[Study on the clinicopathological correlation in the secondary acute respiratory distress syndrome]. / Estudio sobre la correlación clínico-patológica en el síndrome de distrés respiratorio agudo secundario.

OBJECTIVE: This study has aimed to study the clinicopathological correlation of patients with secondary acute respiratory distress syndrome (ARDS), specifically having extrapulmonary causes. SETTING: A 22 beds intensive care unit. DESIGN: An observational study of case series. PATIENTS: Seventeen patients whose death was caused by acute respiratory distress syndrome were included. INTERVENTION: A systematic histopathological study was made of all the pulmonary lobes of patients who died in our ICU with the clinical diagnosis of secondary ARDS, who had undergone an autopsy between 1999 and 2009. The Kappa analysis was used to analyze the grade of correlation between the clinical and the pathological diagnosis. RESULTS: The autopsy confirmed to cases of false positive in 17 patients with ARDS (11%). The kappa value was 0.77, so that the concordance analysis was considered to be satisfactory. CONCLUSIONS: The clinical criteria for ARDS correlate well with acute alveolar damage (AAD) in the autopsy study in patients with secondary ARDS, although some false positive cases can be observed.

Aberrant gene methylation and bronchial dysplasia in high risk lung cancer patients.

INTRODUCTION: The risk for lung cancer is incremented in high degree dysplasia (HGD) and in subjects with hypermethylation of multiple genes. We sought to establish the association between them, as well as to analyze the DNA aberrant methylation in sputum and in bronchial washings (BW). METHODS: Cross sectional study of high risk patients for lung cancer in whom induced sputum and autofluorescence bronchoscopy were performed. The molecular analysis was determined on DAPK1, RASSF1A and p16 genes using Methylation-specific PCR. RESULTS: A total of 128 patients were enrolled in the study. Dysplasia lesions were found in 79 patients (61.7%) and high grade dysplasia in 20 (15.6%). Ninety eight patients out of 128 underwent molecular analysis. Methylation was observed in bronchial secretions (sputum or BW) in 60 patients (61.2%), 51 of them (52%) for DAPK1, in 20 (20.4%) for p16 and in three (3.1%) for RASSF1A. Methylated genes only found in sputum accounted for 38.3% and only in BW in 41.7%, and in both 20.0%. In the 11.2% of the patients studied, HGD and a hypermethylated gene were present, while for the 55.1% of the sample only one of both was detected and for the rest of the subjects (33.6%), none of the risk factors were observed. CONCLUSIONS: Our data determines DNA aberrant methylation panel in bronchial secretions is present in a 61.2% and HGD is found in 15.6%. Although both parameters have previously been identified as risk factors for lung cancer, the current study does not find a significative association between them. The study also highlights the importance of BW as a complementary sample to induced sputum when analyzing gene aberrant methylation.

Prognostic impact of mutated K-ras gene in surgically resected non-small cell lung cancer patients.

Mutated K-ras oncogenes have been detected in a third of lung adenocarcinomas, located usually in codon 12, its presence correlating negatively with survival. To further define the role of K-ras point mutations in non-small cell lung cancer, we studied the presence of mutated K-ras genes in surgical specimens from 66 patients. Polymerase chain reaction was performed from sections of formalin-fixed paraffin-embedded tissue. We screened for point mutations in codons 12, 13 and 61 of the K-ras gene by dot blot hybridization analysis with mutation-specific oligonucleotide probes. Ras gene mutations were present in 13 of 66 carcinomas (20%), nine in codon 12 and four in codon 61. Three squamous cell carcinomas harbored two different point mutations in K-ras codon 12. Mutated K-ras genes were found more frequently in squamous cell carcinomas (eight of 38) than in adenocarcinoma (three of 22). Analysis of nucleotide sequence disclosed a multifarious mutation pattern of K-ras codon 12, where the most common conversion was from glycine (GGT) to valine (GTT). K-ras point mutation positive subset had poorer survival, nine of the 13 patients died during the follow-up period as compared with 22 of 53 patients with no mutation in the K-ras gene (P = 0.01). The difference was also strikingly significant when stratified according to node status.

Overexpression of Ki-67 and cyclins A and B1 in JC virus-infected cells of progressive multifocal leukoencephalopathy.

Both SV40 and JC virus (JCV) appropriate the host cell replicative machinery to attend to their own reproductive needs. SV40 large T antigen is able to induce the expression of cyclins A, B1, and E (but not of cylin D1) in transfected diploid cells. Whether JCV infection influences cyclin expression in a similar fashion in the setting of progressive multifocal leukoencephalopathy (PML) remains unknown. Brain lesions from 7 PML cases (4 autopsies and 3 biopsies) were immunohistochemically investigated for the expression of Ki-67 and cyclins A, B1, and D1. All 7 cases showed strong positivity for Ki-67 and cyclins A and B1 in JCV-infected oligodendrocytes and astrocytes, the nuclear immunolocalization of cyclin A being in strong contrast to the cytoplasmic distribution of cyclin B1. No immunostaining for cyclin D1 was obtained in any of the 7 cases. These findings suggest that JCV infection is associated with overexpression of Ki-67 and cyclins A and B1 in PML host glial cells. Since cyclin changes in JCV-infected cells recapitulate SV40 T antigen-associated cyclin fluctuations, it appears reasonable to think that JCV T antigen shares some of the previously described capabilities of SV40 T antigen to alter cyclin expression for the sake of viral replication.

Accumulation of wild-type p53 protein in progressive multifocal leukoencephalopathy: a flow of cytometry and DNA sequencing study.

p52 protein accumulation in JC virus (JCV)-infected cells of progressive multifocal leukoencephalopathy (PML) has been previously shown. Since many viral proteins are known to bind and stabilize p53, we are addressing the question of whether p53 protein accumulation in PML is the result of its sequestration by JCV and not the outcome of a p53 gene mutation which would prolong its half-life. We have investigated the status of the p53 gene in frozen autopsy brain samples from five PML patients. After isolating genomic DNA, p53 gene exons 2 through 9 were amplified and sequenced. No discrepancies were found in the DNA sequences of exons 2 through 9 and their intron/exon barriers when compared to those published for wild-type p53. On the other hand, dual (p53/DNA) flow cytometry analysis revealed p53 expression above that of the isotypic controls for each case. No aneuploid populations could be identified, however, which seems at odds with the aneuploid status normally associated with mutation-induced p53 dysfunction. These results indicate that the p53 gene harbors no mutations in PML and provide further evidence of p53 protein accumulation in this condition. Since p53 protein buildup in JCV-infected cells is not the consequence of a mutagenic interaction between JCV and the cell genome, we propose instead that p53 accumulation results from its binding and stabilization by JCV T protein.

DNA amplification in glial cells of progressive multifocal leukoencephalopathy: an image analysis study.

JC virus (JCV), the agent of progressive multifocal leukoencephalopathy (PML), has been shown by both immunohistochemistry and flow cytometry to be associated with p53 protein stabilization. Since stabilization/inactivation of p53 is associated with the development of genomic instability, abnormal cell DNA contents are to be expected in JCV-infected cells of PML. This work explores that possibility by image analysis evaluation of DNA content in PML-infected oligodendrocytes and bizarre astrocytes. Brain paraffin sections of PML lesions from five adult male patients with the acquired immune deficiency syndrome (AIDS) were treated with the Feulgen technique to obtain a stochiometric staining of DNA and analyzed with a microscope image processor. Inclusion-bearing oligodendrocytes exhibited near tetraploid DNA indices in each of the five cases, whereas atypical astrocytes were in the hypertetraploid range in all cases and were polyploid in four instances. This evidence of DNA amplification in PML glial cells is congruent with the functional abolition of p53 protein in association with JCV infection and lends further support to the role of p53 as a keeper of diploid status and guardian of genomic stability.

Giant cell myocarditis: monocytic immunophenotype of giant cells in a case associated with ulcerative colitis.

Giant cell myocarditis (GCM) is a rare condition whose histologic hallmark, the multinucleate giant cell, is of debated origin (monocytic v myogenic). We report the case of a 46-year-old woman with a previous diagnosis of ulcerative colitis who rapidly deteriorated and died as the result of refractory ventricular tachyarrhythmias. Postmortem examination showed a diffuse infiltration of the myocardium by round cells and multinucleate giant cells. Immunohistochemically, round cells were demonstrated to be T lymphocytes admixed with monocytes. Multinucleate giant cells expressed monocytic markers (MAC 387, lysozyme) and were negative for muscle markers (actin, desmin, myoglobin). This case illustrates the monocytic and macrophagic nature of multinucleate giant cells and lends support to the autoimmune hypothesis of GCM by the concurrence of the latter with ulcerative colitis.

Proliferating cell nuclear antigen expression in normal, regenerative, and neoplastic liver: a fine-needle aspiration cytology and biopsy study.

Information about a tissue's proliferative activity can be obtained from the immunocytochemical investigation of proliferating cell nuclear antigen (PCNA), an auxiliary protein of DNA polymerase delta expressed by cycling cells. To determine whether a relationship exists between morphology and PCNA expression in normal, regenerative, and malignant neoplastic hepatocytes, this study was undertaken on 48 fine-needle aspiration cytology (FNAC) cell blocks from eight normal livers, eight cirrhotic livers, and 32 hepatocellular carcinomas (HCCs), as well as on 41 needle or wedge biopsy specimens from 10 normal livers, 13 cirrhotic livers, one focal nodular hyperplastic liver, and 17 HCCs. Anti-PCNA monoclonal antibody PC10 was applied to formalin-fixed, paraffin-embedded tissue using the avidin-biotin method. Proliferating cell nuclear antigen immunoreactivity was evaluated as follows: absent; minimal, less than 5% positive nuclei; grade 1, 5% to 25% positive nuclei; grade 2, 26% to 50% positive nuclei; grade 3, 51% to 75% positive nuclei; and grade 4, 76% to 100% positive nuclei. In both the FNAC and biopsy series normal and regenerative livers were either completely negative or minimally immunoreactive (under 5% positive nuclei). In contrast, all well-differentiated HCC cases exhibited over 15% positive nuclei. Most well-differentiated HCCs were grade 1 (85.7% in the FNAC series and 76.92% in the biopsy series) and the majority of moderately differentiated HCCs were grade 3 (63.63% in the FNAC series, but only 50% in the biopsy series). Therefore, absent or minimal PCNA immunoreactivity seems to be a useful adjuvant to discriminate normal/regenerative liver from HCC, whose degree of differentiation tends to correlate with the level of PCNA expression. These observations apply to both the FNAC and biopsy series, which yielded very similar data.

p53 and proliferating cell nuclear antigen expression in JC virus-infected cells of progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML), a demyelinating disease of the central nervous system (CNS) caused by infection with JC papova virus (JCV), is characterized by marked atypical changes in the glial cells. The JCV T protein binds cellular p53 (a tumor suppressor gene product), which as a result loses its normal down regulating influence on the cell cycle. We hypothesized that this binding would stabilize p53 and prolong its half life, leading to its immunohistochemical detection. To prove our theory combined JCV DNA:DNA in situ hybridization (ISH) and glial fibrillary acidic protein (GFAP) immunohistochemistry (IHC) as well as p53/GFAP double IHC were performed on routinely processed sections of five brains obtained at autopsy and two cerebral biopsy specimens from seven patients with PML. All specimens showed JCV infected oligodendrocytes and bizarre looking astrocytes that immunostained strongly for p53. In addition, because loss of p53 function results in proliferating cell nuclear antigen (PCNA) overexpression PCNA/GFAP double IHC was carried out, and a positive immunoreaction was obtained in JCV infected cells in the two biopsy specimens. The evidence of p53 immunoreactivity in JCV harboring glial cells seems to indicate a link between the JCV induced stabilization/inactivation of p53 and the striking tumorlike glial changes seen in PML. Proliferating cell nuclear antigen overexpression in these cells further supports this pathogenetic construct.

Role of CD44 in the invasiveness of glioblastoma multiforme and the noninvasiveness of meningioma: an immunohistochemistry study.

CD44 is a polymorphic family of cell adhesion molecules that seems to be instrumental in the mechanism of tumor invasion and metastasis. Tumor cell expression of CD44, or lack thereof, may be one of the factors conditioning the highly disparate ability to penetrate the brain extracellular matrix (ECM) exhibited by glioblastoma multiforme (GM) and conventional meningioma. To assess the presence of CD44 in these two tumor types we have immunohistochemically investigated the expression of CD44 standard form (CD44s) and the variant isoforms containing the domain encoded by variant exon 3 (CD44v3) and variant exon 6 (CD44v6) in paraffin-embedded tissue from 10 conventional meningiomas and 10 GMs. A CD44s-/CD44v-phenotype was discerned in the meningioma cases, whereas GMs featured a CD44s+/CD44v- expression profile. Consequently, the growth patterns of meningioma and GM seem to be, at least in part, a reflection of their CD44 expression status. Paucity of CD44 in meningioma cells would render them unable to infiltrate the brain ECM, whereas CD44-rich glioma cells would successfully migrate through it. Conversely, lack of CD44v expression would contribute to explain the lack of metastatic potential characterizing both conventional meningioma and GM.

Desmin myopathy: a multisystem disorder involving skeletal, cardiac, and smooth muscle.

Myopathy associated with desmin-type intermediate filaments is an uncommon disorder of skeletal and/or cardiac muscle. The present study focuses on a 28-year-old man with generalized muscular atrophy, cardiomyopathy, and intestinal malabsorption and pseudo-obstruction. Abundant sarcoplasmic granular and filamentous aggregates that were ultrastructurally continuous with Z lines or dense bodies and exhibited intense immunostaining for desmin were present throughout the skeletal musculature, myocardium, and smooth muscle of the intestine. Moreover, neurofilament-immunoreactive axonal spheroids were identified in the spinal cord and roots. These widely distributed findings illustrate the multisystemic character of desmin myopathy, which in this instance first adds intestinal smooth muscle involvement to its already known skeletal and cardiac muscle manifestations. The additional presence of neurofilament aggregates in the spinal cord and roots constitutes an extremely rare conjunction of intermediate filament pathology of the neuromuscular system.

Expression of CD15 in normal and metaplastic Paneth cells of the digestive tract.

AIMS: To substantiate that incubation with monoclonal antibody CD15 (C3D-1) elicits a distinctive immunoreaction in normal small intestinal Paneth cells, normal and metaplastic Paneth cells along the digestive tract were assessed to determine whether they are also immunoreactive to CD15. METHODS: Paneth cells in paraffin wax embedded specimens of normal small intestine, appendix and proximal ascending colon, and from cases of chronic gastritis and ulcerative colitis were investigated immunohistochemically for lysozyme and CD15 antigen expression by means of the avidin-biotin peroxidase complex method. RESULTS: CD15 antibody reacted with a high proportion of both normal and metaplastic Paneth cells. Paneth cell immunoreactivity to CD15, however, was less intense and less extensive than to antilysozyme antibody, though the latter also stained many other cell types and was more commonly associated with nonspecific background staining. CONCLUSIONS: CD15 seems to be a valuable adjuvant for the study of Paneth cells in the normal and diseased digestive tract. Furthermore, as CD15 has been shown to be involved in activation of phagocytes, its expression in Paneth cells reinforces their proposed role as antimicrobial agents and regulators of the intestinal flora.

Differential expression of CD44v6 in adenocarcinoma of the pancreas: an immunohistochemical study.

Alternative splicing gives rise to numerous CD44 isoforms, some of which seem to have a role in tumour metastasis. Specifically, a variant form of CD44 with sequences encoded by exon v6 (CD44v6) confers metastatic potential when transfected into a nonmetastasizing cell line of rat pancreatic adenocarcinoma. This study has investigated standard CD44 (CD44s) and CD44v6 expression immunohistochemically in 6 samples of normal pancreatic tissue, 4 of tissue affected by chronic pancreatitis, and 24 of tissue from metastasizing and nonmetastasizing pancreatic adenocarcinomas. In addition, 18 samples from lymph node or visceral metastases were included in the study. CD44s was expressed in nonneoplastic tissue and in tissue from pancreatic adenocarcinomas. In contrast, CD44v6 was not detected in any of the normal tissue or chronic pancreatitis specimens, whereas 54% of pancreatic adenocarcinomas and 55% of metastases expressed this variant exon. Although it is not clear whether CD44 isoforms containing exon v6 play a part in malignant progression in the human exocrine pancreas, it seems plausible that the expression of multiple isoforms containing this and other variant exon confers a selective advantage on pancreatic adenocarcinoma.

Alzheimer's disease and the cystatin C gene polymorphism: an association study.

Cystatin C is an amyloidogenic protein that colocalizes with beta-amyloid (Abeta) within arteriolar walls in Alzheimer disease (AD) brains. Recently, a coding polymorphism in the cystatin C gene (CST3) has been claimed to confer risk for the development of late-onset AD. In the present work we have tested the frequencies of CST3-A and CST3-G alleles and used chi-square and logistic regression analyses to assess the association among the CST3 polymorphism, apolipoprotein E4 (APOE4), and AD in a series of 159 AD patients and 155 controls. The CST3-A allele was seen to be an accumulation risk factor for early-onset AD. Furthermore, a synergistic association among the CST3-A allele, APOE4 and AD was found in AD patients whose ages were between 60 and 74 years.

[Hepatic changes in patients with isolated immunodeficiency virus infection and AIDS]. / Alteraciones hepáticas en los pacientes con infección aislada por el virus de la inmunodeficiencia humana y con SIDA.

BACKGROUND: The aim of the present study was to describe the spectrum of liver disease in isolated infection by the human immunodeficiency virus and in the acquired immunodeficiency syndrome as well as evaluate whether clinical and/or biological data exist to permit specific diagnosis in liver biopsy. METHODS: Liver biopsy was performed in 39 patients with the human immunodeficiency virus (34 via parenteral drug addicts), 22 of whom had established acquired immunodeficiency syndrome. RESULTS: In 29 cases (74%) a specific histologic diagnosis was obtained with the changes most frequently found being the presence of granulomas (11 patients), mainly in patients with stablished AIDS, and chronic active hepatitis non A non B (10 patients), specially in the cases with isolated infection by the human immunodeficiency virus. Obtaining of a specific diagnosis was associated with an increase in GOT and a decrease of the CD4 lymphocytes and the CD4/CD8 quotient (p = 0.002 in all cases). The existence of established AIDS or prolonged fever was associated with the finding of hepatic granulomas (p = 0.02 and p = 0.002, respectively). The increase in GPT, the absence of stablished AIDS and the absence of prolonged fever was associated to the presence of chronic active hepatitis (p = 0.01, p = 0.002 and p = 0.0002, respectively). CONCLUSIONS: In patients with infection by the human immunodeficiency virus liver biopsy provides diagnostic information in a high percentage of cases. The presence of established AIDS, prolonged fever, and hypertransaminasemia may point towards possible histologic diagnosis.

Rarity of JC virus DNA sequences and early proteins in human gliomas and medulloblastomas: the controversial role of JC virus in human neurooncogenesis.

JC virus (JCV), the agent of progressive multifocal leucoencephalopathy (PML), exerts an oncogenic effect in several laboratory animal models. Moreover, JCV genomic DNA and early viral protein T-antigen have been detected in various types of human central nervous system (CNS) neoplasms. To further explore this association we have studied paraffin-embedded brain biopsy tissue from 60 neoplasms (55 gliomas and five medulloblastomas) and 15 reactive gliosis cases for the presence of JCV DNA sequences and proteins. Four post mortem cases of HIV-associated PML were used as positive controls. Samples were assessed by polymerase chain reaction (PCR) amplification of early (large T antigen) and late (virion protein 3) sequences and immunohistochemistry (IHC) with both PAb 2024 and anti-SV40 large T antigen monoclonal antibodies. Five cases (three neoplasms and two reactive gliosis instances) showed low viral DNA levels when PCR-tested for VP3 or large T, while no case was immunoreactive for any of the two antibodies used. The four PML cases yielded positive results with both PCR and IHC. Additionally, IHC with both antibodies was applied to a tissue micro-array including 109 CNS tumours and 21 reactive gliosis samples. No immunoreactivity was detected in any of these tissue micro-array samples. The rarity of JCV DNA sequences and early proteins in our brain tumours enriches the controversy over the role of JCV in human neurooncogenesis, whose clarification is in need of further molecular and epidemiologic studies.

Decreased immunoreactivity for cell-cycle regulator p27(Kip1) in Kaposi's sarcoma correlates with higher stage and extracutaneous involvement.

A consistent relationship has been established between the development of Kaposi's sarcoma (KS) and human herpes virus-8 (HHV8) infection. HHV8-encoded v-cyclin, through its complexing with cyclin-dependent kinase 6, contributes to the phosphorylation and proteasome-mediated degradation of p27(Kip1). On the other hand, down-regulation of p27(Kip1) expression seems to facilitate metastatic dissemination in a variety of human neoplasms. Although the neoplastic nature of KS remains controversial, it has been repeatedly demonstrated that in some patients KS may behave as a malignant neoplasm and follow an ominous course, especially in HIV-positive patients and when associated with extracutaneous involvement. To determine whether decreased p27(Kip1) levels are also related to more aggressive behaviour in KS, it was decided to investigate p27(Kip1) immunoreactivity in KS biopsy specimens and its possible changes in relation to cutaneous versus extracutaneous involvement and HIV serological status. Forty-nine cases of KS (29 AIDS-related and 21 classical) corresponding to 30 cutaneous biopsy specimens (ten macules, seven plaques, and 13 tumours) and 19 extracutaneous biopsy specimens were immunostained to determine the expression of p27(Kip1) and the proliferation marker Ki-67 antigen. The mean percentages of p27(Kip1)-positive cells were significantly higher in biopsy specimens from skin lesions (77.8+/-21.1) than in those from extracutaneous locations (42.0+/-26.0). Amongst cutaneous lesions, p27(Kip1) expression was significantly higher in macules (83.8+/-18.5) and plaques (91.4+/-6.4) than in tumours (65.8+/-22.6). Ki-67 immunoreactivity showed no correlation with any of the variables studied. These results lend support to the hypothesis that decreased levels of p27(Kip1), which may have been brought about by HHV8 infection, play a role in KS progression through its various histopathological stages, to its eventual extracutaneous spread.

Differential expression of alpha-synuclein isoforms in dementia with Lewy bodies.

Dementia with Lewy bodies (DLB) is characterized by the widespread presence of Lewy bodies (LBs) in the brain. alpha-Synuclein, the main component of LBs, is expressed as two main isoforms (112 and 140), but little is known about their differential expression in the brain. We compared alpha-synuclein 112 and alpha-synuclein 140 expression levels in the prefrontal cortices of six DLB patients, eight Alzheimer disease (AD) patients, and six control subjects. Relative alpha-synuclein 112 and alpha-synuclein 140 expression levels were determined by real-time polymerase chain reaction with competimer technology using a LightCycler System. Whereas total alpha-synuclein levels were just marginally elevated in DLB in comparison with the other groups, alpha-synuclein 112 was seen to be markedly increased in DLB compared with AD cases and controls. In contrast, alpha-synuclein 140 levels were significantly diminished in both neurodegenerative disorders in comparison with controls. These results show differential overexpression of alpha-synuclein 112 in DLB, a finding that could be of importance in DLB pathogenesis.

p53 immunoreactivity in hepatocellular adenoma, focal nodular hyperplasia, cirrhosis and hepatocellular carcinoma.

The prolonged half-life of mutant p53 makes feasible its immunocytochemical detection. In order to assess the pathogenetic role of mutant p53 in regenerative and neoplastic liver disease we studied its immunohistochemical expression in cases of hepatic cirrhosis, hepatocellular carcinoma (HCC), cirrhosis with areas of HCC, hepatocellular adenoma and focal nodular hyperplasia. The study included needle and wedge biopsies of 50 cirrhotic livers, 59 HCCs (36 of them with associated cirrhosis), six adenomas and two focal nodular hyperplasias. Sixty-five HCC fine-needle cytology specimens were also included in the study. There was no immunohistochemical evidence of mutant p53 expression in any of the cases of cirrhotic liver (except for one instance associated with HCC) adenoma or focal nodular hyperplasia. In contrast p53 was detected in 8.5% of HCC cases in the biopsy series and 24% of HCC cases in the fine needle aspiration series. In addition, mutant p53 expression in HCC was positively correlated with tumour grade. According to grade, the distribution of p53 positive immunoreactivity among HCCs was as follows: Grade I-II, 0% of cases in the biopsy series and 9% in the fine needle aspirates; Grade III, 18% in the biopsy series and 55% in the fine needle aspirates; and Grade IV, 40% in the biopsy series. Therefore, mutant p53 expression does not seem to be associated with benign liver lesions but seems to correlate with the progression of HCC through various grades of increasing malignancy.