Treatment with the antigranulocyte monoclonal antibody RB6-8C5 impairs resistance of mice to gastrointestinal infection with Listeria monocytogenes

Treatment with the antigranulocyte monoclonal antibody (MAb) RB6-8C5 increased the severity of infection in mice intragastrically inoculated with Listeria monocytogenes. Most MAb RB6-8C5-treated mice died when inoculated intragastrically with as few as 4 x 10(4) L. monocytogenes bacteria, whereas most control mice survived intragastric inoculation with 4 x 10(8) L. monocytogenes bacteria. The increased severity of infection in MAb RB6-8C5-treated mice appeared to result from listerial multiplication in the spleen and liver rather than from local proliferation in the intestinal tract or mesenteric lymph nodes.     

2-deoxy-D-glucose-induced metabolic stress enhances resistance to Listeria monocytogenes infection in mice

OBJECTIVE: Bacteria are considered to be important in the pathogenesis of several forms of arthritis. The goal of this study was to apply the 16S ribosomal RNA (rRNA)-polymerase chain reaction method for the detection of bacterial DNA in synovial fluid (SF) and synovial tissue (ST) from inflamed joints. METHODS: Samples from 5 patients with septic arthritis and from 7 with osteoarthritis or arthritis secondary to joint trauma were used as controls. Samples from 6 patients with spondylarthropathy (SpA) and from 20 with undifferentiated arthritis (UA) were analyzed for the presence of bacterial DNA using universal 16S rRNA primers. Automated sequencing and comparative data analysis were performed to identify the species. RESULTS: In the positive control group, the bacterial species cultured from the synovium could be identified in all cases. No bacterial DNA was detected in the SF and ST from patients in the negative control group. In 4 of 6 patients with SpA and 7 of 20 with UA, the analysis of joint samples revealed the presence of bacterial DNA. Sequence analysis indicated the presence of multiple species, which was confirmed by sequencing of cloned products. CONCLUSION: When the the above techniques were used with a stringent regimen, we were able to demonstrate that it is possible to collect and analyze joint samples without contaminating bacterial DNA. The accumulation of phagocytic cells that contain bacterial DNA of various species could play a role in the pathogenesis of both SpA and UA.     

Elimination of resident macrophages from the livers and spleens of immune mice impairs acquired resistance against a secondary Listeria monocytogenes infection

To determine the kinetics of tissue macrophage and microglial engraftment after bone marrow (BM) transplantation, we have developed a model using the ROSA 26 mouse. Transplanted ROSA 26 cells can be precisely identified in recipient animals because they constitutively express beta-galactosidase (beta-gal) and neomycin resistance. B6/129 F2 mice were irradiated and transplanted with BM from ROSA 26 donors and their tissues (spleen, marrow, brain, liver, and lung) examined at various time points to determine the kinetics of engraftment. Frozen sections from transplanted animals were stained histochemically for beta-gal to identify donor cells. At 1, 2, 6, and 12 months posttransplantation, 98% to 100% of granulocyte-macrophage colonies were of donor (ROSA 26) origin determined by beta-gal staining and by neomycin resistance. Splenic monocytes/macrophages were 89% donor origin by 1 month confirming quick and complete engraftment of hematopoietic tissues. At this time, only rare ROSA 26 tissue macrophages or microglia were observed. Alveolar macrophage engraftment was evident by 2 months and had increased to 61% of total tissue macrophages at 1 year posttransplantation. The kinetics of liver Kupffer cell engraftment were similar to those seen in the lung. However, donor microglial engraftment remained only 23% of total microglia at 6 months and increased to only 30% by 1 year. Also, donor microglia were predominantly seen at perivascular and leptomeningeal, and not parenchymal, sites. The data show that microglia derive from BM precursors but turn over at a significantly slower rate than other tissue macrophages. No clinical or histological graft-versus-host disease was observed in the recipients of ROSA 26 BM. These kinetics may impact strategies for the gene therapy of lysosomal storage diseases. Because individual donor cells can be identified in situ, the ROSA 26 model should have many applications in transplantation biology including studies of homing and differentiation.     

Interleukin-6-deficient mice are highly susceptible to Listeria monocytogenes infection: correlation with inefficient neutrophilia

We have produced interleukin-6 (IL-6)-deficient mice to examine, in vivo, the wide variety of biological activities attributed to this multifunctional cytokine. To investigate the role of IL-6 during infectious disease, IL-6-deficient mice were challenged with sublethal doses of Listeria monocytogenes, a facultative intracellular bacterium. While normal control animals were able to clear the infection, mutant animals exhibited a high mortality rate and showed uncontrolled replication of the bacteria in the spleen and liver at 2 and 3 days postinfection. Sections of infected tissues showed an increase in the number and severity of inflammatory foci. All aspects of this phenotype in the mutant animals were completely reverted upon administration of recombinant murine IL-6 (rIL-6). Various parameters of natural killer (NK) cell and macrophage function were unaffected in the challenge of the mutant animals. However, IL-6-deficient animals failed to mount peripheral blood neutrophilia in response to listeriosis, whereas control animals displayed a prominent neutrophilia in the blood at 24 and 48 h postinfection. Additionally, we analyzed the efficacy of rIL-6 in protecting animals devoid of lymphocytes or devoid of neutrophils during listeriosis. Administration of rIL-6 was protective to animals devoid of lymphocytes, suggesting that the rIL-6 protective effect was not mediated through lymphocytes. In contrast, control and mutant animals depleted of neutrophils were refractory to the rIL-6 protective effect. These data suggest that IL-6 is critical early during listeriosis, perhaps acting by stimulating neutrophils either directly or indirectly. Additionally, these data show a promising therapeutic potential for rIL-6 administration during opportunistic infection.     

Bovine mastitis caused by Listeria monocytogenes: kinetics of antibody responses in serum and milk after experimental infection

The kinetics of antibodies in serum and milk directed against proteins from Listeria monocytogenes were studied using 4 lactating cows after infection was experimentally induced in the udder with four strains of serotypes 4b or 1/2a. Antibodies (IgG and IgA) in samples of composite quarter milk and serum of the cow were measured by indirect ELISA. Microtiter plates were coated with proteins obtained from the culture supernatant of L. monocytogenes 4b. After challenge, an IgG response in serum and milk to listerial infections in the udder occurred for all cows, although the response varied among cows. In sera, the IgG titers reached a peak at 9 to 13 wk after challenge and remained elevated until 21 to 33 wk after challenge. In milk, the IgG titer increased significantly 3 wk after the challenge for all cows. A weak and nonpersistent increase in IgA antibodies also occurred. These results indicate that IMI by L. monocytogenes induced an increase of antibodies in milk, which could be detected with an ELISA test using our antigenic preparation. Therefore, this antigenic preparation could be used for the evaluation of a new method of diagnosis for bovine mastitis caused by L. monocytogenes.     

Prevention of diabetes but not insulitis in NOD mice injected with antibody to CD4

Non-obese diabetic (NOD) mice were injected with a rat monoclonal antibody to CD4 from birth every two weeks through 6 months of age. These animals gained weight normally but < 11% of their spleen T cells were CD4+, compared with 28% of CD4+ in controls injected with polyclonal rat IgG. The reduction in CD4 cell percentage was associated with a reduction in the number of cells in the thymus and spleen following the injection. CD4+ cells which survived the injections were nevertheless able to enter cell cycle when stimulated by Con A. None of the CD4-treated NOD mice became diabetic by 6 months of age and none of the animals studied histologically at this time had insulitis. At 9 months of age (three months after stopping the CD4 injections) the mice made antibody to human IgG. At 1 year of age most of the male mice had insulitis, although none of the male or female mice had become spontaneously diabetic. Two thirds of animals injected with cyclophosphamide at 16 months became diabetic within 3 weeks. The results confirm that treatment with CD4 antibody in the first 6 months suffices to reduce the incidence of diabetes in NOD mice. The treatment does not prevent the subsequent development of insulitis in injected mice and does not prevent the accumulation of cells capable of causing overt diabetes after cyclophosphamide injection.     

Prolongation of survival of skin allograft with anti-Thy-1 monoclonal antibody in mice

Skin allograft rejection is mainly mediated by T lymphocytes. We investigated the cytotoxicity of anti-Thy-1 McAb to murine thymocytes in vitro, and evaluated the prolonging effect on the survival of murine skin allografts (BALB/c--C57BL/6) in vivo with the McAb. The results showed murine thymocytes were destroyed in vitro by the McAb with complement; the survival of skin allograft was prolonged in vivo with the McAb. Lymphocyte infiltration in skin allografts was inhibited. These results provide a valuable reference for the clinical usefulness of anti-CD3 McAb in prolonging survival of human skin allografts in burn patients.     

Protective activity of a murine monoclonal antibody against European bat lyssavirus 1 (EBL1) infection in mice

A mouse model was designed to test in vivo the efficacy of rabies immune globulins and specific neutralizing monoclonal antibodies to prevent European bat lyssavirus 1 infection. Human or equine rabies immune globulins previously found to contain variable amounts of neutralizing bat lyssavirus crossreactive antibodies were passively transferred to mice receiving intramuscularly a lethal dose of bat lyssavirus type 1. Immune globulins did not protect mice well against bat lyssavirus 1 whereas they reduced the mortality caused by rabies virus. In contrast, mice inoculated with bat lyssavirus 1 or rabies virus survived when passively immunized with bat lyssavirus 1 specific monoclonal antibody (mAb 8-2). This monoclonal antibody, an IgG2 alpha, recognized an epitope located in the antigenic site IIa of rabies glycoprotein. A mutation replacing the lysine 198 by glutamate in a rabies variant abrogated sensitivity to this neutralizing antibody. Because of its broad neutralizing spectrum against wild virus isolates, including European bat lyssaviruses, this monoclonal antibody should be a good candidate for rabies immune globulin replacement. It could improve efficacy of rabies vaccination, used either alone or in conjunction with human rabies immune globulins or monoclonal antibody cocktail to supplement their lack of crossreactivity to European bat lyssavirus 1.     

Genetics of resistance to Trypanosoma congolense in inbred mice: efficiency of apparent clearance of parasites correlates with long-term survival

To study the genetic parameters of resistance to Trypanosoma congolense infection, highly susceptible BALB/c and relatively resistant C57BL/6 mice were crossed to produce reciprocal F1 and F2 offspring. Mice were infected with T. congolense and monitored for parasitemia within the first 2 wk and also for their survival periods. BALB/c mice showed unrestrained parasite growth to the time of death (median survival period, msp = 12.0 days), whereas in C57BL/6 mice, parasitemia reached an initial peak on day 6 and was followed by a rapid apparent clearance of the parasites in an average period of 3 days. Their msp was 163.0 days. The F1 mice cleared the parasites, following the first peak of parasitemia, in an average period of 4 days and had an msp of 69.5 days. Thus, the F1 offspring displayed an intermediate phenotype between susceptible and resistant parents in terms of parasite clearance and survival period. Resistance in F2 mice, as measured by survival times, was inherited as a polygenic trait. Among F2 mice, there was an inverse correlation between the time taken to clear the initial wave of parasitemia and the survival period, r = -0.58; P < 0.05. Thus, the pattern of control of the parasites following the first peak of parasitemia appears to be a good predictive factor for the survival period of mice infected with T. congolense.     

Natural resistance to Marek''s disease at hatching in chickens lacking maternal antibody, and relationship between this early resistance and resistance acquired with age

A double-blind cross-over trial was conducted with 20 healthy paid volunteers for the evaluation of the subacute effects of chlorpromazine (CPZ) and sulpiride, in oral doses used for anxious outpatients, on psychomotor skills related to driving. Psychomotor performance was measured on the 7th and 14th days of treatment at 30, 90 and 150 min after the intake of 0.5 g/kg of an alcoholic or placebo drink. After the neuroleptics alone, reaction and coordination skills, but not attention, were slightly impaired, CPZ differing significantly from the placebo on the 14th day. Both drugs interacted additively with alcohol. The combined administration of CPZ and alcohol led to inaccuracy, a slowing of reactions and impaired proprioception and coordination. The combination of sulpiride and alcohol increased the error rate in the choice reaction test and impaired coordination in the coordination test driven at a free speed. It is concluded that the psychomotor decrement that occurs after 2 weeks of treatment with small doses of CPZ may effect the ability to control a motor vehicle. The concurrent administration of alcohol during treatment with CPZ or sulpiride may cause some extra risk in traffic or occupational life.     

Successful induction of adjuvant arthritis in mice by treatment with a monoclonal antibody against IL-4

Adjuvant arthritis (AA) is an experimental model of autoimmune disease in rats induced by immunization with Mycobacterium tuberculosis (MT). Induction of AA in other species, including mice, has been shown to be difficult. In the present study, we found that AA could be induced in mice if the animals were treated with a mAb (11B11 mAb) against IL-4. Histologically, the joints exhibited synovial edema with infiltration of many neutrophils in the early phase of inflammation. In its late phase, there were proliferation of synovium, cell infiltrate in which mononuclear cells predominated, and destruction of cartilage and subchondral bone. The joint inflammation was passively transferred to normal syngeneic recipient mice with lymphoid cells but not with sera from mice immunized with MT followed by treatment with the anti-IL-4 Ab. Delayed-type hypersensitivity (DTH) and proliferative responses of lymphoid cells to purified protein derivative were markedly augmented in 11B11 mAb-treated mice. Furthermore, the induction of arthritis was associated with a marked decrease in IL-4 secretion but a significant increase in IFN-gamma and IL-2 production. Thus, the neutralization of IL-4 by an anti-IL-4 Ab appears to be required for the induction of AA in mice.     

Failure of treatment with interleukin-2 receptor-specific monoclonal antibody in acute coxsackievirus B3 myocarditis in mice

T cell activation is assumed to play a crucial role in many viral infections. An important marker for the activation of T cells is the interleukin-2 receptor (IL-2R); resting T lymphocytes do not bear detectable amounts of IL-2R. AMT13, a rat monoclonal antibody against mouse IL-2R, inhibits interleukin-2-dependent cell growth both in vitro and in vivo. Therefore, to clarify the effects of anti-IL-2R antibody treatment upon coxsackievirus B3 (CB3)-infected C3H/He mice, AMT13, 1 microg/mouse per day, was administered, subcutaneously, starting on day 0 (group 2) in experiment I or on day 7 (group 4) in Experiment II for 7 days, respectively. Groups 1 and 3 were examined as infected controls. In both experiments, there was no significant difference in mortality or in the severity of myocarditis between the treated and the untreated groups. Also, myocardial CB3 titers on day 7 did not differ significantly between groups 1 and 2. In addition, the distribution of activated T cell subsets in the inflamed myocardium was not changed by the treatment, and the paucity of myocardial IL-2R-positive cells was confirmed in all groups. Effects of the antibody treatment were confirmed by a decrease in delayed type hypersensitivity. Although some reports have shown that anti-IL-2R antibody has been successfully applied to ameliorate acute renal graft-versus-host disease, to enhance survival of skin allografts, and to suppress diabetic insulitis, it did not exert a beneficial effect on acute CB3 myocarditis in mice.     

Unstable expression and thermal instability of a species-specific cell surface epitope associated with a 66-kilodalton antigen recognized by monoclonal antibody EM-7G1 within serotypes of Listeria monocytogenes grown in nonselective and selective broths

Conditions that resulted in unstable expression and heat instability of a cell surface epitope associated with a 66-kDa antigen in Listeria monocytogenes serotypes were identified with the probe monoclonal antibody (MAb) EM-7G1 in an enzyme-linked immunosorbent assay. This epitope appeared to be absent in three serotypes (serotypes 3b, 4a, and 4c), which did not react with MAb EM-7G1 irrespective of the enrichment broth tested. The remaining 10 serotypes were detected by MAb EM-7G1 only when cells were grown in nonselective brain heart infusion broth (BHI) or selective Listeria enrichment broth (LEB). When cells were grown in Listeria repair broth (LRB), only 6 of the 13 serotypes were detected by MAb EM-7G1, and recognition of serogroup 4 was completely lost. None of the 13 serotypes was detected by MAb EM-7G1 when cells were grown in two other commonly used Listeria-selective media, UVM1 broth and Fraser broth (FRB), indicating that possible loss of epitope expression occurred under these conditions. MAb EM-7G1 maintained species specificity without cross-reacting with live or heat-killed cells of six other Listeria spp. (Listeria ivanovii, Listeria innocua, Listeria seeligeri, Listeria welshimeri, Listeria grayi, and Listeria murrayi) irrespective of the enrichment conditions tested. Due to heat instability of the cell surface epitope when it was exposed to 80 or 100 degrees C for 20 min, MAb EM-7G1 is suitable for detection of live cells of L. monocytogenes in BHI or LEB but not in LRB, UVM1, or FRB enrichment medium.     

Induction of protective T cells against Listeria monocytogenes in mice by immunization with a listeriolysin O-negative avirulent strain of bacteria and liposome-encapsulated listeriolysin O

Only listeriolysin O (LLO)-producing strains of Listeria monocytogenes generate protective immunity in mice. Based on the findings that endogenous gamma interferon (IFN-gamma) production was induced only by such strains and that purified LLO could induce IFN-gamma from NK cells, we have postulated that LLO may play a pivotal role in the induction of Th1-type protective T cells, which are highly dependent on IFN-gamma. In this study, mice were immunized with L. monocytogenes ATCC 15313, an LLO-nonproducing avirulent strain, along with LLO encapsulated in liposome (LLO-liposome). LLO-liposome was highly potent in the induction of various cytokines, including IFN-gamma. Immunization of mice with either LLO-liposome or the viable strain ATCC 15313 alone did not induce protection against challenge infection. In contrast, the combination of LLO-nonproducing bacteria plus LLO-liposome induced a significant level of protective immunity mediated mainly by Th1-type cells capable of producing a large amount of IFN-gamma in an antigen-specific manner. The protection afforded by the combination was not dependent on LLO-specific cytotoxic T cells. These results support the idea that the inability of an LLO-nonproducing avirulent strain or killed bacteria to induce the generation of protective T cells is due not to the lack of a central T-cell epitope(s) but to the lack of ability to induce the production of endogenous cytokine during the early stage of immunization; the results also suggest that an appropriate use of LLO at least in an animal model may be effective in the induction of antigen-specific Th1-dependent protective immunity to various kinds of intracellular parasitic bacteria.     

Influence of maternal antibodies on vaccine responses: inhibition of antibody but not T cell responses allows successful early prime-boost strategies in mice

The transfer of maternal antibodies to the offspring and their inhibitory effects on active infant immunization is an important factor hampering the use of certain vaccines, such as measles or respiratory syncytial virus vaccine, in early infancy. The resulting delay in protection by conventional or novel vaccines may have significant public health consequences. To define immunization approaches which may circumvent this phenomenon, experiments were set up to further elucidate its immunological bases. The influence of maternal antibodies on antibody and T cell responses to measles hemagglutinin (MV-HA) were analyzed following MV-HA immunization of pups born to immune or control BALB/c mothers using four different antigen delivery systems: live or inactivated conventional measles vaccine, a live recombinant canarypox vector and a DNA vaccine. High levels (> 5 log10) of maternal anti-HA antibodies totally inhibited antibody responses to each of the vaccine constructs, whereas normal antibody responses were elicited in presence of lower titers of maternal antibodies. However, even high titers of maternal antibodies affected neither the induction of vaccine-specific Th1/Th2 responses, as assessed by proliferation and levels of IFN-gamma and IL-5 production, nor CTL responses in infant mice. On the basis of these unaltered T cell responses, very early priming and boosting (at 1 and 3 weeks of age, respectively) with live measles vaccine allowed to circumvent maternal antibody inhibition of antibody responses in pups of immune mothers. This was confirmed in another immunization model (tetanus toxoid). It suggests that effective vaccine responses may be obtained earlier in presence of maternal antibodies through the use of appropriate immunization strategies using conventional or novel vaccines for early priming.     

Inheritance of lipopolysaccharide-enhanced nonspecific resistance to infection and of susceptibility to endotoxic shock in lipopolysaccharide low-responder mice

In a previous study, we demonstrated that lipopolysaccharide (LPS) and other bacterial immunostimulants, in contrast to their activity in a closely related high-responder subline, failed to elicit nonspecific resistance in LPS low-responder mice against Klebsiella pneumoniae infection. To investigate the type of inheritance controlling the LPS-induced nonspecific resistance to infection, the present study was performed in low- and high-responder C3H sublines and in F1 and F2 hybrids. In addition, F1 mice were backcrossed to each parental type. Inheritance of susceptibility to endotoxin was also tested in both sublines and their hybrids and backcross progeny. For these latter assays, mice were previously adrenalectomized because removal of this gland considerably enhances their sensitivity. Our present findings are consistent with the hypothesis that LPS enhances nonspecific resistance to infection and that susceptibility to endotoxin shock in the absence of corticoids may be determined by a single autosomal dominant gene.     

Inhibition of tumor necrosis factor alpha alters resistance to Mycobacterium avium complex infection in mice

Increased production of tumor necrosis factor alpha (TNF-alpha) appears to play an important role in the progression of human immunodeficiency virus disease. One treatment strategy being explored is the use of TNF-alpha inhibitors. TNF-alpha also appears to be important in conferring resistance to infections, and the inhibition of this cytokine may exacerbate the emergence of opportunistic pathogens, such as Mycobacterium avium complex (MAC). The present study examines the possibility that inhibition of TNF-alpha will increase the progression of disease in mice infected with MAC. C57BL/6 beige (bg/bg) mice have been shown to be highly susceptible to infection with MAC and are routinely used for testing of antimycobacterial drugs. However, bg/bg mice are known to exhibit impaired phagocyte and natural killer cell function. Since these cell types are important sources of TNF-alpha, the susceptibility of the bg/bg strain to infection with MAC was compared with those of the heterozygous (bg/+) and wild-type (+/+) strains of C57BL/6 mice. The susceptibilities of the bg/bg and bg/+ strains of mice infected with MAC were found to be comparable. The +/+ strain was the least susceptible. Mycobacterial burden and serum TNF-alpha levels increased over time in all the strains of mice tested. The bg/+ strain of C57BL/6 mice was then chosen to measure the activity of TNF-alpha antagonists. Treatment with dexamethasone decreased serum TNF-alpha levels and increased mycobacterial burden. Treatment with anti-TNF-alpha antibody or pentoxifylline did not significantly alter serum TNF-alpha levels but increased mycobacterial burden. Treatment with thalidomide neither consistently altered mycobacterial burden in the spleens or livers of infected mice nor affected serum TNF-alpha levels.