Listeriolysin O is essential for virulence of Listeria monocytogenes: direct evidence obtained by gene complementation.

The role of listeriolysin O in the intracellular multiplication of Listeria monocytogenes and, therefore, its pathogenicity was questioned through a genetic complementation study. A nonhemolytic mutant was generated by inserting a single copy of transposon Tn917 in the bacterial chromosome. This insertion was localized by DNA sequence analysis in hlyA, the gene coding for listeriolysin O. As was another mutant that we previously characterized, this mutant was avirulent in the mouse. It was transformed with a plasmid carrying only hlyA, able to replicate in L. monocytogenes, and stably maintained in vitro and in vivo. The complemented strain displayed a hemolytic phenotype identical to that of the wild-type strain and was fully virulent, therefore attributing a crucial role to listeriolysin O in virulence and excluding the hypothesis of a polar effect of the transposon insertion on genes adjacent to hlyA and possibly involved in virulence.     

Apical mitochondria-rich cells in the human epididymis: an ultrastructural, enzymohistochemical, and immunohistochemical study.

An ultrastructural, enzymohistochemical, and immunohistochemical study of the ductus epididymis in normal men was undertaken to investigate the characteristics of the apical mitochondria-rich cells (AMRCs). These cells, which differ morphologically from the principal cells (PCs), appear in isolation in the caput epididymidis (5.8 +/- 1.7 cells per cross-sectional duct) and only occasionally in the corpus epididymidis. The morphologic appearance of AMRCs varies from slender cells extending from the basement membrane to the lumen to apical cells without apparent contact with the basement membrane. The former display a round pale nucleus located in the middle of the epithelium; the apical cells have a dark nucleus, which, surrounded by a narrow cytoplasmic band, protrudes into the lumen. The cytoplasm of AMRCs is electron-dense and contains numerous mitochondria surrounded by rough endoplasmic reticulum cisternae. In the apical portion, there are lysosomes, vesicles with an electron-dense granule, and vacuoles showing a variable size and content. The stereocilia are shorter and less numerous than those of the PCs. The AMRCs are similar to the PCs in the intensely positive reaction for the enzymatic activity acid phosphatase, as well as in the lack of reaction for alkaline phosphatase and phosphorylase activities. AMRCs differ from PCs in: (1) a more intense reaction to the enzymatic activities ATPase, NADP, and succinic dehydrogenease, (2) a more intense immunostaining by AE1/AE3 and Ks4.62 anti-cytokeratin antibodies, and anti-estradiol receptor protein (D5) antibodies, and (3) a lower staining affinity for epithelial membrane antigen (EMA) antibodies. No positive immunostaining for the anti-cytokeratin Ks8.6 antibodies was observed in either AMRCs or PCs.     

Sublingual immunotherapy for hazelnut food allergy: a randomized, double-blind, placebo-controlled study with a standardized hazelnut extract

BACKGROUND: Food allergy may be life-threatening, and patients affected need to receive accurate diagnoses and treatment. Hazelnut has often been implicated as responsible for allergic reactions, and trace quantities can induce systemic reactions. OBJECTIVE: The aim of this study was to evaluate the efficacy and tolerance of sublingual immunotherapy with a standardized hazelnut extract in patients allergic to hazelnut. METHODS: This was a randomized, double-blind, placebo-controlled study. Inclusion criteria were a history of hazelnut allergy and positive skin prick test and double-blind placebo-controlled food challenge results. Patients were then randomly assigned into 2 treatment groups (hazelnut immunotherapy or placebo). Efficacy was assessed by double-blind, placebo-controlled food challenge after 8 to 12 weeks of treatment. Blood samples were drawn for measurement of specific IgE, IgG(4), and serum cytokines before and after treatment. RESULTS: Twenty-three patients were enrolled and divided into 2 treatment groups. Twenty-two patients reached the planned maximum dose at 4 days. Systemic reactions were observed in only 0.2% of the total doses administered. Mean hazelnut quantity provoking objective symptoms increased from 2.29 g to 11.56 g (P = .02; active group) versus 3.49 g to 4.14 g (placebo; NS). Moreover, almost 50% of patients who underwent active treatment reached the highest dose (20 g), but only 9% in the placebo. Laboratory data showed an increase in IgG(4) and IL-10 levels after immunotherapy in only the active group. CONCLUSION: Our data confirm significant increases in tolerance to hazelnut after sublingual immunotherapy as assessed by double-blind, placebo-controlled food challenge, and good tolerance to this treatment.     

The distribution and cellular localization of the serotonin 1C receptor mRNA in the rodent brain examined by in situ hybridization histochemistry. Comparison with receptor binding distribution

The regional distribution and cellular localization of mRNA coding for the serotonin 1C receptor were investigated in tissue sections of mouse and rat brain by in situ hybridization histochemistry. Several 32P-labelled riboprobes derived from mouse genomic clones were used. The serotonin 1C receptor binding sites were visualized autoradiographically and quantified using [3H]mesulergine as ligand, in the presence of spiperone to block serotonin 1C receptors. Strong hybridization signal was observed in the choroid plexus of all brain ventricles. High levels of hybridization were also seen in the anterior olfactory nucleus, pyriform cortex, amygdala, some thalamic nuclei, especially the lateral habenula, the CA3 area of the hippocampal formation, the cingulate cortex, some components of the basal ganglia and associated areas, particularly the nucleus subthalamicus and the substantia nigra. The midbrain and brainstem showed moderate levels of hybridization. The distribution of the serotonin 1C receptor mRNA corresponded well to that of the serotonin 1C receptors. The highest levels of serotonin 1C receptor binding were observed in the choroid plexus. In addition, significant levels of the serotonin 1C receptor binding were seen in the anterior olfactory nucleus, pyriform cortex, nucleus accumbens, ventral aspects of the striatum, paratenial and paracentral thalamic nuclei, amygdaloid body and substantia nigra pars reticulata. The cingulate and retrosplenial cortices as well as the caudal aspects of the hippocampus (CA3) were also labelled. Binding in brainstem and medulla was low and homogeneously distributed. No significant binding was seen in the habenular and subthalamic nuclei. Similar findings were obtained in rat brain. These results demonstrate that, in addition to their enrichment in the choroid plexus, the serotonin 1C receptor mRNA and binding sites are heterogeneously distributed in the rodent brain and thus could be involved in the regulation of many different brain functions. The combination of in situ hybridization histochemistry with receptor autoradiography opens the possibility of examining the regulation of the serotonin 1C receptor synthesis after pharmacological or physiological alterations.     

Presence of the cfxA gene in Bacteroides distasonis

In this study we investigated the presence of the cfxA gene (encoding a class A beta-lactamase) in 73 strains of the Bacteroides fragilis group belonging to the species B. distasonis (34), B. vulgatus (14), B. thetaiotaomicron (8), B. merdae (6), B. caccae (9) and B. ovatus (2) isolated from human intestinal microflora of healthy children and adults. Employing specific primers to the cfxA gene, a 312-bp amplified fragment was obtained in 2 strains of B. vulgatus and 9 strains, the majority from children, of B. distasonis. The expression of this enzyme was analysed by determining the MICs to cefoxitin and cefotaxime and values varied from 2 to >256 microg/ml of both cefoxitin and cefotaxime. Sequence analysis of the amplicons corresponding to the cfxA gene from B. distasonis and B. vulgatus revealed identical sequences between these isolates and high similarity with other beta-lactamase genes of anaerobes such as cfxA of B. vulgatus (99%) and cfxA2 of Prevotella intermedia (99%), both sequences of which deposited in Genbank under accession numbers U38243 and AF118110, respectively. However, a fragment obtained from a B. distasonis strain (EC17-4) showed a unique RFLP profile and 87% nucleotide similarity with cfxA and cfxA2 genes. These results seem to suggest a dissemination of these resistance determinants among Bacteroides species.     

Tissue microarray immunohistochemical expression analysis of mismatch repair (hMLH1 and hMSH2 genes) in endometrial carcinoma and atypical endometrial hyperplasia: relationship with microsatellite instability.

Alterations in the mismatch repair genes (hMLH1 and hMSH2) play an important role in the development of microsatellite instability in sporadic endometrial cancer. Tissue microarray technology allows molecular profiling of tumor samples at the DNA, RNA, and protein levels. We analyzed hMLH1 and hMSH2 expression by immunohistochemistry in a group of atypical endometrial hyperplasias (n = 10), endometrioid endometrial carcinomas (n = 58), and nonendometrioid endometrial carcinomas (n = 27) on tissue microarray. The results were correlated with microsatellite instability status as evaluated by BAT-25 and BAT-26. Overall, 29.4% of lesions showed microsatellite instability. Loss of nuclear hMLH1 and hMSH2 protein expression was seen in 22.3% and 6.5% of cases, respectively. Immunohistochemistry for hMLH1 and hMSH2 showed lack of protein expression in 64% and 16.6% of microsatellite instability-positive endometrial lesions, respectively. Taken together, hMLH1 or hMSH2 protein expression was absent in 18 of 24 microsatellite instability-positive cases (75% sensitivity). A high level of concordance was found between immunohistochemistry for hMLH1 and hMSH2 and microsatellite instability status evaluated by BAT-25 and BAT-26 (kappa value of 0.7). Of the 57 cases found to be microsatellite instability negative, 53 showed normal expression of both proteins (93% specificity). The observed predictive value of absence of expression of hMLH1 for predicting microsatellite instability-positive status was 82%. The predictive value of normal expression of both proteins for predicting microsatellite instability-negative status was 90%. These results are consistent with those previously reported in whole tissue sections. Therefore, immunohistochemical analysis of hMLH1 and hMSH2 expression on tissue microarray provides an accurate technique for screening for tumors with microsatellite instability. Tissue microarrays represent an ideal approach for comparing different diagnostic or predictive markers with one another in consecutive tissue microarray sections.     

An interleukin-4-dependent precursor clone is an intermediate of the differentiation pathway from an interleukin-3-dependent precursor clone into myeloid cells as well as B lymphocytes

An interleukin-3-dependent progenitor clone LyD9 and its interleukin-4-dependent derivative clone K-4 were shown to differentiate into myeloid cells as well as B lymphocytes by coculture with bone marrow stroma cells. The K-4 clone is an intermediate between myeloid/lymphoid cells and the LyD9 clone that requires interleukin-4 for differentiation into B lymphocytes and myeloid cells. Granulocyte-macrophage colony stimulating factor-dependent derivatives (LS-1 and K-GM) were also established from induced LyD9 cells. LS-1 and K-GM were myeloid-committed cells.     

Management of chronic viral hepatitis in HIV-infected patients: Spanish Consensus Conference. October 2000

Co-infection by human immunodeficiency virus and hepatitis B and C viruses is quite common because they share similar routes of transmission. The introduction of highly active antiretroviral therapy has significantly improved the life expectancy of HIV-infected patients in the last few years. However, chronic viral hepatitis represents an emerging cause of morbidity and mortality in this population, either as a result of end-stage liver disease or as a consequence of hepatotoxicity induced by antiretroviral drugs. The main goal of the Consensus Conference was to establish specific recommendations for the management of chronic viral hepatitis B and C in HIV-infected patients. The role of orthotopic liver transplantation for co-infected individuals with end-stage liver disease was also assessed.     

Dopamine receptors in human brain: autoradiographic distribution of D1 sites

The distribution of dopamine D1 receptors has been determined in post mortem human brain tissues using in vitro receptor autoradiography, with ([3H]N-methyl) SCH 23390 as ligand. The highest densities of dopamine D1 sites were seen in the nucleus caudatus, putamen, globus pallidus pars medialis and substantia nigra. Intermediate densities were associated with the amygdala, mammillary bodies, cerebral cortex and CA1. The remaining part of the hippocampus as well as the diencephalon, brainstem and cerebellum contained low levels of [3H]SCH 23390 binding sites. The distribution of D1 receptors in the human brain closely resembles that reported for the rat brain. In addition, there was a good correlation between the anatomical localization of D1 sites and the distribution of dopaminergic nerve terminals in the central nervous system. The densities of D1 receptors in the human brain were observed to markedly decrease with age during the first decades of life. However, no further modifications were found beyond the age of 40 years. We did not observe any significant influence of other parameters such as gender and post mortem delay in our samples.