Autoimmune lymphoproliferative syndrome in a patient with a new minimal deletion in the death domain of the FAS gene

We present a case of autoimmune lymphoproliferative syndrome (ALPS) caused by a previously undescribed minimal deletion in the death domain of the FAS gene. ALPS is an uncommon disease associated with an impaired Fas-mediated apoptosis. The patient presented with a history of splenomegaly since 4 months of age, associated with cervical lymphadenopathy, which improved with oral corticosteroid treatment. Relevant laboratory findings were the presence of anemia, thrombocytopenia, and positive direct and indirect Coombs tests. He was not an offspring of consanguineous parents. Two cervical lymph node biopsies were performed, at 4 years and at 6 years of age. In both lymph nodes, there was marked paracortical expansion by lymphocytes in variable stages of immunoblastic transformation and a very high cell proliferating index. Some clear cells were also present, raising the suspicion of malignant lymphoma. In one of the lymph nodes, there was also a focus rich in large histiocytes with round nuclei and emperipolesis, consistent with focal Rosai-Dorfman disease. Immunostaining showed numerous CD3+ cells, many of which were double-negative (CD4- CD8-) and expressed CD57, especially around the follicles. Molecular studies of the lymph node biopsy showed a point deletion (4-base pair deletion) in exon 9 of the FAS gene (930del TGCT), which results in 3 missense amino acids. (c) 2008 Elsevier Inc. All rights reserved.

Myosin Va phosphorylated on ser(1650) is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcription

Nuclear actin and nuclear myosins have been implicated in the regulation of geneexpression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser(1650) MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine(1650) and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser(1650) MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser(1650) MVa to nucleoli, as well as separating a fraction of phosphoser(1650) MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation.

Galectin-3 Overexpression in Invasive Laryngeal Carcinoma, Assessed by Computer-assisted Analysis

The larynx is the most common site of malignancy in the upper aerodigestive tract. In Brazil, malignant laryngeal lesions represent 2% of all cancers, with similar to 3000 annual deaths. The association between human papillomavirus (HPV) and laryngeal cancer is still controversial. The aim of the present retrospective study was to determine the expression of galectin-3 immunoperoxidase in laryngeal carcinoma by examining paraffin-em bedded larynx biopsies from 65 patients, 10 in situ laryngeal carcinomas, 27 laryngeal carcinomas without metastases, and 28 with metastases. Twenty-eight cervical lymph nodes from patients with metastatic lesions were also evaluated. Nested PCR was performed to detect and type HPV DNA. Galectin-3 expression was assessed by immunohistochemistry using a computer-assisted system. Among 65 patients, 55 (84.6%)were positive to beta-globin (internal control); 10 (15.4%) patients were beta-globin negative and were excluded from the HPV evaluation. Thus, 7 (12.7%) out of 55 patients were HPV positive and 48 (87.3%) out of 55 patients were HPV negative. High expression of galectin-3 was observed in invasive laryngeal tumors, suggesting that galectin-3 could be associated with the invasiveness and aggressiveness of laryngeal carcinomas. (J Histochem Cytochem 57:665-673, 2009)

BUBR1 expression in benign oral lesions and squamous cell carcinomas: Correlation with human papillomavirus

Oral squamous Cell carcinoma (OSCC) is the most common head and neck cancer. Only in Brazil, the estimate is for 14,160 new cases in 2009. HPV is associated with increasing risk of oral cancer, but its role in carcinogenesis is still controversial. BUBR1, all important protein in the mitotic spindle assembly checkpoint (SAC), has been associated with some virus-encoded proteins and cancer. The aim of the present study was to evaluate the expression of BUBR1 in non-malignant oral lesions and OSCC with and without metastasis associated with HPV infection. We performed immunohistochemistry for BUBR1 in 70 OSCC biopsies divided into three groups (in situ tumors, invasive tumors without metastasis and invasive tumors with metastasis) with their respective lymph nodes from samples with metastasis and in 16 non-malignant oral lesions. PCR was performed in order to detect HPV DNA. Significantly higher BUBR1 expression associated with shorter survival (p=0.0479) was observed in malignant lesions. There was also it significant correlation (r=1.000) with BUBR1 expression in lesions with metastasis and their lymph nodes. Ninety percent of OSCC and 100% of benign lesions were HPV positive. HPV16 and HVP18 were present in 13 and 24% of HPV-positive OSCC samples, respectively. HPV was more prevalent (76%) in samples with a high BUBR1 expression and the absence of viral DNA had no influence oil BUBR1 expression. These findings suggest that HPV could be associated with overexpression of BUBR1 in OSCC. but not in benign oral lesions.

Development of chronic cardiomyopathy in canine Chagas disease correlates with high IFN-gamma, TNF-alpha, and low IL-10 production during the acute infection phase

When infected with Trypanosoma cruzi, Beagle dogs develop symptoms similar to those of Chagas disease in human beings, and could be an important experimental model for a better understanding of the immunopathogenic mechanisms involved in chronic chagasic infection. This study evaluates IL-10, IFN-gamma and TNF-alpha production in the sera, culture supernatant, heart and cervical lymph nodes and their correlation with cardiomegaly, cardiac inflammation and fibrosis in Beagle dogs infected with T. cruzi. Pathological analysis showed severe splenomegaly, lymphadenopathy and myocarditis in all infected dogs during the acute phase of the disease, with cardiomegaly, inflammation and fibrosis observed in 83% of the animals infected by T. cruzi during the chronic phase. The data indicate that infected animals producing IL-10 in the heart during the chronic phase and showing high IL-10 production in the culture supernatant and serum during the acute phase had lower cardiac alterations (myocarditis, fibrosis and cardiomegaly) than those with high IFN-gamma and TNF-alpha levels. These animals produced low IL-10 levels in the culture supernatant and serum during the acute phase and did not produce IL-10 in the heart during the chronic phase of the disease. Our findings showed that Beagle dogs are a good model for studying the immunopathogenic mechanism of Chagas disease, since they reproduce the clinical and immunological findings described in chagasic patients. The data suggest that the development of the chronic cardiac form of the disease is related to a strong Th1 response during the acute phase of the disease, while the development of the indeterminate form results from a blend of Th1 and Th2 responses soon after infection, suggesting that the acute phase immune response is important for the genesis of chronic cardiac lesions. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Genes that code for T cell signaling proteins establish transcriptional regulatory networks during thymus ontogeny

Gene expression profiling by cDNA microarrays during murine thymus ontogeny has contributed to dissecting the large-scale molecular genetics of T cell maturation. Gene profiling, although useful for characterizing the thymus developmental phases and identifying the differentially expressed genes, does not permit the determination of possible interactions between genes. In order to reconstruct genetic interactions, on RNA level, within thymocyte differentiation, a pair of microarrays containing a total of 1,576 cDNA sequences derived from the IMAGE MTB library was applied on samples of developing thymuses (14-17 days of gestation). The data were analyzed using the GeneNetwork program. Genes that were previously identified as differentially expressed during thymus ontogeny showed their relationships with several other genes. The present method provided the detection of gene nodes coding for proteins implicated in the calcium signaling pathway, such as Prrg2 and Stxbp3, and in protein transport toward the cell membrane, such as Gosr2. The results demonstrate the feasibility of reconstructing networks based on cDNA microarray gene expression determinations, contributing to a clearer understanding of the complex interactions between genes involved in thymus/thymocyte development.

SAGE analysis demonstrates increased expression of TOSO contributing to Fas-mediated resistance in CLL

To identify novel genes involved in the molecular pathogenesis of chronic lymphocytic leukemia (CLL) we performed a serial analysis of gene expression (SAGE) in CLL cells, and compared this with healthy B cells (nCD19(+)). We found a high level of similarity among CLL subtypes, but a comparison of CLL versus nCD19(+) libraries revealed 55 genes that were over-represented and 49 genes that were down-regulated in CLL. A gene ontology analysis revealed that TOSO, which plays a functional role upstream of Fas extrinsic apoptosis pathway, was over-expressed in CLL cells. This finding was confirmed by real-time reverse transcription-polymerase chain reaction in 78 CLL and 12 nCD19(+) cases (P <.001). We validated expression using flow cytometry and tissue microarray and demonstrated a 5.6-fold increase of TOSO protein in circulating CLL cells (P =.013) and lymph nodes (P =.006). Our SAGE results have demonstrated that TOSO is a novel overexpressed antiapoptotic gene in CLL.

Experimental autoimmune encephalomyelitis evolution was not modified by multiple infections with Strongyloides venezuelensis

P>According to the hygiene hypothesis, the increased incidence of allergic and autoimmune diseases in developed countries is mainly explained by the decreased contact between the human population and certain environmental agents as lactobacillus, mycobacteria and helminths. In this study, we evaluated the effect of multiple infections with Strongyloides venezuelensis on the development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats. Multiple infections before EAE induction were not able to change the evolution of the disease. No alterations were observed in weight loss, clinical score and inflammation intensity at the central nervous system. The presence of significant levels of parasite-specific IgG1 but not IgG2b suggested a Th2 polarization. However, the percentage and absolute number of CD4+CD25+Foxp3+ T cells were not changed, being their levels in the spleen and lymph nodes of infected rats comparable to the ones found in normal animals. These results suggest that a Th2-polarized response without concomitant expansion of Foxp3+ regulatory T cells was not able to modify EAE progression. Even though these results do not threaten the hygiene hypothesis, they suggest that this paradigm might be an oversimplification. They also emphasize the need of a study to compare the immunoregulatory ability associated with different helminth spp.

Tc-99m-labeled dextran for mammary lymphoscintigraphy in dogs

Lymphoscintigraphy is the technique of choice for sentinel lymph node detection in women with early breast cancer, but there is limited information evaluating the value of this technique in animals. We investigated mammary lymphatic drainage in 25 young female mongrel dogs by intramammary injection of 18.5 MBq of Tc-99m-dextran (70,000 Da). Lymph node anatomical referencing was obtained using an external marker, bone scintigraphy, or scintiscanning the body contour. Cranial and caudal thoracic mammary glands drained into the cranial sternal lymph node and axillary lymph center. The cranial thoracic mammary gland also drained into the superficial cervical lymph node in two of five animals. The cranial abdominal gland was drained by the axillary lymph center. The caudal abdominal mammary gland was drained by the superficial inguinal lymph node in all animals and simultaneously by medial iliac lymph nodes in four of five animals. In one dog, this mammary gland was also drained by the mediastinal and the superficial cervical lymph nodes. The inguinal mammary gland was drained by superficial inguinal lymph nodes and simultaneously via the medial iliac lymph node in one animal. Lymphatic communications between lymph nodes were identified in 11 of 25 (44%) animals. Tc-99m-dextran mammary lymphoscintigraphy was easy and rapid to perform and may provide valuable information for further studies.

Experimental infection with Rangelia vitalii in dogs: Acute phase, parasitemia, biological cycle, clinical-pathological aspects and treatment

Recently we conducted the molecular characterization of Rangelia vitalii, a protozoan with high pathogenicity for young dogs in southern Brazil. To date, the descriptions of the disease have been restricted to natural infection cases. Therefore, this study aimed to evaluate the parasitemia, biological cycles and clinical-pathological findings in dogs experimentally infected with R. vitalii in the acute phase of disease, and also aimed to test a therapeutic protocol based on the diminazene aceturate. For this study, we used 12 young dogs (females), separated into two groups. Group A was composed of healthy dogs, not-infected (n = 5), and Group B consisted of animals infected with R. vitalii (n = 7). After infection, the animals were monitored by blood smear examinations, which showed intra-erythrocytic forms of the parasite 5 days post-infection (PI). Parasitemia increased progressively in these animals and had the highest peak of circulating parasites between 9 and 11 days PI. Subsequently, the parasitemia reduced and the protozoan was seen inside the leukocytes in days 17, 19 and 21 PI. The most prominent clinical signs observed at the 20 day PI of experiment were lethargy, fever and anorexia. We observed a decrease of hematocrit of infected animals compared with not-infected dogs, featuring a moderate anemia. Pathological evaluation of one dog in Group B at day 21 PI revealed splenomegaly, hepatomegaly, lymphadenopathy, and hemorrhages at necropsy. Histological examination showed only follicular hyperplasia in the spleen and lymph nodes, and the etiologic agent in the vascular endothelium. At 21 days PI, it was performed the treatment of dogs in Group B (n = 6) with a single dose of diminazene aceturate, which showed a curative efficacy of 100% in cleaning R. vitalii from blood of infected dogs. (C) 2011 Elsevier Inc. All rights reserved.

Squamous Cell Carcinoma of the Tongue and Floor of the Mouth: Analysis of Survival Rate and Independent Prognostic Factors in the Amazon Region

Oral squamous cell carcinoma (OSCC) accounts for more than 95% of all malignant neoplasms in the oral cavity. Although several studies have shown the epidemiology of this cancer in Brazil, there do not seem to be any studies that describe the prognostic factors related to OSCC in the Amazon region. Therefore, the aim of this study was to determine the survival rate and prognostic significance of different factors in patients from this region affected by OSCC. Data from 85 patients with histologically confirmed squamous cell carcinoma of the tongue and floor of the mouth identified from the Ofir Loyola Hospital archives were collected and analyzed using univariate (log-rank test) and multivariate (Cox proportional hazard model) tests. The overall 5-year survival rate was found to be 27%. Univariate analysis showed that the 5-year survival rate was significantly higher for younger (<= 45 y) female patients, patients with T1-2 tumors and clinically clear neck nodes (N0), patients with early stage cancers (AJCC stage I-II), and patients treated with surgical procedures. However, multivariate analysis showed that the 5-year survival rate was significantly higher only in the younger patients and those who underwent surgical treatment. The age of the patient at the moment of diagnosis and treatment with surgical procedures were the only independent prognostic factors that affected the 5-year survival rate of the patients in this region.

Downregulation of CD9 protein expression is associated with aggressive behavior of oral squamous cell carcinoma

Squamous cell carcinoma of the oral cavity (OSCC) is a malignancy characterized by a high degree of local aggression and metastasis to cervical lymph nodes. Tetraspanins are proteins with functional roles in a wide array of cellular processes and are reported to be associated with tumor progression. The present study investigated the expression of the CD9, CD37, CD63, CD81 and CD82 tetraspanins in OSCC using immunohistochemistry (IHC) and quantitative Real Time-PCR (qRT-PCR). Tissue microarray (TMA) analysis of samples from 179 cases of OSCC and 10 normal samples oral mucosa were evaluated immunomorphologically. We analyzed CD9 and CD82 expression by qRT-PCR in 66 OSCC cases and 4 normal samples of oral mucosa. Expression of CD63, CD37 and CD81 was not detected in the samples studied. CD82 was downregulated or negative in 127 of 179 (80%) specimens; no correlation was observed between CD82 expression, clinicopathological parameters, disease-free survival and 5-year overall survival. CD9 expression was downregulated or negative in 75 of 129 (42%) OSCC samples. Loss of CD9 expression in OSCC samples correlated with the incidence of lymph node metastasis (p = 0.017). Disease-free survival and the 5-year overall survival of patients with downregulated or negative CD9 expression were significantly lower than in patients with positive CD9 expression (p = 0.010 and p = 0.071, respectively). No correlation was found between CD9 or CD82 expression and clinicopathological parameters by qRT-PCR. Our results suggest that the downregulation or lack of expression of the CD9 protein might indicate a more aggressive of OSCC. (C) 2009 Elsevier Ltd. All rights reserved.

The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis

Background Prolonged exposure of the lip to sunlight may cause actinic cheilitis (AC) and squamous cell carcinoma (SCC). Maspin is a serpin with tumor suppressor functions. This work analyzed the presence and distribution of maspin in AC and lip SCC. Methods Sections from 36 cases diagnosed as AC (18 cases with mild epithelial dysplasia, 11 with moderate and 7 with severe), 18 cases diagnosed as lip SCC and 7 specimens containing normal lip vermillion epithelium were submitted for immunohistochemical analysis to detect maspin. Results All AC cases with mild and two cases with moderate dysplasia were scored 3. The remaining nine cases with moderate dysplasia were identified as score 2, whereas all cases with severe dysplasia were scored 1. Positive staining for maspin decreased from the basal layer to the surface. Among the 18 lip SCCs studied, 15 cases showed abundant staining for maspin. Epithelium adjacent to the SCCs also showed intense positive staining in all cells. Conclusions Our results suggest that the loss of maspin expression occurs from the basal layer to the surface. Lip SCCs related to solar radiation show an intense presence of maspin protein in almost all tumor cells as well as the neighboring epithelium. Fontes A, Sousa SM, Santos E, Martins MT. The severity of epithelial dysplasia is associated with loss of maspin expression in actinic cheilitis.

Precise Insertion of Orthodontic Miniscrews with a Stereolithographic Surgical Guide Based on Cone Beam Computed Tomography Data: A Pilot Study

Purpose: Orthodontic miniscrews are commonly used to achieve absolute anchorage during tooth movement. One of the most frequent complications is screw loss as a result of root contact. Increased precision during the process of miniscrew insertion would help prevent screw loss and potential root damage, improving treatment outcomes. Stereo lithographic surgical guides have been commonly used for prosthetic implants to increase the precision of insertion. The objective of this paper was to describe the use of a stereolithographic surgical guide suitable for one-component orthodontic miniscrews based on cone beam computed tomography (CBCT) data and to evaluate implant placement accuracy. Materials and Methods: Acrylic splints were adapted to the dental arches of four patients, and six radiopaque reference points were filled with gutta-percha. The patients were submitted to CBCT while they wore the occlusal splint. Another series of images was captured with the splint alone. After superimposition and segmentation, miniscrew insertion was simulated using planning software that allowed the user to check the implant position in all planes and in three dimensions. In a rapid-prototyping machine, a stereolithographic guide was fabricated with metallic sleeves located at the insertion points to allow for three-dimensional control of the pilot bur. The surgical guide was worn during surgery. After implant insertion, each patient was submitted to CBCT a second time to verify the implant position and the accuracy of the placement of the miniscrews. Results: The average differences between the planned and inserted positions for the ten miniscrews were 0.86 mm at the coronal end, 0.71 mm at the center, and 0.87 mm at the apical tip. The average angular discrepancy was 1.76 degrees. Conclusions: The use of stereolithographic surgical guides based on CBCT data allows for accurate orthodontic mini screw insertion without damaging neighboring anatomic structures. INT J ORAL MAXILLOFAC IMPLANTS 2011;26:860-865

Absence of functional TLR4 impairs response of macrophages after Candida albicans infection

Candida albicans is recognized by phagocytic cells through a set of recognition receptors patterns. Recently, we showed the importance of TLR2 in the regulation of neutrophil survival after C. albicans infection. In the present work, we analyzed the involvement of TLR4 in the recognition of C. albicans by neutrophils and macrophages. Our results show that the absence of functional TLR4 resulted in lower chemotaxis of neutrophils to the site of infection, lower levels of TNF-alpha, CXCL1 and nitric oxide, and dissemination and persistence of the pathogen in lymph nodes and spleen. In vitro, the phagocytic activity, nitric oxide production and myeloperoxidase activity, CXCL1, IL-1 beta production by neutrophils from TLR4-defective mice were not changed. In contrast, macrophages from TLR4-defective mice demonstrated lower phagocytosis and lower levels of CXCL1, IL-1 beta and TNF-alpha. Together, these data demonstrate that TLR4 signals are important for the recognition of C. albicans by macrophages and their absence allows persistence of the infection.