Related factors to human toxocariasis in a rural community of Argentina

The objective of this study was to evaluate the relationship between toxocariasis frequency and demographic, environmental, sanitary variables, eosinophylia, and other intestinal parasites in a rural population of Argentina. Serological examination of 100 individuals was carried out by using ELISA technique for the detection of anti-toxocara antibodies. Eosinophiles in peripheral blood, presence of intestinal parasites, and demographic, environmental, and socio-cultural data were evaluated. Eighty-one feces samples of dogs belonging to the studied people were analyzed to detect eggs of Toxocara canis. Thirty of them were from 30 dogs and 51 were pools from dog feces. Samples of dirt from around the homes (n: 47) and from public park (n: 4) were taken. To determine the associations, the c² and Fisher tests were used. The seroprevalence was 23%. Eosinophilia in peripheral blood was detected in 86.95% seropositive individuals and in 37.66% seronegative individuals (p < 0.001, OR = 11.03). Of the 23 people with positive serology, 69.56% had at least one intestinal parasite. All individuals with positive serology had dogs in their homes. Among the dog owners there was a significant association between the presence of anti-toxocara antibodies and home flooding. Eggs of T. canis were detected in the feces of 5/81 dogs and three of these dogs belonged to individuals with positive serology. Eggs of Toxocara spp. were found in 41.17% of the dirt samples, eight of which came from the area surrounding the homes of individuals with positive serology (p = 0.032; OR = 4.36). Taking into account all the variables influencing the frequency of toxocariasis in this population, the implementation of Public Health programs specifically focused on anti-parasitic treatment of dogs is recommended.

High expression of co-stimulatory and adhesion molecules are observed on eosinophils during human Schistosoma mansoni infection

Herein we have focused attention on major phenotypic features of peripheral blood eosinophils from chronic Schistosoma mansoni-infected patients. For this purpose, detailed immunophenotypic profiles of a range of cell surface markers were performed, including activation markers (CD23/CD69/CD25/HLA-DR), co-stimulatory molecules (CD28/CD80/CD86), chemokine receptors (CXCR1/CXCR2/CCR3/CCR5) besides L-selectin-CD62L and adhesion molecules (CD18/CD54). Our major findings pointed out increased frequency of CD23+-cells, besides decreased percentages of CD69+-eosinophils, suggesting a chronic activation status with low frequency of early activated eosinophils in chronic S. mansoni-infected patients (INT) in comparison to non-infected individuals (NI). Moreover, a dichotomic expression of beta-chemokine receptors was observed during human schistosomiasis mansoni with higher CCR5 and lower levels of CCR3 observed between groups. Enhanced expression of co-stimulatory receptors (CD28/CD86) and adhesion molecules (CD54/CD18), besides striking lower frequency of L-selectin+ were reported for eosinophils from INT group as compared to NI. Interestingly, the frequency of CD62L+-eosinophils and a range of cell activation related molecules pointed out an opposite pattern of association in NI and INT, where only INT patients that display lower frequency of CD62L+-eosinophils (first CD62L tertile) kept the unusual relationship between the expression of L-selectin and the CD23 activation marker. These findings suggest that distinct dynamic of activation markers expressed by eosinophils may occur during chronic S. mansoni infection.

The sample processing time interval as an influential factor in flow cytometry analysis of lymphocyte subsets

The objective of this paper is to propose a protocol to analyze blood samples in yellow fever 17DD vaccinated which developed serious adverse events. We investigated whether or not the time between sample collection and sample processing could interfere in lymphocyte subset percentage, for it is often impossible to analyze blood samples immediately after collection due to transport delay from collection places to the flow cytometry facility. CD4+CD38+ T, CD8+CD38+ T, CD3+ T, CD19+ B lymphocyte subsets were analyzed by flow cytometry in nine healthy volunteers immediately after blood collection and after intervals of 24 and 48 h. The whole blood lysis method and gradient sedimentation by Histopaque were applied to isolate peripheral blood mononuclear cells for flow cytometry analyses. With the lysis method, there was no significant change in lymphocyte subset percentage between the two time intervals (24 and 48 h). In contrast, when blood samples were processed by Histopaque gradient sedimentation, time intervals for sample processing influenced the percentage in T lymphocyte subsets but not in B cells. From the results obtained, we could conclude that the whole blood lysis method is more appropriate than gradient sedimentation by Histopaque for immunophenotyping of blood samples collected after serious adverse events, due to less variation in the lymphocyte subset levels with respect to the time factor.

Junctional adhesion molecule-2 (JAM-2) promotes lymphocyte transendothelial migration.

The molecular mechanisms underlying lymphocyte extravasation remain poorly characterized. We have recently identified junctional adhesion molecule-2 (JAM-2), and have shown that antibodies to JAM-2 stain high endothelial venules (HEVs) within lymph nodes and Peyer patches of adult mice. Here we show that mouse lymphocytes migrate in greater numbers across monolayers of endothelioma cells transfected with JAM-2. The significance of these findings to an understanding of both normal and pathologic lymphocyte extravasation prompted us to clone the human homologue of JAM-2. We herein demonstrate that an anti-JAM-2 antibody, or a soluble JAM-2 molecule, blocks the transmigration of primary human peripheral blood leukocytes across human umbilical vein endothelial cells expressing endogenous JAM-2. Furthermore, we show that JAM-2 is expressed on HEVs in human tonsil and on a subset of human leukocytes, suggesting that JAM-2 plays a central role in the regulation of transendothelial migration.

Identification of a novel 45-kDa cell surface molecule involved in activation of the human Jurkat T cell line.

This report describes a surface molecule, Tp45, which appears to be involved in interleukin 2 production and Ca2+ mobilization by Jurkat cells. The Tp45 molecule was identified by a monoclonal antibody, MX13, on the surface of either T3/TCR+ or T3/TCR- human T cell lines. Biochemical data showed that mAb MX13 precipitated a single polypeptide chain of 45 kDa both under reduced and nonreduced conditions from lysates of 125I-surface-labeled cells. Sequential immunodepletion experiments using lysates of 125I-labeled T3/TCR+ cells showed that Tp45 was distinct from the alpha chain of the TCR complex. However, incubation of such cells with either anti-T3 or anti-TCR monoclonal antibody induced complete modulation of both the T3/TCR complex and Tp45. Conversely, complete modulation of both Tp45 and the T3/TCR complex was observed after incubation with anti-Tp45 antibody. Functional studies showed that anti-Tp45 antibody induced high levels of interleukin 2 production in Jurkat cells. In addition, incubation of these cells with the antibody resulted in Ca2+ mobilization from internal stores. Anti-Tp45 antibody reacted with 3-19% peripheral blood (E-rosette-positive) T cells in individual donors. The magnitude of the proliferative response elicited by anti-Tp45 antibody for peripheral blood T cells was lower than that induced by an anti-T3 antibody. This observation is compatible with the idea that only a subpopulation of T cells is reactive with anti-Tp45. Multicolor flow cytometry analysis showed that the Tp45+ cells belong preferentially to the T8 subset.

First encounter of subclinical human Leishmania (Viannia) infection in State of Rio Grande do Sul, Brazil

The objective of the present study was to evaluate the specificity of the Montenegro skin test (MST) in an area in Brazil, state of Grande do Sul State (RS), which was considered to be non-endemic for leishmaniasis. Sixty subjects presented a positive MST and were reevaluated by clinical examination, serology and polymerase chain reaction (PCR) of peripheral blood for the detection of subclinical Leishmania infection. None of the subjects presented clinical signs or symptoms of current leishmaniasis or a history of the disease.Leishmania (Viannia) DNA was detected in blood by PCR and hybridization in one subject. The PCR skin test-positive individual remained asymptomatic throughout the study. Clinical examination showed no scars suggestive of past cutaneous leishmaniasis. Human subclinical infection with Leishmania (Viannia) in RS was confirmed by PCR. This is the first report of subclinical infection with this parasite in the human population of this area.

A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 µg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 µM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 µ M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 µM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.

Host immune response to Toxoplasma gondii and Ascaris lumbricoides in a highly endemic area: evidence of parasite co-immunomodulation properties influencing the outcome of both infections

Toxoplasmosis and ascaridiasis evoke polar Th-1 and Th-2 host immune responses, respectively. A study to investigate the specific cytokine profile production by in vitro cultures of peripheral blood mononuclear cells from individuals living under precarious sanitary conditions in a highly endemic area for the parasites Toxoplasma gondii and Ascaris lumbricoides was conducted. High levels of both IFN-³ (Th-1) and IL-13 (Th-2) were observed in groups of co-infected individuals presenting toxoplasmic ocular lesions. Significantly lower IL-10 and TGF-² levels were produced by co-infected individuals in comparison with groups of individuals not infected with A. lumbricoides and either positive or negative for T. gondii living under good sanitary conditions (control groups). The possible influence of co-parasitism on the clinical presentation of ocular toxoplasmosis is discussed.

Circulating natural killer and γδ T cells decrease soon after infection of rhesus macaques with lymphocytic choriomeningitis virus

Rhesus macaques infected with the WE strain of lymphocytic choriomeningitis virus (LCMV-WE) serve as a model for human infection with Lassa fever virus. To identify the earliest events of acute infection, rhesus macaques were monitored immediately after lethal infection for changes in peripheral blood mononuclear cells (PBMCs). Changes in CD3, CD4, CD8 and CD20 subsets did not vary outside the normal fluctuations of these blood cell populations; however, natural killer (NK) and γδ T cells increased slightly on day 1 and then decreased significantly after two days. The NK subsets responsible for the decrease were primarily CD3-CD8+ or CD3-CD16+ and not the NKT (primarily CD3+CD56+) subset. Macaques infected with a non-virulent arenavirus, LCMV-Armstrong, showed a similar drop in circulating NK and γδ T cells, indicating that this is not a pathogenic event. V³9 T cells, representing the majority of circulating γδ T cells in rhesus macaques, displayed significant apoptosis when incubated with LCMV in cell culture; however, the low amount of cell death for virus-co-cultured NK cells was insufficient to account for the observed disappearance of this subset. Our observations in primates are similar to those seen in LCMV-infected mice, where decreased circulating NK cells were attributed to margination and cell death. Thus, the disappearance of these cells during acute hemorrhagic fever in rhesus macaques may be a cytokine-induced lymphopenia common to many virus infections.

Association between IL-18 gene polymorphisms and biopsy-proven giant cell

INTRODUCTION: The objective was to investigate the potential implication of the IL18 gene promoter polymorphisms in the susceptibility to giant-cell arteritis GCA). METHODS: In total, 212 patients diagnosed with biopsy-proven GCA were included in this study. DNA from patients and matched controls was obtained from peripheral blood. Samples were genotyped for the IL18-137 G>C (rs187238), the IL18-607 C>A (rs1946518), and the IL18-1297 T>C (rs360719) gene polymorphisms with polymerase chain reaction, by using a predesigned TaqMan allele discrimination assay. RESULTS: No significant association between the IL18-137 G>C polymorphism and GCA was found. However, the IL18 -607 allele A was significantly increased in GCA patients compared with controls (47.8% versus 40.9% in patients and controls respectively; P = 0.02; OR, 1.32; 95% CI, 1.04 to 1.69). It was due to an increased frequency of homozygosity for the IL18 -607 A/A genotype in patients with GCA (20.4%) compared with controls (13.4%) (IL18 -607 A/A versus IL18 -607 A/C plus IL18 -607 C/C genotypes: P = 0.04; OR, 1.59; 95% CI, 1.02 to 2.46). Also, the IL18-1297 allele C was significantly increased in GCA patients (30.7%) compared with controls (23.0%) (P = 0.003; OR, 1.48; 95% CI, 1.13 to 1.95). In this regard, an increased susceptibility to GCA was observed in individuals carrying the IL18-1297 C/C or the IL18-1297 C/T genotypes compared with those carrying the IL18-1297 T/T genotype (IL18-1297 C/C plus IL18-1297 T/C versus IL18-1297 T/T genotype in GCA patients compared with controls: P = 0.005; OR, 1.61; 95% CI, 1.15 to 2.25). We also found an additive effect of the IL18 -1297 and -607 polymorphisms with TLR4 Asp299Gly polymorphism. The OR for GCA was 1.95 for combinations of genotypes with one or two risk alleles, whereas carriers of three or more risk alleles have an OR of 3.7. CONCLUSIONS: Our results show for the first time an implication of IL18 gene-promoter polymorphisms in the susceptibility to biopsy-proven GCA. In addition, an additive effect between the associated IL18 and TLR4 genetic variants was observed.

Radiation induced apoptosis and initial DNA damage are inversely related in locally advanced breast cancer patients.

Background. DNA-damage assays, quantifying the initial number of DNA double-strand breaks induced by radiation, have been proposed as a predictive test for radiation-induced toxicity. Determination of radiation-induced apoptosis in peripheral blood lymphocytes by flow cytometry analysis has also been proposed as an approach for predicting normal tissue responses following radiotherapy. The aim of the present study was to explore the association between initial DNA damage, estimated by the number of double-strand breaks induced by a given radiation dose, and the radio-induced apoptosis rates observed. Methods. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radio-induced apoptosis at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. Results. Radiation-induced apoptosis increased in order to radiation dose and data fitted to a semi logarithmic mathematical model. A positive correlation was found among radio-induced apoptosis values at different radiation doses: 1, 2 and 8 Gy (p < 0.0001 in all cases). Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). A statistically significant inverse correlation was found between initial damage to DNA and radio-induced apoptosis at 1 Gy (p = 0.034). A trend toward 2 Gy (p = 0.057) and 8 Gy (p = 0.067) was observed after 24 hours of incubation. Conclusions. An inverse association was observed for the first time between these variables, both considered as predictive factors to radiation toxicity.

The immunomodulator PSK induces in vitro cytotoxic activity in tumour cell linesvia arrest of cell cycle and induction of apoptosis

BACKGROUND Protein-bound polysaccharide (PSK) is derived from the CM-101 strain of the fungus Coriolus versicolor and has shown anticancer activity in vitro and in in vivo experimental models and human cancers. Several randomized clinical trials have demonstrated that PSK has great potential in adjuvant cancer therapy, with positive results in the adjuvant treatment of gastric, esophageal, colorectal, breast and lung cancers. These studies have suggested the efficacy of PSK as an immunomodulator of biological responses. The precise molecular mechanisms responsible for its biological activity have yet to be fully elucidated. METHODS The in vitro cytotoxic anti-tumour activity of PSK has been evaluated in various tumour cell lines derived from leukaemias, melanomas, fibrosarcomas and cervix, lung, pancreas and gastric cancers. Tumour cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of PSK on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in PSK-treated cells. RESULTS PSK showed in vitro inhibition of tumour cell proliferation as measured by BrdU incorporation and viable cell count. The inhibition ranged from 22 to 84%. Inhibition mechanisms were identified as cell cycle arrest, with cell accumulation in G0/G1 phase and increase in apoptosis and caspase-3 expression. These results indicate that PSK has a direct cytotoxic activity in vitro, inhibiting tumour cell proliferation. In contrast, PSK shows a synergistic effect with IL-2 that increases PBL proliferation. CONCLUSION These results indicate that PSK has cytotoxic activity in vitro on tumour cell lines. This new cytotoxic activity of PSK on tumour cells is independent of its previously described immunomodulatory activity on NK cells.

A fatal overdose of cocaine associated with coingestion of marijuana, buprenorphine, and fluoxetine. Body fluid and tissue distribution of cocaine and its metabolites determined by hydrophilic interaction chromatography-mass spectrometry(HILIC-MS).

Chromatographic separation of highly polar basic drugs with ideal ionspray mass spectrometry volatile mobile phases is a difficult challenge. A new quantification procedure was developed using hydrophilic interaction chromatography-mass spectrometry with turbo-ionspray ionization in the positive mode. After addition of deuterated internal standards and simple clean-up liquid extraction, the dried extracts were reconstituted in 500 microL pure acetonitrile and 5 microL was directly injected onto a Waters Atlantis HILIC 150- x 2.1-mm, 3-microm column. Chromatographic separations of cocaine, seven metabolites, and anhydroecgonine were obtained by linear gradient-elution with decreasing high concentrations of acetonitrile (80-56% in 18 min). This high proportion of organic solvent makes it easier to be coupled with MS. The eluent was buffered with 2 mM ammonium acetate at pH 4.5. Except for m-hydroxy-benzoylecgonine, the within-day and between-day precisions at 20, 100, and 500 ng/mL were below 7 and 19.1%, respectively. Accuracy was also below +/- 13.5% at all tested concentrations. The limit of quantification was 5 ng/mL (%Diff < 16.1, %RSD < 4.3) and the limit of detection below 0.5 ng/mL. This method was successfully applied to a fatal overdose. In Switzerland, cocaine abuse has dramatically increased in the last few years. A 45-year-old man, a known HIV-positive drug user, was found dead at home. According to relatives, cocaine was self-injected about 10 times during the evening before death. A low amount of cocaine (0.45 mg) was detected in the bloody fluid taken from a syringe discovered near the corpse. Besides injection marks, no significant lesions were detected during the forensic autopsy. Toxicological investigations showed high cocaine concentrations in all body fluids and tissues. The peripheral blood concentrations of cocaine, benzoylecgonine, and methylecgonine were 5.0, 10.4, and 4.1 mg/L, respectively. The brain concentrations of cocaine, benzoylecgonine, and methylecgonine were 21.2, 3.8, and 3.3 mg/kg, respectively. The highest concentrations of norcocaine (about 1 mg/L) were measured in bile and urine. Very high levels of cocaine were determined in hair (160 ng/mg), indicating chronic cocaine use. A low concentration of anhydroecgonine methylester was also found in urine (0.65 mg/L) suggesting recent cocaine inhalation. Therapeutic blood concentrations of fluoxetine (0.15 mg/L) and buprenorphine (0.1 microg/L) were also discovered. A relatively high concentration of Delta(9)-THC was measured both in peripheral blood (8.2 microg/L) and brain cortex (13.5 microg/kg), suggesting that the victim was under the influence of cannabis at the time of death. In addition, fluoxetine might have enhanced the toxic effects of cocaine because of its weak pro-arrhythmogenic properties. Likewise, combination of cannabinoids and cocaine might have increase detrimental cardiovascular effects. Altogether, these results indicate a lethal cocaine overdose with a minor contribution of fluoxetine and cannabinoids.

The Presence of Precursors of Benign Pre-B Lymphoblasts (Hematogones) in the Bone Marrow of a Paediatric Patient with Cytomegalovirus Infection

Hematogones are normal B-lymphoid precursors that multiply in the bone marrow of small children and of adults with ferropenic anaemia, neuroblastoma or idiopathic thrombocytopenic purpura. They are not normally found in peripheral blood, and the immunophenotype is virtually indistinguishable from that of B lymphoblasts. We discuss the case of a 3-month infant with an active cytomegalovirus infection, with hepatitis and pancytopenia associated with 13% hematogones in the bone marrow

REPETIDO_A new extract of the plant calendula officinalis produces a dual in vitro effect: cytotoxic anti-tumor activity and lymphocyte activation

BACKGROUND Phytopharmacological studies of different Calendula extracts have shown anti-inflammatory, anti-viral and anti-genotoxic properties of therapeutic interest. In this study, we evaluated the in vitro cytotoxic anti-tumor and immunomodulatory activities and in vivo anti-tumor effect of Laser Activated Calendula Extract (LACE), a novel extract of the plant Calendula Officinalis (Asteraceae). METHODS An aqueous extract of Calendula Officinalis was obtained by a novel extraction method in order to measure its anti-tumor and immunomodulatory activities in vitro. Tumor cell lines derived from leukemias, melanomas, fibrosarcomas and cancers of breast, prostate, cervix, lung, pancreas and colorectal were used and tumor cell proliferation in vitro was measured by BrdU incorporation and viable cell count. Effect of LACE on human peripheral blood lymphocyte (PBL) proliferation in vitro was also analyzed. Studies of cell cycle and apoptosis were performed in LACE-treated cells. In vivo anti-tumor activity was evaluated in nude mice bearing subcutaneously human Ando-2 melanoma cells. RESULTS The LACE extract showed a potent in vitro inhibition of tumor cell proliferation when tested on a wide variety of human and murine tumor cell lines. The inhibition ranged from 70 to 100%. Mechanisms of inhibition were identified as cell cycle arrest in G0/G1 phase and Caspase-3-induced apoptosis. Interestingly, the same extract showed an opposite effect when tested on PBLs and NKL cell line, in which in vitro induction of proliferation and activation of these cells was observed. The intraperitoneal injection or oral administration of LACE extract in nude mice inhibits in vivo tumor growth of Ando-2 melanoma cells and prolongs the survival day of the mice. CONCLUSION These results indicate that LACE aqueous extract has two complementary activities in vitro with potential anti-tumor therapeutic effect: cytotoxic tumor cell activity and lymphocyte activation. The LACE extract presented in vivo anti-tumoral activity in nude mice against tumor growth of Ando-2 melanoma cells.