Gastroduodenal opportunistic infections and dyspepsia in HIV-infected patients in the era of Highly Active Antiretroviral Therapy

Background and Aim: Dyspeptic symptoms are frequently reported by human immuno-defficiency virus (HIV)-infected patients under highly active antiretroviral therapy. Whether opportunistic infections are a cause of dyspepsia is still unknown. In this study we prospectively compare the prevalence of gastrointestinal opportunistic infections in dyspeptic versus non-dyspeptic HIV-infected patients with advanced immunodeficiency. Patients and Methods: Six hundred and ninety HIV-infected patients under highly active antiretroviral therapy underwent esophagogastroduodenoscopy with mucosal biopsies from the stomach and duodenum. Group 1: 500 patients (161 women, 339 men; mean age 38.8 years; mean CD4 count 154.3 cells/mm(3) with dyspeptic symptoms such as epigastric pain, nausea, vomiting and fullness. Group 2: 190 patients (169 men, 21 women; mean age 40.7 years; mean CD4 count 171.6 cell/mm(3)) with no dyspeptic symptoms. Results: Group 1: Gastrointestinal opportunistic infections were observed in eight (1.6%), and non-opportunistic parasites in two (0.4%), patients. They were: Cytomegalovirus (four patients), Cryptosporidium sp. (two patients), Schistosoma mansoni sp. (one patient), Strongyloides stercoralis (one patient) and Giardia sp. (two patients). In five patients esophagogastroduodenoscopy showed no mucosal lesions. Group 2: Giardia sp. was detected in two patients (1.1%: P = 0.07947). Conclusion: Gastrointestinal opportunistic infections were shown in a small number of HIV-infected patients under highly active antiretroviral therapy with advanced immunodeficiency. Although gastrointestinal opportunistic infections were detected exclusively in the dyspeptic patient group, they could not be related to these symptoms, since the number of infected patients was not statistically significant. To correctly diagnose opportunistic infections, multiple biopsy specimens may be necessary even from normal-appearing mucosa.

Molecular and ultrastructural studies of the fibrotic lesions of bovine focal proliferative fibrogranulomatous panniculitis (Lechiguana)

This work aimed to investigate some aspects related to the pathogenicity of Lechiguana, a bovine fibroproliferative lesion characterized by rapid collagen accumulation. Light and transmission electron microscopy and in situ hybridization studies were performed in order to elucidate the fibrogenic activity of this lesion. The characterization of fibroblastic plasticity in the lesion was done by immunohistochemical study for alpha-smooth-muscle cell actin. The ovoid-shaped cells presented positive reaction for alpha-smooth-muscle cell actin in their cytoplasm and, at the electron-microscopic level demonstrated basal lamina-like material adjacent to the external surface and collagen fibrils that corresponded to a cell population phenotypically similar to the myofibroblast. We also investigated alpha 1 collagen type I mRNA at different times of evolution of Lechiguana lesions, using isotopic and non-isotopic in situ hybridization. The results strongly suggest the involvement of a myofibroblast-like cell population that expresses mRNA for type I collagen and is probably associated with the increase of collagen deposition.

Increased mRNA expression of collagen V gene in pulmonary fibrosis of systemic sclerosis

Background Collagen V shows promise as an inducer of interstitial lung fibrosis in experimental systemic sclerosis (SSc). Materials and methods Remodelling of the pulmonary interstitium was evaluated based on the clinical data and open lung biopsies from 15 patients with SSc. Normal lung tissues obtained from eight individuals who died of traumatic injuries were used as control group. Immunofluorescence, immunohistochemistry, morphometry, tri-dimensional reconstruction and a real-time polymerase chain reaction were used to evaluate the quantity, structure and molecular chains of collagen V. The impact of these markers was tested on clinical data. Results The main difference in collagen V content between SSc patients and the control group was an increased, abnormal and distorted fibre deposition in the alveolar septa and the pre-acinar artery wall. The lungs from SSc patients presented [alpha 1(V)] and [alpha 2(V)] mRNA chain expression increased, but [alpha 2(V)] was proportionally increased compared with the control group. High levels of collagen V were inversely associated with vital capacity (r = -0.72; P = 0.002), forced vital capacity (r = -0.76; P < 0.001), forced expiratory volume in 1-s (r = -0.89; P < 0.001) and diffusing capacity for carbon monoxide (r = -0.62; P = 0.04). Conclusions Abnormal collagen V fibres are overproduced in lungs from SSc patients and may play an important role in the pathogenesis of the disease as this molecule regulates tissue collagen assembly. The aberrant histoarchitecture observed in SSc can be related to the overexpression of the [alpha 2(V)] gene of unknown origin.

Differential immunoreactivity of glucocorticoid receptors and vasopressin in neurons of the anterior and medial parvocellular subdvisions of the hypothalamic paraventricular nucleus

arginine-vasopressin in the parvocellular neurons of the hypothalamic paraventricular nucleus is known to play an important role in the control of the hypothalamo-pituitary-adrenal axis. In the present study, we verify plasma corticosterone levels, the distribution of glucocorticoid receptor- and arginine-vasopressin-positive neurons, and the co-localization of both glucocorticoid receptors and arginine-vasopressin in neurons in the anterior and medial parvocellular subdivisions of the paraventricular nucleus after manipulations of the hypothalamus-pituitary-adrenal axis. Normal, sham surgery, and adrenalectomized male rats were subjected to intraperitoneal injections of saline or dexamethasone to measure plasma corticosterone levels by a radioimmunoassay. We also examined arginine-vasopressin and glucocorticoid receptor immunofluorescence in sections from the paraventricular nucleus. Our results showed that the immunoreactivity of arginine-vasopressin neurons increased in the anterior parvocellular subdivision and decreased in the medial parvocellular subdivision from adrenalectomized rats treated with dexamethasone. On the other hand, we showed that the immunoreactivity of glucocorticoid receptors increased in the anterior and medial parvocellular subdivisions of these same animals. However, the immunoreactivity of glucocorticoid receptors is higher in the medial parvocellular than anterior parvocellular subdivision. The co-localization of arginine-vasopressin and glucocorticoid receptors was found only in the medial parvocellular subdivision. These findings indicate that glucocorticoids have direct actions on arginine-vasopressin-positive neurons in the medial parvocellular but not anterior parvocellular subdivision. There is a differentiated pattern of arginine-vasopressin-positive neuron expression between the anterior and medial parvocellular subdivisions. (C) 2010 Elsevier Inc. All rights reserved.

Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alpha v beta 3-integrin and low levels of RHOC. Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified. Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library. Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Suppression subtractive hybridization analysis reveals expression of conserved and novel genes in male accessory glands of the ant Leptothorax gredleri

Background: During mating, insect males eject accessory gland proteins (Acps) into the female genital tract. These substances are known to affect female post-mating behavior and physiology. In addition, they may harm the female, e. g., in reducing its lifespan. This is interpreted as a consequence of sexual antagonistic co-evolution. Whereas sexual conflict abounds in non-social species, the peculiar life history of social insects (ants, bees, wasps) with lifelong pair-bonding and no re-mating aligns the reproductive interests of the sexes. Harming the female during mating would negatively affect male fitness and sexual antagonism is therefore not expected. Indeed, mating appears to increase female longevity in at least one ant species. Acps are presumed to play a role in this phenomenon, but the underlying mechanisms are unknown. In this study, we investigated genes, which are preferentially expressed in male accessory glands of the ant Leptothorax gredleri, to determine which proteins might be transferred in the seminal fluid. Results: By a suppression subtractive hybridization protocol we obtained 20 unique sequences (USs). Twelve had mutual best matches with genes predicted for Apis mellifera and Nasonia vitripennis. Functional information (Gene Ontology) was available only for seven of these, including intracellular signaling, energy-dependent transport and metabolic enzyme activities. The remaining eight USs did not match sequences from other species. Six genes were further analyzed by quantitative RT-PCR in three life cycle stages of male ants. A gene with carboxy-lyase activity and one of unpredicted function were significantly overexpressed in accessory glands of sexually mature males. Conclusions: Our study is the first one to investigate differential gene expression in ants in a context related to mating. Our findings indicate that male accessory glands of L. gredleri express a series of genes that are unique to this species, possibly representing novel genes, in addition to conserved ones for which functions can be predicted. Identifying differentially expressed genes might help to better understand molecular mechanisms involved in reproductive processes in eusocial Hymenoptera. While the novel genes could account for rapidly evolving ones driven by intra-sexual conflict between males, conserved genes imply that rather beneficial traits might get fixed by a process described as inter-sexual cooperation between males and females.

Sudan Black B treatment reduces autofluorescence and improves resolution of in situ hybridization specific fluorescent signals of brain sections

Interference by autofluorescence is one of the major concerns of immunofluorescence analysis of in situ hybridization-based diagnostic assays. We present a useful technique that reduces autofluorescent background without affecting the tissue integrity or direct immunofluorescence signals in brain sections. Using six different protocols, such as ammonia/ethanol, Sudan Black B (SBB) in 70% ethanol, photobleaching with UV light and different combinations of them in both formalin-fixed paraffin-embedded and frozen human brain tissue sections, we have found that tissue treatment of SBB in a concentration of 0.1% in 70% ethanol is the best approach to reduce/eliminate tissue autofluorescence and background, while preserving the specific fluorescence hybridization signals. This strategy is a feasible, non-time consuming method that provides a reasonable compromise between total reduction of the tissue autofluorescence and maintenance of specific fluorescent labels.

Validation of dot blot hybridization and denaturing high performance liquid chromatography as reliable methods for TP53 codon 72 genotyping in molecular epidemiologic studies

Background: Mutations in TP53 are common events during carcinogenesis. In addition to gene mutations, several reports have focused on TP53 polymorphisms as risk factors for malignant disease. Many studies have highlighted that the status of the TP53 codon 72 polymorphism could influence cancer susceptibility. However, the results have been inconsistent and various methodological features can contribute to departures from Hardy-Weinberg equilibrium, a condition that may influence the disease risk estimates. The most widely accepted method of detecting genotyping error is to confirm genotypes by sequencing and/or via a separate method. Results: We developed two new genotyping methods for TP53 codon 72 polymorphism detection: Denaturing High Performance Liquid Chromatography (DHPLC) and Dot Blot hybridization. These methods were compared with Restriction Fragment Length Polymorphism (RFLP) using two different restriction enzymes. We observed high agreement among all methodologies assayed. Dot-blot hybridization and DHPLC results were more highly concordant with each other than when either of these methods was compared with RFLP. Conclusions: Although variations may occur, our results indicate that DHPLC and Dot Blot hybridization can be used as reliable screening methods for TP53 codon 72 polymorphism detection, especially in molecular epidemiologic studies, where high throughput methodologies are required.

In situ hybridization detection of homeobox genes reveals distinct expression patterns in oral squamous cell carcinomas

Aims: To analyse the expression of three homeobox genes (HOXA7, PITX1 and PRRX1) in oral squanous cell carcinomas (OSCC) and the relationship of such expression to certain distinct histopathological features of OSCC and in comparison to adjacent non-neoplastic epithelium (NT). Methods and results: Digoxigenin-labelled riboprobes that are specific for each homeobox gene were generated and in situ hybridization was carried out on frozen sections. In NT samples, HOXA7 and PITX1 transcripts were found more frequently in all epithelial layers, while PRRX1 was expressed in the basal layer. With OSCC samples, expression of the three genes was associated with all histological features. However, the HOXA7 and PITX1 signals were more intense in sheets and nests and PRRX1 in small nests and isolated cells. Conclusion: HOXA7, PIXT1 and PRRX1 homeobox genes have different patterns of expression in OSCC depending on its histological features.

Differential display and suppression subtractive hybridization analysis of the pulp of ripening banana

The fruit of banana undergoes several important physico-chemical changes during ripening. Analysis of gene expression would permit identification of important genes and regulatory elements involved in this process. Therefore, transcript profiling of preclimacteric and climacteric fruit was performed using differential display and Suppression subtractive hybridization. Our analyses resulted in the isolation of 12 differentially expressed cDNAs, which were confirmed by dot-blots and northern blots. Among the sequences identified were sequences homologous to plant aquaporins, adenine nucleotide translocator, immunophilin, legumin-like proteins, deoxyguanosine kinase and omega-3 fatty acid desaturase. Some of these cDNAs correspond to newly isolated genes involved in changes related to the respiratory climacteric, or stress-defense responses. Functional characterization of ripening-associated genes could provide information useful in controlling biochemical pathways that would have an impact on banana quality and shelf life. (C) 2009 Elsevier B.V. All rights reserved.

Transcriptional Response of Peripheral Lymphocytes to Early Fibrosarcoma: A Model System for Cancer Detection Based on Hybridization Signatures

Since circulating leukocytes, mainly B and T cells, continuously maintain vigilant and comprehensive immune surveillance, these cells could be used as reporters for signs of infection or other pathologies, including cancer. Activated lymphocyte clones trigger a sensitive transcriptional response, which could be identified by gene expression profiling. To assess this hypothesis, we conducted microarray analysis of the gene expression profile of lymphocytes isolated from immunocompetent BALB/c mice subcutaneously injected with different numbers of tumorigenic B61 fibrosarcoma cells. Flow cytometry demonstrated that the number of circulating T (CD3(+)CD4(+) or CD3(+)CD8(+)) or B (CD19(+)) cells did not change. However, the lymphocytes isolated from tumor cell-injected animals expressed a unique transcriptional profile that was identifiable before the development of a palpable tumor mass. This finding demonstrates that the transcriptional response appears before alterations in the main lymphocyte subsets and that the gene expression profile of peripheral lymphocytes can serve as a sensitive and accurate method for the early detection of cancer. Exp Biol Med 234:802-812, 2009

A study of adrenocortical tumors in children and adolescents by a comparative genomic hybridization technique

Adrenocortical tumors (ACT) are rare neoplasms of the adrenal glands accounting for 0.2% of all pediatric cancers. However, the incidence of ACT in South Brazilian children is 10 to 15 times greater than the worldwide incidence. Comparative genomic hybridization studies have revealed the presence of a high degree of chromosomal instability in ACT. We evaluated 16 ACT, 8 of them carcinomas and 8 adenomas. The presence of changes in DNA copy numbers was determined by comparative genomic hybridization, and the findings were validated by real-time polymerase chain reaction on the basis of IGF-II gene expression. The adenomas showed a mean of 19.7 imbalances per case, with the most frequent gain and loss being 4p15.1-p15.3 and 20p11.2-p13.2, respectively. The carcinomas presented with a mean of 35.5 imbalances per case, with the more frequent gain being 2q14.1-q24.3 and the more frequent losses being 3q21-q26.2, 20q12-qter, and 22q11.2-q13.3. The most frequent imbalances in both adenomas and carcinomas were gains of 1p21-p31.2, 2p12-p21 and loss of 20p11.2-p12. The expression of IGF-II mRNA (11p15.5) was higher in samples that presented with a gain of this region. It has been established that great genomic instability exists in pediatric ACT.

Reliability of short comparative genomic hybridization in fibroblasts and blastomeres for a comprehensive aneuploidy screening: first clinical application

BACKGROUND: Comparative genomic hybridization (CGH) is a valuable alternative to fluorescence in situ hybridization (FISH) for preimplantation genetic screening (PGS) because it allows full karyotype analysis. However, this approach requires the cryopreservation of biopsied embryos until results are available. The aim of this study is to reduce the hybridization period of CGH, in order to make this short-CGH technique suitable for PGS of Day-3 embryos, avoiding the cryopreservation step. METHODS: Thirty-two fibroblasts from six aneuploid cell lines (Coriell) and 48 blastomeres from 10 Day-4 embryos, discarded after PGS by FISH with 9 probes (9-chr-FISH), were analysed by short-CGH. A reanalysis by the standard 72 h-CGH and FISH using telomeric probes was performed when no concordant results between short-CGH and FISH diagnosis were observed. The short-CGH was subsequently applied in a clinical case of advanced maternal age. RESULTS: In 100% of the fibroblasts analysed, the characteristic aneuploidies of each cell line were detected by short-CGH. The results of the 48 blastomeres screened by short-CGH were supported by both 72 h-CGH results and FISH reanalysis. The chromosomes most frequently involved in aneuploidy were 22 and 16, but aneuploidies for the other chromosomes, excepting 1, 10 and 13, were also detected. Forty-one of the 94 aneuploid events observed (43.6%) corresponded to chromosomes which are not analysed by 9-chr-FISH. CONCLUSIONS: We have performed a preliminary validation of the short-CGH technique, including one clinical case, suggesting this approach may be applied to Day-3 aneuploidy analysis, thereby avoiding embryo cryopreservation and perhaps helping to improve implantation rate after PGS.

Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments

To evaluate the checkerboard DNA-DNA hybridization method for detection and quantitation of bacteria from the internal parts of dental implants and to compare bacterial leakage from implants connected either to cast or to pre-machined abutments. Nine plastic abutments cast in a Ni-Cr alloy and nine pre-machined Co-Cr alloy abutments with plastic sleeves cast in Ni-Cr were connected to Branemark-compatible implants. A group of nine implants was used as control. The implants were inoculated with 3 mu l of a solution containing 10(8) cells/ml of Streptococcus sobrinus. Bacterial samples were immediately collected from the control implants while assemblies were completely immersed in 5 ml of sterile Tripty Soy Broth (TSB) medium. After 14 days of anaerobic incubation, occurrence of leakage at the implant-abutment interface was evaluated by assessing contamination of the TSB medium. Internal contamination of the implants was evaluated with the checkerboard DNA-DNA hybridization method. DNA-DNA hybridization was sensitive enough to detect and quantify the microorganism from the internal parts of the implants. No differences in leakage and in internal contamination were found between cast and pre-machined abutments. Bacterial scores in the control group were significantly higher than in the other groups (P < 0.05). Bacterial leakage through the implant-abutment interface does not significantly differ when cast or pre-machined abutments are used. The checkerboard DNA-DNA hybridization technique is suitable for the evaluation of the internal contamination of dental implants although further studies are necessary to validate the use of computational methods for the improvement of the test accuracy. To cite this article:do Nascimento C, Barbosa RES, Issa JPM, Watanabe E, Ito IY, Albuquerque Junior RF. Use of checkerboard DNA-DNA hybridization to evaluate the internal contamination of dental implants and comparison of bacterial leakage with cast or pre-machined abutments.Clin. Oral Impl. Res. 20, 2009; 571-577.doi: 10.1111/j.1600-0501.2008.01663.x.

Use of the DNA Checkerboard hybridization method for detection and quantitation of Candida species in oral microbiota

The DNA Checkerboard method enables the simultaneous identification of distinct microorganisms in a large number of samples and employs up to 45 whole genomic DNA probes to gram-negative and gram-positive bacterial species present in subgingival biofilms. Collectively, they account for 55%-60% of the bacteria in subgingival biofilms. In this study, we present the DNA Checkerboard hybridization as an alternative method for the detection and quantitation of Candida species in oral cavities. Our results reveal that DNA Checkerboard is sensitive enough and constitutes a powerful and appropriate method for detecting and quantifying Candida species found in the oral cavity.