Effects of Russell's viper venom on mediator production in cultured human umbilical vein endothelial cells.

Hemodynamic alterations in Russell's viper envenomation are the result of interactions of various vasoactive mediators and perhaps proinflammatory cytokines. Since vascular endothelium is likely to be exposed to high concentrations of the venom and the endothelial cell itself not only plays an important role in the physiologic control of the circulation, but also play a role in inflammation with the synthesis and secretion of proinflammatory cytokines. It was therefore, the objective of this study to determine the effects of Russell's viper venom (RVV) on proinflammatory cytokine production by cultured human umbilical vein endothelial cells (HUVEC) and the release of endothelium-derived substances. Endothelial cells were isolated from freshly obtained human umbilical cord vein and grown in tissue culture to confluence as a homogeneous population. Cells were then incubated at 37 degrees C under 5 per cent CO2 with RVV (0.2, 1.0, 5.0, and 25.0 microg/ml) or lipopolysaccharide (LPS, 10 microg/ml) for 3, 6, 12 and 24 hours. After an indicated time, the levels of endothelin-1 (ET-1); 6-keto-PGF1alpha (a stable metabolite of PGI2) tumor necrosis factor-alpha (TNF-alpha); interleukin-1beta (IL-1beta); and interleukin-6 (IL-6) in supernatants were measured by using ELISA or EIA. The effect of RVV or LPS on cell viability was also measured using MIT assay. The results showed copious amounts of ET-1 production irrespectively with the presence of RVV or LPS. Whereas, production of PGI2 (measured as 6-keto-PGF1alpha, a stable metabolite) was increased significantly higher in the RVV- and LPS-treated EC than in the control EC. However, TNF-alpha and IL-6 productions were not different among these groups. The levels of IL-1beta were very low, although IL-1beta was detectable in the group treated with RVV at a concentration of 25.0 microg/ml. In conclusion, RVV upto 25 microg/ml stimulated PGI2 production by cultured HUVEC. This effect was unlikely related to production of proinflammatory cytokines since LPS or RVV is not sufficient per se to elevate a substantial amount of EC-derived cytokines. The higher amount of IL-6 compared to TNF-alpha and IL-1beta may be produced through other pathways apart from production via a cascade of cytokines. This is the first report showing that RVV up to 25 microg/ml has no effect on prominent proinflammatory cytokine production by HUVEC. However, in blood circulation, the major source of cytokines production is monocyte-macrophage lineage cell. Thus, RVV in blood circulation may activate the production of proinflammatory cytokines mainly from those cells and subsequently induce toxicity.

Potassium status of Northeast Thai constructors in three different geographic locations.

Sudden and unexpected death of young adults during sleep is a phenomenon among Southeast Asians and particularly young Northeast (NE) Thailand constructors in Singapore. Survivor of sudden unexplained death syndrome (SUDS) without structural heart disease with idopathic ventricular fibrillation (VF) has been documented. Low plasma potassium (K) and depletion of K can occur simply through a reduction of K intake and are associated with increased risk of VF. The K-status of the populations was evaluated in the NE (Group 1, n=30), Bangkok (Group 2, n=48) and Singapore (Group 3, n=46). Groups 2 and 3 were further subdivided into Group 2A (worked in Bangkok < or = 1 year, n=8), Group 2B (worked in Bangkok > 1 year, n=40), Group 3A (consumed self-prepared or ready-to-buy meals, n=25) and Group 3B (regularly consumed foods provided free-of-charge by construction companies, n=21). Thirty-four male healthy university personnels from the NE and Bangkok served as the control--Group 4. Two 24-h urine samples and a fasting blood sample were collected from each subject. Dietary-K from food was determined by duplicated meal analysis. All these samples were then analyzed for their K-content. Group 3A had the lowest K-status: their K-intake, serum-K, and urinary-K level were 29 +/- 5.8 mmol/day (% low K-intake=100), 3.43 +/- 0.34 mmol/L (% hypokalemia=48) and 19.23 +/- 8.2 mmol/day (% hypokaliuria=87.5), respectively. Among the construction workers, average K-intake, serum-K and urinary-K levels were 45.5 +/- 6.1 mmol/day (% low K-intake = 37.5), 3.93 +/- 0.2 mmol/L (% hypokalemia = 2.5) and 39.6 +/- 9.2 mmol/day (% hypokaliuria = 12.5), respectively. The values of Group 2B were similar to Group 4. In addition, when the data from all of the groups were compared, there was a positive correlation between dietary-K (intake) and urinary-K (excretion) (r=0.881, p<0.001). In conclusion, NE Thailand constructors from various locations exhibited low K status with low dietary-K, high incidence of hypokalemia, and low urinary-K. From the present study, this low K status may be an important trigger factor for VF in construction workers and associated with increase risk of SUDS.

Molecular cloning of phospholipase A2 from a Thai Russell's viper venom gland cDNA library.

Snake venom contains several toxins. Russell's viper (D. russellii, RV) is a venomous snake prevalent in northern and central Thailand. RV bites can cause disseminated coagulation, hemolysis, and edema of the bitten limbs. To identify protein components of RV venom, we made a cDNA library from RV venom glands, and randomly sequenced cloned cDNA. We were able to clone a cDNA encoding RV phospholipase A2 (PLA2). PLA2 is an active enzyme found in several species of snake venom worldwide. PLA2 is thought to be toxic to cell membrane, thereby, can cause local cell and tissue damage, as well as systemic effects in snake bite victims. This PLA2 cDNA clone would facilitate in vivo studies of the pathophysiology of RV bite.

Cell-mediated immunosuppression in mice by street rabies virus not restored by calcium ionophore or PMA.

It is known that rabies virus can suppress the host immune system. In this study we demonstrate a depression of cell-mediated immunity in mice, peripherally infected with Thai street rabies virus. The cell-mediated cytolysis of spleen cells from mice increased transiently on day 5 after infection and declined rapidly thereafter until death. The proliferation of spleen cells stimulated with a T-cell mitogen such as phytohemagglutinin or concanavalin A, was significantly suppressed during the course of infection. There was also a marked suppression of IL-2 secretion in parallel with a decrease of the T-cell proliferative response to mitogen. The suppression of T-cell proliferation was not restored by treatment with a calcium ionophore (A 23187) or phorbol 12-myristate-13 acetate (PMA).

Comparison of separated-lymphocyte and whole-blood lymphocyte proliferation assays for evaluating rabies-induced cellular immunity.

A whole-blood lymphocyte proliferation assay was compared to a standard method requiring the isolation of peripheral blood mononuclear cells (PBMC). Both methods were used to measure the cell-mediated immune responses to rabies in rabies-vaccine recipients. Whole-blood cells gave moderately higher lymphoproliferative responses in terms of stimulation indices than did separated-PBMC. The results obtained from these two methods can be considered equivalent for the purpose of quantitating cellular reactivity to rabies. The use of whole blood has advantages over the standard isolated-PBMC method.

Maternal antibodies against rabies in Thai puppies. A preliminary study.

Serum antibodies to rabies virus were measured in 32 Thai puppies before primary vaccination. Only five showed evidence of rabies antibody by ELISA testing and they had no rabies virus neutralizing activity detectable by RFFIT. Immunologic ignorance of these young dogs leaves a large part of the canine population susceptible to rabies. This could be one reason for the failure of canine rabies control efforts in Thailand.

Radioactive and non-radioactive lymphocyte proliferation assays for measuring rabies-specific cellular immunity.

We compared the radioactive lymphocyte proliferation assay for measuring rabies specific cell-mediated immunity in a group of 42 rabies vaccine recipients with a new technique using rabies-stimulated lymphocytes in a colorimetric test kit (CellTiter 96, Promega Corporation, USA). Results of both tests were good agreement (Kappa = 0.68), however, they did not show good correlation in degree (magnitude) of positivity. In addition, the conventional assay showed a higher degree of sensitivity.

Management of cobra bite by artificial respiration and supportive therapy.

During the period when the hospital ran out of cobra antivenom, 4 patients bitten by cobra with neuromuscular symptoms and respiratory depression were treated with artificial respiration. Complete recovery was noted within 36 to 72 hours. The data are interpreted to indicate the reversible binding of the venom to receptors. Artificial ventilation appears to be another alternative to specific antivenom treatment and may be used when the antivenom is not available or if there is antivenom hypersensitivity.

Angiotensin converting enzyme inhibitor and chronic renal failure.

To evaluate the renal protection of enalapril a study was made in 37 patients with chronic renal failure and hypertension. Sixteen patients had diabetic nephropathy with the serum creatinine ranging from 2.0 to 4.0 mg/dl. Twenty-one patients had non-diabetic chronic renal failure with serum creatinine from 2.2 to 6.3 mg/dl. Of 16 patients with diabetic nephropathy, 6 served as control and 10 received enalapril. Nine patients in the non-diabetic chronic renal failure group served as controls, while 12 were given enalapril. The control patients received alpha methyldopa (500 mg/day) for blood pressure control. In the studied patients enalapril was given at the dose of 5-10 mg/day. Over a period of 2 yrs, enalapril attenuated progression of renal failure in patients with diabetic nephropathy at the serum creatinine level from 2 to 2.9 mg/dl (creatinine clearance 21.5-38.4 ml/min) when compared with control patients. At the serum creatinine of 3 to 4 mg/dl progression of renal failure did not differ from control patients. In non-diabetic renal failure progression of renal failure was delayed in patients with serum creatinine level ranging from 2.2 to 5 mg/dl (creatinine clearance 18-42 ml/min); patients with serum creatinine level ranging from 5.5 to 6.3 mg/dl had deterioration of renal function as control patients. Proteinuria was decreased in all patients on enalapril. Thus, for preventing progression of renal failure, enalapril should be given in chronic renal failure with milder degree of renal function impairment; in diabetic nephropathy it should be started earlier at the lower serum creatinine level than in non-diabetic chronic renal failure, yet with comparable creatinine clearance.

Seasonal changes in serum and erythrocyte potassium among renal stone formers from northeastern Thailand.

Hypocitraturia, hypokalemia and low urinary excretion of potassium are common findings in nephrolithiasis in Northeastern Thailand. However, intracellular potassium has not been studied. We measured serum potassium, erythrocyte potassium, 24-hour urinary excretion of sodium, potassium, citrate, ammonium, titratable acid and pH in 17 nephrolithiasis patients from Northeastern Thailand during 2 seasons: the cool and hot months. There were no significant differences in urinary pH, ammonium, titratable acid, citrate and potassium during these 2 seasons. However, hypocitraturia and hypokaliuria were observed in the majority of cases in both periods. Seasonal variation in serum potassium, erythrocyte potassium, urine volume and urinary excretion of sodium was observed. Values were significantly lower in the hot months. In the cool months, the urinary excretion of citrate correlated linearly with that of potassium (r = 0.696, p < 0.002). Such a correlation was not found in the hot months. We concluded that extracellular and intracellular potassium depletion might be present in these patients and is greater during the hot than during the cool months. The pathogenesis may be multi-factorial.

Abnormal erythrocyte Na, K-ATPase activity in a northeastern Thai population.

We studied the cellular membrane enzyme responsible for potassium transport in different Thai populations. We measured plasma and intraerythrocytic concentrations of sodium and potassium, activities of erythrocytic membrane Na, K-activated adenosine triphosphatase (Na, K-ATPase), ouabain-insensitive ATPase, total ATPase and the activity ratio of Na, K-ATPase/total ATPase in 25 healthy blood donors at Khon Kaen University Hospital, Khon Kaen (group 1), and in 32 donors at the National Blood Center, Thai Red Cross Society, Bangkok (group 2). Group 1 subjects had significantly higher concentrations of erythrocyte sodium (p < 0.001) and lower activity of Na, K-ATPase (p < 0.001) than group 2. When data of these 2 groups were combined, erythrocyte Na+ correlated inversely with Na, K-ATPase and the activity ratio of Na, K-ATPase/total ATPase. Our study suggests that there is a defect in membrane transport enzymes for sodium/potassium in certain northeast Thai populations.

Plasma amino acid patterns in normal Thais and in patients with chronic renal failure.

The effect of chronic renal failure (CRF) on the pattern of plasma free amino acid concentrations was studied in 22 healthy controls (group 1); 43 CRF patients of which serum creatinine levels were 2-4.9 mg/dl (group 2, n = 11), 5-10 mg/dl (group 3, n = 10), more than 10 mg/dl (group 4, n = 9), and chronically hemodialysed patients (group 5, n = 13). In all renal failure groups, plasma concentrations of eight free essential amino acids-isoleucine, leucine, lysine, methionine, threonine, tryptophan, tyrosine and valine and those of three non-essential amino acids-alanine, glutamate and serine were significantly lower than those in controls. Plasma concentrations of free arginine, cystine, glutamate and serine were significantly higher in CRF patients. Patterns of change of plasma aminogram were similar among CRF patients regardless of the stages of renal function or dialytic treatment. Stepwise changes of some plasma free amino acids were observed as renal function became worse. The molar ratios of plasma free valine/glycine, serine/glycine and tyrosine/phenylalanine were decreased accordingly. Our study confirms the presence of abnormal plasma aminogram, specifically that of essential amino acids, in CRF. Therapeutic intervention is warranted but still needs further investigations.

The use of oral essential amino acids in hemodialysis patients.

Fifteen patients with chronic renal failure, stabilized on twice weekly hemodialysis, received oral essential amino acid therapy (6.3 g/day) over a period of 12.3 months. Clinical and laboratory improvement was observed with respect to body weight, tricep skin fold thickness, mid upper arm circumference serum albumin, C3 and plasma essential amino acids. Serum triglyceride was decreased. The patients felt well with increased appetite. Essential amino acids were well tolerated without side effects.

Measurements of urinary state of saturation with respect to calcium oxalate and brushite (CaHPO4.2H2O) in renal stone formers.

Urinary supersaturation with respect to calcium oxalate and/or brushite (CaHPO4.2H2O) is critical for the formation of calcium stones. The aim of this study is to use concentration product ratio (CPR) as a tool to assess the state of urine saturation with respect to calcium oxalate and brushite. One 24-h urine specimen from each of 16 healthy city dwellers (GI), 18 healthy villagers (GII) and 28 villagers with renal stones (GIII) was collected and analyzed for calcium, sodium, potassium, phosphate, uric acid, citrate and oxalate. The CPRs of calcium and oxalate and of calcium and phosphate before and after equilibration of the urine with the corresponding seeding crystals were also determined. Urinary volume and the excretion rate of calcium, potassium, uric acid, citrate and oxalate of GII and of sodium, phosphate, uric acid and citrate of GIII were significantly less than those of GI. The CPRs for calcium oxalate and brushite were 2.9 +/- 0.3 and 1.7 +/- 0.2 for GI, 2.7 +/- 0.2 and 1.3 +/- 0.1 for GII and 2.5 +/- 0.2 and 1.1 +/- 0.1 for GIII, respectively. The CPR values indicated that urine of all groups was generally supersaturated with respect to calcium oxalate salt (CPRs were above 1) and were not different among the groups. With regard to brushite, urine was also supersaturated but the state of supersaturation was less than that of calcium oxalate. Furthermore, instead of being supersaturated, brushite in many urine specimens of GIII was undersaturated and its mean CPR was even significantly less than that of GI (P less than 0.01).

Low specific gravity urine with crystalluria as discriminant index for nephrolithiasis.

A community-based study for crystalluria in morning urine (MU) specimens was carried out under light microscopy. The MU specimens were collected from 29 males with renal stones (GI), 36 age-and sex-matched normal controls (GII) and 27 household members of GI who did not have stones (GIII). The findings can be summarized as follows. 1. In the groups as a whole, almost all crystal and crystal aggregate found was oxalate type and with highest prevalence in GI. 2. In urine with low specific gravity (SG) i.e. less than or equal to 0.010, prevalence of oxalate crystals in GI (57.7%) was significantly higher (p less than 0.05) than in both GII (5.9%) and GIII (13%). Furthermore, at this range of SG, 15 per cent of the MU specimens in GI showed aggregation of oxalate crystals, whereas, the condition was neither found in GII nor GIII. 3. Our data suggest urine supersaturation with respect to calcium oxalate was found in both renal stone patients and normal subjects but more frequently in the former and also suggests more deficiency or lack of inhibitors for oxalate crystal nucleation and aggregation in urine of renal stone patients. The occurrence of oxalate crystals and crystal aggregates in urine of low SG may be useful as an index to discriminate stone patients from normal subjects or as an index to indicate the high risk group in the community.

Identification of urinary metabolites and quantitative measurement of creatinine by a proton nuclear magnetic resonance spectrometry.

1H-NMR spectra of 60 human urine specimens were recorded without pretreatment by a JEOL FX 90 Q spectrometer operating at 89.55 MHz. The signals of the methyl protons of creatinine (3.04 +/- 0.02 ppm) were observed in all spot fasting morning urine samples collected from 7 healthy persons, 10 patients with nephrotic syndrome and 43 patients with diabetes mellitus. The concentrations of creatinine measured by NMR spectroscopy (Y) and the chemical assay based on the Jaffe reaction (X), over the range of 19-190 mg/dl, were compared by the least-squares linear regression analysis (Y = 6.7799 + 0.6717 X). The mean urinary creatinine concentration by NMR spectroscopy appeared to be lower than that obtained by the Jaffe reaction at the normal and high normal levels. In the urine of 20 diabetic patients with an average blood glucose of 251.30 +/- 50.26 (SD) mg/dl typical spectra of the multiple large signals of glucose protons at position from 3.13 +/- 0.04 to 4.04 +/- 0.12 (SD) ppm were shown. Moreover, some urinary metabolites and amino acids spectra were occasionally detected at one time.