Liver tissue schistosomiasis mansoni vaccine, as a third generation livestock immunogenes

To assess the effects of cholestasis during pregnancy on fetal and neonatal mRNA expression, protein mass, and function of the Na+/taurocholate cotransporting polypeptide (Ntcp), common bile duct ligation (BDL) was performed in pregnant rats on day 14 of pregnancy (maternal cholestasis [MC] group), and livers were harvested at days 20 and 21 of fetal life, as well as at days 4, 7, 14, 21, and 28 after birth. Sham-operated rats and their litters were used as controls. Ntcp steady-state mRNA levels, protein mass, and function were determined by Northern blotting, immunoblotting, and taurocholate (TC) transport studies in isolated short-term cultured hepatocytes, respectively. In addition, protein mass and function of the organic anion transporting polypeptide (Oatp1), another sinusoidal bile acid transporter, were studied at 4 weeks of age. The majority of pregnant cholestatic rats (94%) were able to carry pregnancy to term. Body and liver weights of the offspring from the MC group were lower than those from sham-operated animals at all postnatal time points. Ntcp steady-state mRNA levels, protein mass, and function were unaffected by MC. The ontogenic pattern of expression was identical in offspring from MC and controls with detection of the Ntcp mRNA at day 21 of fetal life. There was a significant increase in mRNA postnatally, reaching adult levels by 7 days of age. Protein mass and function of Ntcp as well as of Oatp1 were similar in offspring from MC and control groups at 4 weeks of age. In conclusion, maternal obstructive cholestasis during the last third of pregnancy does not affect the fetal/neonatal expression of the basolateral bile acid transporters, Ntcp and Oatp1. This suggests that the impaired bile acid excretion described in this experimental model is not related to altered uptake of bile acids in the affected neonate.     

Macrophages as effector cells of protective immunity in murine schistosomiasis. III. Loss of susceptibility to macrophage-mediated killing during maturation of S. mansoni schistosomula from the skin to the lung stage

Newly transformed skin-stage and lung-stage schistosomula were compared in terms of their susceptibility to killing mediated by activated mouse macrophages in vitro. Although skin-stage schistosomula were readily killed by macrophages activated as a consequence of either BCG or Schistosoma mansoni infection and used either as cell monolayers or in suspension, lung-stage larvae appeared to be totally resistant to this effector mechanism and survived normally when reinjected into mice. Resistance of schistosomula to in vitro damage by macrophages was evident as early as 18 hr after host infection and was complete in worms recovered at 42 hr. The insusceptibility of lung-stage larvae is apparently not due to a defect in effector cell-target contact, because the induction of extensive macrophage adherence to the worms by the addition of anti-mouse red blood cell antisera to the cultures had no effect on parasite viability. These findings provide additional support for the concept that schistosomula during their development to the lung stage undergo a generalized change affecting their susceptibility to a variety of different immunologic effector mechanisms.     

Eosinophil recruitment to the lung in a murine model of allergic inflammation. The role of T cells, chemokines, and adhesion receptors

Eosinophil accumulation is a distinctive feature of lung allergic inflammation. Here, we have used a mouse model of OVA (ovalbumin)-induced pulmonary eosinophilia to study the cellular and molecular mechanisms for this selective recruitment of eosinophils to the airways. In this model there was an early accumulation of infiltrating monocytes/macrophages in the lung during the OVA treatment, whereas the increase in infiltrating T-lymphocytes paralleled the accumulation of eosinophils. The kinetics of accumulation of these three leukocyte subtypes correlated with the levels of mRNA expression of the chemokines monocyte chemotactic peptide-1/JE, eotaxin, and RANTES (regulated upon activation in normal T cells expressed and secreted), suggesting their involvement in the recruitment of these leukocytes. Furthermore, blockade of eotaxin with specific antibodies in vivo reduced the accumulation of eosinophils in the lung in response to OVA by half. Mature CD4+ T-lymphocytes were absolutely required for OVA-induced eosinophil accumulation since lung eosinophilia was prevented in CD4+-deficient mice. However, these cells were neither the main producers of the major eosinophilic chemokines eotaxin, RANTES, or MIP-1alpha, nor did they regulate the expression of these chemokines. Rather, the presence of CD4+ T cells was necessary for enhancement of VCAM-1 (vascular cell adhesion molecule-1) expression in the lung during allergic inflammation induced by the OVA treatment. In support of this, mice genetically deficient for VCAM-1 and intercellular adhesion molecule-1 failed to develop pulmonary eosinophilia. Selective eosinophilic recruitment during lung allergic inflammation results from a sequential accumulation of certain leukocyte types, particularly T cells, and relies on the presence of both eosinophilic chemoattractants and adhesion receptors.     

Calomys callosus: an alternative model to study fibrosis in schistosomiasis mansoni. The pathology of the acute phase

Twenty Calomys callosus, Rengger, 1830 (Rodentia-Cricetidae) were studied in the early stage of the acute schistosomal mansoni infection (42nd day). The same number of Swiss Webster mice were used as a comparative standard. Liver and intestinal sections, fixed in formalin-Millonig and embedded in paraffin, were stained with hematoxilin and eosin, PAS-Alcian Blue, pH = 1.0 and 2.5, Lennert's Giemsa, Picrosirius plus polarization microscopy, Periodic acid methanamine silver, Gomori's silver reticulin and resorcin-fuchsin. Immunohistological study (indirect immunofluorescence and peroxidase labeled extravidin-biotin methods) was done with antibodies specific to pro-collagen III, fibronectin, elastin, condroitin-sulfate, tenascin, alpha smooth muscle actin, vimentin and desmin. The hepatic granulomas were small, reaching only 27% of the volume of the hepatic Swiss Webster granuloma. They were composed mainly by large immature macrophages, often filled by schistosomal pigment, characterizing an exsudative-macrophage granuloma type. The granulomas were situated in the parenchyma and in the portal space. They were often intravascular, poor of extracellular matrix components, except fibronectin and presented, sometimes alpha smooth muscle actin and vimentin positive cells. The C. callosus intestinal granulomas were similar to Swiss Webster, showing predominance of macrophages. Therefore, the C. callosus acquire very well the Schistosoma mansoni infection, without developing strong hepatic acute granulomatous reaction, suggesting lack of histopathological signs of hypersensitivity.     

Immunoglobulin A response in murine schistosomiasis: stimulatory role of egg antigens

The immunoglobulin G (IgG) and IgA antibody responses to different Schistosoma mansoni antigens have been determined in chronically infected mice as well as in unisexually infected animals. With a panel of enzyme-linked immunosorbent assays (ELISAs), soluble antigens from furcocercariae, adult worms, and eggs were probed with sera collected at 3-week intervals. Bisexually infected animals developed significant IgG and IgA antibody responses to the antigens tested, which increased after egg deposition. In unisexual infections no significant differences were recorded in the IgG antibody profile for furocercaria and adult worm antigens, whereas the IgA antibody response was impaired. Both the IgA and IgG antibody responses toward egg antigens were reduced compared with those in a bisexual infection. Furthermore, a specific mucosal IgA antibody response was observed only in the bisexually infected animals. Histological analysis performed on bisexually infected mice led to the observation of eggs and granulomatous lesions within the Peyer's patch follicles, which are essential sites for the induction of mucosal immunity in the intestine. These data suggest a relationship between egg deposition and the induction of the IgA antibody response toward schistosomes.     

Identification of the immunodominant T cell epitope of p38, a major egg antigen, and characterization of the epitope-specific Th responsiveness during murine schistosomiasis mansoni

A recently cloned major Schistosoma mansoni egg Ag p38 induced and elicited strong Th1-type responsiveness in mice of H-2k haplotype. Now, we have identified the immunodominant T cell epitope of p38 and analyzed the dynamics of epitope-specific Th responsiveness during murine schistosomiasis mansoni. Overlapping recombinant and synthetic peptides that encompassed the full-length 354 amino acid of p38 were tested for proliferation and cytokine production in peptide- or p38-sensitized mice. The immunodominant T cell epitope of p38 that elicited pulmonary granuloma formation was localized within peptide P4 (amino acids 235-249). The P4-specific cytokine response of splenocytes that had been sensitized s.c. with p38, P4 or soluble egg Ags in IFA, or i.p. with 3000 eggs was predominantly as the Th1 type, with strong IL-2 and IFN-gamma, but trace amounts of IL-4 and IL-5 secretion. At 6.5 wk of infection, splenocytes and mesenteric lymph node cells responded to p38/P4 peptides with predominantly Th1-type responsiveness. This response did not switch to a Th2-type pattern from 8 wk onwards; rather, it underwent down-modulation. Moreover, the hepatic granuloma lymphocytes at 6.5 wk responded to p38/P4 predominantly with Th1-type cytokine production, indicating that they participate in early granuloma formation. From 8 wk onwards an immune deviation to the p38-specific response was observed that was manifested by rising IgG1, IgE, and IgG2a Ab production as opposed to declining Th1- and Th2-type cytokine secretion.     

Anti-viral immunity: spotting virus-specific T cells

Historically, quantitation of virus-specific CD8+ T cells has been accomplished by limiting dilution analysis of cytotoxic precursor cells. Recent studies have shown that this technique greatly underestimates the actual number of antigen-specific cells and have provided new insight into anti-viral immune responses.     

Hycanthone dose-response in treatment of schistosomiasis mansoni in St. Lucia

Clinical trials of hycanthone (single intramuscular dose) were undertaken in schistosomiasis mansoni patients in St. Lucia at five dose levels: 3.0, 2.5, 2.0, 1.5, and 1.0 mg/kg body weight. The most common side effect, vomiting, decreased in frequency from 51% at the highest dose to 3% at the lowest; minor side effects showed a similar trend. Three fecal specimens were examined before and at 6 months after treatment by qualitative, quantitative, and hatching techniques. All dose levels caused reductions in egg excretion of 89 to 98%. Rates of cure (absence of eggs by all three methods) according to dose (descending), pretreatment egg output (0-19, 20-49, 50-399, 400+ eggs/ml feces), and age (0-7, 8-14, 15-29, 30+ years) were analyzed to estimate the effect of each variable if the others had been constant. For dose, the standardized percentage success rates were 53.9%, 62.0%, 51.2% 54.0%, and 27.4%; for egg output, 67.0%, 51.8%, 43.2%, and 21.7%; and for age, 25.2%, 34.5%, 59.3% and 57.4%. Logit regression analysis shows a significant difference in cure rate (a) between the lowest dose and all others, among which latter there was no difference, (b) between patients excreting 0 to 49 eggs/ml before treatment and those excreting 50+ eggs/ml, and (c) between the age groups 0 to 14 and 15+ years. All dose levels caused some regression in enlargement of liver or spleen. A dose of 1.5 to 2.0 mg/kg body weight is considered to be as effective as one of 3.0 mg/kg and more acceptable for a control program because of the marked reduction in side effects.     

Cytokine profiles of lesional and splenic T cells in Porphyromonas gingivalis infection in a murine model

T cell cytokine profiles in the spleens and Porphyromonas gingivalis-induced lesions of P. gingivalis-immunized mice were examined. BALB/c mice were immunized with P. gingivalis outer membrane (OM) antigens/mouse weekly for 3 weeks followed by challenge with live organisms 2 weeks after the final immunization. Control mice were immunized with PBS. Spleens were excised at 0 and 4 days and lesions at 1, 4, and 7 days after challenge. Splenic and lesional CD4 and CD8 cells were stained for intracytoplasmic interleukin (IL)-4, interferon (IFN)-gamma, and IL-10. More than 50% of the T cells in the spleens of immunized mice were IFN-gamma positive at day 0 which was significantly higher than for IL-4 or IL-10, these levels decreasing significantly 4 days after challenge. Less than 6% of the T cells in sham immunized mice were cytokine positive at day 0, although at day 4, there was a significant increase in the percent IL-10 positive CD4 cells and IL-4 and IL-10 positive CD8 cells. There were no differences in the percent IL-4, IFN-gamma, or IL-10 positive T cells in the lesions of immunized mice, but there was a dramatic decrease at day 7 to very low levels in control mice. In conclusion, the results of the present study show a predominant Th1 response in the spleens of BALB/c mice after immunization with P. gingivalis OM antigens, suggesting that a protective immune response to P. gingivalis may involve a strong IFN-gamma response.     

Some genetic, clinical and immunologic interrelations in schistosomiasis mansoni

The aim of the present work was to study the possible association of some class I, II MHC gene products with variations in the clinico-pathological outcome of human schistosomiasis mansoni as well as with the variability in immune responsiveness. The study was carried out on 47 patients with schistosomiasis mansoni and 20 healthy volunteers served as control group for the immunological parameters and 200 subjects for the genetic studies. The following were determined: class I, II HLA typing, serum IgG, IgM, C3c, immediate intradermal test and passive haemagglutination using S mansoni worm antigen, T lymphocyte subsets, delayed intradermal test and leukocyte migration inhibition using phytohaemagglutinin (PHA) and soluble egg antigen (SEA) of S mansoni. A statistically significant association was found between HLA-B5 and DR3 and with the occurrence of hepatosplenic disease; this phenotype also correlated with changes in T lymphocyte subsets and high immune reactivity, both humoral and cell mediated. HLA-DQI was also associated with failure to develop hepatosplenic disease. The present study consolidates also the view of the important role of host immune reactivity in the clinical outcome of schistosomiasis mansoni and demonstrates the contribution of the genetic impact on both clinical and immunological heterogeneity of the disease.     

The role of T cells in polyethylene particulate induced inflammation

OBJECTIVE: To investigate the role of T lymphocytes in ultra-high molecular weight polyethylene (UHMWPE) induced inflammation in joint arthroplasty. METHOD: We address the role of T cells in wear induced inflammation by injecting the knee joints of both immune competent rats and mice and severe combined immunodeficient (SCID) mice with UHMWPE. Histological and immunohistochemical analysis of the synovial tissues was compared. Interaction between human T cells and UHMWPE particles was examined in vitro using T cell activation assays. RESULTS: Histological and immunohistochemical analysis of the knees of the immune competent animals showed significant UHMWPE induced inflammation. In contrast, the tissue in the SCID mice knee joints showed very little inflammatory response to UHMWPE despite phagocytosis of the particulate. Since the SCID mice have no functional T or B lymphocytes, it is highly likely that the lack of inflammation in knee joints may be due to the absence of mouse T cells, as the infiltration of T cells into the joint tissue may enhance the inflammatory response to UHMWPE particles. T cell activation assays showed that T cells were not directly activated by UHMWPE particles and the nature of the interaction was not revealed from these experiments. CONCLUSIONS: Although T cells are not directly involved in UHMWPE particle induced inflammation, as shown by the T cell activation assays, the histological data from the mice studies clearly show differences in the amplitude of inflammation from animals with and without functional T cells. Our studies suggest that the T cells may enhance the inflammatory response due to a bystander effect. Since the macrophages upon ingestion of UHMWPE particles release several cytokines including tumor necrosis factor-alpha, interleukin 1, and IL-6, it is possible that T cells in the vicinity of these macrophages may become attracted to the knee joint and activated due to cytokine release.     

Local immunity in lung-associated lymph nodes in a murine model of pulmonary histoplasmosis

Local immunity against acute pulmonary histoplasmosis was studied in the lung-associated lymph nodes of normal nonimmune mice infected intratracheally with live Histoplasma capsulatum yeasts. The phenotypes and distribution of cells in lung-associated lymph nodes and spleens were determined by flow cytometry. In addition, the immune responsiveness of these cells was evaluated by in vitro blastogenesis. Anti-H. capsulatum antibodies in serum and H. capsulatum antigen in tissue were measured by immunoassays. Cellular immune responses were greater in the lymph nodes than in the spleens. In lymph nodes 7 days after infection, a marked increase in the number of B lymphocytes caused the percentage to rise to 43%, compared with 26% in controls, and it remained elevated throughout the course of infection. A CD3+ cell that did not express CD4 or CD8 increased in number until it constituted 21% of lymph node cells, compared with 5% in controls, by day 14. The numbers of CD4+ and CD8+ T lymphocytes were modestly increased from days 7 to 35, but their percentages dropped because of the greater numbers of B lymphocytes and CD3+4-8- cells. Macrophages consistently constituted 2 to 3% of lymph node cells during the study. In spleens 7 days after infection, the percentage of macrophages in infected mice rose to 21%, compared with 9% in controls, but the total spleen cell number did not increase until day 14, when all cell subsets were nearly double in number. The in vitro blastogenic response of lymph node cells to H. capsulatum peaked at day 7, but spleen cell response was minimal during the course of infection. Histoplasma-specific serum immunoglobulin G antibodies reached peak levels by day 21 and remained high to the end of the study. In contrast, levels of antigen-specific immunoglobulin M antibodies were very low. These data suggest that antigen-specific immune responses occur in lung-associated lymph nodes and that this draining lymph node response may be an important component in host defense against Histoplasma lung infection.     

Neutrophils as effector cells of T-cell-mediated, acquired immunity in murine listeriosis

The control of the infections caused by Listeria monocytogenes, considered an example of an intracellular parasite, is thought to involve co-operation between antigen-specific T cells and activated macrophages. Here we investigated the participation of polymorphonuclear leucocytes in the mechanisms of resistance during the immune phase of the antimicrobial response to L. monocytogenes infection. We found that BALB/c mice were unable to express T-cell-mediated (acquired) immunity to this pathogen in the absence of granulocytes. We propose that neutrophils should be included in the concept of cell-mediated immunity and that their antimicrobial role is not exclusively expressed during the early phases of a primary infection.     

Patterns of infection and transmission of human schistosomiasis mansoni and schistosomiasis haematobium in White Nile Province, Sudan

The local effect of salbutamol and N6,2'-O-dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2 cyclic AMP) on the rat pleural inflammation caused by allergen was investigated. Antigen (ovalbumin, 12 micrograms/cavity) intrathoracically administered to immunized rats led to a marked pleural protein extravasation and leukocyte infiltration, as attested by the quantification of protein and enumeration of leukocytes recovered from the pleural cavity. Salbutamol (10-40 micrograms/cavity) and the cell-permeable cyclic AMP analogue, Bt2 cyclic AMP (20-160 micrograms/cavity), injected 1 h and 5 min before the antigen, respectively, inhibited the exudation occurring within 30 min, and neutrophil and eosinophil accumulation occurring 4 and 24 h, respectively. The late eosinophilia was also markedly attenuated by salbutamol administered 10 min post-challenge, when mast cells had already been degranulated. Pretreatment with the beta-adrenoceptor antagonist propranolol (1 mg/kg, i.v.) failed to modify the inhibitory effect of Bt2 cyclic AMP, but abolished the blockade caused by salbutamol of leukocyte infiltration under conditions where the salbutamol anti-exudatory activity was impaired to about 80%. In another set of experiments, salbutamol (20 and 40 micrograms/cavity) markedly inhibited the exudation caused by histamine and 5-hydroxytryptamine (5-HT) which, though to a lesser extent, was also sensitive to Bt2 cyclic AMP (80 micrograms/cavity). As observed with allergic pleurisy, propranolol impaired the inhibition by salbutamol of histamine- and 5-HT-induced exudation, whereas the Bt2 cyclic AMP inhibition was not affected. We conclude that salbutamol and Bt2 cyclic AMP share the ability to inhibit pleural exudation and leukocyte recruitment caused by allergen in immunized rats, suggesting that the anti-inflammatory effect of salbutamol may be mediated by a cyclic AMP signaling pathway, probably via beta 2-adrenoceptor activation.     

Virus-specific immunity after gene therapy in a murine model of severe combined immunodeficiency

Human severe combined immunodeficiency (SCID) can be caused by defects in Janus kinase 3 (JAK3)-dependent cytokine signaling pathways. As a result, patients are at high risk of life-threatening infection. A JAK3 -/- SCID mouse model for the human disease has been used to test whether transplant with retrovirally transduced bone marrow (BM) cells (JAK3 BMT) could restore immunity to an influenza A virus. The immune responses also were compared directly with those for mice transplanted with wild-type BM (+/+ BMT). After infection, approximately 90% of the JAK3 BMT or +/+ BMT mice survived, whereas all of the JAK3 -/- mice died within 29 days. Normal levels of influenza-specific IgG were present in plasma from JAK3 BMT mice at 14 days after respiratory challenge, indicating restoration of B cell function. Influenza-specific CD4(+) and CD8(+) T cells were detected in the spleen and lymph nodes, and virus-specific CD8(+) effectors localized to the lungs of the JAK3 BMT mice. The kinetics of the specific host response correlated with complete clearance of the virus within 2 weeks of the initial exposure. By contrast, the JAK3 -/- mice did not show any evidence of viral immunity and were unable to control this viral pneumonia. Retroviral-mediated JAK3 gene transfer thus restores diverse aspects of cellular and humoral immunity and has obvious potential for human autologous BMT.     

Gene therapy with a single chain interleukin 12 fusion protein induces T cell-dependent protective immunity in a syngeneic model of murine neuroblastoma

A major goal of tumor immunotherapy is the effective eradication of established metastases associated with the induction of a T cell-mediated protective immunity. We achieved this in a poorly immunogenic murine neuroblastoma model by gene therapy with a single chain interleukin 12 (scIL-12) fusion protein that assures equal expression of its p35 and p40 subunits. Thus, NXS2 hybrid neuroblastoma cells (C1300 x dorsal root ganglion cells), which form experimental bone marrow and liver metastases in syngeneic A/J mice, were transduced with a gene encoding murine interleukin 12, monomerized by introduction of a protein linker between the p35 and p40 protein chains of this heterodimeric cytokine. We demonstrate for the first time that subcutaneous vaccination with these transduced cells induces a protective immunity, as indicated by the complete absence of liver and bone marrow metastasis after challenge with NXS2 wild-type tumor cells. Furthermore, vaccination of animals with established liver and bone marrow metastases completely eradicated liver metastases and suppressed bone marrow metastases. The local and systemic immune response against scIL-12-transduced NXS2 cells is largely dependent on CD8(+) T cells. This was demonstrated in vivo by depletion of immunocompetent A/J mice with monoclonal anti-CD4 and anti-CD8 antibodies and in vitro by specific major histocompatibility complex, class I-restricted CD8(+) T cell-mediated killing of NXS2 and their parental C1300 neuroblastoma cells. In conclusion, we demonstrate successful anti-tumor immunotherapy with an scIL-12 fusion protein that could facilitate clinical application of interleukin 12 gene therapy.     

CD8 T cells in HIV infection: mechanisms of immunity

CD8 lymphocytes probably play a key role in the immunologic defense against HIV. The evidence is that they control viral replication by at least two mechanisms: direct antigen-specific cytolysis, which appears to be required for optimal suppression, and release of soluble antiviral factors. New therapeutic strategies are taking into account mechanisms by which the virus may evade CD8 control.