Immunostimulatory effects of polar glycopeptidolipids of Mycobacterium chelonae for inactivated rabies vaccine.

Humoral and cellular immune responses were analyzed with Fuenzalida-Palacios rabies vaccine associated with pGPL-Mc, polar glycopeptidolipids extracted from Mycobacterium chelonae, aiming at its use as adjuvant. These results were compared to those obtained with BCG, a well-known immunostimulator, under the same conditions. Rabies vaccine plus pGPL-Mc (2.5 mg/kg) induced a significant increase in serum neutralizing activity, in vitro lymphocyte proliferation (spontaneous, specific and mitogen stimulation) and delayed type hypersensibility. In addition, pGPL-Mc, as well as BCG, enhanced the vaccine potency. Our results support further studies to encourage the use of pGPL-Mc as an immunostimulator of veterinary vaccines, before consideration for human vaccines.

Pregnancies modulate B lymphopoiesis and myelopoiesis during murine ageing.

We recently reported that pregnancy affects age-related changes in the distribution of lymphoid and macrophage populations in the spleen of C57Bl/6 mice. In the present study, we examined the influence of pregnancies on the generation of various developmental B-cell subsets and granulocyte/macrophage lineage cells during murine ageing. Using flow cytometry, changes in lymphoid (mature and early B-cell precursors: B220high, B220low, surface immunoglobulin M (sIgM) mu chain +/-) and myeloid (monocyte/macrophage Mac-1/CD11b, granulocyte Gr-1/Ly-6G) compartments were monitored in the bone marrow of young (2 months) and 15- and 23-month-old mice including male, multiparous and virgin female mice. Pregnancies delayed the age-related decline in murine B lymphopoiesis and maintained B-cell reserve capacity during ageing. We also found an increased production of myeloid cells induced by pregnancies at middle (15 months) and advanced (23 months) ages. This comparative study provides new information on changes in marrow lymphopoiesis and myelopoiesis with age. Our data emphasizes that the onset, magnitude and kinetics of age-related changes in the haematopoietic marrow are parity dependent. These changes could influence the incidence of age-related diseases and may account for the greater longevity of females.

[Aging and the immune system. Experimental aspects]. / Vieillissement et système immunitaire. Quelques aspects expérimentaux.

So far no comparative studies have been conducted to know whether physiological influences related to sex hormonal differences affect the age-related changes of the immune system. The aim of this study was to investigate whether pregnancies and sex influence the age-related changes in the peripheral lymphoid compartment and functions of T cells in mice. Using flow cytometry, we examined changes in (Thy1.2+) T cells, (B220+) B cells and (CD11b/Mac-1+) macrophages in the spleen of multiparous and virgin females and males at 2, 8, 15 and 23 months of age. The development of naive (CD44low) and memory (CD44high) cells were investigated in CD4+ and CD8+ T cell subsets. To analyze the age-related changes in functions of T cells, we examined the secretion of some T cell immunoregulatory cytokines (IL-2, IL-4, gamma-interferon and GM-CSF) of in vitro Concanavalin A-activated spleen cells of C57BL/6 mice. Both short term (8 months) and long term (15-23 months) effects of pregnancies were obvious in the age-related changes of the immune system. Short term effect included delayed appearance of memory CD4+ cells and the preserved IL-2 production. At eight months, shortly after pregnancies, both parameters were higher in multiparous females. Later effects of pregnancies were evidenced by a higher level of macrophages (Mac-1+) than in other groups throughout life. The increased gamma-interferon, IL-4 and GM-CSF productions appeared earlier, at 15 months, IL-4 and GM-CSF levels remained higher in multiparous females than in virgin females and males in late adulthood. Sex differences were also noticed: males exhibited lower macrophage levels after one year and gamma-interferon secretion capacity than females in late life. This study underlines that the onset, magnitude and kinetics of the age-related changes in the distribution of immune cells and T cell functions are parity- and sex-dependent. These changes may influence the incidence of age-related diseases and may explain the greater longevity of women, especially the multiparous ones.

Sex and parity modulate cytokine production during murine ageing.

We have previously shown that physiological hormone differences related to pregnancy or sex affect the age-related distribution of mononuclear cell populations during murine ageing. To determine whether such changes are involved in the age-related changes in functions of T cells, we examined the secretion of major T cell immunoregulatory cytokines (IL-2, IL-4, interferon-gamma (IFN-gamma), IL-3, IL-6 and granulocyte-macrophage colony-stimulating factor (GM-CSF)) of in vitro concanavalin A-activated spleen cells of C57B1/6 mice. The study included multiparous and virgin females and males at 2, 8, 15 and 23 months of age. Short-term effects of parity (8 months) were evidenced by the decrease of IFN-gamma and the preserved IL-2 production in multiparous females (8 months), while IFN-gamma was unchanged and IL-2 decreased in virgin mice. The increase in IL-4 production appeared earlier in multiparous females (15 months) than in virgin mice (23 months). The increase in IL-4/IFN-gamma and IL-4/IL-2 ratios at 8 and 15 months, respectively, in multiparous females, suggests that pregnancy modifies the Th1/Th2 equilibrium. In late adulthood (15 months), IL-6 and GM-CSF production was higher in multiparous females than in virgin males or females. Sex differences were also noticed: IFN-gamma secretion capacity was lower in males than in females during ageing. This study underlines that the onset, magnitude and kinetics of the age-related changes in cytokine production are parity- and sex-dependent. These changes probably influence the incidence of age-related diseases and may explain the greater longevity of females.

[Calicivirus infection of the hare and human calicivirus]. / Calicivirose du lièvre et calicivirus de l'homme.

The calicivirus outbreak in hares which occurred in Center West of France in the fall of 1996 illustrates the pathogenic role of caliciviruses in animals and in humans. A comparison of these different viruses, based on the RNA nucleic sequences is presented. The aspect of interspecies transmission is also discussed.

[Helicobacter infections of man and of domestic carnivores: comparative data]. / Hélicobactérioses de l'homme et des carnivores domestiques: quelques données comparatives.

The role of Helicobacter pylori in generating of the chronic gastritis and in the maintaining of the gastroduodenal ulcerous disease, has been a major medical discovery of these past years in human gastroenterology. More recently in Man, studies have showed that the gastric tumours (adenocarcinoma, lymphoma) are epidemiologically associated with the H. pylori infection. Although the H. pylori infection is the one of the most frequent in the word, the epidemiologic and ecologic aspects of this infections are still not very well known. Thanks to phylogenic studies using the new molecular biology techniques and to fundamental experimental studies, we know more about helicobacteria in domestic carnivores as well as their morphologic characteristic, their taxonomia and more importantly details concerning their ecological niche. Few clinical studies have been made to this day, but the ones that have been undertaken are interesting in confirming the extensive prevalence of Helicobacter infections in domestic carnivores and in underlining their role in the genesis of the inflammatory gastropathies observed in these species. Recent observations have demonstrated the ubiquitous character of these helicobacteria by showing their presence in the stomach of man, dogs and cats. This ubiquitous character has led some scientists to consider the potential zoonotic risk of the human infection by Helicobacter heilmannii, felis or pylori. Finally, the Helicobacter infection of animals seems to be an interesting model not only in the study of the affections caused by these bacteria, but also in the elaboration of a future vaccine against the H. pylori infection in man.

[Cat scratch disease and associated infections]. / Maladie des griffes du chat et infections associées.

Cat scratch disease (CSD) was first described in France by Debré et al. in 1950, yet the causative bacterial agent of CSD remained obscure until 1992, when Bartonella (formerly Rochalimaea) henselae was implicated in CSD by serological and microbiologic studies. B. henselae had been linked initially to bacillary angiomatosis (BA), but also bacillary peliosis, relapsing bacteremia and endocarditis. Cats are healthy carriers of B. henselae and B. clarridgeiae, and can be bacteremic for months to years. Cat to cat transmission of the organism involves the cat flea in absence of direct contact transmission. Present knowledge on the etiology, clinical features and epidemiological characteristics of cat scratch disease/bacillary angiomatosis are presented.

Surface antigen expression in spleen cells of C57B1/6 mice during ageing: influence of sex and parity.

So far all studies on the murine ageing process have been conducted on virgin mice. Immune ageing may be influenced by sex hormone differences related to sex or pregnancies. The aim of this study was to investigate whether pregnancies and gender influence the cell changes observed during ageing in a peripheral lymphoid compartment of C57B1/6 mice. Using flow cytometry, changes in (Thy1.2+) T cell, (B220+) B cell and (CD 11b/Mac-1) macrophage spleen populations were monitored in 2, 8 (3 months after last pregnancy) 15 and 23-month-old mice including males, virgin and multiparous females. The development of naive (CD44(low)), memory (CD44(high)), activated/memory (MEL-14, CD62L) cells were investigated in CD4+ and CD8+ T cell subsets. Both short term (at 8 months) and long term (at 15 and 23 months) effects of multiparity were obvious in the lymphocyte/macrophage population changes associated with the ageing process. Short-term effects included delayed appearance of CD4+CD44(high) memory lymphocytes and increased numbers of both CD4+MEL-14(1ow) activated/memory cells and Mac-1+ macrophages when compared with virgin control mice. Later effects of multiparity were increased CD8alpha(dull) populations and increased T/B cell ratios and the ratio of memory to naive CD4+ cells (CD44+(high)/CD44+(low). A sex effect was noticed: males exhibited lower Mac-1+ levels and memory/naive ratio in CD4+ subset than virgin females throughout life. These results suggest that gender and/or pregnancies affect the age-related distribution of lymphoid and macrophage cell populations in the spleen of C57B1/6 mice.

Stimulatory effects of polar glycopeptidolipids of Mycobacterium chelonae on murine haematopoietic stem cells and megakaryocyte progenitors.

The effects of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) on haematopoietic stem cells and on megakaryocyte progenitors in bone marrow (BM) and spleen were investigated in mice. We studied the in vivo spleen colony-forming ability and marrow repopulating ability of pGPL-Mc by assays of colony-forming units-spleen (CFU-S). The number of CFU-S was increased in BM when both donors and recipients were treated with pGPL-Mc. In contrast, a single treatment of donors induced enhancement of spleen CFU-S. The number of pre-CFU-S was not significantly increased by pGPL-Mc injection. Megakaryocyte (Meg) progenitors were determined in vitro with a quantitative cultural analysis of bone marrow and spleen cells in agar in the presence of spleen-conditioned medium. A statistically significant increase in BM and spleen CFU-Meg was observed two days after the last administration of pGPL-Mc. This experiment points out the ability of pGPL-Mc to induce substantial stimulation of megakaryocytopoiesis and slight proliferation of stem cells in BM, but which is more pronounced in spleen. This molecule therefore appears to be a potential adjuvant of chemo- and radiotherapy in order to palliate the cytotoxic side effects of these cancer therapeutic modalities.

Adjuvanticity of pGPL-Mc and LRS in the immune responses of monkeys to oral immunization with diphtheria and tetanus toxoids.

Experiments were carried out to examine the adjuvanticity of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) or the London rocket seed (LRS) when combined with diphtheria and tetanus toxoids in an oral immunization of the African green monkey. The results showed that none of the monkeys receiving diphtheria and tetanus toxoids combined with 25 mg/kg of pGPL-Mc showed an increase in the the level of diphtheria antitoxin (DA) on the third and sixth weeks following the first and the second immunizations. One monkey from this group responded with increased seroneutralizing antibodies 3 weeks after the third feeding. On the other hand, one monkey, 3 weeks after the first immunization, and three monkeys, 3 weeks after the second and third oral vaccinations, showed an increase in specific anti-diphtheria antibody responses when the toxoids were combined with 25 mg/kg of LRS. The anti-diphtheria antitoxin responses of monkeys receiving diphtheria and tetanus toxoids combined with 50 mg/kg of pGPL-Mc or 50 mg/kg of LRS were significantly enhanced compared to the groups administered 25 mg/kg of the two adjuvants. The increase was observed in four out of five pGPL-Mc administered and in three out of five LRS-receiving monkeys. The results show that pGPL-Mc induced the highest titres of anti-diphtheria antitoxin compared to LRS, whereas the level of anti-diphtheria antitoxin titre of the two monkeys receiving the toxoids alone was less than 0.1 i.u./ml of serum throughout the experiment. According to the statistical analyses, no significant differences were recorded between the diphtheria antitoxin responses of monkeys following the first, second or third administration of LRS-adjuvated diphtheria and tetanus toxoids. However, a significant difference (P < or = 0.05) was observed in the diphtheria antitoxin response between the first and the second immunization of monkeys administered with toxoids adjuvated with 50 mg/kg of pGPL-Mc. The tetanus antitoxin responses of all monkeys were less than 0.1 i.u. of antitoxin per millilitre of serum throughout the study, which is considered not to be protective. However, we have recorded an anti-tetanus antitoxin titre of more than 0.2 i.u./ml of serum in one monkey that received diphtheria and tetanus toxoids combined with 50 mg/kg of pGPL-Mc.

Effects of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) on haematopoietic regeneration and resistance to infection of sublethally irradiated mice.

The influence of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) treatment on the reversal of irradiation-induced leukopenia (granulocytopenia, monocytopenia) and thrombocytopenia and its ability to protect mice against lethal infections were investigated in this study. The administration of pGPL-Mc to irradiated mice significantly accelerated the recovery of leukocyte and thrombocyte numbers in the peripheral blood. Granulocytes and monocytes were the principal cells of the leukocyte population that responded to the potent stimulus of this product. The reversal of granulocytopenia and monocytopenia in treated mice was achieved on day 14 and reached a peak value on day 20. Responses in mice receiving 100 mg/kg of pGPL-Mc was about 40-fold compared to controls and about 4-fold compared to the rhG-CSF-treated group. Normal levels of thrombocytes were reached by day 17 in mice treated with 100 mg/kg and by day 20 in those receiving 25 mg/kg of pGPL-Mc. The administration of pGPL-Mc to mice with irradiation-induced granulocytopenia was characterized by highly significant protection of these animals against lethal Klebsiella pneumoniae or Escherichia coli infections. Therefore, pGPL-Mc appears to possess a considerable potential for improvement of the outcome of radiotherapy and may contribute to the successful avoidance of irradiation-induced toxicities.

Effects of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) on granulomacrophage progenitors.

Effects of polar glycopeptidolipids of Mycobacterium chelonae (pGPL-Mc) in the in vivo stimulation of haematopoietic growth and differentiation of murine bone marrow and spleen cells was investigated in this study. Progenitors were determined with a quantitative cultural analysis of bone marrow and spleen cells in methylcellulose using rmGM-CSF and rmIL3. Injection of pGPL-Mc produced a significant time-related increase in the number of bone marrow and spleen CFUs. pGPL-Mc treatment, in particular, increased the number of bone marrow and splenic CFU-GMs, CFU-Gs and CFU-Ms during and after three intraperitoneal administrations. The greatest myeloid stimulation of bone marrow CFU-GMs, CFU-Gs and CFU-Ms was observed between days 7 and 14, with maximal values on days 12 and 14. Highly significant stimulation of splenic CFU-GMs, CFU-Gs and CFU-Ms was observed between days 7 and 10 with maximal values on day 10, while the initial stimulation of these progenitors was observed starting from day 1 in bone marrow and day 7 in spleen. These effects of pGPL-Mc were associated with an increase in granulocyte, monocyte and thrombocyte counts in the peripheral blood. Granulocyte and monocyte counts remained high up until day 12, while those of thrombocytes were prolonged until day 18. May-Grünwald-Giemsastained colony samples and differential white blood cell counts demonstrated that the granulocyte population is composed almost entirely of neutrophils. pGPL-Mc is therefore a broad-spectrum haematopoietic growth factor with a highly promising application in the reversal of chemotherapy- and/or radiotherapy-induced myelo-suppression.

Mycobacterial polar glycopeptidolipids enhance resistance to experimental murine candidiasis.

Intraperitoneal administration of polar glycopeptidolipids extracted from Mycobacterium chelonae (pGPL-Mc) greatly increased the resistance of mice against a lethal disseminated Candida albicans infection. This enhanced resistance was demonstrated by an increase in the number of survivors and the prolongation of the mean survival time of animals following a lethal challenge. These effects were dependent upon the infective dose of Candida albicans, the dose of pGPL-Mc and the timing of its administration. This enhanced resistance was correlated with the development and persistence of a hyperleukocytosis, associated with a long lasting increase in the number of polymorphonuclear neutrophils. On the contrary, no candidacidal effect of the serum collected from pretreated mice was observed; suggesting that the ability of pGPL-Mc to increase resistance against Candida albicans infection is likely to be mediated by polymorphonuclear neutrophils. These results confirm previously described immunostimulating properties of pGPL-Mc and open the way for the evaluation of its effect in the prevention of opportunistic infections in neutropenic patients.

Quantitative and qualitative changes in CD44 and MEL-14 expression by T cells in C57BL/6 mice during aging.

Aging is associated with a decrease in the functional activity of T cells. We have explored age-related alterations in CD44 and MEL-14 expression by spleen cells bearing the Thy1.2, CD4 or CD8 antigens in C57BL/6 mice at 2, 8, 15 and 23 months of age. The membrane expression of CD44 and MEL-14 molecules can be used to distinguish naive (CD44low, MEL-14high) from preactivated/memory (CD44high, MEL-14low) T cells. Our results show that the proportion of CD4+ splenic cells begins to decrease at an intermediate age (8-month-old mice), whereas the proportion of CD8+ cells remains unaltered. The proportion of CD4+ and CD8+ splenic cells with the CD44high memory phenotype was increased at an early stage of aging (in 8-month-old mice) without a concomitant change in MEL-14 expression. In older mice, MEL-14 expression decreased on CD4+ but not on CD8+ subsets. Recent studies have reported that following activation, the expression of CD44 molecules containing additional, so-called variable exons can be detected. By PCR, we observed an increase in CD44 transcripts containing the v6 or v7 variable exons in murine lymph nodes at the age of 15 months. Our results suggest that v6- or v7-containing variants of CD44 may be involved in the development of memory cells. Taken together, these results suggest that the trafficking of memory T cells in aging may be altered by quantitative and/or qualitative differences in the expression of molecules involved in lymphocyte recirculation.