or lifestyle changes or because of the stress they experience after their infant's birth and for the first few months of infant caretaking. This study examined anxiety, depression, and immune cell phenotypes in 30 mothers of VLBW infants and in 30 mothers of healthy term infants over the first 4 postpartal months to determine if mothers of preterm VLBW infants differed from mothers of healthy term infants in psychological and immunologic parameters. Additionally, lymphocyte proliferation and natural killer cell functional assays were performed in a subset of mothers. Mothers of VLBW infants had increased anxiety and decreased lymphocyte proliferation compared to mothers of term infants. When lymphocyte and monocyte subsets were compared over time between the two groups of mothers differences were found in CD8, CD20, CD3-/CD56+, CD14, and HLA class II Ia on monocytes. Mothers with high-fat diets had lower percentages of some monocytes (CD14), lymphocytes (CD4+/CD45RA+), and natural killer cells (CD3-/CD57+) during the first 4 postpartal months.
Chronic administration of dexamethasone results in Fc receptor up-regulation and inhibition of class I antigen expression on macrophages from MRL/lpr autoimmune mice
Authors: Zuckerman S H , Evans G F , Bryan N
Published: January 2020

The MRL/lpr mouse develops, after approximately 8 weeks of age, a severe autoimmune syndrome with many features resembling human systemic lupus erythematosus, including autoantibodies against DNA and basement membranes resulting in immune complexes, vasculitis, and multiorgan disease. While this murine model of lupus has been used for the identification of therapeutics with potential efficacy in human autoimmune disease, the long-term impact of chronic immunosuppressive therapy on macrophage function in this paradigm is not understood. To this end, MRL/lpr mice were treated prophylactically with dexamethasone at 0.01, 0.1, and 1 mg/kg of body weight for 20 weeks or were allowed to develop autoimmune disease and, at 15 weeks of age, treated therapeutically with 1-mg/kg dexamethasone for 8 additional weeks. Analysis of surface antigens on resident peritoneal macrophages demonstrated a progressive loss in class I expression with a concomitant increase in Fc receptor expression. Neither phagocytosis nor CD11b expression was modulated with chronic steroid treatment. Furthermore, dexamethasone treatment was associated with a reduction in anti-DNA antibodies and total immunoglobulin G and yet an elevation in serum cholesterol due to an increase in high-density lipoproteins. Therefore, the MRL/lpr mouse serves not only as a small-animal model of autoimmune disease but also as one in which the negative and positive sequelae associated with chronic immunosuppression can be further understood.
Evaluation of a poliovirus-binding inhibition assay as an alternative to the virus neutralization test
Authors: Herremans M M , Reimerink J H , Ras A , Van Der Avoort H G , Kimman T G , Van Loon A M , Conyn-Van Spaendonck M A , Koopmans M P
Published: January 2020

An enzyme-linked immunosorbent assay (ELISA)-based poliovirus-binding inhibition (PoBI) test to detect and quantify antibodies to polioviruses was optimized and evaluated for use in population studies as an alternative to the virus neutralization test (NT) in tissue culture. The sensitivities of the inhibition ELISA compared with the NT in an inactivated poliovirus vaccine (IPV)-vaccinated population were 98.6, 97.4, and 92.1% for serotypes 1, 2, and 3, respectively. The specificities of the PoBI test, as determined with sera from nonvaccinated persons, were also high for all three serotypes (99.0, 95.8, and 100%, respectively). Antibodies to other enteroviruses did not cross-react in the serotype 1 and 3 PoBI, and only levels of cross-reactivity were found for serotype 2. We found high correlations between the PoBI and NT titers for serotypes 1 and 2 in IPV-vaccinated blood donors (0.97 and 0.95), in oral poliovirus vaccine (OPV)-vaccinated blood donors (0.91 and 0.95), and in naturally immune persons (0.90 and 0.87). The correlation coefficient for serotype 3, however, was significantly lower in OPV-vaccinated blood donors (0.73) and in naturally immune persons (0.76) than in IPV-vaccinated persons (0.94; P < 0.01). These results indicate that the antibody response to serotype 3 poliovirus in IPV recipients is different from that in OPV recipients and naturally infected persons. We conclude that the PoBI test is a suitable alternative to the NT for estimating the seroprevalence of neutralizing antibodies to poliovirus, especially in large-scale population studies.
High prevalence of antibodies to Mycoplasma penetrans in human immunodeficiency virus-seronegative and -seropositive populations in Brazzaville, Congo
Authors: Tuppin P , Delamare O , Launay V , Gueguen M , Samba M C , Pambou L , Montagnier L , Grau O
Published: January 2020

In industrialized countries, the prevalence of antibodies to Mycoplasma penetrans is higher among human immunodeficiency virus (HIV)-seropositive homosexuals than other HIV-seropositive and HIV-seronegative groups. In an African heterosexual population, we found a higher prevalence of M. penetrans antibodies in HIV-seronegative blood donors (15.5%) than in France (0.9%) or the United States (0.3%) and a prevalence of 13.4% in HIV-seropositive individuals. HIV-seropositive individuals with less than 5% CD4 cells had a higher prevalence of M. penetrans antibodies than individuals with 5% or more CD4 cells (25.0 versus 8.5%).
Adaptive mutation and cocolonization during Helicobacter pylori infection of gnotobiotic piglets
Authors: Akopyants N S , Eaton K A , Berg D E
Published: January 2020

Clinical isolates of Helicobacter pylori, the gastric pathogen implicated in gastritis, peptic ulcers, and gastric cancer in humans, are diverse in traits likely to be important for colonization and disease. Here we report studies using a gnotobiotic piglet-H. pylori infection model to test for host-specific adaptation and to detect cocolonization by different strains. First, an H. pylori strain that initially had grown only weakly in piglets was adapted to them by spontaneous mutation and selection during 12 serial passages; this resulted in an increase in yield from about 10(3) to > 10(7) bacteria per g of mucosa. Second, piglets were fed mixtures of two different well-adapted strains and the presence of one or both strains was monitored by restriction analysis of a PCR-amplified flagellar (flaA) gene segment. The restriction fragment patterns from pools of bacteria indicated that both strains had colonized most piglets and that both strains were present at more than half of the individual biopsy sites, although often at unequal ratios. This suggests a microcolonial mode of growth with limited migration of bacteria between neighboring sites in the gastric mucosa. We propose that the gnotobiotic piglet-H. pylori infection model will be useful for testing how spontaneous mutation, selection, and DNA transfer between strains during mixed infection may each contribute to adaptation to specific hosts and the evolution of virulence of this important pathogen.
Isolation of extracytoplasmic proteins from Serpulina hyodysenteriae B204 and molecular cloning of the flaB1 gene encoding a 38-kilodalton flagellar protein
Authors: Gabe J D , Chang R J , Slomiany R , Andrews W H , McCaman M T
Published: January 2020

Extracytoplasmic proteins were released from Serpulina (Treponema) hyodysenteriae (strain B204) by treatment of whole cells with a nonionic detergent (Tween 20). Centrifugation of the Tween 20-released proteins at 100,000 x g sedimented 10 major extracytoplasmic proteins with approximate molecular masses of 44, 43.5, 42, 39, 38, 34, 33.5, 33, 31, and 29 kDa. Treatment of the sedimented fraction with 6 M urea solubilized all of the proteins except the 39-kDa protein. Peptide sequences were obtained for the purified 42-, 39-, 38-, 34-, 31-, and 29-kDa proteins. The peptide sequences of the 42-, 38-, and 31-kDa proteins indicate that they likely are components of the periplasmic flagella. The amino-terminal peptide sequence of the 38-kDa protein was used to design an oligonucleotide probe and to clone an S. hyodysenteriae DNA fragment containing the gene encoding this protein. The predicted 290-amino-acid protein sequence derived from the cloned gene was highly homologous to those of several other bacterial flagellar proteins and is preceded by consensus sigma D nucleotide sequences found upstream of other flagellar genes. On the basis of its similarity to the FlaB proteins of other spirochetes, we propose to designate the cloned S. hyodysenteriae gene flaB1 and its encoded protein FlaB1. Vaccination of pigs with FlaB1 or its recombinant counterpart did not protect them from an experimental challenge.
Revised sequence of the Porphyromonas gingivalis prtT cysteine protease/hemagglutinin gene: homology with streptococcal pyrogenic exotoxin B/streptococcal proteinase
Authors: Madden T E , Clark V L , Kuramitsu H K
Published: January 2020

The prtT gene from Porphyromonas gingivalis ATCC 53977 was previously isolated from an Escherichia coli clone possessing trypsinlike protease activity upstream of a region encoding hemagglutinin activity (J. Otogoto and H. Kuramitsu, Infect. Immun. 61;117-123, 1993). Subsequent molecular analysis of this gene has revealed that the PrtT protein is larger than originally reported, encompassing the hemagglutination region. Results of primer extension experiments indicate that the translation start site was originally misidentified. An alternate open reading frame of nearly 2.7 kb, which encodes a protein in the size range of 96 to 99 kDa, was identified. In vitro transcription-translation experiments confirm this size, and Northern (RNA) blot experiments indicate that the protease is translated from a 3.3-kb mRNA. Searching the EMBL protein database revealed that the amino acid sequence of the revised PrtT is similar to sequences of two related proteins from Streptococcus pyogenes. PrtT is 31% identical and 73% similar over 401 amino acids to streptococcal pyrogenic exotoxin B. In addition, it is 36% identical and 74% similar over 244 amino acids with streptococcal proteinase, which is closely related to streptococcal pyrogenic exotoxin B. The similarity is particularly high at the putative active site of streptococcal proteinase, which is similar to the active sites of the family of cysteine proteases. Thus, we conclude that PrtT is a 96- to 99-kDa cysteine protease and hemagglutinin with significant similarity to streptococcal enzymes.
Reconstituted high-density lipoprotein neutralizes gram-negative bacterial lipopolysaccharides in human whole blood
Authors: Parker T S , Levine D M , Chang J C , Laxer J , Coffin C C , Rubin A L
Published: January 2020

We have tested hypotheses relating lipoprotein structure to function as measured by the relative ability to neutralize endotoxin by comparing natural human lipoproteins, a chemically defined form of reconstituted high-density lipoprotein (R-HDL), and a lipid emulsion (Intralipid). The human whole-blood system was used as an in vitro model of lipopolysaccharide (LPS) binding protein and CD14-dependent activation of cytokine production. When lipoproteins were compared on the basis of protein content, R-HDL was most effective in reducing tumor necrosis factor alpha (TNF-alpha) production followed in order by very low density lipoprotein, low-density lipoprotein, Intralipid, and natural HDL. However, when these particles were compared by protein, phospholipid, cholesterol, or triglyceride content by stepwise linear regression analysis, only phospholipid was correlated to effectiveness (r2 = 0.873; P < 0.0001). Anti-CD14 monoclonal antibodies MY4 and 3C10 inhibited LPS binding protein and CD14-dependent activation of TNF-alpha production by LPS at LPS concentrations up to approximately 1.0 ng/ml. R-HDL (2 mg of protein per ml) blocked TNF-alpha production by LPS from both smooth- and rough-type gram-negative bacteria at concentrations up to 100 ng of LPS per ml but had little effect on heat-killed gram-positive Staphylococcus aureus and no effect on other LPS-independent stimuli tested. These results support our hypothesis that LPS is neutralized by binding to phospholipid on the surface of R-HDL and demonstrate that R-HDL is a potent inhibitor of the induction of TNF-alpha by LPS from both rough- and smooth-form gram-negative bacteria in whole human blood.
Knockout mutants of Actinobacillus pleuropneumoniae serotype 1 that are devoid of RTX toxins do not activate or kill porcine neutrophils
Authors: Jansen R , Briaire J , Smith H E , Dom P , Haesebrouck F , Kamp E M , Gielkens A L , Smits M A
Published: January 2020

The Actinobacillus pleuropneumoniae RTX-toxins ApxI, ApxII, and ApxIII are important virulence factors of this swine pathogen. It is hypothesized that the Apx toxins are deleterious to defense cells of the host, enabling the bacterium to infect the host. To confirm this, we studied the effect on porcine polymorphonuclear neutrophils of mutant strains of A. pleuropneumoniae that were devoid of Apx toxins. For this purpose, we developed a system for targeted mutagenesis of A. pleuropneumoniae based on the conditionally replicating plasmid pVE6063 and insertional mutagenesis by homologous recombination. Employing this system on the reference strain of serotype 1, a strain that secretes ApxI and ApxII, we generated mutant strains that were devoid of ApxI and/or ApxII. We compared the ability of the parent strain and the mutant strains to provoke an oxidative burst in porcine neutrophils and to kill these cells. The parent strain and mutants that secreted either ApxI or ApxII provoked an oxidative burst and killed the neutrophils, whereas mutant strains that were devoid of ApxI and ApxII did not. These experiments indicate the importance of ApxI and ApxII to these profound effects on neutrophils and emphasize the importance of ApxI and ApxII in pathogenesis.
Noninhibitory binding of human interleukin-2-activated natural killer cells to the germ tube forms of Candida albicans
Authors: Arancia G , Molinari A , Crateri P , Stringaro A , Ramoni C , Dupuis M L , Gomez M J , Torosantucci A , Cassone A
Published: January 2020

During incubation in vitro with yeast or germ tube forms of Candida albicans, only 2 to 6% of freshly isolated human natural killer (NK) cells (> 85% CD16+, CD56+, CD3-; < 15% CD3+; cytolytic for the NK-susceptible target K562 but not for the NK-resistant target DAUDI), were seen to interact with the fungal cells. As seen under the electron microscope, the contact area had a limited extent and was narrow, and neither the surface nor the intracytoplasmic organization of the NK cell was altered. In contrast, more than 30% of interleukin-2-activated NK (LAK) cells (> 96% CD16+, CD56+, CD3-; 1.5% CD3+; cytolytic for both K562 and DAUDI targets) interacted closely with the fungus. This interaction was particularly extensive with the surface of the fungal germ tube that was intimately enveloped by villous protrusions from the lymphocyte surface. The fungus-interacting LAK cell also showed a remarkable redistribution of surface microvilli and polarization of cytoplasmic organelles, such as the Golgi apparatus, centrioles, and granules, toward the area of fungal contact. Together with the elevated cytolytic potential against the K562 and DAUDI targets, all the morphological data suggested the presence of a potentially active lytic machinery in the fungus-interacting LAK cell. Nonetheless, two independent assays for anticandidal activity did not show consistent killing or fungal growth inhibition by either fresh NK or LAK cells. While offering direct evidence of the strong interaction between human LAK cells and the germ tubes, precursors of tissue-invasive hyphal forms of C. albicans, our observations also suggest that this interaction may not be sufficient to kill the fungus or arrest its growth.
Rickettsia conorii entry into Vero cells
Authors: Teysseire N , Boudier J A , Raoult D
Published: January 2020

The entry of rickettsiae into eukaryotic cells is mediated by an induced phagocytosis, but rickettsiae have never been observed in a closed phagocytic vacuole. In this study, Rickettsia conorii entry into Vero cells was observed by transmission electron microscopy during a period of 3 to 20 min after bacterium-cell contact. The entry occurred within 3 min after bacterium-cell contact, and R. conorii was observed in the process of engulfment, within a phagocytic vacuole, or free in the cytosol. Escape from the phagosome is a very rapid step since phagosome lysis was only occasionally observed. By 12 min, 90% of bacteria were internalized and half were free in the cytosol. This report confirms that rickettsiae penetrate nonphagocytic cells by induced phagocytosis and is the first demonstration of rickettsiae within a complete phagocytic vacuole.
Fetal outcome in murine Lyme disease
Authors: Silver R M , Yang L , Daynes R A , Branch D W , Salafia C M , Weis J J
Published: January 2020

Lyme disease is an inflammatory syndrome caused by infection with Borrelia burgdorferi. Although this syndrome has important implications for human pregnancy, little is known about gestational infection with B. burgdorferi. Fetal death occurred in 33 of 280 gestational sacs (12%) in 39 C3H/HeN female mice infected by intradermal injection of B. burgdorferi 4 days after mating (acute infection), compared with 0 of 191 sacs in 25 control mice (P = 0.0001). Forty-six percent of acutely infected mice suffered at least one fetal death, compared with none of the control animals (P = 0.0002). There were no fetal deaths in 18 C3H/HeN mice infected 3 weeks prior to mating (chronic infection). A sensitive PCR technique detected B. burgdorferi DNA in the uteri of acutely infected mice but did not detect DNA in the uteri of controls or chronically infected mice. Spirochete DNA was only rarely detected in fetal tissues, and its presence was not required for fetal death. The inclusion of an internal competitive PCR target indicated that the lack of B. burgdorferi sequences in fetal DNA was not due to the presence of a PCR inhibitor. Histologic analysis of gestational tissues from infected animals demonstrated nonspecific pathology consistent with fetal death. These findings indicate an association between murine fetal death and acute infection with B. burgdorferi early in gestation but not with chronic infection. Our data suggest that fetal death is due to a maternal response to infection rather than fetal infection. These findings could provide an explanation for observations in humans in which sporadic cases of fetal death in women infected with B. burgdorferi during pregnancy have been reported, while previous infection has not been associated with fetal death.
Human immunoglobulin G (IgG) Fc receptor IIA (CD32) polymorphism and IgG2-mediated bacterial phagocytosis by neutrophils
Authors: Sanders L A , Feldman R G , Voorhorst-Ogink M M , de Haas M , Rijkers G T , Capel P J , Zegers B J , van de Winkel J G
Published: January 2020

Human immunoglobulin G (IgG) Fc receptor IIa (Fc gamma RIIa; CD32) is expressed on phagocytes, triggers phagocytosis, and represents the sole Fc receptor for IgG (Fc gamma R) capable of interaction with IgG2, the main IgG subclass induced in response to bacterial capsular polysaccharides. The two genetically determined structurally different allotypes of human Fc gamma RIIa, the products of the Fc gamma RIIa-R131 and IIa-H131 alleles, have functionally different reactivities with human IgG2. In humans, the Fc gamma RIIa-H131 allotype is known to interact efficiently with complexed human IgG2, whereas the IIa-H131 allotype does so only poorly. This polymorphism may therefore have implications for IgG2-mediated phagocytosis of encapsulated bacteria and susceptibility to bacterial infections. Phagocytosis of IgG2-opsonized bacteria by homozygous Fc gamma RIIa-R/R131, heterozygous IIa-H/R131, and homozygous IIa-H/H131 polymorphonuclear cells (PMN) was compared. A higher phagocytic capacity of IgG2-opsonized group B type III streptococci by PMN of homozygous H/H131 individuals compared with PMN from homozygous R/R131 individuals was observed (P = 0.001), while heterozygous IIa-H/R131 PMN showed intermediate phagocytosis. In this model system, IgG2-mediated phagocytosis was independent of the Fc gamma RIIIb-NA1/NA2 allelic polymorphism.
Fibronectin-binding antigen 85 and the 10-kilodalton GroES-related heat shock protein are the predominant TH-1 response inducers in leprosy contacts
Authors: Launois P , N'Diaye M N , Cartel J L , Mane I , Drowart A , Van Vooren J P , Sarthou J L , Huygen K
Published: January 2020

Peripheral blood mononuclear cells from 27 healthy leprosy contacts were analyzed for lymphoproliferation and TH-1 cytokine secretion (interleukin-2 and gamma interferon) in response to heat shock proteins with molecular masses of 65, 18, and 10 kDa from Mycobacterium leprae and the 30-32-kDa antigen 85 (Ag 85) from Mycobacterium bovis BCG. Cells from 18 and 19 of 19 lepromin-positive contacts proliferated or produced TH-1 cytokines in response to the M. leprae 10-kDa protein and to Ag 85, respectively. Limiting-dilution analysis for two lepromin-positive contacts indicated that about one-third of M. leprae-reactive T cells displayed specificity to the M. leprae 10-kDa protein and Ag 85. The M. leprae 65- and 18-kDa proteins were less potent TH-1 response inducers: gamma interferon and interleukin-2 could be measured in 14 and 19 lepromin-positive contacts, respectively. In contrast, very low or undetectable proliferative and cytokine responses were found for 8 lepromin-negative contacts. Our data demonstrate that the fibronectin-binding Ag 85 and the 10-kDa GroES homolog are powerful mycobacterial TH-1 response inducers in the vast majority of lepromin-positive contacts and suggest that they might be valuable candidates for a future subunit vaccine.
Analysis of expression of CagA and VacA virulence factors in 43 strains of Helicobacter pylori reveals that clinical isolates can be divided into two major types and that CagA is not necessary for expression of the vacuolating cytotoxin
Authors: Xiang Z , Censini S , Bayeli P F , Telford J L , Figura N , Rappuoli R , Covacci A
Published: January 2020

Colonization of the mucosa of the stomach and the duodenum by Helicobacter pylori is the major cause of acute and chronic gastroduodenal pathologies in humans. Duodenal ulcer formation strongly correlates with the expression of an antigen (CagA) that is usually coeexpressed with the vacuolating cytotoxin (VacA), a protein that causes ulceration in the stomach of mice. However, the relationship between these two virulence factors is unknown. To define whether CagA and VacA are coexpressed in all clinical isolates and their relationships, we collected 43 clinical isolates of H. pylori and studied their genetic and phenotypic properties. Based on this analysis, most of the strains could be classified into two major types. Type I bacteria had the gene coding for CagA and expressed the CagA protein and the vacuolating cytotoxin. Type II bacteria did not have the gene coding for CagA and did not express either the CagA protein or the vacuolating cytotoxin. Type I and type II bacteria represented 56 and 16%, respectively, of the 43 clinical isolates, while the remaining 28% had an intermediate phenotype, expressing CagA independently of VacA or vice versa. This finding shows that although it is present in most cytotoxic strains, CagA is not necessary for the expression of the vacuolating cytotoxin.
Differential effects of monoclonal antibody blockade of adhesion molecules on in vivo susceptibility to soft tissue infection
Authors: Garcia N , Mileski W J , Lipsky P
Published: January 2020

Leukocyte adherence to endothelial cells has been implicated in the pathogenesis of microvascular injury as well as in host defense against various infectious microorganisms. Administration of monoclonal antibodies directed against the beta chain of the leukocyte integrins inhibits leukocyte-endothelial-cell adherence and has been reported to modulate ischemia-reperfusion and inflammatory injury. However, such inhibition of adhesion molecule function adversely affects resistance to infection. The following studies were carried out to determine whether monoclonal antibodies to other adhesion molecules, including L-selectin (CD62L), and CD11a (the alpha chain of LFA-1), also increase susceptibility to infection. New Zealand White rabbits were shaved and given subcutaneous injections on their dorsa with 10(9) CFU of Staphylococcus aureus ATCC 25923 at two sites and with 10(8) CFU at two sites. A second set of rabbits were given subcutaneous injections with 10(8) CFU of P. aeruginosa ATCC 27853 at two sites and with 10(7) CFUs at two sites. The animals were monitored for 1 week. There were three blinded experimental groups: controls given saline and two groups given blocking monoclonal antibodies to either L-selectin (Dreg-200) or CD11a (R7.1). In contrast to monoclonal antibodies to CD18, none of the monoclonal antibodies significantly increased the risk of abscess formation by S. aureus, although inhibition of CD11a increased the rate of abscess formation by P. aeruginosa.
Nitric oxide production during murine Lyme disease: lack of involvement in host resistance or pathology
Authors: Seiler K P , Vavrin Z , Eichwald E , Hibbs J B , Weis J J
Published: January 2020

The murine model of Lyme disease was used to determine the role of inflammatory induced nitric oxide (NO) during infection by the spirochete Borrelia burgdorferi. The outer surface lipoproteins of B. burgdorferi are potent stimulators of inflammatory cytokines and NO production by cultured macrophages in vitro. The addition of NO to cultures of B. burgdorferi prevents growth, suggesting a protective role of NO for the infected host. NO is also a crucial effector in some models of arthritis. Therefore, the involvement of NO in controlling B. burgdorferi infection and its participation in pathological development of arthritis were investigated. Both mildly arthritic (BALB/c) and severely arthritic (C3H/HeJ) strains of mice systemically produced high levels of NO 1 week after infection with B. burgdorferi, as determined by urinary nitrate. NO production remained high throughout the infection in BALB/c mice, while in C3H/HeJ mice NO production returned rapidly to uninfected levels. The in vivo inhibitor of the NO synthase enzyme NG-L-monomethyl arginine (LMMA) was given to mice to investigate whether decreasing NO production would alter the course of disease. LMMA effectively blocked NO production in infected mice; however, there was no significant difference in arthritis development, spirochete infection of tissues, or production of specific antibody in LMMA-treated mice. These results indicate that B. burgdorferi is able to persist in the host even in the presence of high levels of NO. Furthermore, NO is not involved in the control of spirochete infection of tissues, nor is it involved in the development of arthritis. The potent activity of NO against intracellular pathogens and the in vivo resistance of B. burgdorferi to NO suggest that this organism is not located in an intracellular compartment during an essential portion of its infection of the mammalian host.
Molecular and biochemical characterization of a Coccidioides immitis-specific antigen
Authors: Pan S , Cole G T
Published: January 2020

Results of earlier investigations have indicated that the saprobic phase of Coccidioides immitis produces a heat-stable, 19-kDa antigen with serine proteinase activity which has been suggested to be specific for this pathogenic fungus. In the present study we have determined the N-terminal and partial internal amino acid sequences of the purified, 19-kDa antigen, cloned the gene which encodes this polypeptide, and confirmed that the secreted proteinase is a Coccidioides-specific antigen (CS-Ag). Both the genomic and cDNA sequences are reported and reveal that the csa gene which encodes this antigen has no introns. A 543-bp open reading frame encodes a 181-amino-acid-containing protein with a predicted molecular mass of 19.8 kDa and an isoelectric point of 8.3. The csa gene was localized on chromosome I of three representative C. immitis clinical isolates on the basis of Southern hybridizations. Expression of the csa gene in Escherichia coli using the pET21a plasmid vector yielded a recombinant protein that was recognized in immunoblot assays by antibody raised to the purified 19-kDa CS-Ag. Secretion of the native antigen is suggested to occur by cleavage of a putative 23-residue signal peptide. The native CS-Ag showed a low degree of glycosylation. Analysis of the carbohydrate composition of the CS-Ag revealed xylose, mannose, galactose, and glucose. However, the purified antigen showed no affinity for concanavalin A. A PCR method with specificity and high sensitivity for detection of C. immitis genomic DNA, using a pair of synthetic oligonucleotide primers whose sequences were based on that of the csa gene, was developed. A 520-bp product was amplified only when C. immitis genomic DNA was used as the template. The lower limits of DNA detection using this PCR method were 1 pg of C. immitis genomic DNA by ethidium bromide staining and 100 fg after Southern hybridization. The csa gene-based PCR method for detection of C. immitis DNA is useful for culture identification and may have clinical applications for the diagnosis of coccidioidal infections.
Lack of a role for natural killer cells in early control of Brucella abortus 2308 infections in mice
Authors: Fernandes D M , Benson R , Baldwin C L
Published: January 2020

Studies were conducted to determine if natural killer (NK) cells are important for early control of the virulent strain Brucella abortus 2308 following infection of mice with high or low challenge doses. Splenocytes from C57BL/10 and BALB/c mice that had been infected with the lower dose of B. abortus displayed increased cytotoxicity against YAC-1 cells during the first week after infection, while infection of C57BL/10 mice with the higher challenge dose either did not alter the level of NK cytotoxic activity or decreased it, depending upon the time postinfection. In vivo depletion of NK cells by monoclonal antibody anti-NK1.1 or polyclonal anti-asialoGM1 antiserum did not result in an increase in the number of brucellae recovered from the spleens or livers of the brucella-resistant C57BL/10 mice or from the spleens of the susceptible BALB/c mice during the first week after infection. Treatment of control mice with the NK-reactive antibodies, however, decreased killing of the NK-sensitive target YAC-1, indicating that the NK cell depletion regimes were effective. Our results suggest that NK cells are not crucial for early control of B. abortus 2308 even though they may be activated following infection. Further experiments indicated that treatment of C57BL/10 mice with poly(A:U) did not decrease the number of brucellae recovered from their spleens although it did decrease the CFU in livers of mice infected with the high challenge dose.
Molecular cloning and expression of a 70-kilodalton heat shock protein of Candida albicans
Authors: La Valle R , Bromuro C , Ranucci L , Muller H M , Crisanti A , Cassone A
Published: January 2020

By screening an expression library of the yeast form of Candida albicans with a serum directed against whole fungal cells, a cDNA (2,325 bp) encoding a stress protein of C. albicans was cloned and sequenced. The cloned sequence (CaRLV130) identified a single open reading frame with a length of 1,968 bp coding for a protein containing 656 amino acid residues (70 kDa). The deduced amino acid sequence was 84% similar to the sequence of the Saccharomyces cerevisiae SSA1 gene, which encodes one member of the 70-kDa heat shock protein (Hsp70) family. The relevant gene (C. albicans HSP70 gene [CaHSP70]) was localized on the highest-M(r) (R1; approximately 3.8 Mb) chromosome of C. albicans as determined by pulse-field electrophoresis. CaHSP70 was expressed after heat shock, as demonstrated by Northern (RNA) blotting and reverse transcriptase-PCR with specific pairs of oligonucleotide sequences and gene probes. A recombinant protein was obtained in Escherichia coli after cloning of the full coding sequence into the BamHI site of the pDS56/RBSII6xhisE- plasmid and purification by nickel chelate affinity chromatography. The recombinant protein (6xhis-CaHsp70) was efficiently recognized in immunoblots by a monoclonal antibody directed against a common epitope of eukaryotic Hsp70 proteins, as well as by sera from normal human subjects. Moreover, immune mouse sera against the purified recombinant protein recognized native, heat-inducible constituents with sizes of around 70 kDa in whole-cell protein extracts of C. albicans. Overall, our data demonstrate that CaHSP70 encodes one member of a family of proteins (Hsp70) which usually represent highly conserved immunodominant antigens of infectious agents.
Activation of human endothelial cells by viable or heat-killed gram-negative bacteria requires soluble CD14
Authors: Noel R F , Sato T T , Mendez C , Johnson M C , Pohlman T H
Published: January 2020

In response to bacterial lipopolysaccharides (LPS; endotoxin), endothelial cells are converted to an activation phenotype expressing both proinflammatory and procoagulant properties that include the induction of leukocyte adhesion molecules and tissue factor expression. LPS-induced endothelial cell activation requires a soluble form of the monocyte LPS receptor, sCD14. We evaluated the capacity of multiple strains of gram-negative and gram-positive bacteria to induce endothelial E-selectin and tissue factor expression through sCD14-dependent pathways with cultured human umbilical vein endothelial cells (HUVE). Both viable and heat-killed gram-negative bacteria (Bacteroides fragilis, Enterobacter cloacae, Haemophilus influenzae, and Klebsiella pneumoniae) but not viable or heat-killed gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae) induced prominent E-selectin surface expression detected by enzyme-linked immunosorbent assay. Tissue factor activity on HUVE, indicated by factor X activation, was induced in response to gram-negative bacteria but not in response to gram-positive bacteria. Gram-negative bacteria induced transcriptional activation in HUVE, indicated by the appearance of E-selectin-specific mRNA and by the demonstration of activation of NF-kappa B, a trans-activating factor necessary for E-selectin and tissue factor gene transcription. In contrast, neither E-selectin mRNA nor activation of NF-kappa B was detected in HUVE treated with gram-positive bacteria. Endothelial cell activation by gram-negative bacteria in each of these assays was inhibited with a monoclonal antibody (60bd) against CD14. Furthermore, CHO-K1 cells, transfected with human recombinant CD14, responded to all strains of gram-negative bacteria (viable or heat killed), indicated by CHO-K1 NF-kappa B activation. We conclude that gram-negative bacteria induce endothelial cell activation through a common sCD14-dependent pathway.
AIDS-associated mycoplasmas possess phospholipases C in the membrane
Authors: Shibata K , Sasaki T , Watanabe T
Published: January 2020

Phosphatidylinositol- and phosphatidylcholine-hydrolyzing activities existed in the cell lysates of AIDS-associated Mycoplasma species, including Mycoplasma fermentans IID 812, M. fermentans incognitus, and M. penetrans GTU-54-6A1. These activities were found to be attributable to phospholipases C, because the water-soluble product from phosphatidylcholine digested by the cell lysate of M. fermentans IID 812 was phosphorylcholine. M. fermentans IID 812 was examined for localization of these enzymes, and it was found that they were associated with the membrane.
Interleukin-8 production by human mesothelial cells after direct stimulation with staphylococci
Authors: Visser C E , Steenbergen J J , Betjes M G , Meijer S , Arisz L , Hoefsmit E C , Krediet R T , Beelen R H
Published: January 2020

Mesothelial cells (MC) are able to produce interleukin-8 (IL-8) after stimulation with IL-1 beta or tumor necrosis factor alpha. The aim of our study was to investigate whether MC are able to produce IL-8 after direct stimulation with clinically relevant bacteria. We observed a significant IL-8 response by the MC which were directly stimulated with viable staphylococci.
The V-region repertoire of Haemophilus influenzae type b polysaccharide antibodies induced by immunization of infants
Authors: Chung G H , Kim K H , Daum R S , Insel R A , Siber G R , Sood S , Gupta R K , Marchant C , Nahm M H
Published: January 2020

Haemophilus influenzae type b (Hib) is a significant pathogen for young children, and three Hib vaccines (named PRP-OMPC, HbOC, and PRP-T) are currently available for young children. Extensive studies of anti-Hib polysaccharide (PS) antibodies (Abs) have shown that the V regions of Abs against the Hib PS comprise a VH gene in the VH3 gene family and a VL gene from various K kappa and V lambda subgroups. To study immunogenic properties of the three vaccines in young children, we determined the VL subgroups and avidities of anti-Hib-PS Abs induced by the three clinically available conjugate vaccines. Ab avidity was measured by determining the concentration of a Hib-PS oligomer that abrogates half of the binding of immunoglobulin G anti-Hib-PS Abs to microwells. The PRP-OMPC vaccine induced lower-avidity Abs than the prelicensure HbOC vaccine (P = 0.05). When we compared anti-Hib-PS Abs expressing V kappa Ia, V kappa II, and V lambda subgroups, a greater Ab response was induced by the prelicensure HbOC vaccine than other vaccines (P < 0.05). When anti-Hib-PS Abs with the V kappa III subgroup were compared, however, both PRP-T and prelicensure HbOC vaccines induced a comparable response, which in turn was greater than those induced by the PRP-OMPC or the postlicensure HbOC vaccine (P < 0.001). The VL repertoire of Abs induced with the prelicensure HbOC or PRP-T vaccine in young children is dominated (about 80%) by anti-Hib-PS Abs using subgroup V kappa II. However, anti-Hib-PS using V kappa II VL accounts for only about 40% of the total anti-Hib-PS Abs induced with the PRP-OMPC vaccine or the postlicensure HbOC. Our data suggest that immunogenic properties of Hib vaccines in young children vary depending on the vaccine preparations as well as the vaccine types.