Protective effects of nutritional supplementation with arginine and glutamine on the penis of rats submitted to pelvic radiation.

Radiotherapy is widely used to treat pelvic malignancies, but normal tissues near the target tumour are often affected. Our aims were thus to determine whether the structural organization of the rat penis is altered by radiation, and whether supplementation with L-arginine (ARG) or L-glutamine (GLN) would have protective effects against these alterations. Groups of rats were treated with: no intervention (CONTR); pelvic radiation, followed by sacrifice 7 (RAD7) or 15 (RAD15) days later; and pelvic radiation, daily supplementation with ARG or GLN, followed by sacrifice 7 (RAD7+ARG, RAD7+GLN) or 15 (RAD15+ARG, RAD15+GLN) days after radiation. Structural components in the corpus cavernosum (CC), tunica albuginea of the corpus spongiosum (TACS) and urethral epithelium (UE) were analysed using stereological and immunohistochemical methods. The results showed that in the CC, connective tissue was increased by 18% in RAD15 (p < 0.04), but this change was partially prevented in RAD15+GLN (p < 0.05) and RAD15+ARG (p < 0.04). The fibrous matrix of the CC trabeculae stained evenly for collagen type I. In RAD15, the intensity of the labelling was increased, whereas in RAD15+GLN and RAD15+ARG the staining was similar to that of CONTR. No staining changes were seen in the groups that were sacrificed 7 days after radiation. Cavernosal elastic fibre content in RAD15 was increased by 61% (p < 0.004), and this was prevented in RAD15+ARG (p < 0.004) but not in RAD15+GLN. In TACS, the amino acids protected (p < 0.02) against the radiation-induced 92% increase in elastic fibre content, but only in RAD15. Cell density in the UE, as well as UE thickness, were reduced by 30% in RAD15 (p < 0.004), and there were protective effects of both amino acids. In conclusion, radiation-induced alterations in penile structures tend to be more pronounced 15 days after radiation session. Both ARG and GLN have protective effects against these changes, with the former being slightly more effective.

Nutritional assessment of very low birth weight infants: relationships between anthropometric and biochemical parameters.

OBJECTIVE: Analyze the importance of biochemical data and their relationship with anthropometric data in the longitudinal nutritional assessment of very low birth weight infants. METHODS: A prospective cohort study was performed on 55 very low birth weight preterm infants (birth weight < 1.500 g and < 37 weeks of gestational age). Measurements of weight, length, head and mid-arm circumferences, mid-arm circumference: head circumference ratio, ponderal index, and body mass index. Serum prealbumin and retinol-binding protein were studied as biochemical parameters. All variables were collected at birth and days 14 and 28 of life. RESULTS: The infants presented a mean birth weight of 1,076.7 +/- 286 g and mean gestational age of 30.7 +/- 2.1 weeks. At birth, the mean serum prealbumin was 7.0 +/-1.7 mg/dl and mean retinol-binding protein was 1.3 +/- 0.4 mg/dl. There was a significant increase in all variables studied from birth to day 28. According to nutritional adequacy, there were no differences between appropriate and small for gestational age infants neither in the anthropometric nor in the biochemical data. The anthropometric measurements did not correlate with biochemical parameters. The serum protein concentrations were converted to serum protein mass (SPM) as follows: SPM = serum protein concentration X (100 X weight) X (1- hematocrit)] since the studied proteins are largely intravascular and the protein mass would be a more accurate index of nutritional status. The SPM of both protein and anthropometric parameters were correlated, except for the ponderal index. CONCLUSIONS: The serum protein mass of the prealbumin and the retinol-binding protein were better nutritional markers in the serial nutritional assessment of very low birth weight infants during neonatal period than the serum protein levels.

Increased and localized accumulation of chondroitin sulphate proteoglycans in the hyperplastic human prostate.

OBJECTIVE: To analyse by immunohistochemistry the expression of chondroitin sulphate (CS) (detected in the hyperplastic prostate and possibly affecting the proliferation of prostate cells) in benign prostatic hyperplasia (BPH) to determine its distribution and location. MATERIALS AND METHODS: Samples of BPH were obtained from 11 patients (aged 58-83 years) and controls consisting of the transitional zone of five prostates from young men aged 19-27 years. Tissue sections were labelled with antibodies against CS, perlecan, type IV collagen, laminin, and smooth muscle cell (SMC) alpha-actin. The amount of CS immunostaining was estimated by semi-quantitative scoring and correlated with prostate-specific antigen (PSA) level and prostate size. RESULTS: The anti-CS antibody faintly stained the stroma of normal prostates, but in BPH samples the staining was intense and concentrated around acini, including the periphery of adjacent SMCs. This staining pattern was totally absent in the normal samples. Type IV collagen, perlecan and laminin were homogeneously distributed in the whole stroma of both normal and BPH samples. There was no significant correlation between intensity of CS staining and either PSA or prostate size. CONCLUSIONS: The expression of CS proteoglycans is increased in BPH, where they co-locate with basement membranes of the acinar epithelium and of peri-acinar SMCs. This enhanced expression is specific for these proteoglycans, as other basement membrane components are unaffected, and this may result from the regulatory effects of local factors that are active in BPH.

Organization and relative content of smooth muscle cells, collagen and elastic fibers in the corpus cavernosum of rat penis.

PURPOSE: The corpus cavernosum smooth muscle and extracellular matrix are essential for normal penile erection and are implicated in erectile dysfunction. Although investigations of these issues have used the rat corpus cavernosum, organization of its components is to date not well known. We characterized and quantified the smooth muscle cells and the main extracellular matrix components of the rat corpus cavernosum. MATERIALS AND METHODS: Collagen, elastic fibers and smooth muscle cells were stained on paraffin sections of rat penises using sirius red and Gomori's reticulin, Weigert's resorcin-fuchsin and an anti-smooth muscle cells alpha-actin antibody, respectively. Stained components were then quantified by computer aided morphometry. RESULTS: Smooth muscle cells were restricted to the subendothelial space of corpus cavernosum and had a volumetric density of 9.1%. Collagen was thick, usually in transversely oriented bundles and was the most abundant component of the trabeculae with a volumetric density of 62.7%. Gomori's reticulin disclosed a meshwork of fibrils also in the subendothelial space but did not stain the thicker bundles. Volumetric density of elastic fibers was 4.9%, and at the periphery of the corpus cavernosum the fibers were parallel to the long axis of the penis, while in deeper regions most of them were transversely oriented and at different directions from those of collagen. CONCLUSIONS: Rat corpus cavernosum differs from that of humans by lesser amounts of smooth muscle cells, greater amounts of collagen and the presence of fibrillar collagen and smooth muscle cell subendothelial layers. Therefore, these differences should be considered when using the rat penis for studies on erection.

Proximal insertion of gubernaculum testis in normal human fetuses and in boys with cryptorchidism.

PURPOSE: We determine how the proximal gubernaculum testis is attached to the testis and epididymis in human fetuses, and compare these data with findings in boys who had undergone surgery for cryptorchidism. MATERIALS AND METHODS: We analyzed 280 testes and epididymides with the gubernacula of 140 well preserved, fresh human fetuses ranging from 10 to 35 weeks after conception with no detectable congenital malformations and 36 undescended testes of 28 boys 2 to 15 years old (mean age 6.8) who had undergone surgery for cryptorchidism. In both groups the different conformations of the relationship among the proximal gubernaculum, testis and epididymis were classified according to a system used for patients with cryptorchidism. In group A the gubernaculum is attached to the testis and epididymis, in group B the gubernaculum is attached only to the testis with a tail disjunction epididymal anomaly, in group C the gubernaculum is attached only to the testis with total disjunction of the epididymis, in group D the gubernaculum is attached only to the epididymal tail and in group E there are no attachments among gubernaculum, testis and epididymis. RESULTS: Of the 280 fetal testes studied 194 (69.2%) were in the abdomen, 38 (13. 57%) in the inguinal canal and 48 (17.14%) in the scrotum. There were 277 cases (98.9%) in group A and 3 (1.1%) in group B. Of the 36 undescended testes analyzed 2 (5.6%) were abdominal and 34 (94.4%) were inguinal. There were 26 cases (72.2%) in group A, 8 (22.2%) in group B and 2 in group D. CONCLUSIONS: In fetuses without congenital malformations or epididymal alterations, such as tail disjunction or elongated epididymis, the proximal portion of the gubernaculum was attached to the testis and epididymis in all cases. In undescended testes there was an increased incidence of paratesticular structure malformations accompanied by gubernacular attachment anomalies compared to the testes in normal fetuses.

Collagen and elastic fibers in the penis of human fetuses at 28 weeks postconception.

OBJECTIVES: The extracellular matrix is a key element in penile function and pathology, yet little is known of its development. Herein we investigated the morphological organization of collagen and elastin in the corpora cavernosa and tunica albuginea of human fetuses. MATERIAL AND METHODS: The penises from 5 fresh human fetuses at 28 weeks postconception (WPC) were routinely fixed and embedded, and all staining procedures were carried out on paraffin sections. Collagen was evidenced by staining with: (1) Gomori's trichrome; (2) sirius red, followed by observation under polarized light, and (3) an antihuman collagen type-III antibody. Elastin and the whole elastic system were revealed using an antihuman elastin antibody and Weigert's resorcin fucsin, respectively. RESULTS: At this stage of fetal development, the albuginea is formed predominantly by dense bundles of collagen. Near the corpora cavernosa, the presence of type-III collagen was also observed. Weigert staining showed numerous fibers of the elastic system in the albuginea. Type-III collagen was found to be strongly positive in the cavernous trabeculae and in the connective sheath surrounding the central artery. Using Weigert staining and an immunolabeling method with primary antibody against human elastin, we found an important quantity of elastic system fibers in the trabeculae of the corpora cavernosa. CONCLUSION: In fetuses at 28 WPC the albuginea is formed predominantly by dense bundles of collagen. The trabecular structures of the corpora cavernosa present a significant quantity of type-III collagen and elastic system fibers.

Detection and analysis of apoptosis in peripheral blood cells from breast cancer patients.

Apoptosis is a well-known specific process of cell death that normally occurs in physiological situations such as tissue or organ development and involution. During tumor growth there is a balance between proliferation and cell death which involves apoptotic mechanisms. In the present study genomic DNAs from 120 breast tumor biopsies were analyzed by agarose gel electrophoresis and none of them presented the fragmentation pattern characteristic of the apoptosis process. However, 33% of the 105 breast cancer patients clearly showed the apoptotic pattern when DNA from blood cells was analyzed. None of the DNAs from healthy volunteer blood cells showed any trace of apoptosis. Since the breast cancer patients were not receiving chemo- or hormone therapy, the possible relationship between blood cortisol levels and the apoptotic pattern found in patient blood cells was investigated. Using a chemoluminescence immunodetection assay, similar cortisol levels were observed in breast cancer patient sera presenting or not apoptotic blood cells and in healthy volunteer sera. Analysis of the clinical data obtained from 60 of these patients showed that patients bearing tumors of smaller size (under 20 mm) were more susceptible to the apoptotic effect in blood cells. According to the Elston grade, it was observed that 7 of 12 patients with grade III tumors (58%) presented apoptotic peripheral blood cells, in contrast to 10 of 48 patients with grade I and grade II tumors. These observations may reflect the immunosuppression characteristic of some breast cancer patients, which may contribute to tumor growth. Therefore, further studies are necessary to elucidate the factor(s) involved in such massive blood cell death.

Detection and analysis of apoptosis in peripheral blood cells from breast cancer patients

Apoptosis is a well-known specific process of cell death that normally occurs in physiological situations such as tissue or organ development and involution. During tumor growth there is a balance between proliferation and cell death which involves apoptotic mechanisms. In the present study genomic DNAs from 120 breast tumor biopsies were analyzed by agarose gel electrophoresis and none of them presented the fragmentation pattern characteristic of the apoptosis process. However, 33 per cent of the 105 breast cancer patients clearly showed the apoptotic pattern when DNA from blood cells was analyzed. None of the DNAs from healthy volunteer blood cells showed any trace of apoptosis. Since the breast cancer patients were not receiving chemo- or hormone therapy, the possible relationship between blood cortisol levels and the apoptotic pattern found in patient blood cells was investigated. Using a chemoluminescence immunodetection assay, similar cortisol levels were observed in breast cancer patient sera presenting or not apoptotic blood cells and in healthy volunteer sera. Analysis of the clinical data obtained from 60 of these patients showed that patients bearing tumors of smaller size (under 20 mm) were more susceptible to the apoptotic effect in blood cells. According to the Elston grade, it was observed that 7 of 12 patients with grade III tumors (58 per cent) presented apoptotic peripheral blood cells, in contrast to 10 of 48 patients with grade I and grade II tumors. These observations may reflect the immunosuppression characteristic of some breast cancer patients, which may contribute to tumor growth. Therefore, further studies are necessary to elucidate the factor(s) involved in such massive blood cell death.

[Respiratory distress in low birth weight: influence of intrauterine growth retardation]. / Insuficiência respiratória em recém-nascidos de baixo peso: influência do retardo de crescimento intra-uterino.

The aim of this study is to evaluate the influence of intra-uterine growth retardation (IUGR), as well as it's types and severity on the development of respiratory distress among low-birth-weight infants. A total of 673 neonates were studied, the small-for-dates infants (SFD), 40% of total, were divided according to the type of IUGR, in proportionate and disproportionate, and according to the severity, in birth weight below 3rd and between 3rd and 10th percentile. Respiratory distress was more frequent among the appropriate for gestational age (57.3%) compared to the SFD infants (33.7%), (p < 0.0001), and among males (52.6%) compared to females (47.4%) (p = 0.01). There was an inverse relationship between gestational age, as well as birth weight and respiratory distress. It occurred in 90.6% of very-low-birth-weight infants and in 39% of the others, with a predominance among the appropriate for gestational age newborns. Respiratory distress occurred in 80% of neonates below 34 weeks of gestational age and in 26% of the neonates above it (p < 0.0001). Regarding to the small-for-dates infants, respiratory distress occurred more frequently among the disproportionate (42.5%), when compared to proportionate infants (28.1%) (p = 0.03). The severity of IUGR had no influence on these results. The authors concluded that among low birth weight infants, the groups with increased risk for respiratory distress are the appropriate for gestational age and, among small-for-dates and disproportionate infants, those weighing less than 1500 g.

Compositional and structural alterations of arterial glycosaminoglycans associated with the complications brought about by thalassemia major. A case report.

A biochemical analysis of glycosaminoglycans was performed in arteries of a fifteen-year old white male who died of beta thalassemia major. The patient presented the severe clinical complications resulting from hemochromatosis, which was evidenced at autopsy and by histologic examination. The arteries under study comprised the thoracic and abdominal aortas and the iliac and pulmonary arteries, which were compared with the same arteries from normal individuals. Data on total glycosaminoglycan and total collagen, including the determination of the relative contents of the different glycosaminoglycans, suggest an as yet undescribed fibrotic process in the thalassemic arteries. Also altered were the proportions of the disaccharides making up chondroitin sulfate and heparin sulfate. A reduction in the molecular weight of arterial heparin sulfate, presumably with free radical involvement, was also detected. All these changes in the extracellular matrix may be ascribed to the presence of large amounts of iron in the tissue, and as such they should be expected in other disorders with chronic iron overload.

Arterial connective tissue alterations in thalassemia major: a biochemical study of a case with severe iron overload.

A biochemical analysis of glycosaminoglycans and collagen was performed in arteries of a 15-year old teenager who died of beta thalassemia major. The patient presented the severe clinical complications resulting from hemochromatosis, which was evidenced at autopsy and by histological examination. The arteries under study comprised the thoracic and abdominal aortas and the iliac and pulmonary arteries, which were compared with the same arteries from normal individuals. Data on total glycosaminoglycan and total collagen, including the determination of the relative contents of the different glycosaminoglycans, suggest an as yet undescribed fibrotic process in the thalassemic arteries. Also altered were the proportions of the disaccharides making up chondroitin sulfate and heparan sulfate. A reduction in the molecular weight of arterial heparan sulfate, presumably with free radical involvement, was also detected. These changes in the extracellular matrix may be ascribed to the presence of large amounts of iron in the tissue, and as such they should be expected in other disorders with chronic iron overload.

Glycosaminoglycan fractions from human arteries presenting diverse susceptibilities to atherosclerosis have different binding affinities to plasma LDL.

The topographic distribution of atherosclerotic lesions is influenced by biochemical factors intrinsic to the arterial wall. In the present work we have investigated whether the composition/chemical structure of glycosaminoglycans constitutes one of these factors. Normal human arteries were obtained at necropsy, and in order of decreasing susceptibility to atherosclerosis, consisted of the abdominal and thoracic aortas and the iliac and pulmonary arteries. The results showed similar concentrations of total glycosaminoglycan and collagen. Of the glycosaminoglycans known to interact with low-density lipoprotein (LDL), dermatan sulfate was present in all arteries in comparable concentrations, but the aortas had a 30% higher content of chondroitin 4/6-sulfate, which in turn was slightly enriched in 6-sulfated disaccharide units. LDL-affinity chromatography with dermatan sulfate+chondroitin 4/6-sulfate fractions demonstrated that increasing affinity to LDL matched an increasing susceptibility to atherosclerosis. Analysis of glycosaminoglycans in the eluates indicated a positive correlation between affinity to LDL and increasing molecular weight and the existence of a fraction of glycosaminoglycans of high affinity to LDL in the aortas only. These results suggest that arterial glycosaminoglycans participate in the multifactorial mechanisms that modulate the differential localization of atherosclerotic lesions.

A comparative analysis of glycosaminoglycans from human umbilical arteries in normal subjects and in pathological conditions affecting pregnancy.

BACKGROUND: Vascular cells express different phenotypes in adult and fetal vessels, and the extracellular matrix they synthesize should reflect these differences. Alterations of vascular proteoglycan/glycosaminoglycan is verified in disorders such as hypertension and diabetes, and when occurring during pregnancy, they bring about structural changes to fetal vessels that often lead to impaired fetus growth. Yet there is little data about the extracellular matrix of an important human fetal vessel, the umbilical artery. EXPERIMENTAL DESIGN: This study involved the biochemical characterization of the extracellular matrix of normal umbilical arteries, umbilical arteries from complicated pregnancies (maternal hypertension and diabetes and intrauterine growth retardation syndrome), and, for purpose of comparison, normal adult arteries (aorta and iliac and pulmonary arteries). Although the collagen types I:III ratio was determined in some cases, emphasis was placed on analysis of glycosaminoglycans. RESULTS: Normal umbilical arteries differ from normal adult arteries in that they contain greater concentrations of hyaluronic acid and lesser concentrations of heparan sulfate and chondroitin 4- and 6-sulfate. The umbilical artery also differs from adult arteries in the disaccharide composition of its chondroitin and heparan sulfates and in the molecular weight of this latter glycosaminoglycan. The glycosaminoglycan distribution in umbilical arteries derived from complicated pregnancies is roughly similar to that of controls. However, total glycosaminoglycan and collagen were significantly reduced, and the collagen I:III ratio was increased in the umbilical arteries from hypertension-complicated pregnancies. CONCLUSIONS: The glycosaminoglycan composition of the normal umbilical artery, a fully differentiated tissue, differs in many aspects from that of normal adult arteries. Of the cases of complicated pregnancies studied, the extracellular matrix of umbilical arteries was altered only in maternal hypertension. The changes, notably a mild fibrosis, were not very pronounced and should not impair hemodynamic properties of the vessel.