Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK) in ME49 strain experimentally infected cats

We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5 x 10(2) mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK). In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.     

Stereologic study of the sinoatrial node of rats -- age related changes

Changes in the sinoatrial node represent the major mechanism of sudden death in humans, and because of the sparse knowledge about the effects of aging on this structure, light microscopic and quantitative studies of the sinoatrial node were undertaken. Twenty-one hearts were studied, seven rat hearts from each of the following age groups: three months of age, twelve months of age and eighteen months of age. In the stereo logic study, the following parameters were studied: Vv_[nc] and Vv_{[interstitium]} % (the volume densities of the nodal cell and interstitium, determined by the point-couting method), and Nv_[nc](1/mm³) (the numerical density of the nodal cell, determined by the disector method). The mean volume of the nodal cell (V_[nc]) (μm³) was also determined. The comparisons showed that in the oldest animals, the volume density of the nodal cells decreased, while the volume density of the interstitium increased. Although numerical density of the nodal cell per volume of sinus node decreased, the nodal cells displayed increased mean volume with age. In conclusion, the aging process implies changes in the cell and fiber content of the sinoatrial node. This revised version was published online in July 2006 with corrections to the Cover Date.     

Neutralizing antibodies as a potential secondary protective mechanism during chronic SHIV infection in CD8+ T-cell-depleted macaques

OBJECTIVE: To directly examine the role of CD8+ T cells in controlling viremia and disease during chronic, low-level primate immunodeficiency virus infection in DNA prime/protein boost-vaccinated macaques. BACKGROUND: A cohort of macaques, vaccinated with either a DNA prime/HIV-1 gp160 boost regimen or with gp160 alone was previously protected partially from sequential challenges with non-pathogenic and pathogenic strains of chimeric simian/human immunodeficiency virus (SHIV). In this study, the effect of temporary ablation of CD8+ T cells in these animals was examined. METHODS: Animals were treated with an anti-CD8 antibody and CD8+ T-cell levels in peripheral blood, plasma viral loads, peripheral blood mononuclear cell-associated virus levels, neutralizing antibody (nAb) titers and simian immunodeficiency virus Gag-specific CD8+ T-cell numbers were followed. RESULTS: Plasma viremia rose sharply in direct synchrony with a rapid but transient drop in CD8+ T cells. However, although levels of cell-associated virus also rose concomitantly, peak levels were much lower than those in virus-challenged, naive animals. In addition, despite a rise of pathogenic SHIV89.6P RNA levels in three animals, CD4+ T-cell counts remained unchanged. In each of these animals, neutralizing antibody titers against the pathogenic SHIV89.6P strain were high. CONCLUSIONS: The results indicate that CD8+ T cells play a key role in suppressing viremia in a chronically infected host. In addition, the results suggest that in the absence of CD8+ T cells, nAb may act as an effective second line of defense by limiting both the spread of infectious virus to new target cells and CD4+ T-cell loss.     

Evaluation of an antigen from Taenia crassiceps cysticercus for the serodiagnosis of neurocysticercosis

We report here the evaluation of an antigen from Taenia crassiceps cysticercus as a potential reagent in an enzyme-immunoelectrotransfer blotting assay (EITB) and an enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of neurocysticercosis (NC) using clinical specimens obtained from patients in different phases of the disease. Serum and cerebrospinal fluid (CSF) samples from 64 patients suspected of having NC according to clinical manifestation and brain computed tomography were tested by ELISA with Taenia solium total saline antigen (ELISA-Tso) and by immunoblotting with T. crassiceps glycoproteins antigen (EITB-gpTcra). Forty-five serum samples were also tested immunoblotting with T. solium glycoproteins antigen (EITB-gpTso) and 30 were tested by ELISA with T. crassiceps 14 kDa glycoprotein (ELISA-gp14Tcra). Serum samples from apparently healthy individuals without any parasitic disease and from patients with other parasitic diseases were included as controls. The results of ELISA-Tso analysis with CSF obtained from 64 patients with NC showed that 53 (83%) were reactive. EITB-gpTcra analysis with serum from the same group of patients showed a sensitivity of 91%. Results of EITB-gpTso and EITB-gpTcra analysis with serum samples demonstrated an agreement of 100% between both tests. ELISA-gp14Tcra was positive in 23 (77%) sera, 22 with paired CSF positive. When ELISA-gp14Tcra results were compared to EITB-Tso results, a relative sensitivity of 95% was observed. All serum samples from the control group were negative in ELISA-gp14Tcra and only one serum from an individual with Taenia saginata was reactive in this assay, showing a specificity of 99% for ELISA-gp14Tcra. This fraction was purified in only one step with a good yield for use in immunoassays. We suggest that the gp14Tcra antigen can be used for detecting anti-cysticercus antibodies in serum samples for epidemiological investigation purposes and also for diagnostic screening of NC patients.     

A recombinant cysteine proteinase from Leishmania (Leishmania) chagasi suitable for serodiagnosis of American visceral leishmaniasis

A recombinant protein, rLdccys1, which was produced by expression of the gene encoding a 30 kDa cysteine proteinase from Leishmania (Leishmania) chagasi, was used for detection of antibodies in sera from patients with active visceral leishmaniasis (VL) in enzyme-linked immunosorbent assays. Analysis of the predicted amino acid sequence of rLdccys1 showed that it contains all the characteristics of a cysteine proteinase. The ability of the protein to react with sera from humans with VL was also shown by Western blotting. The sensitivity for detection of specific antibodies to L. (L.) chagasi bodies using rLdccys1, L. (L.) chagasi promastigote lysates, and amastigote lysates was 80%, 98%, and 99%, respectively. No cross-reactivity between rLdccys1 and Chagas disease was observed, and there was little positive reactivity with sera from patients with cutaneous leishmaniasis and tuberculosis, compared with promastigote and amastigote extracts. Our findings indicate that rLdccys1 from L. (L.) chagasi constitutes a potential tool for the diagnosis of American VL.     

The cross-talk between angiotensin and insulin differentially affects phosphatidylinositol 3-kinase- and mitogen-activated protein kinase-mediated signaling in rat heart: implications for insulin resistance

Insulin and angiotensin II (AngII) may act through overlapping intracellular pathways to promote cardiac myocyte growth. In this report insulin and AngII signaling, through the phosphatidylinositol 3-kinase (PI 3-kinase) and MAPK pathways, were compared in cardiac tissues of control and obese Zucker rats. AngII induced Janus kinase 2 tyrosine phosphorylation and coimmunoprecipitation with insulin receptor substrate 1 (IRS-1) and IRS-2 as well as an increase in tyrosine phosphorylation of IRS and its association with growth factor receptor-binding protein 2. Simultaneous treatment with both hormones led to marked increases in the associations of IRS-1 and -2 with growth factor receptor-binding protein 2 and in the dual phosphorylation of ERK1/2 compared with the administration of AngII or insulin alone. In contrast, an acute inhibition of both basal and insulin-stimulated PI 3-kinase activity was induced by both hormones. Insulin stimulated the phosphorylation of MAPK equally in lean and obese rats. Conversely, insulin-induced phosphorylation of Akt in heart was decreased in obese rats. Pretreatment with losartan did not change insulin-induced activation of ERK1/2 and attenuated the reduction of Akt phosphorylation in the heart of obese rats. Thus, the imbalance between PI 3-kinase-Akt and MAPK signaling pathways in the heart may play a role in the development of cardiovascular abnormalities observed in insulin-resistant states, such as in obese Zucker rats.     

二尖瓣成形术与二尖瓣置换术治疗退行性二尖瓣关闭不全的远期疗效比较研究

背景退行性变是西方国家二尖瓣关闭不全常见的病因,与二尖瓣置换术(MVR)相比,二尖瓣成形术(MVP)不仅无需长期抗凝,避免了抗凝相关并发症,而且能够有助于左心功能的维护。目前MVR与MVP治疗退行性变的远期疗效尚不明确。目的比较MVP与MVR治疗退行性二尖瓣关闭不全的远期疗效。方法选取2003—2008年在俄罗斯巴库列夫心血管外科研究中心接受瓣膜手术的退行性二尖瓣关闭不全患者274例为研究对象。根据手术方式的不同,将患者分为MVP组(129例)和MVR组(145例)。收集患者一般资料,统计患者5年、10年累积生存率、无脑卒中发生率及再次瓣膜手术情况。分别根据患者随访终点时死亡情况、脑卒中发生情况,将患者分为死亡组(42例)和非死亡组(232例)、脑卒中组(30例)和非脑卒中组(244例)。采用单因素分析和多因素Cox回归分析探讨退行性二尖瓣关闭不全患者死亡、发生脑卒中的影响因素。结果MVP组患者体外循环时间、主动脉阻断时间、术后住院时间长于MVR组(P<0.05)。死亡组患者体外循环时间、呼吸机使用时间长于非死亡组,MVP率低于非死亡组(P<0.05)。脑卒中组患者入院时血肌酐、入院时左心室射血分数(LVEF)高于非脑卒中组,MVP率低于非脑卒中组(P<0.05)。多因素Cox回归分析结果显示,MVP是退行性二尖瓣关闭不全患者死亡[HR=0.053,95%CI(0.005,0.585)]、发生脑卒中[HR=0.095,95%CI(0.013,0.698)]的保护因素(P<0.05)。MVP组患者5年累积生存率低于MVR组,10年累积生存率高于MVR组(P<0.05)。MVP组患者5年、10年累积无脑卒中发生率低于MVR组(P<0.05)。MVP组和MVR组患者再次瓣膜手术率比较,差异无统计学意义(P>0.05)。结论MVP是退行性二尖瓣关闭不全患者死亡、发生脑卒中的保护因素,对于退行性二尖瓣关闭不全患者,行MVP者的远期预后优于行MVR者,临床应积极推广MVP。     

Immunohistochemical detection of polycyclic aromatic hydrocarbon-DNA damage in human blood vessels of smokers and non-smokers

The presence of cigarette smoking-related polycyclic aromatic hydrocarbon (PAH)-DNA adducts was investigated in human arterial tissue using an immunoperoxidase staining method with a monoclonal antibody that recognizes benzo(a)pyrene and structurally related PAH diol epoxide-DNA adducts. This is the first time that immunohistochemical methods for detection of PAH-DNA damage have been applied to human endothelial and smooth muscle cells of blood vessels. Internal mammary artery specimens from a total of 37 smokers and non-smokers were tested. Positive nuclear staining was observed in both endothelial and smooth muscle cells, with higher staining in the endothelium. The correlation between smoking status, available for 33 subjects, and detectable PAH-DNA adducts in endothelium did not reach statistical significance (odds ratio=3.38, 95% confidence interval is 0.47–27.60) in this small series. While no causal role can be inferred from our results, they support the theory that endothelial injury caused by cigarette smoking and other environmental exposures may be an early event in the process of atherosclerosis.     

Dermatophyte susceptibilities to antifungal azole agents tested in vitro by broth macro and microdilution methods

The in vitro susceptibility of dermatophytes to the azole antifungals itraconazole, fluconazole and ketoconazole was evaluated by broth macro and microdilution methods, according to recommendations of the CLSI, with some adaptations. Twenty nail and skin clinical isolates, four of Trichophyton mentagrophytes and 16 of T. rubrum were selected for the tests. Itraconazole minimal inhibitory concentrations (MIC) varied from < 0.03 to 0.25 microg/mL in the macrodilution and from < 0.03 to 0.5 microg/mL in the microdilution methods; for fluconazole, MICs were in the ranges of 0.5 to 64 microg/mL and 0.125 to 16 microg/mL by the macro and microdilution methods, respectively, and from < 0.03 to 0.5 microg/mL by both methods for ketoconazole. Levels of agreement between the two methods (+/- one dilution) were 70% for itraconazole, 45% for fluconazole and 85% for ketoconazole. It is concluded that the strains selected were inhibited by relatively low concentrations of the antifungals tested and that the two methodologies are in good agreement especially for itraconazole and ketoconazole.     

Primary cutaneous lymphomas: applicability of current classification schemes (European Organization for Research and Treatment of Cancer, World Health Organization) based on clinicopathologic features observed in a large group of patients.

Classification of primary cutaneous lymphomas (PCLs) is the subject of ongoing controversy. Based on a series of 556 patients, the applicability of the European Organization for Research and Treatment of Cancer (EORTC) classification for PCLs was assessed and compared to the proposed World Health Organization (WHO) classification of hematologic malignancies. The large majority of patients could be properly classified according to the scheme proposed by the EORTC. Comparison of estimated 5-year survival for specific diagnostic categories of PCLs demonstrated nearly complete concordance of the present results with those of the EORTC study for most of the indolent cutaneous T-cell lymphomas and cutaneous B-cell lymphomas, whereas differences were found for mycosis fungoides-associated follicular mucinosis and Sezary syndrome. A few patients with newly described entities (CD8(+) epidermotropic cytotoxic T-cell lymphoma, primary cutaneous natural killer/T-cell lymphoma) could not be classified according to the EORTC scheme. Comparison of the EORTC with the WHO classification showed that the EORTC scheme allows a more precise categorization of the patients, especially for cutaneous B-cell lymphoma. In conclusion, the study confirmed that the EORTC classification allows a better management of patients with PCL. Small amendments to that classification should be carried out to account for recently described entities and to unify some of the diagnostic categories.