Anti-TCRß mAb induces long-term allograft survival by reducing antigen-reactive T cells and sparing regulatory T cells.

TCR specific antibodies may modulate the TCR engagement with antigen-MHC complexes, and in turn regulate in vivo T cell responses to alloantigens. Herein, we found that in vivo administration of mAbs specific for mouse TCRß (H57-597), TCRα or CD3 promptly reduced the number of CD4(+) and CD8(+) T cells in normal mice, but H57-597 mAb most potently increased the frequency of CD4(+) Foxp3(+) Treg cells. When mice were injected with staphylococcal enterotoxin B (SEB) superantigen and H57-597 mAb, the expansion of SEB-reactive Vß8(+) T cells was completely abrogated while SEB-nonreactive Vß2(+) T cells remained unaffected. More importantly, transient H57-597 mAb treatment exerted long-lasting effect in preventing T cell responses to alloantigens, and produced long-term cardiac allograft survival (>100 days) in 10 out of 11 recipients. While Treg cells were involved in maintaining donor-specific long-term graft survival, T cell homeostasis recovered over time and immunity was retained against third party allografts. Moreover, transient H57-597 mAb treatment significantly prolonged survival of skin allografts in naïve recipients as well as heart allografts in skin-sensitized recipients. Thus, transient modulation of the TCRß chain by H57-597 mAb exhibits potent, long-lasting therapeutic effects to control alloimmune responses.

The effect of endotoxin on functional parameters of mammary CID-9 cells.

The effect of endotoxin on mammary CID-9 cells, which differentiate in culture and express beta-casein, was investigated. Cells in culture supplemented with lactogenic hormones and dripped with EMS-Matrix (EMS-drip), were treated daily with endotoxin (0.5-500 microg/ml). Endotoxin at concentrations of less or equal to 10 microg/ml did not affect cell growth and viability up to 5 days post endotoxin treatment. Endotoxin (0.01-10 microg/ml) was added to the culture medium, upon confluence, and functional parameters were examined within 48 h post endotoxin treatment. Nuclear factor-kappaB (NF-kappaB) (p52) increased in nuclear extracts from endotoxin-stimulated cells within 1 h of treatment, while beta-casein mRNA and protein expression decreased in a concentration-dependent manner at 24 and 48 h post treatment. Zymography showed that the 72 and 92 kDa gelatinase activity increased in cells at 24 and 48 h post endotoxin treatment at 10 and 50 microg/ml. At the latter concentration, the active form of 72 kDa gelatinase was induced at 48 h. Interleukin-6 and tumor necrosis factor-alpha levels increased at 1-3 h post endotoxin treatment and peaked at 6 h in cells on plastic and EHS-drip. Nerve growth factor (NGF) levels increased in control and endotoxin-treated cells in a time-dependent manner, and endotoxin increased NGF levels in culture at 6 and 9 h post endotoxin treatment. This study shows that endotoxin activated NF-kappaB, suppressed beta-casein expression and upregulated gelatinases, cytokines and NGF. This model could be used to investigate the role of mammary cells in initiating and propagating inflammation and to test candidate molecules for potential anti-inflammatory properties.

Determination of shelf life of concentrated yogurt (labneh) produced by in-bag straining of set yogurt using hazard analysis.

Strained yogurt, labneh, produced by straining cow's milk set yogurt in cloth bags, was stored at 5, 15, and 25 degrees C, and changes in microbial counts, pH, titratable acidity, percentage of free whey, and sensory attributes were monitored during storage. Counts of total aerobes, psychrotrophic yeasts, yeasts and molds, and lactic acid bacteria, except in samples stored at 25 degrees C, increased irrespective of storage temperature. The pH of samples decreased, titratable acidity and percentage of free whey increased, and texture defects were detected at a later stage than flavor changes during storage. Shelf-life data of labneh was adequately described by the Weibull distribution. The nominal shelf life determined using sensory changes and yeast counts as failure criteria ranged from 8.5 to 10.5, 4.7 to 5.8 and 2.3 to 2.7 d at 5, 15 and 25 degrees C, respectively. Q10 (shelf life at T degrees C/shelf life at T+10 degrees C) for flavor quality loss was 1.98 at 5 degrees C, and the corresponding activation energy was 11.3 kcal/mol.

Urothelial synthesis of prostanoids in the ovine ureter.

The purpose of this study was to investigate the site of production (urothelium/smooth muscle) and quantitative release of prostanoids in the ureter. Quantitative analysis of prostacyclin (PGI2) was by its metabolite, 6-keto PGFalpha, thromboxane (TXB2) and prostaglandin F2alpha. Synthesis by radiometry and radioimmunoassay was performed in vitro in sheep ureter specimens before and after removal of the inner epithelial layer and after addition of indomethacin. The major prostanoids present in the ureter were PGI2 and TXB2; PGI2 was quantitatively the largest component. Removal of the thin inner epithelial layer (urothelium) reduced mostly PGI2; addition of arachidonic acid significantly augmented PGI2 only in ureters with intact urothelium but did not alter TXB2 levels. The main source of prostanoid synthesis (PGI2) of the ureter is to be found in the urothelium. The functional role of the prostanoids may be related to coordination of peristalsis.

FtsI and FtsW are localized to the septum in Escherichia coli.

The localization of FtsI (PBP3), a penicillin-binding protein specifically required for cell division in Escherichia coli, was investigated by immunofluorescence microscopy and found to localize to the septum. The localization of FtsI was not observed in ftsZ or ftsA mutants, indicating that it was dependent on the prior localization of these proteins. Addition of furazlocillin, a specific inhibitor of FtsI, prevented localization of FtsI even though FtsZ and FtsA localization occurred. Interestingly, the localization of FtsN was also prevented by furazlocillin. FtsZ displayed limited localization in furazlocillin-treated cells, whereas it was efficiently localized in FtsI-depleted cells. FtsW, another essential cell division protein, was also localized to the septum.

Overexpression of the hslVU operon suppresses SOS-mediated inhibition of cell division in Escherichia coli.

A multicopy clone was isolated which conferred resistance to the SOS inducer nitrofurantoin in an Escherichia coli lon mutant. Plasmid pHL1 was found to contain a 7-8 kbp HindIII DNA insert from a region of the chromosome at 88.5 minutes. Further characterisation of pHL1 revealed that resistance to nitrofurantoin was due to the overexpression of the hslV-hslU operon which encodes an ATP-dependent protease complex in E. coli. The overexpression of hslVU also conferred resistance to ultraviolet irradiation in the lon mutant. It is proposed that when overproduced, the HslV-HslU protease complex can degrade SulA which is an endogenous inhibitor of the essential cell division protein FtsZ. The ability of HslVU to degrade SulA in vivo suggests that Lon and HslVU may share a range of substrates. Furthermore, the suppression of lon could be used as a simple genetic test of proteolytic activity of cloned HslVU.

ftsW is an essential cell-division gene in Escherichia coli.

In the absence of exogenous promoters, plasmid-mediated complementation of the temperature-sensitive ftsW201 allele requires the presence of the full coding sequence of ftsW plus upstream DNA encompassing the C-terminus of mraY and the full coding sequence of murD. We used molecular and genetic techniques to introduce an insertional inactivation into the chromosomal copy of ftsW, in the presence of the plasmid-borne wild-type ftsW gene under the control of P(BAD). In the absence of arabinose, the ftsW-null strain is not viable, and a shift from arabinose- to glucose-containing liquid medium resulted in a block in division, followed by cell lysis. Immunofluorescence microscopy revealed that in ftsW-null filaments, the FtsZ ring is absent in 50-60% of filaments, whilst between one and three Z-rings per filament can be detected in the remainder of the population, with the majority of these containing only one Z-ring per filament. We also demonstrated that the expression of only ftsWS (the smaller of two ftsW open reading frames) from P(BAD) is sufficient for complementation of the ftsW-null allele. We conclude that FtsW is an essential cell-division protein in Escherichia coli, and that it plays a role in the stabilization of the FtsZ ring during cell division.

Two polypeptide products of the Escherichia coli cell division gene ftsW and a possible role for FtsW in FtsZ function.

Two new mutations in the cell division gene ftsW have been isolated and characterized. The ftsW263(Ts) mutation results in a block to division at the initiation stage, similar to that previously observed with the ftsW201(Ts) mutation. The ftsW1640(Ts) mutation, however, causes a block to division at a later stage. The ftsW201 and ftsW263 mutants were shown to be phenotypically sensitive to the genetic background and growth conditions and are possibly relA dependent. Immunofluorescence microscopy showed that the FtsZ protein can localize to presumptive division sites in strains carrying ftsW(Ts) mutations at the nonpermissive temperature, suggesting that FtsW is unlikely to be specifically required for the localization of FtsZ to the division site. Examination of the localization of FtsZ in an ftsW rodA double mutant (lemon-shaped cells) revealed several classes of cells ranging from a common class where an FtsZ ring structure is absent to a class where FtsZ forms a complete ring at the midpoint of a lemon-shaped cell, suggesting a role for FtsW in the establishment of a stable FtsZ-based septal structure. We further demonstrate that two FtsW peptides, FtsWL (large) and FtsWS (small), can be identified and that the expression of ftsWS is sufficient for complementation of ftsW(Ts) mutations.

Identification of FtsW and characterization of a new ftsW division mutant of Escherichia coli.

The product of the ftsW gene has been identified as a polypeptide that, like the related RodA protein, shows anomalous mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. FtsW is produced at low levels that can be increased by altering the translation initiation region of the mRNA. Overproduction of FtsW strongly inhibits cell growth. A new mutant allele, ftsW201, causes a temperature-dependent block in the initiation stage of cell division which is similar to the division block in ftsZ mutants. The block in initiation of division in the ftsW201 allele is shown to be independent of FtsZ or the FtsZ inhibitor, SulA. In addition, the ftsW201 mutant is hypersensitive to overproduction of the division initiation protein FtsZ at the permissive temperature. Our results suggest a role for FtsW in an early stage of division which may involve an interaction with FtsZ.

Aqueous extracts of garlic (Allium sativum) inhibit prostaglandin synthesis in the ovine ureter.

The prostaglandins (PGs) synthesized from C14-arachidonic acid by the homogenized sheep ureter were identified as being prostacyclin (PGI2), PGF2 alpha and thromboxane B2 (TXB2). The radioimmunoassay (RIA) estimation of 6-keto-PGF1 alpha, a stable metabolite of PGI2, confirms that it was the major metabolite of arachidonic acid. Aqueous extracts of fresh garlic (5, 12.5, 25 and 50 mg/ml) were shown to inhibit the synthesis of the prostanoids in a dose dependent manner. Fresh garlic extracts (1, 2.5, 5 and 10 mg/ml) also dose dependently inhibited spontaneous rhythmic contractions of the isolated ureter. Boiled garlic (5, 12.5, 25 and 50 mg/ml) had no effect on either ureteral motility or the PG synthesizing capacity of the sheep ureter.

Psychosocial and toxicological profile of drug misuse in male army conscripts in Kuwait.

The pattern of drug use among 2183 young Kuwaiti conscripts was studied using the Drug Abuse Screening Test (DAST) and urine analysis. Drug misuse rates were low, compared with similar set-ups elsewhere as independently confirmed by urine test (0.7% amphetamine and 2.8% other street drugs; alcohol was not detected in this sample) and self-report (7.9% on DAST). This may be attributed to the influence of the Islamic traditions on lifestyles. Significant correlations were seen between self-report of multiple drug use and positive urine results for both amphetamine and other street drugs, while positive urine amphetamine was associated with families seeking help for treatment, problems with family and/or work and having or being hospitalized for drug-related medical problems. Amphetamine as the major identified individual misused drug was significantly associated with older age group but negatively correlated with marital status, higher education and employment. The discrepancy between self-report of drug misuse-related problems and objective proof of this may reflect the inappropriateness of some of DAST items to this sample, culturally desired responses or both.

Evaluation of outpatient prescribing for substance misusers in Kuwait: the need for a rational approach.

The outpatient follow-up care of 140 substance misusers in Kuwait was evaluated. Treatment has been traditionally based on substitution pharmacotherapy with no set objectives or criteria for outcome evaluation. Patients' structural characteristics, mode of referral, clinical diagnoses, medication prescribed and screening urine samples for drugs were examined for meaningful associations between these variables. Benzodiazepines were by far the most prescribed (94%) medication. Only 40% of the urine test results were positive for drugs containing different combinations of medications and street drugs. Unlike patients who received benzodiazepine, those who received tricyclic antidepressant medication were significantly more likely to associate with predictors of improvement. There is a need for better training of doctors and careful selection of patients in order to implement treatment strategies based on good doctor-patient relationships and well defined objectives for treatment and clear criteria of outcome.

Escherichia coli mutant Y16 is a double mutant carrying thermosensitive ftsH and ftsI mutations.

The Escherichia coli mutant Y16, which shows thermosensitive colony formation and filamentation with reduced amounts of penicillin-binding protein 3 (PBP3), has mutations in the ftsI gene encoding PBP3 and in the ftsH gene. The ftsI mutation markedly reduces the amount of PBP3 at 42 degrees C, whereas the amount of the ftsH single mutant is slightly reduced.

The effect of indomethacin and metamizole on ureteral motility and urine flow in sheep.

The objective of this study was to evaluate the effect of two non-steroid anti-inflammatory drugs, indomethacin and metamizole, on ureteral peristalsis during acute occlusion similar to the situation in renal colic. In 12 pentobarbital anesthetized sheep, both ureters were cannulated and the frequency of ureteral contractions, urine flow, mean ureteral pressure and blood pressure were recorded during 10-min control and i.v. drug administration periods. Both indomethacin (1-2 mg/kg) and metamizole (60-120 mg/kg) showed a dose dependent reduction in peristaltic frequency without reduction of the mean pressure. In addition, the pressure amplitude of the peristaltic waves was also lowered, particularly with indomethacin. Only indomethacin reduced the urine flow. Arterial blood pressure was elevated by both drugs, particularly after the first dose of indomethacin. It can be concluded that indomethacin and metamizole reduce ureteral peristaltic frequency, probably blocking the impulse transmission at the ureteropelvic junction.

Toxicological screening: a three year experience in poisonings in Kuwait.

The results of toxicological screening of body fluids (urine, blood and gastric lavage) from pediatric patients were analyzed for the period January 1985 - December 1988. Of the 119 cases, approximately one half were positive for at least one foreign substance. In about one fifth of the cases, multiple substances were detected. The most commonly implicated drugs were those acting on the central nervous system including the barbiturates, tricyclic antidepressants, phenothiazines, benzodiazepines and carbamazepine.

The effect of glucocorticosteroids on in vitro motility of the ureter of the sheep.

1. The effects of three glucocorticosteroids, hydrocortisone, methylprednisolone and dexamethasone, as well as two non-steroidal anti-inflammatory agents (NSAIDs) indomethacin and diclofenac sodium were tested in vitro on the spontaneously rhythmic contracting ureteral preparation of the sheep. 2. The NSAIDs and the steroids methylprednisolone (10(-7)-10(-4) M) and dexamethasone (10(-8) - 10(-4) M), dose-dependently inhibited ureteral motility. Hydrocortisone caused a cessation of spontaneous contractions only at the high concentration of 10(-4) M. 3. Pretreatment of ureteral strips with the protein synthesis inhibitor cycloheximide (10(-6) M) abolished the inhibitory action of the corticosteroids on peristalsis, consistent with the suggestion that the action of steroids on the ureter is mediated via the synthesis of the anti-phospholipase protein, lipocortin. 4. The potency of the steroids in descending order was found to be dexamethasone greater than methylprednisolone greater than hydrocortisone.

The effect of ureteral distension on peristalsis. Studies on human and sheep ureters.

Isolated sheep and human ureteral preparations (from patients with bilharzia) were subjected in-vitro to graded elongation and the effect on tension and spontaneous peristaltic frequency was assessed. Sheep specimens were obtained from three locations: the intra- and extrarenal portion of the pelvis and distal ureter. Elongation (stretch) induced an increase in spontaneous frequency only in pelvic ureteric specimens, but not in the distal ureter. Basal tension increased exponentially with stretch and most markedly in the distal sheep ureter and also in human preparations. Active tension (amplitude of phasic contractions) increased with stretch in specimens from all locations and reached a maximum at 110-115% elongation. These data suggest that acute distension of the ureter increases frequency of peristaltic waves only in the intrarenal parts of the ureter. Acute obstruction in renal colic can induce hypermotility in terms of increased frequency and force of contraction.

Correlates of alcohol-related casualty in Kuwait.

This paper reports the extent to which alcohol consumption is associated with casualties in a country in which alcohol is prohibited religiously and culturally. Selective screening of particular casualty groups by blood test established qualitative evidence of alcohol use in 107 out of 1058 patients (10%) examined at a general hospital and a traumatology hospital in Kuwait. The rate of alcohol-associated casualty was significantly higher in traumatology hospital patients (15%) than in general hospital (7%), stressing the association between alcohol and trauma. Absence of differences by place of casualty disproves lay beliefs that motor vehicle casualty is the major alcohol-associated risk in Kuwait. Other casualties, particularly work and domestic injuries, should be regarded as being affected by alcohol consumption. Statistically significant high alcohol-associated casualty rates characterized Kuwaitis (14%), the unemployed/retired (20%) and coma cases (22%) suggesting that these categories may suffer from serious alcohol problems.