Long synthetic peptides encompassing the Plasmodium falciparum LSA3 are the target of human B and T cells and are potent inducers of B helper, T helper and cytolytic T cell responses in mice.

We synthesized 17 long synthetic peptides (LSP) spanning the whole 200-kDa Plasmodium falciparum liver stage antigen-3 (LSA3), an antigen that induces protection in chimpanzee, and analyzed their immunogenicity in BALB/c mice and their antigenicity in individuals living in a hyper-endemic malaria area. Our findings show that both specific antibodies and T cell proliferation against most LSA3-LSP develop in malaria-exposed adults. All individuals studied had detectable antibodies against a minimum of 6 and a maximum of 15 polypeptides. It is noteworthy that antibody prevalence and titers were as high against non-repeat as repeat regions. Although the extent of T cell reactivity was lower than that observed for B cells, most of the sequences contained at least one T helper epitope, indicating that the majority of LSA3-LSP contain both B and T cell epitopes within the same sequence. Injection of LSA3-LSP with SBSA2 adjuvant in mice, showed strong immunogenicity for most of them, eliciting both T cell responses and specific antibody production. While all the peptides were immunogenic for B cells, different patterns of T cell responses were induced. These peptides were thus classified in three sets according to the levels of the T cell proliferative and of the IFN-gamma-specific responses. Importantly, antibodies and T cells against some of the LSP were able to recognize LSA3 native protein on P. falciparum sporozoites. Additionally, some LSP (44-119, 1026-1095, 1601-1712) also contained epitopes recognized by H-2(d) class I-restricted T cells. These results led to the identification of numerous domains that are highly antigenic and immunogenic within the LSA3 protein, and underline the value of the LSP approach for vaccine development.

Apoptosis modulation in mononuclear cells recovered from individuals exposed to Plasmodium falciparum infection.

In endemic areas, asymptomatic infection by the malaria parasite Plasmodium falciparum was found associated with elevated percentages of human host's mononuclear cell spontaneous in-vitro apoptosis. In Dielmo, a village where malaria is holoendemic, apoptosis was age-and parasite-dependent. In-vitro exposure of peripheral blood mononuclear cells (PBMC) to the parasite extract induced a marked increase in the mononuclear cell membrane expression of functional CD95 antigen: a 3-h exposure of the mononuclear cells to anti-CD95 antibodies led to a detectable increase in the mean percentage of apoptotic nuclei found in the cultures carried out in the presence of P. falciparum extracts compared to control cultures. IL-2, IL-4, IL-6 and IL-10 promoted the viability of PBMC in cultures while IL-1alpha or IFN-gamma had no obvious impact and TNFalpha gave conflicting results. IL-2 was the most efficient cytokine at rescuing PBMC from cell death and this effect was associated with a strong increase in T cell activation. In contrast, IL-4 and IL-10 had no such effect on T cell activation, hence they acted as survival factors and not through their mitogenic activity. Taken together, these different observations suggested that the levels of in-vitro apoptosis observed were not only associated with parasite infection, but also potentially modulated by the human host through different pathways.

Reduced immune activation and T cell apoptosis in human immunodeficiency virus type 2 compared with type 1: correlation of T cell apoptosis with beta2 microglobulin concentration and disease evolution.

This study analyzes the degree of immune activation and characterizes apoptosis in lymphocytes from healthy West African donors or patients infected with human immunodeficiency virus (HIV)-1 or -2. The lower decline of CD4 T cells in HIV-2- compared with HIV-1-infected donors is associated with lower levels of immune activation, evaluated by HLA-DR expression on lymphocytes and sera concentrations of IgG and beta2 microglobulin (beta2m). Ex vivo apoptosis was found in both infections in all lymphocyte subsets, including CD4 and CD8 T cells, as well as B cells, but was lower in HIV-2 than in HIV-1 infection. Interestingly, high correlations were found in HIV-2- and HIV-1-infected donors between the level of CD4 T cell apoptosis and beta2m concentration and progression of the disease. These observations support the hypothesis that long-term activation of the immune system, weaker in HIV-2 infection, significantly contributes to T cell deletion and disease evolution.

Pattern of immunoglobulin isotype response to Plasmodium falciparum blood-stage antigens in individuals living in a holoendemic area of Senegal (Dielmo, west Africa).

Three cross-sectional studies were conducted in a representative cohort of individuals living continuously in an area holoendemic for malaria in Senegal. Plasma from 145 children and adults were tested. The pattern of antimalarial immunoglobulin class (IgM and IgG) and subclass (IgG1 to IgG4) antibody distribution was determined by enzyme-linked immunosorbent assay using a crude blood-stage antigen of Plasmodium falciparum-infected red blood cells. Adults had higher levels of specific antibodies than children, and IgM, IgG2, and IgG3 accounted for the highest difference (2.9, 6.5, and 4.5 times, respectively). Differences in antibody levels were significant for IgG1 to IgG4 between the lowest and the highest transmission season. No particular isotype distribution pattern could be found associated with any given parasitemia level. The relationship between the optical density (OD) values of each isotype and the risk of clinical malaria attack was tested using a Poisson regression model. Only the IgG3 OD increases were found associated with a significantly reduced risk of malaria attack. These seroepidemiologic data suggest that whereas the total IgG-specific activity is not indicative of any given level of protection against malaria, the level of IgG3 was significantly associated with the relative susceptibility to clinical P. falciparum malaria attacks.

Acute Plasmodium falciparum infection is associated with increased percentages of apoptotic cells.

The impact of acute malaria infection on the level of spontaneous apoptosis, i.e., the percentage of apoptotic cells detectable in lymphocytes cultured without any exogenous stimulus for 3 days in vitro, was evaluated. Quantitation of apoptosis was performed by staining of lymphocyte nuclei with propidium iodide and analysis of the fluorescence by cytometry. The mean apoptosis of 23 HIV-negative patients (15 Africans and 8 Europeans) determined during a confirmed Plasmodium falciparum attack was 27.2% (95% confidence interval (CI) = 23.5-30.7%) i.e., 2.2 times the mean level found in 49 controls (12.4%, CI = 11.1-13.6). These controls included age- and sex-matched Africans (n = 37) and Europeans (n = 12) differing only by their previous level of exposure to P. falciparum. Naive (European) as well as previously exposed (African) subjects showed dramatically elevated levels of spontaneous apoptosis during the malaria attack (mean = 22.5%, CI = 20.7-24.4 for Europeans; mean = 29.7%, CI = 24.6-34.7 for Africans). Such unusually raised levels were observed for at least 1.5 months and were probably detectable for longer periods as suggested by the fact that the mean level of spontaneous apoptosis in healthy Africans was basically higher (13.8%, CI = 12.5-15) than the one found in healthy Europeans (8.2%, CI = 6.3-10.1) (P = 0.0001). Selective immunomagnetic cell isolations carried out immediately before apoptosis quantitation showed that this process affected not only the alpha beta T cells (CD4+ T cells as well as CD8+ T cells) but also the gamma delta T cells and the B-lymphocyte subset.(ABSTRACT TRUNCATED AT 250 WORDS)

Near infrared spectroscopic analysis of forage samples digested in situ (nylon bag).

The objective was to investigate the feasibility of using near infrared reflectance spectroscopy to determine the composition of samples generated in situ. Five alfalfa and five orchardgrass hays of differing maturities were incubated for 0, 6, 24, 48, and 72 h (Experiment 1) or for 0, 2, 4, 8, 12, 24, 48, and 96 h (Experiment 2) in rumen-fistulated, lactating cows, using nylon bags. After washing to remove rumen contents, samples were analyzed using a nonrotating circular cell in a scanning monochromator. All samples then were dried at 55 degrees C and analyzed for CP and ADF by wet chemistry and rescanned in the dry state. The degree of DM digestion of the original sample was calculated from duplicate bags. Results for spectral analysis of dried samples (Experiment 1), with one-half the samples for validation, were typical of results found for dry forages. The results for scanning wet samples were less accurate than for dry ones. Analysis of samples from Experiment 2 by equations developed in Experiment 1 often resulted in extremely large biases, but these were corrected by including six samples of each forage from Experiment 2 in the calibration set (from Experiment 1) and redeveloping the equations. Although it is possible to use near infrared reflectance spectroscopy to determine the composition of wet samples generated in situ, results are more accurate if the samples are scanned after drying.