Plastic compression of a collagen gel forms a much improved scaffold for ocular surface tissue engineering over conventional collagen gels

We compare the use of plastically compressed collagen gels to conventional collagen gels as scaffolds onto which corneal limbal epithelial cells (LECs) are seeded to construct an artificial corneal epithelium. LECs were isolated from bovine corneas (limbus) and seeded onto either conventional uncompressed or novel compressed collagen gels and grown in culture. Scanning electron microscopy (SEM) results showed that fibers within the uncompressed gel were loose and irregularly ordered, whereas the fibers within the compressed gel were densely packed and more evenly arranged. Quantitative analysis of LECs expansion across the surface of the two gels showed similar growth rates (p > 0.05). Under SEM, the LECs, expanded on uncompressed gels, showed a rough and heterogeneous morphology, whereas on the compressed gel, the cells displayed a smooth and homogeneous morphology. Transmission electron microscopy (TEM) results showed the compressed scaffold to contain collagen fibers of regular diameter and similar orientation resembling collagen fibers within the normal cornea. TEM and light microscopy also showed that cell–cell and cell–matrix attachment, stratification, and cell density were superior in LECs expanded upon compressed collagen gels. This study demonstrated that the compressed collagen gel was an excellent biomaterial scaffold highly suited to the construction of an artificial corneal epithelium and a significant improvement upon conventional collagen gels.

Molecular forms of porphobilinogen deaminase in acute intermittent porphyria. A study by Western immunoblotting

Acute intermittent porphyria is an inborn error of haem synthesis which is transmitted as a dominant character with variable phenotypic expression. The disorder is caused by a partial deficiency of porphobilinogen deaminase in all tissues so far studied. The nature of the enzymatic deficiency of porphobilinogen deaminase in haemolysates from patients with acute intermittent porphyria was examined by the use of monospecific antibody probes. In affected heterozygotes from three British pedigrees of diverse ancestry, the catalytic deficiency of porphobilinogen deaminase was accompanied by diminished enzyme protein, as determined by radial immunodiffusion. No evidence of functionally attenuated enzyme was demonstrable by kinetic studies. The molecular forms of the residual enzyme were investigated in red cell extracts and in lysed preparations of reticulocytes by a sensitive Western blotting procedure. This revealed the presence of reduced amounts of porphobilinogen deaminase polypeptide co-migrating with wild type enzyme (Mr approximately 40,000), and no evidence of variant forms in situ. The studies show that porphobilinogen deaminase deficiency in acute intermittent porphyria is commonly associated with a CRM-phenotype. The residual activity under these circumstances is thus related to expression of a single normal allele, since sensitive techniques detected neither aberrant nor degraded forms of the enzyme in erythroid tissues.

Motile sperm organelle morphology examination (MSOME): intervariation study of normal sperm and sperm with large nuclear vacuoles

Background: Although the motile sperm organelle morphology examination (MSOME) was developed only as a selection criterion, its application as a method for classifying sperm morphology may represent an improvement in evaluation of semen quality, with potential clinical repercussions. The present study aimed to evaluate individual variations in the motile sperm organelle morphology examination (MSOME) analysis after a time interval.Methods: Two semen samples were obtained from 240 men from an unselected group of couples undergoing infertility investigation and treatment. Mean time interval between the two semen evaluations was 119 +/- 102 days. No clinical or surgical treatment was realized between the two observations. Spermatozoa were analyzed at greater than or equal to 8400 x magnification by inverted microscope equipped with DIC/Nomarski differential interference contrast optics. At least 200 motile spermatozoa per semen sample were evaluated and percentages of normal spermatozoa and spermatozoa with large nuclear vacuoles (LNV/one or more vacuoles occupying >50% of the sperm nuclear area) were determined. A spermatozoon was classified as morphologically normal when it exhibited a normal nucleus (smooth, symmetric and oval nucleus, width 3.28 +/- 0.20 mu m, length 4.75 +/- 0.20 mu m/absence of vacuoles occupying >4% of nuclear area) as well as acrosome, post-acrosomal lamina, neck and tail, besides not presenting cytoplasm around the head. One examiner, blinded to subject identity, performed the entire study.Results: Mean percentages of morphologically normal and LNV spermatozoa were identical in the two MSOME analyses (1.6 +/- 2.2% vs. 1.6 +/- 2.1% P = 0.83 and 25.2 +/- 19.2% vs. 26.1 +/- 19.0% P = 0.31, respectively). Regression analysis between the two samples revealed significant positive correlation for morphologically normal and for LNV spermatozoa (r = 0.57 95% CI: 0.47-0.65 P < 0.0001 and r = 0.50 95% CI: 0.38-0.58 P < 0.0001, respectively).Conclusions: The significant positive correlation and absence of differences between two sperm samples evaluated after a time interval with respect to normal morphology and LNV spermatozoa indicated that MSOME seems reliable (at least for these two specific sperm forms) for analyzing semen. The present result supports the future use of MSOME as a routine method for semen analysis.

Efficacy of hyaluronic acid binding assay in selecting motile spermatozoa with normal morphology at high magnification

Background: The present study aimed to evaluate the efficacy of the hyaluronic acid (HA) binding assay in the selection of motile spermatozoa with normal morphology at high magnification (8400x).Methods: A total of 16592 prepared spermatozoa were selected and classified into two groups: Group I, spermatozoa which presented their head attached to an HA substance (HA-bound sperm), and Group II, those spermatozoa that did not attach to the HA substance (HA-unbound sperm). HA-bound and HA-unbound spermatozoa were evaluated according to the following sperm forms: 1-Normal morphology: normal nucleus (smooth, symmetric and oval configuration, length: 4.75+/-2.8 mu m and width: 3.28+/-0.20 mu m, no extrusion or invagination and no vacuoles occupied more than 4% of the nuclear area) as well as acrosome, post-acrosomal lamina, neck, tail, besides not presenting a cytoplasmic droplet or cytoplasm around the head; 2-Abnormalities of nuclear form (a-Large/small; b-Wide/narrow; c-Regional disorder); 3-Abnormalities of nuclear chromatin content (a-Vacuoles: occupy >4% to 50% of the nuclear area and b-Large vacuoles: occupy >50% of the nuclear area) using a high magnification (8400x) microscopy system.Results: No significant differences were obtained with respect to sperm morphological forms and the groups HA-bound and HA-unbound. 1-Normal morphology: HA-bound 2.7% and HA-unbound 2.5% (P = 0.56). 2-Abnormalities of nuclear form: a-Large/small: HA-bound 1.6% vs. HA-unbound 1.6% (P = 0.63); b-Wide/narrow: HA-bound 3.1% vs. HA-unbound 2.7% (P = 0.13); c-Regional disorders: HA-bound 4.7% vs. HA-unbound 4.4% (P = 0.34). 3. Abnormalities of nuclear chromatin content: a-Vacuoles >4% to 50%: HA-bound 72.2% vs. HA-unbound 72.5% (P = 0.74); b-Large vacuoles: HA-bound 15.7% vs. HA-unbound 16.3% (P = 0.36).Conclusions: The findings suggest that HA binding assay has limited efficacy in selecting motile spermatozoa with normal morphology at high magnification.

Anti-neutrophil cytoplasmic antibodies (ANCA) in the clinical forms of leprosy

Anti-neutrophil cytoplasmic antibodies (ANCA) are autoantibodies against enzymes present in primary granules of neutrophils and lysosomes of monocytes detected in systemic vasculitis and in other diseases, including infections, ANCA are markers of active Wegener granulomatosis, which presents some anatomo-pathologic and immune response features similar to those of leprosy. Thus, we raised the hypothesis that ANCA may be present in leprosy as markers specifically linked to the presence of vasculitis. The aim of this study was to determine the presence of ANCA in leprosy and its correlation with the clinical forms of the disease. Sera from 60 normal individuals and from 59 patients with different clinical forms of leprosy were studied. The patients were also allocated into reactional and nonreactional groups. By indirect immunofluorescence, ANCA were positive, an atypical pattern A-ANCA, in 28.8% of the patient sera. A-ANCA predominated, although not significantly (p >0,05), in the reactional groups (37.9% vs 20.0%), and in those at the lepromatous pole (41.6% vs 20.0%). There was no correlation between ANCA positivity and either disease duration, disease activity, or therapeutic regimen (p >0.05), An interesting finding was the correlation between ANCA and gender: 94.1% of ANCA-positive patients were males (p <0.01), a feature that so far has not been reported in ANCA-related diseases and for which there is no explanation at the moment. By ELISA, the sera of the lepromatous leprosy patients did not show activity against either PR3, MPO, HLE, the most common ANCA antigens. Because A-ANCA are nonspecific, this finding requires further investigation for the determination of the responsible antigen(s), in conclusion, A-ANCA are present in 28.8% of leprosy patients but are not related to vasculitis in the erythema nodosum leprosum reaction and are not a marker of a specific clinical form.

Serology of paracoccidioidomycosis. II. Correlation between class-specific antibodies and clinical forms of the disease

Untreated and previously treated patients with paracoccidioidomycosis were studied for: (i) serum levels of total IgG, IgM and IgA immunoglobulins, by radial immunodiffusion and Paracoccidioides brasiliensis (Pb) antibodies, by indirect immunofluorescence; (ii) correlation between their levels with the clinical forms of the disease; (iii) correlation between the serum titres obtained by tube precipitin with those of anti-Pb IgG, IgM and IgA. In the untreated group, serum IgG levels were significantly increased in patients with the more systemic forms of the disease, especially the acute progressive form. Serum IgA levels were significantly increased in all patients with no statistical difference between clinical forms. Serum IgM levels were normal in all patients. Anti-Pb IgG, IgA and IgM were detected in 97·5%, 32·5% and 45·0% of all cases, respectively. There was a sharp tendency towards higher levels of anti-Pb IgG among those with the acute progressive form (83·4%) in relation to the chronic, more localized forms, mixed form (68·0%) and isolated organic form (55·5%). In the untreated and previously treated group sera, there was positive correlation between the level of anti-Pb IgG and positivity for the tube precipitin test, suggesting that the precipitin-type antibodies are of the IgG class. Broadly, the present data demonstrate a polyclonal activation of the humoral immune system in paracoccidioidomycosis, with a positive relationship between serological results and severity of the disease. © 1984.

Correlation between cell-mediated immunity and clinical forms of paracoccidioidomycosis

Cellular immune response to specific and non-specific stimulants was investigated, both in vivo and in vitro, in 29 healthy controls and in 53 previously untreated patients with the chronic isolated organic form (CIOF), the chronic mixed form (CMF) and the acute progressive form (APF) of paracoccidioidomycosis. The study included skin tests to Paracoccidioides brasiliensis antigen (PbAg) and phytohaemagglutinin (PHA), DNCB sensitization, determination of T lymphocytes and complement rosette-forming cells, lymphocyte transformation and leucocyte migration inhibition tests using PbAg and PHA. Patients displayed staggered cutaneous response to PHA and to PbAg, with marked decrease in intensity in the APF group. DNCB sensitization test and proliferative response of lymphocytes to PHA and PbAg were severely depressed in most of the patients. Leucocyte migration inhibition indices to PbAg were highly positive, while response to PHA was slightly decreased regardless of the clinical form. The number of T lymphocytes was reduced in most of patients and in them the number of complement-rosette forming cells was normal. The distribution of patients according to a suppression index, based in the results of the tests employed, revealed a tendency towards an increased degree of cellular immunosuppression from the least severe (CIOF) to the most severe (APF) clinical form of the disease. On the whole, the present study demonstrated a gamut of immunological reactivity in paracoccidioidomycosis. © 1985.

Miniversal deformations of matrices of bilinear forms

Arnold [V.I. Arnold, On matrices depending on parameters, Russian Math. Surveys 26 (2) (1971) 29-43] constructed miniversal deformations of square complex matrices under similarity; that is, a simple normal form to which not only a given square matrix A but all matrices B close to it can be reduced by similarity transformations that smoothly depend on the entries of B. We construct miniversal deformations of matrices under congruence. (C) 2011 Elsevier Inc. All rights reserved.

Primary antiphospholipid syndrome: morphofunctional penile abnormalities with normal sperm analysis

Objective: To perform a global gonadal and sexual functions assessment in primary antiphospholipid syndrome (PAPS) patients. Methods: A cross-sectional study was conducted in 12 male PAPS patients and 20 healthy controls. They were assessed by demographic data, clinical features, systematic urological examination, sexual function, testicular ultrasound, seminal parameters according to the World Health Organization (WHO), seminal sperm antibodies, and hormone profile, including follicle stimulating hormone (FSH), luteinizing hormone (LH), morning total testosterone, and thyroid hormones. Results: The median of current age and age of spermarche were similar in PAPS patients and controls (37.5 vs. 32.4 years, p = 0.270, and 13.1 vs. 12.85 years, p = 0.224, respectively), with a higher frequency of erectile dysfunction in the former group (25% vs. 0%, p = 0.044). Further analysis of PAPS patients with and without previous arterial thrombosis demonstrated that the median penis circumference was significantly lower in PAPS with arterial thrombosis than in PAPS without this complication (8.1 [6-10] vs. 10.2 [10-11] cm, p = 0.007). In addition, the median penis circumference was significantly lower in PAPS patients with erectile dysfunction than in patients without this complication (7.5 [6-9.5] vs. 9.5 [7.5-11] cm, p = 0.039). Regarding seminal analysis, the median sperm concentration, sperm motility, and normal sperm forms by WHO guidelines were comparable in PAPS patients and controls (141.5 [33-575] vs. 120.06 [34.5-329] x 106/ml, p = 0.65; 61.29 [25-80] vs. 65.42 [43-82]%, p = 0.4; 21.12 [10-42.5] vs. 23.95 [10-45]%, p = 0.45, respectively), and none of them had oligo/azoospermia. No differences were observed between PAPS patients and controls regarding the frequency of antisperm antibodies, testicular volume by ultrasound, or hormone profile (FSH, LH, morning total testosterone, and thyroid hormone) (p > 0.05). Conclusions: Normal testicular function has been identified in PAPS patients, in spite of morphofunctional penile abnormalities. Previous arterial thrombosis may underlie penile anthropometry alteration. Lupus (2012) 21, 251-256.

Monogenic forms of hypertension

Arterial hypertension in childhood is less frequent as compared to adulthood but is more likely to be secondary to an underlying disorder. After ruling out more obvious causes, some patients still present with strongly suspected secondary hypertension of yet unknown etiology. A number of these children have hypertension due to single gene mutations inherited in an autosomal dominant or recessive fashion. The finding of abnormal potassium levels (low or high) in the presence of suppressed renin secretion, and metabolic alkalosis or acidosis should prompt consideration of these familial diseases. However, mild hypertension and the absence of electrolyte abnormalities do not exclude hereditary conditions. In monogenic hypertensive disorders, three distinct mechanisms leading to the common final pathway of increased sodium reabsorption, volume expansion, and low plasma renin activity are documented. The first mechanism relates to gain-of-function mutations with a subsequent hyperactivity of renal sodium and chloride reabsorption leading to plasma volume expansion (e.g., Liddle's syndrome, Gordon's syndrome). The second mechanism involves deficiencies of enzymes that regulate adrenal steroid hormone synthesis and deactivation (e.g., subtypes of congenital adrenal hyperplasia, apparent mineralocorticoid excess (AME)). The third mechanism is characterized by excessive aldosterone synthesis that escapes normal regulatory mechanisms and leading to volume-dependent hypertension in the presence of suppressed renin release (glucocorticoid remediable aldosteronism). Hormonal studies coupled with genetic testing can help in the early diagnosis of these disorders.

Specific, Reversible Cytostatic Protection of Normal Cells Against Negative Effects of Chemotherapy

Chemotherapy is a common and effective method to treat many forms of cancer. However, treatment of cancer with chemotherapy has severe side effects which often limit the doses of therapy administered. Because some cancer chemotherapeutics target proliferating cells and tissues, all dividing cells, whether normal or tumor, are affected. Cell culture studies have demonstrated that UCN-01 is able to reversibly and selectively arrest normal dividing cells; tumor cells lines do not undergo this temporary arrest. Following UCN-01 treatment, normal cells displayed a 50-fold increase in IC50 for camptothecin; tumor cells showed no such increased tolerance. We have examined the response of the proliferating tissues of the mouse to UCN- 01 treatment, using the small bowel epithelium as a model system. Our results indicate that UCN-01 treatment can cause a cell cycle arrest in the gut epithelium, beginning 24 hours following UCN-01 administration, with cell proliferation remaining suppressed for one week. Two weeks post-UCN-01 treatment the rate of proliferation returns to normal levels. 5-FU administered during this period demonstrates that UCN-01 is able to provide protection to normal cells of the mouse within a narrow window of efficacy, from three to five days post-UCN-01. UCN-01 pretreated mice displayed improved survival, weight status and blood markers following 5-FU compared to control mice, indicating that UCN-01 can protect normal dividing tissues. The mechanism by which UCN-01 arrests normal cells in vivo was also examined. We have demonstrated that UCN-01 treatment in mice causes an increase in the G1 phase cell cycle proteins cdk4 and cyclin D, as well as the inhibitor p27. Phosphorylated Rb was also elevated in the arrested cells. These results are a departure from cell culture studies, in which inhibition of G1 phase cyclin dependent kinases led to hyposphosphorylation of Rb. Future investigation will be required to understand the mechanism of UCN-01 action. This is important information, especially for identification of alternate compounds which could provide the protection afforded by UCN-01.

BLyS/BAFF-R signaling pathway in human aggressive non Hodgkin's lymphoma B cell and normal peripheral blood B lymphocytes

B-lymphocyte stimulator (BLyS also called BAFF), is a potent cell survival factor expressed in many hematopoietic cells. BLyS levels are elevated in the serum of non-Hodgkin lymphoma (NHL) patients, and have been reported to be associated with disease progression, and prognosis. To understand the mechanisms involved in BLyS gene expression and regulation, we examined expression, function, and regulation of the BLyS gene in B cell non-Hodgkin's lymphoma (NHL-B) cells. BLyS is constitutively expressed in aggressive NHL-B cells including large B cell lymphoma (LBCL) and mantle cell lymphoma (MCL) contributing to survival and proliferation of malignant B cells. Two important transcription factors, NF-κB and NFAT, were found to be involved in regulating BLyS expression through at least one NF-κB and two NFAT binding sites in the BLyS promoter. Further study indicates that the constitutive activation of NF-κB and BLyS in NHL-B cells forms a positive feedback loop contributing to cell survival and proliferation. In order to further investigate BLyS signaling pathway, we studied the function of BAFF-R, a major BLyS receptor, on B cells survival and proliferation. Initial study revealed that BAFF-R was also found in the nucleus, in addition to its presence on plasma membrane of B cells. Nuclear presentation of BAFF-R can be increased by anti-IgM and soluble BLyS treatment in normal peripheral B lymphocytes. Inhibition of BLyS expression decreases nuclear BAFF-R level in LBCL cells. Furthermore, we showed that BAFF-R translocated to nucleus through the classic karyopherin pathway. A candidate nuclear localization sequence (NLS) was identified in the BAFF-R protein sequence and mutation of this putative NLS can block BAFF-R entering nucleus and LBCL cell proliferation. Further study showed that BAFF-R co-localized with NF-κB family member, c-rel in the nucleus. We also found BAFF-R mediated transcriptional activity, which could be increased by c-rel. We also found that nuclear BAFF-R could bind to the NF-κB binding site on the promoters of NF-κB target genes such as BLyS, CD154, Bcl-xL, Bfl-1/A1 and IL-8. These findings indicate that BAFF-R may also promote survival and proliferation of normal B cells and NHL-B cells by directly functioning as a transcriptional co-factor with NF-κB family member. ^

DETERMINAÇÃO DAS FORMAS DO ARCO DENTÁRIO INFERIOR NA OCLUSÃO NORMAL NATURAL

Com a finalidade de determinar as formas do arco dentário inferior de maior incidência na oclusão normal natural, utilizou-se um método matemático associado ao emprego de uma função polinomial, o qual foi aplicado a 63 modelos de arcadas inferiores selecionados a partir de 6118 adolescentes. Todos os indivíduos eram portadores de dentição permanente, incluindo os segundos molares, e oclusão normal natural. Em cada dente foi fixada uma esfera de vidro, que teve a função de simular o acessório do aparelho ortodôntico, sendo utilizada na medição das distâncias entre o centro da imagem dessas esferas aos eixos x e y. Após a digitalização dos modelos de gesso, as imagens foram plotadas em um programa de computador, a fim de se obterem a função polinomial de sexto grau e o gráfico dessa função para os 126 segmentos de curva, originados das secções das imagens em lado direito e esquerdo. A seguir organizaram-se esses segmentos, de acordo com as características da curvatura anterior dos arcos dentários, em oito grupos diferentes de formas, que receberam as denominações de Forma A, Forma B, Forma C, Forma D, Forma E, Forma F, Forma G, Forma H. Cada grupo foi, então, dividido em três subgrupos, conforme os tamanhos pequeno, médio e grande. Os resultados indicaram 23 formas representativas do arco dentário inferior e uma forma média para a oclusão normal natural.

DETERMINAÇÃO DAS FORMAS DO ARCO DENTÁRIO INFERIOR NA OCLUSÃO NORMAL NATURAL

Com a finalidade de determinar as formas do arco dentário inferior de maior incidência na oclusão normal natural, utilizou-se um método matemático associado ao emprego de uma função polinomial, o qual foi aplicado a 63 modelos de arcadas inferiores selecionados a partir de 6118 adolescentes. Todos os indivíduos eram portadores de dentição permanente, incluindo os segundos molares, e oclusão normal natural. Em cada dente foi fixada uma esfera de vidro, que teve a função de simular o acessório do aparelho ortodôntico, sendo utilizada na medição das distâncias entre o centro da imagem dessas esferas aos eixos x e y. Após a digitalização dos modelos de gesso, as imagens foram plotadas em um programa de computador, a fim de se obterem a função polinomial de sexto grau e o gráfico dessa função para os 126 segmentos de curva, originados das secções das imagens em lado direito e esquerdo. A seguir organizaram-se esses segmentos, de acordo com as características da curvatura anterior dos arcos dentários, em oito grupos diferentes de formas, que receberam as denominações de Forma A, Forma B, Forma C, Forma D, Forma E, Forma F, Forma G, Forma H. Cada grupo foi, então, dividido em três subgrupos, conforme os tamanhos pequeno, médio e grande. Os resultados indicaram 23 formas representativas do arco dentário inferior e uma forma média para a oclusão normal natural.

ESTUDO DAS FORMAS E DIMENSÕES LINGUAIS DAS ARCADAS DENTÁRIAS EM INDIVÍDUOS BRASILEIROS COM OCLUSÃO NORMAL

A preocupação com o estudo das formas e dimensões das arcadas dentárias sempre esteve presente na ciência ortodôntica. Para a Ortodontia Lingual, que surgiu no final da década de 70, o primeiro artigo publicado foi o Fujita, onde relatou sobre a forma do arco a ser utilizado nesta técnica, a forma de cogumelo. Apesar de estar sendo divulgada de uma maneira mais intensa nestes últimos anos como uma solução estética definitiva e eficaz, o enfoque dos estudos sobre esta técnica tem sido a fabricação de novos materiais, técnicas de montagem do aparelho lingual e soluções clínicas, com poucas menções sobre a morfologia das arcadas dentárias. O presente trabalho tem a finalidade de estudar as formas e dimensões linguais das arcadas dentárias de indivíduos leucodermas com oclusão normal. Foram utilizados 47 pares de modelos de gesso de oclusão normal digitalizados pela face olcusal, previamente desgastadas até o terço médio da coroa para proporcionar melhor visualização. Por meio do programa CorelDraw 12 foram determinados pontos de referências e criados alguns pontos virtuais necessários para a realização das medidas. Os resultados determinaram três formas das arcadas dentárias linguais: cogumelo, árvore de Natal e mista. A maior prevalência foi a forma árvore de Natal, mas quando analisadas separadamente as arcadas dentárias linguais, encontrados no superior, maior prevalência da forma de cogumelo e no inferior da forma árvore de Natal. Conseqüentemente, esta foi a combinação mais prevalente entre as arcadas dentárias linguais superiores e inferiores. Propusemos diagramas para conformação de arcos ortodônticos linguais com base nos valores obtidos da amostra, determinando-se o quartil 1, mediana e quartil 3, como definidores dos tamanhos pequeno, médio e grande.