Role of gelatinases MMP-2 and MMP-9 in tissue remodeling following acute lung injury

Acute lung injury is characterized by a severe disruption of alveolo-capillary structures and includes a variety of changes in lung cell populations. Evidence suggests the occurrence of rupture of the basement membranes and interstitial matrix remodeling during acute lung injury. The dynamic equilibrium of the extracellular matrix (ECM) under physiological conditions is a consequence of the balance between the regulation of synthesis and degradation of ECM components. Matrix metalloproteinases (MMPs) represent a group of enzymes involved in the degradation of most of the components of the ECM and therefore participate in tissue remodeling associated with pathological situations such as acute lung injury. MMP activity is regulated by proteolytic activation of the latent secreted proenzyme and by interaction with specific tissue inhibitors of metalloproteinases. This review details our knowledge of the involvement of MMPs, namely MMP-2 and MMP-9, in acute lung injury and acute respiratory distress syndrome.

Standardization of a fluorimetric assay for the determination of tissue angiotensin-converting enzyme activity in rats

The tripeptide Hip-His-Leu was used to standardize a fluorimetric method to measure tissue angiotensin-converting enzyme (ACE) activity in rats. The fluorescence of the o-phthaldialdehyde-His-Leu adduct was compared in the presence and absence of the homogenate (25 µl) to determine whether the homogenate from different tissues interfered with the fluorimetric determination of the His-Leu product. Only homogenates from lung and renal medulla and cortex showed significantly altered fluorescence intensity. To overcome this problem, the homogenate from these tissues were diluted 10 times with assay buffer. The specificity of the assay was demonstrated by the inhibition of ACE activity with 3 µM enalaprilat (MK-422). There was a linear relationship between product formation and incubation time for up to 90 min for homogenates of renal cortex and medulla and liver, for up to 60 min for ventricles and adrenals and for up to 30 min for the aorta, lung and atrium homogenates. In addition, there was a linear relationship between product formation and the amount of protein in the homogenates within the following range: lung, 30-600 µg; renal cortex and medulla, 40-400 µg; atrium and ventricles, 20-200 µg; adrenal, 20-100 µg; aorta, 5-100 µg; liver, 5-25 µg. No peptidase activity against the His-Leu product (31 nmol), assayed in borate buffer (BB), was detected in the different homogenates except the liver homogenate, which was inhibited by 0.1 mM r-chloromercuribenzoic acid. ACE activity in BB was higher than in phosphate buffer (PB) due, at least in part, to a greater hydrolysis of the His-Leu product in PB. ACE activity of lung increased 20% when BB plus Triton was used. Enzyme activity was stable when the homogenates were stored at -20o or -70oC for at least 30 days. These results indicate a condition whereby ACE activity can be easily and efficiently assayed in rat tissue samples homogenized in BB using a fluorimetric method with Hip-His-Leu as a substrate.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

Expression and biological activity of two recombinant polypeptides related to subunit 1 of the interferon-a receptor

Abnormal production of interferon alpha (IFN-a) has been found in certain autoimmune diseases and can be also observed after prolonged therapy with IFN-a. IFN-a can contribute to the pathogenesis of allograft rejection in bone marrow transplants. Therefore, the development of IFN-a inhibitors as a soluble receptor protein may be valuable for the therapeutic control of these diseases. We have expressed two polypeptides encoding amino acids 93-260 (P1) and 261-410 (P2) of the extracellular domain of subunit 1 of the interferon-a receptor (IFNAR 1-EC) in E. coli. The activities of the recombinant polypeptides and of their respective antibodies were evaluated using antiproliferative and antiviral assays. Expression of P1 and P2 polypeptides was achieved by transformation of cloned plasmid pRSET A into E. coli BL21(DE3)pLysS and by IPTG induction. P1 and P2 were purified by serial sonication steps and by gel filtration chromatography with 8 M urea and refolded by dialysis. Under reducing SDS-PAGE conditions, the molecular weight of P1 and P2 was 22 and 17 kDa, respectively. Polyclonal anti-P1 and anti-P2 antibodies were produced in mice. P1 and P2 and their respective polyclonal antibodies were able to block the antiproliferative activity of 6.25 nM IFN-aB on Daudi cells, but did not block IFN-aB activity at higher concentrations (>6.25 nM). On the other hand, the polypeptides and their respective antibodies did not inhibit the antiviral activity of IFN-aB on Hep 2/c cells challenged with encephalomyocarditis virus.

Treatment of cytomegalovirus retinitis with an intraocular sustained-release ganciclovir implant

The objective of this prospective study was to evaluate the efficacy and complications of the use of an intraocular sustained-release ganciclovir implant for the treatment of active cytomegalovirus (CMV) retinitis in AIDS patients. Thirty-nine eyes of 26 patients were submitted to ocular surgery. All patients underwent complete ocular examination before and after surgery. The surgical procedure was always done under local anesthesia using the same technique. The mean time for the surgical procedure was 20 min (range, 15 to 30 min). The average follow-up period was 3.7 months. Of all patient, only 4 presented recurrence of retinitis after 8, 8, 9 and 2 months, respectively. Three of them received a successful second implant. All 39 eyes of the 26 patients presented healing of retinitis as shown by clinical improvement evaluated by indirect binocular ophthalmoscopy and retinography. Retinitis healed within a period of 4 to 6 weeks in all patients, with clinical regression signs from the third week on. Six (15.4%) eyes developed retinal detachment. None of the patients developed CMV retinitis in the contralateral eye. The intraocular implant proved to be effective in controlling the progression of retinitis for a period of up to 8 months even in patients for whom systemic therapy with either ganciclovir or foscarnet or both had failed. The intraocular sustained-release ganciclovir implant proved to be a safe new procedure for the treatment of CMV retinitis, avoiding the systemic side effects caused by the intravenous medications and improving the quality of life of the patients.

Amifostine (WR-2721), a cytoprotective agent during high-dose cyclophosphamide treatment of non-Hodgkin's lymphomas: a phase II study

Clinical trials indicate that amifostine may confer protection on various normal tissues without attenuating anti-tumor response. When administered prior to chemotherapy or radiotherapy, it may provide a broad spectrum of cytoprotection including against alkylating drugs. The mechanism of protection resides in the metabolism at normal tissue site by membrane-bound alkaline phosphatase. Toxicity of this drug is moderate with hypotension, nausea and vomiting, and hypocalcemia being observed. We report a phase II study using amifostine as a protective drug against high-dose cyclophosphamide (HDCY) (7 g/m2), used to mobilize peripheral blood progenitor cells (PBPC) and to reduce tumor burden. We enrolled 29 patients, 22 (75.9%) affected by aggressive and 7 (24.1%) by indolent non-Hodgkin's lymphoma (NHL), who were submitted to 58 infusions of amifostine and compared them with a historical group (33 patients) affected by aggressive NHL and treated with VACOP-B followed by HDCY. The most important results in favor of amifostine were the reduction of intensity of cardiac, pulmonary and hepatic toxicity, and a significant reduction of frequency and severity of mucositis (P = 0.04). None of the 29 patients died in the protected group, while in the historical group 2/33 patients died because of cardiac or pulmonary toxicity and 2 patients stopped therapy due to toxicity. Amifostine did not prevent the aplastic phase following HDCY. PBPC collection and hematological recovery were adequate in both groups. The number of CFU-GM (colony-forming units-granulocyte/macrophage) colonies and mononuclear cells in the apheresis products was significantly higher in the amifostine group (P = 0.02 and 0.01, respectively). Side effects were mild and easily controlled. We conclude that amifostine protection should be useful in HDCY to protect normal tissues, with acceptable side effects.

Familial predisposition to hypertension and the association between urinary sodium excretion and blood pressure in a population-based sample of young adults

The reasons for the inconsistent association between salt consumption and blood pressure levels observed in within-society surveys are not known. A total of 157 normotensive subjects aged 18 to 35 years, selected at random in a cross-sectional population-based survey, answered a structured questionnaire. They were classified as strongly predisposed to hypertension when two or more first-degree relatives had a diagnosis of hypertension. Anthropometric parameters were obtained and sitting blood pressure was determined with aneroid sphygmomanometers. Sodium and potassium excretion was measured by flame spectrophotometry in an overnight urine sample. A positive correlation between blood pressure and urinary sodium excretion was detected only in the group of individuals strongly predisposed to hypertension, both for systolic blood pressure (r = 0.51, P<0.01) and diastolic blood pressure (r = 0.50, P<0.01). In a covariance analysis, after controlling for age, skin color and body mass index, individuals strongly predisposed to hypertension who excreted amounts of sodium above the median of the entire sample had higher systolic and diastolic blood pressure than subjects classified into the remaining conditions. The influence of familial predisposition to hypertension on the association between salt intake and blood pressure may be an additional explanation for the weak association between urinary sodium excretion and blood pressure observed in within-population studies, since it can influence the association between salt consumption and blood pressure in some but not all inhabitants.

Time sequence of changes in the responsiveness of glycogen breakdown to adrenergic agonists in perfused liver of rats with insulin-induced hypoglycemia

The time-course changes of the responsiveness of glycogen breakdown to a- and ß-adrenergic agonists during insulin-induced hypoglycemia (IIH) were investigated. Blood glucose levels were decreased prior to the alteration in the hepatic responsiveness to adrenergic agonists. The activation of hepatic glucose production and glycogenolysis by phenylephrine (2 µM) and isoproterenol (20 µM) was decreased in IIH. The changes in the responsiveness of glycogen catabolism were first observed for isoproterenol and later for phenylephrine. Hepatic ß-adrenergic receptors showed a higher degree of adrenergic desensitization than did a-receptors. Liver glycogen synthase activity, glycogen content and the catabolic effect of dibutyryl cyclic AMP (the ß-receptor second messenger) were not affected by IIH.

The hemolytic component of cancer anemia: effects of osmotic and metabolic stress on the erythrocytes of rats bearing multifocal inoculations of the Walker 256 tumor

Cancer anemia is classified as an anemia of chronic diseases, although it is sometimes the first symptom of cancer. Cancer anemia includes a hemolytic component, important in the terminal stage when even transfused cells are rapidly destroyed. The presence of a chronic component and the terminal complications of the illness limit studies of the hemolytic component. A multifocal model of tumor growth was used here to simulate the terminal metastatic dissemination stage (several simultaneous inoculations of Walker 256 cells). The hemolytic component of anemia began 3-4 days after inoculation in 100% of the rats and progressed rapidly thereafter: Hb levels dropped from 14.9 ± 0.02 to 8.7 ± 0.06 from days 7 to 11 (~5 times the physiologically normal rate in rats) in the absence of bleeding. The development of anemia was correlated (r2 = 0.86) with the development of other systemic effects such as anorexia. There was a significant decrease in the osmotic fragility of circulating erythrocytes: the NaCl concentration causing 50% lysis was reduced from 4.52 ± 0.06 to 4.10 ± 0.01 (P<0.01) on day 7, indicating a reduction in erythrocyte volume. However, with mild metabolic stress (4-h incubation at 37oC), the erythrocytes showed a greater increase in osmotic fragility than the controls, suggesting marked alteration of erythrocyte homeostasis. These effects may be due to primary plasma membrane alterations (transport and/or permeability) and/or may be secondary to metabolic changes. This multifocal model is adequate for studying the hemolytic component of cancer anemia since it is rapid, highly reproducible and causes minimal animal suffering.

The use of filter paper plasticized with polyvinyl alcohol-glutaraldehyde in ELISA

F1-antigen purified from Yersinia pestis was covalently linked to 5-mm diameter filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde. These discs were used both for ELISA and dot-ELISA for the detection of anti-F1 IgG in rabbits. The best conditions were achieved using 1.25 µg of F1 antigen/disc, 3% w/v skim milk in PBS as blocking agent, anti-IgG peroxidase conjugate diluted 12,000 times, and serum from rabbits immunized or not against Y. pestis, diluted 6,400 times. The absorbance values obtained from the comparative study between this procedure and conventional ELISA were not significantly different but the low cost of the reagents employed in ELISA using the filter paper discs plasticized with polyvinyl alcohol-glutaraldehyde makes this method economically attractive.

Infusions of AP5 into the basolateral amygdala impair the formation, but not the expression, of step-down inhibitory avoidance

We evaluated the effects of infusions of the NMDA receptor antagonist D,L-2-amino-5-phosphonopentanoic acid (AP5) into the basolateral nucleus of the amygdala (BLA) on the formation and expression of memory for inhibitory avoidance. Adult male Wistar rats (215-300 g) were implanted under thionembutal anesthesia (30 mg/kg, ip) with 9.0-mm guide cannulae aimed 1.0 mm above the BLA. Bilateral infusions of AP5 (5.0 µg) were given 10 min prior to training, immediately after training, or 10 min prior to testing in a step-down inhibitory avoidance task (0.3 mA footshock, 24-h interval between training and the retention test session). Both pre- and post-training infusions of AP5 blocked retention test performance. When given prior to the test, AP5 did not affect retention. AP5 did not affect training performance, and a control experiment showed that the impairing effects were not due to alterations in footshock sensitivity. The results suggest that NMDA receptor activation in the BLA is involved in the formation, but not the expression, of memory for inhibitory avoidance in rats. However, the results do not necessarily imply that the role of NMDA receptors in the BLA is to mediate long-term storage of fear-motivated memory within the amygdala.

Protective effect of policosanol on atherosclerotic lesions in rabbits with exogenous hypercholesterolemia

Policosanol is a mixture of higher aliphatic alcohols purified from sugar cane wax, with cholesterol-lowering effects demonstrable in experimental models and in patients with type II hypercholesterolemia. The protective effects of policosanol on atherosclerotic lesions experimentally induced by lipofundin in rabbits and rats and spontaneously developed in stumptail monkeys have been described. The present study was conducted to determine whether policosanol administered orally to rabbits with exogenous hypercholesterolemia also protects against the development of atherosclerotic lesions. Male New Zealand rabbits weighing 1.5 to 2 kg were randomly divided into three experimental groups which received 25 or 200 mg/kg policosanol (N = 7) orally for 60 days with acacia gum as vehicle or acacia gum alone (control group, N = 9). All animals received a cholesterol-rich diet (0.5%) during the entire period. Control animals developed marked hypercholesterolemia, macroscopic lesions and arterial intimal thickening. Intima thickness was significantly less (32.5 ± 7 and 25.4 ± 4 µm) in hypercholesterolemic rabbits treated with policosanol than in controls (57.6 ± 9 µm). In most policosanol-treated animals, atherosclerotic lesions were not present, and in others, thickness of fatty streaks had less foam cell layers than in controls. We conclude that policosanol has a protective effect on the atherosclerotic lesions occurring in this experimental model.

Novel methods for the encapsulation of meglumine antimoniate into liposomes

The antimonial drug, meglumine antimoniate, was successfully encapsulated in dehydration-rehydration vesicles and in freeze-dried empty liposomes (FDELs). High encapsulation efficiencies (from 28 to 58%) and low weight ratios of lipids to encapsulated antimony (from 1:0.15 to 1:0.3) were achieved. These formulations, contrary to those obtained by conventional methods, can be stored as intermediate lyophilized forms and reconstituted just before use. The efficacy of FDEL-encapsulated meglumine antimoniate was evaluated in hamsters experimentally infected with Leishmania chagasi. A significant reduction of liver parasite burdens was observed in animals treated with this preparation, when compared to control animals treated with empty liposomes. In contrast, free meglumine antimoniate was found to be inefficient when administered at a comparable dose of antimony. This novel liposome-based meglumine antimoniate formulation appears to be promising as a pharmaceutical product for the treatment of visceral leishmaniasis.

Dietary sodium intake induced myenteric neuron hypertrophy in Wistar rats

In the present study we investigated the effect of salt intake on myenteric neuron size of the colon of adult male Wistar rats. The animals were placed on either a high-salt (HS; 8%; 12 animals) or a low-salt diet (LS; 0.15%; 12 animals) for 15 or 52 weeks and blood pressure was measured. The sizes of myenteric neurons of the distal colon from both groups were measured. No difference in neuron size was observed between the HS and LS groups after 15 weeks. After 52 weeks on HS, neuron size was increased (P<0.005) when compared with the LS group. The rats also presented hypertension, which was significantly different at 52 weeks (142 ± 11 vs 119 ± 7 mmHg). These results suggest that a long time on an HS diet can significantly increase myenteric nerve cell size.

Mapping cancer, cardiovascular and malaria research in Brazil

This paper presents performance indicators for the Brazilian cancer, cardiovascular and malaria research areas from 1981 to 1995. The data show an increasing number of papers since 1981 and author numbers indicate a continuous growth of the scientific community and suggest an expected impact of scientific activity on biomedical education. The data also characterize cardiovascular research as a well-established area and cancer research as a faster growing consolidating field. The 1989-1994 share of Brazilian articles among world publications shows a growing trend for the cancer (1.61) and cardiovascular (1.59) areas, and a decrease for the malaria area (0.89). The burden of the three diseases on society is contrasted by the small number of consolidated Brazilian research groups, and a questionable balance of thematic activity, especially with regard to malaria. Brazilian periodicals play an important role in increasing the international visibility of science produced in the country. Cancer and cardiovascular research is strongly concentrated in the Southeastern and in Southern regions of Brazil, especially in São Paulo (at least one address from São Paulo in 64.5% of the 962 cancer articles and in 66.9% of the 2250 cardiovascular articles, the second state being Rio de Janeiro with at least one address in 14.1 and 11% of those articles, respectively). Malaria research (468 articles) is more evenly distributed across the country, following the pattern of the endemic distribution of the disease. Surveying these national indicator trends can be useful to establish policies in the decision process about health sciences, medical education and public health.