Inhibitory activities of epidermal growth factor receptor tyrosine kinase-targeted dihydroxyisoflavone and trihydroxydeoxybenzoin derivatives on Sarcocystis neurona, Neospora caninum, and Cryptosporidium parvum development.

Several gene sequences of parasitic protozoa belonging to protein kinase gene families and epidermal growth factor (EGF)-like peptides, which act via binding to receptor tyrosine kinases of the EGF receptor (EGFR) family, appear to mediate host-protozoan interactions. As a clue to EGFR protein tyrosine kinase (PTK) mediation and a novel approach for identifying anticoccidial agents, activities against Sarcocystis neurona, Neospora caninum, and Cryptosporidium parvum grown in BM and HCT-8 cell cultures of 52 EGFR PTK inhibitor isoflavone analogs (dihydroxyisoflavone and trihydroxydeoxybenzoine derivatives) were investigated. Their cytotoxicities against host cells were either absent, mild, or moderate by a nitroblue tetrazolium test. At concentrations ranging from 5 to 10 microg/ml, 20 and 5 analogs, including RM-6427 and RM-6428, exhibited an in vitro inhibitory effect of > or = 95% against at least one parasite or against all three, respectively. In immunosuppressed Cryptosporidium parvum-infected Mongolian gerbils orally treated with either 200 or 400 mg of agent RM-6427/kg of body weight/day for 8 days, fecal microscopic oocyst shedding was abolished in 6/10 animals (P of <0.001 versus untreated controls) and mean shedding was reduced by 90.5% (P of <0.0001) and 92.0% (P of <0.0001), respectively, higher levels of inhibition than after nitazoxanide (200 mg/kg/day for 8 days) or paromomycin (100 mg/kg/day for 8 days) treatment (55.0%, P of <0.001, and 17.5%, P of >0.05, respectively). After RM-6427 therapy (200 mg/kg/day for 8 days), the reduction in the ratio of animals with intracellular parasites was nearly significant in ileum (P = 0.067) and more marked in the biliary tract (P < 0.0013) than after nitazoxanide or paromomycin treatment (0.05 < P < 0.004). RM-6428 treatment at a regimen of 400 mg/kg/day for 12 days inhibited oocyst shedding, measured using flow cytometry from day 4 (P < 0.05) to day 12 (P < 0.02) of therapy, when 2/15 animals had no shedding (P < 0.0001) and 11/15 were free of gut and/or biliary tract parasites (P < 0.01). No mucosal alteration was microscopically observed for treated or untreated infected gerbils. To our knowledge, this report is the first to suggest that the isoflavone class of agents has the potential for anticoccidial therapy.

Association between chronic urticaria and thyroid autoimmunity: a prospective study involving 99 patients.

BACKGROUND: The association between chronic urticaria and thyroid autoimmunity has been a subject of debate. However, this link was suggested in studies searching thyroid microsomal antibodies (TMA), which are less sensitive and less specific than anti-thyroperoxidase antibodies, moreover these studies did not measure anti-TSH receptor antibodies, nor did they use a control group. As a consequence, the results of these studies are difficult to interpret. OBJECTIVE: The aim of this study was to determine whether chronic urticaria is statistically associated with thyroid autoimmunity. METHODS: In a prospective case-control study, we compared the frequency of thyroid autoantibodies in 45 patients with chronic urticaria and in 30 healthy adult volunteers; we also compared the frequency of chronic urticaria in 32 patients with thyroid diseases with thyroid autoantibodies and in 22 patients with thyroid diseases without thyroid autoantibodies. Thyroid autoantibodies and thyroid hormones were measured in all the subjects; antinuclear antibodies, rheumatoid factors, complement, IgE were assessed and routine laboratory tests were done in patients with chronic urticaria. Fisher's exact statistics were used to test our hypothesis. RESULTS: The frequency of thyroid autoantibodies was significantly higher in patients with chronic urticaria than in healthy controls (26.7%/3.3%; p < 0.01). All the patients with thyroid autoantibodies had thyroid hormone concentrations within the normal limits. The frequency of chronic urticaria was not significantly different (12.5%/9.1%; p = 0.7) in patients with thyroid diseases with or without thyroid antibodies. The rest of the biological investigations revealed only 1 patient with connective tissue disease. CONCLUSION: This study shows a significant association between chronic urticaria and thyroid autoimmunity, and that tests to detect thyroid autoantibodies are relevant in patients with chronic urticaria, whereas extensive laboratory tests are not.

Fetal distress increases interleukin-6 and interleukin-8 and decreases tumour necrosis factor-alpha cord blood levels in noninfected full-term neonates.

OBJECTIVE: To assess the influence of fetal distress on interleukin-1beta, interleukin-6, interleukin-8 and on tumour necrosis factor-alpha blood levels in noninfected full-term neonates. STUDY DESIGN: In a multicentre prospective study, cord blood samples were obtained at time of delivery from 234 noninfected full-term neonates for the purposes of measuring serum levels of interleukin-1beta, interleukin-6, interleukin-8 and tumour necrosis factor-alpha using immunoassays. Women were classified into four groups according to the mode of delivery (vaginal delivery or caesarean section) and the presence or absence of fetal distress. The role of labour was also investigated. RESULTS: No significant relationship was found between cytokine cord blood levels and the mode of delivery. Fetal distress was associated with an increase in interleukin-6 (P = 0.01) and interleukin-8 (P < 0.001) levels, and a decrease in tumour necrosis factor-alpha (P < 0.001). Labour was also associated with a significant increase in interleukin-6 and interleukin-8 cord blood levels (P = 0.01 and P < 0.001, respectively). CONCLUSION: Fetal distress and labour were each associated with elevated interleukin-6 and interleukin-8 cord blood levels in noninfected full term neonates while only fetal distress was associated with decreased tumour necrosis factor-alpha levels.

Cryptosporidium parvum infection stimulates the secretion of TGF-beta, IL-8 and RANTES by Caco-2 cell line.

Cryptosporidium parvum is a common cause of diarrhea in humans. Although mild inflammatory mucosal infiltrate is usually observed, limited information is currently available on the pathogenic mechanisms involved in this phenomenon. The aim of this work was to investigate in vitro the influence of C. parvum infection on the secretion of lymphocyte-targeted chemokines (RANTES. MIP-1alpha, MIP-1beta, IL-8), proinflammatory cytokines (TNF-alpha, GM-CSF and IL-6) and TGF-beta by human enterocytic Caco-2 cells. C. parvum infection stimulates IL-8, RANTES and TGF-beta secretion by both the basal and apical side of caco-2 cells. A slight increase in TNF-alpha production by infected cells was observed in the apical compartment. Data suggest that enterocytic chemokines and/or TGF-beta are involved in the initiation and regulation of the mucosal response to C. parvum infection.

Detection by the comet assay of apoptosis induced in lymphoid cell lines after growth factor deprivation.

Dysregulation of apoptosis contributes to various diseases such as neurodegenerative or aging disorders, autoimmune syndromes or cancers. Numerous experimental paradigms have been explored to characterize molecular and cellular modulators of apoptosis. Similarly, numerous techniques have been described for detecting and/or quantifying accurately cells committed to apoptosis. Besides the conventional techniques, we describe in this report that the comet assay, which detects DNA single- and double-strand breaks in situ, at the cellular level, is relevant for the characterization of apoptotic cells. The comet assay is very sensitive and detects DNA fragmentation occurring in the apoptotic process as early as exposure of phosphatidylserine residues on the outer leaflet. Thus the comet assay can be used for the recognition of apoptosis that follows the death signal caused, for example, by genotoxic stress as well as lack of survival signal as in growth factor deprivation.

Transcriptional and post-transcriptional mechanisms induce cyclin-D1 over-expression in B-chronic lymphoproliferative disorders.

Cyclin D1 participates in cell-cycle control, in the progression through the G(1) phase and in the transition from the G(1) to the S phase. The CCND1 locus, located in 11q13, is amplified and cyclin-D1 protein is over-expressed in a wide range of human solid tumors. In some B-lymphoid malignancies, the t(11;14)(q13;q32) translocation joins the Ig heavy-chain locus to the CCND1 locus and leads to cyclin-D1 over-expression. In this study, a series of 127 patients presenting a B-chronic lymphoproliferative disorder (B-CLPD) was analyzed using a competitive RT-PCR designed to detect cyclin-D1-mRNA over-expression. Cyclin-D1 mRNA was expressed in patients with mantle-cell lymphoma (MCL; 10/10), hairy-cell leukemia (HCL; 3/5), B-chronic lymphoid leukemia (B-CLL; 4/111) and B large-cell lymphoma (BLCL; 1/1). Densitometric analysis of RT-PCR products and Western-blot autoradiograms, in addition to cytogenetic data, indicated that activation of the cyclin-D1 gene occurred independently of the t(11;14)(q13;q32) translocation in patients with HCL. Indeed, a normal-sized protein of 36 kDa exhibiting a level incompatible with gene activation by a translocation mechanism was detected in lymphoid cells with a normal karyotype. Moreover, we found a discrepancy between cyclin-D1 mRNA and protein levels in MCL and B-CLL, which suggested that some regulatory mechanisms acting at a post-transcriptional level persist in tumor cells.

Enzyme immunoassay detection of Cryptosporidium parvum inhibition by sinefungin in sporozoite infected HCT-8 enterocytic cells.

Complete parasite development was obtained in differentiated human enterocytic HCT-8 cells infected at confluence with Cryptosporidium parvum sporozoites, and evaluated in a quantitative enzyme immunoassay. Forty-eight hours after infection, a linear correlation was found between optical density values and the number of parasites determined in an immunofluorescent assay. Sinefungin exerted an inhibitory effect when added within 4 h after sporozoite addition to HCT-8 cultures (MIC50 = 38 mumol L-1), while the inhibitory effects of paromomycin and pentamidine dimethanesulfonate were also easily detected (MIC50 = 0.87 mumol L-1 and 0.27 mumol L-1, respectively). Except for high pentamidine dimethanesulfonate concentrations, no alteration in optical microscopy morphology or trypan blue exclusion of HCT-8 cells was observed in the presence of anticryptosporidial agents, which suggests that they were primarily active against developing parasites. Data suggest that EIA detection of C. parvum development in sporozoite-infected HCT-8 cells provides an accurate and convenient model for in vitro evaluation of parasite infectivity, growth and response to anticryptosporidial agents.

In vitro evaluation of B-CLL cells apoptotic responses to irradiation.

Defective apoptosis is a mechanism which could possibly explain B chronic lymphocytic leukemia (B-CLL) cell accumulation. Differences in evolution and prognosis of B-CLL patients may be due to heterogeneity in apoptotic cell death. We studied the apoptotic response to in vitro gamma radiation of blood mononuclear cells from 18 untreated B-CLL patients. In cells irradiated with 2, 4 or 8 Gy and then cultured for 20 hours, the percentage of trypan blue excluding (viable) cells was not modified (>92%). An apoptotic response to irradiation was detected in the majority of the patients, but the individual percentage of apoptotic cells varied widely (8 to 81% after 8 Gy irradiation) in individual cases. The flow cytometric analysis of nick-end DNA labeling demonstrated a dose effect of irradiation, particularly in patients with an apoptotic response of over 20%. In the future, a valuable clue to the selection of irradiation regimens for B-CLL patients may be the investigation of correlations between in vitro radiation-induced apoptosis and the in vivo response to radiation therapy.

IgE multiple myeloma.

IgE multiple myeloma is a rare disease characterized by a high frequency of Bence-Jones proteinuria and plasma cell leukaemia when compared to other isotypes of monoclonal proteins. Only 35 cases have been reported. We describe a 70-year-old woman with a stage III IgE kappa multiple myeloma presenting with a sacral plasmacytoma. Immunological and biochemical studies showed IgE kappa producing tumoral plasma cells. Serum total IgE was high without clinical symptoms suggesting an hyperIgE syndrome or mast cell activation. The patient underwent surgical removal of the sacral tumor and monthly melphalan-prednisone treatment together with intravenous pamidronate infusions. Magnetic Resonance Imaging (MRI) of the dorsolumbar spine revealed an epidural process leading to T6-T9 radiotherapy. Bone densitometry showed a decreased bone mineral content supporting the management of myeloma-related osteoporosis with bisphosphonate infusions. A good partial response with plateau-phase and increase of bone mineral content was achieved after 1 year of treatment and still persists after a 28 months follow-up.

Hydrogen peroxide gas plasma sterilization is effective against Cryptosporidium parvum oocysts.

The aim of this work was to evaluate in an immunosuppressed rat cryptosporidiosis model a new method that combines vacuum and low-temperature hydrogen peroxide gas plasma for sterilization of endoscopic material contaminated by Cryptosporidium parvum. Rats were challenged with oocysts either air-dried or air-dried and treated with vacuum alone or associated with gas plasma. No rat was found infected after gas plasma exposure of oocysts, whereas vacuum or air-drying alone resulted only in a decreased infectivity.

High frequency of IgG antagonizing follicle-stimulating hormone-stimulated steroidogenesis in infertile women with a good response to exogenous gonadotropins.

OBJECTIVE: To investigate the presence of FSH-blocking IgG in infertile women. DESIGN: Retrospective study. Sera from patients and controls were processed for IgG purification, and purified IgG were tested at various concentrations for their ability to inhibit the recombinant human FSH-induced P production in vitro by human granulosa cells. SETTING: Departments of Endocrinology, and Obstetrics and Gynecology, University of Caen. PATIENT(S): Fifty-seven infertile women including 14 women with premature ovarian failure (POF), 29 women with a poor response to IVF-ET, and 14 women with a good response to IVF-ET. Controls consisted of 22 healthy age-matched women. INTERVENTION(S): IVF-ET allowed human granulosa cell pooling and culture for FSH bioassay. MAIN OUTCOME MEASURE(S): Inhibition by purified IgG of the in vitro recombinant human FSH-induced P production by human granulosa cells. RESULT(S): Blocking IgG were identified in only 3 of 14 POF and in 2 of 29 women with a poor response to IVF-ET. In contrast, IgG from women with a good response to IVF-ET inhibited significantly P production, and blocking IgG were detected in 85% women with a good response to IVF-ET. CONCLUSION(S): This study identified FSH-blocking IgG in a high proportion of women with a good response to IVF-ET. The significance of this remains questionable.

Respiratory tract cryptosporidiosis in immunosuppressed rat is associated with an epithelial metaplasia.

Cryptosporidium parvum is an opportunistic protozoa that chronically infects the digestive tract of immunocompromised hosts. Respiratory cryptosporidiosis, which was reported in AIDS patients, is an uncommon feature of mammalian cryptosporidiosis models. In this study, we document the respiratory lesion; observed in an immunosuppressed rat model of cryptosporidiosis. Twenty rats were immunosuppressed with corticosteroids and low protein diet. They were challenged intratracheally with 10(6) C. parvum sporozoites. Lungs and ileums were examined on D3, D6, D10, D14. On D10 and D14, C. parvum were present in the respiratory tract of all animals in association with the progressive appearance of an immature malpighian metaplasia. On D14, an intestinal infection was also detected in 2/4 animals. The respiratory tract appears to be a fully permissive area for the protozoa in immunosuppressed rats. Introduction of parasites on the respiratory mucosa seems a requisite to induce respiratory cryptosporidiosis. This experimental protocol yields a low mortality rate, and so modelizes late and/or chronic stages of respiratory cryptosporidiosis.

An immunosuppressed rat model for evaluation of anti-Cryptosporidium activity of sinefungin.

Cryptosporidium parvum causes life-threatening diarrhoea in immunocompromised, especially AIDS patients and the efficiency of proposed anti-cryptosporidial therapies is limited or doubtful. An immunosuppressed adult rat model of C. parvum infection was developed for screening molecules candidate for curative and preventive activity in human cryptosporidiosis. Among 31 drugs tested, lasalocid (2-10 mg/kg/24 h), and sinefungin (2-10 mg/kg/24 h), exhibited some activity against C. parvum infection. Oral sinefungin therapy resulted in a dose related suppression of oocysts shedding, which correlated with oocyst disappearance from ileum sections and was also efficient in preventing infection. Relapses were observed after discontinuation of curative sinefungin therapy, which suggests that the biliary tract, a major location and parasite reservoir which sustains persisting infection, was not cleared of parasites by the drug. Improved therapeutic procedures with sinefungin (or analogues) will result from current pharmacological studies.

[Cell-mediated immunity and protection against blood stages of Plasmodium falciparum]. / Immunité à médiation cellulaire et protection contre les stades sanguins de Plasmodium falciparum.

In recent years, cell-mediated immunity against malaria has been the subject of intensive investigation either in humans from malaria endemic areas, or experimental models. Cellular immune mechanisms have been regarded as secondary to humoral immunity but, there is increasing evidence that shows its critical role in protection against blood stage plasmodium parasites. In the context of a large humoral-cellular interaction, T helper lymphocytes and monocytes/macrophages may play a key role in the elimination of plasmodial blood stages, particularly P. falciparum. IL-2, IL-4, IL-5, IFN-gamma cytokines secreted principally by CD4+ T lymphocytes and oxygen and nitrogen radicals produced by activated macrophages, are involved in the control of plasmodial infection. The spleen also plays a very important function in the anti-malarial protection by its increased capacity for filtration/destruction of parasitized red blood cells and by induction of B and T memory lymphocytes. Successful vaccination against malaria needs a choice of plasmodial antigens or B and T immunodominants epitopes able to stimulate plasmodium-specific lymphocytes and functional modification in the spleen.

Virus recovery from stools of patients undergoing bone marrow transplantation.

Diarrhea in marrow transplant recipients is a frequent complication attributable to non-infectious events such as acute GVHD or infectious events such as viral gastroenteritis. Rotavirus and enteric adenovirus are the most frequent viral pathogens. To determine the frequency of these infections, we prospectively examined the stool specimens of 94 patients who underwent autologous BMT (34 cases) or allogeneic BMT (60 cases). Stool specimens were examined from patients twice weekly. Nineteen of the 94 patients were infected with viral pathogens. This study showed: (1) an incidence of viral gastroenteritis identical in autologous and allogeneic BMT (20%), (2) a persistent risk despite treatment in laminar air flow rooms, (3) a significant association with severe acute GVHD, and (4) a significant risk of multiple viral infections in autologous BMT recipients. Rotavirus and adenovirus are a cause of enteritis involvement in patients undergoing BMT and they may be underdiagnosed and confused with GVHD. Screening of stool specimens after BMT should be directed to prevention and treatment of these viral infections to decrease the morbidity and mortality associated with BMT.

Decreased blood TcR gamma delta+ lymphocytes in AIDS and p24-antigenemic HIV-1-infected patients.

The significance of blood TcR gamma delta+ lymphocyte level was evaluated in the context of immunodeficiency and infections in 209 HIV-1-infected patients. Blood TcR gamma delta+ lymphocyte values were found higher in patients belonging to the CDC group II/III than those in the CDC groups IV C1 and IV D (P < 0.001) and P < 0.01, respectively). TcR gamma delta+ lymphocyte counts were lower in patients with oral candidiasis (P < 0.01), and in association with pneumocystosis or toxoplasmosis (P < 0.001). In 81 patients with a detectable HIV-1 p24 antigenemia, TcR gamma delta+ lymphocyte counts were lower than those in nonantigenemic patients (P < 0.001). In the CDC II/III group, p24-antigenemic patients exhibited lower TcR gamma delta+ cell counts than those in patients without antigenemia (P = 0.06). Data suggest that depletion of the TcR gamma delta+ lymphocyte subset characterizes HIV-1-infected patients with oral candidiasis, pneumocystosis, toxoplasmosis, and/or HIV-1-antigenemia.

Curative and preventive anticryptosporidium activities of sinefungin in an immunosuppressed adult rat model.

An immunosuppressed rat model was used to investigate the anti-Cryptosporidium parvum activity of sinefungin. In infected animals, oral sinefungin therapy resulted in a dose-related suppression of oocyst shedding, which correlated with oocyst disappearance from ileal sections. When administered prior to or on the day of oocyst challenge, sinefungin successfully prevented infection. These data suggest that sinefungin could be considered as a candidate molecule in the treatment of human cryptosporidiosis, considered to be the most significant enteric opportunistic infection in AIDS.

[Leukocytic cytomegalic antigen. A new diagnostic method of cytomegalovirus infection after transplantation]. / Antigène cytomégalique leucocytaire. Nouvelle méthode diagnostique de l'infection à cytomégalovirus après transplantation.

The occurrence of cytomegalovirus (CMV) viremia after transplantation is predictive of visceral lesions. Three-hundred and sixty blood samples were collected from 21 transplant recipients and examined. Direct CMV antigen detection was positive in 41 samples (11.4 percent), rapid viral isolation in 24 samples (6.7 percent) and conventional cell culture in 9 cases (2.5 percent). Direct detection of CMV antigen in blood leucocytes is as specific as, and more sensitive and rapid than isolation in culture. In 50 percent of secondary infections the antigenaemia assay and serology were equally sensitive, and antigenemia appeared earlier in 2 primary infections.