Human visceral leishmaniasis expresses Th1 pattern in situ liver lesions

The architectural and infiltrate pattern of liver human visceral leishmaniasis (HVL) have been systematically classified as typical, fibrogenic or nodular. Despite this histopathological classification, the immune response based on cytokines and cellular phenotypes have never been performed. The aim of this study was to determine the immunophenotypic pattern and cytokine profile of the nodular involvement of the Liver in HVL. We evaluated nine cases of the nodular form of HVL. In situ immune response was studied through cytokine analysis and immunohistochemical study for phenotype markers: IL-1, IL-4, IL-1 0, TNF-alpha, IFN-gamma, CD4+ T cells, CD8+ T cells, CD20, CD68, CD57 and macrophage activation was determined by evaluation of iNOS activity. HVL seems to be related to a better immune response. Amastigotes were rarely found on liver sections. Leishmania antigen expression was also rare and located in the inflammatory nodules. The lower expression of IL-4 and IL-10, moderate expression of TNF-alpha and IFN-gamma demonstrate a panorama of Th1 phenotype. The increased expression of NK cells could help in sustaining this model of response. This pattern of immune response is probably responsible for improvement in the parasite`s clearance from liver tissue and it is a prognostic marker of human visceral leishmaniasis. (C) 2008 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Reappraisal of the immunopathogenesis of disseminated leishmaniasis: in situ and systemic immune response

Disseminated leishmaniasis (DL) is an emerging form of Leishmania braziliensis infection characterised by multiple cutaneous lesions on different parts of the body and a high rate of mucosal involvement. Systemic production of TNF alpha and IFN-gamma in DL patients is lower than in cutaneous leishmaniasis (CL) caused by L braziliensis, which may account for parasite dissemination due to the decreased ability to control parasite growth. In this study, the systemic and in situ immune response of DL and CL patients was characterised through evaluation of chemokine and cytokine production. In situ evaluation showed similar production of IFN gamma, TNF alpha, IL-10, transforming growth factor-beta (TGF beta), chemokine (C-C motif) ligand 2 (CCL2), CCL3, CCL11 and chemokine (C-X-C motif) ligand 10 (CXCL10) in papular and ulcerative lesions from DL as well as in ulcerated lesions from CL. Serum levels of CXCL9, a chemokine that attracts 1-cells, was higher in serum from DL than from CL These data indicate that a decrease in the type 1 immune response in peripheral blood of DL patients is due to attraction of Leishmania antigen-activated T-cells to the multiple cutaneous lesions. This may account for the absence of or few parasites in the lesions and for the development of ulcers similar to those observed in CL (C) 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Polyploidy in atypical grade II choroid plexus papilloma of the posterior fossa

Cytogenetic studies of choroid plexus tumors, particularly for atypical choroid plexus papillomas, have been rarely described. In the present report, the cytogenetic investigation of an atypical choroid plexus papilloma occurring at the posterior fossa of a 16-year-old male is described. Comparative genome hybridization analysis demonstrated gains of genetic material from almost all chromosomes. Chromosome losses involved 19p, regional losses at chromosome X and loss of chromosome Y. The presence of polyploid cells was confirmed by fluorescence in situ hybridization analysis with probes directed to centromeric regions. Furthermore, the microscopic analysis of cultures showed nuclear buds, nucleoplasmic bridges, and micronuclei in 23% of tumor cells suggesting the presence of complex chromosomal abnormalities. Previous cytogenetic studies on choroid plexus papillomas showed either normal, hypodiploid or hyperdiploid karyotypes. To the best of our knowledge, this is the first report of polyploidy in choroid plexus papilloma of intermediate malignancy grade. Although the mechanisms beneath such genome duplication remain to be elucidated, the observed abnormal nuclear shapes indicate constant restructuring of the tumor`s genome and deserves further investigation.

Monoclonal B-cell lymphocytosis in first-degree relatives of patients with sporadic (non-familial) chronic lymphocytic leukaemia

Although biological similarities have been described among monoclonal B-cell lymphocytosis (MBL) and chronic lymphocytic leukaemia (CLL), the relationships between these two conditions are not fully understood, and new epidemiological studies in different populations and different countries continue to be reported. Here, we investigated 167 first-degree relatives from 42 families of patients with non-familial (sporadic) CLL, using four-colour flow cytometry. MBL was found in seven of 167 subjects (4.1%). Monoclonality was detected in all cases either by light-chain restriction or by polymerase chain reaction. Fluourescence in situ hybridization did not show any chromosomal abnormality. The prevalence of MBL according to age was 0 (0/54) in individuals aged less than 40 years, 2.5% (2/81) between 40 and 60 years, and 15.6% (5/32) in individuals over 60 years. The prevalence of MBL cases in individuals over 60 years was similar to that found in familial CLL relatives at the same age group. This suggests that in older first-degree relatives of patients with sporadic CLL, the risk of MBL detection is as high as in older first-degree relatives from CLL families, which could render these individuals belonging to `sporadic CLL families` as susceptible as individuals from `familial CLL` to the development of clinical CLL.

Translocations t(X;14)(q28;q11) and t(Y;14)(q12;q11) in T-cell prolymphocytic leukemia

P>We report a case of T-cell prolymphocytic leukemia (T-PLL) in a 41-year-old male. Classical cytogenetic, spectral karyotyping (SKY) and fluorescence in situ hybridization (FISH) studies of a blood sample obtained at diagnosis revealed the co-existence of t(X;14)(q28;q11), t(Y;14)(q12;q11) and a ring chromosome derivated from i(8)(q10). Immunophenotypic studies revealed involvement of T-cell lineage, with proliferation of CD4(-) CD8(+). The co-existence of two translocations involving both sex chromosomes in a case of T-PLL is rare. Chromosomal instability associated with the disease progression may have allowed the emergence of cell clones with translocations involving the sex chromosomes and the ring chromosome observed.

Segmental amplification of MLL gene associated with high expression of AURKA and AURKB genes in a case of acute monoblastic leukemia with complex karyotype

We report a case of acute monoblastic leukemia showing a jumping translocation with the MLL gene in a 17-year-old male. Classic cytogenetic and spectral karyotyping revealed a complex karyotype, and fluorescence in situ hybridization (FISH) demonstrated amplification of the MLL gene followed by translocation to chromosomes 15q, 17q, and 19q. In addition, molecular analyses showed a high expression of AURKA and AURKB genes. It is already known that overexpression of Aurora kinases is associated with chromosomal instability and poor prognosis. The formation of jumping translocations is a rare cytogenetic event and there is evidence pointing toward preferential involvement of the heterochromatin region of donor chromosomes and the telomere ends of recipient chromosomes. Jumping translocation with the MLL gene rearrangement is an uncommon phenomenon reported in leukemia cytogenetics. (C) 2010 Elsevier Inc. All rights reserved.

Serotonergic neurotransmission in the dorsal raphe nucleus recruits in situ 5-HT2A/2C receptors to modulate the post-ictal antinociception

The post-ictal immobility syndrome is followed by a significant increase in the nociceptive thresholds in animals and humans. The aim of this study was to assess the involvement of the dorsal raphe nucleus (DRN) in the post-ictal antinociception. The second aim was to study the role of serotonergic intrinsic mechanisms of the DRN in this hypo-algesic phenomenon. Pentylenetetrazole (PTZ), an ionophore GABA-mediated Cl- influx antagonist, was peripherally used to induce tonic-clonic seizures in Wistar rats. The nociceptive threshold was measured by the tail-flick test. Neurochemical lesions of the DRN, performed with microinjection of ibotenic acid (1.0 mu g/0.2 mu L), caused a significant decrease of tonic-clonic seizure-induced antinociception, suggesting the involvement of this nucleus in this antinociceptive Process. Microinjections of methysergide (1.0 and 5.0 mu g/0.2 mu L), a non-selective serotonergic receptor antagonist, into DRN caused a significant decrease in the post-ictal antinociception in seizing animals, compared to controls, in all post-ictal periods Presently studied. These findings were corroborated by microinjections of ketanserin (at 1.0 and 5.0 mu g/0.2 mu L) into DRN. Ketanserin is an antagonist with large affinity for 5-HT2A/2C serotonergic receptors, which, in this Case, Caused a significant decrease in the tail-flick latencies in seizing animals, compared to controls after the first 20 min following tonic-clonic convulsive reactions. These results indicate that serotonergic neurotransmission of the DRN neuronal clusters is involved in the organization of the post-ictal hypo-algesia. The 5-HT2A/2C receptors of DRN neurons seem to be critically involved in the increase of nociceptive thresholds following tonic-clonic seizures. (c) 2008 Elsevier Inc, All rights reserved.

Downregulation of Melanocortin-4 Receptor during Refeeding and its Modulation by Adrenalectomy in Rats

Melanocortin system and corticotropin releasing hormone (CRH) are implicated in the control of feeding behavior. Besides its anorexigenic effect on food intake, CRH is one of the most important regulators of hypothalamic-pituitary-adrenal (HPA) axis activity. Therefore, there could be an interplay between HPA axis activity and melanocortin system. We investigated the expression of melanocortin-4 receptor (MC4-R) mRNA in the hypothalamus of rats after 14 days of food restriction or after a fasting-refeeding regimen, in sham or adrenalectomized rats. Male Wistar rats were subjected to free access to food or food ingestion restricted for 2 h a day (8-10 AM) during 14d, when plasma corticosterone, ACTH, insulin, leptin concentrations, and MC4-R mRNA expression were determined before and after refeeding. Another set of rats was fasted for 48 h, followed by refeeding during 2 or 4 h on the seventh day after adrenalectomy (ADX) or sham surgery. On the day of the experiment, rats were anesthetized and perfused and the brain processed for MC4-R mRNA by in situ hybridization. Long-term reduction of food intake, either secondary to food restriction or adrenalectomy, reduced body weight gain and also leptin and insulin plasma concentrations. Food ingestion reduced MC4-R expression in the paraventricular nucleus in naive rats subjected to food restriction and also in sham rats fasted for 48 h. However, after ADX, MC4-R expression was not changed by refeeding. In conclusion, the present data indicate that MC4-R expression is downregulated by food ingestion and this response could be modulated by glucocorticoid withdrawal.

Allelic status of 1p and 19q in oligodendrogliomas and glioblastomas: multiplex ligation-dependent probe amplification versus loss of heterozygosity

Identification of the 1p/19q allelic status in gliomas, primarily those with a major oligodendroglial component, has become an excellent molecular complement to tumor histology in order to identify those cases sensitive to chemotherapy. In addition to loss of heterozygosity (LOH), fluorescence in situ hybridization (FISH), or comparative genomic hybridization (CGH), multiplex ligation-dependent probe amplification (MLPA) has been shown to be an alternative methodology to identify deletions of those chromosome arms. We used MLPA to explore the 1p and 19q glioblastomas, and a series of 76 gliomas: 41 tumors with a major oligodendroglial component, 34 glioblastomas, and one low-grade astrocytoma. We compared the MLPA findings of the oligodendroglial cases with those previously obtained using LOH in the same samples. Thirty-eight of 41 oligodendrogliomas displayed identical findings by both LOH and MLPA, and losses at either 1p and/or 19q were identified in 12 of 35 (34%) astrocytic tumors. These findings agree with data previously reported comparing MLPA versus FISH or CGH in gliomas and suggest that MLPA can be used in the identification 1p/19q allelic deletions on these brain neoplams. (c) 2009 Elsevier Inc. All rights reserved. reserved.

Tetrasomy 8 in a patient with chronic lymphocytic leukemia

We report a case of a 47-year-old man diagnosed with chronic lymphocytic leukemia (CLL) with two extra copies of chromosome 8. Classical cytogenetic analysis by the immunostimulatory combination of DSP30 and interleukin 2 showed tetrasomy of chromosome 8 in 60% of the metaphase cells (48,XY,+8,+8[12]/46,XY[8]). Spectral karyotype analysis confirmed the abnormality previously seen by G banding. Additionally, interphase fluorescence in situ hybridization using an LSI CEP 8 probe performed on peripheral blood cells without any stimulant agent showed tetrasomy of chromosome 8 in 54% of analyzed cells (108 of 200). To our knowledge, tetrasomy 8 as the sole chromosomal abnormality in CLL has not been previously described. The prognostic significance of tetrasomy 8 in CLL remains to be elucidated. However, the patient has been followed up in the outpatient hospital since 2004 without any therapeutic intervention and has so far remained stable. (C) 2010 Elsevier Inc. All rights reserved.

Diazepam decreases leukocyte-endothelium interactions in situ

High doses of diazepam reduce the inflammatory paw edema in rats. This effect was attributed to an action of diazepam on the Translocator Protein (TSPO). We evaluated the effects of diazepam (10 mg/kg, intraperitoneally) on leukocyte rolling and migration. In carrageenan-induced acute inflammation, diazepam decreased the interaction of leukocytes with endothelial cells (rolling) and the number of leukocytes in the mesentery (migration). RU486 (antagonist of glucocorticoid receptors) reduced the effects of diazepam on leukocyte rolling and migration, suggesting a participation of endogenous corticosteroids. We also showed that the effects of diazepam on leukocyte-endothelium interactions are mediated by nitric oxide (NO), since prior treatment with l-arginine (precursor of NO) partially precludes the inhibitory effects of diazepam; conversely, pretreatment with L-NAME (false substrate of the NO synthase) somewhat potentiates the effects of diazepam. The pathways that underlie the effects of diazepam remain to be further elucidated, but we believe that both local and systemic mechanisms may overlap to explain the influence of diazepam on leukocyte-endothelium interactions.

Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos

Bovine Herpesvirus type-5 (BoHV-5), which is potentially neuropathogenic, was recently described to be related with reproductive disorders in cows. The objective was to elucidate mechanisms involved in propagation of BoHV-5 in embryonic cells. For this purpose, bovine embryos produced in vitro were assayed for apoptotic markers after experimental infection of oocytes, in vitro fertilization, and development. Host DNA fragmentation was detected with a TUNEL assay, expression of annexin-V was measured with indirect immunofluorescence, and viral DNA was detected with in situ hybridization. Infective BoHV-5 virus was recovered from embryos derived from exposed oocytes after two consecutive passages on Madin-Darby bovine kidney (MDBK) cells. The viral DNA corresponding to US9 gene, localized between nucleotides 126243 to 126493, was detected in situ and amplified. There was no significant difference between the ratio of TUNEL stained nuclei and total cells in good quality blastocysts (0.87 +/- 0.05, mean SD), but there were differences (P < 0.05) between infected (0.18 +/- 0.05) and uninfected blastocysts (0.73 +/- 0.07). The Annexin-V label was more intense in uninfected embryos (0.79 +/- 0.04; P < 0.05). The quality of infected and uninfected embryos was considered equal, with no significant effect on embryonic development. In conclusion, we inferred that BoHV-5 infected bovine oocytes, replicated, and suppressed some apoptotic pathways, without significantly affecting embryonic development. (C) 2010 Elsevier Inc. All rights reserved.

Detection of TGIF1 homeobox gene in oral squamous cell carcinoma according to histologic grading

Objective. TGIF1 homeobox gene involvement in oral cancer has not yet been investigated. This study analyzed the expression of TGIF1 transcripts and protein in oral squamous cell carcinoma (OSCC). Study design. Snap-frozen samples from 16 patients were taken from both OSCC and nontumoral adjacent epithelium (NT) for in situ hybridization (ISH). Forty-six paraffin-embedded samples of OSCC were submitted to immunohistochemistry (IHC). A descriptive analysis of the transcript signal detection was accomplished, and TGIF1 immunoexpression was carried out considering protein levels, localization, and cellular differentiation. Results. ISH reactions showed TGIF1 transcripts with a signal that was frequently intense in NT, and generally weak in OSCC, and that had stronger transcript signal in well-differentiated areas of OSCC when compared with poorly differentiated ones. IHC reactions had poorly differentiated cases associated with TGIF1 protein expression in both the nucleus and cytoplasm (P = .05, Fisher test). Conclusions. TGIF1 gain or loss of function might possibly play a role in oral cancer cell differentiation. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011; 111: 218-224)

Differential Shh, Bmp and Wnt gene expressions during craniofacial development in mice

In this study, Bmp-4, Wnt-5a and Shh gene expressions were compared during early craniofacial development in mice by comparative non-isotopic in situ hybridization. Wild-type C57BL/6J mice were studied at various stages of embryonic development (from 8.5- to 13.5-day-old embryos - E8.5-13.5). During early odontogenesis, transcripts for Bmp-4, Shh and Wnt-5a were co-localised at the tooth initiation stage. At E8.5, Shh mRNA expression was restricted to diencephalon and pharyngeal endoderm. Before maxillae and mandible ossification, Bmp-4 and Wnt-5a signals were detected in the mesenchymal cells and around Meckel`s cartilage. During palatogenesis, Shh was expressed only in the epithelium and Wnt-5a only in the mesenchyme of the elevating palatal shelves. During tongue development, Shh expression was found in mesenchyme, probably contributing to tongue miogenesis, while Wnt-5a signal was in the epithelium, possibly during placode development and papillae formation. Taken together, these findings suggest that Bmp-4, Shh and Wnt-5a gene expressions may act together on the epithelial mesenchymal interactions occurring in several aspects of the early mouse craniofacial development, such as odontogenesis, neuronal development, maxillae and mandible ossification, palatogenesis and tongue formation. (C) 2009 Elsevier GmbH. All rights reserved.

Influence of Cavity Preparation with Er:YAG Laser on Enamel Adjacent to Restorations Submitted to Cariogenic Challenge In Situ: A Polarized Light Microscopic Analysis

Background and Objectives: Er:YAG laser has been used for caries removal and cavity preparation, using ablative parameters. Its effect on the margins of restorations submitted to cariogenic challenge has not yet been sufficiently investigated. The aim of this study was to assess the enamel adjacent to restored Er:YAG laser-prepared cavities submitted to cariogenic challenge in situ, under polarized light microscopy. Study Design/Materials and Methods: Ninety-one enamel slabs were randomly assigned to seven groups (n = 13): I, II, III-Er:YAG laser with 250 mJ, 62.5 J/cm(2), combined with 2, 3, and 4 Hz, respectively; IV, V, VI-Er:YAG laser with 350 mJ, 87.5 J/cm(2), combined with 2, 3, and 4 Hz, respectively; VII-High-speed handpiece (control). Cavities were restored and the restorations were polished. The slabs were fixed to intra-oral appliances, worn by 13 volunteers for 14 days. Sucrose solution was applied to each slab six times per day. Samples were removed, cleaned, sectioned and ground to polarized light microscopic analysis. Demineralized area and inhibition zone width were quantitatively assessed. Presence or absence of cracks was also analyzed. Scores for demineralization and inhibition zone were determined. Results: No difference was found among the groups with regard to demineralized area, inhibition zone width, presence or absence of cracks, and demineralization score. Inhibition zone score showed difference among the groups. There was a correlation between the quantitative measures and the scores. Conclusion: Er:YAG laser was similar to high-speed handpiece, with regard to alterations in enamel adjacent to restorations submitted to cariogenic challenge in situ. The inhibition zone score might suggest less demineralization at the restoration margin of the irradiated substrates. Correlation between the quantitative measures and scores indicates that score was, in this case, a suitable complementary method for assessment of caries lesion around restorations, under polarized light microscopy. Lasers Surg. Med. 40:634-643, 2008. (c) 2008 Wiley-Liss, Inc.