Cumulative incidence of femoral localized periosteal thickening (beaking) preceding atypical femoral fractures in patients with rheumatoid arthritis.

The incidence of localized periosteal thickening (LPT, also termed beaking) of the lateral cortex that often precedes an atypical femoral fracture (AFF) was not high in patients with rheumatoid arthritis (RA) but incomplete AFFs developed in two patients. Higher-dose prednisolone was a significant risk factor for LPT in patients with RA. INTRODUCTION: Atypical femoral fractures (AFFs) are stress fractures; bisphosphonate (BP) use is a major risk factor for the development of such fractures. Localized periosteal thickening (LPT, also termed beaking) of the lateral cortex often precedes a complete or incomplete AFF. We evaluated the incidence of latent LPT in patients with rheumatoid arthritis (RA), to evaluate LPT progression, and to define LPT risk factors. METHODS: A total of 254 patients with RA were included; all underwent annual X-ray evaluation, dual-energy X-ray absorptiometry, and analyses of serum and bone metabolic markers for 2-3 years. LPT of the lateral cortex was sought in femoral X-rays. RESULTS: The incidence of LPT was 2.4% (6/254). Among patients on both BP and prednisolone (PSL) at enrollment, the incidence was 2.3% (3/131). Two femurs of two patients with LPT developed incomplete AFFs; LPT was extensive and associated with endosteal thickening. One patient had been on BP and PSL and microscopic polyangiitis was comorbidity. The other was on a selective estrogen receptor modulator and PSL. A daily PSL dose >5 mg (OR 11.4; 95%CI 2.15-60.2; p = 0.004) and higher-dose methotrexate (OR 1.22; 95%CI 1.01-1.49; p = 0.043) were significant risk factors for LPT. CONCLUSIONS: The incidence of latent LPT was not high (2.4%) but incomplete AFFs developed in two RA patients. Higher-dose PSL because of a comorbid disease requiring glucocorticoid treatment other than RA or refractory RA were risk factors for LPT; X-ray screening for latent LPT would usefully prevent complete AFFs.

Characterization of patients with systemic lupus erythematosus who meet the diagnostic criteria for TAFRO syndrome.

Purpose TAFRO syndrome is a novel disorder manifesting as fever, anasarca, thrombocytopenia, renal insufficiency and organomegaly, and its etiology has not been clarified. The aim of this study was to elucidate similarities and differences between systemic lupus erythematosus (SLE) and TAFRO syndrome. Methods We examined 46 consecutive patients diagnosed with SLE and determined whether they meet the proposed diagnostic criteria for TAFRO syndrome (2015 version). Results Of the 46 patients with SLE, four (8.7%) also met the TAFRO syndrome criteria (TAFRO-like group). All patients in the TAFRO-like group were males, and their mean age was significantly higher than that of the non-TAFRO group (67.5 ± 8.7 vs. 39.3 ± 18.1 years, p = 0.004). C-reactive protein and γ-glutamyl transpeptidase levels were significantly higher, and frequencies of anti-dsDNA and anti-Sm antibodies were significantly lower in the TAFRO-like than non-TAFRO group. Elder cases (onset age ≥ 50 years) met significantly more categories of the diagnostic criteria for TAFRO syndrome than did those with younger cases. Conclusions Several patients with SLE, especially elder cases, showed features similar to those of TAFRO syndrome. Although exclusion of SLE is needed in the diagnostic criteria for TAFRO syndrome, TAFRO syndrome-like SLE should be considered.

DAX1 Overexpression in Pediatric Adrenocortical Tumors: A Synergic Role with SF1 in Tumorigenesis.

DAX1 transcription factor is a key determinant of adrenogonadal development, acting as a repressor of SF1 targets in steroidogenesis. It was recently demonstrated that DAX1 regulates pluripotency and differentiation in murine embryonic stem cells. In this study, we investigated DAX1 expression in adrenocortical tumors (ACTs) and correlated it with SF1 expression and clinical parameters. DAX1 and SF1 protein expression were assessed in 104 ACTs from 34 children (25 clinically benign and 9 malignant) and 70 adults (40 adenomas and 30 carcinomas). DAX1 gene expression was studied in 49 ACTs by quantitative real-time PCR. A strong DAX1 protein expression was demonstrated in 74% (25 out of 34) and 24% (17 out of 70) of pediatric and adult ACTs, respectively (χ(2)=10.1, p=0.002). In the pediatric group, ACTs with a strong DAX1 expression were diagnosed at earlier ages than ACTs with weak expression [median 1.2 (range, 0.5-4.5) vs. 2.2 (0.9-9.4), p=0.038]. DAX1 expression was not associated with functional status in ACTs. Interestingly, a positive correlation was observed between DAX1 and SF1 protein expression in both pediatric and adult ACTs (r=0.55 for each group separately; p<0.0001). In addition, DAX1 gene expression was significantly correlated with SF1 gene expression (p<0.0001, r=0.54). In conclusion, DAX1 strong protein expression was more frequent in pediatric than in adult ACTs. Additionally, DAX1 and SF1 expression positively correlated in ACTs, suggesting that these transcription factors might cooperate in adrenocortical tumorigenesis.

MHC class I and II expression in juvenile dermatomyositis skeletal muscle.

OBJECTIVE: To assess MHC I and II expressions in muscle fibres of juvenile dermatomyositis (JDM) and compare with the expression in polymyositis (PM), dermatomyositis (DM) and dystrophy. PATIENTS AND METHODS: Forty-eight JDM patients and 17 controls (8 PM, 5 DM and 4 dystrophy) were studied. The mean age at disease onset was 7.1+/-3.0 years and the mean duration of weakness before biopsy was 9.4+/-12.9 months. Routinehistochemistry and immunohistochemistry (StreptABComplex/HRP) for MHC I and II (Dakopatts) were performed on serial frozen muscle sections in all patients. Mann-Whitney, Kruskal Wallis, chi-square and Fisher's exact statistical methods were used. RESULTS: MHC I expression was positive in 47 (97.9%) JDM cases. This expression was observed independent of time of disease, corticotherapy previous to muscle biopsy and to the grading of inflammation observed in clinical, laboratorial and histological parameters. The expression of MHC I was similar on JDM, PM and DM, and lower in dystrophy. On the other hand, MHC II expression was positive in just 28.2% of JDM cases and was correlated to histological features as inflammatory infiltrate, increased connective tissue and VAS for global degree of abnormality (p<0.05). MHC II expression was similar in DM/PM and lower in JDM and dystrophy, and it was based on the frequency of positive staining rather than to the degree of the MCH II expression. CONCLUSIONS: MHC I expression in muscle fibres is a premature and late marker of JDM patient independent to corticotherapy, and MHC II expression was lower in JDM than in PM and DM.

Fulminant hepatic failure in northern Brazil: morphological, immunohistochemical and pathogenic aspects of Lábrea hepatitis and yellow fever.

A morphological evaluation of histopathological liver samples from 42 fulminant hepatic failure cases from the Amazon Basin was undertaken in order to differentiate yellow fever (YF) from Lábrea hepatitis (LH) and other entities. The pattern and distribution of liver cell death as well as ballooning degeneration and midzonal apoptotic bodies were the most distinctive features of YF, whereas morula cells were the major finding for LH. Nineteen well characterised cases were further submitted to immunohistochemical studies for expression of YF antigen, hepatitis B surface antigen (HBsAg) and delta virus antigen. In both diseases, but particularly in LH, portal vein branch phlebitis was evident and might have played a role in the pathogenesis of hepatic injury, leading to hepatic extinction and portal tract approximation. The regeneration pattern was remarkable: a high proliferative index was detected in YF, whereas in LH multinucleation and pseudoacinar transformation, associated with portal type I collagen deposition and portal elastic fibre proliferation, was seen. In conclusion, midzonal apoptosis, portal phlebitis and a high proliferative index in patients without evidence of previous liver injury was the dominant picture in YF. On the other hand, LH cases showed extensive, predominantly lytic hepatocytic necrosis, portal and hepatic vein phlebitis and morula cells in patients with a morphological background of chronic liver disease.

Association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult patients with diffuse astrocytomas.

Clarification of TP53 alterations is important to understand the mechanisms underlying the development of diffuse astrocytomas. It has been suggested that the alleles of TP53 at codon 72 differ in their ability to induce apoptosis in human cancers. The aim of this study was to analyze the possible association of TP53 mutation, p53 overexpression, and p53 codon 72 polymorphism with susceptibility to apoptosis in adult Brazilian patients with diffuse astrocytomas. We analyzed 56 surgical specimens of diffuse astrocytomas for alterations of TP53, using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) direct sequencing. p53 and cleaved caspase 3 protein expression were assessed by immunohistochemistry. We found TP53 mutations in 19.6% (11 out of 56) of tumors tested, with the lowest mutation rate found in the cases of glioblastomas (8.8%) (p = 0.03). Only 16.1% of tumors tested showed cleaved caspase 3-positive staining, demonstrating that apoptosis is very inhibited in these tumors. All tumors having TP53 mutation and p53 accumulation had no expression of cleaved caspase 3. Additionally, no association was observed in tumors having proline and arginine alleles and expression of cleaved caspase 3. We concluded that clarification of the TP53 alterations allows a better understanding of the mechanisms involved in the progression of diffuse astrocytomas, and the allele status at codon 72 was not associated with apoptosis in these tumors.

Comparison of sequences of cDNA clones obtained from oligo-capping cDNA libraries with those from unigene.

We compared in detail the characteristics of the sequences of the cDNA clones obtained by the oligo-capping method (oligo-capping clones) with that of the sequences in the UniGene database. To compare the completeness of the sequences, three new variables, "fullness-proportion of clones" (the ratio of complete clones to total clones in a library), "fullness-proportion of genes" (the ratio of complete genes to total genes in a library), and "fullness-proportion of database" (the ratio of complete genes to total genes in a database sampled from a library), were defined. The fullness-proportion of clones of oligo-capping clones was 57.3%, 2.2 times larger than that of UniGene (25.9%). The fullness-proportion of genes of oligo-capping clones was 41.8%, 2.4 times larger than that of UniGene (17.8%). When gene length was restricted to > or = 1.5 kb, the fullness-proportion of genes of oligo-capping clones was four times larger than that of UniGene. The fullness-proportion of database of oligo-capping clones was approximately the same as that of UniGene. By simulating the clone redundancy, this coincidence was found to be due to the large redundancy of the UniGene database. Consequently, the cDNA sequence database of oligo-capping clones enabled high throughput selection of full-length cDNA clones.

Basaloid squamous carcinoma of oral cavity: a histologic and immunohistochemical study.

Basaloid squamous carcinoma (BSC) is an aggressive variant of squamous cell carcinoma (SCC) with a predilection for the upper aerodigestive tract. In the English literature, approximately 40 cases of BSC have been described in the oral cavity. BSC has frequently been confused with adenoid cystic carcinoma (ACC), basal cell adenocarcinoma, and undifferentiated SCC. The purpose of the investigation was to examine the histological features and immunohistochemical expression of differentiation-related substances, including cytokeratin (CK) subtypes, vimentin, S-100, chromogranin, laminin, and type IV collagen, for the characterization of biological features of these tumours. We studied three cases of BSC of the oral cavity, three cases of ACC, and one case of basal cell adenocarcinoma. Well-differentiated and undifferentiated SCCs were also studied for comparison. The BSCs showed many histopathologic similarities to cases previously reported. Among the CK subtypes analyzed, CK14 was the only subtype expressed by all basaloid cells of BSC. Potentially useful for the differential diagnosis was the finding of CKs 7 and 19 expression in the basaloid cells of ACC, and CKs 7 and 8 in basal cell adenocarcinoma. In BSCs, laminin and type IV collagen were found in the microcystic spaces between basaloid cells, but neither ACCs nor basal cell adenocarcinoma showed this feature. These data suggest that immunohistochemical findings are helpful in distinguishing BSC of the oral cavity from other histopathologically similar tumours.

Demonstration of mycobacterial antigens in skin biopsies fron suspected leprosy cases in the absence of bacilli.

Skin-biopsies from fifty-six patients suspected of early leprosy from Bahia State, Brazil, were examined histopathologically. The Fite-Faraco staining failed to demonstrate acid-fast bacilli in this material. The prominent features of the lesions were inflammation of the neurovascular bundles and sometimes inflammation of the skin appendages. The non-specific infiltrate was predominantly composed of histiocytes and lymphocytes. In 41 cases (73.2%) epidermal atrophy was also present. The avidin-biotin peroxidase technique was used with primary antibodies to detect bacillary antigens (anti-BCG serum) and nerve branches (anti-S-100 protein serum). Immunohistochemical detection of bacillary antigens using the anti-BCG serum was positive in 28 cases (50%). A positive staining for S-100 protein was observed in 40 cases (71.4%) in dendritic antigen-presenting cells of the skin. The detection of bacillary antigens, together with the clear demonstration of nerve bundles enhanced our capacity to fulfill morphologic criteria for the diagnosis of early leprosy. Our observations indicate that the use of immunohistochemical methods represent a useful tool for the early diagnosis of leprosy.

Detection of human parvovirus B19 in a patient with hepatitis.

Parvovirus B19 has been associated by some investigators with cases of severe hepatitis. The aim of the present study was to determine the presence of active parvovirus B19 infection among 129 Brazilian patients with non-A-E hepatitis. The patients were assayed for antibodies against parvovirus B19, IgM class, by ELISA. In IgM-positive cases, parvovirus B19 DNA was assayed by PCR in serum and liver tissue and parvovirus VP1 antigen in liver tissue was assayed by immunohistochemistry. Antibodies against parvovirus B19, IgM class, were detected in 3 (2.3%) of 129 patients with non-A-E hepatitis. Previous surgery and blood transfusions were reported by these 3 patients. One patient was a 56-year-old female with severe hepatitis, with antimitochondrial antibody seropositivity and submassive necrosis at liver biopsy, who responded to corticosteroid therapy. Strong evidence for active parvovirus B19 infection was found in this patient, with parvovirus B19 DNA being detected by PCR in liver tissue. Furthermore, parvovirus VP1 antigen was also detected in liver tissue by immunohistochemistry. The other two IgM-positive patients were chronic hepatitis cases, but active infection was not proven, since neither viral DNA nor antigen were detected in their liver tissues. This and other reports suggest a possible relation between parvovirus B19 infection and some cases of hepatitis.

Leptospiral antigens in the liver of experimentally infected guinea pig and their relation to the morphogenesis of liver damage.

In order to investigate the morphogenes of experimental leptospirosis by morphologic and immunohistologic methods, 24 guinea-pigs were inoculated intraperitoneally with L. interrogans serogroup Icterohaemorrhagiae. They were divided in 6 groups, sacrificed from the 1st to the 6th day of infection. Semiquantitative analyses of histopathological liver lesions were performed in 1 micron sections of tissue embedded in glycol-methacrylate. The distribution of leptospiral antigen (L. Ag) and its glycolipoprotein (GLP) was demonstrated by peroxidase-antiperoxidase on paraffin embedded tissue. Significant lesions appeared at the 4th day of infection, progressing to a peak on the 6th day. Inflammation was associated with injury of the portal triad. Liver cells showed either swelling or acidophilic degeneration and necrosis, together with loss of cell cohesion, leading to disarray of liver cell plates. Mitochondria were found progressively enlarged and irregularly distributed. L. Ag expression was parallel to the morphological changes. Portal distribution was significant at the 4th day and on later stages centrilobular localization became predominant. Spiral forms suggestive of intact leptospires were initially found but, chiefly at the 6th day, L. Ag was seen in granules, probably resulting from phagocytosis. GLP staining was similar to granular L. Ag in morphology, and distribution. Cytokeratin condensation was seen in liver cells with acidophilic necrosis and was marked in areas of disorganization of cell plates. Our findings lead us to hypothesize a direct leptospiral cytotoxic effect on endothelial and on liver-cell membranes. At first, leptospires themselves would induce subcellular changes acting mainly on membrane permeability. Afterwards, their granular forms, including GLP, would act as adjuvant factors. These findings demonstrate that the disarray of liver cell plates at the late phase of the disease is genuine.

[Glycoproteins from Leptospira interrogans serogroup icterohaemorrhagiae: distribution in the liver and kidney of experimentally infected guinea pigs]. / Glicolipoproteína de Leptospira interrogans sorogrupo icterohaemorrhagiae: distribuição em fígado e rim de cobaias experimentalmente infectadas.

Tissue damage in leptospirosis has been ascribed to direct effect of the microorganisms and/or their virulence, including products synthetized by leptospires or released during their lysis. This study aimed at chemical extraction of the glycolipoprotein (GLP) from virulent leptospires, production of a rabbit anti-GLP and analysis of its distribution in liver and kidney of inoculated guinea-pigs, sacrificed sequentially from the 1st to 6th day of infection, covering the whole, spectrum of acute leptospirosis. The comparison of GLP expression to local injuries aimed at new pathogenetic data. GLP was detected in liver and kidney in 2 out of 6 guinea-pigs on the 5th day and in all 6 animals on the 6th day of infection. Granular forms were seen in the cytoplasm of macrophages, free in interstitium or adhered to endothelial and parenchymal cell membranes, especially in the most damaged sites. These findings lead us to the hypothesis of GLP as a toxic factor resulting from leptospiral lysis by macrophages. Although it was not proved as a promoter of initial lesions, it seems to be related to the enhancement of tissue damage late in the course of the disease.

Differential gene expression profiles of hepatocellular carcinomas associated or not with viral infection.

Chronic hepatitis B (HBV) and C (HCV) virus infections are the most important factors associated with hepatocellular carcinoma (HCC), but tumor prognosis remains poor due to the lack of diagnostic biomarkers. In order to identify novel diagnostic markers and therapeutic targets, the gene expression profile associated with viral and non-viral HCC was assessed in 9 tumor samples by oligo-microarrays. The differentially expressed genes were examined using a z-score and KEGG pathway for the search of ontological biological processes. We selected a non-redundant set of 15 genes with the lowest P value for clustering samples into three groups using the non-supervised algorithm k-means. Fisher's linear discriminant analysis was then applied in an exhaustive search of trios of genes that could be used to build classifiers for class distinction. Different transcriptional levels of genes were identified in HCC of different etiologies and from different HCC samples. When comparing HBV-HCC vs HCV-HCC, HBV-HCC/HCV-HCC vs non-viral (NV)-HCC, HBC-HCC vs NV-HCC, and HCV-HCC vs NV-HCC of the 58 non-redundant differentially expressed genes, only 6 genes (IKBKbeta, CREBBP, WNT10B, PRDX6, ITGAV, and IFNAR1) were found to be associated with hepatic carcinogenesis. By combining trios, classifiers could be generated, which correctly classified 100% of the samples. This expression profiling may provide a useful tool for research into the pathophysiology of HCC. A detailed understanding of how these distinct genes are involved in molecular pathways is of fundamental importance to the development of effective HCC chemoprevention and treatment.

Comparison of disposition parameters of quinidine and quinine in the rat.

The difference in disposition of quinidine (Qd) and its diastereomer quinine (Qn) after intravenous administration was examined in rats at doses ranging from 5 to 20 mg/kg. Dose-dependent kinetics in total clearance and in distribution volume of tissue based on a two-compartment model was observed for Qd; there was no evidence of nonlinearity for Qn. However, there was no significant difference between Qd and Qn for blood clearance at doses of 5 and 10 mg/kg, at which the blood clearances were almost equal to hepatic blood flow for both Qd and Qn since the excretion of Qd and Qn into the urine and bile was minimal. This indicates the elimination of these diastereomers to be non-restrictive in the liver. A concentration dependence in unbound volume of tissue distribution and in plasma protein binding was observed for Qd; there was no concentration dependence for Qn. Although affinity of the drug for components on or within the blood cells was not concentration-dependent for either Qd or Qn, a significantly higher binding capacity for Qn than for Qd was observed attributable to blood cell binding. Based on these results, it is suggested that a larger number of binding sites exist for Qn than for Qd in the body. However, the dissociation binding constant for Qd is much lower than for Qn, resulting in a higher binding of Qd and Qn at low concentrations, with a reversal at high concentrations.

Indeterminate leprosy: histopathologic and histochemical predictive parameters involved in its possible change to paucibacillary or multibacillary leprosy.

In an attempt to find clinical, bacteriological, histopathological, and immunohistochemical parameters to predict the progress of indeterminate leprosy patients to either paucibacillary (PB) or multibacillary (MB) leprosy, skin biopsies from 51 patients with indeterminate leprosy were retrieved from the files of the São Paulo Health Institute (Brazil). All of these patients had progressed to either PB or MB leprosy over a period of time which varied from 2 months to 24 years. Clinical records were examined, and new sections were cut from the paraffin blocks and stained by hematoxylin-eosin and Fite-Faraco stains; the avidin-biotin peroxidase technique was used with primary antibodies to detect bacillary antigens (anti-BCG serum) and nerve branches (anti-S-100 protein anti-serum). A moderate (++) or strongly positive ( ) Mitsuda skin test was observed in some patients progressing to PB leprosy. Noteworthy is that even patients initially Mitsuda negative may evolve to PB leprosy. a) A 2+ bacterial index and/or the presence of bacilli, even though few in number, in various dermal structures; b) multiple positive antigen sites as detected by anti-BCG anti-serum; and c) dermal nerve involvement, when evaluated as single parameters, correlated with a progression indeterminate to MB leprosy. An index resulting from the summation of the above three parameters identified 13 (72%) of 18 of these cases which progressed to MB leprosy.

Treated-cured indeterminate leprosy: a search for predictive histopathological and immunohistochemical parameters in skin biopsies taken from patients at admission and at clinical discharge.

In a previous study an index (sigma 3) resulting from the summation of three parameters, i.e., presence of bacilli, even in small numbers, in various dermal structures, multiple positive antigen sites as detected by anti-BCG antiserum and dermal nerve involvement, identified 72.22% of cases of indeterminate leprosy which progressed to multibacillary leprosy. The present study was undertaken to investigate possible parameters which might be indicative of indeterminate leprosy which would persist unchanged or be cured (treated cured patients). Thirty treated cured indeterminate leprosy patients were selected from the files of the São Paulo Health Institute and studied by histopathological, immunohistochemical and statistical methods similar to those employed in the previous study. The sigma 3 index was 4.10 +/- 0.60, a finding that places this group of patients in a position close to that of patients changing to paucibacillary leprosy but statistically different from that of patients progressing to multibacillary leprosy. Moreover, it was found that patients belonging to this group have heterogeneous single parameters, some of them suggestive of multibacillary and others of paucibacillary leprosy. Immunologically based techniques mainly employing rabbit anti-BCG serum as the primary antibody have proved to be valuable to detect antigen sites in biopsies from indeterminate leprosy patients and should be used together with the bacillary index during the follow up and clinical discharge control of such patients. In the present study, we show that clinical discharge of these patients did not mean a complete clearance of bacillary antigens.

[The importance of fixation in immunohistochemistry: distribution of vimentin and cytokeratins in samples fixed in alcohol and formol]. / A importância da fixação em imuno-histoquímica: a distribuição de vimentina e citoqueratinas em amostras fixadas em alcool e formol.

The fixation in ethanol or formalin for trypsin digestion in immunohistochemical detection of cytokeratins and vimentin was assessed in a case of ovarian cystadenofibrocarcinoma. Superior reactivity for both markers was achieved in ethanol-fixed sections, even in samples stocked up to 60 days. Cytokeratin reaction in formalin-fixed sections was better when trypsin was used. However, this digestion was deleterious to vimentin detection. These data are presented to alert surgeons and oncologists on the relevance of fixation of specimens suspicious for neoplasia, since different epitopes may require different fixatives and the inadequate choice in the operative room may impart difficulties when immunohistochemistry is necessary.

An unusual case of muscular limb-girdle dystrophy and mitochondrial myopathy.

Limb-girdle muscular weakness and wasting could be caused by different diseases (inflammatory and hereditary myopathies, muscular dystrophies and neurogenic atrophies). Among these, Limb-Girdle Muscular Dystrophy (LGMD) is an heterogeneous group of pathologies that have progressive proximal limbs and girdle weakness, with some dystrophic features by the muscle biopsy. We studied a case of LGMD in an adult man with a typical histological and histochemical profile, associated to a mitochondrial deficit characterized by presence of ragged-red fibers, a histochemical Cytochrome Oxidase deficiency and abnormal mitochondria by ultrastructure.