Soluble antigens from group B streptococci induce cytokine production in human blood cultures

Group B streptococcal antigens stimulated tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), and IL-6 production in human blood cultures in a concentration- and time-dependent fashion. The minimal concentrations of type-specific polysaccharides, lipoteichoic acid, and group-specific polysaccharide required to produce these effects were, respectively, 0.01, 1, and 10 microg/ml. Cell separation experiments indicated that monocytes were the cell type mainly responsible for cytokine production. Time course studies indicated that TNF-alpha was released before the other cytokines. TNF-alpha, however, did not appear to directly induce IL-1beta, as shown by blockade experiments with anti-TNF-alpha antibodies. IL-6 levels were moderately but significantly decreased by anti-TNF-alpha. These data indicate that several products from group B streptococci are able to directly stimulate human monocytes to release TNF-alpha, IL-1beta, and IL-6. These findings may be clinically relevant, since proinflammatory cytokines can mediate pathophysiologic changes during sepsis.     

The induction and expression of IgA anti-DNP antibodies in rat bile and secretions

Pregnant rats were immunized with dinitrophenylated type III-pneumococcal vaccine by the intravenous, gastric, or intramammary routes. Anti-DNP antibody responses in the IgA, IgG and IgM isotypes were measured in serum, secretions and bile. Gastric intubation was most effective at eliciting IgA antibody responses in bile and secretions, whereas the other routes were more effective at inducing IgG responses in serum and bile. IgM antibody responses were infrequent and were found in fluids most closely associated with the immunization route. Isoelectric focusing studies of IgA antibodies appearing in secretions and bile revealed that the gastric route consistently elicited antibody spectrotypes with shared components. Intravenous and intramammary immunization resulted in IgA spectrotypes possessing less homology, suggesting that these protocols lead to independent antibody responses triggered in spleen, draining lymph nodes, or secretory sites. After gastric stimulation, the appearance of IgA antibodies with identical spectral components in secretions and bile favours the concept that IgA precursor cells with identical clonotype potential migrate from the gastrointestinal area to secretory sites. Antibody expression in bile appears to result from the selective transfer of IgA populations gaining access to serum after synthesis at a secretory site.     

Deletion of repeats in the alpha C protein enhances the pathogenicity of group B streptococci in immune mice

The alpha C protein is a protective surface-associated antigen of group B streptococci (GBS). The prototype alpha C protein of GBS (strain A909) contains nine identical tandem repeats, each comprising 82 amino acids, flanked by N- and C-terminal domains. Clinical isolates of GBS show variable numbers of repeats with a normal distribution and a median of 9 to 10 repeats. Here, we show that escape mutants of GBS expressing one-repeat alpha C protein were 100-fold more pathogenic than GBS expressing wild-type nine-repeat alpha C protein in neonatal mice whose dams were immunized with antiserum elicited to nine-repeat alpha C protein (50% lethal doses of 1.6 x 10(3) and 1.8 x 10(5), respectively; P = 0.0073). There was no difference in pathogenicity in nonimmune mice. Enzyme-linked immunosorbent assay inhibition showed that nine-repeat but not one-repeat alpha C protein is readily available for antibody binding on the surface of intact GBS. Immune electron microscopy studies with antibodies to the capsular polysaccharide (CPS) and to the alpha C protein demonstrated localization of the nine-repeat alpha C protein and the CPS at similar distances from the cell wall. The one-repeat alpha C protein was visualized poorly and only in close proximity to the cell wall, thus suggesting that antibody binding to the protein was hindered by CPS or other cell surface components. We concluded that deletion in the repeat region of the alpha C protein enhanced the pathogenicity of GBS in immune mice by (i) loss of a protective (conformational) epitope(s) and (ii) loss of antibody binding to the alpha C protein due to a decrease in antigen size relative to cell wall components and/or CPS.     

Decreased capacity for type-specific-antigen synthesis accounts for high prevalence of nontypeable strains of group B streptococci in Mexico

The low incidence of group B streptococcal (GBS) invasive neonatal disease in Mexico has been attributed to the low prevalence of serotype III strains, a major serotype in developed countries. In addition, nontypeable strains account for 12% of the isolates in Mexico and < 1% of the isolates in the United States. In this study, 57 GBS isolates (28 nontypeable by the Lancefield procedure) from carrier and infected neonates and women from Mexico were also examined for the presence of type-specific antigen by an enzymatic procedure using N-acetylmuramidase digestion of the cell wall to release soluble type-specific antigen. Of the 28 nontypeable strains from Mexico, 23 were typeable by the enzyme extraction procedure, with serotype III being the predominant serotype in invasive disease. These results suggest that nontypeable isolates of GBS should be further examined by the enzymatic extraction procedure to determine the presence of type-specific antigen. Furthermore, these limited results suggest that serotype III is likely a major serotype in invasive disease also in Mexico.     

Colonization with group B streptococci in neonates and premature infants from selected neonatal departments

Group B streptococci are considered an important etiological agent of sepsis and meningitis in neonates and, particularly, in premature infants. There is a close correlation between colonization with these bacteria and the frequency of symptomatic infection. It is estimated that symptomatic infections occur in 1.0% of colonised neonates. The purpose of this work was to investigate the frequency of neonate colonization with group B streptococci for determination of the risk of symptomatic infection.     

Virus-neutralizing antibodies of immunoglobulin G (IgG) but not of IgM or IgA isotypes can cure influenza virus pneumonia in SCID mice

The ability of monoclonal antibodies (MAbs) to passively cure an influenza virus pneumonia in the absence of endogenous T- and B-cell responses was investigated by treating C.B-17 mice, homozygous for the severe combined immunodeficiency (SCID) mutation, with individual monoclonal antiviral antibodies 1 day after pulmonary infection with influenza virus PR8 [A/PR/8/34 (H1N1)]. Less than 10% of untreated SCID mice survived the infection. By contrast, 100% of infected SCID mice that had been treated with a single intraperitoneal inoculation of at least 175 micrograms of a pool of virus-neutralizing (VN+) antihemagglutinin (anti-HA) MAbs survived, even if antibody treatment was delayed up to 7 days after infection. The use of individual MAbs showed that recovery could be achieved by VN+ anti-HA MAbs of the immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgG3 isotypes but not by VN+ anti-HA MAbs of the IgA and IgM isotypes, even if the latter were used in a chronic treatment protocol to compensate for their shorter half-lives in vivo. Both IgA and IgM, although ineffective therapeutically, protected against infection when given prophylactically, i.e., before exposure to virus. An Fc gamma-specific effector mechanism was not an absolute requirement for antibody-mediated recovery, as F(ab')2 preparations of IgGs could cure the disease, although with lesser efficacy, than intact IgG. An anti-M2 MAb of the IgG1 isotype, which was VN- but bound well to infected cells and inhibited virus growth in vitro, failed to cure. These observations are consistent with the idea that MAbs of the IgG isotype cure the disease by neutralizing all progeny virus until all productively infected host cells have died. VN+ MAbs of the IgA and IgM isotypes may be ineffective therapeutically because they do not have sufficient access to all tissue sites in which virus is produced during influenza virus pneumonia.     

Cytologic diagnosis of Chlamydia in cervicovaginal secretions. Use of a Papanicolaou stain modification with buffered wright solution

OBJECTIVE: To carry out a prospective study on 1,569 females to evaluate the diagnostic utility of adding buffered Wright solution to Papanicolaou stain for observing cytoplasmic inclusions in cervicovaginal cytology. STUDY DESIGN: Group A had multivacuolated cells and group B, granules (not in a vacuole) in the cytoplasm of low-intermediate epithelial or parabasal cells. There were 7 patients in group A and 16 in group B. Two duplicate plates of cervicovaginal secretions were obtained from each patient before and after treatment; one was stained with Papanicolaou stain and the other with our variant. The trial of therapy consisted of doxycycline (effectiveness for statistical analysis = 0.9) in oral doses of 100 mg every 12 hours for 7 days; 7 days after the beginning of treatment, check samples were obtained. RESULTS: In group A, six patients had cytoplasmic inclusions with the variant stain, and two had cytoplasmic inclusions with Papanicolaou stain. The binomial test revealed that probably six of the seven patients had infections with Chlamydia (P = .372) and that in 100% of cases, the variant stain showed cytoplasmic inclusions, while Papanicolaou stain was observed in 33% of cases. These findings were morphologically and statistically proven (P = .124) on control slides with posttreatment absence of multivacuolated cells. CONCLUSION: In relation to the selection criteria for B group, the intracytoplasmic granules found in parabasal and low intermediate cells had no relation to Chlamydia. The study demonstrated the superiority of our variant of Papanicolaou stain for cervicovaginal Chlamydia diagnosis.     

Distribution of monoclonal antibodies in intestinal and urogenital secretions of mice bearing hybridoma ''backpack'' tumours

Primary Epstein-Barr virus (EBV) infection may manifest itself as a benign lymphoproliferative disorder, infections mononucleosis (IM). EBV infection has been characterized in lymphoreticular tissues from nine patients with IM using the abundantly expressed EBV-encoded nuclear RNAs (EBERs) as a marker of latent infection. Expression of the virus-encoded nuclear antigen (EBNA) 2 and of the latent membrane protein (LMP) 1 was seen in variable proportions of cells in all cases. Double labelling revealed heterogeneous expression patterns of these proteins. Thus, in addition to cells revealing phenotypes consistent with latencies I (EBNA2-/LMP1-) and III (EBNA2+/LMP1+), cells displaying a latency II pattern (EBNA2-/LMP1+) were observed. Cells expressing EBNA2 but not LMP1 were also detected; whilst this may represent a transitory phenomenon, the exact significance of this observation is at present uncertain. EBER-specific in situ hybridization in conjunction with immunohistochemistry revealed expression of the EBERs mainly in B-lymphocytes, many of which showed features of plasma cell differentiation. By contrast, convincing evidence of latent EBV infection was not found in T-cells, epithelial or endothelial cells. Double-labelling immunohistochemistry revealed expression of the replication-associated BZLF1 protein in small lymphoid cells, often showing plasmacytoid differentiation. There was no unambiguous expression of this protein in other cell types. These results suggest that B-cells are the primary target of EBV infection and that plasma cells may be a source of infectious virus found in the saliva of IM patients.     

CpG DNA can induce strong Th1 humoral and cell-mediated immune responses against hepatitis B surface antigen in young mice

Successful neonatal immunization of humans has proven difficult. We have evaluated CpG-containing oligonucleotides as an adjuvant for immunization of young mice (1-14 days old) against hepatitis B virus surface antigen. The protein-alum-CpG formulation, like the DNA vaccine, produced seroconversion of the majority of mice immunized at 3 or 7 days of age, compared with 0-10% with the protein-alum or protein-CpG formulations. All animals, from neonates to adults, immunized with the protein-alum vaccine exhibited strong T helper (Th)2-like responses [predominantly IgG1, weak or absent cytotoxic T lymphocytes (CTL)]. Th2-type responses also were induced in young mice with protein-CpG (in 1-, 3-, and 7-day-old mice) and protein-alum-CpG (in 1- and 3-day-old mice) but immunization carried out at older ages gave mixed Th1/Th2 (Th0) responses. DNA vaccines gave Th0-like responses when administered at 1 and 7 days of age and Th1-like (predominantly IgG2a and CTL) responses with 14-day-old or adult mice. Surprisingly, the protein-alum-CpG formulation was better than the DNA vaccine for percentage of seroconversion, speed of appearance, and peak titer of the antibody response, as well as prevalence and strength of CTL. These findings may have important implications for immunization of human infants.     

Change in susceptibility of group B streptococci to penicillin G from 1968 through 1975

This report describes a study of 212 isolates of group B streptococci from sore throats over an 8-year period. A small but increasing percentage showed increased resistance to penicillin G when tested in an in vitro system.     

Incidence, technique of isolation, and treatment of group B streptococci in obstetric patients

A prospective study was performed to determine the incidence of group B streptococci cultured from the cervix in a series of obstetric clinic patients at St. Margaret's Hospital. Cultures were taken during each trimester and planted to both a blood agar plate and a selective broth medium. Of a total of 812 cultures, 18 were positive, an over-all incidence of 2.2 per cent. During the first, second, and third trimesters, 2.2 per cent, 2.5 per cent, and 1.9 per cent of cultures were positive, respectively. Women with positive cultures were treated with a 10 day course of oral penicillin or a suitable alternative; four of 16 had persistent positive cultures at the end of the first treatment period, three of these four responded to a second course of antibiotics, and the remaining patient responded after a third treatment course. The total number of positive cultures by selective broth was 24, compared to nine in blood agar, indicating a 2.7-fold increase in pickup by the selective broth.     

Detection of carriers of group B streptococci in pregnant women with absorbed charcoal culture. Streptococcal Diseases Study Group

For prevention of group B streptococcal (GBS) infection in neonates, GBS carriers of pregnant women should be detected. Procedures for detection of carriers were needed to get higher positive rate and to be handled with easy. Urine samples were shown to detect higher rates than vaginal smear samples in pregnant women. GBS in the urine were adsorbed to charcoal granula for easier transporting. These adsorbed charcoal were tested as to whether they could be preserved with different temperatures and days. Urine with 10(1) CFU/ml of GBS added could be detected after 7 days at temperatures of 4, 25, 37, 42 degrees C. The cultures of this procedure with the cultures of urine sediment of pregnant women were compared. Detecting rates of this procedure and urine sediment were 177 (26.2%) and 136 (20.1%) of 676 pregnant women, respectively. These data suggested that adsorbed charcoal culture was useful with higher positive rates for detection of the GBS carriers in pregnant women.     

Use of a DNA probe for the rapid detection of group B streptococci in obstetric patients

OBJECTIVE: To determine the accuracy of a DNA probe as a rapid diagnostic test for detecting colonization of the female genital tract by group B streptococci during pregnancy. METHODS: Two rayon-tipped applicators were used to collect secretions from the posterior vaginal wall of 440 pregnant women. One of the applicators was inoculated into selective Todd-Hewitt broth and used as the reference standard for identification of group B streptococci. The other applicator was used for analysis with the DNA probe, preceded by either 2.5 hours of incubation for the initial 75 patients, or 3.5 hours' incubation for the remaining women. Following hybridization with an acridinium-labeled probe, chemiluminescence was measured with a luminometer. RESULTS: The prevalence of positive cultures was 20%. For the initial 75 patients whose cultures were amplified by incubation for 2.5 hours, the DNA probe had a sensitivity of 44%, specificity 94%, positive predictive value 79%, and negative predictive value 77%. For the cultures that were incubated for 3.5 hours, respective values were 71, 90, 61, and 94%. All vaginal specimens that had an average initial cell count of 1.5 x 10(3) cells/mL were accurately detected by the probe after 3.5 hours' growth amplification. False-positive results occurred primarily when the specimens were grossly contaminated with blood (26 of 39). The mean time required to perform the assay, including 3.5 hours of growth amplification, was 4.3 hours. CONCLUSIONS: The DNA probe demonstrated good overall sensitivity and gave no false-negative results when group B streptococci were present in concentrations of 1 x 10(4) cells/mL or greater. Sensitivity improved significantly with 3.5 hours' growth amplification as compared with 2.5 hours (P < .05), reflecting better identification of lightly colonized patients.     

Production of sequence specific polyclonal antibodies to human parathyroid hormone 1-37 by immunization with multiple antigenic peptides

To generate site-specific antibodies to the N-terminal bioactive fragment of the parathyroid hormone hPTH 1-37, multiple antigenic peptide systems (MAP) for immunization were used. Two 10 residue fragments and a 14 residue fragment derived from knowledge of the secondary structure of hPTH 1-37 were selected to be synthesized as MAPs. Each peptide (hPTH 1-10, hPTH 9-18, and hPTH 24-37) was synthesized directly onto a branching heptalysine core matrix by automated solid phase synthesis. The hPTH 1-10 and the hPTH 24-37 MAP were highly immunogenic in rabbits. Ten polyclonal antisera obtained from rabbits were characterized by epitope mapping. Antigenic determinants were found as follows: 1) Sera K1-K3 raised to MAP 1-10 showed a predominant binding sequence at hPTH 1-5. 2) Sera K4-K6 raised to MAP 8-18 preferentially bound to residues 9-14. 3) Immunizing with hPTH 24-37 MAP led to antisera characterized as follows: serum K7 recognized residues 24-37, the sequence used for immunization, sera K8, K9 and K10 bound to residues 24-37 and 26-34. In summary, the favoured regions as deduced from the secondary structure of hPTH 1-37 were covered by the produced antibodies.     

Synergistic effects of human B and T lymphocyte mitogens induce uniform, high levels of immunoglobulin secretion among normal donors

Synergistic effects of B-cell mitogen Staphylococcus bacteria strain Cowan I (Cowan I) plus T-cell activator pokeweed mitogen (PWM) in generating immunoglobulin-secreting cells (ISC) from human peripheral blood mononuclear cells (MNC) were investigated. ISC were assayed by reverse plaque-forming cells uing protein A-coated red blood cells. Low concentrations of PWM plus Cowan I gave superadditive effects on ISI induction, generating 3-20 times as many ISC as optimal amounts of either mitogen alone. The mitogens together and separately showed similar kinetics of ISC; synergy was observed at every day tested. IgM ISC represented 10% of initial MNC from all cell donors tested even if the donors were low responders to either mitogen alone. The numbers of ISC obtained are higher than previous reports and more uniform among donors, making this a superlative method for studies on normal human immunoglobulin secretion.     

IgA class reticulin antibodies in relatives of patients with coeliac disease

IgA class reticulin antibodies were not found in patients with Crohn's disease, ulcerative colitis, and hiatus hernia despite a significant incidence of IgG class reticulin antibodies. None of 56 normal healthy subjects was positive. In contrast, 13 (76%) of the sera from 17 patients with coeliac disease on normal diet were positive for IgA class antibodies as were 19 (20%) of 93 first degree relatives. Seventy-three relatives underwent jejunal biopsy. Grade III (flat) histology was found in 13 and, of these patients, 10 (77%) showed IgA class reticulin antibody in their serum. It is suggested that determination of IgA class reticulin antibodies was a useful test to determine which relative must be biopsied.     

Risk of preterm delivery in pregnant women with group B streptococcal urinary infections or urinary antibodies to group B streptococcal and E. coli antigens

OBJECTIVE: To establish whether there is an association between preterm delivery and either group B streptococcal urinary infection or the presence of urinary antibodies to group B streptococcal or E. coli antigens. DESIGN: A prospective study with urine culture and antibody measurement performed at the first antenatal visit and at 28 weeks gestation. SETTING: Ninewells Hospital, Dundee. SUBJECTS: Two thousand and forty-three women registering consecutively at an antenatal clinic. MAIN OUTCOME MEASURE: Delivery at less than 37 weeks gestation. RESULTS: No increase in preterm delivery was observed in women with positive urine cultures for group B streptococci either at booking or at 28 weeks, even when confirmed by positive repeat cultures. Preterm delivery was more common in women with elevated urinary antibodies to E. coli antigens at booking (relative risk 1.81, 95% CI 1.22-2.68, P = 0.005) and at 28 weeks (relative risk 2.36, 95% CI 1.60-3.48, P < 0.0001) and to group B streptococcal antigens at 28 weeks (relative risk 2.24, 95% CI 1.46-3.43, P = 0.0003). CONCLUSIONS: These data do not support previous reports that positive urine cultures for group B streptococci are associated with an increased risk of preterm delivery. Our report of an association between elevated levels of urinary antibodies and preterm delivery is a new finding consistent with the possibility that a local inflammatory response to uro-genital infection may be important in stimulating the onset of preterm labour. The results suggest that screening for urinary antibodies at 28 weeks gestation might help to identify a group of women at increased risk of prematurity.     

Comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women

To determine which mucosal immunization routes may be optimal for induction of antibodies in the rectum and female genital tract, groups of women were immunized a total of three times either orally, rectally, or vaginally with a cholera vaccine containing killed Vibrio cholerae cells and the recombinant cholera toxin B (CTB) subunit. Systemic and mucosal antibody responses were assessed at 2-week intervals by quantitation of CTB-specific antibodies in serum and in secretions collected directly from mucosal surfaces of the oral cavity, rectum, cervix, and vagina with absorbent wicks. The three immunization routes increased levels of specific immunoglobulin G (IgG) in serum and specific IgA in saliva to similar extents. Rectal immunization was superior to other routes for inducing high levels of specific IgA and IgG in rectal secretions but was least effective for generating antibodies in female genital tract secretions. Only vaginal immunization significantly increased both specific IgA and specific IgG in both the cervix and the vagina. In addition, local production of CTB-specific IgG in the genital tract could be demonstrated only in vaginally immunized women. Vaginal immunization did not generate antibodies in the rectum, however. Thus, generation of optimal immune responses to sexually transmitted organisms in both the rectal and the genital mucosae of women may require local immunization at both of these sites.     

SXT and Taxo A disks for presumptive identification of group A and B streptococci in throat cultures

A bacitracin (0.04 units, BBL)-SXT (trimethoprim, 1.25 mg, plus sulfamethoxazole, 23.75 mg, BBL) susceptibility test was 94% accurate in presumptively identifying streptococci as either group A, B or not group A and B.