bluuebunny/crossref_metadata_2025 huggingface dataset:
The level of lipopolysaccharide-binding protein is significantly increased in plasma in patients with the systemic inflammatory response syndrome
Authors: Myc A , Buck J , Gonin J , Reynolds B , Hammerling U , Emanuel D
Published: January 2020

Currently, there is no way to predict with a high degree of sensitivity and specificity which patients are likely to develop systemic inflammatory response syndrome (SIRS) following systemic infection, trauma, organ rejection, or blood loss. The level of human lipopolysaccharide-binding protein (LBP) was determined in the plasma of 22 patients with a clinical diagnosis of early SIRS. Twenty-nine plasma samples from healthy volunteers were used as controls. The mean level of LBP in the plasma of healthy volunteers was 7.7 micrograms/ml (standard deviation, 6.2 micrograms/ml). Twenty-one of 22 patients (95%) with SIRS had an LBP level on admission at least 2 standard deviations above the mean LBP level for a healthy volunteer control group (range, 4.9 to 114.2 micrograms/ml; mean, 36.6 micrograms/ml; standard deviation, 22.2 micrograms/ml; P < 0.0001). The level of LBP in the plasma of the majority of patients with early SIRS is significantly increased compared to that in healthy controls. The sensitivity, specificity, and predictive value of elevated plasma LBP levels in patients with SIRS remain to be determined.
Immunoregulatory effects of morphine on human lymphocytes
Authors: Nair M P , Schwartz S A , Polasani R , Hou J , Sweet A , Chadha K C
Published: January 2020

It is now well established that parenteral drug abuse is a significant risk factor for contracting human immunodeficiency virus type 1 (HIV-1) infection and subsequently developing AIDS. Earlier studies have shown that morphine can modulate various immune responses and therefore support the premise that morphine is a cofactor in susceptibility to and progression of HIV infection. Dysregulation of interferon (IFN) production, nonspecific apoptosis of T cells, and the immune response to soluble HIV gene products have been associated with potential mechanisms of pathogenesis in HIV disease. The present study was undertaken to examine the immunomodulatory role of morphine on HIV protein-induced lymphocyte proliferative responses, Sendai and Newcastle disease virus-induced alpha IFN (IFN-alpha) and IFN-beta production by lymphocytes and fibroblast cells, respectively, and induction of apoptosis of normal lymphocytes in vitro. Our results demonstrate that HIV protein-induced human lymphocyte proliferative responses were significantly inhibited by morphine in a dose-dependent manner. Furthermore, morphine significantly inhibited both IFN-alpha and IFN-beta production by normal lymphocytes and fibroblasts but induced apoptosis of normal lymphocytes. Inhibition of IFN-alpha production by morphine could be reversed by the opiate receptor antagonist naloxone. This suggests that the immunomodulatory effects of morphine are mediated through the opioid receptor. These studies support a role of morphine as a cofactor in the pathogenesis of HIV infection and describe some of the possible pathologic mechanisms which underlie the immunoregulatory effects of morphine.
Serological cross-reactions between Coxiella burnetii and Legionella micdadei
Authors: Musso D , Raoult D
Published: January 2020

Coxiella burnetii and Legionella micdadei are both gram-negative bacteria potentially responsible for identical clinical syndromes resembling upper respiratory infections. These infections, quite common in immunocompromised patients, are usually diagnosed by serology with a microimmunofluorescence assay. We found that 34.5% of Q fever patients had a significant titer of antibodies against L. micdadei. Cross-reactions involved immunoglobulin G antibodies and were demonstrated by a cross-adsorption study and protein immunoblotting. Western blot analysis performed after treatment with proteinase K indicated that cross-reactions were probably due to both protein and lipopolysaccharide antigens. It is critical that the existence of this cross-reaction be recognized, as misdiagnosis of either condition may lead to incorrect and ineffective treatment.
Immune response to the mannose-sensitive hemagglutinin in patients with cholera due to Vibrio cholerae O1 and O0139
Authors: Qadri F , Jonson G , Begum Y A , Wennerås C , Albert M J , Salam M A , Svennerholm A M
Published: January 2020

The mannose-sensitive hemagglutinin (MSHA) is a type 4 pilus present in Vibrio cholerae O1 strains of the El Tor biotype, as well as in strains of serogroup O139. It has been shown to be a colonization antigen in animal models. The aim of this study was to investigate systemic and local antibody responses to MSHA in adult patients with cholera due to V. cholerae O1 and O139. Twenty-four of 28 (86%) patients with O1 cholera and 11 of 17 (65%) patients with O139 cholera showed significant increases in MSHA-specific immunoglobulin A (IgA) and IgM antibody-secreting cells (ASCs) 7 days after the onset of disease. However, the magnitude of the ASC response in O1 cholera patients was significantly higher than that in the O139 cholera patients in both IgA-producing (P = 0.015) and IgM-producing (P = 0.029) cells. Both groups of patients responded with antibody responses to MSHA in plasma, seroconverting with both IgA (63 to 70% of patients) and IgG (43 to 59% of patients) antibodies. Compared to the MSHA-specific antibody levels determined in healthy controls (n = 10), more than 90% of O1 and O139 cholera patients showed responses to MSHA of both the IgA and the IgG isotypes. About 70% of the patients in both groups also had antibody responses to MSHA in their feces. In summary, we demonstrated that MSHA is immunogenic, giving rise to both systemic and local antibodies in patients with cholera due to both O1 and O139 serogroups.
Anti-endothelial cell antibody, thrombomodulin, and von Willebrand factor in idiopathic inflammatory myopathies
Authors: Salojin K V , Bordron A , Nassonov E L , Shtutman V Z , Guseva N G , Baranov A A , Targoff I N , Youinou P
Published: January 2020

Sera from 19 patients with idiopathic inflammatory myopathy (IIM) were examined for the presence of anti-endothelial cell antibodies (AECA) by an immunoglobulin G-specific cellular enzyme-linked immunosorbent assay. The mean binding index of AECA was found to be 37.7% +/- 26.5% for the patients, compared with a mean of 7.2% +/- 2.7% for normal controls (P < 0.04). Levels of thrombomodulin, von Willebrand factor antigen, and serum creatine kinase were also shown to be augmented. Interestingly, positive correlations between AECA on the one hand and Raynaud's phenomenon and interstitial lung disease on the other were demonstrated. Given that the pathogenesis of IIM remains uncertain, these findings may be of importance.
Reactivities of Lewis antigen monoclonal antibodies with the lipopolysaccharides of Helicobacter pylori strains isolated from patients with gastroduodenal diseases in Japan
Authors: Amano K , Hayashi S , Kubota T , Fujii N , Yokota S
Published: January 2020

We have examined the reactivity of monoclonal antibodies (MAbs) specific for Lewis antigens (Le(x), Le(y), Le(a), and Le(b)) with Helicobacter pylori lipopolysaccharides (LPS) by immunoblot analysis and enzyme-linked immunosorbent assay (ELISA). Sixty-eight strains of H. pylori were isolated from patients with chronic gastritis, gastric and duodenal ulcers, and gastric cancer in Japan. The cells were treated with proteinase K, and the resulting fractions were used as a source of LPS for the immunoassays. In the immunoblot analysis, 28 isolates (41%) and 29 isolates (42%) reacted with anti-Le(x) and anti-Le(y) MAbs, respectively, while 4 isolates (6%) and 7 isolates (10%) reacted with anti-Le(a) and anti-Le(b) MAbs. On the other hand, in ELISA, the number of isolates that reacted with anti-Le(x) MAbs fell significantly to 21 isolates (30%) but the number of isolates that reacted with the other anti-Lewis antigen MAbs remained relatively unchanged. These data show that the immunoblotting technique is more sensitive than the ELISA technique for the detection of immunocomplexes of anti-Le(x) MAbs and components of H. pylori LPS. Furthermore, human serum was found to react with the synthetic Lewis antigens regardless of the status of the individual's H. pylori infection. This means that humans may naturally possess antibodies against Lewis antigens in the absence of H. pylori infection.
ICAM-1, soluble-CD23, and interleukin-10 concentrations in serum in renal-transplant recipients with Epstein-Barr virus reactivation
Authors: Hornef M W , Bein G , Wilhelm D , Fricke L , Kirchner H
Published: January 2020

Primary and reactivated Epstein-Barr virus (EBV) infections after organ transplantation are associated with the development of posttransplant lymphoproliferative malignancies. Since viral reactivation frequently stays asymptomatic, early diagnosis and treatment are challenges during posttransplant patient monitoring. Both soluble-CD23 (sCD23) and intercellular adhesion molecule 1 (ICAM-1) cell surface expression as well as interleukin-10 (IL-10) production are closely associated with viral gene expression. Therefore, immunoglobulin M (IgM), IgG, IgA, sCD23, ICAM-1, and IL-10 concentrations were measured in serum samples from patients during EBV reactivation (n = 14) and were compared with those in samples from patients without EBV reactivation (n = 10) following renal transplantation. In addition, serum sCD23, ICAM-1, and IL-10 concentrations were measured longitudinally in weekly to biweekly samples from 10 patients with EBV reactivation for at least 20 weeks following transplantation. A significant elevation of sCD23 was found during viral reactivation (P < 0.05), whereas ICAM-1 levels showed a nonsignificant increase. The finding of a highly significant elevation of the serum IL-10 concentration during EBV reactivation (P < 0.001) may support speculations about its role in EBV-induced lymphoproliferation and in the development of opportunistic infections and secondary malignancies. Maximum serum IL-10 levels at the time of EBV reactivation were found in 7 of 10 patients. Well-defined ICAM-1 and sCD23 concentration peaks were found in 9 of 10 and 8 of 10 patients, respectively. Although both markers are not specific for EBV reactivation and therefore may not be useful for primary diagnosis, sCD23 and ICAM-1 might be potent tools for the clinical monitoring of EBV activity and virus-induced lymphoproliferation.
Humoral immune responses of Brucella-infected cattle, sheep, and goats to eight purified recombinant Brucella proteins in an indirect enzyme-linked immunosorbent assay
Authors: Letesson J J , Tibor A , van Eynde G , Wansard V , Weynants V , Denoel P , Saman E
Published: January 2020

Brucellosis research is currently focused on the identification of nonlipopolysaccharide (LPS) antigens which could potentially be useful for the specific serologic diagnosis of brucellosis as well as for vaccinal prophylaxis. On the basis of previous reports, we selected eight Brucella proteins (OMP36, OMP25, OMP19, OMP16, OMP10, p17, p15, and p39) as candidate antigens to be further evaluated. The genes encoding these proteins were cloned, sequenced, and overexpressed in Escherichia coli. The recombinant proteins were purified with a polyhistidine tag and metal chelate affinity chromatography and evaluated in an indirect enzyme-linked immunosorbent assay (iELISA). The specificity of the iELISA was determined with sera from healthy cattle, sheep, and goats and ranged from 95 to 99%, depending on the recombinant antigen and the species tested. Sera from experimentally infected, and from naturally infected, animals were used to evaluate the sensitivity of the iELISA. The antiprotein antibody response was often delayed when compared to the anti-smooth LPS (S-LPS) response and was limited to animals which developed an active brucellosis infection (experimentally infected pregnant animals and sheep and goats from areas where brucellosis is still endemic). Among the recombinant antigens, the three cytoplasmic proteins (p17, p15, and p39) gave the most useful results. More than 80% of the animals positive in S-LPS serology were also positive with one of these cytoplasmic proteins alone or a combination of two of them. None of the recombinant antigens detected experimentally infected nonpregnant cows and sheep or naturally infected cattle. This study is a first step towards the development of a multiprotein diagnostic reagent for brucellosis.
Rapid identification of rough Brucella isolates by a latex coagglutination assay with the 25-kilodalton outer membrane protein and rough-lipopolysaccharide-specific monoclonal antibodies
Authors: Bowden R A , Verger J M , Grayon M , Cloeckaert A
Published: January 2020

A latex coagglutination assay was developed to identify rough (R) isolates of Brucella. Latex beads were coated, via protein A, with either an anti-Brucella rough-lipopolysaccharide (R-LPS) monoclonal antibody (MAb) or an anti-Brucella 25-kDa outer membrane protein (Omp25) MAb. Slide agglutination tests were done for 68 strains of Brucella spp., including type strains of all biovars as well as field isolates. Latex beads coated with MAb to R-LPS coagglutinated only R strains, whereas latex beads coated with MAb to Omp25 coagglutinated all the R Brucella isolates except Brucella ovis. Coagglutination was easier to read than agglutination with rabbit R-Brucella-specific antiserum. Thus, this assay accurately differentiates B. ovis from other R Brucella isolates. The latex coagglutination assay can substitute, to advantage, for the current anti-Brucella (R) rabbit monospecific serum.
Antibody responses of splenectomized patients with non-Hodgkin's lymphoma to immunization with polyvalent pneumococcal vaccines
Authors: Petrasch S , Kühnemund O , Reinacher A , Uppenkamp M , Reinert R , Schmiegel W , Lütticken R , Brittinger G
Published: January 2020

The serum antibody responses of splenectomized patients with non-Hodgkin's lymphoma (NHL) who had been immunized with a polyvalent pneumococcal vaccine (Pneumovax 23) were evaluated by an enzyme-linked immunosorbent assay with the 23-valent pneumococcal vaccine as the antigen. A response to immunization, defined as a twofold-or-higher rise of the prevaccination titer of antibodies against Streptococcus pneumoniae polysaccharide, was elicited in 5 of 11 patients with NHL. No significant difference in the level of antibodies against S. pneumoniae polysaccharide between lymphoma patients and patients who had undergone splenectomy for other reasons was detected (P = 0.83 and 0.87 before and after vaccination, respectively). NHL patients who did not respond to the first immunization received a booster dose of the polysaccharide vaccine. This injection did not increase the pneumococcal-antibody titer significantly (P = 0.7). We conclude that vaccination with pneumococcal polysaccharides in splenectomized patients with NHL elicits an adequate antibody response in 45.4% of the cases and should therefore be administered. Revaccination of the nonresponders does not further increase the pneumococcal-antibody levels.
Evaluation of the high-density agglutination test for Coxiella burnetii antibodies in animals
Authors: Nguyen S V , To H , Minamoto N , Ogawa M , Yamaguchi T , Fukushi H , Hirai K
Published: January 2020

The usefulness of the high-density particle agglutination (HDPA) test as a potential tool for the detection of anti-Coxiella burnetii antibodies in animal sera was studied by using 619 cow, 589 dog, and 150 cat serum samples and antisera from rabbits, guinea pigs, and mice. The sensitivity and specificity of the test versus those of the reference microimmunofluorescence test were determined at two different threshold titer values. At the cutoff value of 1:16, the sensitivities of the HDPA test for cow, dog, and cat sera were 94.3, 95, and 91.3%, respectively, and the specificities were 95.5, 95.3, and 91.3%, respectively. At the cutoff value of 1:32, the sensitivities were 86.7, 88.3, and 82.6%, respectively, and the specificities were 99, 99.2, and 98.4%, respectively. For the group of immune laboratory animals all samples from rabbits, guinea pigs, and mice were positive by the HDPA test. The erythrocyte-sensitizing substance from phase II C. burnetii was found to contain protein and carbohydrate, and both fractions are immunoreactive. The study results show that the HDPA test is a useful tool in the epizootiological survey of Coxiella infection in animals.
Phagocytosis and oxidative-burst response of planktonic Staphylococcus epidermidis RP62A and its non-slime-producing variant in human neutrophils
Authors: Heinzelmann M , Herzig D O , Swain B , Mercer-Jones M A , Bergamini T M , Polk H C
Published: January 2020

The ability of bacterial organisms to produce an extracellular polysaccharide matrix known as slime has been associated with increased virulence and delayed infections in various prosthetic implants. Within a biofilm, this slime may protect the embedded bacteria from host defense mechanisms, especially phagocytosis by polymorphonuclear leukocytes. To determine whether planktonic Staphylococcus epidermidis is protected in a similar way, a novel flow cytometric assay was performed, measuring ingestion and adherence during phagocytosis and the production of superoxide during oxidative burst. Hydrophobicity was determined by hydrophobic interaction chromatography. Slime-producing S. epidermidis RP62A and its phenotypic variant, non-slime-producing RP62A-NA, were compared. The results showed increased phagocytosis of RP62A at 2, 5, 10, and 30 min; increased adherence of RP62A at 30 s and 30 min; and increased superoxide production of RP62A after 2 min. Decreased hydrophobicity of RP62A over RP62A-NA was correlated with a hydrophilic slime coat. The data argue that the host aggressively combats slime-producing S. epidermidis. This biological phenomenon is potentially important during bacteremia to prevent further adhesion, accumulation, and the genesis of a bacterial biofilm on implants or tissue surfaces.
Monophosphoryl lipid A behaves as a T-cell-independent type 1 carrier for hapten-specific antibody responses in mice
Authors: Myers K R , Beining P , Betts M , Snippe H , Inman J , Golding B
Published: January 2020

It is known that the lipopolysaccharide (LPS) of gram-negative bacteria, in addition to being a potent adjuvant, is an effective carrier for covalently associated haptens. However, the toxic nature of most forms of LPS precludes their use as adjuvants or carriers for human vaccines. 4'-Monophosphoryl lipid A (MLA), a derivative of LPS with attenuated toxicity, is currently being tested in humans as an immunological adjuvant. In this study, MLA was tested for its ability to function as a carrier for a small hapten, the trinitrophenyl group (TNP). MLA was first modified by addition of 6-aminocaproic acid to the 6' position of the disaccharide backbone (Cap-MLA). TNP was then attached to Cap-MLA via the free amino group, yielding TNP-Cap-MLA. Immunization of normal mice with TNP-Cap-MLA resulted in high-titer anti-TNP responses of immunoglobulin M and all immunoglobulin G subclasses. Furthermore MLA, like other T-cell-independent type 1 (TI-1) carriers, induced responses in athymic and X-linked immunodeficient mice. In all cases, immunization with either MLA alone or TNP-Cap plus MLA failed to induce measurable anti-TNP antibodies of any isotype, indicating that covalent association of MLA and hapten was necessary for MLA's carrier activity to be manifested. These properties of MLA make it a potential candidate as a carrier for vaccine subunit components, such as small peptides, especially for situations in which T-cell help is impaired, as occurs following human immunodeficiency virus type 1 infection.
A 135-kilodalton surface antigen of Mycoplasma hominis PG21 contains multiple directly repeated sequences
Authors: Ladefoged S A , Birkelund S , Hauge S , Brock B , Jensen L T , Christiansen G
Published: January 2020

A monoclonal antibody was used to characterize a 135-kDa surface-located membrane protein (Lmp1) generally present in Mycoplasma hominis strains. The monoclonal antibody, 552, was applied to identify the corresponding gene in an expression library of M. hominis PG21 DNA. The M. hominis PG21 lmp1 gene was sequenced, and its gene product was characterized with the goal of elucidating the structure and function of Lmp1. A total of 7,196 bp in the lmp1 region was sequenced. An open reading frame of 4,032 bp, encoding a protein of 1,344 amino acids with a calculated molecular weight of 147,000, was identified. Analysis of the deduced amino acid sequence predicted a hydrophilic protein with a basic pI (10.0). The N-terminal 24 amino acids were a typical leader sequence. Downstream from the first 726 nucleotides, six similar direct repeats of 471 nucleotides were found. In repeat 7, a single-base substitution, C-->A, gave rise to the stop codon of lmp1. Thus, the C-terminal 945 amino acids were encoded by the 471-bp direct repeats. As evidenced by Southern blot analysis, the gene encoding the 135-kDa antigen is part of a multigene family. One of the genes, lmp2, was situated directly downstream from lmp1 where the direct repeats continued.
Induction of transforming growth factor beta 1 by purified protein derivative of Mycobacterium tuberculosis
Authors: Toossi Z , Young T G , Averill L E , Hamilton B D , Shiratsuchi H , Ellner J J
Published: January 2020

We examined the ability of purified protein derivative (PPD) of Mycobacterium tuberculosis to induce transforming growth factor beta 1 (TGF-beta 1), a potent immunosuppressive and macrophage-deactivating molecule, in blood monocytes from healthy individuals. TBF-beta 1 activity in PPD-induced monocyte supernatants was identified by Western immunoblot analysis and was not inhibited by polymyxin B, an inhibitor of bacterial lipopolysaccharide (LPS). Furthermore, PPD at equivalent amounts in weight to LPS was as potent in stimulation of monocyte production of TGF-beta 1 at 24 h of culture, as quantified by enzyme-linked immunosorbent assay. The inducing effect of PPD, in contrast to that of LPS, was sustained at later time points of culture (72 h). PPD enhanced the constitutive expression of TGF-beta 1 steady-state mRNA in monocytes at 24 and 48 h of culture. In contrast, neither mycobacterial heat shock protein (64-kDa protein of M.bovis) nor LPS induced TGF-beta 1 mRNA. Decay studies suggested a transcriptional rather than a posttranscriptional effect of PPD on TGF-beta 1 gene expression.
Tumor necrosis factor (TNF)-dependent shedding of the p55 TNF receptor in a baboon model of bacteremia
Authors: Redl H , Schlag G , Adolf G R , Natmessnig B , Davies J
Published: January 2020

We have tested the hypothesis that the tumor necrosis factor (TNF) plays a significant role in vivo in TNF receptor shedding and studied the release of TNF-binding protein 1 (TNF-BP1), the soluble fragment of the 55- to 60-kDa TNF alpha (TNF) receptor, in a baboon model of Escherichia coli bacteremia, using three different doses of bacteria in acute infection (8 h) experiments (n [animals] = 11) and a single dose in a subchronic infection (72 h) experiment. In the subchronic infection study, one group of animals (n = 6) was pretreated with a neutralizing murine monoclonal antibody to TNF (CB0006). Concentrations of TNF and TNF-BP1 in plasma were determined in specific, monoclonal antibody-based immunoassays. Untreated animals (n = 6) showed undetectable TNF concentrations (< 10 pg/ml at baseline), whereas TNF-BP1 levels in plasma were in the range of 2 ng/ml, similar to concentrations observed in humans. Infusion of bacteria resulted in a rapid, dose-dependent increase in plasma TNF concentrations that reached a maximal level after 2 h and returned to baseline within 6 h. TNF-BP1 concentrations also showed a dose-dependent increase to peak concentrations three- to fivefold above baseline within 2 h but, in contrast to TNF levels, remained significantly elevated for up to 48 h. In animals pretreated with antibody CB0006, circulating TNF was completely neutralized, and TNF-BP1 was significantly reduced. We conclude that TNF-BP1 is released in bacteremia and that release in vivo is partially dependent on the presence of TNF.
Role of endotoxin in acute inflammation induced by gram-negative bacteria: specific inhibition of lipopolysaccharide-mediated responses with an amino-terminal fragment of bactericidal/permeability-increasing protein
Authors: Kohn F R , Kung A H
Published: January 2020

A recombinant 23-kDa amino-terminal fragment of human bactericidal/permeability-increasing protein (rBPI23), a potent lipopolysaccharide (LPS)-binding/neutralizing protein, was used as a probe to assess the role of endotoxin in the acute inflammatory responses elicited by gram-negative bacteria in rat subcutaneous air pouches. In initial experiments, rBPI23 prevented the Escherichia coli O111:B4 LPS-induced accumulation of polymorphonuclear leukocytes (PMN), tumor necrosis factor alpha (TNF-alpha), and nitrite (a stable end product of nitric oxide formation) in exudate fluids. Significant inhibition of TNF-alpha production was still evident when rBPI23 treatment was delayed for 30 min after LPS instillation. In subsequent experiments, rBPI23 also prevented the nitrite and early (2-h) TNF-alpha accumulation induced by three different strains of formaldehyde-killed gram-negative bacteria (E. coli O7:K1, E. coli O111:B4, and Pseudomonas aeruginosa 12.4.4) but did not inhibit the PMN or late (6-h) TNF-alpha accumulation induced by these bacteria. As with LPS challenge, a significant inhibition of early TNF-alpha production was still evident when rBPI23 treatment was delayed for 30 to 60 min after instillation of killed bacteria. The results indicate that in this experimental model the NO and early TNF-alpha responses to gram-negative bacterial challenge are mediated predominantly by endotoxin, whereas the PMN and late TNF-alpha responses may be mediated by other bacterial components. Moreover, the results indicate that rBPI23 can inhibit the bacterially induced production of certain potentially harmful mediators (TNF-alpha and NO) without entirely blocking the host defense, i.e., PMN response, against the bacteria.
A Cryptosporidium parvum genomic region encoding hemolytic activity
Authors: Steele M I , Kuhls T L , Nida K , Meka C S , Halabi I M , Mosier D A , Elliott W , Crawford D L , Greenfield R A
Published: January 2020

Successful parasitization by Cryptosporidium parvum requires multiple disruptions in both host and protozoan cell membranes as cryptosporidial sporozoites invade intestinal epithelial cells and subsequently develop into asexual and sexual life stages. To identify cryptosporidial proteins which may play a role in these membrane alterations, hemolytic activity was used as a marker to screen a C. parvum genomic expression library. A stable hemolytic clone (H4) containing a 5.5-kb cryptosporidial genomic fragment was identified. The hemolytic activity encoded on H4 was mapped to a 1-kb region that contained a complete 690-bp open reading frame (hemA) ending in a common stop codon. A 21-kDa plasmid-encoded recombinant protein was expressed in maxicells containing H4. Subclones of H4 which contained only a portion of hemA did not induce hemolysis on blood agar or promote expression of the recombinant protein in maxicells. Reverse transcriptase-mediated PCR analysis of total RNA isolated from excysted sporozoites and the intestines of infected adult mice with severe combined immunodeficiency demonstrated that hemA is actively transcribed during the cryptosporidial life cycle.
Pertussis toxin export genes are regulated by the ptx promoter and may be required for efficient translation of ptx mRNA in Bordetella pertussis
Authors: Baker S M , Masi A , Liu D F , Novitsky B K , Deich R A
Published: January 2020

The gene products from an 8-kb region adjacent to the 3' end of the ptx operon are required by Bordetella pertussis for the export of pertussis holotoxin. At least one of these gene products (PtlC) is specifically required for the export of assembled holotoxin from the periplasmic space. ptlC mutants exhibit a 20-fold reduction in the amount of holotoxin present in the culture supernatant but have no effect upon the assembly or steady-state level of holotoxin present in the periplasmic space. Impaired export of holotoxin from the ptlC strain blocks expression of toxin at a posttranscriptional level, and wild-type levels of ptx mRNA are detected in the mutant strain. The transcription of ptl is subject to modulation by MgSO4 in the same manner as ptx is; however, in B. pertussis strains containing an E. coli tac promoter in place of the native ptx promoter, wild-type levels of ptx mRNA are present and holotoxin is synthesized and exported even in the presence of MgSO4. Promoter mapping of the region extending from the ptxS3 coding region to the ptlC coding region failed to detect the ptl transcription initiation site. Additional RNase protection experiments with ptx promoter deletion and substitution strains indicate that the ptl operon is transcribed from the ptx promoter as part of a > 11-kb mRNA.
Surface exposure of outer membrane protein and lipopolysaccharide epitopes in Brucella species studied by enzyme-linked immunosorbent assay and flow cytometry
Authors: Bowden R A , Cloeckaert A , Zygmunt M S , Bernard S , Dubray G
Published: January 2020

Seven surface-exposed outer membrane proteins (OMPs) in Brucella supp. have been previously described (A. Cloeckaert, P. de Wergifosse, G. Dubray, and J. N. Limet, Infect. Immun. 58:3980-3987, 1990). OMPs were shown to be more accessible to monoclonal antibodies (MAbs) on rough (R) Brucella melitensis and B. abortus strains than to MAbs on their smooth (S) counterparts. In this work, we have extended this study to representatives of the main Brucella species, using MAbs specific for OMPs and S and R lipopolysaccharides (S-LPS and R-LPS). Enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunoelectron microscopy showed important differences between strains in the binding of OMP- and R-LPS-specific MAbs which were in part related to the particular expression of S-LPS, irrespective of the species. Results indicated that both the amount and the length of O polysaccharide on S-LPS greatly influenced the accessibility of OMP and R-LPS epitopes to MAbs. S-R B. melitensis EP and S B. suis 40, for instance, which express O-polysaccharide chains in small amounts and with short mean length, respectively, bound a greater number of OMP- and R-LPS-specific MAbs than the other S Brucella strains. The major 31- to 34-kDa OMP was the most exposed OMP on S strains of B. melitensis and B. suis. In most cases, flow cytometry results agreed with those of ELISA and supplied additional data, such as the homogeneity or heterogeneity of OMP expression at the strain level. However, there were some discordances between flow cytometry and ELISA results concerning the surface exposure of the 25- to 27-kDa and 31- to 34-kDa OMPs on S strains and that of minor OMPs in vaccine strain B. melitensis Rev.1. Immunoelectron microscopy confirmed the poor accessibility of OMPs to MAbs on the surface of S Brucella strains. The naturally R pathogenic species B. ovis and B. canis bound the majority of OMP-specific MAbs as well as the R-LPS-specific MAbs. Therefore, the conserved OMP and R-LPS epitopes could play a role as targets of protective antibody-mediated immunity in infections caused by naturally R B. ovis and B. canis.
T-cell suppression and selective in vivo activation of TH2 subpopulation by the Entamoeba histolytica 220-kilodalton lectin
Authors: Talamás-Rohana P , Schlie-Guzmán M A , Hernández-Ramírez V I , Rosales-Encina J L
Published: January 2020

A 220-kDa surface protein (L220) with lectin activity from Entamoeba histolytica trophozoites has been characterized previously (J. L. Rosales-Encina, I. Meza, A. López-de-León, P. Talamás-Rohana, and M. Rojkind, J. Infect. Dis. 156:790-797, 1987). This molecule is involved in the adhesion process (I. Meza, F. Cázares, J. L. Rosales-Encina, P. Talamás-Rohana, and M. Rojkind, J. Infect. Dis. 156:798-805, 1987) and is very immunogenic. In this work, we studied both the humoral and the cellular immune responses to L220. We compared L220 with L220-derived components, such as a fusion peptide (M-11) and chemically obtained peptides (by treating the 220-kDa molecule with N-chlorosuccinimide-urea). Spleen cells from L220-immunized mice were unable to proliferate in vitro when stimulated with the protein. However, a proliferative response was obtained when mice were immunized with the L220-derived fusion peptide or the cleaved lectin. To find out if there was a correlation between the observed responses and TH1 or TH2 activation, we analyzed patterns of cytokine secretion (interleukin-2 [IL-2], IL-4, IL-10, and gamma interferon). Cells from mice immunized with peptides that induced cell proliferation (100, 80, and 47 kDa) with the peptides (P < 0.01) and with the intact molecule secreted IL-2 and gamma interferon, showing a TH1-subset pattern. Conversely, cells from mice immunized with the intact 220-kDa molecule secreted IL-4 and IL-10, typical of a TH2 subpopulation; however, antibodies from each group recognized the 220-kDa molecule as determined by Western blotting (immunoblotting). These results suggest that various epitopes in the 220-kDa molecule generate different response patterns, suppressing or activating T-cell responses.
Pseudomonas aeruginosa invasion of and multiplication within corneal epithelial cells in vitro
Authors: Fleiszig S M , Zaidi T S , Pier G B
Published: January 2020

Pseudomonas aeruginosa is usually considered an extracellular pathogen. Using assays to determine intracellular survival in the presence of gentamicin, we have previously demonstrated that P. aeruginosa is able to invade corneal cells during infectious keratitis in mice. In vitro, P. aeruginosa was found to enter the following cells: human corneal cells removed by irrigation; epithelial cells in the cornea of rats, mice, and rabbits; and primary corneal epithelial cells cultured from rat and rabbit eyes. The level of invasion was related to the level of adherent or associated bacteria. In general, invasion was more efficient with cultured epithelial cells than with cells tested in situ. Invasion did not occur when assays were performed at 4 degrees C. Cytochalasin D but not colchicine inhibited bacterial invasion, suggesting that bacterial entry was an endocytic process dependent on actin microfilaments but not microtubules. Bacteria that invaded cultured corneal epithelial cells were found to multiply within cells. The ability of P. aeruginosa to invade and multiply within corneal epithelial cells may contribute to the virulence of this organism during infectious keratitis, since intracellular bacteria can evade host immune effectors and antibiotics commonly used to treat infection.
Candida albicans cell walls contain the fluorescent cross-linking amino acid dityrosine
Authors: Smail E H , Briza P , Panagos A , Berenfeld L
Published: January 2020

Several clinical and laboratory isolates of Candida albicans have a natural blue surface fluorescence when cultured and observed with sensitive optics. The localization and color of the fluorescence are similar to those of the natural fluorescence of sporulated Saccharomyces cerevisiae which is caused by the generation and surface deposition of the cross-linking amino acid dityrosine. In S. cerevisiae, dityrosine production results from the direct action of at least two genes and is responsible for resistance of the ascospores to lytic enzymes and physicochemical trauma. Among the criteria for the identification of dityrosine is pH sensitivity of the fluorescence intensity and a highly characteristic shift of the fluorescence excitation maximum with a change in pH. Video microscopy of whole Candida organisms revealed the characteristic dityrosine intensity maximum at pH approximately 10 and the intensity minimum at pH approximately 2. Separation of an acid hydrolysate of Candida cell walls by reverse-phase high-performance liquid chromatography revealed a fluorescence peak that coelutes with the reagent dityrosine. At pH approximately 10, this peak has a fluorescence excitation maximum of 320 to 325 nm, while at pH approximately 2, the excitation maximum is 285 to 290 nm. This excitation maximum shift and the observed emission maximum of approximately 410 nm are characteristic of dityrosine. Two separate strains of C. albicans were injected intraperitoneally into mice and harvested at 24 h. Blue surface fluorescence was observed, suggesting that dityrosine generation occurs in vivo as well as in vitro. This is the first report of the presence of dityrosine in a human fungal pathogen.
Experimental immunization with Borrelia burgdorferi induces development of antibodies to gangliosides
Authors: Garcia-Monco J C , Seidman R J , Benach J L
Published: January 2020

Patients with neuroborreliosis produce antibodies, mostly of the immunoglobulin M (IgM) class, to gangliosides, particularly to those with Gal(beta 1-3)GalNac terminal sequences. Lewis rats were immunized with a nonpathogenic strain of Borrelia burgdorferi and with a chloroform-methanol extract (nonprotein) of this organism (CM) to determine whether antibodies to B. burgdorferi also recognized gangliosides. Rats were also immunized with asialo-GM1 to determine whether the elicited antibodies recognized antigens in B. burgdorferi. Rats immunized with B. burgdorferi produced low levels of IgM antibodies that cross-reacted with asialo-GM1 and GM1. Rats immunized with CM had marked IgM reactivity to asialo-GM1 and GM1. Immunization with asialo-GM1 resulted in antibodies that cross-reacted with B. burgdorferi antigens. Although antibodies to B. burgdorferi were of both the IgM and IgG classes, those to CM and to asialo-GM1 and GM1 were predominantly in the IgM fraction. Reactivity of the IgM antibodies decreased after adsorption with the heterologous and the homologous antigens, indicating bidirectional cross-reactivity between CM, asialo-GM1, and GM1 and that immunization with one produces antibodies to the other. There was no in vivo deposition of Ig in peripheral nerves, nor was there nerve pathology as a result of immunizations, but IgM antibodies to asialo-GM1 and CM recognized homologous antigens in the nodes of Ranvier of peripheral nerves from nonimmunized rats. This immunization model suggests that antibodies to gangliosides in Lyme disease have a microbial origin and are potentially relevant in pathogenesis.
Contact-dependent transfer of the galactose-specific lectin of Entamoeba histolytica to the lateral surface of enterocytes in culture
Authors: Leroy A , De Bruyne G , Mareel M , Nokkaew C , Bailey G , Nelis H
Published: January 2020

In a study to investigate early interactions of Entamoeba histolytica with epithelial cell monolayers, we found that a monoclonal antibody (MAb), CD6, against an ameba surface antigen recognized the lateral surface of epithelial cells after coculture with trophozoites. Display of the CD6 antigen on the epithelial cells necessitated contact with active trophozoites. It was found neither at 4 degrees C, nor with prefixed trophozoites, nor with trophozoite-conditioned media, nor when a filter prevented direct contact. Monolayers exposed to amebic sonicates or detergent lysates showed random immunostaining. Access to the antigenic site was limited, as immunostaining occurred predominantly with subconfluent monolayers. CD6 epithelial cell binding was first observed after 5 min of coculture; trophozoite-mediated target cell lysis was not detected until 15 min following coculture. Epithelial cell immunostaining occurred with some other ameba-specific antibodies but not with MAbs raised against the 170-kDa subunit of the galactose-N-acetylgalactosamine (Gal/GalNAc)-specific lectin. The CD6 MAb as well as some other ameba-specific antibodies immunoprecipitated from trophozoite lysates the same bands as the MAbs against the cysteine-rich domain of the 170-kDa subunit of the Gal/GalNAc-specific lectin. Why the latter MAbs failed to stain epithelial cells in the vicinity of attached trophozoites is presently unknown. We concluded that E. histolytica trophozoites transferred the intact amebic Gal/GalNAc-specific lectin or a portion of it to the lateral surface of epithelial cells. This juxtacrine transfer preceded killing of target cells.
In vitro influence of Mycoplasma penetrans on activation of peripheral T lymphocytes from healthy donors or human immunodeficiency virus-infected individuals
Authors: Sasaki Y , Blanchard A , Watson H L , Garcia S , Dulioust A , Montagnier L , Gougeon M L
Published: January 2020

Mycoplasma penetrans is a mycoplasma species newly isolated from the urine of human immunodeficiency virus (HIV)-infected individuals and presents the only case in which an association has been found between antibodies against a mycoplasma and HIV infection. To further explore the effects of M. penetrans on the immune system, we studied the influence of this mycoplasma on peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-infected individuals. M. penetrans induced, in addition to blastogenesis of PBMCs, a significant proliferative response associated with the expression of some activation markers such as CD69, HLA-DR, and CD25. This M. penetrans-dependent lymphocyte activation was observed not only in healthy donors but also in HIV-infected persons at different stages of the disease. In addition, our study revealed that both CD4+ and CD8+ T lymphocytes were responsive to M. penetrans. Interestingly, the mitogenic activity of M. penetrans was associated with mycoplasma cells but not with the supernatants of mycoplasma culture. The potent stimulating activity of M. penetrans on T lymphocytes from HIV-infected individuals is of particular interest in view of the supposed contribution of immune activation to HIV replication and disease progression.
Campylobacter jejuni motility and invasion of Caco-2 cells
Authors: Szymanski C M , King M , Haardt M , Armstrong G D
Published: January 2020

We investigated the influence of motility on Campylobacter jejuni binding and invasion of Caco-2 cells. C. jejuni was motile in soft agar at basic (pH 8.5) and neutral pH values representative of the intestinal environment. However, C. jejuni was immobilized at pH 5.0. The inability of C. jejuni to swarm on soft agar at pH 5.0 was not related to flagellar depolymerization or loss of viability. In tissue culture medium, C. jejuni displayed typical periods of straight swimming punctuated by tumbling behavior. This behavior was altered when the viscosity of the medium was adjusted to mimic the viscosity of intestinal mucus. C. jejuni showed longer periods of straight swimming with significantly increased velocity followed by pauses instead of tumbles. The binding and invasion of C. jejuni in Caco-2 cells also increased significantly in high-viscosity growth medium. We speculate that the swimming behavior of C. jejuni in a viscous environment may be an important factor in the interaction of these organisms with host epithelial cells. The pH, which affects C. jejuni motility, may also influence the tropism of these organisms.
Immune response to Schistosoma mansoni phosphoglycerate kinase during natural and experimental infection: identification of a schistosome-specific B-cell epitope
Authors: Lee K W , Thakur A , Karim A M , LoVerde P T
Published: January 2020

A cDNA clone encoding Schistosoma mansoni phosphoglycerate kinase (SmPGK) was previously identified by affinity-purified antibodies which are specific for 3-h-old schistosomula tegumental antigens. Antibodies to the recombinant SmPGK which has enzymatic activity were localized to various tissues including the tegument of the 3-h-old schistosomula and 42-day-old adult worms. In this study, we show that SmPGK is an immunogenic molecule in both natural infection in humans and experimental vaccination in animals. To understand the role that a highly conserved molecule like SmPGK played during schistosome infection, we affinity purified antibodies to SmPGK from patients with chronic schistosomiasis and demonstrated that they did not cross-react with human PGK. However, affinity-purified rabbit anti-SmPGK antibodies did show immunoreactivity to both human PGK and rabbit PGK. Thus, during natural infection antibodies that cross-react with human PGK are not produced; however, as a result of active immunization with an intact conserved molecule, such cross-reacting antibodies are produced. Immunological analysis of cyanogen bromide digests of SmPGK with monoclonal antibodies that recognize SmPGK but not human PGK identifies a B-cell epitope on a 12.2-kDa fragment represented by amino acids 61 to 174.
Helicobacter pylori cytotoxin: importance of native conformation for induction of neutralizing antibodies
Authors: Manetti R , Massari P , Burroni D , de Bernard M , Marchini A , Olivieri R , Papini E , Montecucco C , Rappuoli R , Telford J L
Published: January 2020

We have attempted to express the Helicobacter pylori vacuolating cytotoxin in Escherichia coli. Although the 95-kDa VacA polypeptide was expressed abundantly, it completely lacked any biological activity. In addition, this material failed to induce neutralizing antibodies after immunization of rabbits. In contrast, highly purified high-molecular-mass cytotoxin from the supernatant of H. pylori cultures was active in a HeLa cell assay and effectively induced a neutralizing response in rabbits. Neutralizing sera were shown to contain a high proportion of antibodies which recognized conformational epitopes found only on the native toxin. The data indicate that toxin-neutralizing epitopes are conformational and that potential vaccines based on the cytotoxin may benefit from the use of the intact molecule.
The broad-range phospholipase C and a metalloprotease mediate listeriolysin O-independent escape of Listeria monocytogenes from a primary vacuole in human epithelial cells
Authors: Marquis H , Doshi V , Portnoy D A
Published: January 2020

Intracellular growth of Listeria monocytogenes begins after lysis of the primary vacuole formed upon bacterial entry into a host cell. Listeriolysin O (LLO), a pore-forming hemolysin encoded by hly, is essential for vacuolar lysis in most cell types. However, in human epithelial cells, LLO- mutants are capable of growth, suggesting that gene products other than LLO are capable of mediating escape from a vacuole. In this study, we investigated the role of other bacterial gene products in lysis of the primary vacuole in the human epithelial cell line Henle 407. Double internal in-frame deletion mutants were constructed by introducing a mutated hly allele into strains harboring deletions in either of the phospholipase C (PLC)-encoding genes or a metalloprotease-encoding gene. Bacterial escape from the primary vacuole, intracellular growth, and cell-to-cell spread were evaluated in Henle 407 cells. The results indicated that, in the absence of LLO, the broad-range PLC and the metalloprotease were both required for lysis of the primary vacuole in Henle 407 cells. Although phosphatidylinositol-specific PLC was not required, the efficiency of escape was reduced in an LLO phosphatidylinositol-specific PLC double mutant. These observations suggest that the relative importance of LLO, the phospholipases, and the metalloprotease may vary in different cell types or in cells from different species. In addition, these studies provide insight into the mechanisms of action of virulence determinants involved in the lysis of vacuolar membranes.