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Biomedical subjects

D R Webb

Publications and source records attributed to D R Webb.

At least 37 records · Page 2Linked to original sources
PubMed1995

A 69-kDa membrane protein associated with lipopolysaccharide (LPS)-induced signal transduction in the human monocytic cell line THP-1.

Lipopolysaccharide (LPS) triggers a wide range of cellular responses in mammalian cells. Several proteins, including CD14, have been reported to possess LPS binding capacity. However, the signal transduction molecule(s) and pathway(s) through which LPS induces cellular responses have not been identified. A rabbit antiserum (5299), which had been made against the N-terminal 20-amino-acid sequence of the cytokine, soluble immune response suppressor, unexpectedly activated the human monocyte cell line THP-1 as assessed by tumor necrosis factor (TNF alpha) production. Normal rabbit serum or IgG caused no TNF production, while antiserum 5299 induced TNF at a 1:10(4) dilution. The activity of antiserum 5299 was not due to endotoxin contamination, since antiserum 5299 contained less endotoxin than required for activation of THP-1 and the TNF-inducing activity disappeared when the antiserum was boiled. Moreover, the activity was recovered in purified immunoglobulin G fraction from the antiserum (5299 IgG). These results suggest that antiserum 5299 contains an antibody which recognizes a signal-transducing molecule. THP-1 desensitized by incubation with LPS for 24 hr could not respond to LPS or antiserum 5299 while responsiveness to phorbol ester remained, suggesting that the signals induced by the antiserum 5299 and LPS are transduced through at least a partially common pathway. Western blotting with antiserum 5299 showed a 69-kDa protein which rapidly appeared in Triton X-100-insoluble fraction after stimulation with LPS or 5299 IgG, suggesting an association of 69-kDa protein with the cytoskeleton. Furthermore, two mutant cell lines were isolated (T-15 and T-25) from gamma-irradiated THP-1 which lack responsiveness to LPS. In these mutant cell lines, the 69-kDa protein was not observed in Triton X-100-insoluble fraction after stimulation with LPS. It is proposed that this protein may play an important role in LPS-stimulated signal transduction.

Blotting, Western↗
PubMed1995

Characterization of the epitope recognized by a mAb that reacts differentially with murine suppressor T cells.

Although reliable antibodies are available that distinguish human suppressor T (Ts) cells from CTL and other T cells, few are available for murine Ts cells. We have developed a mAb (984D4.6.5) that, in the presence of complement, depletes alloantigen-specific Ts cells but not CTL. This antibody recognizes activated Ts cells but not their precursors. In these studies, flow cytometric analysis demonstrates that 984D4.6.5 reacts with several Ts cell hybridomas, cloned Ts cell lines and WEHI-3 (a myelomonocytic tumor cell line). Reactivity was not detected with BW5147, Th cell hybridomas, cloned Th cells, CTL lines and hybridomas, B cell lines, thymocytes, splenocytes, bone marrow cells nor a variety of tumor cells. Among 984D4.6.5 positive lines, expression is heterogeneous and the number of cells expressing high levels of the epitope is increased when the hybridomas are maintained at a relatively high cell density. Neuriminidase and pronase deplete the epitope recognized by mAb 984D4.6.5. Protein synthesis and glycosylation inhibitors also reduce expression of this epitope. These observations suggest that the epitope recognized by 984D4.6.5 is a carbohydrate linked to a polypeptide. This antibody was tested by ELISA for binding to a large panel of carbohydrates and glycolipids coupled to BSA. The only one that bound 984D4.6.5 was LS tetrasaccharide c (NeuNAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc), an O-linked carbohydrate. Comparative analysis shows that both the sequence and the linkage of these sugars are essential to the reactivity with the 984D4.6.5 antibody. This epitope is expressed by a glycoprotein of approximately 200 kDa, as shown by Western blots. The identity of this glycoprotein remains to be determined, but indirect evidence suggests that it is not CD45.

Animals↗
PubMed1995

Evidence for a calcium regulated, bidirectional intronic promoter in the murine TCR V alpha 1 gene.

Previous studies of the TCR alpha chain gene have located promoter elements 5' to the start of the various V alpha genes. The only fully characterized enhancer for the entire alpha chain gene (V, J and C genes) has been located approximately 3 kb from the 3' end of C alpha. We now report the existence of additional regulatory elements located in the introns of several murine V alpha genes (V alpha 1, V alpha 3 and V alpha B6.2.16). In the case of V alpha 1, this element appears to be a promoter with bidirectional activity that is not T cell specific. Interestingly, upstream of the promoter in the antisense strand, an open reading frame has been found that codes for a small molecular weight protein (approximately 60 amino acids) that contains a proline-rich region and a tyrosine-isoleucine motif that has homology to Ig beta (the B29 gene product). A rabbit antiserum made against this sequence has confirmed its existence by Western blot and immunoprecipitation. Thus this V alpha 1 intronic promoter has the potential not only to induce the formation of a truncated V alpha 1 gene product, but also regulates the expression of a small molecular weight protein that may be involved in lymphocyte antigen receptor signaling. The activity of this promoter is regulated by changes in intracellular calcium. In the presence of ionomycin the promoter is down-regulated in the sense direction and its activity is enhanced in the antisense direction. This result suggests that this promoter can act differentially to produce two very different gene products. The bidirectional V alpha 1 promoter appears to be the first in the Ig superfamily to induce potentially functional proteins in both directions.

Amino Acid Sequence↗
PubMed1995

Microsurgical vasovasostomy in military personnel.

This paper details the operative techniques, results and reasons for reversals of vasectomy in military personnel. Fifty-two reversals were performed by two surgeons (AC & DW) over a 7 year period. Data collection was by (i) retrospective analysis of service documents, surgical registers and laboratory records and (ii) response to a questionnaire. The interval between original vasectomy and reversal varied from 5 months to 16 years. Change of sexual partner was the most common reason for requesting the procedure. There were two recorded postoperative infections and no postoperative mortality. Post-reversal semen analysis proved anastomotic success in 49 out of 51 cases (96%). Semen analysis was not returned for the remaining case. Of the couples who had been trying for at least 18 months, 28 out of 42 (67%) had conceived, including one miscarriage. A further seven cases had been trying for less than 18 months, or had no opportunity due to physical separation. Two of these seven patients ended their relationship soon after operation. Pregnancy outcome was unknown in three cases.

Adult↗
PubMed1994

The Ca2+/calmodulin-activated, phosphoprotein phosphatase calcineurin is sufficient for positive transcriptional regulation of the mouse IL-4 gene.

We have studied the TCR mediated signal transduction pathways involved in transcriptional regulation of the mouse IL-4 gene. The sequences extending from base pair -766 to +63 of the IL-4 gene were inserted upstream of a luciferase indicator gene. Transcriptional activity was observed when the construct, [pIL-4(-766)], was transfected into either the IL-4 producing cell line, EL-4, or the IL-4 non-producing T cell hybridoma, 68-41, but not in the L929 fibroblast cell line. By analysis of deletion mutants of pIL-4(-766), we identified a transcriptional regulatory element that is tightly associated with a signal coming from the TCR and which controls inducible activation of the IL-4 promoter. By analysis of deletion mutants of pIL-4(-766), this latter element was found between base pairs -147 to -17. Electrophoretic mobility shift assays indicated that expression of a nuclear binding protein with binding sites between base pairs -84 and -55 could be induced. By competition and mutation analysis, the binding motif of this protein was determined to be AAAATTTTCC. Stimulation with ionomycin alone was sufficient to induce activity in pIL-4(-766). Cyclosporin A inhibited both the IL-4 promoter activity and activation of the inducible nuclear protein. Transient over-expression of a constitutively active form of the Ca2+/calmodulin-regulated protein phosphatase, calcineurin was sufficient to cause activation of pIL-4(-766) without any additional stimulus. These results indicate that the signaling requirements for activation of upstream positive regulatory elements of the IL-4 gene are distinct from those of the IL-2 gene. Ca2+ mobilization is sufficient to activate the IL-4 promoter, whereas IL-2 gene transcription requires both Ca2+ mobilization and protein kinase C activation.

Animals↗
PubMed1994

Laparoscopic ureterolithotomy: the results of an initial series, and an evaluation of its role in the management of ureteric calculi.

OBJECTIVE: To evaluate the place of laparoscopic ureterolithotomy in the management of ureteric calculi for which extracorporeal shockwave lithotripsy (ESWL) and endourological techniques are unsuitable. PATIENTS AND METHODS: Laparoscopic ureterolithotomy was attempted in nine patients (eight men, one woman) with an age range of 26-81 years (mean 55.5) who had large, long standing and impacted calculi in the upper and mid ureter. The stone size ranged from 5 to 28 mm (mean 13.2) and stone duration ranged from one to 24 months (mean 8.2). The transperitoneal route was used in six patients and the extraperitoneal route in three but was converted to a transperitoneal approach in two. RESULTS: The stone was successfully removed in all nine patients. The operative time ranged from 80 to 260 minutes (mean 158). No intra-operative complication was encountered and no patient required a blood transfusion. Post-operative complications included urinary leak and fever. The post-operative stay ranged from 2 to 13 days (mean 5.2). CONCLUSIONS: Laparoscopic ureterolithotomy has definite advantages over open ureterolithotomy. Furthermore, the more difficult a stone is for treatment by ESWL and endourological techniques, the more suitable it is for laparoscopic removal. For large, hard, long-standing and impacted ureteric calculi, one laparoscopic ureterolithotomy as initial therapy may be preferable to multiple endourological and ESWL procedures.

Adult↗
PubMed1994

Management of children with slightly elevated blood lead levels.

OBJECTIVE: To determine whether counseling of parents reduced blood lead levels in their young children. PARTICIPANTS AND METHODS: A lead exposure study was conducted in the vicinity of a defunct lead smelter. A total of 827 volunteers including 490 children younger than 6 years participated in the study. The mean venous blood lead level in the 490 children was 0.33 mumol/L (6.9 micrograms/dL), with a range of 0.03 to 1.94 mumol/L (0.7 to 40.2 micrograms/dL). A total of 78 (16%) children had blood lead levels > or = 0.48 mumol/L (10 micrograms/dL). However, only 5 (1%) of the children had blood lead levels of > or = 1.21 mumol/L (25 micrograms/dL). The blood lead levels in this community were much lower than levels reported in earlier studies in many areas of the United States, when lead levels in air and in food were higher. Most houses in the study community were built before 1920 and had high lead paint levels. High levels of lead were also found in many house dust and soil samples. Intervention consisted of home visits and counseling of parents of children with blood lead levels of > or = 0.48 mumol/L (10 micrograms/dL). RESULTS: In children with initial blood lead levels of > or = 0.48 mumol/L (10 micrograms/dL) the blood lead test was repeated 4 months later. The arithmetic mean blood lead level 4 months later showed a decrease from 0.72 mumol/L (15 micrograms/dL) to 0.38 mumol/L (7.8 micrograms/dL). An additional blood sample was taken 1 year after the first sample in a subset of 30 children. These children showed a mean blood lead level of 0.43 mumol/L (9 micrograms/dL) at the 1-year follow-up, whereas their mean blood lead level at the 4-month follow-up had been 0.38 mumol/L (8 micrograms/dL). CONCLUSION: Educating parents proved a very effective tool. Further studies are needed in larger populations to evaluate the effectiveness of this approach.

Child, Preschool↗
PubMed1993

Saying the 'S' word in public.

The mechanism by which self-reactive, peripheral lymphocytes are prevented from causing overt autoimmune disease is the subject of much debate. A recent meeting examined the evidence to support a role for T-cell mediated suppression in maintenance of peripheral tolerance.

Animals↗
PubMed1993

A 91-day feeding study in rats with caprenin.

Caprenin, a randomized triglyceride primarily comprising caprylic (C8:0), capric (C10:0), and behenic (C22:0) acids, was administered in a semi-purified diet to weanling Sprague-Dawley rats (25/sex/group) at dose levels of 5.23, 10.23 or 15.00% (w/w) for 91 days. Corn oil was added at 8.96, 5.91 and 3.00%, respectively, to provide essential fatty acids and digestible fat calories. Corn oil alone (12.14%) and a blend of medium-chain triglyceride (MCT) oil plus corn oil (11.21 and 3.13%, respectively) served as controls. All diets were formulated to provide about 4000 kcal/kg of diet and 26.8% of digestible calories from fat by assuming that corn oil, MCT oil, and caprenin provided 9, 7 and 5 kcal/g, respectively. Survival, clinical signs, body weight, feed consumption, feed efficiency, organ weights, organ-to-body-weight ratios, organ-to-brain-weight ratios, haematological values and clinical chemistry parameters were evaluated in all groups. Histopathology of a full complement of tissues was evaluated in the corn oil and MCT oil control groups as well as the high-dose caprenin group. Additional rats (n = 5/sex/group) were included in the study to determine whether there was marked storage of C22:0 in heart, liver or perirenal fat at the end of the 91-day feeding period. No significant differences in body weight gain were measured with the balanced caloric diets, although feed conversion efficiency was reduced in the high-dose caprenin group. No adverse effects from the ingestion of caprenin were detected, nor were significant amounts of C22:0 present in the fat extracted from the selected fat depot sites. These results establish a no-observable-adverse-effect level (NOAEL) of more than 15% (w/w) caprenin in the diet (or more than 83% of total dietary fat), which is equal to a mean exposure level of more than 13.2 g/kg/day for male rats and more than 14.6 g/kg/day for female rats.

Adipose Tissue↗
PubMed1993

Extraperitoneal laparoscopy: early experience and evaluation.

This paper evaluates the use of various balloon dilators in the flank, iliac fossa and retro-pubic regions to create extraperitoneal spaces for subsequent extraperitoneal laparoscopy. Eight patients were investigated, three undergoing pelvic surgery, four renal surgery (two nephrectomies) and one laparoscopic ureterolithotomy. Extraperitoneal balloon dilatation was simple, safe and easy and provided large tamponaded bloodless cavities and excellent vision. Port access was slightly restricted by the lateral peritoneal reflections and bony prominences of the thorax and pelvis. This approach for laparoscopy is safe, simple and deserving of further evaluation.

Female↗
PubMed1993

Intraduodenal delivery of intrinsically and extrinsically labelled CaCO3 in the rat: effect of solubilization on calcium bioavailability.

The dissolution of CaCO3 before intraduodenal administration was found to be an important factor determining calcium (Ca) bioavailability. Extrinsically and intrinsically labelled 47CaCO3 preparations were sequentially dissolved by serial additions of HCl. Aliquots of these preparations were collected before (no HCl added) and during the solubilization process and administered intraduodenally to rats. Whole body 47Ca retention 72 h post-dose was used as a measure of Ca bioavailability. Although dissolution of CaCO3 significantly increased Ca bioavailability (P < 0.001), Ca from both intrinsically and extrinsically labelled CaCO3 was absorbed and retained to some extent without prior acid dissolution. Due to a disproportionately high concentration of 47Ca on the particle surface, extrinsically labelled 47CaCO3 overestimated bioavailability when unsolubilized or partially solubilized CaCO3 preparations were used (P < 0.05). These data indicate that dissolution is a determining factor for Ca bioavailability from CaCO3. Incomplete dissolution will significantly limit but not completely prevent Ca bioavailability. The disintegration and dissolution characteristics of commercial CaCO3 preparations, which vary widely, may produce important differences in Ca absorption.

Animals↗
PubMed1993

Endopyelotomy for pelvi-ureteric junction obstruction in children.

Endopyelotomy or percutaneous pyeloplasty has become an attractive alternative to open pyeloplasty in adult patients in recent years. However, the experience and reports of this procedure in the paediatric population have been limited to date. We report our experience in paediatric patients undergoing this procedure. Seventeen children with primary (14) or secondary (3) pelvi-ureteric junction (PUJ) obstruction were consented for endopyelotomy over a 3-year period. Patient age ranged from 4 months to 16 years, and the male to female ratio was 11:6. Percutaneous nephrostomy was established in 5 patients prior to endopyelotomy either for diagnosis or for drainage of an acutely obstructed upper tract. The remaining 12 had endopyelotomy as a one-stage procedure. The average duration of the procedure was 103 min (range 60-150 min) and the average hospital stay was 5 days (range 3-9 days). Follow-up averaged 15 months (range 3-36 months). Endopyelotomy was successful in 10/13 patients (77%), unchanged in 1, and failed in the remaining 2. We believe that percutaneous pyeloplasty can be safely and successfully performed in children with primary and secondary PUJ obstruction.

Adolescent↗
PubMed1992

Stone-forming potential of milk or calcium-fortified orange juice in idiopathic hypercalciuric adults.

Milk and orange juice (OJ) fortified with calcium-citrate-malate (CCM; calcium-citrate-malate salt is a coined term to describe a trademarked calcium delivery system found only in Citrus Hill Plus Calcium) were evaluated for their potential to alter urine chemistries and crystallization during the course of an eleven week, crossover study. Six male and six female, non-stone-forming, idiopathic hypercalciuric adults consumed 600 mg per day as beverage calcium for four consecutive weeks. The results show that both milk and CCM-fortified OJ (CCM-OJ) were well tolerated by male and female hypercalciurics since few significant changes occurred in overall urinary chemistry profiles. CCM-OJ did, however, significantly (P less than or equal to 0.05) increase urinary pH relative to milk in both sexes and urinary citrate concentration in women only. Both of these urinary parameters were significantly increased by CCM-OJ in both sexes relative to the basal period. The combined pH and citrate effects are recognized to protect against calcium oxalate stone formation through formation of the soluble calcium citrate salt. These data indicate that either milk or CCM-fortified orange juice can be consumed, at least within the limits tested, as a dietary source of calcium without increasing the risk of stone formation in a calcium-sensitive subpopulation. That the urine chemistries and crystallization of such calcium-sensitive individuals does not significantly change with the consumption of either beverage implies that the urine of normal individuals would be exceedingly unlikely to change as well.

Adolescent↗
PubMed1991

Involvement of protein kinase C in competence induction of macrophages to generate T suppressor cells.

We have previously described an Ia-expressing macrophage hybridoma clone, termed clone 59, which attains the ability to induce Ts cells after activation with murine rIFN-gamma. In this report, we show that a protein kinase C (PKC) activator, PMA (10 ng/ml) can replace IFN-gamma in inducing this form of macrophage competence. IFN-gamma-induced cellular competence was abrogated specifically by a PKC inhibitor but not by inhibitors that have specificity for cyclic nucleotide-dependent protein kinases. Furthermore, PGE2 known to induce protein kinase A in murine macrophages also failed to induce competence. In contrast, the ability to induce Th responses was neither dependent on IFN-gamma nor inhibited by prior treatment with protein kinase inhibitors. Furthermore, PKC depletion of macrophages by treatment with high doses (100 ng/ml) of PMA abrogated their ability to induce Ts cells. In addition, PKC-depleted macrophages failed to regain the ability to stimulate Ts cells after further treatment with IFN-gamma. The ability of IFN-gamma to modulate macrophage-mediated induction of Ts cells does not clearly correlate with an increased Ia expression as inducible expression of Ia was not consistently abrogated by PKC inhibitor treatment. In addition, PKC inhibitors failed to prevent the production of the cytokines IL-1 and IL-6. However, incubation of IFN-gamma or PMA-treated macrophages with antibodies recognizing the putative IJ ligand blocked the ability to induce Ts cells, suggesting the expression of these determinants on accessory cells is responsible for Ts induction.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Involvement of two distinct regulatory T cell populations in the antigen-specific suppression of cytolytic T cell generation.

Alloantigen-specific, radiation-resistant T cells generated in mixed-lymphocyte cultures inhibited the generation of allospecific CTL responses in vitro. This regulatory T cell population was studied using mAb generated to Ag-specific suppressor factors that regulate the response to the synthetic terpolymer L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT). Both monoclonal 984 D4.6.5 and a pool of four mAb 2441, when added in the presence of complement, eliminated alloantigen-specific inhibition of the CTL response. When separate cell cultures treated with mAb 984 or 2441 plus complement were recombined, inhibition was reestablished, suggesting that two or more populations of cells are required for active inhibition. Furthermore, neither the mAb 984 nor the mAb 2441 plus complement had any effect on any stage of CTL development. This suggests that the inhibition of the CTL response was not the result of cytolytic activity via the regulatory T cells. Experiments in which these antibodies were added without complement treatment showed that the mAb 2441 neutralized the inhibitory activity, whereas mAb 984 augmented inhibition. It is concluded from these studies that regulatory T cells originally identified in humoral immune responses also regulate cell-mediated immune responses. Suppressor epitopes are displayed on the surface of these cells that allow them to be distinguished from other T cells. These data also show the utility of the mAb 984 and 2441 raised against specific suppressor T cell products in different experimental models of immunity. These studies suggest that phenotypically distinct Ts cell populations can play a normal regulatory role in both cell-mediated and humoral immunity.

Animals↗
PubMed1991

Distinct mechanisms of regulation of protein kinase C epsilon by hormones and phorbol diesters.

In this study, we examined the effects of T cell activators on the regulation of protein kinase C (PKC) isozymes present in thymocytes. Using affinity-purified anti-PKC antisera, we determined that the major PKC isoforms in murine thymocytes are PKC beta and PKC epsilon. The CD4+/CD8+ thymocyte subset expressed high levels of both PKC beta and PKC epsilon, whereas the CD4-/CD8- subset expressed much less of both. PKC beta was down-regulated following treatment of thymocytes with phorbol 12-myristate acetate (PMA) (2 x 10(-8) M) or ionomycin (0.4 microM). In contrast, PMA did not induce the down-regulation of PKC epsilon. Ionomycin alone, however, induced PKC epsilon down-regulation, similar to its effect on PKC beta. Similar observations were made on a promonocytic cell line, U937, which expresses PKC alpha, PKC beta (Strulovici, B., Daniel-Issakani, S., Oto, E., Nestor, J., Jr., Chan, H., and Tsou, A.-P. (1989) Biochemistry 28, 3569-3576), and PKC epsilon. To facilitate the study of PKC beta and PKC epsilon, we established a Chinese hamster ovary cell line which expresses murine PKC epsilon in addition to endogenous PKC alpha and PKC beta. Both PKC isoforms (beta and epsilon) were mostly in particulate form. PMA treatment left the majority of immunoreactive PKC epsilon intact. By contrast, thrombin treatment caused the disappearance of particulate and cytosolic PKC epsilon (60% by 10 min and 80% by 1 h). PMA and thrombin promoted the down-regulation of PKC beta with similar kinetics (100% down-regulation by 3 h). These results indicate that: 1) thymocytes express PKC epsilon; and 2) this isozyme exhibits a novel form of regulation distinct from the other PKC isozymes.

Amino Acid Sequence↗