Paola Minoprio

Polyclonal lymphocyte responses to murine Trypanosoma cruzi infection. I. Quantitation of both T- and B-cell responses.

Lymphoid activity was studied in spleen and lymph node cells from Trypanosoma cruzi‐infected mice. Blast transformation in each lymphocyte class was assessed by dual parameter analysis for size and surface markers by both FACS and conventional immunofluorescence, while proliferative activity was measured by tritiated thymidine uptake, autoradiography, and analysis of DNA content in single cells. Acute infection results m rapid Mast transformation and proliferative activity of all three lymphocyte classes (Ig+, L3T4+, and Lyt 2+). At 2 weeks of infection most cells in these organs are enlarged and more than half are dividing. By 2 and 6 months after infection (chronic phase of resistant strains), large numbers of activated B lymphocytes and. to a lesser extent, of Lyt 2+T cells are still detected. Similar results were obtained in C57BL/(resistant) and C3H/HeJ (susceptible) mouse strains. The implications of this massive polyclonal lymphocyte response to the parasite for the physiopathology of acute and chronic infection are discussed.

A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase

Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.

Most B Cells in Acute Trypanosoma cruzi Infection Lack Parasite Specificity

The specificity of B lymphocytes activated in the acute phase of murine Trypanosoma cruzi infection was analysed in a panel of immunoglobulin‐secreting hybridomas derived by fusion of lymph node cells 7 days after intraperitoneal parasite inoculation. The immunoglobulin isotype distribution of the hybrids reflected the total plaque‐forming cell (PFC) response in the animal at this point, with a predominance of IgG2a, IgM, and IgG2b. Screening of the hybridoma antibodies on parasite antigens by three independent methods (western blot, ELISA, and immunofluorescence) revealed only one (out of a total of 51) that bound a parasite molecule with an apparent molecular mass of 180 kDa. In contrast, antibodies of both IgM and IgG classes were found to react with a panel of autologous antigens. These results establish that most B cells activated by T. cruzi infection are not specific for parasite antigens and therefore indicate the relevance of analysing the totality of host responses to infection.

Drug discovery targeting amino acid racemases.

Drug Discovery Targeting Amino Acid Racemases Paola Conti, Lucia Tamborini, Andrea Pinto, Arnaud Blondel, Paola Minoprio, Andrea Mozzarelli, and Carlo De Micheli* Dipartimento di Scienze Farmaceutiche “P. Pratesi”, via Mangiagalli 25, 20133 Milano, Italy Institut Pasteur, Unit e de Bioinformatique Structurale, CNRS-URA 2185, D epartement de Biologie Structurale et Chimie, 25 rue du Dr. Roux, 75724 Paris, France Institut Pasteur, Laboratoire des Processus Infectieux a Trypanosoma; D epartement d’Infection et Epid emiologie; 25 rue du Dr. Roux, 75724 Paris, France Dipartimento di Biochimica e Biologia Molecolare, via G. P. Usberti 23/A, 43100 Parma, Italy Istituto di Biostrutture e Biosistemi, viale Medaglie d’oro, Roma, Italy

Trypanosoma cruzi proline racemases are involved in parasite differentiation and infectivity.

Polyclonal lymphocyte activation is one of the major immunological disturbances observed after microbial infections and among the primary strategies used by the parasite Trypanosoma cruzi to avoid specific immune responses and ensure survival. T. cruzi is the insect‐transmitted protozoan responsible for Chagas’ disease, the third public health problem in Latin America. During infection of its mammalian host, the parasite secretes a proline racemase that contributes to parasite immune evasion by acting as a B‐cell mitogen. This enzyme is the first described eukaryotic amino acid racemase and is encoded by two paralogous genes per parasite haploid genome, TcPRACA and TcPRACB that give rise, respectively, to secreted and intracellular protein isoforms. While TcPRACB encodes an intracellular enzyme, analysis of TcPRACA paralogue revealed putative signals allowing the generation of an additional, non‐secreted isoform of proline racemase by an alternative trans‐splicing mechanism. Here, we demonstrate that overexpression of TcPRAC leads to an increase in parasite differentiation into infective forms and in its subsequent penetration into host cells. Furthermore, a critical impairment of parasite viability was observed in functional knock‐down parasites. These results strongly emphasize that TcPRAC is a potential target for drug design as well as for immunomodulation of parasite‐induced B‐cell polyclonal activation.

Is TNFα involved in early susceptibility of Trypanosoma cruzi-infected C3H/He mice?

Abstract Early wasting and subsequent mortality may occur in mice of some inbred strains following infection with Trypanosoma cruzi. It was hypothesized that TNFα/cachectin might be involved in this process. Thus, sera collected from mice of strains differing in their susceptibility or resistance to Trypanosoma cruzi infection were checked for the presence and level of TNFα, a cytokine able to exert acute toxic effects. C3H/HeJ or C3H/HePas (susceptible), BALB/c (intermediate) and C57BL/6 (resistant) mice were infected with the CL or Colombian strain of Trypanosoma cruzi, and TNF activity was measured in the sera during the acute phase of the infection. Only serum collected from infected C3H/He mice contained TNF activity. However, TNF activity could be measured in serum of all strains, following LPS infection, indicating that the infection was able to prime macrophages of infected mice to secrete TNFα. The TNFα/cachectin release in the sera of C3H mice may play a role in the early wasting and death of these mice after Trypanosoma cruzi infection.

Susceptible mice present higher macrophage activation than resistant mice during infections with myotropic strains of Trypanosoma cruzi

Summary The kinetics of macrophage activation were compared among inbred strains of mice (C3H, BALB, B6 and BIO.A) that are known to differ in their relative resistance to infections with the myotropic strains (Colombian and CL) of Trypanosoma cruzi. The parameters utilized to measure macrophage activation were rapid spreading on glass surfaces, hydrogen peroxide release and tumour necrosis factor/cachectin production. Macrophages obtained from C3H (susceptible), BALB (intermediate) and B6 or BIO.A (resistant) mice infected with both strains of T. cruzi began to spread rapidly at the onset of parasitaemia. Surprisingly, the amount of hydrogen peroxide released by peritoneal cells obtained from the more susceptible mouse strain (C3H) was significantly higher than in the other mouse strains. Also, only in the serum of C3H mice was tumour necrosis factor/cachectin detected. These results suggest that resistance against infections with myotropic strains of T. cruzi does not correlate with enhanced macrophage activation. It is also shown that the acquired macrophage activation is largely dependent on T‐lymphocytes bearing the phenotypic marker CD4 (helper/inducer), since all parameters of macrophage activation were significantly inhibited in athymic mice or in C3H mice treated in vivo with monoclonal antibody anti‐CD4+ T‐cells.

Ig-Isotype Patterns of Primary and Secondary B Cell Responses to Plasmodium Chabaudi Chabaudi Correlate with IFN-γ and IL-4 Cytokine Production and with CD45RB Expression by CD4+ Spleen Cells

In this work, the authors analysed T and B lymphocyte subsets and cytokine production in the spleen of BALB/c mice during polyclonal lymphocyte activation (primary infection) and parasite‐specific response to Plasmodium chabaudi chabaudi (secondary infection). The secondary response was evaluated in fully immunoprotected animals, 60 days after a chloroquine‐cured infection. The authors observed that in polyclonal lymphocyte activation antibody‐secreting cells of all isotypes increased, with predominance of IgG2a and IgG3 classes. At that time, IFN‐γ was largely produced, but IL‐4/IL‐5 were just slightly enhanced. In mice re‐infected after 60 days, the Ig‐isotype pattern was restricted to IgG1 and only IL–4/IL‐5 were produced. In both responses, however, the levels of IL‐2 were greatly reduced, while those of IL‐10 were enhanced to similar levels. The different involvement of Th1 and Th2 cells in both responses was confirmed through analysis of CD45RB expression by CD4+ cells. The authors observed that CD45RBhigh cells were the major CD4+ subpopulation in primary infected mice, while CD45RBlow cells predominated in 60 days re‐infected animals. Moreover, the great majority of activated (large) CD4+ cells in the primary infection belonged to the CD45RBhigh subset, while after re‐infection most of the CD4+ large had a CD45RBlow phenotype.

Trypanosoma vivax infections: pushing ahead with mouse models for the study of Nagana. I. Parasitological, hematological and pathological parameters.

African trypanosomiasis is a severe parasitic disease that affects both humans and livestock. Several different species may cause animal trypanosomosis and although Trypanosoma vivax (sub-genus Duttonella) is currently responsible for the vast majority of debilitating cases causing great economic hardship in West Africa and South America, little is known about its biology and interaction with its hosts. Relatively speaking, T. vivax has been more than neglected despite an urgent need to develop efficient control strategies. Some pioneering rodent models were developed to circumvent the difficulties of working with livestock, but disappointedly were for the most part discontinued decades ago. To gain more insight into the biology of T. vivax, its interactions with the host and consequently its pathogenesis, we have developed a number of reproducible murine models using a parasite isolate that is infectious for rodents. Firstly, we analyzed the parasitical characteristics of the infection using inbred and outbred mouse strains to compare the impact of host genetic background on the infection and on survival rates. Hematological studies showed that the infection gave rise to severe anemia, and histopathological investigations in various organs showed multifocal inflammatory infiltrates associated with extramedullary hematopoiesis in the liver, and cerebral edema. The models developed are consistent with field observations and pave the way for subsequent in-depth studies into the pathogenesis of T. vivax - trypanosomosis.

Significant association between the skewed natural antibody repertoire of Xid mice and resistance to Trypanosoma cruzi infection.

The Xid mutation predominantly affects the development of B cells and consequently the levels and composition of natural antibodies in sera. In contrast to the congenic and susceptible BALB/c strain, immunodeficient BALB.Xid mice display a resistant phenotype both to acute Trypanosoma cruzi infection and to the development of severe cardiopathy. Because natural antibodies are known to be basically self‐antigen driven, IgM and IgG natural antibody repertoires (NAR) were compared before and during infection in these two strains. The analysis revealed fundamental alterations of IgM and IgG NAR in pre‐ and post‐infected Xid mice. In particular, relatively increased natural (pre‐existing) autoreactive IgG, dominated by the unique recognition of a single band in autologous heart extracts, was typical for uninfected Xid mice. This natural autoreactive IgG directed to heart antigens disappeared early after infection not only in Xid, but also in individual BALB/c mice that survived the acute infection. Conversely, the subgroup of BALB/c mice that died early after infection presented the most pronounced instances of the rapid, relative increase of IgM reactivies to self and non‐self proteins. These results suggest that self‐reactive NAR may play a role in an immunoregulatory mechanism relevant for the determination of susceptibility/resistance to infections. This may act either by influencing specific responses, or by modulating the self‐aggressive components responsible for pathology.