The adjuvant LT-K63 can restore delayed maturation of follicular dendritic cells and poor persistence of both protein- and polysaccharide-specific antibody-secreting cells in neonatal mice.

Ab responses in early life are low and short-lived; therefore, induction of protective immunity requires repeated vaccinations. One of the major limitations in early-life immunity is delayed maturation of follicular dendritic cells (FDCs), which play a central role in mediating the germinal center (GC) reaction leading to production of Ab-secreting cells (AbSCs). We assessed whether a nontoxic mutant of Escherichia coli heat-labile enterotoxin (LT-K63) and CpG1826 as model adjuvants could accelerate FDC maturation and immune response in neonatal mice, using a pneumococcal polysaccharide of serotype 1 conjugated to tetanus toxoid (Pnc1-TT) as a model vaccine. In neonatal NMRI mice, a single dose of Pnc1-TT coadministered with LT-K63 enhanced Pnc1-TT-induced GC reaction. In contrast, CpG1826 had no effect. Accordingly, LT-K63, but not CpG1826, accelerated the maturation of FDC networks, detected by FDC-M2(+) staining, characteristic for adult-like FDCs. This coincided with migration of MOMA-1(+) macrophages into the GCs that can enhance GC reaction and B cell activation. The FDC-M2(+) FDC networks colocalized with enhanced expression of TNF-α, which is critical for the maintenance of mature FDCs and is poorly expressed in neonates. The accelerated maturation of FDC networks correlated with increased frequency and prolonged persistence of polysaccharide- and protein-specific IgG(+) AbSCs in spleen and bone marrow. Our data show for the first time, to our knowledge, that an adjuvant (LT-K63) can overcome delayed maturation of FDCs in neonates, enhance the GC reaction, and prolong the persistence of vaccine-specific AbSCs in the BM. These properties are attractive for parenteral vaccination in early life.

Effect of IL-15 on IgG versus IgE antibody-secreting cells in vitro.

Immunoglobulin E (IgE) antibodies are major contributors to the pathology of atopic and allergic diseases as well as to immune response to helminth infections. Development of an adequate immunoglobulin G (IgG) immune response against infectious agents and vaccine antigens is considered in most cases as crucial for protection from disease. In vivo and in vitro production of IgE and IgG depends on cytokines and other soluble factors. Recently it has been shown that IgG antibody secreting cells (ASCs) can be generated by in vitro maturation of blood cells with Interleukin- (IL-)15 and CpG DNA or other stimulation cocktails, while IgE-ASCs develop upon cultivation with anti-CD40 and IL-4. In the present study we employed an enzyme linked immunospot assay (ELISPOT) to assess the capacity of individuals to develop into either IgE-ASCs or IgG-ASCs upon stimulation with different combinations of stimulation cocktails in order to investigate the influence of cytokines that are dysregulated in IgE-mediated immune reactions on ASC generation. Furthermore, we modified the method to assess IgG- and IgE-ASCs specific for two model antigens causing allergic rhinitis in humans. We demonstrate that IL-15, which is important for development of IgG-ASCs, decreases the number of IgE-ASCs when added to media commonly used for in vitro development of IgE-ASCs. We show that our method is suitable for the detection of specific and non-specific IgE-ASCs and IgG-ASCs and allows the investigation of the interplay between IgG-ASCs and IgE-ASCs in different populations.

Decreased CD4+CD25+ cells and increased dimeric IgA-producing cells in tonsils in IgA nephropathy.

BACKGROUND: The relationship between tonsillar autoimmune response and the pathogenesis of IgA nephropathy (IgAN) has been previously demonstrated. However, the role of CD4+CD25+ cells, which play critical roles in maintaining peripheral tolerance and preventing autoimmunity, has not yet been defined in IgAN. METHODS: The lymphocytes from tonsils of all subjects (including 37 IgAN cases and 37 controls without renal diseases) were cultured for 72 hours without stimulation, or with stimulation by alpha-hemolytic streptococcus (HS) isolated from the tonsillar crypts of cases (the HS-IgAN) or controls (HS-controls). The CD4+CD25+ cells were measured by flow cytometry. Expression of J chain mRNA was analyzed by in situ hybridization (ISH) and the dimeric IgA-producing cells were identified by immunofluorescence and fluorescent ISH. RESULTS: The number of CD4+CD25+ cells was significantly lower in cases than in controls (0.98% +/- 0.204% vs. 3.58% +/- 0.554%, 1.37% +/- 0.214% vs. 5.78% +/- 0.562%, and 1.43% +/- 0.202% vs. 6.05% +/- 0.521%, for nonstimulation, HS-controls and HS-cases, respectively). CD4+CD25+ cells from cases showed a significantly lower stimulation index (SI) when stimulated with HS-controls and HS-IgAN than controls (p<0.05), whereas the number of dimeric IgA-producing cells was significantly higher in cases than controls (11.9% +/- 3.1% vs. 6.5% +/- 1.5%, 33.5% +/- 5.7% vs. 13.9% +/- 1.2%, and 35.1% +/- 6.2% vs. 13.9% +/- 1.2%, for nonstimulation, HS-controls and HS-cases, respectively). The dimeric IgA-producing cells from patients with IgAN showed a significantly higher SI when stimulated with HS-controls, or HS-IgAN than those from patients without renal disease (p<0.01). The SI of CD4+CD25+ cells was negatively correlated with that of dimeric IgA-producing cells. CONCLUSION: The results suggest that CD4+CD25+ cells and dimeric IgA-producing cells in tonsils may be related to the pathogenesis of IgAN.

Effect of probiotics and breastfeeding on the bifidobacterium and lactobacillus/enterococcus microbiota and humoral immune responses.

OBJECTIVE: To assess impact of probiotics and breastfeeding on gut microecology. STUDY DESIGN: Mothers were randomized to receive placebo or Lactobacillus rhamnosus GG before delivery, with treatment of the infants after delivery. We assessed gut microbiota, humoral immune responses, and measured soluble cluster of differentiation 14 (sCD14) in colostrum in 96 infants. RESULTS: Fecal Bifidobacterium and Lactobacillus/Enterococcus counts were higher in breastfed than formula-fed infants at 6 months; P <.0001 and P=.01, respectively. At 3 months, total number of immunoglobulin (Ig)G-secreting cells in breastfed infants supplemented with probiotics exceeded those in breastfed infants receiving placebo; P=.05, and their number correlated with concentration of sCD14 in colostrum. Total numbers of IgM-, IgA-, and IgG-secreting cells at 12 months were higher in infants breastfed exclusively for at least for 3 months and supplemented with probiotics as compared with breastfed infants receiving placebo; P=.005, P=.03 and P=.04, respectively. Again, sCD14 in colostrum correlated with numbers of IgM and IgA cells; P=.05 in both. CONCLUSIONS: We found an interaction between probiotics and breastfeeding on number of Ig-secreting cells, suggesting that probiotics during breastfeeding may positively influence gut immunity.

Immunogenicity of intranasally administered meningococcal native outer membrane vesicles in mice.

Colonization of the human nasopharyngeal region by Neisseria meningitidis is believed to lead to natural immunity. Although the presence of bactericidal antibody in serum has been correlated with immunity to meningococcal disease, mucosal immunity at the portal of entry may also play an important role. This study was undertaken to examine in mice the possibility of safely using native outer membrane vesicles (NOMV) not exposed to detergent as an intranasal (i.n.) vaccine. The mucosal and systemic responses of mice to intranasal and intraperitoneal (i.p.) vaccination with NOMV were compared over a range of doses from 0.1 to 20 microgram. Intranasal vaccination of mice with NOMV induced a strong systemic bactericidal antibody response, as well as a strong local immunoglobulin A immune response in the lung as determined by assay of lung lavage fluid by enzyme-linked immunosorbent assay and lung antibody secreting cells by enzyme-linked immunospot assay. However, 8- to 10-fold-higher doses of NOMV were required i.n. compared to i.p. to elicit an equivalent bactericidal antibody response in serum. Some NOMV vaccine was aspirated into the lungs of mice during i.n. immunization and resulted in an acute inflammatory response that peaked at 1 to 2 days postimmunization and was cleared by day 7. These results indicate that i.n. delivery of meningococcal NOMV in mice is highly effective in eliciting the production of both a mucosal immune response and a systemic bactericidal antibody response.

Intrathecal synthesis of immunoglobulin G and Mycobacterium tuberculosis-specific humoral immune response in tuberculous meningitis.

Local synthesis of immunoglobulin G (IgG) in the central nervous system was investigated in 10 patients with tuberculous meningitis (TBM), 15 patients with aseptic meningitis (AM), and 15 patients with pulmonary tuberculosis only (PTBO). The IgG synthesis rate for patients with TBM was 56.4 +/- 18.9 mg/day (mean +/- standard deviation), which was significantly higher than that for patients with AM (8.0 +/- 6.7 mg/day, P < 0.001) and that for patients with PTBO (7.5 +/- 4.4 mg/day, P < 0.001). Therefore, the increased IgG synthesis rate in the central nervous system provided supporting evidence for differentiating the diagnosis of TBM from that of AM (sensitivity, 100%; specificity, 83.3%). Simultaneous measurement by enzyme-linked immunosorbent assay of IgG seroreactivity to lipoarabinomannan and purified protein derivative antigens in cerebrospinal fluid (CSF) demonstrated seropositivity in all 6 patients with TBM, 4 of 15 patients with AM, and 4 of 10 patients with PBTO. All patients showing false-positive reactivity in CSF demonstrated seropositivity in sera and normal ranges for IgG synthesis rates in CSF. Also, the semiquantitive measurement of IgG antibody (Ab) titers in these patients demonstrated higher IgG Ab titers in serum than in CSF except for one patient with a highly elevated albumin quotient, suggesting a leaky blood-brain barrier. The results strongly suggested that the Mycobacterium tuberculosis-specific IgG Abs were diffusible through the blood-brain barrier, which addresses the pitfall of serological tests for the early diagnosis of TBM. The serological detection of IgG Abs to lipoarabinomannan and purified protein derivative antigens in CSF could be misleading in the presence of simultaneously elevated of IgG Abs in serum.

Anticuerpos contra productos extracelulares del estreptococo grupo A: importancia diagnóstica en la fibre reumática aguda / Antibodies against extracellular products of group a streptococcus. its diagnostic value in acute rheumatic fever

La infección estreptocóccica de la faringe es condición sine qua non el desarrollo de la fiebre reumática (FR), La demostración de esa infección suele requerir métodos inmunoserológicos que detectan anticuerpos contra productos extracelulares del estreptococo (PEE). Se evaluó la respuesta inmune humoral contra PEE en niños y adultos con y sin diagnóstico de FR. Se estableció que la distribución de valores para anti-estreptolisina O (AEL-O) no es gaussiana y que el valor de referencia debe manejarse como percentila. En adultos la percentila 97 es 227, en niños la percentila 90 es 451. En caso de FR, todos los enfermos excepto uno tuvo valores superiores. Una prueba de aglutinación que reconoce otros anticuerpos a PEE (Estreptozima mr), mostró en menores de 15 años que 15/28 los tenían a título bajo, en cambio en el grupo con FR sólo 1 mostró ausencia de esos anticuerpos. A mayor título de AEL-O mayor título en Estreptozima. Los métodos probados son eficientes para reconocer la respuesta inmune humoral contra PEE

Mecanismos inmunológicos en las enfermedades infecciosas / Immunologycal mecanisms of infectious diseases

Los mecanismos de inmunidad contra las infecciones bacterianas, parasitarias y virales son extremadamente complejos y tienen como principal función la eliminación de los microorganismos patógenos. Los sistemas de defensa no específicos contra las bacterias, son proporcionados por los granulocitos, que ingieren y matan a la mayoría de los patógenos. Sin embargo, se necesita de la inmunidad específica contra bacterias encapsuladas o intracelulares y contra parásitos y virus para eliminar a éstos, lo cual requiere del desarrollo de anticuerpos a través de la inmunidad humoral y de la inmunidad celular que puede desencadenar la actividad microbicida de los macrófagos. En muchas infecciones humanas, todos los mecanismos inmunológicos (anticuerpos, complemento, linfocitos, granulocitos y macrófagos) son esenciales para desarrollar una inmunidad protectora contra muchos microorganismos patógenos

The suppressive effect of deoxyspergualin on the differentiation of human B lymphocytes maturing into immunoglobulin-producing cells.

Deoxyspergualin, an analog of spergualin, has been known as a novel immunosuppressive agent with strong immunosuppressive activity in in vivo experimental systems. In the present study, we examined the effect of deoxyspergualin (DSG) and methyldeoxyspergualin (MeDSG) on the proliferation and differentiation of human B lymphocytes in vitro. Highly purified B cells from human tonsil samples were isolated by Percoll density gradient from nonrosetted cells and were used as target cells. Both agents had little effect on the proliferative response of resting or activated B lymphocytes. However, they suppressed the immunoglobulin synthesis of B lymphocytes not only in a T cell-dependent, but also in a T cell-independent system. The inhibition of antibody synthesis was manifested in the early stage of B cell differentiation. Both drugs also suppressed Ig secretion, but not proliferation, of an EBV-transformed human B lymphoblastoid cell line. These results indicate that DSG and MeDSG have a selective immunosuppressive effect on the differentiation pathway of B lymphocytes.

VP4-specific intestinal antibody response to rotavirus in a murine model of heterotypic infection.

We have adapted a murine model of heterotypic rotavirus infection for the purpose of evaluating the intestinal antibody response to an infection that mimics human vaccination. Neonatal mice were infected with the rhesus rotavirus (RRV). The enzyme-linked immunospot assay was used in order to avoid common artifacts in the quantitation of intestinal immune responses inherent in measurements of luminal or serum immunoglobulins and to obtain easily quantifiable data in a flexible and convenient format. Functionally active lymphocytes were harvested from the spleen, small intestinal lamina propria, Peyer's patches, and mesenteric lymph nodes and processed into single-cell suspensions. Antibody-secreting cells (ASC) were quantitated from 5 to 50 days after infection for total, RRV-specific, baculovirus-expressed VP4-specific, and single-shell RRV-specific ASC secreting either immunoglobulin G (IgG), IgM, or IgA. The response to VP4 constituted less than 1.5% of the total virus-specific response, which was located almost exclusively in the gut and was 90% IgA. Intestinal ASC were directed overwhelmingly toward proteins incorporated in the single-shell particle, predominantly VP2 and VP6. We conclude that the antibody response to VP4, thought to be the site of the important neutralization sites conserved among several rotavirus serotypes, is an extremely small portion of the overall antibody response in the intestinal tract.

[A virulence study of tick-borne encephalitis virus strains isolated in the southern Soviet Far East]. / Izuchenie virulentnosti shtammov virusa kleshchevogo éntsefalita, izolirovannykh na iuge Sovetskogo Dal'nego Vostoka.

Study of virulence for white mice and Syrian hamsters of 115 tick-borne encephalitis virus strains isolated in Maritime Territory showed virulence to be a complex biological manifestation of pathogenic properties of tick-borne encephalitis virus. The virulent properties of strains may have individual manifestations in each species of specific causative agent hosts and susceptible experimental biological models.

In vitro regulation of IgA subclass production. III. Selective transformation of IgA1 producing cells by Epstein-Barr virus.

In past experiments, using limited dilution analysis, we have demonstrated that a high percentage of immunoglobulin-secreting clones derived from Epstein-Barr virus- (EBV) stimulated lymphocytes secrete IgA. To further characterize the IgA produced by these clones, the IgA subclass of supernatants from clones stimulated 4 to 6 wk previously with EBV was determined by radioimmunoassay. All of 17 IgA-producing clones secreted IgA1; none secreted IgA2. Because we have shown that surface IgM+ (sIgM+) B cells are an enriched source of IgA2 plasma cell precursors, panning techniques were used to purify sIgM+ B cells from tonsils. Of 103 clones derived from these sIgM+ B cells, 102 secreted IgA1 and only one secreted IgA2. The relative absence of IgA2-producing clones could not be attributed to an absence of EBV receptors on IgA2 cells. A mean of 84 +/- 4% of freshly isolated IgA2 B cells and 78 +/- 6% of IgA1 B cells could be stained with a monoclonal antibody binding the EBV receptor; and there was no failure of EBV to infect IgA2 plasma cells precursors. Of IgA2 plasma cells derived from peripheral blood lymphocytes stimulated 7 days previously with EBV, 54 +/- 7% were positive for the EBV nuclear antigen, compared with 54 +/- 18% of IgA1 plasma cells from the same cultures. Seven days after EBV stimulation, a mean of 25% of the total IgA plasma cells were positive for cytoplasmic IgA2, whereas by 21 days after stimulation only 7% were positive for IgA2. This shift in the proportions of IgA1 and IgA2 plasma cells could be attributed to a failure of the IgA2 plasma cell number to increase after 10 days in culture. There was no evidence for selective suppression of IgA2 production by T cells or selective lysis of IgA2 plasma cells by infectious EBV particles. These results demonstrate that although precursors for both IgA1- and IgA2-producing cells can be stimulated to differentiate in response to EBV, there is preferential transformation of IgA1-producing cells.

[Mycoplasma infection as a possible cause of hybridoma instability]. / Zarazhenie mikoplazmami gibridom kak vozmozhnyi faktor ikh nestabil'nosti.

Cytogenetic analysis of mouse hybridoma producing monoclonal antibodies to diphtheria toxin and of its derivative, that lost secretory activity at the third passage in vivo, has been carried out. 58% cells of antibody secreting cell lines belonged to a modal class (76-79 chromosomes per cll). The modal chromosomal number of the subline that has stopped producing antibodies decreased to 63-66 per cell and the stem line of this derivative consisted of 30% of cell population only. Chromosome aberrations were much more frequent in hybridoma cells, that ceased to secrete antibodies, than in cells of original hybridoma: 32.3% of aberrant metaphases (1.38 break per cell) and 6.3% of aberrant metaphases (0.1 break per cell), respectively. Mycoplasma infection was found in the hybridoma subline that stopped producing antibodies as defined by the microbiological and cytochemical techniques. Mice might be the possible source of infection. By means of cloning of hybridoma variant, that did not secrete immunoglobulins, several sublines with the recovered secretory function were obtained.

[Effect of the influenza virus and its structural components on the animal immunocompetence system]. / Vliianie virusa grippa i ego strukturnykh komponentov na immunokompetentnuiu sistemu zhivotnykh.

The authors studied the effect of the influenza virus A (PR8/34) and of its structural components on the immunological reactivity of mice. The enzyme of the external coat of the influenza virus--neuraminidase--possessed an immunodepressive action. Administration of neuraminidase led to the elimination of sialic acids from the surface of lymphocytes and to the reduction of their electrophoretic mobility. The mechanism of the immunodepressive action of neuraminidase is discussed.