Morphological characterization of the false vocal cords as larynx-associated lymphoid tissue.

The lymphoid follicles (LF) found in the false vocal cords (FVC) protect the upper air tracts, similar to the lymphoid tissue associated to the respiratory mucosas. However, studies that characterize the phenotype of cells like larynx-associated lymphoid tissue (LALT) are lacking. We analyzed the FVC of autopsied adults according to morphometric and immunohistochemical criteria and defined their possible role as LALT. We analyzed 249 FVC. Primary antibodies, CD68+ macrophages, CD20+, CD3+, and FDC+ were used for the evaluation of inflammatory cell phenotypes. In 40.6% of the cases, there was an inflammatory reaction. In 42.2% of the cases, LF were identified in the submucosa. In 17.3% of the cases, neither LF nor mononuclear cells were identified in the FVC, and these patients were from an older age group (p=0.013). A significant increase in the number of all LF cell phenotypes was observed in patients with pulmonary inflammation; the difference in both T- and B-lymphocytes was statistically significant (p=0.010). The morphological findings of LF suggest a probable participation of the FVC in the protection of the larynx and lungs, and similarity to LALT.

Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146+ perivascular cells and fibroblasts.

OBJECTIVE: The relationship of multipotent mesenchymal stromal cells (MSC) with pericytes and fibroblasts has not been established thus far, although they share many markers of primitive marrow stromal cells and the osteogenic, adipogenic, and chondrogenic differentiation potentials. MATERIALS AND METHODS: We compared MSCs from adult or fetal tissues, MSC differentiated in vitro, fibroblasts and cultures of retinal pericytes obtained either by separation with anti-CD146 or adhesion. The characterizations included morphological, immunophenotypic, gene-expression profile, and differentiation potential. RESULTS: Osteogenic, adipocytic, and chondrocytic differentiation was demonstrated for MSC, retinal perivascular cells, and fibroblasts. Cell morphology and the phenotypes defined by 22 markers were very similar. Analysis of the global gene expression obtained by serial analysis of gene expression for 17 libraries and by reverse transcription polymerase chain reaction of 39 selected genes from 31 different cell cultures, revealed similarities among MSC, retinal perivascular cells, and hepatic stellate cells. Despite this overall similarity, there was a heterogeneous expression of genes related to angiogenesis, in MSC derived from veins, artery, perivascular cells, and fibroblasts. Evaluation of typical pericyte and MSC transcripts, such as NG2, CD146, CD271, and CD140B on CD146 selected perivascular cells and MSC by real-time polymerase chain reaction confirm the relationship between these two cell types. Furthermore, the inverse correlation between fibroblast-specific protein-1 and CD146 transcripts observed on pericytes, MSC, and fibroblasts highlight their potential use as markers of this differentiation pathway. CONCLUSION: Our results indicate that human MSC and pericytes are similar cells located in the wall of the vasculature, where they function as cell sources for repair and tissue maintenance, whereas fibroblasts are more differentiated cells with more restricted differentiation potential.

Morphological, immunohistochemical and chromosome in situ hybridization in the differential diagnosis of Hydatidiform Mole and Hydropic Abortion.

OBJECTIVE: To explore the utility of histological, immunohistochemical and chromosome in situ hybridization (CISH) test in the differential diagnosis of Complete Hydatidiform Mole (CHM), Partial Hydatidiform Mole (PHM) and Hydropic Abortion (HA). STUDY DESIGN: We analyzed the histological characteristics, p57kip2 and Factor VIII expression and CISH test in 38 cases with some diagnostic concerns, comprising 13 CHM, 14 PHM and 11 HA. RESULTS: Our results indicate that p57kip2 expression and the ploidy assessed by CISH were essential for the reclassification of 2 cases, one from CHM to PHM and another from PHM to HA, as well as for confirming the previous diagnosis in cases where there were conflicting features. p57kip2 expression is diagnostic if no cells at all present it (CHM) or when there are over 10% of cells expressing it (PHM and HA). CONCLUSIONS: We concluded that there is no single criterion for the distinction of CHM, PHM and HA. So p57kip2 expression and CISH test can be used in association with the histological findings for the differential diagnosis of the three conditions in cases presenting some concern for definitive diagnosis.

Brain tissue fragments in the amniotic fluid of rats with neural tube defect.

AIMS: Brain tissue nodules are occasionally seen in the lungs of neural tube defect (NTD) cases. We looked for brain tissue fragments in amniotic fluid of rats with NTD as it is the basis for the aspiration hypothesis. METHODS: Eighty-seven pregnant rats were randomly divided into experimental (n=58) and control (n=29) groups. Experimental rats received 100,000 U of vitamin A in 1 mL of corn oil on gestational days 8, 9 and 10, while control rats received corn oil. On gestational days 15, 18, 19, 20 and 21, amniotic fluid was drawn from three control animals and five experimental animals and analysed. RESULTS: NTD was found in 22.75% of experimental fetuses and in no control fetuses. Brain tissue fragment number and volume fraction increased between gestational days 18 and 20, falling on day 21. CONCLUSIONS: Excessive doses of vitamin A induce a high rate of early fetal death and development of NTD. Brain tissue fragments in the amniotic fluid reflect the evolution from exencephaly to anencephaly and could support the aspiration hypothesis. However, as it is a late event in the rat, this model may not reproduce the brain tissue nodules in the lung.

Fluorescent in situ hybridization in liver cell touch preparations from autopsy.

The objective of the present study was to develop a simplified and low-cost protocol for the investigation of congenital anomalies of chromosomal etiology by fluorescent in situ hybridization (FISH) using probes for chromosomes X, 18, 13/21 in liver cell touch preparations obtained from autopsies performed at the University Hospital of the Faculty of Medicine of Ribeirão Preto. Liver touch preparations were obtained from 11 autopsy cases and fixed in 95% ethanol or methanol:acetic acid (3:1). The FISH technique was carried out according to the protocol of Pinkel with modifications, using probes for chromosomes X, 18, 13/21. There was no significant difference in labeling intensity, quantity of nuclei, or number of signals present per nucleus between the materials fixed with the two fixatives. Similar results were obtained with different times of storage up to 14 months at -20 degrees C. We concluded that the use of touch preparations pretreated with acetic acid and fixed in 95% ethanol represents an efficient, practical, and low-cost method of cell preparation for FISH analysis.

Ultrastructural and histological findings on examination of skin in osteogenesis imperfecta: a novel study.

Osteogenesis imperfecta (OI) is a heterogeneous group of inherited disorders of bone formation, resulting in low bone mass and an increased propensity for fractures. It is a variable condition with a range of clinical severities. The histological and ultrastructural findings in the skin of patients with OI have not been described in detail in the previously published literature. Although protein analysis of cultured fibroblasts has historically been used in the diagnostic work-up of OI patients, other aspects of skin examination are not routinely performed as part of the diagnostic pathway in patients with OI. The aims of this study were to perform histological and ultrastructural examination of skin biopsies in patients with OI. This was to identify common and distinguishing features in the numerous genetically distinct subtypes of OI and compare the findings with those in patients who did not present with fractures, and to enable the use of the results thus obtained to aid in the diagnostic work-up of patients with OI. As part of a larger research study set-up to identify clinical features and natural history in patients with atypical features of OI, skin biopsy and examination (histology and electron microscopy) were undertaken. Genetic analysis and ancillary investigations were also performed to identify similarities within this group and to differentiate this group from the 'normal' population. At the end of this study, we were able to demonstrate that the histological and electron microscopic findings on a skin biopsy may be an indicator of the likelihood of identifying a pathogenic mutation in type 1 collagen genes. This is because patients with specific findings on examination, such as elastic fibre area fraction (on histological analysis), collagen fibril diameter variability, deviation from the expected mean and collagen flowers (on electron microscopy), are more likely to be positive on genetic analyses. This has, in turn, provided more insight into the pathways to direct gene testing and has reinforced the need for accurate phenotyping before undertaking further genetic investigations. The morphometric assessment of elastic fibre area fraction and ultrastructural findings from this study have provided us with a better understanding of OI and insights into the possible mechanism of these changes in the skin. Correlation of skin findings with the clinical phenotype as well as genetic testing has enabled understanding of the molecular pathogenesis and translation of changes at the genomic level to clinical phenotype.

Increased number of fetal nucleated red blood cells in the placentas of term or near-term stillborn and neonates correlates with the presence of diffuse intradural hemorrhage in the perinatal period.

Release of nucleated red blood cells (nRBCs) into the peripheral blood occurs in stillbirths/neonates with a probable hypoxic mode of death and antenatal stress. We correlated the number of nRBCs in the placenta with the occurrence of intradural (IDH) and subdural hemorrhage (SDH) and the potential link with fetal hypoxia. Two groups of 22 cases each of nonmacerated term or near-term (≥36 weeks of gestational age) stillborn or newborns dying in the 1st day of life were studied. One group had IDH (with or without SDH) and the other did not have IDH or SDH. In each case, the number of nRBCs was determined in 10 consecutive placental fields at ×40. Data were analyzed with Fisher exact test, receiver operating characteristic (ROC) curve analysis, and logistic regression. There was a significant association between the diffuse IDH and increased number of nRBCs (Fisher exact test P  =  0.0165). An ROC curve analysis showed that the cut-off number of nRBCs with the highest accuracy was 2.15 nRBCs/high-power field, with 79% sensitivity and 67% specificity. The presence of diffuse IDH was associated with SDH (Fisher exact test, P  =  0.002). The absence of hypoxic brain change was associated with the absence of diffuse IDH (odds ratio 0.308; P  =  0.039). We established a significant correlation between the release of nRBCs into the placental circulation and the occurrence of diffuse IDH and between diffuse IDH and the presence of SDH.

Genotype-phenotype study in type V osteogenesis imperfecta.

Type V osteogenesis imperfecta (OI) presents with moderate-to-severe skeletal deformity and is characterized by hyperplastic callus formation at fracture sites and calcification of the interosseous membranes of the forearm and lower leg. The facial dysmorphism is not well characterized and has not been described in previous reports. Inheritance is autosomal dominant, although the genetic aetiology remained unknown until very recently. The aims of this study were to establish the genetic aetiology in patients with type V OI and further characterize patients with this condition, and to ascertain whether they have a similar clinical phenotype and facial dysmorphism. Three families (one mother-daughter pair and two singletons) were identified with the above features and further investigations (molecular genetic analysis and skin biopsy including electron microscopy, histology and collagen species analysis) were performed. Accurate phenotyping of patients with type V OI was performed. PCR amplification was performed using the Sheffield Diagnostic Genetics Service pyrosequencing assay for the interferon-induced transmembrane protein-5 (IFITM5) gene. All the patients had been confirmed to have a heterozygous variant, c.[-14C>T];[=], in the 5'-UTR of the IFITM5 gene, which is located in the transcribed region of this gene. This recurrent mutation, in IFITM5, also known as bone-restricted interferon-induced transmembrane protein-like protein or BRIL, encodes a protein with a function in bone formation and plays an important role in osteoblast formation. All four patients in this study appear to have similar clinical features and facial dysmorphism, including a short, up-turned nose, a small mouth, a prominent chin and greyish-blue sclerae. Skin biopsy in one patient showed clumping of elastic fibres and normal biochemical analysis of collagen. We have been able to characterize patients with type V OI further and confirm the genetic aetiology in this distinct form of OI. Accurate phenotyping (including describing the common facial features) before investigations is important to enable the useful interpretation of genetic results and/or target-specific gene testing.

Ischemic myocardial injuries after cardiac malformation repair in infants may be associated with oxidative stress mechanisms.

BACKGROUND: Despite advances in pediatric cardiac surgery, perioperative myocardial injury can be the major determinant of postoperative dysfunction after cardiac surgery. This study investigated the pathology-related differences in 29 infants with congenital heart disease that led to death. The infants were treated at the University Hospital of Ribeirão Preto, Brazil. METHODS: The patients were divided into four groups: Group 1, 16 infants who underwent operations for congenital heart disease on cardiopulmonary bypass; Group 2, four infants who underwent off-cardiopulmonary bypass operations for congenital heart disease; Group 3, nine infants who died from congenital heart disease prior to surgical treatment; and Group 4 (control group), five infants with no congenital heart disease and who died from other causes. The myocardial injuries and oxidative stress mechanisms were assessed by histopathology and immunohistochemistry and were quantified by morphometrical analyses. RESULTS: Contraction band necrosis and dystrophic calcification were found primarily in infants of Group 1. Coagulation necrosis and healing were prominent in Group 2, while infants without repair (Group 3) showed mainly colliquative myocytolysis. Apoptotic cells were more prominent in the operative groups. The control group showed no significant myocardial lesions. Lipid peroxidation was the principal mechanism of oxidative stress accounting for the myocardial lesions. CONCLUSION: The diversity of the lesions observed in these hearts seemed to indicate a large spectrum of cell damage due to inadequate myocardial perfusion, especially when these infants underwent surgery. Oxidative mechanisms could be a common mediator in the pathogenesis of myocardial injuries, mediated by peroxidation of the membrane phospholipids and resulting in changes in the permeability of the cell membrane, cell death, and intracellular calcium overload. Furthermore, an immature and often hypertrophied myocardium may promote unfavorable conditions, leading to heart failure and a lethal outcome.

Evaluation of clinical and functional abnormalities in Pompe disease: Case Report / Avaliação das anomalias clínico-funcionais na doença de Pompe: Relato de Caso

The patient was a 15 year-old girl who turned out at the Physical Therapy Clinic presenting progressiv escoliosis and angle of 50º Coob by X-Ray. She complained of back pain, headache and weakness of shoulder and pelvic girdle. Physical therapy evaluation came across features of delayed motor development and undernourishment, together with generalized muscle weakness (grade = 4) which was observed by the Kendall test. Lung vital capacity was 40.5%. Clinical Changes: studies of the enzymes with acid alpha-glucosidase assay kits used on filter paper and leukocytes showed low enzyme activity, suggesting a late form of the Pompe disease. The molecular studies proved that the patient had amutation associated with late-onset Pompe disease. Acid alpha-glucosidase enzyme assay studies performed in skin fibroblasts showed a reduction of the enzymatic activity of the acid alpha-glucosidase, confirming the previous results. On account of the results, Pompe disease induced important changes inclinical and functional, as well as metabolic changes, decreased strength and muscle action potentially, biomechanical changes in the spine and changes in respiratory capacity. Furthermore, this case of Pompe disease illustrates the importance of adequate physical therapy evaluation as it can be the starting point of investigation of serious conditions such as late onset Pompe disease

Paciente do sexo feminino com 15 anos, apresentou-se na Clínica de Fisioterapia, devido à presença de escoliose progressiva com ângulo de Coob de 50º pelo Raio-X. Apresentou queixa de dor na coluna e na cabeça, fraqueza de cintura escapular e pélvica. Na avaliação fisioterapêutica observou-se um quadro semelhante ao atraso do desenvolvimento motor e desnutrição, com fraqueza muscular generalizada(grau = 4) observada pelo teste de Kendall. Na função pulmonar a capacidade vital apresentou com 40,5%. Estudos enzimáticos com dosagem da alfa-glicosidade ácida em papel-filtro e leucócitos evidenciaram baixa atividade enzimática, sugestivo de forma tardia da doença de Pompe. No estudo molecular, comprovou-se que a paciente possuía mutação associada à forma tardia da doença; estudos enzimáticos da alfa-glicosidase ácida em fibroblastos cultivados a partir de biópsia de pele evidenciaram redução da atividade enzimática da alfa-glicosidase ácida, confirmando estudos enzimáticos prévios. Perante os resultados, a doença de Pompe apresentou alterações clínicas e funcionais importantes como alteração do metabolismo, diminuição de força e do potencial de ação da musculatura, alterações biomecânicas na coluna e na capacidade respiratória. Adicionalmente, o caso ilustra a importância da avaliação fisioterapêutica adequada, pois ele pode ser o ponto de partida da investigação de doenças graves como o presente caso

Síndrome da Morte Súbita na Infância / Sudden Infant Death Syndrome

A Síndrome da Morte Súbita da Infância (SIDS) é uma entidade ainda pouco diagnosticada em nosso meio, mas que se constitui na principal causa de morte no primeiro ano de vida, nos países industrializados. Acomete crianças aparentemente hígidas, bem nutridas e cuidadas, no primeiro ano de vida, porém com nítido pico entre o terceiro (3º) e o quarto (4º) mês de vida. Nesta revisäo, procuramos mostrar a evoluçäo histórica desta síndrome, bem como analisar as principais hipóteses e fatores de risco ligados a ela, além de mostrar o panorama atual no Brasil e no mundo.

Contribuiçäo ao estudo das afecções pulmonares nas autópsias pediátricas / Contribution to the study of pulmonary pediatric pathology in autopsies

Modelo do estudo: estudo retrospectivo. Objetivos: determinar a freqüência de algumas das principais afecções pulmonares, pediátricas, com base na casuística de autópsias do Serviço de Patologia do HCFMRP-USP, comparando-a com a literatura, a fim de produzir um texto com informações atualizadas, que possa servir de orientaçäo ao estudo dos problemas, principalmente para alunos de graduaçäo. Metodologia: foram analisados os dados de quinhentas e cinco (505) autópsias pediátricas, entre junho de 1993 e junho de 1996; sexo, idade gestacional, idade no momento da morte, peso corporal, causa da morte, peso e alterações pulmonares. O programa Epi-Info, versäo 6, foi usado para a tabulaçäo dos dados e análise estatística, e os resultados comparados com a literatura. Resultados: o sexo masculino predominou (53,7 por cento) sobre o feminino (45,9 por cento) e indeterminado (0,4 por cento). A maior parte dos óbitos ocorreu no período neonatal precoce (47 por cento), intra-uterino (19 por cento) e neonatal tardio (7 por cento), somando 73 por cento de todos os casos. A idade gestacional variou de dezesseis (16) a quarenta e três (43) semanas. Prematuridade foi a causa de morte mais freqüente (29,7 por cento), seguida pelas anomalias congênitas (10,9 por cento), anoxia intra-uterina (8,3 por cento), infecções (8,1 por cento), causas perinatais (2,4 por cento), isoimunizaçäo (1,8 por cento), neoplasias (0,4 por cento) e outras (38,4 por cento). As afecções pulmonares mais comuns foram: petéquias (49,1 por cento), hemorragia maciça (34,5 por cento), derrame pleural (29,5 por cento), doença da membrana hialina (25,6 por cento), atelectasia (24,6 por cento), pneumonia (16,8 por cento), pneumotórax (10,8 por cento), lobaçäo anormal (8,6 por cento), enfisema intersticial (5 por cento), hipoplasia (4,4 por cento) e displasia broncopulmonar (2,4 por cento). Conclusões: as afecções pulmonares säo muito freqüentes nas autópsias pediátricas, representando, muitas vezes, a causa da morte e estäo, de forma geral, de acordo com as freqüências encontradas na literatura, havendo, no entanto, algumas variações que, possivelmente, refletem diferenças na atençäo pré e perinatal, nos diferentes países. As alterações pulmonares identificadas estäo, em sua maioria, relacionadas à prematuridade e anomalias congênitas, constituindo-se nos grupos cuja reduçäo traria os maiores benefícios médicos e sócio-econômicos