Intranasal Immunization with Heat-Inactivated Streptococcus pneumoniae Protects Mice against Systemic Pneumococcal Infection

In order to study the mucosal and serum antibody response to polysaccharide-encapsulated bacteria in mice, a preparation of heat-inactivated Streptococcus pneumoniae type 4 was administered, with and without cholera toxin, at various mucosal sites. It appeared that intranasal immunization of nonanesthesized animals was superior to either oral, gastric, or colonic-rectal antigen delivery with regard to the induction of serum immunoglobulin G (IgG) and IgA, as well as saliva IgA antibodies specific for pneumococci. The marked IgA antibody response in feces after intranasal, but not after oral or gastric, immunization is suggestive of a cellular link between the nasal induction site and the distant mucosal effector sites. Intranasal immunization also induced antibodies in serum and in mucosal secretions against type-specific capsular polysaccharide. IgA and IgG antibody levels in pulmonary lavage fluids correlated well with saliva IgA and serum IgG antibodies, respectively. Antibody determinations in pulmonary secretions may therefore be redundant in some cases, and the number of experimental animals may be reduced accordingly. After intraperitoneal challenge with type 4 pneumococci, mice immunized intranasally were protected against both systemic infection and death, even without the use of cholera toxin as a mucosal adjuvant. Thus, an efficient intranasal vaccine against invasive pneumococcal disease may be based on a very simple formulation with whole killed pneumococci.     

肺炎链球菌自溶酶（LytA）黏膜免疫小鼠的生物安全性评价

目的开展肺炎链球菌自溶酶（LvtA）生物安全性的动物实验研究,为其申报生物制品鉴定提供实验数据。方法制备纯化的重组表达LytA蛋白,以6．5μg、13．0μg和19．5μg3种剂量黏膜免疫BALB／c小鼠,根据《中华人民共和国国家标准食品安全性毒理学评价程序》（GB15193．1—1994）进行LytA的90d喂养试验、传统致畸试验及遗传毒性试验检测,以分析其生物安全性。结果90d喂养试验结果显示各实验组小鼠与对照组小鼠的体重增长趋势一致,且各时间点检测的小鼠体重均无显著性差异（P〉0．05）。传统致畸试验检测发现各实验组小鼠与对照组小鼠心脏、肺、肝脏、肾、脾、胃、子宫重量、红细胞和白细胞数目、谷丙转氨酶、尿素氮、总胆固醇、甘油三脂、血糖、总蛋白、自蛋白及球蛋白等指标均元明显差异（P〉0．05）。小鼠骨髓嗜多染红细胞微核试验结果显示LytA经鼻腔黏膜免疫小鼠无遗传毒性。结论利用90d喂养试验、传统致畸试验及遗传毒性试验证明LytA鼻腔黏膜免疫BALB／c小鼠安全、毒副作用不明显,有良好的生物安全性,值得进一步研究。     

Intranasal Administration of Synthetic Recombinant Peptide-Based Vaccine Protects Mice from Infection by Schistosoma mansoni

Schistosomiasis is the cause of a chronic debilitating disease which accounts for significant mortality and morbidity every year, especially in tropical and subtropical areas. An epitope derived from the protective surface protein 9B-Ag of Schistosoma mansoni, designated 9B peptide-1, was previously showed to be protective in mice when conjugated to bovine serum albumin and administered subcutaneously in complete Freund's adjuvant. In this work, this protective peptide was expressed in the flagellin of a Salmonella vaccine strain, and the isolated recombinant flagella were used for immunization of mice. Since during the invasion of the parasite into the host the schistosomula migrate first to the lungs, the intranasal route of administration was employed in order to halt the parasite at an early stage of the infection. Such intranasal immunization with this peptide expressed in flagellin, without the addition of adjuvants, resulted in a significant humoral response and also led to protection against challenge infection, manifested as a reduction of the worm burden by an average of 42%.     

Immunization with Recombinant Pneumolysin Induces the Production of Antibodies and Protects Mice in a Model of Systemic Infection Caused by Streptococcus pneumoniae

Bulletin of Experimental Biology and Medicine - Immunogenic and protective activity of recombinant pneumolysin was studied in experiments on male BALB/c mice. The mice were immunized...     

Role of Capsule and Suilysin in Mucosal Infection of Complement-Deficient Mice with Streptococcus suis

Virulent Streptococcus suis serotype 2 strains are invasive extracellular bacteria causing septicemia and meningitis in piglets and humans. One objective of this study was to elucidate the function of complement in innate immune defense against S. suis. Experimental infection of wild-type (WT) and C3^−/− mice demonstrated for the first time that the complement system protects naive mice against invasive mucosal S. suis infection. S. suis WT but not an unencapsulated mutant caused mortality associated with meningitis and other pathologies in C3^−/− mice. The capsule contributed also substantially to colonization of the upper respiratory tract. Experimental infection of C3^−/− mice with a suilysin mutant indicated that suilysin expression facilitated an early disease onset and the pathogenesis of meningitis. Flow cytometric analysis revealed C3 antigen deposition on the surface of ca. 40% of S. suis WT bacteria after opsonization with naive WT mouse serum, although to a significantly lower intensity than on the unencapsulated mutant. Ex vivo multiplication in murine WT and C3^−/− blood depended on capsule but not suilysin expression. Interestingly, S. suis invasion of inner organs was also detectable in C5aR^−/− mice, suggesting that chemotaxis and activation of immune cells via the anaphylatoxin receptor C5aR is, in addition to opsonization, a further important function of the complement system in defense against mucosal S. suis infection. In conclusion, we unequivocally demonstrate here the importance of complement against mucosal S. suis serotype 2 infection and that the capsule of this pathogen is also involved in escape from complement-independent immunity.     

Stevioside Protects LPS-Induced Acute Lung Injury in Mice

Stevioside, a diterpene glycoside component of Stevia rebaudiana, has been known to exhibit anti-inflammatory properties. To evaluate the effect and the possible mechanism of stevioside in lipopolysaccharide (LPS)-induced acute lung injury, male BALB/c mice were pretreated with stevioside or dexamethasone 1 h before intranasal instillation of LPS. Seven hours later, tumor necrosis factor-α, interleukin-1β, and interleukin-6 in bronchoalveolar lavage fluid (BALF) were measured by using enzyme-linked immunosorbent assay. The number of total cells, neutrophils, and macrophages in the BALF were also determined. The right lung was excised for histological examination and analysis of myeloperoxidase activity and nitrate/nitrite content. Cyclooxygenase 2 (COX-2), inducible NO synthase (iNOS), nuclear factor-kappa B (NF-κB), inhibitory kappa B protein were detected by western blot. The results showed that stevioside markedly attenuated the LPS-induced histological alterations in the lung. Stevioside inhibited the production of pro-inflammatory cytokines and the expression of COX-2 and iNOS induced by LPS. In addition, not only was the wet-to-dry weight ratio of lung tissue significantly decreased, the number of total cells, neutrophils, and macrophages in the BALF were also significantly reduced after treatment with stevioside. Moreover, western blotting showed that stevioside inhibited the phosphorylation of IκB-α and NF-κB caused by LPS. Taken together, our results suggest that anti-inflammatory effect of stevioside against the LPS-induced acute lung injury may be due to its ability of inhibition of the NF-κB signaling pathway. Stevioside may be a promising potential therapeutic reagent for acute lung injury treatment.     

Importance of Bacterial Replication and Alveolar Macrophage-Independent Clearance Mechanisms during Early Lung Infection with Streptococcus pneumoniae

Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors are less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated, and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model, after infection with a high-dose inoculum of encapsulated S. pneumoniae, alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 min compared to 135 min for alveolar macrophage-dependent clearance. In addition, after a high-dose inoculum, successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 min. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 min for alveolar macrophage-independent and 31 min for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower-dose inoculum, the bacterial doubling time increased to 56 min and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 min and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, and rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae.     

Bakuchiol Protects Against Acute Lung Injury in Septic Mice

Sepsis is a systemic inflammatory reaction that may lead to multiple organ damage and acute lung injury (ALI). Bakuchiol (Bak) has been reported to confer protection against inflammation and oxidative stress. However, its effect on sepsis-induced acute lung injury remains unclear. In the present study, male C57BL/6 mice were subjected to cecal ligation and puncture (CLP), and Bak (15, 30, 60 mg/kg) was administered intragastrically after 0 and 3 h of surgery. Lung water content was detected. Pathologic changes in lung tissues were evaluated via hematoxylin and eosin (H&E) staining. The levels of myeloperoxidase (MPO), IL-1β, IL-6, and TNF-α were evaluated using ELISA. In addition, expression levels of phosphorylated (p)-IκB, ICAM-1, HMGB1, nitrotyrosine (3-NT), claudin-1, and VE-cadherin were detected using Western blot. Further, IL-1β expression was evaluated using immunofluorescence. SOD activity, contents of MDA, and 8-OHdG were detected to determine the level of oxidative stress. Our results suggested that Bak (60 mg/kg) treatment significantly attenuated pathologic changes and edema in lung tissues and attenuated inflammation and oxidative stress in the lung following sepsis. Additionally, Bak treatment alleviated sepsis-induced lung endothelial barrier disruption. In conclusion, Bak treatment attenuates ALI following sepsis by suppressing inflammation, oxidative stress, and endothelial barrier disruption. Our study indicates that Bak is a potential candidate to treat sepsis-induced ALI.     

Lactoferrin Protects Gut Mucosal Integrity During Endotoxemia Induced by Lipopolysaccharide in Mice

The hypothesis that lactoferrin protects mice against lethal effects of bacterial lipopolysaccharide (LPS) is the subject of experimental investigations described in this article. Lipopolysaccharide is a powerful toxin produced by Gram negative bacteria that when injected into humans or experimental animals reproduce many of the pathophysiologic and immune responses caused by live bacteria. Lactoferrin administered intraperitoneally 1 hr prior to injection of LPS significantly enhanced the survival of mice, reducing LPS-induced mortality from 83.3% to 16.7%. Changes in locomotor and other behavioral activities resulting from LPS injection were not present in mice treated with lactoferrin. Also, histological examination of intestine revealed remarkable resistance to injury produced by LPS if mice were pretreated with lactoferrin. Severe villus atrophy, edema and epithelial vacuolation were observed in LPS-treated animals but not in lactoferrin-treated counterparts. Electrophysiological parameters were used to assess secretory and absorptive functions in the small intestine. In mice treated with LPS, transmural electrical resistance was reduced and absorption of glucose was increased. Lactoferrin treatment had no significant influence on basal electrophysiological correlates of net ion secretion or glucose absorption nor on changes induced by LPS. Collectively, these results suggest that lactoferrin attenuates the lethal effect of LPS and modulates behavioral and histopathological sequela of endotoxemia.     

Morphine Disrupts Interleukin-23 (IL-23)/IL-17-Mediated Pulmonary Mucosal Host Defense against Streptococcus pneumoniae Infection

Streptococcus pneumoniae is a pathogen that causes serious respiratory disease and meningitis in the immunocompromised drug abuse population. However, the precise mechanisms by which drug abuse compromises the host immune defense to pulmonary S. pneumoniae infection is not fully understood. Using a well-established murine model of opiate abuse and S. pneumoniae lung infection, we explored the influence of morphine treatment on the interleukin-23 (IL-23)/IL-17 axis and related innate immunity. Impairment of early IL-23/IL-17 production caused by morphine treatment was associated with delayed neutrophil migration and decreased pneumococcal clearance. Furthermore, morphine treatment impaired MyD88-dependent IL-23 production in alveolar macrophages and dendritic cells in response to in vitro S. pneumoniae cell infection. Moreover, morphine treatment significantly inhibited the S. pneumoniae-induced phosphorylation of interferon response factor 3 (IRF3), ATF2, and NF-κBp65. T-cell receptor δ (TCRδ)-deficient mice showed a decrease in IL-17 production and a severely weakened capacity to clear lung S. pneumoniae infection. Finally, morphine treatment resulted in diminished secretion of antimicrobial proteins S100A9 and S100A8/A9 during early stages of S. pneumoniae infection. In conclusion, morphine treatment causes a dysfunction in IL-23-producing dendritic cells and macrophages and IL-17-producing γδT lymphocytes in response to S. pneumoniae lung infection. This leads to diminished release of antimicrobial S100A8/A9 proteins, compromised neutrophil recruitment, and more-severe infection.Immunocompromised individuals are at high risk for Streptococcus pneumoniae pulmonary infection (30). Previous studies have shown that opiate abuse causes immunosuppression by disrupting both innate and adaptive components of the immune system (26, 33). Opiate abuse is a critical risk factor for increasing susceptibility and severity of bacterial infection, including S. pneumoniae (31, 32). However, additional work is needed to understand the precise mechanisms by which opiate abuse increases the susceptibility to S. pneumoniae lung infection.Interleukin-23 (IL-23) has been recently identified as a cytokine closely related to IL-12 (5). The balance between IL-23 and IL-12 controls the outcome of inflammatory responses (16). IL-23 is secreted by activated macrophages and dendritic cells (DCs) and induces memory T-cell proliferation and is the critical factor required for T-cell IL-17 expression in response to bacterial challenge (3). IL-23 release leads to the production of IL-17. Furthermore, IL-17 promotes neutrophilic inflammation by upregulating CXC chemokines and hematopoietic growth factors (13). Several recent studies report the important role of IL-23 and IL-17 in the induction of neutrophil-mediated protective immune response against extracellular bacterial or fungal pathogens, such as Klebsiella pneumoniae (14), Pseudomonas aeruginosa (12), Porphyromonas gingivalis (34), Citrobacter rodentium (20), Bacteroides fragilis (9), and Escherichia coli (27).Generally, the IL-23/IL-17 axis plays an important role in the host defense against bacterial infections (11). Previous studies have demonstrated that IL-17 is critical for the recruitment of phagocytes that leads to the clearance of S. pneumoniae colonization from the mucosal surface of the nasopharynx (18, 35). However, whether the IL-23/IL-17 axis contributes to modulating the innate immunity in response to S. pneumoniae lung infection has not been addressed. Using a well-established opiate abuse and S. pneumoniae lung infection mouse model (31, 32), we demonstrate that S. pneumoniae induces IL-23 and IL-17 expression in the lungs as early as 2 h following infection. Morphine treatment causes a decrease in both IL-23 and IL-17 synthesis during the early stages of infection, leading to delayed neutrophil recruitment. This results in an increased bacterial burden within the lungs and the initiation of systemic disease.     

A Neutralizing Monoclonal IgG1 Antibody of Platelet-Activating Factor Acetylhydrolase SsE Protects Mice against Lethal Subcutaneous Group A Streptococcus Infection

Group A Streptococcus (GAS) can cause life-threatening invasive infections, including necrotizing fasciitis. There are no effective treatments for severe invasive GAS infections. The platelet-activating factor (PAF) acetylhydrolase SsE produced by GAS is required for invasive GAS to evade innate immune responses and to invade soft tissues. This study determined whether the enzymatic activity of SsE is critical for its function in GAS skin invasion and inhibition of neutrophil recruitment and whether SsE is a viable target for immunotherapy for severe invasive GAS infections. An isogenic derivative of M1T1 strain MGAS5005 producing SsE with an S178A substitution (SsE^S178A), an enzymatically inactive SsE mutant protein, was generated. This strain induced higher levels of neutrophil infiltration and caused smaller lesions than MGAS5005 in subcutaneous infections of mice. This phenotype is similar to that of MGAS5005 sse deletion mutants, indicating that the enzymatic activity of SsE is critical for its function. An anti-SsE IgG1 monoclonal antibody (MAb), 2B11, neutralized the PAF acetylhydrolase activity of SsE. Passive immunization with 2B11 increased neutrophil infiltration, reduced skin invasion, and protected mice against MGAS5005 infection. However, 2B11 did not protect mice when it was administered after MGAS5005 inoculation. MGAS5005 induced vascular effusion at infection sites at early hours after GAS inoculation, suggesting that 2B11 did not always have access to infection sites. Thus, the enzymatic activity of SsE mediates its function, and SsE has the potential to be included in a vaccine but is not a therapeutic target. An effective MAb-based immunotherapy for severe invasive GAS infections may need to target virulence factors that are critical for systemic survival of GAS.     

Sortase A Confers Protection against Streptococcus pneumoniae in Mice

Streptococcus pneumoniae sortase A (SrtA) is a transpeptidase that is highly conserved among pneumococcal strains, whose involvement in adhesion/colonization has been reported. We found that intraperitoneal immunization with recombinant SrtA conferred to mice protection against S. pneumoniae intraperitoneal challenge and that the passive transfer of immune serum before intraperitoneal challenge was also protective. Moreover, by using the intranasal challenge model, we observed a significant reduction of bacteremia when mice were intraperitoneally immunized with SrtA, while a moderate decrease of lung infection was achieved by intranasal immunization, even though no influence on nasopharynx colonization was seen. Taken together, our results suggest that SrtA is a good candidate for inclusion in a multicomponent, protein-based, pneumococcal vaccine.Streptococcus pneumoniae colonizes the nasopharynx of humans and represents a leading cause of severe diseases, such as otitis media, pneumonia, and meningitis. S. pneumoniae is one of the major causes of bacterial pneumonia in developing countries (19). It is estimated that each year, nearly 1 million children worldwide die because of pneumococcal diseases (10). Besides children, groups at high risk of pneumococcal infection are immunocompromised subjects and the elderly, for whom a high case fatality rate is also observed. The last decades have seen an increase in investigations of protein antigens, and several protein candidates have been proposed for a vaccine for S. pneumoniae (2) to overcome the problems inherent to the currently available polysaccharide-based vaccines. In fact, the 23-valent polysaccharide pneumococcal vaccine is not effective in children under 2 years of age, whose immune systems are unable to mount a T-independent response to polysaccharides. On the other hand, the 7-valent polysaccharide conjugate vaccine, although efficacious, induces serotype replacement (5, 20). Moreover, while more than 90 S. pneumoniae serotypes are presently known, both polysaccharide pneumococcal vaccines and polysaccharide conjugate vaccines are effective only against the serotypes included in the vaccine. Efforts to identify new S. pneumoniae factors that play a role in colonization and pathogenesis may contribute to the indication of possible targets of either new therapeutic agents or vaccines.Sortase A (SrtA) is a membrane-anchored transpeptidase expressed by gram-positive bacteria (12). The role of SrtA in the processing of sorting signals at the LPXTG motif to anchor surface proteins to the cell wall envelope was first described for Staphylococcus aureus (21), in which an isogenic SrtA mutation resulted in a strongly reduced ability to infect animals (13, 23). SrtA has been shown to participate in the colonization and/or pathogenesis of several Streptococcus species (1, 6, 8, 22, 24).S. pneumoniae SrtA has been described as playing a role in adhesion to human pharyngeal cells in vitro (7), in nasopharyngeal colonization in chinchilla (3), and in pneumonia, bacteremia, and nasopharyngeal colonization in murine models (15). Although SrtA seems to be dispensable in pilus biogenesis, its possible role in repressing pilus islet expression has been very recently proposed (9). SrtA has been found to be widely expressed among S. pneumoniae isolates and highly conserved, with a DNA identity of 99 to 100% (15). Although all of these findings suggest that pneumococcal SrtA might be useful as a protein vaccine, to the best of our knowledge no data have been provided so far on the protective efficacy afforded by SrtA immunization in animal models. Thus, we investigated the protective role of SrtA in murine models of S. pneumoniae infection.     

Animal Model of Mycoplasma pneumoniae Infection Using Germfree Mice

We have attempted to establish a gnotobiotic mouse model monoassociated with Mycoplasma pneumoniae following single or repeated infection to examine the mechanism of pathogenesis following M. pneumoniae infection. M. pneumoniae inoculated into germfree mice colonized equally well at 10⁵ CFU/lung in both single infection and repeated infection. In histopathological observation, repeatedly infected mice showed pneumonia with mild infiltration of mononuclear cells and macrophages. Antibody titers against M. pneumoniae rose in the repeatedly infected mice but not in the singly infected mice. The percentage of CD4-positive, CD8-positive, and CD25-positive lymphocytes infiltrated in the lung was increased in the repeatedly infected mice. In contrast, the lymphocyte subset in the spleen was not significantly different among mock-, singly, and repeatedly infected mice. In the study of cytokine productivity of spleen cells, production of interleukin (IL)-4 and IL-10 was significantly increased and that of gamma interferon was remarkably increased in the mice following repeated infection. These results indicate that a gnotobiotic mouse model monoassociated with M. pneumoniae was established and that immune mechanisms might be involved in the pathogenesis in pneumonia following M. pneumoniae infection.     

A Case of Lung Transplantation Following Mycoplasma pneumoniae Infection

Reported here is a case of severe necrotizing pneumonia following Mycoplasma pneumoniae infection that occurred in a 55-year-old man. The histological changes of lung parenchyma included granulomas and bronchiolitis obliterans. Mycoplasma infection was diagnosed by repeated antibody determination (complement fixation test) and confirmed using the polymerase chain reaction to detect the pathogen from a tracheal aspirate. Prior to this episode of pneumonia, the patient had been healthy, except for Reiter's disease that had been diagnosed 18 years previously. In addition to severe pulmonary involvement, the patient developed rhabdomyolysis with subsequent acute renal failure, Stevens-Johnson syndrome, biochemical pancreatitis, severe anemia, and an effusion of the right knee. Contrary to the symptoms of pulmonary disease, all of the extrapulmonary manifestations except anemia were transient. Due to persistent respiratory insufficiency and long-term failure to wean the patient from a respirator, a lung transplantation was performed. Five weeks after transplantation the patient died as a result of intrapulmonary hemorrhage. To the best of our knowledge, this is the first report of pneumonia due to Mycoplasma pneumoniae leading to lung transplantation. Furthermore, the multiple extrapulmonary manifestations in this case make it exceptional. Electronic Publication     

An OspC-Specific Monoclonal Antibody Passively Protects Mice from Tick-Transmitted Infection by Borrelia burgdorferi B31

A murine monoclonal antibody directed against Borrelia burgdorferi B31 outer surface protein C (OspC) antigen was generated by a method whereby borreliae were inoculated into the mouse via the natural transmission mode of tick feeding. Passive immunization with this antibody resulted in protection of C3H/HeJ and outbred mice from a tick-transmitted challenge infection. Immunofluorescence staining of borrelia cells indicated surface exposure of the OspC epitope reactive with the monoclonal antibody.     

Protective Immunity of Microsphere-Based Mucosal Vaccines against Lethal Intranasal Challenge with Streptococcus pneumoniae

Mucosal vaccination of capsular polysaccharide (PS) of Streptococcus pneumoniae and subsequent creation of the first line of immunological defense in mucosa were examined. Mucosal as well as systemic antibody responses to PS were evoked by peroral or intranasal immunization of BALB/c mice with PS-cholera toxin B subunit (CTB) conjugates entrapped in the alginate microspheres (AM). The bacterial colonization at the lung mucosa was most profoundly inhibited (<95%) by intranasal immunization with the naked conjugate (PS-CTB). The mice vaccinated orally with encapsulated conjugate [AM(PS-CTB)] showed significant reduction on the level of pneumococcal bacteremia (<99%). Eighty percent of the mice perorally immunized with AM (PS-CTB) were protected from lethal intranasal challenge with S. pneumoniae, whereas more than 60% of the mice in the other control groups died of infection. Our novel approach may prove to be important in the development of a mucosal vaccine that will provide protection of mucosal surfaces of host.     

Cytokine Response to Group B Streptococcus Infection in Mice

This study was undertaken to better understand the complex relationship between specific and non-specific host defence mechanisms and group B streptococci (GBS). A comprehensive kinetics analysis of cytokine mRNA expression was performed, by Northern blot assay, in peritoneal exudate cells (PEC) and spleen cells (SC) recovered from CD-1 mice at various times during the course of an intraperitoneal infection with a lethal dose (5 × 10³ microorganisms/mouse) of type Ia GBS, reference strain 090 (GBS-Ia). Analysis of cytokines involved in the development of a specific T_H response shows that GBS-Ia in PEC induce only a weak increase of IL-2 mRNA expression and in SC a cytokine pattern characterized by IL-2, IFN-γ and IL-12 in the absence of IL-4, IL-5 and IL-10. This selected cytokine pattern could provide appropriate conditions for the development of a T_H1 response. Analysis of inflammatory cytokines, which are usually induced early during an in vivo infection, shows that there is a significant expression of mRNA specific for IL-1β, TNFα and IL-6, both in PEC and SC only at 24 h which persists at a high level until 36 h. This delayed cytokine induction, accompanied by the contemporary activation of splenic phagocytic cells, occurs only when the number of GBS-Ia is extremely high. In fact, at 24 h GBS-Ia have heavily colonized all organs. In vitro infection of thioglycollate-elicited peritoneal macrophages confirms that the ability of GBS-Ia to induce a strong inflammatory cytokine response depends strictly on the number of infecting microorganisms. Indeed, macrophages respond to GBS-Ia with a very rapid induction of IL-1β and TNFα mRNA when infected at a ratio of 1:10, but not at 100:1. Two major observations emerged from this study: (1) GBS-Ia, by inducing a cytokine pattern which seems to favour development of a T_H1 response, could evade antibody production essential for resistance to GBS; and (2) inflammatory cytokine response is induced when a heavy microbial invasion of the host has already occurred. These novel features of GBS-Ia could contribute to the development and progression of lethal infection in mice.