Effect of Akt activation and experimental pharmacological inhibition on responses to neoadjuvant chemoradiotherapy in rectal cancer.

BACKGROUND: Neoadjuvant chemoradiotherapy (CRT) is one of the preferred initial treatment strategies for locally advanced rectal cancer. Responses are variable, and most patients still require surgery. The aim of this study was to identify molecular mechanisms determining poor response to CRT. METHODS: Global gene expression and pathway enrichment were assessed in pretreatment biopsies from patients with non-metastatic cT2-4 N0-2 rectal cancer within 7 cm of the anal verge. Downstream Akt activation was assessed in an independent set of pretreatment biopsies and in colorectal cancer cell lines using immunohistochemistry and western blot respectively. The radiosensitizing effects of the Akt inhibitor MK2206 were assessed using clonogenic assays and xenografts in immunodeficient mice. RESULTS: A total of 350 differentially expressed genes were identified, of which 123 were upregulated and 199 downregulated in tumours from poor responders. Mitochondrial oxidative phosphorylation (P < 0·001) and phosphatidylinositol signalling pathways (P < 0·050) were identified as significantly enriched pathways among the set of differentially expressed genes. Deregulation of both pathways is known to result in Akt activation, and high immunoexpression of phosphorylated Akt S473 was observed among patients with a poor histological response (tumour regression grade 0-2) to CRT (75 per cent versus 48 per cent in those with a good or complete response; P = 0·016). Akt activation was also confirmed in the radioresistant cell line SW480, and a 50 per cent improvement in sensitivity to CRT was observed in vitro and in vivo when SW480 cells were exposed to the Akt inhibitor MK2206 in combination with radiation and 5-fluorouracil. CONCLUSION: Akt activation is a key event in the response to CRT. Pharmacological inhibition of Akt activation may enhance the effects of CRT. Surgical relevance Organ preservation is an attractive alternative in rectal cancer management following neoadjuvant chemoradiotherapy (CRT) to avoid the morbidity of radical surgery. Molecular steps associated with tumour response to CRT may provide a useful tool for the identification of patients who are candidates for no immediate surgery. In this study, tumours resistant to CRT were more likely to have activation of specific genetic pathways that result in phosphorylated Akt (pAkt) activation. Pretreatment biopsy tissues with high immunoexpression of pAkt were more likely to exhibit a poor histological response to CRT. In addition, the introduction of a pAkt inhibitor to cancer cell lines in vitro and in vivo led to a significant improvement in sensitivity to CRT. Identification of pAkt-activated tumours may thus allow the identification of poor responders to CRT. In addition, the concomitant use of pAkt inhibitors to increase sensitivity to CRT in patients with rectal cancer may constitute an interesting strategy for increasing the chance of a complete response to treatment and organ preservation.

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.

Annexin A1 subcellular expression in laryngeal squamous cell carcinoma.

AIMS: Annexin A1 (ANXA1) is a soluble cytoplasmic protein, moving to membranes when calcium levels are elevated. ANXA1 has also been shown to move to the nucleus or outside the cells, depending on tyrosine-kinase signalling, thus interfering in cytoskeletal organization and cell differentiation, mostly in inflammatory and neoplastic processes. The aim was to investigate subcellular patterns of immunohistochemical expression of ANXA1 in neoplastic and non-neoplastic samples from patients with laryngeal squamous cell carcinomas (LSCC), to elucidate the role of ANXA1 in laryngeal carcinogenesis. METHODS AND RESULTS: Serial analysis of gene expression experiments detected reduced expression of ANXA1 gene in LSCC compared with the corresponding non-neoplastic margins. Quantitative polymerase chain reaction confirmed ANXA1 low expression in 15 LSCC and eight matched normal samples. Thus, we investigated subcellular patterns of immunohistochemical expression of ANXA1 in 241 paraffin-embedded samples from 95 patients with LSCC. The results showed ANXA1 down-regulation in dysplastic, tumourous and metastatic lesions and provided evidence for the progressive migration of ANXA1 from the nucleus towards the membrane during laryngeal tumorigenesis. CONCLUSIONS: ANXA1 dysregulation was observed early in laryngeal carcinogenesis, in intra-epithelial neoplasms; it was not found related to prognostic parameters, such as nodal metastases.

Study of major genetic factors involved in pituitary tumorigenesis and their impact on clinical and biological characteristics of sporadic somatotropinomas and non-functioning pituitary adenomas.

Genetic and functional aberrations of guanine nucleotide-binding protein, alpha stimulating (GNAS), aryl hydrocarbon receptor interacting protein (AIP), and pituitary tumor transforming gene (PTTG) are among the most prominent events in pituitary tumorigenesis. A cohort of Brazilian patients with somatotropinomas (n=41) and non-functioning pituitary adenomas (NFPA, n=21) from a single tertiary-referral center were evaluated for GNAS and AIP mutations and gene expression of AIP and PTTG. Results were compared to the clinical and biological (Ki67 and p53 expression) characteristics of tumors and their response to therapy, if applicable. Genetic analysis revealed that 27% of somatotropinomas and 4.8% of NFPA harbored GNAS mutations (P=0.05). However, no differences were observed in clinical characteristics, tumor extension, response to somatostatin analog therapy, hormonal/surgical remission rates, Ki67 index, and p53 expression between mutated and non-mutated somatotropinomas patients. PTTG overexpression (RQ mean=10.6, min=4.39, max=11.9) and AIP underexpression (RQ mean=0.56, min=0.46-max=0.92) were found in virtually all cases without a statistically significant relationship with clinical and biological tumor features. No patients exhibited somatic or germline pathogenic AIP mutations. In conclusion, mutations in GNAS and abnormal PTTG and AIP expression had no impact on tumor features and treatment outcomes in this cohort. Our data support some previous studies and point to the need for further investigations, probably involving epigenetic and transcriptome analysis, to improve our understanding of pituitary tumor behavior.

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.

Immunohistochemical and in situ hybridization studies of the liver and kidney in human leptospirosis.

An in situ hybridization (ISH) assay for the detection of leptospiral DNA in tissues was described and its diagnostic and pathogenetic usefulness in combination with immunohistochemistry (IHC) was evaluated in formalin-fixed, paraffin-embedded liver and kidney samples from human fatal cases of leptospirosis. IHC assays with anti-E-cadherin antibodies assessed the liver-plate disarray frequently observed in leptospirosis. Immunohistochemistry detected leptospiral antigen (LAg) in macrophages, both in human liver and kidney. In guinea pigs, in addition to these findings, staining on cell membranes of hepatocytes and, occasionally, in apical membrane of kidney tubular cells was demonstrated. Positive ISH signal was observed chiefly in the nuclei of human hepatocytes and in the cytoplasm and nuclei of liver cells of experimentally infected guinea pigs. Loss of E-cadherin membrane expression is associated with liver-plate disarray. These findings were discussed in the contention that, in leptospirosis, cell membrane damage might be important for the pathogenesis of the disease. Finally, it was suggested that both IHC and/or ISH might be used for both diagnostic and research purposes.

Liver mitochondrial dysfunction and oxidative stress in the pathogenesis of experimental nonalcoholic fatty liver disease.

Oxidative stress and hepatic mitochondria play a role in the pathogenesis of nonalcoholic fatty liver disease. The aim of the present study was to evaluate the role of hepatic mitochondrial dysfunction and oxidative stress in the pathogenesis of the disease. Fatty liver was induced in Wistar rats with a choline-deficient diet (CD; N = 7) or a high-fat diet enriched with PUFAs-omega-3 (H; N = 7) for 4 weeks. The control group (N = 7) was fed a standard diet. Liver mitochondrial oxidation and phosphorylation were measured polarographically and oxidative stress was estimated on the basis of malondialdehyde and glutathione concentrations. Moderate macrovacuolar liver steatosis was observed in the CD group and mild liver steatosis was observed in the periportal area in the H group. There was an increase in the oxygen consumption rate by liver mitochondria in respiratory state 4 (S4) and a decrease in respiratory control rate (RCR) in the CD group (S4: 32.70 +/- 3.35; RCR: 2.55 +/- 0.15 ng atoms of O2 min-1 mg protein-1) when compared to the H and control groups (S4: 23.09 +/- 1.53, 17.04 +/- 2.03, RCR: 3.15 +/- 0.15, 3.68 +/- 0.15 ng atoms of O2 min-1 mg protein-1, respectively), P < 0.05. Hepatic lipoperoxide concentrations were significantly increased and the concentration of reduced glutathione was significantly reduced in the CD group. A choline-deficient diet causes moderate steatosis with disruption of liver mitochondrial function and increased oxidative stress. These data suggest that lipid peroxidation products can impair the flow of electrons along the respiratory chain, causing overreduction of respiratory chain components and enhanced mitochondrial reactive oxygen species. These findings are important in the pathogenesis of nonalcoholic fatty liver disease.

A polymorphism in endostatin, an angiogenesis inhibitor, predisposes for the development of prostatic adenocarcinoma.

We have performed association studies between a novel coding single nucleotide polymorphism (D104N) in endostatin, one of the most potent inhibitors of angiogenesis, and prostate cancer. We observed that heterozygous N104 individuals have a 2.5 times increased chance of developing prostate cancer as compared with homozygous D104 subjects (odds ratio, 2.4; 95% confidence interval, 1.4-4.16). Modeling of the endostatin mutant showed that the N104 protein is stable. These results together with the observation that residue 104 is evolutionary conserved lead us to propose that: (a) the DNA segment containing this residue might contain a novel interaction site to a yet unknown receptor; and (b) the presence of N104 impairs the function of endostatin.

A prospective study of hepatitis B virus markers in patients with chronic HBV infection from Brazilian families of Western and Asian origin.

The purpose of the present study was to determine the frequency of hepatitis B virus (HBV) markers in families of HBsAg-positive patients with chronic liver disease. Serum anti-HBc, HBsAg and anti-HBs were determined by enzyme immunoassay and four subpopulations were considered: genetically related (consanguineous) and non-genetically related (non-consanguineous) Asian subjects and genetically related and non-genetically related Western subjects. A total of 165 and 186 relatives of Asian and Western origin were enrolled, respectively. The occurrence of HBsAg and anti-HBs antibodies was significantly higher (P < 0.0001) in family members of Asian origin (81.8%) than in family members of Western origin (36.5%). HBsAg was also more frequent among brothers (79.6 vs 8.5%; P < 0.0001), children (37.9 vs 3.3%; P < 0.0001) and other family members (33.9 vs 16.7%; P < 0.0007) of Asian than Western origin, respectively. No difference between groups was found for anti-HBs, which was more frequently observed in fathers, spouses and other non-genetic relatives. HBV infection was significantly higher in children of Asian than Western mothers (P < 0.0004). In both ethnic groups, the mothers contributed more to their children's infection than the fathers (P < 0.0001). Furthermore, HBsAg was more frequent among consanguineous members and anti-HBs among non-consanguineous members. These results suggest the occurrence of vertical transmission of HBV among consanguineous members and probably horizontal sexual transmission among non-consanguineous members of a family cluster. Thus, the high occurrence of dissemination of HBV infection characterizes family members as a high-risk group that calls for immunoprophylaxis. Finally, the study showed a high familial aggregation rate for both ethnic groups, 18/19 (94.7%) and 23/26 (88.5%) of the Asian and Western origin, respectively.

Serodiagnosis of hepatitis C virus. Effect of new evaluation of cutoff values for enzyme-linked immunosorbent assay in Brazilian patients.

With the goal of reducing false-positive results in enzyme-linked immunosorbent assay (ELISA) serodiagnosis of hepatitis C virus in clinical practice, a study was undertaken to establish better cutoff values. We examined 277 serum samples from patients with hepatitis (non-A, non-B; B; autoimmune); subjects with antinuclear antibodies or rheumatoid factor, anticytomegalovirus or Epstein-Barr virus IgG or IgM antibodies, or parasitic disease (Chagas disease, leishmaniasis); and healthy volunteers. Concordant positive results in 2 different immunoblot assays in 250 samples were taken as indicative of true-positive, and when negative, of absence of infection. Reactivity in 3 ELISA tests were evaluated for the manufacturer recommended cutoff (CO) and for 2CO, 3CO, and 4CO; and corresponding sensitivity and specificity were calculated for single or combined pairs of ELISA tests. Although CO is adequate for blood bank screening, because it provides maximal sensitivity, the frequently observed false-positive results could be significantly reduced by increasing the cutoff value to 2CO, with no significant loss in sensitivity either in relation to pairs of immunoenzymatic tests or to a single ELISA.

Lung lesions in human leptospirosis: microscopic, immunohistochemical, and ultrastructural features related to thrombocytopenia.

Lung fragments from 12 patients were collected immediately after death and studied by light and electron microscopy and by immunohistochemistry to describe the main morphologic and ultrastructural aspects of the lung and platelets in leptospirosis (Weil's syndrome), to search for the possibility of disseminated intravascular coagulation (DIC), and to assess the relationship between endothelial lesions and local platelet aggregation and the leptospiral antigen distribution, as well as its relationship with the intensity of the lesions. The immunohistochemical results for fibrin aggregates were positive in the lumen and/or on the vascular endothelium in nine cases and on the alveolar surface in seven cases, leading to the diagnosis of the adult respiratory distress syndrome in these seven cases. Test results for leptospiral antigen by immunohistochemistry were positive in eight cases with no direct relationship between antigen deposits in the pulmonary vascular endothelium and intensity of the lesions. The ultrastructural findings were uniform and constant. Capillary lesions were characterized by swelling of endothelial cells, an increase in pinocytotic vesicles, and giant dense bodies in the cytoplasm of these cells. No necrosis, rupture, nor exposed subendothelial collagen was observed outside the hemorrhagic areas, and the intercellular junctions were preserved. The lung involvement in severe human leptospirosis presents as hemorrhagic pneumopathy with septal capillary lesions that are the usual cause of death. The thrombocytopenia that was verified in 11 of 12 patients in our study seems to bear no relationship to DIC and seems to be determined by activation, adhesion, and aggregation of platelets to the stimulated vascular endothelium, with an amorphous electron-dense substance between the endothelial cells and the adherent platelets in places where the subendothelial collagen was not exposed.

Unexpected low prevalence of delta antibodies in the east Amazon region and São Paulo: evidence for regional differences in the epidemiology of delta hepatitis virus within Brazil.

Antibodies (anti-HD) to hepatitis delta virus (HDV) were tested by radioimmunoassay in 207 human serum samples from the eastern Amazon (states of Pará and Amapá) and São Paulo, Brazil. 42 Amazon HBsAg asymptomatic carriers were negative for anti-HD. 84 São Paulo HBsAg asymptomatic carriers were also negative. Among the 81 HBsAg patients from São Paulo with different liver diseases, only one had anti-HD. Liver biopsy of this chronic active hepatitis case was positive for HBsAg, HBcAg and HDAg in liver, by an immunoperoxidase technique. The low prevalence of HDV infections in São Paulo and eastern Amazon was unexpected and contrasts with the recent reports of high prevalence in the western Amazon region. Such regional differences emphasize the need for extensive and precise worldwide epidemiological studies of HDV.

Spontaneous hepatitis B surface antigen clearance in a long-term follow-up study of patients with chronic type B hepatitis. Lack of correlation with hepatitis C and D virus superinfection.

We investigated the frequency of HBsAg clearance and the possible role of viral superinfection in a long-term follow-up of 184 patients with chronic hepatitis B (CHB). Our subjects were 184 patients with chronic hepatitis B and the follow-up was 12-216 months (mean 66.2 +/- 53.7 months). The investigative methods used were: immunoenzymatic assays for HBV, HCV, HDV, and HIV markers; polymerase chain reaction (PCR) for HBV DNA; and liver biopsy and immunoperoxidase. During the follow-up, 20 of the 184 patients cleared serum HBsAg. A comparison of patients with persistent HBsAg(group I) and of those who cleared this marker (group II) showed a significant difference in mortality (P = 0.002) between the two groups and a tendency to a more severe exacerbation (flare) in group II (P = 0.07). Antibodies to hepatitis C and D virus as well as antibodies to HIV were equally distributed in both groups. Thirteen patients (7.9%) from group I, but none from group II, subsequently developed hepatocellular carcinoma. These results suggest that the frequency of spontaneous clearance of HBsAg during chronic HBV infection is low. No determinant factor for the clearance was found, including the presence of liver cirrhosis. Serum HBV DNA was undetectable by PCR after clearance in 16 out of 17 patients.

Hantavirus pulmonary syndrome in the State of São Paulo, Brazil, 1993-1998.

Between 1993 and 1998, 10 cases of clinical hantavirus infection were diagnosed in Brazil. Hantavirus-specific IgM, or positive immunohistochemical analysis for hantavirus antigen, or positive reverse transcription-polymerase chain reaction results for hantavirus RNA were used to confirm nine of these cases; eight were hantavirus pulmonary syndrome (HPS), and one was mild hantavirus disease. The remaining clinical case of hantavirus infection was fatal, and no tissue was available to confirm the diagnosis. During the first 7 months of 1998, five fatal HPS cases caused by a Sin Nombre-like virus were reported from three different regions in the State of São Paulo, Brazil: two in March (Presidente Prudente Region), two in May (Ribeirão Preto Region), and one in July (Itapecerica da Serra Region). Epidemiologic, ecologic, and serologic surveys were conducted among case contacts, area residents, and captured rodents in five locations within the State of São Paulo in June of 1998. Six (4.8%) of 125 case contacts and six (5.2%) of 116 area residents had IgG antibody to Sin Nombre virus (SNV) antigen. No case contacts had a history of HPS-compatible illness, and only one area resident reported a previous acute respiratory illness. A total of 403 rodents were captured during 9 nights of trapping (1969 trap nights). All 27 rodents that were found to be positive for IgG antibody to SNV antigen were captured in crop border and extensively deforested agricultural areas where four of the 1998 HPS case-patients had recently worked. The IgG antibody prevalence data for rodents suggest that Bolomys lasiurus and perhaps Akodon sp. are potential hantavirus reservoirs in this state of Brazil.

Aggressive metastatic follicular thyroid carcinoma with anaplastic transformation arising from a long-standing goiter in a patient with Pendred's syndrome.

In this article we describe detailed pathological and molecular genetics studies in a consanguineous kindred with Pendred's syndrome. The index patient was a 53-year-old female patient with congenital deafness and goiter. Her parents were first-degree cousins. She had a large goiter (150 g) that had been present since childhood. One of her sisters and a niece are also deaf and have goiter as well. The presence of Pendred's syndrome was confirmed by a positive perchlorate test and the demonstration of a Mondini malformation. Thyroid function tests (under levothyroxine [LT4] therapy) were in the euthyroid range with a thyrotropin [TSH] level of 2.8 microU/mL (0.2-3.2), a serum total thyroxine (T4) of 90 nmol/L (54-142), and a serum total triiodothyronine (T3) of 2.7 nmol/L (0.8-2.4). Total thyroidectomy was performed, and the mass in the right lobe was found to have invaded adjacent tissues. The histopathological findings were consistent with a follicular carcinoma with areas of anaplastic transformation and lung metastasis. The patient was treated twice with 100 mCi 131iodine (3,700 MBq) and received suppressive doses of LT4. Postoperatively, the serum thyroglobulin (Tg) levels remained markedly elevated (2,352 to 41,336 ng/mL). The patient died of a sudden severe episode of hemoptysis. Sequence analysis of the PDS gene performed with DNA from the two relatives with Pendred's syndrome revealed the presence of a deletion of thymidine 279 in exon 3, a point mutation that results in a frameshift and a premature stop codon at codon 96 in the pendrin molecule. We concluded that prolonged TSH stimulation because of iodine deficiency or dyshormonogenesis in combination with mutations of oncogenes and/or tumor suppressor genes, may result in the development of follicular thyroid carcinomas that undergo transformation into anaplastic cancers. It is likely that these pathogenetic mechanisms have been involved in the development of aggressive metastatic thyroid cancer in this unusual patient with Pendred's syndrome.

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.

Differential reactivity for galectin-3 in Hürthle cell adenomas and carcinomas.

Hürthle cell carcinomas behave as the most aggressive variant of differentiated thyroid carcinoma of follicular origin, with frequent recurrences and higher morbidity. Its differential diagnosis with Hürthle cell adenoma remains a problem for the clinician and for the pathologist. The vertebrate lectins, galectin-1 and galectin-3 have been implicated in the regulation of cellular growth, differentiation, and malignant transformation in thyroid neoplasms. Galectin-3, a beta-galactoside binding protein, has been recently found to be highly expressed in papillary and follicular carcinomas. The current study was undertaken to investigate immunohistochemical reactivity for galectin-3 of thyroid specimen tissues with Hürthle cell adenomas (n = 14) and carcinomas (n = 17), follicular (n = 14) and papillary (n = 11) carcinomas, colloid goiter (n = 30), Hashimoto's thyroiditis (n = 11), follicular adenoma (n = 9), and normal thyroid tissues (n = 18). Follicular (78.5%) and papillary (82.0%) carcinomas were frequently reactive for galectin-3, more often when some H rthle cells were present. There was no galectin-3 immunostaining in any of the specimens from Hashimoto's thyroiditis, colloid goiters or normal thyroid samples, whereas only one case of follicular adenoma was found positive (11.1%). By contrast, galectin-3 immunostaining in Hürthle cell carcinomas was significantly higher (59%) than in H rthle cell adenomas (7.1), p < 0.05). These results suggest that galectin-3 may potentially serve as a marker in difficult differential diagnosis cases involving Hürthle cell adenomas and Hürthle cell carcinomas.

Nesidioblastosis associated with congenital malformations of the heart. Morphological and immunohistochemical study of 5 necropsy cases.

Pancreatic tissue from 5 children with congenital heart disease (CHD) with morphological alterations of the endocrine components at autopsy were compared in a double-blind semiquantitative study, which included light microscopy and immunocytochemistry, to pancreatic tissue of 5 children of similar age but without CHD. Hyperplasia and hypertrophy of islets of Langerhans, scattered endocrine cells within the exocrinic acini, cluster of endocrine cells budding from ductular epithelium or within interlobular connective tissue, and hyperplasia of ductular epithelium were found to be present in the patients with congenital cardiac defects. The observed findings resembled those present in a surgically resected pancreas from a child with clinically overt nesidioblastosis. Although this study suggests a higher incidence of endocrine pancreas disturbances in certain types of CHD, the clinical significance of these morphological alterations will require further studies.

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.

Human fatal yellow fever. Immunohistochemical detection of viral antigens in the liver, kidney and heart.

An immunohistochemical method to detect yellow fever antigen was developed using immune sera from rabbits and hamsters and hyperimmune ascitic fluid from mice. A search for the antigen was carried out in liver, kidney and heart in three fatal cases of yellow fever. In the liver it was present in the cytoplasm of hepatocytes, Councilman bodies and Kupffer cells. Yellow fever antigen was also detected in renal tubular epithelium and in groups of myocardial fibers. These findings suggest that viral replication occurs at sites other than the liver. Since yellow fever shares many features with other haemorrhagic fevers the use of immunohistochemistry can impart a significant improvement in the accuracy of its histopathological diagnosis.