Analysis of immune response patterns in naïve and Plasmodium berghei-infected young rats following a ferroquine treatment

The direct antimalarial activity of ferroquine (FQ, SSR97193), a chloroquine (CQ) derivative, is well established. To determine whether the FQ anti-parasite activity affects the host immune properties, we have investigated its effect on several immunological parameters in young rats infected with Plasmodium berghei and compared it with that of CQ. In uninfected young rats, treatment with either drug did not show any impairment in the cellular distribution of spleen cells in their response to mitogens and did not induce the production of IL-10 in vivo. After infection, rats treated with CQ or FQ showed no parasitemia and survived with no recrudescence, in comparison with placebo. Nevertheless, FQ cured young rats more rapidly than its parent drug. Analysis of cellular distribution including CD4+TCR+, CD8+TCR+, NK and NKT cells in blood and spleen and the production of specific antibodies did not reveal any alteration of these parameters in infected young rats treated either with CQ or FQ. However, we observed a persistence of CD4+CD25+T-cells in infected CQ-treated rats when compared with infected FQ-treated rats, very likely related to the delay of blood parasite clearance by CQ-treatment. Another significant difference is that the CQ treatment dramatically inhibited the lymphoproliferative response of young infected rats when compared with FQ. Collectively, the absence of any observable immunotoxic effects due to FQ in naïve and infected young rats, together with previous results indicating the susceptibility to FQ of all Plasmodium falciparum field isolates and CQ-resistant strains make it a promising drug for malarial treatment.     

Immune mechanism of rats on Nippostrongylus brasiliensis in vitro. II. The influence of lymphocytes and peritoneal cells

Nippostrongylus were collected from the intestines of rats 6 days p.i. and kept under sterile conditions in cultures. Serum, lymphocytes and peritoneal cells of immune or non-infected animals were added in various combinations to the culture media. The culture media were changed 2-3 times in an experimental period of 10 days, resp. serum and cells were added. The lymphocytes were isolated from the peripheral blood or from the mesenterial lymph nodes whereas the mononuclear cells were obtained from the peritoneal cavity. Serum and lymphocytes from the peripheral blood both from immune and non-infected rats, had no increased lethal effect on Nippostrongylus. The highest lethality rate of adults (65-68%) was achieved in cultures with peritoneal cells and lymphocytes from the lymph nodes of sensitized rats. Serum of infected or non-infected animals had no influence on adult Nippostrongylus in cultures with these cell combinations. In the controls without any cell-supplements the survival rate of the parasites was up to 88%.     

Role of oxidative stress and apoptosis in the placental pathology of Plasmodium berghei infected mice

Placental malaria is a common clinical complication during pregnancy and is associated with abortion, premature delivery, intrauterine growth retardation and low birth weight. The present study was designed to delineate the underlying mechanism of placental pathology during malarial infection with special reference to oxidative stress and apoptosis. Experimentally, pregnant BALB/c mice were infected with Plasmodium berghei infected red blood cells on gestation day 10. The presence of malarial infection in placenta was confirmed by histopathological studies. It was observation that infected placenta had plugged placental sinusoids with parasitized red blood cells and malarial pigments. Interestingly, we found significant increase in the level of malondialdehyde, the index of oxidative stress and decreased activity of catalase, the antioxidant in infected placenta. Furthermore, in infected placenta the oxidative stress mediated apoptosis was determined by DNA fragmentation assay, ethidium bromide/acridine orange staining and caspase activity. It was observed that oxidative stress begin after second day of malarial infection. Interestingly, it was observed that there was down regulation of anti-apoptotic protein Bcl-2 and up regulation of pro-apoptotic protein Bax in infected placenta, suggesting the involvement of mitochondrial pathway of apoptosis which was further confirmed by activation of caspase 9. However, no change in the expression of Fas gene and caspase 8 activity, indicated the absence of death receptor pathway. Thus, it can be concluded that the placental pathology during malarial infection is mediated by mitochondrial pathway of apoptosis occurring due to augmented lipid peroxidation which may in turn jeopardise the materno-fetal relationship.     

Can the surface immobilization antigens of Philasterides dicentrarchi (Ciliophora: Scuticociliatida) be used as target antigens to develop vaccines in cultured fish?

In order to test whether immobilization antigens (i-antigens) of Philasterides dicentrarchi could be suitable antigenic targets against scuticociliatosis, polyclonal olive flounder (Paralichthys olivaceus) sera were raised against P. dicentrarchi by immunization with lysates of ciliates grown using chinook salmon epithelial (CHSE) cells, and the ability of the immune sera to kill the ciliates via classical complement pathway was analyzed in relation to agglutination activity. The immune sera showed clear agglutination activity against the CHSE-cultured ciliates. However, the agglutinated ciliates were not killed but escaped from the agglutinated mass within a few hours. Ciliates isolated from fish artificially infected with the same population of CHSE-cultured ciliates were not agglutinated by the immune sera even at the lowest dilution. In antibody-dependent complement-mediated killing (ADCK), the immune sera completely killed the CHSE-cultured ciliates at relatively higher serum dilutions (showing low or no agglutination activity). However, CHSE-cultured ciliates were not killed completely at lower immune serum dilutions (showing high agglutination activity). In contrast to CHSE-cultured ciliates, the ciliates isolated from infected fish were killed at lower dilutions of the immune sera in spite of no agglutination response. Considering the presence of various i-antigen types, ability to change i-antigen type in response to corresponding antibody, and relatively low ADCK activity at high agglutination titer, i-antigens of P. dicentrarchi may not be good targets for subunit vaccine development. To develop subunit vaccines against scuticociliatosis, other surface antigens expressed constitutively or expressed specifically under the infection state for survival of the ciliates in the host fish might be more favorable to elicit protective antibodies than the surface i-antigens.     

The age-related resistance of rats to Plasmodium berghei infection is associated with differential cellular and humoral immune responses

In this study, we investigated how the age of rats would affect the course of infection of and the immune response to Plasmodium berghei. Both young (4-week-old) and adult rats (8-week-old) can be infected with P. berghei ANKA strain, with significantly higher levels of infected red blood cells in young rats. While 100% of young rats succumbed to infection, adult rats were able to clear blood parasites and no mortality was observed. Analysis of cellular distribution and circulating cytokines demonstrated the persistence of CD4+/CD25+ T cells and high expression of circulating interleukin-10 (IL-10) during the progression of infection in young-susceptible rats, whereas high levels of CD8+ T cells and natural killer T cells are detected in adult-resistant rats. Analysis of antibody isotypes showed that adult rats produced significantly higher levels of interferon-gamma (IFN-gamma)-dependent IgG2c antibodies than young rats during infection. Further evaluation of the role of IL-10, IFN-gamma and of immune cells showed that only the adoptive transfer of spleen cells from adult-resistant rats was able to convert susceptibility of young-susceptible rats to a resistant phenotype. These observations suggest that cell-mediated mechanisms are crucial for the control of a primary infection with P. berghei in young rats.     

Blood groups of crab-eating macaques (Macaca fascicularis) demonstrated by isoimmune rhesus monkey (Macaca mulatta) sera.

Twenty-one isoimmune sera produced in rhesus monkey (Macaca mulatta) containing type-specific antibodies for simian-type red cell antigens were tested for their cross-reactivity with red cells from crab-eating macaques (M. fascicularis). The majority of the antisera gave cross-reactions determining polymorphisms in the red cells of crab-eating macaques, homologous to those of rhesus monkeys. These results attest to the close taxonomic realationship between the two species of macaques, and have the practical implication that isoimmune sera produced for blood typing can also be used for typing red cells from related species, as has been also observed in studies on apes.     

The opsonizing activity of the sera of hamadryas baboons immunized with a preparation of the outer membranes of Francisella tularensis (based on data from the luminol-dependent luminescence method)]

The opsonizing properties of sera obtained from hamadryas baboons immunized with the preparation of F. tularensis outer membranes (OM) were studied with the use of luminol-dependent chemiluminescence (CL) of whole blood. The immunization of monkeys with the OM preparation was shown to lead to the formation of functionally active antibodies possessing opsonizing properties with respect to virulent F. tularensis. Immune sera obtained from the animals immunized with live vaccine and from those immunized with OM preparation had no essential differences in their opsonizing properties. The level of IgG antibodies in immune sera correlated with the CL parameters of whole blood in the presence of F. tularensis opsonized with these sera. Increased CL of phagocytes observed after addition of bacteria and immune sera under test to whole blood taken from a nonimmune donor made it possible to evaluate the functional activity of antibodies, thus permitting its use as a test for the evaluation of the effectiveness of new vaccine preparations.     

One-step concentration of malarial parasite-infected red blood cells and removal of contaminating white blood cells

Background

The aim of this study was to develop site-specific antibodies as a tool to capture Plasmodium falciparum -dihydrofolate reductase (Pf-DHFR) from blood samples from P. falciparum infected individuals in order to detect, in a sandwich ELISA, structural alterations due to point mutations in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.

Methods

A homology model of Pf-DHFR domain was employed to define an epitope for the development of site-specific antibodies against Pf-DHFR. The homology model suggested an exposed loop encompassing amino acid residues 64–100. A synthetic peptide of 37-mers whose sequence corresponded to the sequence of amino acid residues 64–100 of Pf-DHFR was synthesized and used to immunize mice for antibodies. Additionally, polyclonal antibodies recognizing a recombinant DHFR enzyme were produced in rabbits.

Results and conclusions

Serum from mice immunized with the 37-mer showed strong reactivity against both the immunizing peptide, recombinant DHFR and a preparation of crude antigen from P. falciparum infected red blood cells. Five monoclonal antibodies were obtained, one of which showed reactivity towards crude antigen prepared from P. falciparum infected red cells. Western blot analysis revealed that both the polyclonal and monoclonal antibodies recognized Pf-DHFR. Our study provides insight into the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes.     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.     

Serological cross-reactivity between Strongyloides venezuelensis and Syphacia muris in Wistar rats (Rattus norvegicus)

One of the problems frequently faced in laboratory facilities is the possibility of the natural parasitic infection of lab animals, which can interfere with biomedical research results. The present study aimed to evaluate cross-reactivity among serum samples from Wistar rats (Rattus norvegicus) naturally infected with Syphacia muris and experimentally infected with Strongyloides venezuelensis. Forty rats were divided into four groups of ten animals each. Parasite load was evaluated by quantifying the adult worms from both helminthes species recovered from the intestines and the S. venezuelensis eggs eliminated in feces. Serological cross-reactivity by parasite-specific IgG detection was tested via enzyme linked immunosorbent assay (ELISA), immunofluorescence antibody test (IFAT) and immunoblotting. The results demonstrated that the quantity of S. venezuelensis eliminated eggs and parthenogenetic females decreased significantly in cases of co-infection with S. muris. ELISA revealed 100% cross-reactivity of serum samples from both species against the opposing antigen. IgG cross-reactivity was confirmed by IFAT using tissue sections of S. venezuelensis larvae and adult S. muris. Immunoblotting showed that IgG antibodies from the sera of animals infected with S. muris recognized eight antigenic bands from S. venezuelensis saline extract and that IgG antibodies from the sera of animals infected with S. venezuelensis recognized seven bands from S. muris saline extract. These results demonstrate the serological cross-reactivity between S. muris and S. venezuelensis in infected rats.     

Identification of immunologically cross-reactive proteins of Sindbis virus: evidence for unique conformation of E1 glycoprotein from infected cells

Hyperimmune antisera to purified Sindbis (SIN) or Semliki Forest (SF) virus were used to identify alphavirus-specific and cross-reactive proteins in virions and infected cells. The hyperimmune sera participated in homologous and cross-cytolysis of alphavirus-infected cells, and the use of monospecific antisera to SIN structural proteins suggested that E1 and E2 could serve as target proteins in cytolysis. Proteins from purified virions or infected cells were extracted with Nonidet P-40, denatured by procedures for sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose solid supports, and reacted with hyperimmune sera and 125I-labeled protein A (immunoblotting on denatured proteins). Alternatively, native proteins extracted by mild Nonidet P-40 treatment were precipitated with hyperimmune sera before denaturation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After immunoblotting, homologous antiserum reacted with the virus structural proteins E1, E2, capsid extracted from purified virions, and the counterparts of these proteins extracted from infected cells. In addition, PE2 and a 92,000-molecular-weight protein from infected cells reacted with homologous antiserum. These proteins were also immunoprecipitated with homologous antiserum. After immunoblotting, the Sindbis capsid protein was shown to be cross-reactive whether derived from purified virions or from infected cells; no cross-reactivity was observed with PE2 or E2 from either source, and the E1 glycoprotein was shown to be cross-reactive only when obtained from virions. However, the E1 glycoprotein could be cross-immunoprecipitated from infected cells (as well as from disrupted virions), and, in addition, capsid and a 92,000-molecular-weight protein were cross-immunoprecipitated from infected cells. These results suggest that a native conformation of the cell-associated E1 glycoproteins may be required for immunological cross-reactivity (immune precipitation), whereas virion but not cell-associated E1 retains immunological cross-reactivity after denaturation (immunoblot technique). The findings extend our previously published evidence which suggested that alphavirus maturation is accompanied by a change in immunological cross-reactivity with respect to E1.     

Uptake of L-tryptophan by erythrocytes infected with malaria parasites (Plasmodium falciparum)

The initial rates of uptake of L-tryptophan into normal human red blood cells and into cells infected by the malarial parasite Plasmodium falciparum in vitro, were investigated. We find that transport in non-infected cells, which is mediated by the specific saturable T system and the apparently non-saturable L system (Rosenberg, Young and Ellory (1980) Biochim. Biophys. Acta 598, 375-384) is considerably enhanced by blood preservation and culture conditions. This increase is mostly due to an increase in the maximal velocity of the saturable component and of the rate constant of the linear component. Uptake is further enhanced in non-infected cells by factors released from infected cells into the culture medium and, even more so, in infected cells at the advanced stage of intraerythrocytic parasite development. At these stages the susceptibility of the transport system to the non-specific inhibitor phloretin and to the competitive inhibitor phenylalanine, is virtually lost. The effect of the parasite on L-tryptophan uptake by the host cell membrane is exerted only on the maximal velocity of the T system, which is carrying most of the substrate under physiological conditions. The possible implications of these findings to the life of the intraerythrocytic parasite are briefly discussed.     

Presence of IgM Antibodies Which Sensitize HIV-1-Infected Cells to Cytolysis by Homologous Complement in Long-Term Survivors of HIV Infection

Although human cells are resistant to homologous human complement due to the presence of species-specific membrane inhibitors, a naturally occurring IgM antibody which recognizes an asialo-oligosaccharide can sensitize HIV-1-infected cells for complement-mediated cytolysis. Therefore, we investigated whether long-term survivors of HIV-1 infection harbor such antibodies in their sera. Thirty of 31 sera from HIV-1 seropositive hemophilia patients who have survived HIV-1 infection 10 years or more showed appreciable cytolytic activity, while only 2 sera of 10 seropositive patients presumed to have been infected with HIV-1 (due to sexual contact) more recently showed cytolytic activity. On the other hand, only 7 out of 43 sera from seronegative hemophilia patients showed cytolytic activity. Immunofluorescence staining for IgM on HIV-L -infected cells essentially correlated with the cytolytic capacity of the sera. Therefore, naturally occurring IgM antibodies and/or generated IgM antibodies reactive with the HIV-L -infected cells in patients might have been responsible for long-term survival due to complement-mediated immune cytolysis which may, in conjunction with cytotoxic T lymphocytes, synergistically suppress the infected cells in vivo. Therefore, the transfusion of such IgM antibodies could be effective for the treatment of HIV-L -infected individuals.     

Experimental Mycoplasma pulmonis infection of rats suppresses humoral but not cellular immune response

Humoral antibody response to sheep red blood cells and cellular immune response to bovine serum albumin were studied in Mycoplasma pulmonis infected, adult, male Sprague-Dawley rats. The hemagglutinating antibody response to sheep red blood cells was evaluated at 0, 3, 5, 7, 14, 21 and 28 days postinfection. Antibody titers during all days postinfection were depressed significantly (p less than 0.05) in infected rats as compared to noninfected controls. Cellular immune responses were evaluated by a delayed hypersensitivity response. Rats were sensitized at 0, 3, 5, 7, 14, 21 or 28 days postinfection with bovine serum albumin and challenged with heat aggregated bovine serum albumin 7 days later. Cell-mediated immune responses in infected rats were not significantly different at any point from controls. These results indicate that M. pulmonis infection in rats suppresses the humoral antibody response to sheep red blood cells, but not the cellular immune response to bovine serum albumin.     

Chromium(III) Nanoparticles Affect Hormone and Immune Responses in Heat-Stressed Rats

This study was conducted to evaluate the effects of chromium nanoparticles (CrNano) on the hormone and immune responses of rats in heat stress condition. A total of 80 male Sprague–Dawley rats were randomly assigned to four dietary treatment groups (n?=?20). The first group was offered a basal diet as a control. The second, third, and fourth groups received basal diet supplemented with 150, 300, and 450 μg/kg Cr, respectively, in the form of CrNano. At the end of the 8-week trial, growth performance, food utilization, and sera concentrations of hormones, immunoglobulins, and alexins were determined. Lymphocyte proliferation activity, antibody response to injected sheep red blood cells (SRBCs), and phagocytosis of peritoneal macrophages were determined by ³H-thymidine uptake method, plaque-forming cells (PFC) assay, and ingesting chicken red blood cells test, respectively. The results indicated that rats that received CrNano exhibited no changes in growth rate and food efficiency compared to the control group. However, dietary supplementation of 150, 300, and 450 μg/kg Cr from CrNano significantly decreased serum concentrations of insulin and cortisol, increased sera levels of insulin-like growth factor I and immunoglobulin G, and enhanced the lymphoproliferative response, anti-SRBC PFC response, and phagocytic activity of peritoneal macrophages. These results suggest that dietary supplementation of Cr as CrNano affects hormone and immune status in heat-stressed rats.     

Detection of antibodies to Streptobacillus moniliformis in rats by an immunoblot procedure

An immunoblot (IB) technique for detecting antibodies to Streptobacillus moniliformis in rat sera was evaluated. Immune sera to three S. moniliformis strains showed a similar reactivity pattern with both autologous and homologous antigens in the 18-87 kDa range. Using a rat S. moniliformis strain as the antigen, a similar reactivity pattern was found with sera from rats infected experimentally with S. moniliformis and sentinels. Two to five proteins were detected in the 32-55 kDa range. Over a period of 2.5 years, 27/133 rat serum panels submitted for routine monitoring yielded one or more S. moniliformis enzyme-linked immunosorbent assay (ELISA)-positive samples. In one of these 27 panels, sera showed an IB reactivity pattern resembling that observed with immune sera and with sera from infected and exposed rats. S. moniliformis was confirmed in the colony by both culture and polymerase chain reaction (PCR). Sera from the remaining 26 ELISA-positive serum panels frequently showed activity to a 57 kDa antigen but not more than one antigen was detected in the 32-55 kDa range. We conclude that the IB can be used as a confirmatory test for the detection of S. moniliformis infection in ELISA-positive rats.     

Changes in the immunotropic activity of neutrophilokins in the course of experimental staphylococcal infection

The influence of the products secreted by activated neutrophils (neutrophilokins) of mice, both intact and infected with staphylococci, on the activity of mouse spleen cells in the graft-versus-host reaction, immune response to sheep red blood cells and the antigen-presenting function of peritoneal macrophages was studied. Neutrophilokins of intact mice stimulated the activity of immunocompetent cells. Neutrophilokins obtained from infected mice on day 3 after infection produced an immunosuppressing effect. On day 7 after infection the immunostimulating activity of neutrophils was restored and showed practically no difference from the normal level.     

The extracorporeal immunostimulating effect of terrilytin in staphylococcal infection]

The intravenous injection of terrilytin-treated lymphocytes into rats infected with staphylococci enhances the formation of staphylococcal alpha antitoxin in the animals and the development of immune response to T-dependent antigen, such as sheep red blood cells (SRBC), but produces no effect on the development of immune response induced by T-independent antigen (lipopolysaccharide). Terrilytin-treated lymphocytes induce the release of the factor promoting the development of immune response to staphylococcal antigens and SRBC by spleen cells, incapable of adherence to plastic, but have no influence on the development of immune response to lipopolysaccharide in rats infected with staphylococci. At the same time in such rats spleen cells adhering to plastic take part in the transfer of signals from terrilytin-treated lymphocytes to nonadhering spleen cells of recipients.     

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. II. Effect of prior immunization with transplantable tumors on primary tumor incidence

Primary carcinogen-induced (7,12-dimethyl-benz[a]anthracene; DMBA) tumor-bearing SC chickens (B2/B2) frequently showed antibodies in their sera which reacted with cells from their autochthonous tumors, chicken embryo fibroblasts (CEF), tumor cells from some transplantable tumor lines, and from approximately 10% of other primary tumors. Similar results were obtained by ELISA on glutaraldehyde-fixed cells and by immunofluorescence on viable cells. The serum antibody reactivity could be removed by absorption with CEF but not with non-cross-reacting primary tumor cells or a variety of normal tissues. Although sera from normal chickens never showed significant reactivity, a high percentage of sera from chickens that had been injected with DMBA but failed to develop detectable tumors showed antibody activity to a transplantable DMBA-induced tumor and to CEF. On the basis of previously established cross-reactivity patterns in protective immunity to transplantable carcinogen-induced fibrosarcomas, attempts were made to protect against chemical carcinogenesis by prior immunization with selected DMBA-induced transplantable tumors. Tumor-immune chickens showed a significant decrease in the development of tumors during the first 3 mo after injection of DMBA (p = 0.001) or methylcholanthrene (p = 0.033) when compared to controls. This resistance to tumor induction in immune chickens was correlated to the degree of tumor immunity to the immunizing tumor present 1 mo after carcinogen injection (p = 0.046). There was, however, no detectable difference in the incidence of tumors arising later than 3 mo after carcinogen injection. The reduction in tumor incidence in immune as compared to control chickens at 5 mo was therefore less striking than the reduction seen at 3 mo. Immunization with CEF and adjuvants or with adjuvants alone afforded no protection to tumor induction.     

In vitro hemolytic activity ofPlasmodium berghei on red blood cells

A suspension ofPlasmodium berghei obtained by lysis with saponin of red blood cells from an infected rat showed high hemolytic activity, when incubatedin vitro with normal rat red blood cells. The hemolysis was a temperature-dependent process and was dependent on the concentration of the parasite. Plasma ofPlasmodium berghei infected albino rats also possessed lytic activity.