Synergistic effect of mycophenolate mofetil and angiotensin-converting enzyme inhibitor in patients with chronic allograft nephropathy

Experimental data and few clinical non-randomized studies have shown that inhibition of the renin-angiotensin system by angiotensin-converting enzyme (ACE) associated or not with the use of mycophenolate mofetil (MMF) could delay or even halt the progression of chronic allograft nephropathy (CAN). In this retrospective historical study, we investigated whether ACE inhibition (ACEI) associated or not with the use of MMF has the same effect in humans as in experimental studies and what factors are associated with a clinical response. A total of 160 transplant patients with biopsy-proven CAN were enrolled. Eighty-one of them were on ACE therapy (G1) and 80 on ACEI_free therapy (G2). Patients were further stratified for the use of MMF. G1 patients showed a marked decrease in proteinuria and stabilized serum creatinine with time. Five-year graft survival after CAN diagnosis was more frequent in G1 (86.9 vs 67.7%; P < 0.05). In patients on ACEI-free therapy, the use of MMF was associated with better graft survival. The use of ACEI therapy protected 79% of the patients against graft loss (OR = 0.079, 95%CI = 0.015-0.426; P = 0.003). ACEI and MMF or the use of MMF alone after CAN diagnosis conferred protection against graft loss. This finding is well correlated with experimental studies in which ACEI and MMF interrupt the progression of chronic allograft dysfunction and injury. The use of ACEI alone or in combination with MMF significantly reduced proteinuria and stabilized serum creatinine, consequently improving renal allograft survival.

Association of angiotensin-converting enzyme activity and polymorphism with echocardiographic measures in familial and nonfamilial hypertrophic cardiomyopathy

Angiotensin-converting enzyme (ACE) activity and polymorphism contribute significantly to the prognosis of patients with cardiomyopathy. The aim of this study was to determine the activity and type of ACE polymorphism in patients with familial and nonfamilial hypertrophic cardiomyopathy (HCM) and to correlate these with echocardiographic measurements (echo-Doppler). We studied 136 patients (76 males) with HCM (69 familial and 67 nonfamilial cases). Mean age was 41 ± 17 years. DNA was extracted from blood samples for the polymerase chain reaction and the determination of plasma ACE levels. Left ventricular mass, interventricular septum, and wall thickness were measured. Mean left ventricular mass index, interventricular septum and wall thickness in familial and nonfamilial forms were 154 ± 63 and 174 ± 57 g/m² (P = 0.008), 19 ± 5 and 21 ± 5 mm (P = 0.02), and 10 ± 2 and 12 ± 3 mm (P = 0.0001), respectively. ACE genotype frequencies were DD = 35%, ID = 52%, and II = 13%. A positive association was observed between serum ACE activity and left ventricular mass index (P = 0.04). Logistic regression showed that ACE activity was twice as high in patients with familial HCM and left ventricular mass index ≥190 g/m² compared with the nonfamilial form (P = 0.02). No other correlation was observed between ACE polymorphisms and the degree of myocardial hypertrophy. In conclusion, ACE activity, but not ACE polymorphisms, was associated with the degree of myocardial hypertrophy in the patients with HCM.

A DNA enzyme targeting Egr-1 inhibits rat vascular smooth muscle cell proliferation by down-regulation of cyclin D1 and TGF-β1

We have demonstrated that a synthetic DNA enzyme targeting early growth response factor-1 (Egr-1) can inhibit neointimal hyperplasia following vascular injury. However, the detailed mechanism of this inhibition is not known. Thus, the objective of the present study was to further investigate potential inhibitory mechanisms. Catalytic DNA (ED5) and scrambled control DNA enzyme (ED5SCR) were synthesized and transfected into primary cultures of rat vascular smooth muscle cells (VSMCs). VSMC proliferation and DNA synthesis were analyzed by the MTT method and BrdU staining, respectively. Egr-1, TGF-β1, p53, p21, Bax, and cyclin D1 expression was detected by RT-PCR and Western blot. Apoptosis and cell cycle assays were performed by FACS. Green fluorescence could be seen localized in the cytoplasm of 70.6 ± 1.52 and 72 ± 2.73% VSMCs 24 h after transfection of FITC-labeled ED5 and ED5SCR, respectively. We found that transfection with ED5 significantly inhibited cultured VSMC proliferation in vitro after 24, 48, and 72 h of serum stimulation, and also effectively decreased the uptake of BrdU by VSMC. ED5 specifically reduced serum-induced Egr-1 expression in VSMCs, further down-regulated the expression of cyclin D1 and TGF-β1, and arrested the cells at G0/G1, inhibiting entry into the S phase. FACS analysis indicated that there was no significant difference in the rate of apoptosis between ED5- and ED5SCR-transfected cells. Thus, ED5 can specifically inhibit Egr-1 expression, and probably inhibits VSMC proliferation by down-regulating the expressions of cyclin D1 and TGF-β1. However, ED5 has no effect on VSMC apoptosis.

Genetic polymorphism of alcohol-metabolizing enzyme and alcohol dependence in Polish men

Alcohol dependence poses a serious medical and sociological problem. It is influenced by multiple environmental and genetic factors, which may determine differences in alcohol metabolism. Genetic polymorphism of the enzymes involved in alcohol metabolism is highly ethnically and race dependent. The purpose of this study was to investigate the differences, if present, in the allele and genotype frequency of alcohol dehydrogenase 1B (ADH1B), ADH1C and the microsomal ethanol-oxidizing system (MEOS/CYP2E1) between alcohol-dependent individuals and controls and also to determine if these genotypes cause a difference in the age at which the patients become alcohol dependent. The allele and genotype frequencies of ADH1B, ADH1C, and CYP2E1 were determined in 204 alcohol dependent men and 172 healthy volunteers who do not drink alcohol (control group). Genotyping was performed by PCR-RFLP methods on white cell DNA. ADH1B*1 (99.3%) and ADH1C*1 (62.5%) alleles and ADH1B*1/*1 (N = 201) and ADH1C*1/*1 (N = 85) genotypes were statistically more frequent among alcohol-dependent subjects than among controls (99.3 and 62.5%, N = 201 and 85 vs 94.5 and 40.7%, N = 153 and 32, respectively). Differences in the CYP2E1 allele and genotype distribution between groups were not significant. The persons with ADH1C*1/*1 and CYP2E1*c1/*c2 genotypes became alcohol dependent at a considerably younger age than the subjects with ADH1C*1/*2, ADH1C*2/*2 and CYP2E1*c1/*c1 genotypes (28.08, 25.67 years vs 36.0, 45.05, 34.45 years, respectively). In the Polish men examined, ADH1C*1 and ADH1B*1 alleles and ADH1C*1/*1 and ADH1B*1/*1 genotypes favor alcohol dependence. The ADH1B*2 allele may protect from alcohol dependence. However, subjects with ADH1C*1/*1 and CYP2E1*c1/*c2 genotypes become alcohol dependent at a considerably younger age than the subjects with ADH1C*1/*2, ADH1C*2/*2 and CYP2E1*c1/*c1 genotypes.

Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3

Epithelium, a highly dynamic system, plays a key role in the homeostasis of the intestine. However, thus far a human intestinal epithelial cell line has not been established in many countries. Fetal tissue was selected to generate viable cell cultures for its sterile condition, effective generation, and differentiated character. The purpose of the present study was to culture human intestinal epithelial cells by a relatively simple method. Thermolysin was added to improve the yield of epithelial cells, while endothelin-3 was added to stimulate their growth. By adding endothelin-3, the achievement ratio (viable cell cultures/total cultures) was enhanced to 60% of a total of 10 cultures (initiated from 8 distinct fetal small intestines), allowing the generation of viable epithelial cell cultures. Western blot, real-time PCR and immunofluorescent staining showed that cytokeratins 8, 18 and mouse intestinal mucosa-1/39 had high expression levels in human intestinal epithelial cells. Differentiated markers such as sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV also showed high expression levels in human intestinal epithelial cells. Differentiated human intestinal epithelial cells, with the expression of surface markers (cytokeratins 8, 18 and mouse intestinal mucosa-1/39) and secretion of cytokines (sucrase-isomaltase, aminopeptidase N and dipeptidylpeptidase IV), may be cultured by the thermolysin and endothelin-3 method and maintained for at least 20 passages. This is relatively simple, requiring no sophisticated techniques or instruments, and may have a number of varied applications.

Functional changes of dendritic cells in hypersensivity reactions to amoxicillin

A better understanding of dendritic cell (DC) involvement in responses to haptenic drugs is needed, because it represents a possible approach to the development of an in vitro test, which could identify patients prone to drug allergies. There are two main DC subsets: plasmacytoid DC (pDC) and myeloid DC (mDC). β-lactams form hapten-carrier conjugates and may provide a suitable model to study DC behavior in drug allergy reactions. It has been demonstrated that drugs interact differently with DC in drug allergic and non-allergic patients, but there are no studies regarding these subsets. Our aim was to assess the functional changes of mDC and pDC harvested from an amoxicillin-hypersensitive 32-year-old woman who experienced a severe maculopapular exanthema as reflected in interleukin-6 (IL-6) production after stimulation with this drug and penicillin. We also aim to demonstrate, for the first time, the feasibility of this method for dendritic cell isolation followed by in vitro stimulation for studies of drug allergy physiopathology. DC were harvested using a double Percoll density gradient, which generates a basophil-depleted cell (BDC) suspension. Further, pDC were isolated by blood DC antigen 4-positive magnetic selection and gravity filtration through magnetized columns. After stimulation with amoxicillin, penicillin and positive and negative controls, IL-6 production was measured by ELISA. A positive dose-response curve for IL-6 after stimulation with amoxicillin and penicillin was observed for pDC, but not for mDC or BDC suspension. These preliminary results demonstrate the feasibility of this methodology to expand the knowledge of the effect of dendritic cell activation by drug allergens.

Construction of a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen and mouse 4-1BBL genes and its effect on dendritic cells

Our aim was to construct a recombinant adenovirus co-expressing truncated human prostate-specific membrane antigen (tPSMA) and mouse 4-1BBL genes and to determine its effect on dendritic cells (DCs) generated from bone marrow suspensions harvested from C57BL/6 mice for which the effect of 4-1BBL on DCs is not clear, especially during DCs processing tumor-associated antigen. Replication deficient adenovirus AdMaxTM Expression System was used to construct recombinant adenovirus Ad-tPSMA-internal ribosome entry site-mouse 4-1BBL (Ad-tPSMA-IRES-m4-1BBL) and Ad-enhanced green fluorescent protein. Day 7 proliferating DC aggregates generated from C57BL/6 mice were collected as immature DCs and further mature DCs were obtained by lipopolysaccharide activated immature DCs. After DCs were exposed to the recombinant adenovirus with 250 multiplicity of infection, the expression of tPSMA and m4-1BBL proteins were detected by Western blot, and the apoptosis and phenotype of DCs were analyzed by flow cytometry. Cytokines (IL-6 and IL-12) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Proliferation of T cells was detected by allogeneic mixed lymphocyte reactions. The tPSMA and m4-1BBL proteins were expressed correctly. The apoptosis rate of DCs transfected with Ad-tPSMA-IRES-m4-1BBL was 14.6%, lower than that of control DCs. The expression of co-stimulatory molecules [CD80 (81.6 ± 5.4%) and CD86 (80.13 ± 2.81%)] up-regulated in Ad-tPSMA-IRES-m4-1BBL-pulsed DCs, and the level of IL-6 (3960.2 ± 50.54 pg/mL) and IL-12 (249.57 ± 12.51 pg/mL) production in Ad-tPSMA-IRES-m4-1BBL-transduced DCs were significantly higher (P < 0.05) than those in control DCs. Ad-tPSMA-IRES-m4-1BBL induced higher T-cell proliferation (OD450 = 0.614 ± 0.018), indicating that this recombinant adenovirus can effectively enhance the activity of DCs.

The chronic blockade of angiotensin I-converting enzyme eliminates the sex differences of serum cytokine levels of spontaneously hypertensive rats

Sex hormones modulate the action of both cytokines and the renin-angiotensin system. However, the effects of angiotensin I-converting enzyme (ACE) on the proinflammatory and anti-inflammatory cytokine levels in male and female spontaneously hypertensive rats (SHR) are unclear. We determined the relationship between ACE activity, cytokine levels and sex differences in SHR. Female (F) and male (M) SHR were divided into 4 experimental groups each (n = 7): sham + vehicle (SV), sham + enalapril (10 mg/kg body weight by gavage), castrated + vehicle, and castrated + enalapril. Treatment began 21 days after castration and continued for 30 days. Serum cytokine levels (ELISA) and ACE activity (fluorimetry) were measured. Male rats exhibited a higher serum ACE activity than female rats. Castration reduced serum ACE in males but did not affect it in females. Enalapril reduced serum ACE in all groups. IL-10 (FSV = 16.4 ± 1.1 pg/mL; MSV = 12.8 ± 1.2 pg/mL), TNF-α (FSV = 16.6 ± 1.2 pg/mL; MSV = 12.8 ± 1 pg/mL) and IL-6 (FSV = 10.3 ± 0.2 pg/mL; MSV = 7.2 ± 0.2 pg/mL) levels were higher in females than in males. Ovariectomy reduced all cytokine levels and orchiectomy reduced IL-6 but increased IL-10 concentrations in males. Castration eliminated the differences in all inflammatory cytokine levels (IL-6 and TNF-α) between males and females. Enalapril increased IL-10 in all groups and reduced IL-6 in SV rats. In conclusion, serum ACE inhibition by enalapril eliminated the sexual dimorphisms of cytokine levels in SV animals, which suggests that enalapril exerts systemic anti-inflammatory and anti-hypertensive effects.

Maternal protein restriction affects gene expression and enzyme activity of intestinal disaccharidases in adult rat offspring

This study investigated the consequences of intrauterine protein restriction on the gastrointestinal tract and particularly on the gene expression and activity of intestinal disaccharidases in the adult offspring. Wistar rat dams were fed isocaloric diets containing 6% protein (restricted, n = 8) or 17% protein (control, n = 8) throughout gestation. Male offspring (n = 5-8 in each group) were evaluated at 3 or 16 weeks of age. Maternal protein restriction during pregnancy produced offspring with growth restriction from birth (5.7 ± 0.1 vs 6.3 ± 0.1 g; mean ± SE) to weaning (42.4 ± 1.3 vs 49.1 ± 1.6 g), although at 16 weeks of age their body weight was similar to control (421.7 ± 8.9 and 428.5 ± 8.5 g). Maternal protein restriction also increased lactase activity in the proximal (0.23 ± 0.02vs 0.15 ± 0.02), medial (0.30 ± 0.06vs 0.14 ± 0.01) and distal (0.43 ± 0.07vs 0.07 ± 0.02 U·g-1·min-1) small intestine, and mRNA lactase abundance in the proximal intestine (7.96 ± 1.11vs 2.38 ± 0.47 relative units) of 3-week-old offspring rats. In addition, maternal protein restriction increased sucrase activity (1.20 ± 0.02 vs 0.91 ± 0.02 U·g-1·min-1) and sucrase mRNA abundance (4.48 ± 0.51 vs 1.95 ± 0.17 relative units) in the duodenum of 16-week-old rats. In conclusion, the present study shows for the first time that intrauterine protein restriction affects gene expression of intestinal enzymes in offspring.

Activation of endogenous angiotensin converting enzyme 2 prevents early injuries induced by hyperglycemia in rat retina

Diabetic retinopathy (DR) is a serious complication of diabetes mellitus that may result in blindness. We evaluated the effects of activation of endogenous angiotensin converting enzyme (ACE) 2 on the early stages of DR. Rats were administered an intravenous injection of streptozotocin to induce hyperglycemia. The ACE2 activator 1-[[2-(dimethylamino) ethyl] amino]-4-(hydroxymethyl)-7-[[(4-methylphenyl) sulfonyl] oxy]-9H-xanthone 9 (XNT) was administered by daily gavage. The death of retinal ganglion cells (RGC) was evaluated in histological sections, and retinal ACE2, caspase-3, and vascular endothelial growth factor (VEGF) expressions were analyzed by immunohistochemistry. XNT treatment increased ACE2 expression in retinas of hyperglycemic (HG) rats (control: 13.81±2.71 area%; HG: 14.29±4.30 area%; HG+XNT: 26.87±1.86 area%; P<0.05). Importantly, ACE2 activation significantly increased the RCG number in comparison with HG animals (control: 553.5±14.29; HG: 530.8±10.3 cells; HG+XNT: 575.3±16.5 cells; P<0.05). This effect was accompanied by a reduction in the expression of caspase-3 in RGC of the HG+XNT group when compared with untreated HG rats (control: 18.74±1.59; HG: 38.39±3.39 area%; HG+XNT: 27.83±2.80 area%; P<0.05). Treatment with XNT did not alter the VEGF expression in HG animals (P>0.05). Altogether, these findings indicate that activation of ACE2 reduced the death of retinal ganglion cells by apoptosis in HG rats.

Deoxynivalenol (DON) degradation and peroxidase enzyme activity in submerged fermentation

This work aims to evaluate deoxynivalenol degradation by Aspergillus oryzae and Rhizopus oryzae in a submerged fermentation system and to correlate it to the activity of oxydo-reductase enzymes. The submerged medium consisted of sterile distilled water contaminated with 50 μg of DON and 4 × 10(6) spore.mL-1 inoculum of Aspergillus oryzae and Rhizopus oryzae species, respectively in each experiment. Sampling was performed every 24 hours for monitoring the peroxidase specific activity, and every 48 hours for determining mycotoxin levels. Results showed that the fungi species were able to decrease DON levels as the peroxidase activity increased. The 48 hours fermentation interval presented the highest peroxidase specific activity (ΔABS/minute.μg.protein-1), 800 and 198, while the highest DON degradation velocity was 10.8 and 12.4 ppb/hour, respectively in both cases for Rhizopus oryzae and Aspergillus oryzae.

Lipase and esterase: to what extent can this classification be applied accurately?

Enzyme technology is an ever-growing field of knowledge and, in recent years, this technology has raised renewed interest, due to the search for new paradigms in several productive processes. Lipases, esterases and cutinases are enzymes used in a wide range of processes involving synthesis and hydrolysis reactions. The objective of this work was to investigate and compare the specific lipase and esterase activities of five enzymes - four already classified as lipases and one classified as cutinase - in the presence of natural and synthetic substrates. All tested enzymes presented both esterase and lipase specific activities. The highest specific esterase activity was observed for Aspergillus 1068 lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate, while the highest specific lipase activity was observed for Geotrichum sp. lipase in natural substrate and for F. oxysporum cutinase in synthetic substrate. These results display some interface-independent lipolytic activity for all lipases tested. This is in accordance with the rationale that a new and broader definition of lipases may be necessary.

Characterization of polyphenol oxidase in two cocoa (Theobroma cacao L.) cultivars produced in the south of Bahia, Brazil

Abstract The reactions leading to the formation of precursors of chocolate flavor are performed by endogenous enzymes present in the cocoa seed. Polyphenol oxidase (PPO) presence and activity during fermentation of cocoa beans is responsible for the development of flavor precursors and is also implicated in the reduction of bitterness and astringency. However, the reliability of cocoa enzyme activities is complicated due to variations in different genotypes, geographical origins and methods of fermentation. In addition, there is still a lack of systematic studies comparing different cocoa cultivars. So, the present study was designed to characterize the activity of PPO in the pulp and seeds of two cocoa cultivars, PH 16 and TSH 1188. The PPO activity was determined spectrophotometrically and characterized as the optimal substrate concentration, pH and temperature and the results were correlated with the conditions during the fermentation process. The results showed the specificity and differences between the two cocoa cultivars and between the pulp and seeds of each cultivar. It is suggested that specific criteria must be adopted for each cultivar, based on the optimal PPO parameters, to prolong the period of maximum PPO activity during fermentation, contributing to the improvement of the quality of cocoa beans.

Lactose intolerance and cow's milk protein allergy

Abstract Adverse reactions to food intake have very diverse etiology and symptomatology. Regarding milk, its food allergy is presented as lactose intolerance, the sugar in milk, or allergy to milk protein. Despite having different symptomatology, confusions among allergic conditions to dairy and its mediators are common. Milk protein allergy originates from protein components present in milk, causing reactions to either the protein fractions in emulsion (caseins) or in whey (milk albumin). The allergic reaction is type IV mediated by T lymphocytes. The allergic reaction produces severe cellular damage and it triggers physical, mental and emotional symptomatology that may vary in time, intensity and severity. Lactose intolerance is originated by total or partial absence of the enzyme that digests this disaccharide. Lactose intolerance can be primary or congenital and secondary; the former being more rare and severe, the latter being more common. Lactase deficiency can be diagnosed by symptoms associated with cramping and diarrhea. Thus, the objective of this study was to conduct a review of available literature on cow’s milk protein allergy and lactose intolerance.

Influence of the degree of hydrolysis and type of enzyme on antioxidant activity of okara protein hydrolysates

Abstract The objective of this work was to evaluate the antioxidant activity of protein hydrolysates obtained by the enzymatic hydrolysis of okara using an endopeptidase (Alcalase) and exopeptidase (Flavourzyme). The reaction was monitored by the pH-stat procedure in which five aliquots were collected during the hydrolysis by each enzyme, corresponding to different degrees of hydrolysis (DH). The antioxidant activities of the aliquots were evaluated by the ABTS, DPPH and FRAP methods. For the hydrolysates obtained using Alcalase, the antioxidant activities increased from: 68.6 to 99.5% (ABTS), 14.5 to 17.7% (DPPH) and 222.6 to 684.9 µM Trolox (FRAP), when the DH varied from 0 to 33.6%. With respect to Flavourzyme, the results were: 67.2 to 88.2% (ABTS), 9.5 to 18.5% (DPPH) and 168.0 to 360.3 µM Trolox (FRAP), when the DH increased up to 5.8%. The results showed that the protein hydrolysates had antioxidant capacities, which were influenced by the degree of hydrolysis and the type of enzyme.