Development of protective immunity in a murine model of melioidosis is influenced by the source of Burkholderia pseudomallei antigens

Melioidosis is a potentially fatal disease caused by the bacterium, Burkholderia pseudomallei. The current study was carried out to determine the mechanisms involved in the development of protective immunity in a murine model of melioidosis. Following intravenous infection with B. pseudomallei, both C57BL/6 and BALB/c mice demonstrated delayed-type hypersensitivity responses and lymphocyte proliferation towards B. pseudomallei antigens, indicating the generation of B. pseudomallei-specific lymphocytes. Adoptive transfer of these lymphocytes to na?ve C57BL/6 mice was demonstrated by a delayed-type hypersensitivity response. Mice were not protected from a subsequent lethal challenge with a highly virulent strain of B. pseudomallei, suggesting that a single intravenous dose of the bacterium is insufficient to induce a protective adaptive immune response. Attempts to induce resistance in susceptible BALB/c mice used repetitive low-dose exposure to live B. pseudomallei. Immune responses and resistance following subcutaneous immunization with live B. pseudomallei were compared with exposure to heat-killed, culture filtrate and sonicated B. pseudomallei antigens. Compared to heat-killed B. pseudomallei, significant protection was generated in BALB/c mice following immunization with live bacteria. Our studies also demonstrate that the type of immune response generated in vivo is influenced by the antigenic preparation of B. pseudomallei used for immunization.     

Efficacy of interferon-gamma and amphotericin B for the treatment of systemic murine histoplasmosis

The number of cases of systemic histoplasmosis has increased substantially in recent years, and improved therapy is needed. We examined the efficacy of immunomodulation with interferon (IFN)-gamma alone or in combination with a suboptimal regimen of amphotericin B for the treatment of primary systemic murine histoplasmosis. In the first study, BALB/c mice were infected with Histoplasma capsulatum G217B and treated with 10(5) U of IFN given every other day either preinfection and postinfection or only postinfection, alone or in combination with amphotericin B. IFN alone given subcutaneously (s.c.) postinfection prolonged survival over untreated controls (P < 0.01), whereas intravenous (i.v.) administration was ineffective. All combination regimens and amphotericin B alone significantly prolonged survival (P < 0.0001). The combination regimens of amphotericin B and IFN i.v. (pre- and postinfection) or IFN s.c. (postinfection) reduced the fungal burden in the liver and spleen; the latter regimen had superior efficacy in the spleen (P < 0.05) to either amphotericin B or IFN alone. After infection with a low-challenge inoculum, IFN given s.c. (pre- and postinfection) alone caused a significant reduction in fungal burden in the spleen (P < 0.001). In an acutely lethal model, combination regimens of IFN s.c. or i.v. and amphotericin B again prolonged survival (P < 0.01-0.001), with amphotericin B plus IFN given s.c. (pre- and postinfection) superior to all regimens (P < 0.05-0.01). This regimen also showed enhanced efficacy in causing the reduction of fungal burden in the spleen (P < 0.05). These results indicate that IFN in combination with AmB shows enhanced efficacy in the treatment of systemic histoplasmosis and support the potential utility of IFN as an adjunctive therapy.     

Plasmodium berghei: characterization of antigens and their role in inducing immunity to infection

In vitro, Plasmodium berghei infected erythrocytes incorporated 35S-methionine into 31 polypeptides with molecular weights from 21 kd to 300 kd. Hemoglobin and additional smaller molecular weight polypeptides were labelled with 35S-methionine by a population of uninfected, reticulocyte-rich rat erythrocytes. 3H-glucosamine was incorporated into at least 3 components by Plasmodium berghei infected erythrocytes. Uninfected, reticulocyte-rich rat erythrocytes did not incorporate 3H-glucosamine. Rabbit antisera against small, free plasmodia formed complexes which contained between 12 and 22 of the 31 labelled polypeptides in the 35S-methionine labelled antigen preparation. Rabbit antisera against soluble antigens washed from small, free plasmodia formed complexes containing many of the same labelled plasmodial polypeptides, however the reactions were particularly strong with those components which yielded polypeptides with molecular weights of 25 kd and 31 kd. Rabbit origin antisera against the 2 preparations did not form detectable complexes with the 3H-glucosamine labelled plasmodial components. Sera from rats undergoing progressive P. berghei infection formed complexes containing an increasing number of 35S-methionine labelled plasmodial polypeptides. Hyperimmune rat serum, the only serum protective upon passive transfer into mice, formed complexes containing 7 polypeptides with molecular weights of 35 kd, 75 kd, 80 kd, 92 kd, 100 kd, 150 kd and 190 kd. Antigens containing 1 or more of these polypeptides may be important in the induction of a protective antibody response against the parasite.     

Breakdown of mucosal immunity in the gut and resultant systemic sensitization by oral antigens in a murine model for systemic lupus erythematosus

Secreted IgA plays a pivotal role in the mucosal immunity to maintain the front line of body defense. We found that the level of fecal IgA was dramatically decreased in aged (NZB x NZW)F(1) (BWF(1)) mice developing lupus nephritis, whereas levels in similarly aged New Zealand Black (NZB) and New Zealand White (NZW) mice remained unchanged compared with young mice. The number of cells obtained from Peyer's patches was markedly decreased in aged BWF(1) mice. Aged BWF(1) mice showed increased susceptibility to pathogenic bacterial infection. Furthermore, oral administration of OVA failed to inhibit secondary IgG response induced by systemic immunization, suggesting defective oral tolerance in aged BWF(1) mice. A significant amount of orally administered OVA was incorporated directly into the intestinal lamina propria in aged BWF(1) mice whereas it was mainly localized in subepithelial domes and interfollicular region in Peyer's patches in young mice. T cells obtained from renal and pulmonary lymph nodes of aged BWF(1) mice that had been orally administered with OVA showed an Ag-specific T cell proliferation, whereas those from young BWF(1), aged NZB, and aged NZW mice did not. Interestingly, aerosol exposure to OVA of aged BWF(1) mice, which had been orally administered with the same Ag, provoked an eosinophil infiltration in the lung. These results demonstrate that mucosal immunity in the gut is impaired and oral Ags induce systemic sensitization instead of oral tolerance in the development of murine lupus.     

The induction of cellular immunity with soluble murine histocompatibility antigens

Soluble transplantation antigens have been prepared from various lymphoid organs of the mouse strains A and C57BL. These preparations have been partially characterized by gel filtration on Sephadex G-200 and G-100. The distribution of various antigenic activities, such as precipitation with rabbit antisera, inhibition of the cytotoxic reactions of heterologous antisera and of alloantibodies, differed considerably among the chromatographic fractions. The soluble antigen preparations retained their antigenic and immunogenic properties, as demonstrated by their ability to block the cytotoxic reactions of alloantisera and to modify tumor growth in immunized recipients. Immunization of normal recipients with the immunogenic transplantation antigen preparations led to the production of sensitized lymphocytes, capable of destroying allogeneic target cells in vitro. Sensitized lymphocytes appeared in the regional lymph nodes after a single injection of 200–300 μg of the antigen preparation, reaching a peak level between 9 and 12 days. On reimmunization, the cytolytic activity of lymph node cells increased considerably and sensitized lymphocytes also appeared in the spleens of immunized animals.     

Immunoprophylaxis against filarial parasite,Brugia malayi: potential of excretory-secretory antigens in inducing immunity

The role of excretory-secretory antigens in inducing immunity in the host againstBrugia malayi microfilariae and infective larvae was studied byin vitro antibody dependent cell-mediated reaction as well asin vivo inoculation of filarial parasites within a microchamber in the host. The immune sera of jirds raised againstBrugia malayi microfilarial and infective larval excretory-secretory antigens(Bm Mf ESA andBm L₃ ESA) promoted the adherence of peritoneal exudate cells toBrugia malayi microfilariae and infective larvaein vitro and induced cytotoxicity to the parasites within 48 h. The antiBm Mf ESA serum was more effective than antiBm L3 ESA serum in inducing cytotoxicity to microfilariae and both antisera had a similar cytotoxic effect on infective larvae. In the microchambers implanted in the immune jirds, host cells could migrate and adhere to the microfilariae and infective larvae and kill them within 48–72 h. Further,Mastomys natalensis immunized againstBm Mf ESA and L₃ ESA generated a high degree of protective response against circulating microfilariae. These results suggest that excretory-secretory antigens are effective in inducing resistance against filarial parasites and thus have potential in immunoprophylaxis.     

Complement-dependent injury and protection in a murine model of acute dextran sulfate sodium-induced colitis

Complement plays a key role in the pathophysiology of many inflammatory diseases, and in this study, we investigated the role of complement in the pathogenesis of inflammatory bowel disease. Compared to wild-type mice, mice deficient in C3 or factor B were protected from acute dextran sulfate sodium (DSS)-induced colitis. C1q/mannose-binding lectin (MBL) double-deficient mice, however, exhibited more severe colitis than wild-type mice. When mice were allowed to recover after DSS treatment, all C1q/MBL(-/-) mice died by day 2 of recovery period, and, surprisingly, all C3(-/-) and factor B(-/-) mice died by day 5. Serum endotoxin levels were significantly increased in complement-deficient mice prior to death, particularly in C1q/MBL(-/-) mice, and antibiotic treatment prevented the lethal effect of DSS in all complement-deficient mice. In contrast to complement deficiency, targeted complement inhibition with either complement receptor 2 (CR2)-Crry (blocks all pathways at C3 activation) or CR2-factor H (blocks alternative pathway) was highly protective at treating established acute colitis. Endotoxin levels remained low in complement-inhibited mice, and complement inhibition also reduced inflammatory cytokines, leukocyte infiltration, and tissue injury while improving wound repair and mucosal healing. CR2-factor H provided more effective protection than CR2-Crry. Thus, complement has both pathogenic and protective roles in acute DSS-induced colitis, and whereas the alternative pathway appears to play a key role in tissue inflammation and injury, the classical/lectin pathway provides important protection in terms of host defense and wound repair. Targeted inhibition of the alternative pathway may represent a therapeutic modality for treating acute phases of inflammatory bowel disease.     

TNF-alpha antagonism generates a population of antigen-specific CD4+CD25+ T cells that inhibit protective immunity in murine histoplasmosis

In both humans and mice, treatment with TNF-alpha antagonists is associated with serious infectious complications including disseminated histoplasmosis. The mechanisms by which inhibition of endogenous TNF-alpha alter protective immunity remain obscure. Herein, we tested the possibility that neutralization of this cytokine triggered the emergence of T cells that dampen immunity. The lungs of mice given mAb to TNF-alpha contained a higher proportion and number of CD4+CD25+ cells than controls. This elevation was not observed in IFN-gamma- or GM-CSF-deficient mice or in those given a high inoculum. Phenotypic analysis revealed that these cells lacked many of the characteristics of natural regulatory T cells, including Foxp3. CD4+CD25+ cells from TNF-alpha-neutralized mice suppressed Ag-specific, but not nonspecific, responses in vitro. Elimination of CD25+ cells in vivo restored protective immunity in mice given mAb to TNF-alpha and adoptive transfer of CD4+CD25+ cells inhibited immunity. In vitro and in vivo, the suppressive effect was reversed by mAb to IL-10. Thus, neutralization of TNF-alpha is associated with the induction of a population of regulatory T cells that alter protective immunity in an Ag-specific manner to Histoplasma capsulatum.     

Corticosteroid-induced suppression of murine B cell immune response antigens

The quantitative variation in expression of B cell surface immune response-associated antigens (sIa) that is induced by in vivo i.v. administration of dexamethasone was studied by flow microfluorometry. Injection of 40 micrograms of dexamethasone resulted in a 35 to 40% reduction in the expression of sIa within 3 hr, reached its maximum effect within 6 hr, which on average resulted in 75% suppression of control values of sIa, and by 12 hr after injection began returning towards baseline levels. The suppressive effect of dexamethasone on B cell sIa was dose dependent with respect to the length of time required to reach maximal suppression, as well as with respect to the duration of suppression that was attained. When injections of dexamethasone were repeated on consecutive days, no additional increase in the level of sIa suppression achieved was observed. B cell sIa was also diminished after injection of dexamethasone into athymic nude mice, which suggests that the suppressive effect of dexamethasone on B cell expression of sIa is not a T cell-dependent phenomenon. Taken together, these data suggest that the suppression of B cell sIa by corticosteroids may be a means whereby endogenous or exogenous corticosteroids are able to influence the normal as well as abnormal immunologic state.     

Toxoplasma gondii: immunogenicity and protection by P30 peptides in a murine model

Vaccines are promising for the control of toxoplasmosis. Here, we evaluated the immunogenicity of 17 peptides derived from SAG1 surface protein of Toxoplasma gondii in CH3 mice. Only 8 of 16 peptides induced specific antibodies. After a lethal challenge, only the vaccination with 4 of 17 peptides that were from the carboxy terminal end of the protein conferred significant survival. Our work shows that vaccination with peptides from the carboxy-terminal positions of SAG1 major surface protein of Toxoplasma protects mice against a lethal challenge.     

Stromelysin-3 suppresses tumor cell apoptosis in a murine model

Stromelysin-3 (STR-3) is a matrix metalloproteinase with a unique pattern of expression and substrate specificity. During embryogenesis and remodeling of normal adult tissues, STR-3 is produced by stromal cells in direct contact with epithelial cells undergoing regional apoptosis and selective cell survival. STR-3 is also overexpressed by interdigitating stromal cells in primary epithelial malignancies. Although STR-3 does not degrade classic extracellular matrix components, the enzyme promotes the establishment of local tumors in nude mice by as yet undefined mechanisms. STR-3 is induced when malignant epithelial cells come into contact with surrounding stromal elements; the active stromal cell-derived 45 kDa enzyme is subsequently processed to a 35 kDa protein without enzymatic activity. We have generated MCF-7 transfectants expressing wild type or catalytically inactive 45 kDa STR-3 (STR-3wt and STR-3cat-) or secreted 35 kDa STR-3 (35 kDa STR-3sec) and evaluated their implantation and survival in nude mice. Tumors developed significantly more rapidly in animals receiving STR-3wt, rather than vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Most importantly, STR-3wt tumors had a significantly lower percentage of apoptotic cells than tumors derived from vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Taken together, these studies suggest that the active STR-3 enzyme may increase tumor take by suppressing tumor cell apoptosis and that 45 kDa to 35 kDa STR-3 processing limits STR-3 activity at the tumor/stromal interface. Because STR-3 is secreted as an active enzyme rather than a proform, subsequent 45 kDa to 35 kDa STR-3 processing may represent a novel mechanism for regulating enzymatic activity.     

Toxoplasma gondii: an evaluation of diagnostic value of recombinant antigens in a murine model

Laboratory diagnostics of toxoplasmosis depends primarily on serological methods detecting specific antibodies. Since these methods do not always enable specific and sensitive recognition of the infection and phase of toxoplasmosis, the search for new diagnostic tools continues. Recombinant antigens promise a new alternative in diagnostics of Toxoplasma gondii infections. In this work the usefulness of six recombinant T. gondii antigens: GRA1, GRA6, GRA7, p35, SAG1, and SAG2 in the detection of primary murine toxoplasmosis was evaluated. Sera obtained from infected mice differing in their natural susceptibility to T. gondii infection, BALB/c (relatively resistant) and C57BL/6 (relatively susceptible), were tested using ELISA. During acute infection high response to GRA7, GRA6, and p35 antigens was noticed, whereas a strong reactivity with surface antigens SAG1 and SAG2 was characteristic for chronic toxoplasmosis. Our results show that the recombinant antigens are useful in distinguishing between acute and chronic toxoplasmosis regardless of the genetically determined susceptibility of the host.     

Rosiglitazone as a regulator of innate immunity in a cell model of hyperglycemia

Epidemiological studies have shown that severe inflammatory responses occur in patients with hyperglycemia. The molecular nature of these changes is currently under intense investigations. A central role of nuclear receptors PPAR has been shown in the regulation of metabolic changes associated with hyperglycemia, a selective agonist of nuclear receptor PPARγ rosiglitazone is used as a hypoglycemic drug. Rosiglitazone is known to have anti-inflammatory effects, but its properties as an anti-inflammatory drug in hyperglycemic conditions have not been studied. This was an aim of our work. We used a human cell culture model of hyperglycemia: HeLa cells incubated in the conditions of 25 mM glucose for 3 days. Control cells were incubated with 5 mM glucose. The cells were stimulated with lipopolysaccharide (LPS) that is known to trigger innate immune response through activation of Toll-like receptor 4 and influence mRNA expression levels of three of PPAR (α, β/δ, γ) isotypes as well as cyclooxygenase (COX-1 and COX-2). We have shown that under hyperglycemic conditions expression levels of PPARα and PPARβ/δ decreased almost twofold, expression level of COX-2 also decreased, while expression levels of COX-1 and PPARγ remained unchanged compared to those under normal glucose concentration. LPS administration in control cells leads to a 1.5–2.5-fold stimulation of expression of COX-2 and PPAR isotypes. In contrast, under hyperglycemia, LPS exhibited no effect on expression of COX-2 and the PPAR isotypes, which indicates potential mechanisms of hyperglycemia-related alterations in innate immunity. Rosiglitazone, an agonist of PPARγ, decreased expression level of PPARβ/δ and abolished the effect of LPS under hyperglycemia. Rosiglitazone also reduced expression level of COX-1 and COX-2, which indicates on the agonist possible role as an anti-inflammatory agent under high glucose concentrations. These data broaden applicability of rosiglitazone as an anti-inflammatory agent in hyperglycemic conditions.     

Complex requirements for nascent and memory immunity in pulmonary histoplasmosis.

The presence of functional T cells is often required for successful resolution of infections with intracellular pathogens, yet the mechanisms by which they contribute to elimination of the invading pathogen in primary and secondary immunity are only partly understood. We report that increased mortality of naive alpha/beta TCR+ or CD4+ T cell-depleted mice infected with the fungus Histoplasma capsulatum is associated with impairment of IFN-gamma production. Upon secondary infection, mice concomitantly depleted of CD4+ and CD8+ cells exhibit decreased survival beyond day 25 of rechallenge, whereas elimination of either T cell subset or B cell deficiency does not result in accelerated mortality compared with controls. Remarkably, despite a decrease of H. capsulatum CFU in lungs of CD4+ plus CD8+-deficient mice, a progressive increase in spleen CFU is observed. The ability to control fungus growth in lungs is associated with vigorous TNF-alpha, but not IFN-gamma, production by bronchoalveolar lavage cells. In contrast, spleen cells from CD4+ plus CD8+-deficient mice are unable to produce TNF-alpha. Thus, the cellular and molecular requirements for protective immunity vary between primary and secondary infection. Furthermore, in secondary histoplasmosis, a sharp contrast can be drawn between lungs and spleens in their reliance upon T cells to control fungal replication. The opposing activities of these organs can be ascribed in part to differential production of TNF-alpha.     

Allosensitization induced suppression of various murine tumors: role of non-H-2 antigens in antitumor immunity

Presence of alloantigens on various murine tumors was tested by tumor rejection in allosensitized Swiss mice. The results indicated the presence of alloantigen on immunogenic tumors like chemically induced fibrosarcoma (FS), ascitic sarcoma 180 (S 180) and immunogenic variant of lymphosarcoma (LS-A) in Swiss mice, while these antigens could not be detected by this procedure on spontaneous lymphosarcoma (LS). Allosensitization with skin graft was found to offer quantitatively higher antitumor resistance than the allosensitization achieved by allogeneic lymphocytes. Antitumor effect was not seen when tumor cells were inoculated earlier than day 3 of grafting. Further, host immunosuppression with whole body irradiation up to day of 3 of skin grafting abrogated the antitumor effect. H-2 compatible and non-H-2 incompatible skin graft sensitization of host could offer resistance against both S 180 and LS-A. Further, tumor immune mice rejected H-2 compatible, non-H-2 incompatible skin graft significantly earlier.     

Clonorchis sinensis-derived total protein attenuates airway inflammation in murine asthma model by inducing regulatory T cells and modulating dendritic cell functions

Asthma is characterized by Th2-mediated inflammation, resulting in airway hyperresponsiveness (AHR) through airway remodeling. Recent epidemiological and experimental reports have suggested an inverse relationship between the development of allergy and helminth infections. Infection by Clonorchis sinensis, a liver fluke that resides in the bile duct of humans, is endemic predominantly in Asia including Korea and China. Using a murine model for asthma, we investigated the effects of C. sinensis-derived total protein (Cs-TP) on allergen-induced airway inflammation and the mechanism underlying the protective effects of Cs-TP administration on asthma. Treatment with Cs-TP attenuated OVA-induced airway inflammation and methacholine-induced AHR, as well as eosinophilia development, lymphocyte infiltration into the lung, and goblet cell metaplasia. This protective effect of Cs-TP is associated with markedly reduced OVA-specific IgE and Th1/Th2 cytokine production. Moreover, Cs-TP increased the number of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells as well as their suppressive activity. In fact, proliferation of OVA-restimulated splenocytes was suppressed significantly. Cs-TP also inhibited the expression of such co-stimulatory molecules as CD80, CD86, and CD40 in LPS- or OVA-stimulated dendritic cells (DCs), suggesting that Cs-TP could interfere with the capacity of airway DCs to prime naïve T cells. These data demonstrate the capacity of C. sinensis to ameliorate allergic asthma and broaden our understanding of the paradoxical relationship between the allergic immune response and helminth infection.     

Vaccination in murine schistosomiasis with adult worm-derived antigens: variables influencing protection in outbred mice

Previous studies have shown that both permissive (mouse) and partially permissive (rabbit) hosts develop high levels of resistance against Schistosoma mansoni infection after vaccination with a multiple antigen extract (SE) obtained by incubation of living adult worms in saline, plus bacterial adjuvant. To investigate variables influencing SE-induced protection in murine schistosomiasis, a series of distinct vaccination protocols were performed focussing on the immunization dose, carrier systems, route, site and amplitude of challenge infection, and time between immunization and challenge. In addition, a new approach was adopted to evaluate SE protective activity, by means of population analysis of worm burden frequency distributions in a large scale study of vaccination in outbred Swiss mice. Distinct curves of frequency and a drastic difference in worm burden distribution of frequencies from SE-vaccinated x non-vaccinated mice were found. It was shown that SE could generate 75% mean protection in outbred mice even in the absence of adjuvant. In addition SE immunization was also able to induce full protection against lethal infection. SE-induced protection could be modulated by such parameters as dose of SE immunization/challenge interval, and route of cercariae injection. These data show that SE yields very high protective activity in outbred mice, and may provide a further insight for rational design of a vaccine in experimental schistosomiasis.     

T cell integrin overexpression as a model of murine autoimmunity

Integrin adhesion molecules have important adhesion and signaling functions. They also play a central role in the pathogenesis of many autoimmune diseases. Over the past few years we have described a T cell adoptive transfer model to investigate the role of T cell integrin adhesion molecules in the development of autoimmunity. This report summarizes the methods we used in establishing this murine model. By treating murine CD4+ T cells with DNA hypomethylating agents and by transfection we were able to test thein vitro effects of integrin overexpression on T cell autoreactive proliferation, cytotoxicity, adhesion and trafficking. Furthermore, we showed that the ability to inducein vivo autoimmunity may be unique to the integrin lymphocyte function associated antigen-1 (LFA-1). Published: October 24, 2003