bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria
Authors: Batchelor S E , Cooper M , Chhabra S R , Glover L A , Stewart G S , Williams P , Prosser J I
Published: January 2020

The speed of recovery of cell suspensions and biofilm populations of the ammonia oxidizer Nitrosomonas europaea, following starvation was determined. Stationary-phase cells, washed and resuspended in ammoniumfree inorganic medium, were starved for periods of up to 42 days, after which the medium was supplemented with ammonium and subsequent growth was monitored by measuring nitrite concentration changes. Cultures exhibited a lag phase prior to exponential nitrite production, which increased from 8.72 h (no starvation) to 153 h after starvation for 42 days. Biofilm populations of N. europaea colonizing sand or soil particles in continuous-flow, fixed column reactors were starved by continuous supply of ammonium-free medium. Following resupply of ammonium, starved biofilms exhibited no lag phase prior to nitrite production, even after starvation for 43.2 days, although there was evidence of cell loss during starvation. Biofilm formation will therefore provide a significant ecological advantage for ammonia oxidizers in natural environments in which the substrate supply is intermittent. Cell density-dependent phenomena in a number of gram-negative bacteria are mediated by N-acyl homoserine lactones (AHL), including N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). Addition of both ammonium and OHHL to cell suspensions starved for 28 days decreased the lag phase in a concentration-dependent manner from 53.4 h to a minimum of 10.8 h. AHL production by N. europaea was detected by using a luxR-luxAB AHL reporter system. The results suggest that rapid recovery of high-density biofilm populations may be due to production and accumulation of OHHL to levels not possible in relatively low-density cell suspensions.
Use of the pre-pro part of Staphylococcus hyicus lipase as a carrier for secretion of Escherichia coli outer membrane protein A (OmpA) prevents proteolytic degradation of OmpA by cell-associated protease(s) in two different gram-positive bacteria
Authors: Meens J , Herbort M , Klein M , Freudl R
Published: January 2020

Heterologous protein secretion was studied in the gram-positive bacteria Bacillus subtilis and Staphylococcus carnosus by using the Escherichia coli outer membrane protein OmpA as a model protein. The OmpA protein was found to be translocated across the plasma membrane of both microorganisms. However, the majority of the translocated OmpA was similarly degraded in B. subtilis and S. carnosus despite the fact that the latter organism does not secrete soluble exoproteases into the culture medium. The finding that purified OmpA, which was added externally to the culture medium of growing S. carnosus cells, remained intact indicates that newly synthesized and exported OmpA is degraded by one or more cell-associated proteases rather than by a soluble exoprotease. Fusion of the mature part of OmpA to the pre-pro part of a lipase from Staphylococcus hyicus allowed the efficient release of the corresponding propeptide-OmpA hybrid protein into the supernatant and completely prevented the cell-associated proteolytic degradation of the mature OmpA, most likely reflecting an important function of the propeptide during secretion of its natural mature lipase moiety. The relevance of our findings for the biotechnological use of gram-positive bacteria as host organisms for the secretory production of heterologous proteins is discussed.
The Vitek immunodiagnostic assay for detection of immunoglobulin M toxoplasma antibodies
Authors: Candolfi E , Ramirez R , Hadju M P , Shubert C , Remington J S
Published: January 2020

We compared the Vitek immunodiagnostic assay for detection of immunoglobulin M (IgM) toxoplasma antibodies (VIDAS TXM) with an IgM enzyme-linked immunosorbent assay (ELISA) which we developed. A total of 407 serum samples from 391 adults were used for this study. We also examined 17 serial serum samples from four women who had seroconverted during gestation. We observed a sensitivity of 99.5%, a specificity of 93%, a positive predictive value of 93.6%, a negative predictive value of 99.5%, and a global agreement of 96.3%. In each case of seroconversion the VIDAS TXM became positive at the same time as did the IgM ELISA.
Production of interferons in human placental trophoblast subpopulations and their possible roles in pregnancy
Authors: Aboagye-Mathiesen G , Tóth F D , Zdravkovic M , Ebbesen P
Published: January 2020

The human cytotrophoblasts are the first fetal cells to arise during embryogenesis and are the progenitor cells to villous (noninvasive), syncytiotrophoblast (noninvasive), "intermediate" extravillous (invasive), and "anchoring" extravillous (invasive) trophoblast subpopulations. These trophoblast subpopulations were isolated from first- and third-trimester placentae and were stimulated with Sendai virus, granulocyte-macrophage colony-stimulating factors (GM-CSF), and platelet-derived growth factor (PDGF) to produce interferons (IFNs). GM-CSF and PDGF induced very low levels of IFN in first-trimester extravillous and villous trophoblast subpopulations. Highly proliferating and invasive intermediate extravillous trophoblast cultures produced five- to eightfold more IFNs than villous trophoblast cultures and two- to fivefold more IFN than the syncytiotrophoblast cultures when stimulated with Sendai virus. Syncytiotrophoblast cultures produced higher levels of IFNs (up to twofold) than villous trophoblast cultures when stimulated with the same virus. Pretreatment of first-trimester extravillous and villous trophoblast cultures with GM-CSF and PDGF followed by infection with Sendai virus resulted in greater IFN production than when the cultures were stimulated with virus alone. The levels of IFN produced were dependent on the type of trophoblast, the type of inducer, and the stage of differentiation of the trophoblasts. The purified trophoblast IFNs have potent antiviral activities when assayed on human amniotic WISH cells, and they inhibited proliferation of normal trophoblasts and trophoblast-derived malignant cells in vitro without any toxicity. Furthermore, the trophoblast IFNs activated NK cell activity and suppressed mitogen-stimulated lymphocyte proliferation at concentrations of between 10 and 1,000 IU/ml. The possible functions of the trophoblast IFNs during pregnancy are discussed with respect to human placental and fetal protection and development.
Serum antibody response to proteins of Moraxella (Branhamella) catarrhalis in patients with lower respiratory tract infection
Authors: Christensen J J , Renneberg J , Bruun B , Forsgren A
Published: January 2020

We searched for antibodies against Moraxella (Branhamella) catarrhalis proteins in the sera of patients with lower respiratory tract infection. Sera from 48 patients with M. catarrhalis and 39 patients without M. catarrhalis in their lower respiratory tract specimens were studied by a gel electrophoresis-immunoperoxidase technique; sera from 23 healthy adult blood donors were also included. Immunoglobulin G (IgG) antibodies against a 28-kDa protein were found significantly more frequently in patients with M. catarrhalis in lower respiratory tract specimens (71%) than in patients without M. catarrhalis in lower respiratory tract specimens (28%) or healthy adult blood donors (22%). Seroconversion, from the acute to the convalescent stages, occurred in at least eight patients with M. catarrhalis and in one patient without detectable M. catarrhalis. IgG antibodies against other M. catarrhalis proteins were found in most sera, including those obtained from blood donors. By adsorption experiments the 28-kDa protein was demonstrated to be surface exposed. IgM antibodies against an 85-kDa protein were found in serum from one patient from whom M. catarrhalis and Streptococcus pneumoniae were isolated from the lower respiratory tract, while IgA antibodies against M. catarrhalis proteins could not be detected in any serum specimen.
Sensitivity of immune complex-dissociated p24 antigen testing for early detection of human immunodeficiency virus in infants
Authors: Lewis D E , Adu-Oppong A , Hollinger F B , Rosenblatt H M , Hanson I C , Reuben J M , Kline M W , Kozinetz C A , Shearer W T
Published: January 2020

Several investigators have suggested that early diagnosis of human immunodeficiency virus (HIV) infection in infants could be accomplished with a modified, more-sensitive, acid-dissociated p24 antigen enzyme-linked immunosorbent assay (ELISA) technique (p24 antigen immune complex dissociation [ICD]). We compared detection of HIV infection by HIV culture, PCR, and p24 antigen ICD assays in 46 infants by using samples collected independently. The detection sensitivity of the p24 antigen ICD assay was 0% with cord blood samples (2 HIV-positive infants), 38% with plasma samples from infants under 3 months of age (8 HIV-positive infants), and 58% overall (12 HIV-positive infants). By contrast, the sensitivities of HIV culture and PCR were 50% for cord blood samples, 75% for plasma samples from infants under 3 months of age, and 83% overall. These results indicate that the p24 antigen ICD does not offer the sensitivity necessary for this assay to be used as an indicator of HIV infection in infants.
Human antibody reactivity against the lower matrix protein (pp65) produced by cytomegalovirus
Authors: Ohlin M , Plachter B , Sundqvist V A , Steenbakkers P G , Middeldorp J M , Borrebaeck C A
Published: January 2020

The lower matrix protein (pp65) is a major product of many laboratory strains of cytomegalovirus (CMV). It is thus an integral part of many CMV serological assays based on native antigen. Recombinant fragments of pp65 have previously been investigated for their usefulness in more-defined assays. The latter antigens have, however, failed to develop a positive response with serum samples derived from a substantial number of infected individuals. Here we show that the human humoral immune response to CMV pp65 is highly diverse and recognizes at least seven distinct but in some cases partly overlapping epitopes. Most of these epitopes could not be mimicked by any of the investigated recombinant or synthetic antigens. Furthermore, when we investigated the ability of human CMV-seropositive serum samples to block the reactivity of pp65-specific antibodies recognizing five different epitopes within pp65, it was evident that several sera did not contain significant levels of antibodies against any of these or overlapping structures. It was thus concluded that the antibody response against CMV pp65 is weak in some CMV-infected individuals, making this antigen unsuitable for use alone in serological screening systems for CMV infection.
Detection of human immunodeficiency virus type 1-specific memory cytotoxic T lymphocytes in freshly donated and frozen-thawed peripheral blood mononuclear cells
Authors: Huang X L , Fan Z , Liebmann J , Rinaldo C
Published: January 2020

Loss of anti-human immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte (CTLm) responses is associated with disease progression in HIV-1 infection. In this study, nonspecific stimulation of peripheral blood mononuclear cells (PBMC) from HIV-1-infected homosexual men with anti-CD3 monoclonal antibody (MAb) was compared with antigen-specific stimulation with inactivated, autologous B lymphoblastoid cells (B-LCL) infected with a vaccinia virus vector encoding HIV-1 IIIb Gag, Pol, and Env (VV-GPE) for activation of HIV-1-specific CTLm responses in a bulk lysis assay and by precursor frequency analysis. The results show that VV-GPE-infected B-LCL stimulated on average 10-fold greater anti-HIV-1 CTLm activity, as detected in the bulk lysis assay, and 55-fold-greater CTLm precursor frequencies specific for the three HIV-1 structural proteins than did stimulation with anti-CD3 MAb. This effect was noted with both freshly donated and frozen-thawed PBMC. The lysis was mediated by CD8+ T cells and was restricted by the major histocompatibility class I complex. These data indicate that antigen-specific stimulation with VV-GPE-infected B-LCL is a highly efficient method for detection of anti-HIV-1 CTLm responses that is applicable to noncurrent prospective studies with frozen PBMC.
(1-->3) beta-D-glucan as a quantitative serological marker for Pneumocystis carinii pneumonia
Authors: Yasuoka A , Tachikawa N , Shimada K , Kimura S , Oka S
Published: January 2020

We detected (1 --> 3) beta-D-glucan (beta-glucan), which is one of the major components of the cyst wall of Pneumocystis carinii, in sera obtained from patients with P. carinii pneumonia (PCP). We confirmed that beta-glucan was detectable by a beta-glucan detection kit (G test; Seikagaku Corporation) in bronchoalveolar lavage fluids (BALFs). The mean concentration of beta-glucan in BALFs obtained from specific-pathogen-free nude mice infected with P. carinii (n = 7; mean, 2,631 [range, 1,031 to 9,095] pg/ml) was significantly higher (P < 0.001) than that in uninfected, specific-pathogen-free mice (n = 7; 6.5 [range, 4.0 to 8.3] pg/ml). The mean level of beta-glucan in BALFs from PCP patients was significantly higher (P < 0.05) than that in BALFs from patients with other lung diseases (7,268 [range, 1,355 to 15,500] pg/ml [n = 4] versus 242.5 [17 to 615] pg/ml [n = 4]). In sera from six of seven patients with PCP, significant levels of beta-glucan (494.1 [8.5 to 1,135] pg/ml) were detected, while it was undetectable in patients with other lung diseases and in a control group. In five patients at follow-up, the level of beta-glucan decreased with clinical improvement. These results suggest that beta-glucan is detectable in sera from patients with PCP and it is a practical serological marker for monitoring of the disease during treatment.
Lymphocyte subpopulation reference ranges for monitoring human immunodeficiency virus-infected Chinese adults
Authors: Kam K M , Leung W L , Kwok M Y , Hung M Y , Lee S S , Mak W P
Published: January 2020

Two hundred eight healthy human immunodeficiency virus (HIV) type 1- and HIV type 2-seronegative Chinese adults (78 males and 130 females; mean age, 32 years; age range, 18 to 71 years) were analyzed for lymphocyte subsets by a standardized and quality-controlled flow cytometric immunophenotyping technique. While the leukocyte differential values were comparable to those found in studies of Caucasians, the means, medians, and 95% reference ranges of lymphocyte subsets were very different. The 95% reference ranges in absolute counts per microliter of whole blood (percentage of lymphocytes) for CD3+, CD3+ CD4+, CD3+ CD8+, CD3- CD19+ (B), and CD3- with CD16+ and/or CD56+ (NK) cells were 672 to 2,368 (54.8 to 83.0%), 292 to 1,366 (23.1 to 51.0%), 240 to 1,028 (17.9 to 47.5%), 82 to 560 (5.1 to 20.8%), and 130 to 938 (7.1 to 38.0%), respectively. CD3+ CD4+ cells showed significant sex difference (for males, mean of 702 [34.8%] and standard deviation of 258 [7.5%]; for females, mean of 728 [37.3%] and standard deviation of 254 [7.4%]) as well as an increase with age of 42 (1.6%) per decade. Investigations of the NK cell population did not show similar findings. Classification of HIV disease, treatment, and prophylactic regimens based on studies which relied heavily on estimations of lymphocyte subsets alone should be used with special caution for Chinese patients. Provided that adequate quality control measures are taken to ensure comparability of data, we recommend that these ranges be used on a day-to-day basis in laboratories that have not yet established their own reference ranges.
Effect of nicotine on secretory component synthesis by secretory epithelial cells
Authors: Gregory R L , Gfell L E
Published: January 2020

Previously, we reported that secretory component (SC), lactoferrin (LF), and lysozyme (LY) levels were significantly lower in saliva from smokeless tobacco (ST) users than in saliva from control non-tobacco users. However, the levels of salivary immunoglobulin A were significantly higher, albeit with an altered attachment of SC, in ST users than in control subjects. SC, LF, and LY are synthesized by secretory epithelial cells at mucosal sites adjacent to lymphocyte regions. In the present report, HT-29 human epithelial cells, cultured with various concentrations of an ST aqueous extract or pure nicotine (0 to 1 mg/ml) or cotinine (0 to 5 mg/ml), exhibited significantly lower levels of cell-associated cell lysate (CL) and secreted culture supernatant (CS) SC, LF, and LY than cells cultured without ST components. Nicotine significantly decreased (P < or = 0.05) the synthesis of SC by 20 to 100%, LF by 20 to 60%, and LY by 5 to 75% of CL and CS control values. Studies also indicated significant decreases (P < or = 0.05) in SC, LF, and LY levels in both CL and CS of cells cultured with ST aqueous extract or cotinine. Total cell numbers and metabolic activity significantly decreased primarily when cells were incubated with higher concentrations of ST extract, nicotine, or cotinine. The addition of human recombinant interleukin-4 or gamma interferon diminished the effects ST had on HT-29 cell synthesis of SC, LF, and LY. Our data indicate that nicotine, cotinine, and ST have an adverse effect on synthesis and secretion of SC, LF, and LY. These effects were below ST concentrations found to be cytotoxic for secretory epithelial cells. Furthermore, addition of interleukin-4 or gamma interferon reduced the suppressive effect of ST on synthesis or secretion of SC, LF, or LY.
Relation of impaired lymphocyte proliferative function to other major human immunodeficiency virus type 1-induced immunological changes
Authors: Bass H Z , Fahey J L , Nishanian P , Detels R , Cumberland W , Kemeny M , Plaeger S
Published: January 2020

Human immunodeficiency virus (HIV) type 1 (HIV-1) induces impairment of immune function reflected in reduced lymphocyte proliferative responses. Many other immune changes are induced by HIV-1, but their relationship to lymphocyte functional defects is not known. The present study was designed to correlate functional defects with other HIV disease parameters. Cryopreserved samples from 118 HIV-1-positive subjects and 40 seronegative individuals were examined. The main findings were that impaired proliferative responses to mitogens correlated with (i) decreased cell surface expression of the interleukin-2 receptor (CD25), (ii) increased expression of HLA-DR antigens on CD4 cells, (iii) reduced CD4 and increased CD8 cell numbers, and (iv) increased levels of serum immune complex dissociated p24 antigen. However, impaired function was not associated with increased serum neopterin, beta2-microglobulin, or soluble interleukin-2 receptor or with CD38 antigen expression on lymphocytes. In summary, proliferative functional impairment correlated with some, but not all, immunological changes associated with HIV-1 infection. Most of the phenotypic markers that correlated with altered function are cell surface molecules with significant roles in lymphocyte proliferation and were associated primarily with CD4 cells, compatible with the view that dysregulation of CD4 cells is responsible for impaired function.
Value of detecting immunoglobulin E antibodies for the serological diagnosis of Toxoplasma gondii infection
Authors: Gross U , Keksel O , Dardé M L
Published: January 2020

The presence of immunoglobulin E (IgE) antibodies was determined by using the immunosorbent-agglutination assay (ISAGA) with 611 serum samples from patients with different clinical conditions to evaluate its value for the serodiagnosis of acute Toxoplasma gondii infection. By analyzing 43 consecutively drawn serum samples from 10 pregnant women who seroconverted, we could show that specific IgE antibodies seem to appear early after infection and are usually present for less than 3 to 5 months. Therefore, we assumed that IgE antibodies seem to be detectable only during the acute or reactivated stage of infection. According to our studies, the IgE ISAGA has an overall sensitivity of only 79.5%, but a specificity of 98.0%, with positive and negative predictive values of 95.5 and 89.8%, respectively. Detection of IgE antibodies in immunosuppressed patients with reactivation of latent T. gondii infection correlates with disease activity. Despite these encouraging results, one must note that IgE antibodies were not detectable in 4 of 14 patients with very recent infection proven by seroconversion. Therefore, detection of IgE antibodies seems to correlate with early acute or reactivated toxoplasmosis, whereas negative IgE results do not exclude the possibility of the acute stage of toxoplasmosis.
MRP 8/14 as marker for Plasmodium falciparum-induced malaria episodes in individuals in a holoendemic area
Authors: Bordmann G , Burmeister G , Saladin S , Urassa H , Mwankyusye S , Weiss N , Tanner M
Published: January 2020

Presence of Plasmodium falciparum parasites in the peripheral blood of patients in a holoendemic area does not necessarily show that their illness is due to malaria. The aim of the present project was therefore to look for biological markers related to symptomatology or clinical events during a malaria episode. We focused our work on a complex of heterodimeric calcium-binding proteins secreted by stimulated neutrophils and monocytes, named MIF or myeloid-related proteins (MRP 8/14). In a longitudinal study including 51 adults from Ifakara, Tanzania (84.7% prevalence for P. falciparum in adults during the study), the level of MRP 8/14 in the serum was significantly related to the parasite load (Spearman correlation coefficient, 0.52; P < 0.0001). In the serum from children up to 6 years admitted at a health post the MRP 8/14 levels were closely related to parasitemia but also to fever episodes (Spearman correlation coefficients, 0.96 and 0.736; P < 0.0001 and P < 0.001, respectively). Although not specific to malaria, the measurement of MRP 8/14 could be an additional tool in assessing malaria-related morbidity.
Immunological markers of disease progression in patients infected with the human immunodeficiency virus
Authors: Pascale J M , Isaacs M D , Contreras P , Gomez B , Lozano L , Austin E , De Martin M C , Gregory R L , McLaughlin G L , Amador A
Published: January 2020

Identification of inexpensive and technically simple immunological tests useful in predicting the progression to AIDS in human immunodeficiency virus (HIV)-infected patients would be especially welcome in developing countries, in which 80% of HIV-infected patients reside and health budgets are low. In the current study, we evaluated CD4+ and total lymphocyte counts and the concentrations in serum of beta 2-microglobulin, p24 antigen, and immunoglobulin A (IgA) as predictors of disease progression in 74 Panamanian HIV-positive patients and 50 HIV-negative healthy individuals. Total lymphocyte and CD4(+)-cell counts for AIDS patients (1,451 +/- 811 cells/microliters, P < 0.001, and 238 +/- 392 cells/microliters, P < 0.0001, respectively and asymptomatic patients (2,393 +/- 664 cells/microliters, P > 0.05, and 784 +/- 475 cells/microliters, P < 0.001, respectively) were lower than those observed for healthy subjects (2,596 +/- 631 cells/microliters and 1,120 +/- 296 cells/microliters, respectively). The levels of beta 2-microglobulin and IgA in serum were significantly elevated in patients with AIDS (5.7 +/- 3.6mg/liter, P < 0.001, and 541 +/- 265 mg/dl, P < 0.0002, respectively) and asymptomatic infected subjects (3.4 +/- 2.1 mg/liter, P = 0.001, and 436 +/- 216 mg/dl, P < 0.0001, respectively) compared with the levels in healthy subjects (2.2 +/- 0.7 mg/liter and 204 +/- 113 mg/dl, respectively). Nonstatistically significant differences (P > 0.05) for concentrations of p24 antigen between asymptomatic infected patients (29 +/- 13 pg/ml) and AIDS patients (40 +/- 23 pg/ml) were observed. Total lymphocyte counts of 1,750 cells/microliters or less, CD4 counts of 200 cells/microliters or less, beta 2-microglobulin concentrations in serum of 4 mg/liter or higher, concentrations of IgA in serum of 450 mg/dl or higher, and the presence in serum of p24 antigen were correlated with elevated risks for developing AIDS. Monitoring both total lymphocytes and beta 2-microglobulin identified 91% of the AIDS patients; these assays may allow reductions in the annual number of CD4(+)-cell evaluations and the costs associated with monitoring both total lymphocytes and beta 2-microglobulin identified 91% of the AIDS patients; these assays may allow reductions in the annual number of CD4(+)-cell evaluations and the costs associated with monitoring the immune status of HIV-positive patients.
Western immunoblotting analysis of the antibody responses of patients with human monocytotropic ehrlichiosis to different strains of Ehrlichia chaffeensis and Ehrlichia canis
Authors: Chen S M , Cullman L C , Walker D H
Published: January 2020

In order to evaluate the relative sensitivity of the detection of antibodies against various antigenic proteins of Ehrlichia chaffeensis for the diagnosis of the emerging infectious disease human monocytotropic ehrlichiosis, Western immunoblotting was performed with 27 serum samples from convalescent patients with antibodies, as demonstrated by indirect immunofluorescence assay. Among 22 patients with antibodies reactive with the 120-kDa protein, 15 showed reactivity with the 29/28-kDa protein(s) and the proteins in the 44- to 88-kDa range. Two of the serum samples with this pattern reacted with the 29/28-kDa protein(s) of only the 91HE17 strain, and one sample reacted with only that of the Arkansas strain, indicating that the antibodies were stimulated by strain-specific epitopes. Overall, antibodies to the 29/28-kDa protein(s) were detected in only 16 patients' sera, suggesting that this protein is less sensitive than the 120-kDa protein. Two of 12 serum samples from healthy blood donors had antibodies reactive with the 120-kDa protein; one of these samples reacted also with the 29/28-kDa protein(s) of Ehrlichia canis, suggesting that unrecognized ehrlichial infection might have occurred, including human infection with E. canis. A high correlation between reactivity with the 120-kDa protein by Western immunoblotting and the recombinant 120-kDa protein by dot blot supports the potential usefulness of this recombinant antigen in diagnostic serology.
Characterization of an antibody directed against a surface component of normal and pleomorphic cells of Streptococcus sanguis
Authors: Eisenberg R J , Montgomery P C
Published: January 2020

Whole cells of Streptococcus sanguis were utilized as an immunoadsorbent to purify large quantities of an antibody (S1) directed against a cell surface component. The S-1 antibody was isolated from antisera to normal (N) and pleomorphic (O) cells by a similar adsorption-elution procedure. The S-1 antibody isolated from antisera to N cells reacted in gel diffusion in identify with the S-1 antibody to O cells, indicating that the antigen which binds S-1 antibody (Ag-1) may not be radically altered when cells become pleomorphic. The S-1 antibodies directed against both N and O cells had restricted heterogeneity, indicating that for both types of cell Ag-1 may have a simple repeating structure. However, N cells were agglutinated to a greater extent by S-1 antibody than O cells. In addition the distribution of the bound S-1 antibody became altered as the cells became pleomorphic. Utilizing the technique of indirect immunofluorescence we observed that the S-1 antibody was distributed evenly on the surface of N cells. As the cells became pleomorphic, the antibody appeared to bind preferentially at the cell poles (capping). Later, as the cells became more grossly deformed, additional bands of immunofluorescence appeared to bisect the cells. Electron microscopic analysis indicated that the bound antibody was not associated with septal notches. The results suggest that the arrangement rather than the immunological properties of Ag-1 became altered as cells became pleomorphic.
Fimbria-specific antibodies detach Escherichia coli from human cells
Authors: Mett H , Kloetzlen L , Vosbeck K
Published: January 2020

Antibodies obtained from rabbits immunized with purified adhesion-mediating fimbriae of Escherichia coli SS142 were specific for the fimbriae of the homologous strain; they did not cross-react with isolated fimbriae of three different E. coli strains or with protein extracts from nine other adhesive E. coli strains. The antibodies inhibited adhesion to Intestine 407 tissue culture cell monolayers and hemagglutinating activity of E. coli SS142 but not of several other E. coli strains. The antibodies were able not only to prevent but also to reverse the adhesion of E. coli SS142 to Intestine 407 cells or human erythrocytes. Analysis of the kinetics of inhibition suggested that the antibodies did not competitively inhibit adhesion. Such antibodies can be useful for distinguishing different mechanistic steps of bacterial adhesion. Their ability to reverse bacterial adhesion in vitro may be of clinical relevance.
Changes in serum colony-stimulating factor and monocytic progenitor cells during Listeria monocytogenes infection in mice
Authors: Wing E J , Waheed A , Shadduck R K
Published: January 2020

The capacity of a host to produce and mobilize monocytes is an essential component of host defenses during the early phases of infection caused by Listeria monocytogenes. In this study, the concentrations of colony stimulating factor (CSF) and the numbers of monocyte progenitor cells (CFUm) were measured in mice during infection with L. monocytogenes. The concentration of CSF in serum increased sharply during the first 24 h of infection and remained elevated for the next 7 days. The number of CFUm in the bone marrow, however, decreased during the first 4 days after injection of L. monocytogenes. Thereafter, the number increased slowly, returning to normal on day 14. The decrease in marrow progenitor cells did not appear to result from a reduced sensitivity to CSF. In contrast to bone marrow changes, spleen progenitor cells increased greater than 400%, reaching a peak 7 days after bacterial challenge. These data indicate that monocyte production during L. monocytogenes infection is correlated with a rise in serum CSF concentration, depletion of bone marrow CFUm, and an increase in the number of spleen CFUm.
Immune responses of leishmaniasis patients to heat shock proteins of Leishmania species and humans
Authors: Skeiky Y A , Benson D R , Guderian J A , Whittle J A , Bacelar O , Carvalho E M , Reed S G
Published: January 2020

The course of human infection with Leishmania braziliensis is variable, ranging from self-healing infection to chronic disease. It is therefore a useful system in which to study immunoregulatory aspects of leishmaniasis, including the effects of parasite antigens on host responses. In the present study, we report on the cloning of, expression of, and comparative analyses of patient immune response to two different L. braziliensis genes homologous to the genes for the eukaryotic 83- and 70-kDa heat shock proteins. rLbhsp83 contains a potent T-cell epitope(s) which stimulated peripheral blood mononuclear cells (PBMC) from all L. braziliensis-infected individuals to proliferate and to produce interleukin-2 (IL-2) gamma interferon, and tumor necrosis factor alpha. The elicitation of IL-4 and IL-10 mRNAs was found to differ depending on the portion of the rLbhsp83 used to stimulate PBMC. rLbhsp83a, which represents the nearly full-length protein, stimulated IL-10 but not IL-4 mRNA. In contrast, a approximately 43-kDa protein representing the C-terminal region of Lbhsp83 stimulated the production of IL-4 but not IL-10 mRNA. rLbhsp70 stimulated PBMC proliferation from patients with mucosal disease but, unlike rLbhsp83, did not stimulate PBMC from self-healing individuals. PBMC from mucosal patients were not stimulated by rHuhsp70 to either proliferate or produce cytokines. This suggests that the hyperresponsiveness of mucosal patient PBMC to Leishmania heat shock proteins does not involve an auto-immune phenomenon resulting from cross-reactivity with self hsp70. In general, although the cytokine profile of patient PBMC in response to both of these Leishmania heat shock proteins represents a mixed Th1-Th2 pattern, the levels of gamma interferon and IL-2 were significantly higher than those of the Th2 cytokines IL-4 and IL-10. Patients with active mucosal and cutaneous disease but not self-healing individuals had significant anti-immunoglobulin G antibody titers to both rLbhsp83 and rLbhsp70 but not to the homologous rHuhsp70. It therefore appears that differential patient immune responses to Leishmania hsp83 and hsp70 may be of particular significance in the induction of protective immune responses as well as in the development of tissue damage in cases with particularly strong hypersensitive reactions.
Characterization of a human monoclonal immunoglobulin M (IgM) antibody (IgMBEN) specific for Vi capsular polysaccharide of Salmonella typhi
Authors: Liao J , Nickerson K G , Bystricky S , Robbins J B , Schneerson R , Szu S C , Kabat E A
Published: January 2020

A search for human monoclonal antibodies to protective antigens of bacteria revealed an immunoglobulin M lambda chain [IgM(lambda); designated IgMBEN] reactive with the Vi capsular polysaccharide of Salmonella typhi. Vi, a linear homopolymer of alpha(1-->4)GalApNAc that is O acetylated at C-3, is a licensed vaccine for typhoid fever. Immunologic properties of IgMBEN were compared to those of burro globulin prepared by intravenous injections of S. typhi (B339-340). IgMBEN and B339-340 yielded identical precipitin lines with Vi by double immunodiffusion. IgMBEN and B339-340 produced similar precipitation results with Vi and its derivatives prepared by de-O-acetylation, carboxyl reduction, and removal or replacement of the N-acetyl at C-2 with O-acetyl. B339-340 yielded maximal precipitation with Vi (0.41 mg of antibody per ml with 1.4 micrograms of Vi); next was carboxyl-reduced, O-acetylated Vi, which precipitated 0.325 mg of antibody per ml with 2.5 micrograms of Vi. IgMBEN yielded maximal precipitation with de-O-acetylated, carboxyl-reduced Vi (approximately 11.0 mg of antibody per ml with approximately 1.3 micrograms of antigen); next were de-O-acetylated Vi (9.89 mg/ml) and Vi (9.19 mg/ml). The precipitin curves and equivalence points of these three antigens were similar. Pneumococcus type 1, which contains GalApNAc, did not precipitate with Vi or its derivatives. These slight differences in specificity between IgMBEN and B339-340 were related to our proposed structure of Vi. We plan to use IgMBEN as a reference for measurement of vaccine-induced Vi antibodies.
Tumor necrosis factor alpha activity in genital tract secretions of guinea pigs infected with chlamydiae
Authors: Darville T , Laffoon K K , Kishen L R , Rank R G
Published: January 2020

Previous studies using the guinea pig model of chlamydial genital infection demonstrated that primary infection is associated with a marked acute inflammatory response early on, while chronic inflammation appears later, at a time when the level of infection is reduced. Challenge infections result primarily in a chronic inflammatory response. The stimuli that initiate inflammation and lead to tissue damage have not been defined. We investigated the possibility that tumor necrosis factors (TNFs) play a role in the inflammatory response to chlamydial genital tract infection. Cytotoxicity assays for TNF were performed on genital tract secretions collected from female guinea pigs during infection with the Chlamydia psittaci agent of guinea pig inclusion conjunctivitis. During the early days of primary infection, high levels of TNF-alpha were detected in genital tract secretions from inbred S2 strain and outbred Hartley strain guinea pigs. Significantly lower levels of TNF-alpha were detected in secretions from both strains during challenge infection. In general, the intensity of the TNF-alpha response was proportional to the intensity of infection. High TNF-alpha levels were present during primary infection at a time of marked neutrophil influx. Thus, TNF-alpha may play an important role in the response to primary chlamydial genital tract infection.
Role of C5a-ase in group B streptococcal resistance to opsonophagocytic killing
Authors: Takahashi S , Nagano Y , Nagano N , Hayashi O , Taguchi F , Okuwaki Y
Published: January 2020

Type III group B streptococci (GBS) can be subdivided into three subtypes, RDP III-1, III-2, and III-3, on the basis of numerical analysis of HindIII restriction endonuclease digestion patterns (HindIII RDP) with their chromosomal DNAs. In the present study, the effect of C5a on opsonophagocytic killing of a representative strain from each RDP type was investigated by using a novel optical method for determining opsonophagocytic killing, and the effect of C5a-ase treatment of C5a on opsonophagocytic killing was also investigated. Pre-stimulation of polymorphonuclear leukocytes (PMNs) with C5a significantly increased opsonophagocytic killing of all three strains. The increase in killing was abolished by pretreating the C5a with GBS that express C5a-ase, a treatment that also destroyed the chemoattractant activity of the C5a. The kinetics of killing of the RDP III-2 strain differed from those of the other two strains. The survival of the RDP III-2 bacteria continued to decline over the entire 60-min incubation of the opsonophagocytic assay when PMNs were prestimulated with C5a or with C5a that had been inactivated with GBS C5a-ase (dC5a). In contrast, killing of the RDP III-1 and III-3 strains almost ceased after 20 or 60 min when PMNs were prestimulated with dC5a or C5a, respectively. A difference in bacterial killing between the III-2 strain and the III-1 and III-3 strains therefore became increasingly apparent with prolonged incubation time. The percentage of bacteria surviving in the extracellular fluid was approximately the same as the percentages of bacteria surviving in both intracellular and extracellular locations when PMNs were prestimulated with either C5a or dC5a. These data imply that the majority of bacterial killing occurred following phagocytosis and suggest that the enhanced killing of GBS following prestimulation of PMNs with C5a resulted from increased ingestion of the bacteria.
Human lipopolysaccharide-binding protein potentiates bactericidal activity of human bactericidal/permeability-increasing protein
Authors: Horwitz A H , Williams R E , Nowakowski G
Published: January 2020

Human bactericidal/permeability-increasing protein (BPI) from neutrophils and a recombinant amino-terminal fragment, rBPI23, bind to and are cytotoxic for gram-negative bacteria both in vitro and ex vivo in plasma or whole blood. To function in vivo as an extracellular bactericidal agent, rBPI23 must act in the presence of the lipopolysaccharide-binding protein (LBP), which also binds to but has no reported cytotoxicity for gram-negative bacteria. LBP, which is present at 5 to 10 micrograms/ml in healthy humans and at much higher levels in septic patients, mediates proinflammatory host responses to gram-negative infection. On the basis of these previous observations, we have examined the effect of recombinant LBP (rLBP) on the bactericidal activity of rBPI23 against Escherichia coli J5 in vitro. Physiological concentrations of rLBP (5 to 20 micrograms/ml) had little or no bactericidal activity but reduced by up to approximately 10,000-fold the concentration of BPI required for bactericidal or related activities in assays which measure (i) cell viability as CFUs on solid media or growth in broth culture and (ii) protein synthesis following treatment with BPI. LBP also potentiated BPI-mediated permeabilization of the E. coli outer membrane to actinomycin D by about 100-fold but had no permeabilizing activity of its own. Under optimal conditions for potentiation, fewer than 100 BPI molecules were required to kill a single E. coli J5 bacterium.
Biological activity of toxic shock syndrome toxin 1 and a site-directed mutant, H135A, in a lipopolysaccharide-potentiated mouse lethality model
Authors: Stiles B G , Krakauer T , Bonventre P F
Published: January 2020

A recombinant of toxic shock syndrome toxin 1 (TSST-1) which contains a single histidine-to-alanine mutation at residue 135 (H135A) was analyzed for toxicity and vaccine potential in a lipopolysaccharide (LPS)-potentiated mouse lethality model. The 50% lethal dose (LD50) of TSST-1 in BALB/c mice was 47.2 micrograms/kg, but H135A was not lethal when tested at a dose equivalent to 10 LD50s of TSST-1. Levels of tumor necrosis factor (TNF) and gamma interferon (IFN-gamma) in serum were, respectively, 10- and 50-fold higher in LPS-potentiated mice injected with 15 LD50s of TSST-1 than in mice given H135A. Mice injected with only TSST-1 did not have elevated levels of TNF or IFN-gamma in serum, while H135A plus LPS or LPS alone elicited identical, yet very low, levels of TNF and IFN-gamma. An enzyme-linked immunosorbent assay of H135A and TSST-1 with anti-TSST-1 serum yielded very similar dose-response curves, which strongly suggests that H135A serologically and conformationally resembles the native toxin. Mice immunized with H135A developed antibodies that recognized TSST-1 in an enzyme-linked immunosorbent assay and afforded protection against a 15-LD50 challenge of TSST-1 plus LPS. The pooled sera of mice immunized with either TSST-1 or H135A also prevented lymphocyte proliferation due to TSST-1.
Human T-cell responses to secreted antigen fractions of Mycobacterium tuberculosis
Authors: Boesen H , Jensen B N , Wilcke T , Andersen P
Published: January 2020

The T-cell response of human donors to secreted antigen fractions of Mycobacterium tuberculosis was investigated. The donors were divided into five groups: active pulmonary tuberculosis (TB) patients with minimal and with advanced disease, Mycobacterium bovis BCG-vaccinated donors with and without contact with TB patients, and nonvaccinated individuals. We found that patients with active minimal TB responded powerfully to secreted antigens contained in a short-term culture filtrate. The response to secreted antigens was mediated by CD4+ Th-1-like lymphocytes, and the gamma interferon release by these cells was markedly higher in patients with active minimal TB than in healthy BCG-vaccinated donors. Patients with active advanced disease exhibited depressed responses to all preparations tested. The specificity of the response to secreted antigens was investigated by stimulating lymphocytes with narrow-molecular-mass fractions of short-term culture filtrate obtained by the multielution technique. Considerable heterogeneity was found within the donor groups. Patients with active minimal TB recognized multiple secreted targets, but interestingly, six of eight patients demonstrated a predominant recognition of a low-mass (< 10-kDa) protein fraction which induced high levels of gamma interferon release in vitro. Only a few of 12 previously characterized secreted antigens were recognized by T cells isolated from TB patients, suggesting the existence of a number of as yet undefined antigenic targets among secreted antigens.
Detection of muramic acid in a carbohydrate fraction of human spleen
Authors: Hoijer M A , Melief M J , van Helden-Meeuwsen C G , Eulderink F , Hazenberg M P
Published: January 2020

In previous studies, we showed that peptidoglycan polysaccharides from anaerobic bacteria normally present in the human gut induced severe chronic joint inflammation in rats. Our hypothesis is that peptidoglycan from the gut flora is involved in perpetuation of idiopathic inflammation. However, in the literature, the presence of peptidoglycan or subunits like muramyl peptides in blood or tissues is still a matter of debate. We were able to stain red pulp macrophages in all six available human spleens by immunohistochemical techniques using a monoclonal antibody against gut flora-derived antigens. Therefore, these human spleens were extracted, and after removal of most of the protein, the carbohydrate fraction was investigated for the presence of muramic acid, an amino sugar characteristic for peptidoglycan. Using three different methods for detection of muramic acid, we found a mean of 3.3 mumol of muramic acid with high-pressure liquid chromatography, 1.9 mumol with a colorimetric method for detection of lactate, and 0.8 mumol with an enzymatic method for detection of D-lactate per spleen (D-lactate is a specific group of the muramic acid molecule). It is concluded that peptidoglycan is present in human spleen not as small muramyl peptides as were previously searched for by other investigators but as larger macromolecules probably stored in spleen macrophages.
Use of tissue culture and animal models to identify virulence-associated traits of Haemophilus ducreyi
Authors: Alfa M J , Stevens M K , DeGagne P , Klesney-Tait J , Radolf J D , Hansen E J
Published: January 2020

To identify virulence-associated properties of Haemophilus ducreyi, 34 strains of this sexually transmitted pathogen were evaluated for in vitro phenotypic characteristics of potential relevance to chancroid pathogenesis and for their ability to produce lesions in the temperature-dependent animal model for chancroid. Of the 34 strains tested, all but three produced a cytopathic effect on human foreskin fibroblasts (HFF) and all but six strains formed large microcolonies on HFF monolayers. A subset of 12 selected strains underwent more extensive analyses and, when evaluated for both their cytadherence kinetics and growth in the presence of HFF monolayers, it was found that several of these strains had a very limited ability to attach to HFF cells. When the same 12 strains were tested in the temperature-dependent rabbit model, only the seven strains which were positive in all of these in vitro-based tests readily produced lesions. In contrast, the five strains that were noted to be deficient in one or more of the phenotypic characteristics scored in the in vitro systems did not produce lesions. This association between the traits measured in vitro and the ability to produce dermal lesions was significant (P = 0.0012). These results suggest that in vitro behavior may be used to predict the virulence potential of H. ducreyi strains. Moreover, the phenotypic characteristics described in this study are appropriate focal points for efforts to determine the molecular basis of the virulence of this pathogen.
Molecular analysis of neutralizing epitopes on outer surface proteins A and B of Borrelia burgdorferi
Authors: Ma J , Gingrich-Baker C , Franchi P M , Bulger P , Coughlin R T
Published: January 2020

The neutralizing epitopes of the major outer surface proteins A and B (OspA and OspB) of Borrelia burgdorferi B31 were investigated by epitope mapping using overlapping synthetic peptides, encompassing full-length OspA and OspB, and antiborrelial monoclonal antibodies (MAbs). OspA MAb N4B12 and OspB MAbs N5G5, W7C2, and P4D1 displayed a complement-independent antiborrelial activity, and complement failed to enhance the antiborrelial activity, as measured by a sensitive colorimetric assay. A combination of N4B12 with N5G5 displayed a higher antiborrelial activity than did the MAbs individually. OspA MAbs B3G11 and L3B5, however, exhibited a significant antiborrelial activity only in the presence of complement. Epitope mapping showed that B3G11 bound to one OspA synthetic peptide with the sequence of amino acids 247 to 256 (QYDSNGTKLE) and produced more than sixfold-higher reactivity than with other sequences, as measured by an enzyme-linked immunosorbent assay. OspB MAb N5G5 bound to an OspB peptide with the sequence of amino acids 211 to 220 (TLKREIEKDG), yielding at least threefold-higher reactivity than with other sequences. These two peptide sequences were found to contain neutralizing epitopes. Other MAbs had weak binding activities with the synthetic peptides, and their specific epitopes remain to be further analyzed. Thus, this study demonstrated both complement-independent and complement-dependent antiborrelial MAbs and identified the linear epitopes on OspA and OspB capable of inducing neutralizing antibody responses.