Kinetic study of the antibody response during the blood meal of Ixodes ricinus: implication on plasma cell maturation in vivo and for anti-Ixodes vaccination.

Anti-tick vaccination could be an ideal solution to prevent pathogen transmission, but none is currently available against Ixodes ticks. Recently, we showed that adult Ixodes ricinus infestation on mice decreases the specific antibody production to BSA injected during infestation. Here, a kinetic study of seric levels of BSA-specific antibodies was performed to evaluate the B memory cell differentiation in Balb/c mice and the capacity of specific B memory cells to respond to BSA during infestation. We concluded that the tick blood meal inhibits or impairs the local differentiation of mature B cells into plasma cells, but does not alter the formation of memory B cell. Accordingly, this mechanism should not be an impediment to anti-Ixodes vaccination.

Influence of the Ixodes ricinus tick blood-feeding on the antigen-specific antibody response in vivo.

The blood meal of hard ticks such as Ixodes ricinus lasts several days. This is made possible by tick salivary factors that inhibit inflammation, haemostasis and the host immune response. We assessed the latter on a model of immune response in vivo. A significant reduction of specific IgM and IgG levels was observed in BALB/c mice infested 5 days before injection with bovine serum albumin (BSA) and QuilA but not in mice infested 5 days after the immunization. This effect was not observed in mock-infested mice and could not be attributed to the use of anesthetics. The antibody response was not merely delayed and the Th(1)/Th(2) balance appeared not altered. T-dependent zones and germinal centers in lymph nodes draining the tick bite site showed no apparent morphological alterations or shift in T cell subpopulations. However, the spleens of tick-infested mice had also an enlarged red pulp, indicating an increased extramedullary haematopoietic activity.

Induction of apoptosis in human promyelocytic leukemia cells by a natural trachylobane diterpene.

BACKGROUND: Trachylobane diterpenes are secondary metabolites, quite rare in nature, and their bioactivities are poorly understood. Recently, we have described the cytotoxic activity of ent-trachyloban-3beta-ol isolated from the leaves of Croton zambesicus, a plant used in African folk medicine. MATERIALS AND METHODS: Cell viability on several cell lines, cell morphology, DNA laddering, annexin Vand caspase-3 activation experiments were undertaken in order to analyse the cytotoxicty of trachylobane diterpene and to determine if this compound is able to induce apoptosis. RESULTS: ent-Trachyloban-3beta-ol exerts a dose-dependent cytotoxic effect, which varies between cell lines. Induction of apoptosis in HL-60 cells could be detected at a concentration of 50 microM after 24-h treatment. CONCLUSION: We show here, for the first time, that a trachylobane diterpene is able to induce apoptosis in human promyelocytic leukemia cells via caspase-3 activation in a concentration-dependent manner.

FDC-B1: a new monoclonal antibody directed against bovine follicular dendritic cells.

Follicular dendritic cells (FDCs) are a unique population of accessory cells located in the light zone of the germinal centres of lymphoid follicles. Their involvement in the generation of humoral immune responses implies a potential role for these cells in many disorders. Indeed, in prion diseases, FDCs seem to be the major sites of extraneuronal cellular prion protein expression and the principal sites of the infectious agent accumulation in lymphoid organs. The identification of FDC is useful for the analysis of their distribution in reactive lymphoid tissue as well as in pathological conditions. The production and characterisation of a new mouse monoclonal antibody directed against bovine follicular dendritic cells (FDC-B1) is reported. The antigen detected by FDC-B1 is expressed exclusively on the surface of FDCs in ruminant lymphoid organs. The antigen has an approximate molecular weight of 28 kDa.

DNA immunisation. New histochemical and morphometric data.

Splenic germinal center reactions were measured during primary response to a plasmidic DNA intramuscular injection. Cardiotoxin-pretreated Balb/c mice were immunized with DNA plasmids encodmg or not the SAG1 protein, a membrane antigen of Toxoplasma gondii. Specific anti-SAG1 antibodies were detected on days 16 and 36 after injection of coding plasmids. The results of ELISAs showed that the SAG1-specific antibodies are of the IgG2a class. Morphometric analyses were done on serial immunostained cryosections of spleen and draining or non-draining lymph nodes. This new approach made it possible to evaluate the chronological changes induced by DNA immunisation in the germinal centres (in number and in size). Significant increases in the number of germinal centres were measured in the spleen and only in draining lymph nodes after plasmid injection, the measured changes of the germinal centers appeared to result from the adjuvant stimulatory effect of the plasmidic DNA since both the coding and the noncoding plasmid DNA induced them. No measurable changes were recorded in the T-dependent zone of lymph organs.

Spermine induces precocious development of the spleen in mice.

Spermine is a low molecular weight polyamine involved in the postnatal maturation of the gut. When it is administered orally to suckling rats, it induces maturation of the intestinal tract (liver, pancreas and small intestine). Here we show that this polyamine is able to induce precocious intestinal and splenic development in suckling mice. In fact, in 15-day-old mice which had received spermine orally twice daily for 3 days we observed an increase in the ratio of white pulp surface to total spleen surface in comparison with untreated mice. The two macrophage subsets of the marginal zone and the B-cell population were more developed and reached the development level of 5- or 10-week-old mice. The proliferation rate of B-cells was increased by spermine administration to pups. These observations suggest that spermine might play a role in immune system development; further investigation of its effects are intended, namely the evaluation of its capacity to enhance defence during the neonatal period.

Isolation of bovine follicular dendritic cells allows the demonstration of a particular cellular prion protein.

As interaction of cellular prion protein (PrPc) and the infectious agent (PrPres) appears to be a crucial pathogenic step promoted by homology, variation in PrPc isoforms on bovine immune cells may explain the absence of infectivity in most bovine lymph organs. In this study, we examined PrPc expression in bovine lymph organs (tonsils and lymph nodes) and on isolated follicular dendritic cells (FDCs). We used a panel of different monoclonal antibodies (MoAbs) raised against different epitopes of prion protein. Two MoAbs recognise amino acids 79-92 (SAF 34 and SAF 32 Mo-Abs); the 6H4 antibody reacts with a specific peptide comprising the 144-152 amino acids, and the 12F10 MoAb recognises the sequence 142-160. After immunolabelling of frozen sections of lymph organs with 6H4 or 12F10 MoAbs, we detected cellular prion protein in germinal centres. However, using the SAF 34 or SAF 32 antibodies, PrPc was revealed outside the lymphoid tissues. No PrPc was observed in the germinal centres. Therefore, we adapted the method of FDC isolation, making it suitable for the study of PrPc expression on their surface. Using electron microscopy, the presence of PrPc on the surface of FDCs was demonstrated only with 6H4 MoAb. These results suggest that bovine follicular dendritic cells express a particular form of prion protein. Either the N-terminal part of PrPc is cleaved or the accessibility of the specific epitope (79-92) of SAF 34 MoAb is abolished by interaction with other molecules. This particular isoform of PrPc on bovine FDCs might be related to the apparent absence of infectivity in lymph organs in cattle affected by bovine spongiform encephalopathy.

Mechanism of colon cancer cell apoptosis mediated by pyropheophorbide-a methylester photosensitization.

Pyropheophorbide-a methylester (PPME) is a second generation of photosensitizers used in photodynamic therapy (PDT). We demonstrated that PPME photosensitization triggered apoptosis of colon cancer cells as measured by using several classical parameters such as DNA laddering, PARP cleavage, caspase activation and mitochondrial release of cytochrome c. Preincubation of cells with N-acetyl cysteine (NAC) or pyrolidine dithiocarbamate (PDTC) protected against apoptosis mediated by PPME photosensitization showing that reactive oxygen species (ROS) are involved as second messengers. On the other hand, photosensitization carried out in the presence of deuterium oxide (D2O) which enhances singlet oxygen (1O2) lifetime only increases necrosis without affecting apoptosis. Since PPME was localized in the endoplasmic reticulum (ER)/Golgi system and lysosomes, other messengers than ROS were tested such as calcium, Bid, Bap31, phosphorylated Bcl-2 and caspase-12 but none was clearly identified as being involved in triggering cytochrome c release from mitochondria. On the other hand, we demonstrated that the transduction pathways leading to NF-kappaB activation and apoptosis were clearly independent although NF-kappaB was shown to counteract apoptosis mediated by PPME photosensitization.

Evidence of rainbow trout prolactin interaction with its receptor through unstable homodimerisation.

This study aims to characterise Prolactin receptor (PRLR) in rainbow trout for which no information is available despite the availability of Salmonid PRL preparations. By screening a freshwater rainbow trout intestine cDNA library with a probe corresponding to the extracellular domain (ECD) of tilapia PRLR, we have cloned a 2.5 kb insert coding for the PRLR. The mature protein of 614 amino acid residues is similar to PRLR isolated in tilapia and also the long form of mammalian PRLR. Analysis of PRLR gene expression in osmoregulatory organs revealed the presence of a unique transcript, thus confirming the involvement of this hormone in the control of osmoregulation in this fish species. By using surface plasmon resonance (SPR) technology, kinetic measurement of interaction between trout PRL and its receptor ECD was studied. This approach allowed us to demonstrate the formation of a transient, unstable homodimeric complex. This unstability could explain the inability to perform binding experiments using homologous PRL. In contrast, heterologous lactogenic ligands were able to interact through a more stable complex. Whether these characteristics of PRL-receptor interaction in rainbow trout are different to what occurs in tilapia where a homologous radioreceptor assay was developed would require further studies.

Differential expression of cellular prion protein on human blood and tonsil lymphocytes.

BACKGROUND AND OBJECTIVE: The expression of cellular prion protein (PrPc) on the surface of peripheral lymphocytes has been previously reported, but little is known about its expression on lymphoid cells from secondary lymph organs. In this report, we compare the surface expression of PrPc on human blood lymphocytes and tonsil lymphocytes. DESIGN AND METHODS: This analysis was performed by cytometry on live lymphocytes isolated from healthy donors or from the tonsils of adults or children. RESULTS: Human peripheral lymphocytes and tonsillar lymphoid cells, but not erythrocytes or granulocytes, express PrPc at their surfaces. Interestingly, we found significantly less PrPc on freshly isolated tonsil lymphocytes, both B and T, than on blood cells. Although tonsil cells bear less PrPc than circulating blood lymphocytes, they are able to express high quantities of PrPc on their surface when placed in culture. However, contrary to previous results, mitogen stimulation does not affect this expression on B- or T-cells. INTERPRETATION AND CONCLUSIONS: We suggest that the PrPc expression by lymphocytes may be modified by interactions occurring during intratissular migration or during cell-to-cell contacts. Whether PrPc plays a role in intracellular communication at this location, as it does in the nervous system, remains an open question.