Severity of group B streptococcal arthritis in selected strains of laboratory mice.

The susceptibilities of C3H/HeN, BALB/c, and C57BL/6N mouse strains to group B streptococci (GBS) infection were evaluated. C3H/HeN mice developed severe polyarthitis; mild lesions and no lesions were observed in BALB/c and C57BL/6N mice, respectively. A correlation between the severity of arthritis, the number of GBS in the joints, and local interleukin-6 and interleukin-1beta production was evident.

Severity of group B streptococcal arthritis is correlated with beta-hemolysin expression.

Septic arthritis is a clinical manifestation of group B streptococcal (GBS) infection in neonates and adults. To examine the potential role of GBS beta-hemolysin in joint injury, mice were infected with 2 wild-type strains or with nonhemolytic (NH) or hyperhemolytic (HH) variants derived by transposon mutagenesis. Compared with mice infected with the parent strains, mice infected with the NH mutants had decreased mortality and bacterial proliferation. A reduced LD(50) and a higher microbial load were obtained in mice infected with the HH mutants. Greater degrees of joint inflammation and damage were observed in the HH mutant-infected animals than in those infected with the parental strains. NH mutant-infected mice manifested only a mild and transient arthritis. Systemic and local levels of interleukin-6 mirrored the observed differences in virulence and severity of arthritis. These data support a direct correlation of GBS beta-hemolysin expression with mortality and severity of articular lesions.

Influence of interferon-gamma administration on the severity of experimental group B streptococcal arthritis.

OBJECTIVE: To assess the effect of interferon-gamma (IFNgamma) administration on the evolution of systemic infection and septic arthritis induced by group B streptococci (GBS) in mice. METHODS: CD1 mice were inoculated intravenously with arthritogenic strain 1/82 of type IV GBS. Exogenous murine IFNgamma or anti-IFNgamma monoclonal antibodies were administered intravenously either 2 hours (-2 hours) before or 18 hours after infection with 1 x 10(7) GBS. Mice were monitored daily for survival and for signs of arthritis. In a subsequent set of experiments, mice were killed at selected times for examination of bacterial clearance, joint histopathology, and cytokine production. RESULTS: Mortality in mice treated with IFNgamma at -2 hours was 100%, compared with 20% in those treated at 18 hours and with 40% in controls. As indicated by the arthritis score, mice treated with IFNgamma at -2 hours developed early and more severe arthritis, whereas those treated at 18 hours had milder arthritis compared with infected controls. Less severe joint pathology in the mice treated with IFNgamma at 18 hours correlated with low levels of interleukin-6 (IL-6) and IL-1beta and a low bacterial load in the joints, whereas rapid onset and worsening of articular lesions in those treated at -2 hours corresponded to early and sustained levels of IL-6. CONCLUSION: The findings of this study demonstrate that the effects mediated by IFNgamma on GBS-induced arthritis may be detrimental or beneficial, depending on the time of administration of IFNgamma in relation to infection with the antigen.

Role of tumor necrosis factor alpha, interleukin-1beta, and interleukin-6 in a mouse model of group B streptococcal arthritis.

Intravenous inoculation of CD1 mice with 10(7) CFU of type IV group B Streptococcus (GBS IV) results in a high incidence of diffuse septic arthritis. In this study the roles of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in articular pathology were evaluated. Cytokine levels were quantified in the serum and joints by enzyme-linked immunosorbent assay in mice injected with GBS IV and tested or not tested with pentoxifylline (PTF), a methylxanthine that affects cytokine production. PTF was administered intraperitoneally at a dose of 1 mg/mouse (50 mg/kg of body weight) 1 h after GBS infection and then at 24-h intervals for 4 days. High levels of IL-1beta and IL-6, but not TNF-alpha, were detected in the joints of mice injected with GBS IV from 5 to 15 days after infection, when articular lesions were most frequent and severe. IL-1beta and IL-6 concentrations in the joints significantly (P < 0.001) exceeded those detected in the serum, confirming a strong local production. PTF treatment resulted in a strong reduction of cytokine production and in a marked decrease in both the incidence and severity of arthritis. Inoculation of exogenous murine recombinant IL-1beta or IL-6 in mice treated with GBS IV plus PTF resulted in an incidence and severity of articular lesions similar to those obtained with inoculation of GBS IV alone. No significant effect was obtained with TNF-alpha administration. These data show a strong involvement of IL-1beta and IL-6, but not TNF-alpha, in the pathogenesis of GBS arthritis.

Role of group B streptococcal capsular polysaccharides in the induction of septic arthritis.

The ability of different serotypes of group B streptococci (GBS) to induce septic arthritis in mice was compared. Types II, III, IV, V, VI and VII GBS were investigated. A highly capsulate strain of type III GBS, COH1, and its mutants, COH1-11 (lacking capsular sialic acid) and COH1-13 (non-capsulate), obtained by transposon insertional mutagenesis, were used to assess the role of type-specific polysaccharide on the induction of arthritis. At an intravenous dose of 10(7) cfu/mouse, reference strains of types II, III, IV, VI and VII and type III strain COH1 induced arthritis with an incidence ranging from 70 to 90%. For type V and strain COH1-11, 10(8) cfu/mouse was required to obtain a 50% incidence of arthritis; lesions were not evident with strain COH1-13. The presence of the capsule played a major role in the induction of GBS septic arthritis. The presence and amount of sialic acid in capsular polysaccharide influenced the incidence of articular lesions. The bacterial dose affected the manifestations of arthritis; the less virulent strains of GBS also induced articular lesions when an adequate number of micro-organisms reached the joints.

Protective activity of a murine monoclonal antibody against acute and chronic experimental infection with type IV group B streptococcus.

A murine IgM monoclonal antibody (MAb H11) was developed against the type polysaccharide capsular antigen of group B streptococcus (GBS), serotype IV, after intraperitoneal immunisation of BALB/c mice with heat-killed bacteria. MAb H11 reacted in immunodiffusion with the purified polysaccharide in both its sialylated and desialylated form, giving a line of identity, and opsonised type IV GBS strains in an in vitro assay. When administered at the time of intraperitoneal lethal challenge with homologous GBS, or 4 h earlier, MAb H11 protected 90% of the mice. Protection was still observed when MAb H11 was given 4 h after the challenge. This MAb was strongly effective in preventing septic arthritis induced by type IV GBS.

In vivo efficacy of azithromycin in treatment of systemic infection and septic arthritis induced by type IV group B Streptococcus strains in mice: comparative study with erythromycin and penicillin G.

We compared the activities of azithromycin, erythromycin, and penicillin G in a mouse model of systemic infection and septic arthritis induced by type IV group B streptococci (GBS). The in vitro and in vivo efficacy data for these drugs were analyzed relative to the pharmacokinetics of the drugs in sera, joints, and kidneys. Adult CD-1 mice were infected intravenously with 10(7) CFU of type IV GBS. Intraperitoneal drug administration was initiated with different dose regimens at different times after infection. A single dose of azithromycin (100 mg/kg) strongly reduced the incidence of articular lesions with respect to that with erythromycin or penicillin G. Treatment with azithromycin (three intraperitoneal administrations of 50 mg/kg at 12-h intervals) resulted in the complete prevention of arthritis. In contrast, erythromycin was poorly effective and penicillin G was effective only if inoculated 30 min after infection and at high doses (400,000 or 600,000 IU/kg). Furthermore, azithromycin was able to cure about 70% of the mice when administered 7, 8, and 9 days after GBS infection. Azithromycin was much more active than erythromycin and penicillin G with respect to bacterial killing in the joints and kidneys. In fact, cultures from these tissues were always negative no matter what treatment schedule was employed. The pharmacokinetics of azithromycin account for its superior in vivo efficacy against type IV GBS. A longer half-life and higher levels of this drug in serum and tissues with respect to those for erythromycin or penicillin G were achieved. The high affinity of azithromycin for the joints strongly supports its potential value for therapy of septic arthritis, which is a severe and frequent clinical manifestation of GBS infection.

Antibody-independent protection in mice against type Ia group B streptococcus lethal infection.

There is ample evidence that protection against group B streptococcal (GBS) disease, both in experimental animals and in humans, is related to the presence of specific antibodies and complement. However, until now the possibility of increasing resistance to GBS infection by potentiating natural cell-mediated immunity in the host, has not been explored. In this study we examine the effect of administering in vivo MVE-2 (a polymer fraction of 1,2-co-polymer of divinyl ether and maleic anhydride) and inactivated Candida albicans (CA) cells on mouse resistance to the reference strain type Ia 090 GBS (GBS-090) lethal infection. MVE-2 and CA, respectively a synthetic and a microbial biological response modifier (BRM), are strong inducers and activators of natural resistance effectors, such as natural killer (NK) cells, macrophages and polymorphonuclear cells (PMN). The results showed that MVE-2 protected 100% CD-1 mice from a systemic lethal challenge with GBS-090 (5 x 10(3) microorganisms/mouse) when administered 3 days before infection at dose of 50 mg kg-1. CA treatment, in five doses (CA-5d) over 14 days protected 100% mice when administered at 2 x 10(7) cells/mouse and when the last CA injection was given 1 day before the GBS-090 challenge. Instead, when the GBS-090 challenge was performed by intraperitoneal route, protection was obtained with CA-5d treatment but not with MVE-2. The possibility that MVE-2 or CA stimulated a rapid production of specific antibodies against GBS-090 infection was excluded by the ELISA assay. Evidence exists that NK cells do not play a primary role as effectors in the MVE-2 and CA conferred protection since the strong reduction in NK activity, due to in vivo administration of anti-asialo GM1 antibodies before GBS-090 infection, did not influence the BRM-induced protection. Besides, high NK activity levels, induced by in vivo rhIL-2 administration, did not protect the mice against GBS-090 infection. Both studies on in vivo clearance and in vitro microbicidal activity, showed that, after 1 h, immunopotentiated effectors were unable to kill GBS-090, but were highly effective against GBS type VI. These results seem to indicate that intracellular GBS-090 killing is a slow process requiring more than 1 h. This study demonstrates that it is possible to increase resistance to GBS-090 lethal infection by BRMs, by potentiating the antibody-independent microbicidal activity of the phagocytes.

Experimental model of type IV Streptococcus agalactiae (group B streptococcus) infection in mice with early development of septic arthritis.

We have established an experimental murine model to gain insight into the pathogenicity and clinical features of type IV group B streptococcus (GBS) infections. Adult CD-1 mice were challenged intravenously with 10(7) type IV GBS cells, inducing systemic invasion. Most of the animals were able to clear the infection from the blood, brain, and lungs within 2 weeks and from the spleen and liver within 1 month. However, the animals were unable to clear the microorganism from the joints and kidneys during the 60-day observation period. About 80% of the mice challenged intravenously with type IV GBS manifested early septic arthritis, which evolved from an acute exudative synovitis to permanent lesions characterized by irreversible joint damage and ankylosis. Induction of persistent septic arthritis was dependent on the number and viability of microorganisms inoculated and was unrelated to the strain of type IV GBS and the growth phase of the inoculum. Type-specific antibodies of both immunoglobulin M and G classes could be detected by agglutination and enzyme-linked immunosorbent assay from days 7 and 14, respectively; immunoglobulin G antibodies persisted for more than 40 days. Complexes of antibodies and group- and type-specific antigens were detected in mouse sera 24 h after infection and persisted up to day 22. These results were obtained an experimental model of type IV GBS chronic infection with early development of septic arthritis, which could be useful in future studies of pathogenicity and immune mechanisms involved in the host resistance to this microorganism.

Cell wall components of Candida albicans as immunomodulators: induction of natural killer and macrophage-mediated peritoneal cell cytotoxicity in mice by mannoprotein and glucan fractions.

Cell wall components from Candida albicans were compared to intact cells for their ability to induce natural cytotoxic immunoeffectors in the peritoneal cavity of mice. A soluble mannoprotein extract (MP) and an insoluble glucan fraction (GG) strongly stimulated the generation of peritoneal effectors capable of lysing YAC-1 and P-815 tumour cell lines in vitro. The anti-YAC-1 effectors were characterized as natural killer (NK) lymphocytes while the anti-P-815 effectors appeared to be activated macrophages. The activity of each fraction was typically dose-dependent and both fractions differed from whole cells in the kinetics of induction of cytotoxicity. However, the NK and macrophage effectors generated by these materials had similar functional and phenotypic properties, irrespective of the material used as inducer. No mannoprotein was detected in the insoluble glucan fraction GG. Hence, the immunoenhancing activity of GG could not be attributed to the presence of some MP or MP-like component. Mannan-rich fractions with low (less than 3%) protein content (M) or extracted by hot alkaline reagent (M-alk) were inactive as NK and macrophage inducers. Thus, the cell wall of C. albicans contains at least two distinct macromolecular complexes which mediate the induction in murine peritoneal exudates of cytotoxic effectors active against tumour cell lines.

Induction of natural killer cell activity by inactivated Candida albicans in mice.

Injection of merthiolate-inactivated yeast form cells of Candida albicans into the peritoneal cavities of mice induced the appearance of a cytolytic effector population against YAC-1 tumor cell lines. This induction was maximally manifested in 5- to 8-week-old animals 3 to 4 days after injection of 2 X 10(7)C. albicans cells, and the peritoneal lytic population exerted its optimum cytotoxic effect after 4 h of incubation. No significant natural cytotoxic activity was generated by C. albicans in the bone marrow or thymus, whereas there was a slight, transient, but significant depression of natural splenic cytotoxicity. Experiments performed to characterize the natural cytotoxic population elicited by the inactivated yeast showed that the effectors were nonadherent, nonphagocytic cells. Moreover, the anti-YAC-1 lytic activity was partially sensitive to anti-Thy1.2 serum and was completely abrogated by treatment of peritoneal nonadherent cells with monoclonal anti-asialo GM1 antibodies. Finally, the peritoneal population of cytotoxic cells induced by C. albicans was fully susceptible to Ly5.1 plus anti-immunoglobulin G2a and complement lysis. Although different cell populations could be induced by inactivated C. albicans, all of our data support the view that the anti-YAC-1 activity was entirely attributable to natural killer lymphocytes.

Combined effects of chemotherapy and host antitumor response in a murine histocompatible lymphoma model.

Combined effects of 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and host antitumor immune response were studied in mice inoculated intraperitoneally with histocompatible LSTRA leukemia cells carrying virus-induced transplantation antigens. Marked chemo-immune collaborative activity was found to occur when selected schedules of BCNU administration were employed. Moreover, synergist effects were also detected between chemotherapy and both specific and non-specific immunotherapy.

Relationships among tumor load, route of tumor inoculation, and response to immunochemotherapy in a murine lymphoma model.

The combined effects of nonspecific immunostimulation with Candida albicans (CA) and chemotherapy were studied in (BALB/cCr X DBA/2Cr)F1 and (C57BL/6Cr X DBA/2Cr)F1 mice bearing virus-induced LSTRA lymphomas. Paradoxically, animals treated with a relatively high number of tumor cells responded better to therapy with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) than those challenged with a low number of tumor cells. However, the majority of mice subjected to low initial tumor load were cured when they were treated with chemotherapy or chemotherapy plus booster injection of CA at a relatively "late" stage of the disease, i.e., when high tumor load was present in tumor-bearing hosts. It has been shown that this phenomenon, provisionally called high tumor load protection, occurs when the animals are challenged ip but not when they are challenged iv with the tumor and is abolished by total-body gamma-irradiation. Moreover, marked host protection can be attained when immunostimulated mice, inoculated iv with lymphoma cells, are subjected to simultaneous challenge with high inocula of the same tumor ip, followed by BCNU administration. These data stress the importance of the peritoneal cavity for successful CA plus drug treatment and suggest that optimal tumor "antigen load" should be present at the time of CA and/or BCNU administration.

Immunopotentiation of anticancer chemotherapy by Candida albicans, other yeasts and insoluble glucan in an experimental lymphoma model.

Several yeast species in the genera Candida, Saccharomyces and Cryptococcus showed powerful immunoadjuvant, chemotherapy-synergic effects against a histocompatible, virus-induced murine lymphoma. Sensitizing and booster intraperitoneal injections of 2 x 10(7) yeast cells on days -14 and +1 (with respect to tumor challenge on day 0) followed by treatment with antiblastic drugs (on day +5) were required to elicit optimum activity. The antitumor effect was not markedly influenced by the morphological growth form of merthiolate-inactivated C. albicans nor by the nature of the carbon source in the growth medium, except for C. albicans cells grown in a medium containing stearic acid, which were not effective. These cells had a higher ratio of soluble to insoluble cell wall components, as compared to glucose-grown cells, but this finding alone could hardly explain the lack of antitumor effects. Previous observations, suggesting that the alkali-acid insoluble beta-glucan (in the form of cell wall ghosts) is the only component of yeast cell walls endowed with antitumor activity comparable to that of whole cells, were confirmed and extended; the soluble mannan and glucan-protein fractions were unable to replace whole cells and glucan ghosts even as sensitizers or as boosting agents.

Cellular mechanisms underlying the adjuvant activity of Candida albicans in a mouse lymphoma model.

Inactivated Candida albicans (CA) possesses strong anti-tumor activity when combined with cytoreductive chemotherapy in a mouse lymphoma model. In the present study, experiments were performed in order to elucidate the mechanism(s) underlying CA immunoadjuvant activity. In vivo chemotherapy studies proved that the synergistic anti-tumor effects were lost in athymic (nu/nu) mice and were also abrogated by radiations. In vitro tests did not suggest a major involvement of natural cytotoxic effectors such as macrophages and natural killer cells nor did CA effects appear to be mediated by induction of interferon. It was concluded that the immunoadjuvant activity of CA largely relies on host responses against tumor-associated transplantation antigens with no major involvement of natural resistance immune mechanisms.