In vitro parasite antigen-induced antibody responses in human helminth infections

Helminth parasites characteristically induce vigorous antibody responses in human infections, but the immunoregulatory mechanisms determining the level of these responses are not at all understood. To investigate these mechanisms, peripheral blood mononuclear cells were obtained from 10 patients with parasitic helminth infections (three with schistosomiasis, three with onchocerciasis, and four with loiasis), along with six normal controls. These cells were then cultured in vitro and the capacity of the cells to respond to a specific parasite antigenic stimulus was examined by measuring the amount of parasite-specific antibody produced. Parasite antigen alone, without exogenous mitogen, induced an IgG anti-parasite antibody response in vitro. Optimal responses were obtained at extremely low antigen concentrations--concentrations at which little if any polyclonal immunoglobulin production occurred. Additionally, the in vitro induction of parasite-specific antibody was antigen dose-dependent, requiring much lower antigen concentrations than those necessary to induce lymphocyte blastogenesis. Antibody production was shown to require the cooperative interaction of B and T cells. These studies demonstrate that in vitro responses to antigens from naturally acquired parasitic infections, like those in individuals postimmunization, can be utilized to dissect the cellular and humoral factors that regulate antibody production to naturally acquired human pathogens.     

Frequency analysis of IgE-secreting B lymphocytes in persons with normal or elevated serum IgE levels

The immunoregulatory mechanisms that determine the high serum IgE antibody levels in disorders such as helminth parasite infections and the hyper-IgE recurrent infection syndrome (HIE) remain poorly understood. To assess whether elevated serum IgE levels result from an increased number of B lymphocytes committed to IgE production, the proportion of IgE-producing B lymphocytes was determined by a filter immunoplaque assay using PBMC from persons with a broad range of serum IgE levels that included normal persons (n = 9) and patients with loiasis (n = 12), tropical pulmonary eosinophilia (TPE) (n = 6), lymphatic filariasis (n = 28), and HIE (n = 8). PBMC from these persons were assessed for production of in vitro IgE. The geometric mean number of IgE-secreting cells in 10(5) B lymphocytes in PBMC was 0.42 (range 0-2.2) in normal persons, 5.6 (range 0.1-35.5) among patients with loiasis, 9.4 (range 0-53.2) among patients with lymphatic filariasis, 52 (range 31.5-115) among patients with TPE, and 218 (range 56-1404) among patients with HIE. When all study subjects were grouped, there were significant correlations with serum IgE levels (r2 = 0.78; p less than 0.0001) and net spontaneous in vitro IgE production (r2 = 0.8; p less than 0.0001). Estimates of the amount of IgE production per B lymphocyte were similar among normal persons, patients with filarial infections, and patients with TPE (geometric means of 134, 96, and 141 pg/ml/cell, respectively); in contrast, for HIE patients, IgE production by individual B cells was significantly lower (geometric mean 28 pg/ml/cell; p less than 0.001). These observations demonstrate that clonal expansion of IgE-producing B lymphocytes is a major mechanism underlying the elevated serum IgE levels seen in persons with hyper-IgE states.     

Specific Humoral Immunity versus Polyclonal B Cell Activation in Trypanosoma cruzi Infection of Susceptible and Resistant Mice

Background

The etiologic agent of Chagas Disease is Trypanosoma cruzi. Acute infection results in patent parasitemia and polyclonal lymphocyte activation. Polyclonal B cell activation associated with hypergammaglobulinemia and delayed specific humoral immunity has been reported during T. cruzi infection in experimental mouse models. Based on preliminary data from our laboratory we hypothesized that variances in susceptibility to T. cruzi infections in murine strains is related to differences in the ability to mount parasite-specific humoral responses rather than polyclonal B cell activation during acute infection.

Methodology/Principal Findings

Relatively susceptible Balb/c and resistant C57Bl/6 mice were inoculated with doses of parasite that led to similar timing and magnitude of initial parasitemia. Longitudinal analysis of parasite-specific and total circulating antibody levels during acute infection demonstrated that C57Bl/6 mice developed parasite-specific antibody responses by 2 weeks post-infection with little evidence of polyclonal B cell activation. The humoral response in C57Bl/6 mice was associated with differential activation of B cells and expansion of splenic CD21^highCD23^low Marginal Zone (MZ) like B cells that coincided with parasite-specific antibody secreting cell (ASC) development in the spleen. In contrast, susceptible Balb/c mice demonstrated early activation of B cells and early expansion of MZ B cells that preceded high levels of ASC without apparent parasite-specific ASC formation. Cytokine analysis demonstrated that the specific humoral response in the resistant C57Bl/6 mice was associated with early T-cell helper type 1 (Th1) cytokine response, whereas polyclonal B cell activation in the susceptible Balb/c mice was associated with sustained Th2 responses and delayed Th1 cytokine production. The effect of Th cell bias was further demonstrated by differential total and parasite-specific antibody isotype responses in susceptible versus resistant mice. T cell activation and expansion were associated with parasite-specific humoral responses in the resistant C57Bl/6 mice.

Conclusions/Significance

The results of this study indicate that resistant C57Bl/6 mice had improved parasite-specific humoral responses that were associated with decreased polyclonal B cell activation. In general, Th2 cytokine responses are associated with improved antibody response. But in the context of parasite infection, this study shows that Th2 cytokine responses were associated with amplified polyclonal B cell activation and diminished specific humoral immunity. These results demonstrate that polyclonal B cell activation during acute experimental Chagas disease is not a generalized response and suggest that the nature of humoral immunity during T. cruzi infection contributes to host susceptibility.     

Cytokine and chemokine responses in patients co-infected with Entamoeba histolytica/dispar, Necator americanus and Mansonella perstans and changes after anti-parasite treatment

This study examined the impact of concurrent parasite infections (amoebiasis, filariasis, necatoriasis) and the effect of anti-parasite treatment on cytokine and chemokine responses in singly and poly-parasitized patients. Cellular reactivity and parasite-specific Th1- and Th2-type cytokine and chemokine profiles were investigated before and six weeks after treatment. In those patients infected with three parasite species, cellular secretion of interleukin 5 (IL-5) and IL-12p40 by PBMC was strongly diminished (p<0.005) but IL-10 was elevated in parasite-infected patients (p<0.0001) in response to protozoa- and helminth-specific as well as bacteria-specific antigens. Macrophage inflammatory chemokines (MIP-1alpha/CCL3 and MIP-1beta/CCL4), macrophage-derived chemokine (MDC/CCL22) and neutrophil activating chemokine (IL-8/CXCL8) were produced by PBMC in similar amounts in endemic controls and singly and poly-parasitized patients, but thymus and activation-regulated chemokine (TARC/CCL17) was produced the highest by PBMC from patients with triple parasite infections (p<0.0001). Following anti-parasite therapy, secretion of IL-12p40 and IL-5 augmented significantly in treated patients while IL-10, MDC, MIP-1alpha, TARC and IL-8 substantially diminished (all p<10(-5)) when their PBMC were activated with parasite- and bacteria-specific antigens. In summary, PBMC from poly-parasitized patients responded to protozoa- and helminth-specific antigens with a compromised IL-5 and IL-12p40 but high IL-10 and a substantial chemokine release. Chemokines may attract and activate effector cells in peri-parasitic tissues to limit parasite proliferation and dissemination, while depressed IL-5 and IL-12p40 but prominent IL-10 may prevent eosinophil and cytotoxic cell-mediated inflammatory processes and pathogenesis to the host. The changes in this profile following anti-parasite therapy disclosed the dynamics of an immune adaptation associated with parasite accumulation and also with clearance of parasite infections.     

Biological activity and diagnostic use of detergent soluble antigens fromSetaria digitata

Filarial parasite, responsible for filariasis is known to remain in the host for long periods of time. A 29 kDa protein isolated fromSetaria digitata by gel electrophoresis and electroelution of detergent soluble surface antigens showed a 70% inhibition of the cell mediated immune response. On evaluation of its diagnostic application, the same protein was found to be very sensitive in detecting antibody at an antigenic protein concentration as low as 1 ng per μl. The cross reactivity of surface antigen with bancroftian filarial patient’s sera was tested by Dot-ELISA and ELISA. Both the antigen as well as antibody detection tests showed 100% positivity with all types of filariasis cases. It did not produce any positive reaction with non-endemic control sera. However, a proportion of endemic normal subjects showed positivity and this is attributed to the fact that people in endemic areas are exposed to infective mosquito bites. The biological property of inhibition and 100% positivity of filariasis cases in both antibody and antigen detection tests point towards the bifunctional nature of the surface proteins before and after release. The same may be happening under normal conditions also, perhaps at a much lower rate     

Regulatory T cells in human lymphatic filariasis: stronger functional activity in microfilaremics

Infection with filarial parasites is associated with T cell hyporesponsiveness, which is thought to be partly mediated by their ability to induce regulatory T cells (Tregs) during human infections. This study investigates the functional capacity of Tregs from different groups of filarial patients to suppress filaria-specific immune responses during human filariasis. Microfilaremic (MF), chronic pathology (CP) and uninfected endemic normal (EN) individuals were selected in an area endemic for Brugia timori in Flores island, Indonesia. PBMC were isolated, CD4CD25(hi) cells were magnetically depleted and in vitro cytokine production and proliferation in response to B. malayi adult worm antigen (BmA) were determined in total and Treg-depleted PBMC. In MF subjects BmA-specific T and B lymphocyte proliferation as well as IFN-gamma, IL-13 and IL-17 responses were lower compared to EN and CP groups. Depletion of Tregs restored T cell as well as B cell proliferation in MF-positives, while proliferative responses in the other groups were not enhanced. BmA-induced IL-13 production was increased after Treg removal in MF-positives only. Thus, filaria-associated Tregs were demonstrated to be functional in suppressing proliferation and possibly Th2 cytokine responses to BmA. These suppressive effects were only observed in the MF group and not in EN or CP. These findings may be important when considering strategies for filarial treatment and the targeted prevention of filaria-induced lymphedema.     

Biochemical and immunologic characterization of a major IgE-inducing filarial antigen of Brugia malayi and implications for the pathogenesis of tropical pulmonary eosinophilia.

A major allergen of the human filarial parasite Brugia malayi has been identified by two-dimensional immunoblot analysis using a serum pool from patients with tropical pulmonary eosinophilia. The allergen is composed of two Ag with M(r) 23 and M(r) 25 and acidic isoelectric point (Bm23-25). Immunoblots using affinity-purified IgE antibodies to BM23-25 indicated that Bm23-25 is expressed mainly in the microfilarial stage. Digestion of the allergen with endoglycosidases indicates that it has N-linked oligosaccharide chains. Analysis of the reactivity of T cells derived from patients with lymphatic filariasis revealed that the Bm23-25 allergen was capable of stimulating T cell proliferation; Bm23-25 was also shown to induce IgE production in vitro from PBMC derived from patients with either TPE or other filarial symptoms. Bronchoalveolar lavage fluid of patients with TPE contained IgE antibodies that recognized Bm23-25 strongly, an observation suggesting that the microfilarial allergen might be involved in the pathogenesis of the TPE syndrome.     

Immunological and biochemical characterization of an epitope of the transferrin receptor involved in the production of interferon-gamma and B-cell growth factor

We have investigated the effect of a newly developed monoclonal antibody, MoAb NDA9, on human lymphocyte function. This MoAb inhibits the capacity of peripheral blood lymphocytes to display blastogenic responses and to produce immunoglobulins when stimulated in vitro with PWM or with soluble antigens. The inhibitory effect seems to result from the decreased ability of T lymphocytes to produce B cell growth factors (BCGF) in the presence of MoAb NDA9. This antibody also blocks the capacity of polyclonal or monoclonal populations of activated human T cells to produce immune interferon (gamma) but has no direct effect on B cell activation and growth in T-cell-independent systems. Immunochemical studies of the antigen recognized by MoAb NDA9 showed that it is an epitope of the transferrin receptor molecule which is distinct from that recognized by the MoAb OKT9.     

Activation and immunoregulation of antigen-specific human b lymphocyte responses: multifaceted role of the monocyte

The multifaceted role of the monocyte in the induction and modulation of antigen-specific antibody responses by human B cells was delineated. Monocytes were absolutely required for the induction of specific antibody responses to both TT and KLH in an antigen-induced in vitro assay. Monocytes were also required for the PWM induction of specific antibody in immunized subjects. Pulsing monocytes with specific antigen or with PWM consistently stimulated proliferation of T cells in absence of added antigen and could also stimulate specific antibody synthesis although less consistently. Stimulation of specific antibody responses with antigen required fewer numbers of monocytes than did stimulation of specific antibody responses with PWM. Polyclonal antibody synthesis induced by PWM was also dependent on monocytes. However, polyclonal antibody synthesis induced by supraoptimal concentrations of antigen was usually optimal in the absence of monocytes and was actually suppressed when increased numbers of monocytes were added to monocyte-depleted cultures. Monocyte supernatants could not replace the absolute requirements for monocytes in the induction of specific antibody synthesis. However, monocyte supernatants could profoundly modulate the antigen-specific as well as the polyclonal Ig response of lymphocytes to either antigen or PWM stimulation in a manner closely resembling monocytes themselves. Thus, we demonstrated that monocytes and their products play a critical role in the activation and immunoregulation of antigen-specific antibody responses of human B cells.     

Onchocerca volvulus and Wuchereria bancrofti: fluorescent antibody staining of frozen homologous sections for diagnosis

Antibody responses to two filarial diseases of man, onchocerciasis and bancroftian filariasis, were evaluated with the indirect fluorescent antibody technique (IFAT), using antigens derived from the appropriate etiologic agent. Antigenic preparations consisted of frozen cut sections of the adult Onchocerca volvulus and stage III larvae of Wuchereria bancrofti fixed to glass slides. Little difference between the preparations was demonstrated in tests for the diagnosis of onchocerciasis. Of 105 sera from individuals with biopsy-proven infections, 102 (97%) reacted with homologous O. volvulus antigen, and 19 of 22 (86%) with W. bancrofti antigen. In bancroftian filariasis, however, the homologously derived antigen was superior for diagnosis, and the highest seropositive rates occurred in acute, symptomatic infections. All such sera (8) reacted with homologous antigen. In contrast, only 75% (6) reacted with onchocercal antigen. Of those with chronic disease, characterized by long-standing elephantiasis or lymphedema without microfilaremia, 79% (22) were reactors to homologous antigen and 32% (9) to heterologous. The lowest seropositive rates occurred where microfilaremia was unaccompanied by local or systemic symptoms: 38% (3) were positive to homologous antigen and none to onchocercal antigen.Of sera from seven individuals apparently free of bancroftian filariasis, but living in a hyperendemic area, five reacted with bancroftian antigen and four with onchocercal antigen. These reactions could be attributed to occult infections, but more likely resulted from repeated exposure to nondeveloping infective larvae.Cross-reactions in nonfilarial infections were rare with either antigen, and no positive reactions occurred in sera from healthy controls.     

Pathophysiologic analysis of peripheral blood lymphocytes from patients with primary immunodeficiency. I. Ig synthesis by peripheral blood lymphocytes stimulated with either pokeweed mitogen or Epstein-Barr virus in vitro

In the present study 5 patients with common variable hypogammaglobulinemia (CVH) and 4 patients with selective IgA deficiency (IgA-D) were analyzed for the cellular defects responsible for impaired Ig synthesis with use of peripheral blood lymphocytes stimulated with either PWM or EBV in vitro. By the use of co-culture with PWM, all the patients examined had intrinsic B cell defects restricted to the synthesis of Ig class corresponding to the low or absent Ig class(es) in the sera. Two types of excessive suppressor T activity were found, which were abrogated by irradiation. One was isotype-nonspecific and the other was IgA-specific. Moreover, failure of IgA-specific helper T activity was demonstrated. The use of EBV as an agent that polyclonally activates B cells independently of T cells and monocytes should allow a clearer delineation of the level of the B cell defects. When co-cultured with EBV, B cells from 3 patients with CVH produced normal to subnormal quantities of IgM although they could produce no IgM upon co-culturing with normal T cells and PWM. B cells from 2 patients with CVH could produce IgM normally by stimulation with either PWM or EBV; however, there was no restoration to produce IgG or IgA in these patients. In addition, B cells from 2 patients with IgA-D produced not only IgG and IgM but also IgA almost normally at 4 days after in vitro stimulation with EBV.     

Interferon production potentials of various human lymphoblastoid cell lines

A number of human lymphoblastoid cells were examined concerning their ability to produce spontaneously liberated and virus-induced interferon (IFN). It was found that, in addition to B cells, various T and nonT-nonB lymphoblastoid cells responded well to Sendai virus infection to form IFN, the characterization of which has been recently reported (20). One B lymphoblastoid cell line from an infectious mononucleosis (IM) patient produced a large amount of IFN-alpha and might become an alternative source of IFN production. Among 68 cell lines examined, 35 cell lines liberated 10 U/ml or more of IFN spontaneously in culture fluid. The presence of Epstein-Barr virus (EBV) genome or its activation appears to have no correlation with the spontaneous liberation of IFN. Spontaneously produced IFN from three cell lines was characterized as IFN-alpha. Comparatively higher amounts of IFN were produced in cells from IM patients than those from Burkitt's lymphoma cases or healthy adults. Spontaneously produced IFN was detected more easily in cells transformed by EBV alone than in those transformed by EBV and a tumor promoter, TPA.     

A polyclonal assay for T helper function involving T-B cell contact mediated by L3T4 molecules

In the presence of pokeweed mitogen (PWM), T helper (TH) cell clones can induce differentiation of a very high proportion of normal B lymphocytes into plasmocytes. This property can be used to test TH cell function regardless of clonal specificity. We have investigated the role of L3T4 surface antigen in this new assay. Only TH cell clones expressing the L3T4 antigen have effector activity in this PWM-dependent helper assay; L3T4- TH cell variants are inactive. The involvement of L3T4 antigen is further shown by the ability of anti-L3T4 monoclonal antibody to inhibit the PWM-dependent polyclonal B cell differentiation induced by L3T4+ TH cell clones. This inhibition is not the consequence of arrested TH cell activation, nor of a lack of appropriate B cell stimulation by TH cell lymphokines. We show that PWM focuses TH cells on the B cell hybridoma LB15-13, and that anti-L3T4 mAb prevents the T-B cell clustering mediated by PWM. Thus, by a mechanism comparable with the one described for concanavalin A in the cytotoxicity assay, PWM acts by bridging TH cells and B cells; the T cell surface antigen L3T4 is involved in this process.     

Biologic activity of antigen receptors artificially incorporated onto B lymphocytes

We describe a method for incorporating monoclonal antibody molecules onto viable murine lymphocytes and summarize the biologic activity of these artificial receptors on B cells. Mouse spleen cells incubated overnight with palmitate conjugates of a monoclonal anti-DNP IgA (protein 315) in the presence of deoxycholic acid incorporate about 50,000 antibody molecules per cell. When concentrations of deoxycholate and palmitoyl-protein 315 are carefully controlled, this labeling procedure does not affect the viability or the normal functions of the receptor-decorated cells. The incorporated antibody specifically binds DNP-antigens, although it appears to be unable to communicate directly with internal cellular components. Yet when these receptor-decorated, unprimed cells are challenged with any one of several DNP-antigens, up to 42,000 per 10(6) B cells differentiate into Ig-secreting cells. This response is about 23-fold greater than that induced in normal cell cultures and is of the same magnitude as that induced by the polyclonal B cell activator LPS. This, in addition to the observation that only about 3.6% of receptor-decorated B cells responding to DNP-conjugated polymerized flagellin (DNP-POL) produce hapten-specific antibody, demonstrates that these antigens cause polyclonal B cell differentiation. Normal spleen cells in the presence of DNP-POL and irradiated spleen cells bearing the artificial receptors do not exhibit the polyclonal antibody response. Also, the response of receptor-decorated B cell is blocked by high but nontoxic concentrations of the nonimmunogenic hapten DNP-lysine. These observations demonstrate that the polyclonal B cell response in this system requires the binding of antigen to artificial receptors on functionally viable cells. The polyclonal B cell response to a thymus-dependent antigen DNP-conjugated bovine gamma-globulin (DNP-BGG) requires the presence of the carrier-primed T cells. On the other hand, T cell depletion by anti-Thy-1.2 monoclonal antibody and complement causes only a slight reduction in the number of receptor-decorated B cells that respond to the relatively thymus-independent antigen DNP-POL. This type of phenomenon is also seen with natural antigen-specific B cells. Thus, polyclonal activation of receptor-decorated B cells exhibits the same gross helper cell requirements as antigenic activation of natural antigen-specific B cells. The results of this study are discussed in the context of the role of membrane-bound surface Ig in antigen-dependent B cell activation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Human peripheral blood mononuclear cells produce IgA anti-influenza virus antibody in a secondary in vitro antibody response

The function and immunoregulation of human IgA memory B cells producing anti-influenza virus antibody was analyzed in vitro in antigen-stimulated cultures. Peripheral blood mononuclear cells (PBMC) from seven of eight normal adult volunteers naturally immunized to influenza virus produced IgA anti-influenza virus antibody when stimulated in vitro with inactivated A/Aichi/68 [H3N2] influenza virus. This IgA antibody response was approximately one-eighth the IgG antibody response. PBMC from each of five patients with selective IgA deficiency failed to produce any measurable IgA antibody. When tonsillar mononuclear cells (TMC) were studied in a similar manner, a relatively higher IgA antibody response was obtained (one-third the IgG antibody) than with PBMC. Additional studies were undertaken to investigate the immunoregulation of this IgA antibody production and the relatively lower amount produced by PBMC than by TMC. Co-cultures of peripheral blood B cells with irradiated peripheral blood T cells (to possibly inactivate a radiosensitive IgA suppressor cell) did not result in a relative increase in IgA antibody production. Also, co-cultures of B cells with increasing numbers of T cells produced parallel increases of IgG and IgA antibody when plotted on a log scale with slopes of approximately 1, suggesting that a single helper T cell was limiting for both isotypes. Finally, pokeweed mitogen-stimulated co-cultures of peripheral blood and tonsillar B and T cells revealed that the B cell population, but not the T cell population, determined the amount of IgA anti-influenza virus antibody produced. Precursor frequency analyses of tonsillar and peripheral blood B cells in antigen-stimulated cultures confirmed that tonsils contained a higher precursor frequency of B cells for IgA anti-influenza virus antibody production (3.95/10(6) B cells) than did peripheral blood B cells (0.65/10(6) B cells). Thus, IgA memory cells are preferentially found in tonsillar tissue as compared with the peripheral blood, consistent with the role of the tonsils as a mucosal immune organ.     

Identification of 38kDa Brugia malayi microfilarial protease as a vaccine candidate for lymphatic filariasis

A FPLC purified 38kDa protease (Bm mf S-7) isolated from B. malayi microfilarial soluble antigen was identified. It showed pronounced reactivity with sera collected from 'putatively immune' asymptomatic and amicrofilaraemic individuals residing in an endemic area for bancroftian filariasis. Further the immune protective activity of Bm mf S-7 antigen was evaluated in susceptible hosts, jirds (Meriones unguiculatus) against B. malayi filarial infection. The antigen showed 89% cytotoxicity against mf and 87-89% against infective (L3) larvae in in vitro antibody dependent cellular cytotoxicity Assay (ADCC) and in situ micropore chamber methods. Bm mf S-7 immunized jirds after challenge infection showed 81.5% reduction in the adult worm burden. The present study has shown that, the 38kDa microfilarial proteases (Bm mf S-7) could stimulate a strong protective immune response against microfilariae and infective larvae in jird model to block the transmission of filariasis. Analysis of IgG subclasses against Bm mf S-7 revealed a significant increase in IgG2 and IgG3 antibodies in endemic normals. Lymphocyte proliferation to Bm mf S-7 was significantly high in endemic normal group as compared to that in clinical and microfilarial carriers. Significantly enhanced levels of IFN-gamma in the culture supernatant of PBMC of endemic normals followed by stimulation with Bm mf S-7 suggest that the cellular response in this group is skewed towards Th 1 type.     

Dot-ELISA in detection of filarial antibodies: comparison with standard ELISA

The present study deals with the preliminary results of two assays, standard ELISA and dot ELISA compared for their sensitivity in measuring humoral immune responses in filarial patients and normals residing in an area of bancroftian filariasis using a heterologous antigen. Responses were more vigorous in filarial patients than in endemic normals by both assays. Higher percentage of positivity and reciprocal titre of antibody were observed in dot ELISA showing more sensitivity than the standard ELISA. The dot ELISA method required only nanogram amounts of antigen.     

Proinflammatory cytokines secreted by monocytes of filarial patients.

The levels of interleukin 1, granulocyte macrophage colony stimulating factor (GM-CSF) and tumour necrosis factor (TNF)-alpha secreted by the monocytes of filarial patients, such as asymptomatic microfilaremics (MF), chronic pathology (CP), and normal individuals, residing in a Wuchereria bancrofti endemic area (EN) in response to whole Brugia malayi antigen (BmA) and Setaria digitata (Sd-cuticular) and a recombinant filarial antigen (pRJ51) were studied. Stimulation of peripheral blood adherent cells with whole parasite antigen showed marked increase in IL-1 levels in MF as compared to CP or EN. The recombinant antigen stimulation, however, resulted in similar levels of IL-1 in MF and CP. In contrast, stimulation of peripheral blood adherent cells with whole parasite antigen produced high levels of GM-CSF and TNF-alpha in CP as opposed to MF or EN. Recombinant antigen stimulation, however, produced high levels of GM-CSF in EN as compared to MF or CP, while no significant change in the release of TNF-alpha was observed in these patients. These results suggest that monocytes from filarial patients exhibit functional activity similar to that observed by the monocytes of endemic normals (control group).     

Development, Characterization, and Use of Monoclonal and Polyclonal Antibodies against the Myxosporean, Ceratomyxa shasta

Both monoclonal and polyclonal antisera were produced against Ceratomyxa shasta. Ascites containing trophozoites of the parasite was collected from infected fish and used as antigen for immunization of mice. The resulting monoclonal antibodies reacted specifically with trophozoite and sporoblast stages but did not react with C. shasta spores by either indirect fluorescent antibody techniques or in Western blots. This indicates that some C. shasta antigens are specific to certain life stages of the parasite. Polyclonal antiserum was produced in a rabbit by injecting a spore protein electro-eluted from an SDS-polyacrylamide gel. This antiserum reacted with both trophozoites and spores by indirect fluorescent antibody techniques and in Western blots. All antisera were tested for cross-reactivity to trout white blood cells, a contaminant of the ascites, and to other myxosporea. Two monoclonal antibodies reacted with white blood cells and myxosporea of the genera Sphaerospora and Myxobilatus. One hybridoma produced antibodies of high specificity for C. shasta pre-spore stages. This is the first report of a monoclonal antibody produced against a myxosporean parasite.     

Development and evaluation of a rapid flow-through immuno filtration test using recombinant filarial antigen for diagnosis of brugian and bancroftian filariasis

There is an imperative need to develop a rapid antibody test that can be used for diagnosis of clinical cases in travelers and expatriates, primary surveillance in areas of unknown endemicity, detection of early infection in childhood and for monitoring chemotherapeutic programs. A rapid-format, simple and qualitative flow through immuno filtration test has been developed for the identification of total IgG antibodies to recombinant filarial antigen WbSXP-1. This test system employs colloidal gold-protein A reagent as the antibody capture reagent. The sensitivity and specificity of the test was evaluated in a total of 1,230 serum samples. The sensitivity of the test was found to be 90.8% with brugian (n = 70) and 91.4% with bancroftian (n = 140) microfilaraemic subjects. The test showed minimum reactivity (4/10) with Loa loa microfilaria (MF) positive sera and no reactivity (0/20) with Onchocerca MF positive sera. This rapid diagnosis is found to be non-reactive with individuals having other parasitic diseases including schistosomiasis (n = 10), soil-transmitted helminthiases (n = 34) and protozoan infections (n = 33) indicating the potential of this test as a prospective method of diagnosis for both brugian and bancroftian lymphatic filariasis. Stability kinetics was studied at different temperatures and different time periods. The rapid flow-through immuno filtration test is advantageous since it can be stored at room temperature, is user friendly and is particularly applicable in the field as an initial screening method, for epidemiological monitoring of filarial infections in bancroftian and brugian endemic regions of the world.