Olivier Garraud

Evaluation of anti-Plasmodium falciparum antibodies in Senegalese adults using different types of crude extracts from various strains of parasite

To date, no consensus exists on the type of crude Plasmodium falciparum Ags to be used in a standard assay for the evaluation of the overall anti-blood-stage immune response in humans. Comparison of the dose-dependent reactivity of using a pool of hyper-immune Senegalese sera to saponin and water schizont extracts of the Senegalese 07/03 isolate indicated similar reactivity on both types of antigen preparations. Water schizont extracts from three different strains of P. falciparum adapted to in vitro culture probed with a panel of specific mouse antisera and monoclonal antibodies reacting with conserved antigens showed similar antigenic content. Seroreactivity of immune individuals living in three different areas of endemicity was assessed in parallel on water crude extracts. The individual IgG, IgM and IgG subclass antibody responses to the various schizont preparations correlated positively. The specific IgM response was higher on the Senegalese schizont extract than on the FCR3 extract and was highest in Dielmo villagers. The IgG response was similar in all three locations and was strain independent. These results indicate that monitoring IgG antibody levels to the widely distributed FCR3 strain using an easily prepared crude lysate might represent a valuable reference ELISA allowing homogenisation and comparison of data from different laboratories.

Impairment of monocyte functions in advanced head and neck cancer

Depression of cell-mediated immunity is well established in most malignancies and especially in head and neck cancers, and much information is available concerning the defect in helper T lymphocyte function. We now report on impairment of the monocyte-macrophage system. Compared with normal controls we found that patients displayed, on one hand, an increased number of peripheral blood monocytes and, on the other hand, a smaller percentage of HLA-DR+ monocytes. Such peripheral blood monocytes normally failed to secrete factors, including interleukin 1 (IL-1). In addition, we observed that the in vivo induced blastogenesis of peripheral blood lymphocytes from patients, which is spontaneously depressed, is partly restored by medium containing IL-1. We cannot exclude, however, that the observed monocyte dysfunction involves other cytokines. Whether such an immune deficiency is due to secondary malnutrition or to the malignancy (or both) remains unclear.

Manipulating blood T cells and B cells from squirrel monkeys: some technical considerations.

The squirrel monkey Saimiri sciureus is an experimental host for a range of human pathogens, and for the assessment of vaccine candidate antigens and vaccine strategies. This experimental host is thus particularly suitable for the follow-up of humoral responses. To understand some of the mechanisms that underlie the defense against experimental pathogens, there is a need of basic knowledge on cellular immune effectors also. The authors report here their experience in characterizing squirrel monkey blood T and B lymphocytes, and in studying in vitro induced activation and proliferation of T and B cells. Particular emphasis is given to the in vitro differentiation of squirrel monkey B cells into immunoglobulin secreting cells, with respect to Plasmodium falciparum antigens.

Peripheral blood mononuclear cells in the squirrel monkey Saimiri sciureus: Characterization and functional aspects of T lymphocytes

Characterization and functional aspects of squirrel monkey peripheral blood mononuclear cells (PBMC), and mainly T cells, are described in the present paper; this should enable the study of cellular immune responses in an experimental model for malaria. PBMC were obtained from Ficoll-Hypaque gradient separation and fractionated into T cells and non-T cells by means of E-rosetting techniques and adherence to plastic dishes. PBMC subset phenotypes were characterized by means of monoclonal antibodies (mAb) directed against human leukocyte differentiation antigens (Ag), fluoresceinated lectins, anti-surface Ig (squirrel-monkey-specific) antibodies (Ab) and latex bead ingestion assays. PBMC functions were assayed through lymphoblastic transformation tests (LTT) in the presence of either numerous mitogenic, comitogenic and anti-mitogenic lectins or anti-human leukocyte differentiation Ag mAb. We sought to standardize reference values for lymphocyte phenotypes and functions in normal squirrel monkeys (prior to experimental infection). We also present evidence that splenectomy (generally rendered necessary for experimental human malaria infection) performed six months prior to the present investigation did not modify PBMC numbers and functions in the tested animals.

Different Plasmodium falciparum Recombinant MSP119 Antigens Differ in Their Capacities to Stimulate In Vitro Peripheral Blood T Lymphocytes in Individuals from Various Endemic Areas

This study reports on T‐cell proliferative responses to the 19‐kDa C‐terminal domain of the Plasmodium falciparum merozoite surface protein (MSP119). Three different recombinant proteins were used: an Escherichia coli product expressing the first EGF‐like domain and Saccharomyces cerevisiae and baculovirus/insect‐cell‐produced proteins containing both EGF‐like domains, the latter protein being produced with or without N‐glycosylation. Cell donors were P. falciparum‐immune adults with no recent history of clinical malaria and recruited from three Senegalese settings with different epidemiological parasite transmission. Each mononuclear‐blood‐cell preparation was stimulated with a range of concentrations of the three proteins. Most subjects’ mononuclear cells were reactive to at least one protein, but significant differences in lymphoproliferation were seen between the settings and within individual cultures depending on the protein source and concentration. Importantly, lymphoproliferation indices correlated inversely with the intensity of P. falciparum malaria transmission. When purified T lymphocytes were cultured in the presence of MSP119 plus autologous monocytes, Bu2003lymphocytes or a proposed CD1+ dendritic‐cell population as costimulatory cells, significant differences were observed depending on the individuals previous exposure to parasites. This study shows that the stimulation of lymphocyte proliferation in vitro with MSP119 depends on several factors, including epidemiological conditions and protein preparations.

Effect of blood storage on lymphocyte subpopulations

In the evaluation of helper and suppressor T lymphocyte subpopulations testing of the cells may not be carried out until the day following collection of the blood. The possibility that changes occur during overnight storage at 4 degrees C or 22 degrees C has been investigated. No statistical differences in percentages of helper and suppressor T cells between fresh samples and overnight stored samples were found if the blood was kept at 22 degrees C.

Experimental IgG antibody production in vitro by peripheral blood and tonsil surface gamma+ B lymphocytes from Plasmodium falciparum-immune West Africans.

Antigen reactive B cells in tonsil specimens from teenagers from a region moderately exposed to P. falciparum were capable of being differentiated in vitro and producing specific immunoglobulin (Ig)G in up to 33% of individual experiments. Mononuclear cells or purified sγ+ CD19+ B cells from peripheral blood or tonsil specimens from P. falciparum‐immune Senegalese subjects produced antigen‐specific IgG upon appropriate stimulation in vitro. One fraction of this IgG was produced de novo by differentiated B cells and another fraction was likely bound on the surface of circulating or resident CD19+ sγ+ B cells which were found in significantly greater numbers in individuals from rural Senegal as compared to nonimmune European controls. This study further documents the baseline levels of in vitro driven anti‐P. falciparum IgG antibody production by mononuclear cells from blood and tonsils in immune populations exposed to P. falciparum differentially. Furthermore, this study demonstrates the relevance and potential utility of tonsils as a source of B lymphocytes to characterize further specific antibody responses to P. falciparum antigens in immune populations.

Immune responses to P. falciparum-MSP1 antigen: lack of correlation between antibody responses and the capacity of peripheral cellular immune effectors to respond to this antigen in vitro

Protective immunity to P. falciparum blood stage infection is thought to be dependent on IgG antibodies, although the mechanisms that underlie such immunity are not clearly understood. One of the antigens thought to be involved in this protective response is MSP1. The present study has examined the levels and distribution of IgG (and IgM) antibodies to the C-terminal 19 kDa fragment of MSP1 in plasma from P. falciparum immune adult Senegalese and the capacity of the peripheral blood mononuclear cells from these patients to either proliferate or secrete IFN-gamma, IL-10 or IL-4 in vitro in response to this antigen. Specific antibodies were found in 74% of individuals plasma; 44% of mononuclear cells proved capable of proliferating in vitro and IFN-gamma, IL-10 and IL-4 were detected in 37, 23 and 0% of culture supernatants, respectively. No significant association was found between the presence of antibodies and immune cell reactivity under the culture conditions used. This study emphasizes the complexity of the mechanisms responsible for the sustained production of potentially protective antibodies in response to proposed T-cell dependent P. falciparum blood stage antigens.

In vitro production of immunoglobulins of various classes and subclasses by cord blood B cells in African neonates: modeling and assessment of determination.

Cord blood B cells obtained from neonates of healthy Senegalese mothers were assayed in vitro for their capacity to fully differentiate and secrete immunoglobulins (Ig) of various classes and subclasses. Stimulation of mononuclear cells with SAC particles or anti-micro antibodies in the presence of IL-4, or with IL-2 and IL-10 induced a strong production of IgG, provided that an additional CD40/CD40L signal was present, in contrast to adult cell cultures. Cord blood mononuclear cells differentially stimulated with various cytokines in order to lead to Ig heavy chain switching and production of the various classes/subclasses consistently produced IgG1, IgG3, IgG4, IgE and IgA. This system has been applied to immune cells from African neonates that have not been extensively studied previously. Estimation of Ig production as OD ratios could be applied to cultures where cord blood B cells are stimulated with defined antigens of human pathogens to which the fetus immune system was primed in utero.

Imbalanced distribution of IgM and IgG antibodies against Plasmodium falciparum antigens and merozoite surface protein-1 (MSP1) in pregnancy

In malaria endemic areas, pregnancy is assumed to be associated with a specific reduction in immunity to Plasmodium falciparum malaria. To understand some of the mechanisms which underlie such a poor immunity, we have attempted to examine the frequency and distribution of IgM and IgG antibodies to a crude antigenic extract of parasitized erythrocytes and to the merozoite surface protein-1 (MSP1), in a population of mothers compared to control non-pregnant women, all living in Dakar and suburbs. Specifically, this work describes: (i) the responses of mothers and control women; (ii) the balance between IgM and IgG responses; and (iii) responses to malarial antigen and to MSP1. An unexpected balance between P. falciparum-specific IgM and IgG is shown, associated with a substantial increase in anti-MSP1 IgM, and a decrease in anti-MSP1 IgG in parturients.