bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipopolysaccharide-specific antibodies in plasma and stools of children with Shigella-associated leukemoid reaction and hemolytic-uremic syndrome
Authors: Azim T , Qadri F , Ahmed S , Sarker M S , Halder R C , Hamadani J , Chowdhury A , Wahed M A , Salam M A , Albert M J
Published: January 2020

Antibody responses to the lipopolysaccharide (LPS) of shigellae were compared between children with uncomplicated and complicated Shigella dysenteriae 1 infection. One hundred fifteen children between 12 and 60 months of age with S. dysenteriae 1 infection were studied. Of these children, 42 had complications (leukemoid reaction and/or hemolytic-uremic syndrome [complicated shigellosis] and 73 had no complications (uncomplicated shigellosis). Antibodies to the LPS of S. dysenteriae 1 and Shigella flexneri Y were measured in plasma and stools, as were total immunoglobulin A (IgA) and IgG concentrations in plasma and the total IgA concentration in stool, on enrollment and 3 to 5 days later. In the plasma, the concentrations of homologous (IgG) and heterologous (IgA) LPS antibodies on enrollment were higher in children with complicated shigellosis than in those with uncomplicated shigellosis. In stool, the concentrations on enrollment were similar between the two groups of children. There was a rise in antibody concentrations in the plasma (homologous and heterologous) and stool (homologous) between the day of enrollment and 3 to 5 days later in children with uncomplicated shigellosis but not in those with complicated shigellosis. These findings suggest that systemic stimulation is more marked in children with complications, so that a subsequent rise in plasma antibody concentrations does not occur in these children. In contrast, the lack of a rise in stool antibody concentrations in children with complicated shigellosis is suggestive of a lower-level mucosal response. Because the duration of diarrhea before enrollment influenced the homologous antibody concentrations, children were further divided into three subgroups (short [3 to 5 days], medium [6 to 9 days], and long [> 9 days] diarrhea durations before enrollment). Comparisons of homologous antibody concentrations between the two groups of children following such subdivisions showed that in children with complicated shigellosis, antibody concentrations were higher in the plasma of children in the short diarrhea duration subgroup but lower in the stool children in the medium diarrhea duration subgroup. No differences in antibody concentrations were observed in children in the other diarrhea duration subgroups. Thus, complications in shigellosis are associated with an early and strong systemic stimulation without a concomitant stimulation of the mucosal antibody response.
Monoclonal antibodies to surface-exposes proteins of Mycoplasma mycoides subsp. mycoides (small-colony strain), which causes contagious bovine pleuropneumonia
Authors: Kiarie M N , Rurangirwa F R , Perryman L E , Jasmer D P , McGuire T C
Published: January 2020

Outbreaks of bovine pleuropneumonia caused by small-colony strains of Mycoplasma mycoides subsp. mycoides occur in Africa, and vaccination is used for control. Since protein subunits are needed to improve multivalent vaccines, monoclonal antibodies (MAbs) were made to facilitate protein identification and isolation. Eleven immunoglobulin M MAbs derived from mouse spleen donors immunized with disrupted whole organisms bound periodate-sensitive epitopes on externally exposed polysaccharide. Seven of these MAbs caused in vitro growth inhibition of M. mycoides subsp. mycoides; however, reaction with carbohydrate epitopes prevented their use in identifying proteins. Ten additional MAbs from mouse spleen donors immunized with Triton X-114-phase integral membrane proteins reacted with periodate-insensitive, proteinase K-sensitive epitopes. These MAbs were classified into three groups based on immunoblots of Triton X-114-phase proteins. One group reacted with 96-, 16-, and 15-kDa proteins. Another group reacted with 26-, 21-, and 16-kDa proteins, while a third group reacted only with 26- and 21-kDa proteins. One MAb from each group reacted with trypsinsensitive epitopes on live organisms, yet none caused in vitro growth inhibition. Representative MAbs reacted with all small-colony strains in immunoblots and did not react with large colony strains. However, these MAbs were not specific for small-colony strains, as proteins from two other M. mycoides cluster organisms were identified. Nevertheless, MAbs to surface-exposed epitopes on integral membrane proteins will be useful for isolation of these proteins for immunization, since one or more might induce growth-inhibiting antibodies or other protective responses.
Epitope mapping of the variable repetitive region with the MB antigen of Ureaplasma urealyticum
Authors: Zheng X , Lau K , Frazier M , Cassell G H , Watson H L
Published: January 2020

One of the major surface structures of Ureaplasma urealyticum recognized by antibodies of patients during infection is the MB antigen. Previously, we showed by Western blot (immunoblot) analysis that any one of the anti-MB monoclonal antibodies (MAbs) 3B1.5, 5B1.1, and 10C6.6 could block the binding of patient antibodies to MB. Subsequent DNA sequencing revealed that a unique six-amino-acid direct tandem repeat region composed the carboxy two-thirds of this antigen. In the present study, using antibody-reactive peptide scanning of this repeat region, we demonstrated that the amino acids defining the epitopes for MAbs 3B1.5 5B1.1 and 10C6.6 are EQP, GK, and KEQPA, respectively. Peptide scanning analysis of an infected patient's serum antibody response showed that the dominant epitope was defined by the sequence PAGK. Mapping of these continuous epitopes revealed overlap between all MAb and patient polyclonal antibody binding sites, thus explaining the ability of a single MAb to apparently block all polyclonal antibody binding sites. We also show that a single amino acid difference in the sequence of the repeats of serovars 3 and 14 accounts for the lack of reactivity with serovar 14 of two of the serovar 3-specific MAbs. Finally, the data demonstrate the need to obtain the sequences of the mba genes of all serovars before an effective serovar-specific antibody detection method can be developed.
Antibody production against hepatitis C virus core and nonstructural 3 proteins is highly sensitive to deficits in T-cell function
Authors: Ando Y , Sönnerborg A , Barkholt L , Birkett A , Ericzon B G , Sällberg M
Published: January 2020

The influence of suppression of CD4+ and CD8+ T cells on the humoral responses to hepatitis C virus (HCV) core and nonstructural 3 proteins was studied. An increasing viral burden cannot substitute for the lack of functional T cells in maintaining humoral HCV-specific responses.
Molecular karyotype analysis of Cryptosporidium parvum: evidence for eight chromosomes and a low-molecular-size molecule
Authors: Blunt D S , Khramtsov N V , Upton S J , Montelone B A
Published: January 2020

We report improved separation of chromosome-sized DNA molecules of the coccidian parasite Cryptosporidium parvum with contour-clamped homogeneous electric fields (CHEF). We used scanning densitometry to determine that the most likely number of chromosomes is eight. Molecular probes consisting of cloned genes were used to distinguish each of five bands visible on CHEF gels. We have also identified a low-molecular-size DNA molecule possibly related to the 35-kb circular DNAs found in other Apicomplexa.
Recombinant Coccidioides immitis complement-fixing antigen: detection of an epitope shared by C. immitis, Histoplasma capsulatum, and Blastomyces dermatitidis
Authors: Yang M C , Magee D M , Kaufman L , Zhu Y , Cox R A
Published: January 2020

We undertook an investigation to assess the utility of a recombinant Coccidioides immitis complement-fixing (CF) antigen for detecting CF antibody in sera from patients with coccidioidomycosis. Enzyme-linked immunosorbent assays established that recombinant CF antigen and, for comparison, a commercially available coccidioidin were reactive with 19 of 19 sera from patients with active coccidioidomycosis. The recombinant antigen was significantly more sensitive than coccidioidin. The median titer obtained when patients' sera were assayed against recombinant CF antigen was 1:51,200 compared to 1:25,600 with coccidioidin (P < 0.027). The recombinant antigen was also more effective in distinguishing the antibody levels in sera from patients with pulmonary coccidioidomycosis than in sera from those with disseminated disease. Whereas patients with pulmonary disease showed a median antibody titer of 1:25,600, those with multifocal disease showed a median titer of 1:102,400 (P < 0.028). The recombinant CF antigen was found, however, to express an epitope(s) that reacted with sera from 6 of 12 patients with histoplasmosis and 2 of 12 patients with blastomycosis.
Immunodiagnosis of alveolar echinococcosis by enzyme-linked immunosorbent assay using a partially purified Em18/16 enriched fraction
Authors: Ito A , Ma L , Itoh M , Cho S Y , Kong Y , Kang S Y , Horii T , Pang X L , Okamoto M , Yamashita T , Lightowlers M W , Wang X G , Liu Y H
Published: January 2020

An improved enzyme-linked immunosorbent assay (ELISA) system using partially purified Eml8/16 enriched fraction (PP-Em18/16) prepared by isoelectric focusing was evaluated for serodiagnosis of alveolar echinococcosis (AE). The PP-Em18/16-ELISA was compared with Em2plus-ELISA by using sera from AE and cystic echinococcosis (CE) patients in China, where both AE and CE are endemic; sera from CE patients in Australia, where only CE exists; and sera from patients with cysticercosis, paragonimiasis, or sparganosis in Korea, where no indigenous AE or CE exists. We used Em2plus-ELISA as a standard ELISA and found 24.6% (17 of 69 specimens) cross-reactivity with sera from CE. Furthermore, some of the sera from paragonimiasis, sparganosis, and cysticercosis patients were also cross-reactive in the Em2plus-ELISA. When we tested for similar cross-reactivity in the same sera from CE patients by PP-Em18/16-ELISA (23.2%, 16 of 69), it became evident that the specificity of the PP-Em18/16-ELISA was better than that of the Em2plus-ELISA, since no sera from patients with the examined parasitic diseases except CE showed cross-reactivity. Some CE patients from China showed exceptionally high levels of antibody in comparison with those of CE patients from Australia, where no AE occurs. It is speculated that these patients with strongly positive cases of CE from China may have been exposed to both species of Echinococcus.
Improved reactivity of hepatitis C virus core protein epitopes in a conformational antigen-presenting system
Authors: Buratti E , Di Michele M , Song P , Monti-Bragadin C , Scodeller E A , Baralle F E , Tisminetzky S G
Published: January 2020

Recent studies have identified several epitopes in the N-terminal portion of the nucleocapsid protein which are predominantly recognized by sera of patients infected with hepatitis C virus (HCV). The characterization of the sequences recognized by theses antibodies and the evaluation of their reactivities have been performed mainly with synthetic peptides. However, synthetic peptides are notoriously unreliable as antigens when the immune response is directed against conformational epitopes. In order to improve the detection of antibody responses in HCV-infected patients, we have evaluated the reactivities of three immunodominant regions of the HCV core protein (residues 1 to 20, 21 to 40, and 32 to 46) displayed in a conformation-specific manner on the surface of the Flock House virus (FHV) capsid protein. The results obtained with these proteins in the analysis of 94 serum samples positive by anti-HCV enzyme-linked immunosorbent assay where then compared with those obtained with the corresponding synthetic peptides. The sequence most reactive both with the peptide and with the FHV protein was the region from residues 1 to 20, confirming the low conformational requirements for the display of these residues. On the other hand, the already reported conformational nature of residues 32 to 46 is in keeping with its observed high reactivity when displayed by the FHV recombinant protein and with the low reactivity displayed by its corresponding synthetic peptide. Finally, the high reactivity observed for the chimeric protein displaying the region from residues 21 to 40, as opposed to the results obtained with the synthetic peptide, also suggests that this sequence contains one or more conformational epitopes whose structures cannot be mimicked correctly with synthetic peptides.
Levels of stem cell factor and interleukin-3 in serum in acute Plasmodium falciparum malaria
Authors: Burgmann H , Looareesuwan S , Wiesinger E C , Winter W , Graninger W
Published: January 2020

The purpose of the present study was to measure serum concentrations of stem cell factor (SCF) and interleukin-3 (IL-3) in patients with acute Plasmodium falciparum malaria. Serum samples from 15 patients were taken on day of admission and days 7, 14, 21, and 28. Anemia developed in 80% of patients. A transient increase in IL-3 could be observed at the beginning of the disease. It remains controversial whether the measured concentrations of IL-3 and SCF correlate with the grade of anemia. The possibly suppressed IL-3 and SCF production may contribute to the prolonged anemia in P. falciparum malaria, as has been shown for erythropoietin.
Recurrent infectious diseases in human CD53 deficiency
Authors: Mollinedo F , Fontán G , Barasoain I , Lazo P A
Published: January 2020

We report a familiar syndrome of recurrent heterogeneous infectious diseases, caused by bacteria, fungi, and viruses, which has as its only detectable defect the lack of CD53 antigen expression in neutrophils. All other assays ruled out known causes of recurrent infectious diseases due to either leukocyte adhesion or phagocytosis defects. CD53 belongs to the transmembrane-4 superfamily of proteins, which are a novel group of membrane proteins implicated in growth regulation and cell motility and possibly cell adhesion. We postulate that defects in these membrane proteins can be clinically manifested as complex recurrent infections.
Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp
Authors: Velasco J , Díaz R , Grilló M J , Barberán M , Marín C , Blasco J M , Moriyón I
Published: January 2020

Immunological cross-reactions between Brucella spp. and Ochrobactrum anthropi were investigated in animals and humans naturally infected by Brucella spp. and in experimentally infected rams (Brucella ovis infected), rabbits (Brucella melitensis infected), and mice (B. melitensis and Brucella abortus infected). In the animals tested, O. anthropi cytosolic proteins evoked a delayed-type hypersensitivity reaction of a frequency and intensity similar to that observed with B. melitensis brucellin. O. anthropi cytosolic proteins also reacted in gel precipitation tests with antibodies in sera from Brucella natural hosts with a frequency similar to that observed with B. melitensis proteins, and absorption experiments and immunoblotting showed antibodies to both Brucella-specific proteins and proteins common to Brucella and O. anthropi. No antibodies to O. anthropi cytosolic proteins were detected in the sera of Brucella-free hosts. Immunoblotting with sera of Brucella-infected sheep and goats showed immunoglobulin G (IgG) to Brucella group 3 outer membrane proteins and to O. anthropi proteins of similar molecular weight. No IgG to the O-specific polysaccharide of O. anthropi lipopolysaccharide was detected in the sera of Brucella-infected hosts. The sera of sheep, goats, and rabbits infected with B. melitensis contained IgG to O. anthropi rough lipopolysaccharide and lipid A, and B. ovis and O. anthropi rough lipopolysaccharides showed equal reactivities with IgG in the sera of B. ovis-infected rams. The findings show that the immunoresponse of Brucella-infected hosts to protein antigens is not necessarily specific for brucellae and suggest that the presence of O. anthropi or some related bacteria explains the previously described reactivities to Brucella rough lipopolysaccharide and outer membrane proteins in healthy animals.
Antigenic characterization of fimbria preparations from Streptococcus mutans isolates from caries-free and caries-susceptible subjects
Authors: Perrone M , Gfell L E , Fontana M , Gregory R L
Published: January 2020

The adhesion of pathogenic bacteria to the host surface is an essential step in the development of numerous infections, including dental caries. Attachment of Streptococcus mutans, the main etiological agent of human dental caries, to the tooth surface may be mediated by glucan synthesized by glucosyltransferase (GTF) and by cell surface proteins, such as P1, which bind to salivary receptors. Fimbriae on the surfaces of many microorganisms are known to function in bacterial adhesion. Previous studies in this laboratory have initially characterized the fibrillar surface of S. mutans. The purpose of this investigation was the comparison of the antigenic properties of fimbria preparations of S. mutans isolates from five caries-resistant (CR) and six caries-susceptible (CS) subjects. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of S. mutans fimbrial preparations revealed five major protein bands at 200, 175, 157, 86, and 66 kDa in preparations from CR and CS subjects. Immunoblot analysis indicated the presence of the same major bands recognized by anti-S. mutans fimbria antisera. Furthermore, the 175- and 157-kDa bands were recognized by antibodies to P1 and GTF, respectively. Immunoblot analysis with antisera to the fimbria preparation, to P1, or to GTF indicated that the levels of fimbria-reactive components and P1 and GTF antigens were higher in S. mutans fimbria preparations from CS subjects than in those from CR individuals. For example, four of six fimbria preparations from CS patients had demonstrable P1, and all had GTF. In contrast, only two of five CR fimbrial preparations exhibited P1 and GTF. Enzyme-linked immunosorbent assay demonstrated similar results for levels of GTF antigen in the fimbrial preparations from CR and CS subjects. The results suggest that differences between the compositions of S. mutans fimbriae in CR and CS individuals may play an important role in the virulence of this microorganism in dental caries.
L-2-oxothiazolidine-4-carboxylic acid inhibits human immunodeficiency virus type 1 replication in mononuclear phagocytes and lymphocytes
Authors: Ho W Z , Starr S E , Sison A , Douglas S D
Published: January 2020

We investigated the effects of L-2-oxothiazolidine-4-carboxylic acid (OTC; Procysteine), a cysteine prodrug, on human immunodeficiency virus type 1 (HIV-1) expression in both adult peripheral and cord blood mononuclear phagocytes and lymphocytes. OTC suppressed HIV-1 expression in monocyte-derived macrophages (MDM) and lymphocytes in a dose-dependent fashion as determined by HIV-1 reverse transcriptase (RT) activity. This inhibitory effect of OTC occurred with three HIV-1 strains (two laboratory-adapted strains and one primary isolate). Addition of OTC to chronically HIV-1-infected MDM cultures also suppressed RT activity by 40 to 50% in comparison to untreated controls. The inhibitory effects of OTC on HIV-1 were not caused by toxicity to MDM or lymphocytes because there was no change in cell viability or cellular DNA synthesis, as evaluated by trypan blue dye exclusion and [3H]thymidine incorporation, at doses of OTC that inhibit virus replication. These observations indicate that OTC has the potential to limit HIV-1 replication in mononuclear phagocytes and lymphocytes and may be useful in the treatment of HIV-1 infection and AIDS.
Standardization of measurement of immunoglobulin-secreting cells in human peripheral circulation
Authors: Baqar S , Nour El Din A A , Scott D A , Bourgeois A L , Mourad A S , Kleinosky M T , Oplinger M J , Murphy J R
Published: January 2020

A sensitive, and at times the most sensitive, measurement of human vaccine immunogenicity is enumeration of antibody-secreting cells (ASC) in peripheral blood. However, this assay, which is inherently capable of measurement of the absolute number of antigen-specific ASC, is not standardized. Thus, quantitative comparison of results between laboratories is not currently possible. To address this issue, isotype-specific ASC were enumerated from paired fresh and cryopreserved mononuclear cell (MNC) preparations from healthy adult volunteers resident in either the United States (US group) or Egypt (EG group). Analysis of fresh cells from US volunteers revealed mean numbers of ASC per 10(6) MNC of 617, 7,738, and 868 for immunoglobulin M (IgM), IgG, and IgA, respectively, whereas EG volunteers had 2,086, 7,580, and 1,677 ASC/10(6) MNC for the respective isotypes. Cryopreservation resulted in a slight reduction in group mean IgM, IgG, and IgA ASC (maximum reduction in group mean, 14%), but in no instance were results obtained with cryopreserved cells significantly lower than those obtained with fresh cells. To determine if cryopreservation affected the number of bacterial antigen-specific ASC detected, cells from a group of US adult volunteers who received a single oral dose of a mutated Escherichia coli heat-labile enterotoxin (LT(R192G)) were tested. There was no significant difference (P > 0.05) in the number of antigen-specific IgA or IgG ASC detected between fresh and cryopreserved MNC. The results support the views that ASC assays can be standardized to yield quantitative results and that the methodology can be changed to make the test more practical.
Preparation of a monoclonal antibody specific for Entamoeba dispar and its ability to distinguish E. dispar from E. histolytica
Authors: Tachibana H , Kobayashi S , Kaneda Y , Takeuchi T , Fujiwara T
Published: January 2020

A monoclonal antibody (MAb), MAb ED17 (immunoglobulin G2a [IgG2a]), prepared against trophozoites of Entamoeba dispar SAW1734RclAR cultured monoxenically with Crithidia fasciculata, reacted with 25 of 26 isolates of E. dispar by an indirect fluorescent-antibody test. In contrast, the MAb failed to react with any of 20 isolates of E. histolytica or other enteric protozoan parasites. Western blot (immunoblot) analysis showed that the molecular mass of the E. dispar antigen recognized by the MAb was 160 kDa under reduced conditions. Immunoelectron microscopy revealed that the antigen was mainly located on digested C. fasciculata, but not on undigested organisms. Double staining with a mixture of MAb ED17 and MAb 4G6 (an IgG1 MAb which reacts exclusively with E. histolytica), followed by incubation with a mixture of fluorescein isothiocyanate-labeled anti-mouse IgG2a and tetramethylrhodamine isothiocyanate-labeled anti-mouse IgG1 antibodies, simultaneously identified mixed populations of E. dispar and E. histolytica. This method may prove to be useful for the accurate identification of E. dispar and E. histolytica, even in mixed infections.
T-cell, antibody, and cytokine responses to homologs of the 60-kilodalton heat shock protein in Helicobacter pylori infection
Authors: Sharma S A , Miller G G , Peek R A , Pérez-Pérez G , Blaser M J
Published: January 2020

For Helicobacter pylori, the hsp60 heat shock protein encoded by hspB is being considered as a potential candidate for subunit vaccines. We investigated the humoral and cellular responses to H. pylori hsp60 and its cross-reactivity with the homologous Mycobacterium bovis p65 protein and autologous human hsp60 protein. H. pylori-infected persons had significantly higher levels than uninfected persons of serum immunoglobulin G antibodies recognizing H. pylori hsp60, but not M. bovis p65 or human hsp60, as determined by enzyme-linked immunosorbent assay. In contrast, immunoblotting demonstrated cross-reactivity of H. pylori hsp60 with human hsp60. T-cell recognition of H. pylori hsp60 was found in both infected and uninfected subjects, and there was no recognition of human hsp60. T cells from infected and uninfected subjects that had been activated in response to H. pylori hsp60 or M. bovis p65 were phenotypically similar but appeared to secrete different levels of gamma interferon and interleukin-10. These results demonstrate an apparent difference in the epitopes recognized by the T and B cells responding to H. pylori hsp60 in H. pylori-infected persons. In contrast to the T-cell responses, which were highly variable in all subjects and showed no recognition of autologous proteins, a specific B-cell response that may have cross-reactivity to human hsp60 is evident in some infected subjects.
Heterogeneity of tbpB, the transferrin-binding protein B gene, among serogroup B Neisseria meningitidis strains of the ET-5 complex
Authors: Rokbi B , Mignon M , Caugant D A , Quentin-Millet M J
Published: January 2020

ET-5 complex strains of Neisseria meningitidis were traced intercontinentally and have been causing hyperendemic meningitis on a worldwide scale. In an attempt to develop a fully broad cross-reactive transferrin-binding protein B (TbpB)-based vaccine, we undertook to assess the extent of variability of TbpB proteins among strains of this epidemiological complex. For this purpose, a PCR-based method was developed to study the heterogeneity of the tbpB genes from 31 serogroup B N. meningitidis strains belonging to the ET-5 complex. To define adequate primers, the tbpB gene from an ET-5 complex strain, 8680 (B:15:P1.3; isolated in Chile in 1987), was cloned and the nucleotide sequence was determined and compared to two other previously published tbpB sequences. A tbpB fragment was amplified from genomic DNA from each of the 31 strains. By this method, heterogeneity in size was observed and further characterized by restriction pattern analysis with four restriction enzymes and by sequencing tbpB genes from three other ET-5 complex strains. Four distinct tbpB gene types were identified. Fifty-five percent of the strains studied (17/31) harbored tbpB genes similar to that of strain BZ83 (B:15:-) isolated in The Netherlands in 1984. Ten of the 31 strains (32.2%) had tbpB genes close to that of strain M982. Only 3 of the 31 (9.6%) were found to harbor tbpB genes close to that of strain 8680, and finally one strain, 8710 (B:15:P1.3; isolated in Chile in 1987), was found to harbor a tbpB gene different from all the others. These results demonstrated a pronounced variability among tbpB alleles within a limited number of ET-5 complex strains collected over a 19-year period. Despite the genetic heterogeneity observed, specific antisera raised to purified Tbps from ET-5 complex strains showed broad cross-reactivity between different TbpBs both by Western blot analysis and bactericidal assay, confirming that a limited number of TbpB molecules included in a vaccine are likely to induce broadly cross-reactive antibodies against the different strains.
Comparative evaluation of purified Taenia solium glycoproteins and crude metacestode extracts by immunoblotting for the serodiagnosis of human T. solium cysticercosis
Authors: Rodriguez-Canul R , Allan J C , Fletes C , Sutisna I P , Kapti I N , Craig P S
Published: January 2020

A lentil-lectin purified glycoprotein (LL-Gp) and a crude saline extract of Taenia solium metacestodes were compared for the immunodiagnosis of human cysticercosis by immunoblotting. The LL-Gp preparation was 95% sensitive for antibodies against a range of seven antigens with molecular masses of 50 to 13 kDa, whereas the sensitivity of the crude saline extract for the detection of antibodies against two major polypeptide molecules (26 and 8 kDa) was 91%. Specificity was 100% with both sets of diagnostic antigens. Affinity-purified antibodies against the 26-kDa molecule from the crude saline extract recognized the 24-kDa diagnostic region in the LL-Gp-purified extract and vice versa, suggesting that the antigens had common epitopes recognized by cysticercotic sera. In addition, in a preliminary community study of 115 randomly selected people from Bali (Indonesia), seroprevalence by immunoblot assay varied from 7.8% (with the crude saline antigen extract) to 9.6% (with the LL-Gp-purified extract). The results of this study demonstrate that both antigenic preparations are applicable for the immunodiagnosis of T. solium cysticercosis. The crude T. solium metacestode antigen extract was as specific as the purified LL-Gp T. solium metacestode extract and simpler to produce but slightly less sensitive.
Development of an in vitro cloning method for Cowdria ruminantium
Authors: Perez J M , Martinez D , Debus A , Sheikboudou C , Bensaid A
Published: January 2020

Cowdria ruminantium is a tick-borne rickettsia which causes severe disease in ruminants. All studies with C. ruminantium reported so far were carried out with stocks consisting of infective blood collected from reacting animals or from the same stocks propagated in vitro. Cloned isolates are needed to conduct studies on immune response of the host, on genetic diversity of the parasite, and on mechanisms of attenuation and the development of vaccines. A method of cloning based on the particular chlamydia life cycle of Cowdria was developed. Instead of cloning extracellular elementary bodies, it appeared more convenient to clone endothelial cells infected by one morula resulting from the infection of the cell by one elementary body of Cowdria. Two hundred and sixteen clones were obtained by limiting dilution of infected cells. The method was experimentally validated by comparing randomly amplified polymorphic DNA fingerprints from individual clones obtained from endothelial cell cultures coinfected with two different stocks of C. ruminantium.
Molecular characterization of an outbreak of respiratory syncytial virus (subgroup A) in Havana, Cuba, by monoclonal antibodies and restriction mapping (N gene)
Authors: Valdivia A , Chacón D , Savón C , Oropesa S , Sarmiento L , Valdes O , Otero A , Rosario D , Goyenechea A
Published: January 2020

Twenty-one respiratory syncytial virus (RSV) strains isolated from one outbreak in Havana, Cuba (1994 to 1995), were analyzed to determine their relatedness. All isolated strains were classified as subgroup A by monoclonal antibodies. Of 21 RSV strains examined, 20 were classified as having restriction pattern NP4 and only 1 was classified as having restriction pattern NP5.
Interleukin-4 inhibits secretion of interleukin-1beta in the response of human cells to mycobacterial heat shock proteins
Authors: Méndez-Samperio P , Badillo-Flores A , Nuñez-Vazquez A , Hernandez Garay M
Published: January 2020

Cellular activation induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) and heat shock proteins (HSP) leads to the production of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. In this study, we found that IL-4 significantly suppressed IL-1beta secretion induced by BCG and the 70- and 65-kDa HSP. When exogenous recombinant human IL-4 was added to human mononuclear cells, a dose- and time-related inhibition of the 70-kDa HSP- and BCG-induced IL-1beta secretion was observed. IL-1beta secretion was maximally inhibited at 24 h of culture, and this inhibitory effect was sustained at a later time point of culture (120 h). In addition, IL-2, another T-cell-derived cytokine acting on monocytes, had no effect on IL-1beta secretion induced by either BCG or the 70-kDa HSP, indicating that in these experiments not all cytokines could immunoregulate IL-1beta secretion. This inhibitory effect was not due to a cytotoxic effect of IL-4, since the viabilities of human mononuclear cells were comparable in the presence and absence of IL-4. IL-4 was also able to inhibit the secretion of IL-1beta by mycobacterium-stimulated cells from three rheumatoid arthritis patients. This inhibitory effect of IL-4 was reversed with a blocking anti-IL-4 antibody. Finally, IL-4 inhibited IL-6 secretion by mycobacterium-activated human cells. These results suggest that IL-4 may be important in the regulation of the immune response to mycobacterial antigens.
Conservation of fimbriae and the hemagglutinating adhesin HA-Ag2 among Porphyromonas gingivalis strains and other anaerobic bacteria studied by epitope mapping analysis
Authors: Du L , Pellen-Mussi P , Chandad F , Mouton C , Bonnaure-Mallet M
Published: January 2020

Monoclonal antibodies characterized as antifimbria and anti-HA-Ag2 were used in immunoblotting to examine the antigenic distribution of fimbriae and HA-Ag2 among a collection of human and animal Porphyromonas strains and human Prevotella and Bacteroides strains. The results showed that fimbrial and HA-Ag2 antigenic structures are peculiar to the species Porphyromonas gingivalis.
CD4+-T-cell counts, spontaneous apoptosis, and Fas expression in peripheral blood mononuclear cells obtained from human immunodeficiency virus type 1-infected subjects
Authors: Patki A H , Georges D L , Lederman M M
Published: January 2020

We examined the relationships among CD4+-T-cell counts, spontaneous apoptosis, and Fas expression among peripheral blood mononuclear cells obtained from human immunodeficiency virus type 1 (HIV-1)-infected patients. After 2 days of incubation, propidium iodide DNA staining and flow cytometry revealed that peripheral blood mononuclear cells from subjects with the lowest CD4+-cell numbers (0 to 99/microl; n = 20) showed the highest frequency of apoptosis: 22.4% +/- 2.7% (mean +/- standard error) versus 13.8% +/- 1.2% and 12.7% +/- 1.4% among peripheral blood mononuclear cells obtained from patients with 100 to 499 CD4+ cells/microl (n = 19) and >500 CD4+ cells/microl (n = 17), respectively. Each of these means differed significantly from the mean frequency of apoptosis (6.3% +/- 0.7%) of peripheral blood mononuclear cells obtained from HIV-1-seronegative controls (P < 0.001, Student's t test). After incubation, the percentage of peripheral blood mononuclear cells expressing Fas antigen was increased for the HIV-1-infected subjects, and this was most evident for patients with more advanced disease. Among patients with fewer than 100 CD4+ cells/microl, 64.4% +/- 5.4% of peripheral blood mononuclear cells were Fas+, as opposed to 25.8% +/- 3.0% and 14.5% +/- 1.7% Fas+ cells among patients with more than 100 CD4+ cells/microl and healthy controls, respectively (P < 0.05 for each group comparison). Interestingly, in all populations, most apoptotic cells did not express Fas. Thus, apoptosis and Fas expression are increased in incubated peripheral blood mononuclear cells obtained from HIV-1-infected patients and these phenomena are enhanced as disease progresses.
Distribution of immunogenic epitopes on the two major immunodominant proteins (rOmpA and rOmpB) of Rickettsia conorii among the other rickettsiae of the spotted fever group
Authors: Xu W , Raoult D
Published: January 2020

Forty-four monoclonal antibodies were raised against strain Seven, the type strain of Rickettsia conorii. Of these 44 monoclonal antibodies, 13, 27, and 4 were demonstrated to be directed against the 116-kDa protein (rOmpA), the 124-kDa protein (rOmpB), and lipopolysaccharide-like antigen, respectively. The antiprotein monoclonal antibodies were found to be directed against 29 distinct epitopes, which were located on the two major immunodominant proteins discussed above. Further analysis showed that strain-specific epitopes were located on the rOmpA protein and species- and subgroup-specific epitopes were located on the rOmpB protein. R. conorii Manuel, Indian tick typhus rickettsia, and Kenya tick typhus rickettsia also possessed all 29 epitopes, whereas the other rickettsiae of the spotted fever group (SFG) expressed between 3 and 25 epitopes, with the exception of Rickettsia helvetica, R. akari, and R. australis which did not possess any epitopes. Additional analyses by Western immunoblotting confirmed that the epitopes shared among the SFG rickettsiae were located on the same two high-molecular-mass proteins as on R. conorii. However, although epitopes on the R. conorii rOmpB protein were expressed on the rOmpB proteins of most other SFG rickettsiae, some were found on the rOmpA proteins of R. aeschlimannii, R. rickettsii, and R. rhipicephali. Both proteins possessing the common epitopes were found to have different sizes in the SFG rickettsial species. The different distributions of common epitopes in the SFG rickettsiae were also used to build a taxonomic dendrogram, which demonstrated that all the R. conorii strains formed a relatively independent cluster within the SFG rickettsiae and was generally consistent with previously proposed taxonomies.
Western blot analysis of antibody response to pneumococcal protein antigens in a murine model of pneumonia
Authors: Mouneimne H , Juvin M , Beretti J L , Azoulay-Dupuis E , Vallee E , Geslin P , Petitpretz P , Berche P , Gaillard J L
Published: January 2020

To detect new antigen candidates for serological tests, we studied the antibody response to pneumococcal protein antigens in mice infected intratracheally with various Streptococcus pneumoniae strains. Sera were tested by Western blotting against whole-cell protein extracts. Mice developed a detectable immunoglobulin G-type response against a small number of polypeptides. The antibody response was strain dependent: sera from individuals infected with the same strain gave similar banding patterns on immunoblots. The banding patterns varied with the strain used for infection. However, a band at 36 to 38 kDa was recognized by all reactive sera. This band appeared to correspond to a polypeptide that was antigenically well conserved among the different S. pneumoniae serotypes. An antibody response to this antigen developed in mice irrespective of the capsular type, the virulence, and the susceptibility to penicillin G of the infecting strain. Thus, this 36- to 38-kDa protein antigen may be of value for the development of a serological test for humans.
Expression of recombinant exoenzyme S of Pseudomonas aeruginosa
Authors: Kulich S M , Frank D W , Barbieri J T
Published: January 2020

The structural gene for the 49-kDa form of exoenzyme S (exoS) isolated from Pseudomonas aeruginosa 388 was expressed in both Escherichia coli and P. aeruginosa PA103. Expression of exoS in E. coli under the transcriptional regulation of the T7 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 49 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Expression of exoS in P. aeruginosa PA103 under the transcriptional regulation of the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS yielded a nitrilotriacetic acid-inducible extracellular protein with an apparent molecular mass of 49 kDa. Recombinant ExoS (rExoS) reacted with the anti-49-kDa form of exoenzyme S immunoglobulin G, existed as an aggregate as determined by gel filtration chromatography, and ADP-ribosylated soybean trypsin inhibitor at a specific activity that was similar (within twofold) to that of native exoenzyme S. Allelic exchange of exoS with a tetracycline gene cartridge yielded a strain of P. aeruginosa 388 that did not express detectable amounts of either ExoS in an immunoblot analysis using the anti-49-kDa form of exoenzyme S immunoglobulin G or ADP-ribosyltransferase activity under standard enzyme assay conditions. Expression of catalytically active rExoS in E. coli demonstrated that exoS was necessary and sufficient for the factor-activating exoenzyme S-dependent ADP-ribosyltransferase activity of exoenzyme S. Expression of nitrilotriacetic acid-inducible rExoS in P. aeruginosa PA103 demonstrated that the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS encoded a functional exoenzyme S promoter. Expression analysis and allelic exchange experiments suggest that the 49- and 53-kDa forms of exoenzyme S are encoded by separate genes.
Location of the complement factor H binding site on streptococcal M6 protein
Authors: Fischetti V A , Horstmann R D , Pancholi V
Published: January 2020

The surface M protein of group A streptococci binds factor H, a regulatory protein of the alternative complement pathway, which may contribute to the antiphagocytic activity of the M molecules. To locate the factor H binding domain in the alpha-helical coiled-coil structure of the M molecule, the M protein was cleaved with pepsin at pH 5.8, which separates the molecule approximately in half. Western blot (immunoblot), amino acid sequence, and mass spectrometric analyses revealed that factor H bound to a 14.6-kDa C-terminal fragment of the M molecule. Competitive inhibition of factor H binding to the 14.6-kDa fragment with M protein peptides localized the binding site to amino acids 256 to 292. This segment is located within the surface-exposed region of the M6 protein, identified as the C-repeat region, whose sequence is conserved among heterologous M and M-like molecules. These studies also identified a second pepsin-susceptible site with the sequence ELAK located within the cell wall-associated region of the M molecule.
The primary structure of Clostridium septicum alpha-toxin exhibits similarity with that of Aeromonas hydrophila aerolysin
Authors: Ballard J , Crabtree J , Roe B A , Tweten R K
Published: January 2020

The gene for Clostridium septicum alpha-toxin was cloned and expressed in Escherichia coli from C. septicum BX96. The toxin was determined to be 443 amino acids in length, with a 31-residue signal peptide that was removed from the toxin during secretion. No extended hydrophobic regions were observed in the mature toxin sequence. Expression of alpha-toxin in E. coli BL21 resulted in the production of ATpro, which was identical to native toxin from C. septicum with respect to activity and activation. The proteolytic activation site for alpha-toxin was determined to be on the carboxy-terminal side of arginine 398, which lies within the sequence KKRRGKR-398SVD. Previous work showing similarities in activation and mechanism between alpha-toxin and Aeromonas hydrophila aerolysin was extended to the primary structures of both toxins. The DNA-derived primary sequence of alpha-toxin exhibited 27% identity and 72% similarity over a 387-residue region with the primary structure of the A. hydrophila aerolysin toxin, a level of similarity heretofore unobserved between toxins produced by a gram-positive organism and a gram-negative organism.
Lipophosphoglycan blocks attachment of Leishmania major amastigotes to macrophages
Authors: Kelleher M , Moody S F , Mirabile P , Osborn A H , Bacic A , Handman E
Published: January 2020

Promastigotes of the intracellular protozoan parasite Leishmania major invade mononuclear phagocytes by a direct interaction between the cell surface lipophosphoglycan found on all Leishmania species and macrophage receptors. This interaction is mediated by phosphoglycan repeats containing oligomers of beta (1-3)Gal residues specific to L. major. We show here that although amastigotes also use lipophosphoglycan to bind to both primary macrophages and a cell line, this interaction is independent of the beta (1-3)Gal residues employed by promastigotes. Binding of amastigotes to macrophages could be blocked by intact lipophosphoglycan from L. major amastigotes as well as by lipophosphoglycan from promastigotes of several other Leishmania species, suggesting involvement of a conserved domain. Binding of amastigotes to macrophages could be blocked significantly by the monoclonal antibody WIC 108.3, directed to the lipophosphoglycan backbone. The glycan core of lipophosphoglycan could also inhibit attachment of amastigotes, but to a considerably lesser extent. The glycan core structure is also present in the type 2 glycoinositolphospholipids which are expressed on the surface of amastigotes at 100-fold-higher levels than lipophosphoglycan. However, their inhibitory effect could not be increased even when they were used at a 300-fold-higher concentration than lipophosphoglycan, indicating that lipophosphoglycan is the major macrophage-binding molecule on amastigotes of L. major. In the presence of complement, the attachment of amastigotes to macrophages was not altered, suggesting that lipophosphoglycan interacts directly with macrophage receptors.
Molecular and structural requirements of a lipoteichoic acid from Enterococcus hirae ATCC 9790 for cytokine-inducing, antitumor, and antigenic activities
Authors: Takada H , Kawabata Y , Arakaki R , Kusumoto S , Fukase K , Suda Y , Yoshimura T , Kokeguchi S , Kato K , Komuro T
Published: January 2020

Comparison was made between the immunobiological and antigenic properties of two lipoteichoic acid (LTA) fractions (LTA-1 and -2) from Enterococcus hirae ATCC 9790, their glycolipid portions, and synthetic compounds partially mimicking the above bacterial products. The more lipophilic LTA-2 fraction was capable of inducing serum tumor necrosis factor alpha and interleukin-6 in muramyldipeptide-primed mice and serum gamma interferon in those primed with Propionibacterium acnes. The LTA-2 fraction also induced tumor necrosis factor alpha, interleukin-6, and thymocyte-activating factor (essentially interleukin-1) in murine peritoneal macrophage cultures. Consecutive intravenous injections of muramyldipeptide and the LTA-2 fraction in Meth A fibrosarcoma-bearing BALB/c mice caused hemorrhagic necrosis and marked regression leading to complete regression of the tumor with no accompanying weakening or lethal effects. The LTA-2 fraction was at least 10,000-fold less pyrogenic in rabbits than a reference endotoxic lipopolysaccharide. The more hydrophilic LTA-1 fraction, on the other hand, showed at most marginal activity in the in vivo and in vitro assays. Natural glycolipids (NGL-1 and -2) which were prepared from a chloroform-methanol extract of Streptococcus pyogenes and E. hirae cells, and comparable in structure to the lipid moieties of the LTA-1 and -2 fractions, respectively, were practically inactive in all of the assays. None of the test synthetic compounds was immunobiologically active, although synthetic partial counterparts of the structure of LTA proposed by W. Fischer (Handb. Lipid Res. 6:123-234, 1990) reacted with murine monoclonal antibody TS-2, which was raised against OK-432, a penicillin-killed S. pyogenes preparation, and capable of neutralizing the cytokine-inducing activities of the LTA-2 fraction.
Contribution of urease to acid tolerance in Yersinia enterocolitica
Authors: De Koning-Ward T F , Robins-Browne R M
Published: January 2020

The stomach serves as a barrier to enteric infection because of the antibacterial effect of the hydrochloric acid in gastric juice. In this study, we tested the ability of the enteric pathogen Yersinia enterocolitica to tolerate a pH range of 2.0 to 6.0 and found that under the conditions of a normal human fasting stomach (pH < 3 and a gastric emptying time of 2 h), Y. enterocolitica is highly acid resistant, showing approximately 85% survival. The resistance of Y. enterocolitica to acid in vitro depended on the bacterial growth phase and the concentration of urea in the medium, being maximal during stationary phase in the presence of at least 0.3 mM urea. Urease-negative mutants of Y. enterocolitica were constructed by disrupting the urease gene complex of a virulent strain of serogroup O9. Compared with the wild type, these mutants showed an approximately 1,000-fold decrease in the ability to tolerate acid in vitro (< 0.08% survival) and a 10-fold reduction in viability after passage through the stomachs of mice. Complementation of the disrupted urease genes in trans restored the ability of urease-negative mutants to tolerate low pH in vitro and gastric acidity to approximately wild-type levels. These findings indicate that urease is responsible for acid resistance in Y. enterocolitica and suggest that urease contributes to the virulence of Y. enterocolitica by enhancing the likelihood of bacterial survival during passage through the stomach.