Long-term effects of human amniotic membrane in a rat model of biliary fibrosis.

Liver fibrosis is the most common outcome of chronic liver diseases, and its progression to cirrhosis can only be effectively treated with liver transplantation. The amniotic membrane (AM) has been studied as an alternative therapy for fibrosis diseases mainly for its favorable properties, including anti-inflammatory, anti-scaring and immunomodulatory properties. It was recently demonstrated that the AM reduces the progression of biliary fibrosis to its advanced stage, cirrhosis, when applied on the liver for 6 weeks after fibrosis induction. Here, we investigated the effects of AM on rat fibrotic liver, during a prolonged period of time. Fibrosis was induced by bile duct ligation (BDL), and at the same time, a fragment of AM was applied around the liver. After 1, 3, 6, and 9 weeks, the degree of fibrosis was assessed by qualitative Knodell scoring, and by quantitative image analysis to quantify the area of collagen deposition in hepatic tissue. While fibrosis progressed rapidly in untreated BDL animals, leading to cirrhosis within 6 weeks, AM-treated livers showed confined fibrosis at the periportal area with few and thin fibrotic septa, but without cirrhosis. In addition, collagen deposition was reduced to about 36 and 55% of levels observed in BDL at 6 and 9 weeks after BDL, respectively, which shows that the longer the period of AM application, the lower the collagen deposition. These results suggested that AM applied as a patch onto the liver surface for longer periods attenuated the severity of biliary fibrosis and protected against liver degeneration caused by excessive collagen deposition.

Long-term effects of human amniotic membrane in a rat model of biliary fibrosis

Liver fibrosis is the most common outcome of chronic liver diseases, and its progression to cirrhosis can only be effectively treated with liver transplantation. The amniotic membrane (AM) has been studied as an alternative therapy for fibrosis diseases mainly for its favorable properties, including anti-inflammatory, anti-scaring and immunomodulatory properties. It was recently demonstrated that the AM reduces the progression of biliary fibrosis to its advanced stage, cirrhosis, when applied on the liver for 6 weeks after fibrosis induction. Here, we investigated the effects of AM on rat fibrotic liver, during a prolonged period of time. Fibrosis was induced by bile duct ligation (BDL), and at the same time, a fragment of AM was applied around the liver. After 1, 3, 6, and 9 weeks, the degree of fibrosis was assessed by qualitative Knodell scoring, and by quantitative image analysis to quantify the area of collagen deposition in hepatic tissue. While fibrosis progressed rapidly in untreated BDL animals, leading to cirrhosis within 6 weeks, AM-treated livers showed confined fibrosis at the periportal area with few and thin fibrotic septa, but without cirrhosis. In addition, collagen deposition was reduced to about 36 and 55% of levels observed in BDL at 6 and 9 weeks after BDL, respectively, which shows that the longer the period of AM application, the lower the collagen deposition. These results suggested that AM applied as a patch onto the liver surface for longer periods attenuated the severity of biliary fibrosis and protected against liver degeneration caused by excessive collagen deposition.

A putative role for telocytes in placental barrier impairment during preeclampsia.

Preeclampsia (PE) is a major health problem occurring in pregnant women and the principal cause of maternal morbidity and perinatal mortality. It is characterized by alteration of the extravilli trophoblast cell migration toward the endometrial spiral arteries with a concomitant reduction in maternal blood flow in the placenta. This result in a state of ischemia-hypoxia which triggers an oxidative stress stage with production of reactive oxygen species. A cascade of cellular and molecular events leads then to endothelial dysfunction, transduction pathway signal disruption and induction of apoptosis and necrosis mechanisms and therefore a significant reduction in the amount of nutrients required for normal fetal development. Placental anchoring chorionic and stem villi present a skeleton of myofibroblasts arranged in parallel disposition to its longitudinal axis. The intraplacental blood volume is controlled by the contraction/relaxation of these myofibroblasts, promoting the delivery of nutrients and metabolites to the fetus. Recently, a new mesodermal originated cell type has been described in the villous stroma, the so named "telocytes". These cells are strategically located between the smooth muscle cells of the blood vessel wall and the myofibroblasts, and it is reasonable to hypothesize that they may play a pacemaker role, as in the intestine. This study provide new information supporting the notion that the occurrence of oxidative stress in PE is not only related to endothelial dysfunction and apoptosis of the trophoblast cells, but also involves telocytes and its putative role in the regulation of fetal blood flow and the intra-placental blood volume. Some ideas aimed at dilucidating the relationship between placental failure and the behavior of telocytes in pathological organs in adulthood, are also discussed.

Prediction of early and late pre-eclampsia from maternal characteristics, uterine artery Doppler and markers of vasculogenesis during first trimester of pregnancy.

OBJECTIVE: To develop a predictive model for pre-eclampsia using clinical, biochemical and ultrasound markers during the first trimester of pregnancy. METHODS: This was a nested case-control study within a pre-eclampsia screening project that involved 5367 asymptomatic pregnant women undergoing routine transvaginal uterine artery (UtA) Doppler at 11 + 0 to 13 + 6 weeks. Following exclusions, there were 70 pregnant women who later developed pre-eclampsia and 289 control patients enrolled during the first trimester who had serum or plasma samples taken at enrolment available for the purposes of this study. Of these, 17 pregnancies were diagnosed with early-onset (delivery < 34 weeks) pre-eclampsia and 53 with late-onset (delivery ≥ 34 weeks) pre-eclampsia. The lowest, highest and mean of left and right UtA pulsatility indices (PI) were calculated. Blood samples were stored at -84 °C until biochemical analysis for markers of vasculogenesis was performed. The distributions of the lowest UtA-PI and the biochemical markers were adjusted for maternal characteristics, expressed as multiples of the median (MoM), and compared between groups. Logistic regression analysis was used to evaluate if any variable was significantly associated with pre-eclampsia. RESULTS: Pregnancies that later developed pre-eclampsia were associated with higher maternal prepregnancy body mass index. An increased lowest UtA-PI was significantly associated with both early- and late-onset disease. Placental growth factor (PlGF) MoM was significantly reduced in women who later developed early- or late-onset pre-eclampsia compared with controls (median (interquartile range), 0.69 (0.33-1.46) and 1.10 (0.39-1.56), respectively, vs 1.19 (0.65-1.84), P < 0.05). Different combined models were generated by logistic regression analysis, and the detection rate with a fixed 10% false-positive rate was 47% and 29% for early- and late-onset pre-eclampsia, respectively. CONCLUSION: Pregnancies that later developed early or late pre-eclampsia were characterized by impaired placentation and an anti-angiogenic state during the first trimester of pregnancy. Regression models which include maternal characteristics, UtA Doppler and PlGF can apparently predict approximately half of pregnancies that will be complicated by early-onset pre-eclampsia. We believe more research in several areas is needed to aid in the creation of a better and more population-specific screening test for pre-eclampsia during the first trimester of pregnancy.

Specific inhibition of protein kinase Cbeta expression by antisense RNA affects the activation of Jurkat T lymphoma cells.

Antisense RNA technology was employed to specifically inhibit the expression of the protein kinase Cbeta (PKCbeta) isoform in Jurkat cells, to explore its influence on the expression of surface antigens (CD69) and the cytokines interleukin-8 (IL-8), tumour necrosis factor (TNF)-alpha and beta, and to characterise its controversial involvement in the expression of IL-2 and its receptor (IL-2R). Transfection of cells with an antisense PKCbeta construct (as-PKCbeta-pREP3) significantly increased IL-2R/CD25 expression in phorbol 12-myristate 13-acetate (PMA)-stimulated as-PKCbeta-pREP3 transfectants, in contrast to Jurkat cells transfected with a control as-PKCalpha-pREP3 plasmid. IL-2 production, in contrast, was strongly inhibited in both transfectant populations stimulated by PMA plus the calcium ionophore ionomycin. Three clones (asb1/asb2/asb3), selected from as-PKCbeta-pREP3 transfectants, showed decreased PKCbeta protein levels (40 percent, 50 percent and 60 percent, respectively, as determined by western blotting) and mRNA levels. The specific inhibition was confirmed in immunoblots for other PKC (alpha, delta, epsilon, gamma, theta, and lambda lambda/tau) isoforms and in immunoprecipitates from representative (c2/asb2) clones. Stimulation of PKCbeta-depleted clones significantly increased CD25 expression but decreased IL-2 production (similarly to as-PKCbeta-pREP3 transfectants) and IL-2 message levels. CD69 expression and IL-8 secretion were significantly decreased, but TNFbeta message levels were highly increased in asb2/asb3 clones, without affecting TNFalpha secretion. Analysis of the mitogen-activated protein kinase (MAP Kinase) signalling pathway showed unaltered extracellular signal regulated kinase 1/2 (ERK1/2) and p38 phosphorylation but increased activation of c-Jun N-terminal kinase (JNK1) and its substrate, the transcription factor ATF-2 (activated transcription factor-2), which are involved in IL-2 gene expression. Our results revealed new PKCbeta functions, affecting CD69 expression and IL-8 production, and support the requirement for PKCbeta in IL-2 secretion/transcription and IL-2R regulation.

VEGF in the muscular layer of placental blood vessels: immuno-expression in preeclampsia and intrauterine growth restriction and its association with the antioxidant status.

The pathophysiology of preeclampsia (PE), a disorder occurring in 5% of all pregnancies, remains largely unknown, but early placental hypoxia and oxidative stress are known to be involved in the mechanism of the syndrome. Maternal plasma and placental tissue samples were collected from PE, intrauterine growth restriction (IUGR), and normotensive pregnant patients. The immunohistochemical expression of vascular endothelial growth factor (VEGF), malondialdehyde (MDA) production and the activity of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase GSH-Px) were determined in the placental tissue. F2-isoprostane concentration and the ferric reducing ability of plasma (FRAP) were determined in maternal plasma. We found that the PE and IUGR groups showed a higher expression of VEGF in the muscular layer of fetal chorionic vessels. In addition, increased plasma F2 isoprostane levels and a significant reduction of FRAP in the plasma of PE women, as well as a lower activity of SOD in PE placentas and a higher activity of GSH-Px in IUGR placentas were found. Additionally, lower PlGF and higher sFlt1 levels were observed in the maternal plasma of PE and IUGR than control. We concluded that in a hypoxic environment, the placenta expresses VEGF in the muscular layer of fetal vessels. The development of PE could be related to the increased expression of VEGF, with decreased placental SOD activity and a decrease of both plasma F2-isoprostane and FRAP levels. In turn, the development of IUGR could be related to the association of decreased plasma FRAP levels and increased placental GSH-Px activity.

Repercussões da insuficiência renal crônica na capacidade de exercício, estado nutricional, função pulmonar e musculatura respiratória de crianças e adolescentes / Consequences of chronic renal insufficiency on the exercise capacity, nutritional status, pulmonary function and respiratory musculature of children and adolescents

OBJETIVO: Avaliar a capacidade funcional, função pulmonar, musculatura respiratória e estado nutricional de crianças e adolescentes portadores de insuficiência renal crônica (IRC) em tratamento conservador. MÉTODOS: Este estudo foi realizado com 30 voluntários, divididos em dois grupos: Portadores de IRC em tratamento conservador (Grupo IRC) e grupo sem comprometimento da função renal (Grupo Controle). Os voluntários foram submetidos à avaliação fisioterapêutica, espirometria, avaliação de força e resistência da musculatura respiratória, do estado nutricional e da capacidade funcional. Para a análise dos dados, foi utilizado o teste de Mann-Whitney com nível de significância de 5 por cento. RESULTADOS: No Grupo IRC, o índice de Tiffeneau foi significativamente menor (p= 0,003). Em relação à função muscular respiratória, os valores de pressão expiratória máxima foram menores (p= 0,010) e os valores do tempo do teste de resistência, maiores (p= 0,003). Na avaliação funcional, as variáveis que diferiram estatisticamente foram: menor distância caminhada (p< 0,001) e maior pressão arterial média (p< 0,001); freqüência respiratória final (p< 0,001) e escala de Borg (p= 0,048). Quanto ao estado nutricional, todas as variáveis, estatisticamente significativas, foram menores. CONCLUSÕES: Crianças e adolescentes portadores IRC, em tratamento conservador, podem apresentar alterações importantes da capacidade funcional, musculatura respiratória e estado nutricional.

OBJECTIVE: To evaluate functional capacity, pulmonary function, respiratory musculature and nutritional status among children and adolescents with chronic renal insufficiency (CRI) undergoing conservative treatment. METHODS: This study was conducted with 30 volunteers, divided into two groups: a group of children and adolescents with CRI undergoing conservative therapy (CRI Group) and a group without renal disease (Control Group). The volunteers underwent physical therapy evaluation, spirometry, strength and resistance tests on their respiratory musculature, nutritional status evaluation and functional capacity assessment. The data were analyzed using the Mann-Whitney test with a significance level of 5 percent. RESULTS: The Tiffeneau index was significantly lower in the CRI Group (p= 0.003). In relation to respiratory muscle function, the maximum expiratory pressure values were lower (p= 0.010) and the time values of the resistance test were greater (p= 0.003). In the functional assessment, the variables that differed statistically were: lower distance walked (p< 0.001), greater mean arterial pressure (p< 0.001), final respiratory rate (p< 0.001) and Borg scale (p= 0.048). Regarding nutritional status, all the statistically significant variables were lower. CONCLUSIONS: Children and adolescents with CRI undergoing conservative treatment may present significantly impaired functional capacity, respiratory musculature and nutritional status.

Photoacoustic monitoring of the absorption of isotonic saline solution by human mucus.

Viscosity and elasticity are the fundamental rheologic properties of respiratory mucus, and are important determinants of transportability of mucus in the mucociliary system. One technique that permits to monitor indirectly the rheologic properties of any sample is the photoacoustic technique. Using that technique, the absorption of isotonic saline solution by human mucus was monitored. The mucus was obtained from 11 volunteers, divided into two groups: five volunteers presenting pneumology symptoms (group I) and six healthy volunteers (group II). The photoacoustic signal of the mucus absorbing the saline solution was monitored as function of time, with measurements being performed each 10 min, up to 120 min. The resulting curves were fitted to sigmoidal curves to simulate the evolution on time of the photoacoustic signal. A characteristic time for the half saturation of the absorption process was obtained. For group I the time obtained was 23.3+/-5.3 min and for group II the time obtained was 55.0+/-7.7 min, both means being significantly different (Student t-test, p<0.05). This result supports the empirical practice of treating individuals presenting symptoms of airway obstruction with about 30 min of inhalations of isotonic saline solution vapor for the clearance of the airways.

Increased immunohistochemical expression of thrombomodulin at placental perivascular myofibroblast in severe preeclampsia (PE).

The presence of pro-coagulant and anti-coagulant components of the placental vascular endothelium and syncytiotrophoblast are essential for homeostasis. Vascular endothelium prevents blood clot formation in vivo by involving a cell surface thrombin-binding glycoprotein, thrombomodulin (TM), that activates plasma anti-coagulant protein C. The TM levels increase during pregnancy, but the fibrinolytic capacity diminishes. Since vascular lesions with placental coagulation disorders can be associated with preeclampsia (PE), we hypothesized that TM expression in the stem villous vasculature and syncytiotrophoblast of the placenta are impaired in PE. Plasma and placental tissue samples were collected from PE (n=12) and normotensive pregnant patients (n=11). Patient's gestational age was 35.7+/-1.2 (normotensive) and 30.6+/-1.5 weeks (PE). Blood samples were drawn 30 min before delivery. Serum PAI-1 and PAI-2 antigens were determined by enzyme-linked immunoabsorbent assay (ELISA). A monoclonal antibody specific for TM was used for immunohistochemical tissue staining (ABC) and the staining was quantified by semi quantitative scores. Results show no intensity differences at the apical syncytiotrophoblast between the two groups. However, in preeclamptic placenta, TM expression diminished in the endothelium of the stem villi arteries and increased in the perivascular and stromal myofibroblats in cases of severe PE. TM changes were associated with an increased PAI-1/PAI-2 ratio. It is suggested that in severe PE, the decreased placental blood flow may be due to structural and functional impairment of the endothelium of the stem villi vessels and the surrounding perivascular and stromal myofibroblast, by increasing TM expression which may modulate fetal blow flow in the villous tree.

Distribution of choline acetyltransferase immunoreactivity in the brain of an elasmobranch, the lesser spotted dogfish (Scyliorhinus canicula).

Although the distribution of cholinergic cells is remarkably similar across the vertebrate species, no data are available on more primitive species, such as cartilaginous fishes. To extend the evolutionary analysis of the cholinergic systems, we studied the distribution of cholinergic neurons in the brain and rostral spinal cord of Scyliorhinus canicula by immunocytochemistry using an antibody against the enzyme choline acetyltransferase (ChAT). Western blot analysis of brain extracts of dogfish, sturgeon, trout, and rat showed that this antibody recognized similar bands in the four species. Putative cholinergic neurons were observed in most brain regions, including the telencephalon, diencephalon, cerebellum, and brainstem. In the retrobulbar region and superficial dorsal pallium of the telencephalon, numerous small pallial cells were ChAT-like immunoreactive. In addition, tufted cells of the olfactory bulb and some cells in the lateral pallium showed faint immunoreactivity. In the preoptic-hypothalamic region, ChAT-immunoreactive (ChAT-ir) cells were found in the preoptic nucleus, the vascular organ of the terminal lamina, and a small population in the caudal tuber. In the epithalamus, the pineal photoreceptors were intensely positive. Many cells of the habenula were faintly ChAT-ir, but the neuropil of the interpeduncular nucleus showed intense ChAT immunoreactivity. In the pretectal region, ChAT-ir cells were observed only in the superficial pretectal nucleus. In the brainstem, the somatomotor and branchiomotor nuclei, the octavolateral efferent nucleus, and a cell group just rostral to the Edinger-Westphal (EW) nucleus contained ChAT-ir neurons. In addition, the trigeminal mesencephalic nucleus, the nucleus G of the isthmus, some locus coeruleus cells, and some cell populations of the vestibular nuclei and of the electroreceptive nucleus of the octavolateral region exhibited ChAT immunoreactivity. In the reticular areas of the brainstem, the nucleus of the medial longitudinal fascicle, many reticular neurons of the rhombencephalon, and cells of the nucleus of the lateral funiculus were immunoreactive to this antibody. In the cerebellum, Golgi cells of the granule cell layer and some cells of the cerebellar nucleus were also ChAT-ir. In the rostral spinal cord, ChAT immunoreactivity was observed in cells of the motor column, the dorsal horn, the marginal nucleus (a putative stretch-receptor organ), and in interstitial cells of the ventral funiculus. These results demonstrate for the first time that cholinergic neurons are distributed widely in the central nervous system of elasmobranchs and that their cholinergic systems have evolved several characteristics that are unique to this group.

Effect of 1,25(OH)(2)-vitamin D(3) on the activation of natural killer cells: role of protein kinase C and extracellular calcium.

As a first approach for studying the implication of PKC and the steroid hormone 1,25(OH)(2)-vitamin D(3) [1,25(OH)(2)D(3)] on natural killer cell (NK) activity, we analyzed in the YT NK cell line the expression of PKC isoforms and the effects of 1, 25(OH)(2)D(3) on BLT-esterase (a marker of NK lytic granules) activity. Western blot and RT-PCR showed a greater extent of PKC alpha, beta, delta, zeta, epsilon, theta, and lambda and lower levels of PKC mu and eta. In a dose-dependent manner 1, 25(OH)(2)D(3) induced significant increases in BLT-esterase and PKC activities and the stimulatory effect on BLT-esterase activity was mimicked and blocked, respectively, by the PKC activator phorbol ester PMA and PKC inhibitors (H7, PKC(19-36), and N-myristoylated PKC(19-31) peptides). Moreover, the effects of 1,25(OH)(2)D(3) on BLT-esterase could be blocked in a Ca(2+)-free (+EGTA) medium and mimicked by the Ca2+ ionophore A23187. The results suggest that 1, 25(OH)(2)D(3) is a stimulatory factor of NK activity acting through a mechanism involving PKC and extracellular Ca2+.

Inhibition of protein kinase C alpha expression by antisense RNA in transfected Jurkat cells.

Taking the antisense approach to inhibit the expression of specific protein kinase C (PKC) isoforms, we investigated the function of PKC alpha in T cell activation by transfecting Jurkat cells with an episomal vector (pREP3) containing a copy of the corresponding gene in the antisense orientation. Transfected Jurkat cells were selected with hygromycin and cloned by limiting dilution. Two (as1/as2) stably transfected antisense PKC alpha-pREP3 clones (as PKC alpha-pREP3) exhibited consistently reductions (76% and 85%, respectively) of PKC alpha levels when analyzed by immunoblotting and immunoprecipitation and also of PKC alpha mRNA (75%, as determined by Northern blotting) when compared to control clones (C1/C2) containing the pREP3 vector alone. The ability of the as-PKC alpha-pREP3 construct to specifically reduce PKC alpha levels in both clones was demonstrated by Western blots probed with antibodies against the PKC beta isozyme (the form structurally more similar to PKC alpha) and other representative isoenzymes expressed in Jurkat cells (PKC delta, epsilon, theta, and mu). Stimulation of transfected Jurkat clones with phorbol-12-myristate-13 alone or in the presence of ionomycin resulted in significant reduction of IL-2R alpha expression, TNF-alpha production, and the induction of transcriptional activity of a pIL-2/Luc construct in both as PKC alpha-reduced clones. The magnitude of these decrements paralleled the reductions of PKC alpha expression. The loss of the effects in clone as1 after a high number of passages correlated with the recovery of normal levels of PKC alpha protein, suggesting a link between these processes. Thus, the findings of this study demonstrate the essential role that PKC alpha plays in major events of the T lymphocyte activation process.

Endothelial cell markers in chronic uremia: relationship with hemostatic defects and severity of renal failure.

Plasma von Willebrand factor antigen, soluble thrombomodulin, and tissue factor were increased in 31 patients with severe chronic renal failure (creatinine clearance <20 ml/min) under conservative treatment, whereas plasminogen activator inhibitor antigen did not differ significantly from healthy controls. No correlation among plasma levels of these proteins was found. Three patterns of relationship between endothelial cell markers and hemostatic defects were identified: 1) Plasma thrombomodulin, a marker of endothelium damage, was found an independent predictor of bleeding time and platelet aggregation, and secretion defects, and was also related to the severity of renal failure; 2) von Willebrand factor antigen, an index of endothelial cell activation and secretion, was significantly correlated with intravascular markers of thrombin and plasmin generation and with platelet adenosine triphosphate content, but not with plasma creatinine levels; and 3) tissue factor and plasminogen activator inhibitor antigen levels were not statistically correlated with the diverse hemostatic defects. Activation of coagulation and fibrinolysis, secondary to endothelial cell activation, appearing early during the evolution of chronic renal failure, is pathogenically related to the platelet dysfunction, and probably to development of atherosclerosis and thrombotic events in this disease. The progression of chronic renal failure, through endothelial cell damage, would lead to aggravation of the platelet functional defect potentiating the hemorrhagic risk.

Hemostatic disorder of uremia: the platelet defect, main determinant of the prolonged bleeding time, is correlated with indices of activation of coagulation and fibrinolysis.

Several parameters of primary hemostasis and markers of activation of coagulation and fibrinolysis were measured in 48 patients with severe (creatinine clearance < 20 ml/min) chronic renal failure (CRF) without dialysis and disease or drugs affecting hemostasis. Bleeding time (BT) was prolonged in 25/48 patients, and was correlated with age of patients, severity of renal failure, hematocrit, impairment in platelet aggregation-secretion and decrease in platelet ATP content. Defects in von Willebrand factor played no role in the prolongation of the BT. Multivariate analysis showed that only platelet dysfunction and severity of renal disease were independent predictors of the BT in uremia. The platelet functional disorder was significantly correlated with a reduction in platelet ATP and ADP. High levels of plasma thrombin-antithrombin complexes (TAT), prothrombin fragment F1 + 2, fibrinogen and factor VIIc were observed in patients with CRF, as described in prethrombotic states. Plasmin-antiplasmin complexes (PAP), fibrinogen and fibrin degradation products (FgDP, FnDP) were significantly increased, and the activity of plasminogen activator inhibitor (PAI-1) was slightly reduced, denoting an activation of fibrinolysis. A negative correlation was found between platelet levels of ATP and ADP with plasma TAT, F1 + 2 and PAP. Furthermore, plasma PAI-1 activity was negatively correlated with the BT and was lower in patients with prolonged BT as compared with controls and patients with normal BT. These links between primary hemostasis and activation of coagulation and fibrinolysis suggest that increased intravascular generation of thrombin and/or plasmin is an important mediator of the defects in primary hemostasis, prolongation of the BT and, probably, bleeding in CRF.

Markers of T-cell differentiation and maturation in C57BL/6 and BALB/c mice and in the calf: a comparative study.

No significant differences were found between C57BL/6 and BALB/c mice in the levels of Thy 1.2 antigen (a T-cell marker) or the activities of the T-cell maturation-related enzymes adenosine deaminase (ADA, EC 3.5.4.4), serine-esterase (SE, EC 3.4.21), N-acetyl-beta-D-glucosaminidase (NABG, EC 3.2.1.30) and beta-glucuronidase (BG, EC 3.1.1.1), in either unfractionated lymphoid cells or T-lymphocyte-enriched fractions. ADA, SE, NABG and BG activities were much higher (P < 0.01) in the calf than in the corresponding populations in mice. However, the distributions of these activities among thymocyte subpopulations were very similar in mice and the calf. These results provide indirect evidence to suggest that the course of T-cell maturation is similar in mice and the calf.

Clinical and experimental study on the long-term effect of dietary gamma-linolenic acid on plasma lipids, platelet aggregation, thromboxane formation, and prostacyclin production.

Effects of a dietary intake of the polyunsaturated omega-6 essential fatty acids (EFAs) linoleic and gamma-linolenic acids (GLA) on blood lipids, platelet function, and vascular prostacyclin production were studied 12 hyperlipidemic patients (doses of 3 g/day) and 12 male Wistar rats (doses of 3 mg/kg/day) for 4 months. In humans, GLA supplementation decreased plasma triglyceride (TG) levels by 48% (p < 0.001) and increased HDL-cholesterol concentration by 22% (p < 0.01). Total cholesterol and LDL-cholesterol levels were significantly decreased by omega-6 EFAs. Platelet aggregation induced by low concentrations of adenosine diphosphate (ADP) and epinephrine, and serum thromboxane B2 decreased by 45% both in humans and animals after GLA supplementation. Bleeding time increased 40% (p , 0.01). In rats, vascular prostacyclin production measured by radioimmunoassay of 6-keto-PGF1 alpha was enhanced by GLA intake. These effects of omega-6 EFAs may contribute to cardiovascular protection and prevention of the atherosclerotic disease.

Prothymosin alpha and factors from calf thymic cells decrease expression of Thy 1.2 antigen among small thymocytes from C57BL/6 mice.

In this work we studied the effect of Prothymosin alpha (ProT alpha) and other thymic factors on the expression of Thy 1.2 antigen (a T-cell marker) and the activities of adenosine deaminase (ADA, E.C. 3.5.4.4), N-acetyl-beta-D-glucosaminidase (NABG, E.C. 3.2.1.30), beta-glucuronidase (BG, E.C. 3.1.1.1) and serine-esterase (SE, E.C. 3.4.21)., the levels of which change during the T-cell differentiation process among small thymocytes obtained from C57BL/6 mice. Incubation of small thymocytes in the presence of ProT alpha, Thymus Extracts (TE) or supernatants prepared from thymic stromal cells (TSCS) or thymocytes (TS) reduced the proportion of cells killed by anti-Thy 1.2 monoclonal antibodies but did not affect the enzymatic activities studied. This is the first evidence that ProT alpha affects Thy 1.2 expression in vitro.

Inhibition of T cell activation by protein kinase C pseudosubstrates.

PKC pseudosubstrate consensus sequences can be found in the amino terminal region of all the different PKC isoenzymes characterized to date. Here we have used four peptides corresponding to the putative pseudosubstrate sequences from the PKC isoenzymes alpha, gamma, delta, and epsilon. These peptides showed PKC inhibitory activity when tested in a PKC-specific enzyme assay at concentrations of 25 to 100 microM, similar to what has been reported for the myristylated peptide KRTLR. Although the presence of a myristyl group at the amino terminal end of any of these peptides is not essential for their inhibitory activity, myristylation increased the inhibitory activity significantly. By contrast, the myristylated control peptide (GALRQQKNVHEVKN) was not active even at a 100 microM concentration. All of the PKC inhibitory peptides were also able to block PKC activity in a cell assay as demonstrated by their ability to inhibit the induction of IL-2R and TNF-beta expression in Jurkat cells. Finally, we confirmed a previous report of the inhibitory activity of the myristylated peptide KRTLR and showed that other related peptides (N-m-RLTRK, N-m-RRLKT) are also active in these assays.

Distribution of serine esterase activity in the lymphoid system of C57BL/6 mice, effect of aging on the enzymatic activity.

1. The activity of serine esterase (SE) was investigated in the lymphoid system of C57BL/6 mice. SE activity increased in the lymphoid tissues with their content of mature T-lymphocytes, except that high levels were also observed in various populations of bone marrow cells. 2. The maturation of T-lymphocytes in the thymus was accompanied by an increase in their SE activity. 3. Experiments on the influence of age on SE activity showed that while thymocytes were not affected, a three-fold increase in activity occurs in spleen lymphocytes between the ages of 26 and 78 wk.