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Biomedical subjects

W Jacobs

Publications and source records attributed to W Jacobs.

At least 37 records · Page 2Linked to original sources
PubMed1998

Tyramine-amplified immunohistochemical testing using "homemade" biotinylated tyramine is highly sensitive and cost-effective.

Immunohistochemical testing using biotinylated tyramine is a recently described technique that results in a strong amplification of the detection signal using antibodies at very high dilutions. In this report we compare the sensitivity and cost-effectiveness of the technique using "homemade" biotinylated tyramine with those of two commercially available kits. The technique using homemade tyramine was easy to follow and highly sensitive and resulted in a 2.5- to 5-fold reduction in costs compared with the commercial kits. Tyramine-amplified immunohistochemical testing using homemade biotinylated tyramine is a valuable and inexpensive tool for both the diagnostic and experimental histopathology laboratory.

Biotinylation↗
PubMed1997

Expression of intercellular adhesion molecule-1 and lymphocytefunction-associated antigen-1 in experimental Schistosoma mansoni infection and in synchronous periparticular hepatic granulomas in mice: immunohistochemistry, confocal laser scanning microscopy, and immunoelectron microscopy.

Adhesion molecules play an important role in inflammatory and immunological responses. We assessed the expression pattern of intercellular adhesion molecule-1 (ICAM-1) and lymphocytefunction-associated antigen-1 (LFA-1) in the livers of mice experimentally infected with Schistosoma mansoni and in synchronous hepatic granulomas induced by injection of soluble egg antigen (SEA)-coupled Sepharose beads in a mesenteric vein of mice. By immunohistochemistry, confocal laser scanning microscopy, and immunoelectron microscopy, ICAM-1 was localized on endothelial cells, sinusoidal-lining cells (Kupffer cells and sinusoidal endothelium), the hepatocyte cell membrane facing Disse's space, and inflammatory cells in the granuloma. LFA-1 was visualized on the inflammatory cells of the granuloma and on phagocytic sinusoidal-lining cells, most likely Kupffer cells. ICAM-1- and LFA-1-immunoreactive cells were present in the granuloma as early as at 3 days after injection of SEA-coupled beads and persisted with time. As granulomas became older, nonimmunoreactive granuloma cells appeared. We conclude that adhesion molecules play an important role in the genesis of the schistosomal granuloma.

Animals↗
PubMed1997

Inhibitory effects of CAI in glioblastoma growth and invasion.

While distant metastases are rare in patients with primary brain malignancies, local growth and invasion are common and life-threatening. Regional infiltration is responsible for the failure of local therapies, resulting in tumor recurrence, progression, and death. The process of invasion requires cellular adhesion, local proteolysis and migration. CAI, carboxyamide-triazole, is an anticancer agent developed as an inhibitor of selected signal transduction pathways. Studies on the effects of CAI on human glioblastoma growth and invasiveness are presented. CAI inhibited proliferation of 6 of 8 cell lines tested in a dose-dependent fashion in vitro (IC50 range 1.5-44 microM), with no effect on the U373 line. Inhibition of adhesion to tissue culture plastic was observed for the H4, T98G, and U373 lines pretreated with CAI; H4 and T98G were inhibited in adhesion to collagen type IV. Incubation with CAI decreased production of the 72 kDa and 92 kDa type IV collagenases in all cell lines, ranging from 16 to 93% inhibition. These observations show that the effects of CAI on cell line behavior can vary between lines that are similar in origin. Despite variability in the inhibitory effects for proliferation and adhesion, CAI is consistently able to inhibit the invasive phenotype of all glioma cell lines in vitro using the Matrigel barrier assay (IC50 range 13-28 microM). These observations suggest that CAI may have benefit in the treatment of gliomas and high grade astrocytomas.

Antineoplastic Agents↗
PubMed1997

Adult Schistosoma mansoni worms positively modulate soluble egg antigen-induced inflammatory hepatic granuloma formation in vivo. Stereological analysis and immunophenotyping of extracellular matrix proteins, adhesion molecules, and chemokines.

Synchronized liver granulomas were induced by injecting Sepharose beads to which SEA soluble egg antigen (SEA) or the concanavalin A binding fraction of SEA had been coupled into a mesenteric vein in naive, single-sex (35 days) and bisexually (28 days) Schistosoma mansoni-infected and Plasmodium berghei-immunized mice. Stereological analysis revealed that peak granuloma formation was already reached 8 days after injection in single-sex infected mice compared with 16 days in naive animals. No difference in granuloma formation between naive and P. berghei-immunized animals and between unisexually and bisexually S. mansoni-infected mice was observed. This suggests that the positive immunomodulatory effect on the granulomogenesis is worm specific and not likely to be due to arousal of the immune system by unrelated factors, nor is it influenced by the gender or degree of maturation of female worms. At all stages in time, the concanavalin A binding-fraction-induced granulomas reached only 65 to 70% of the volume of SEA-induced granulomas. Immunophenotyping of extracellular matrix proteins around deposited heads revealed that fibronectin was the dominant extracellular matrix protein and that also type I and IV collagen and laminin were deposited. Temporal analysis of the expression of the adhesion molecules ICAM-1, LFA-1, VLA-4, and VLA-6 was performed. Morphological evidence is presented for the role of adhesion molecules in the initiation and maintenance of hepatic granuloma formation. The chemokine monocyte chemoattractant protein-1 was expressed in the granuloma and in hepatic artery branches. From these data, it is concluded that adult S. mansoni worms positively modulate schistosomal hepatic granuloma formation in vivo. Adhesion molecules and chemokines play important roles in schistosomal granuloma formation.

Animals↗
PubMed1996

Identification and molecular characterization of a m5 muscarinic receptor in A2058 human melanoma cells. Coupling to inhibition of adenylyl cyclase and stimulation of phospholipase A2.

We report the identification and biochemical characterization of an endogenous m5 muscarinic acetylcholine receptor (mAChR) in the A2058 human melanoma cell line. This is the first demonstration of a m5AChR outside the central nervous system. The unusual effector coupling of this endogenous m5AChR is presented. The coding region amplified by polymerase chain reaction was identical to the known m5AChR sequence. Binding studies indicated a Kd of 99 +/- 6 pM and a Bmax of 45 +/- 4 fmol/mg membrane protein. This m5AChR coupled to stimulation of arachidonic acid release and to a 50% inhibition of forskolin-stimulated cAMP accumulation. The inhibition of cAMP production was insensitive to pertussis toxin treatment, but was dependent upon extracellular calcium. In contrast to the odd mAChR pattern, no cAMP was produced in response to carbachol (CC) stimulation. Moreover, no release of inositol phosphates could be measured after CC treatment despite the presence of at least 2 phospholipase C isoforms in A2058 cells. CC-stimulated arachidonic acid release (EC50 = 17.8 +/- 0.1 microM) was dependent upon external Ca2+, with marked reduction after coincubation with EGTA, Co2+, or high doses of verapamil (IC50 = 166 microM) or diltiazem (IC50 = 243 microM). Brief exposure to phorbol 12-myristate 13-acetate augmented CC-stimulated arachidonic acid release, whereas prolonged phorbol 12-myristate 13-acetate treatment resulted in down-regulation of release. Activation of the m5AChR resulted in Ca2+ influx that was attenuated by muscarinic antagonism and removal of extracellular Ca2+. A2058 cells exposed to CC had no alteration of cell shape or growth potential in monolayer culture, however, a statistically significant reduction in density-independent growth was observed over the range of CC concentrations from 0.1 to 100 microM. This endogenous m5AChR has a novel signal transduction coupling profile and receptor activation reduces clonogenic potential.

Adenylyl Cyclase Inhibitors↗
PubMed1996

Antiobesity effect of diazoxide in obese Zucker rats.

Hyperinsulinism and insulin resistance are characteristic findings in obese subjects. Obesity in both humans and experimental animals is associated with a reduced number of insulin receptors and a decreased insulin-mediated glucose disposal, whereas sensitivity to insulin's antilipolytic action is unaltered. To evaluate the antiobesity effect of diazoxide (DZ), an inhibitor of glucose-stimulated insulin release, 7-week-old Zucker obese and lean rats were studied. Obese and lean rats were grouped into DZ-treated (150 mg/kg/d) and control (C) groups. DZ-treated obese rats consumed similar amounts of calories per kilogram body weight (BW) compared with C obese animals, but gained less weight (P<.01). Postabsorptive plasma free fatty acids (FFA), cholesterol, and triglycerides were significantly higher in obese versus lean animals (P<.01). DZ treatment reduced plasma triglyceride levels in obese animals (P<.001), but had no significant effect on FFA or cholesterol concentrations. Plasma glucose concentrations in the postabsorptive state and during glucose tolerance tests (GTTs) were significantly lower in DZ obese versus C obese rats (P<.01) despite a decrease in plasma insulin concentrations in DZ-treated animals (P<.01). In contrast, DZ lean rats developed glucose intolerance (P<.05). Sensitivity and responsiveness to the antilipolytic effect of insulin in isolated adipocytes were significantly decreased in DZ obese as compared with C obese rats (P<.01). Moreover, adipocyte specific insulin receptor binding was increased in both DZ lean and DZ obese animals (P<.01). This was accompanied by increased basal and insulin-stimulated glucose transport in both genotypes (P<.01). In conclusion, DZ increased insulin receptor binding and glucose transport while decreasing hyperinsulinemia and insulin sensitivity to the antilipolytic action of insulin. This combined effect resulted in improved glucose tolerance and a decrease in weight gain in obese rats, implying that pharmacologic modification of the disturbed insulin metabolism of obesity may be therapeutically beneficial.

Adipocytes↗
PubMed1996

Stereological evaluation of malignant mesothelioma versus benign pleural hyperplasia.

The differential diagnosis between malignant mesothelioma and benign pleural hyperplasia constitutes a well-known problem. In the present study we examined unbiased stereological techniques to assess the mean nuclear volume (MNV) using the point-sampled intercepts (PSI) in 37 cases of malignant mesothelioma and in 28 cases of benign pleural hyperplasia. Neither the use of different fixatives nor the histological type of malignant mesothelioma produced any significant difference on the measured nuclear volume. The differences observed between the MNV data obtained from benign pleural hyperplasia and those from any of the three types of malignant mesothelioma were found to be highly significant. All lesions with an MNV larger than 250 microns3 were found in our study to correspond to the malignant mesothelioma type, while an MNV that was smaller than 200 microns3 could only be detected in benign specimens. These observations lead us to propose the MNV measurement using PSI as an additional tool to enhance the differential diagnosis of malignant mesothelioma versus benign pleural hyperplasia.

Cell Nucleus↗
PubMed1996

Outpatient reduction mammaplasty: a review of 338 consecutive cases.

Reduction mammaplasty is usually accomplished under general anesthesia, often with an overnight stay. With cost-containment pressures, the ability to perform this surgery in an outpatient setting would have obvious benefits. We present a series of 338 patients who have undergone bilateral reduction mammaplasty over a 3-year period. Two hundred eighty-six cases (84.6%) were performed on an outpatient basis and 52 (15.4%) were inpatients. Patient age ranged from 13 to 82 years. Significant differences were found between the two groups regarding average age (34.3 and 42.4 years, respectively) and average total resection weights (1,486.1 gm and 1,895.6 gm, respectively). The maximum total resection weight was 6,000 gm in the outpatient group and 7,140 gm in the inpatient group. Surgical techniques included inferior pedicle (N = 273), central mound (N = 54), and free nipple graft (N = 11). There were no substantial differences in the incidence of minor complications, including wound separation, seromas, hypertrophic scars, infection, and hypopigmentation. Autologous transfusion was utilized in 18 patients early in the series. Of the 175 patients (52%) who returned a follow-up questionnaire, all considered their outpatient experience a positive one. In addition, 33% of the inpatients who responded (N = 8/24) felt their hospital stay was unnecessary. Outpatient surgery resulted in a savings of $1,500 to $2,500 when compared to an overnight stay. Reduction mammaplasty can be performed safely in an outpatient setting and is not necessarily limited by age or extent of resection.

Adolescent↗
PubMed1995

Induction of pseudoseizures with intravenous saline placebo.

For a 2-year period, all patients admitted to the inpatient adult EEG videotelemetry unit of the University of Miami School of Medicine underwent attempted event induction with intravenous normal saline placebo. Of 175 patients monitored during that period, 101 underwent attempted placebo saline induction, whereas 58 patients were either in the pediatric age group, were undergoing a repeat hospitalization (i.e., depth electrode monitoring), or refused induction. The final diagnosis in each patient was established after review of the history; physical, interictal, and ictal EEG findings; brain imaging studies; interictal and postictal brain single photon emission computed tomography (SPECT) and serum prolactin levels; psychiatric and psychological evaluations; and detailed neuropsychological testing. Final diagnoses were separated into epilepsy alone, pseudoseizures, epilepsy and pseudoseizures, and other (neither epilepsy nor pseudoseizures). No patient with an eventual diagnosis of epilepsy alone was inducible. Forty-one patients with a diagnosis of epilepsy were not inducible. Of 32 patients with an eventual diagnosis of pseudoseizures, 29 were inducible. One of these 29 was also diagnosed with epilepsy. Three patients with an eventual diagnosis of pseudoseizures were not inducible; 90.6% of patients with an eventual diagnosis of pseudoseizures were inducible, i.e., had events identical to those reported by history, after injection of saline placebo. Placebo saline injection is a safe and effective means of distinguishing epilepsy from pseudoseizures.

Adult↗
PubMed1994

Calcium influx modulates expression of matrix metalloproteinase-2 (72-kDa type IV collagenase, gelatinase A).

Altered regulation of metalloproteinases may play a role in a variety of pathologic conditions including cancer. Previous studies have demonstrated transforming growth factor-beta 1 (TGF-beta 1)-mediated stimulation of expression and activation, and phorbol ester-mediated inhibition of matrix metalloproteinase (MMP)-2 (72-kDa type IV collagenase/gelatinase A), indicating a role for transmembrane signal transduction in MMP-2 regulation. We now describe a role for calcium mobilization in the regulation of MMP-2 expression. Receptor-operated calcium influx has been shown to be inhibited by a novel synthetic inhibitor, carboxy amido-triazole (CAI). Incubation of A2058 human melanoma, HT-1080 human fibrosarcoma, and OVCAR3 human ovarian cancer cells with CAI (0-10 microM) resulted in a dose-dependent reduction in MMP-2 latent and activated species activity by zymogram analysis of conditioned medium. This reduction is not due to direct inhibition of the enzyme by CAI or CAI-induced MMP-2 degradation. Decreased quantity of secreted MMP-2 protein in CAI-treated cells was shown by immunoblot and pulse-chase analysis of newly synthesized MMP-2. Cell coincubation with CAI (2 microM) and TGF-beta 1 (5 ng/ml) caused a decrease in the overall amount of latent and activated MMP-2 by zymogram and immunoblot analysis and showed that CAI inhibited TGF-beta 1 stimulation of MMP-2 production at the level of RNA expression. This was confirmed by Northern analysis of A2058 cells treated with CAI (2 microM) for 24 and 48 h and demonstrated a 55% reduction in message for MMP-2 and a 61% reduction in message for MMP-1, 54-kDa interstitial collagenase. Specificity for CAI action was demonstrated by equivalent MMP-2 inhibitory activity from analogs of CAI that retained the ability to inhibit calcium influx and by lack of inhibition by exposure to inactive CAI analogs that could not inhibit calcium influx. As an independent verification of specificity, a marked reduction in MMP-2 gelatinase activity by zymogram was shown after treatment of A2058 cells with SK&F 96365, an unrelated inhibitor of receptor-operated calcium influx. These results suggest a role for calcium-mediated signal transduction in the expression of metalloproteinases.

Biological Transport↗
PubMed1994

Structure-function analysis of signal and growth inhibition by carboxyamido-triazole, CAI.

Evidence is accumulating that calcium homeostasis and calcium-regulated events may be selectively important in generation and maintenance of the malignant phenotype. CAI, a carboxyamido-triazole with a halogenated benzophenone tail, is a novel inhibitor of receptor-operated calcium influx and arachidonic acid release which inhibits malignant proliferation, invasion, and metastasis. The focus of this investigation was structural analysis of CAI and to determine if the inhibition of calcium influx and arachidonic acid release by CAI and its antiproliferative activity were mediated through the same chemical domains. Four families of molecular modifications of the CAI parent were synthesized: (I) modification or substitution of the triazole ring; (II) removal of the substituted benzophenone tail; (III) dehalogenation or partial truncation of the benzophenone moiety; and (IV) removal of the triazole and altered substitutions of the benzophenone tail. Compounds were tested for the inhibition of calcium influx and arachidonic acid release and inhibition of proliferation and colony formation in soft agar using the malignant CHO line transfected with the m5 muscarinic receptor and the A2058 human melanoma cell line. Only CAI and Group I compounds inhibited stimulated calcium influx, arachidonic acid release, and proliferation. Linear regression analysis of the relationship of the 50% inhibitory concentration values for all compounds in inhibition of calcium influx and arachidonate release was statistically significant (r2 = 0.993). Similarly, a linear relationship was demonstrated between inhibition of calcium influx and inhibition of tumor cell proliferation (r2 = 0.971). Groups II-IV had minimal or no signal or growth inhibitory activity. This investigation provides the first evidence for a coordinate link between calcium influx, calcium-mediated arachidonic acid release, and malignant proliferation and metastasis and constitutes the initial analysis of structurally important domains of the CAI molecule.

Aminoimidazole Carboxamide↗
PubMed1994

Proliferation in malignant mesothelioma as determined by mitosis counts and immunoreactivity for proliferating cell nuclear antigen (PCNA).

In order to assess its discriminating and prognostic value, we studied immunoreactivity for proliferating nuclear cell antigen (PCNA) in human malignant mesothelioma (31 cases) and in human non-neoplastic mesothelium (33 cases with reactive mesothelium and 20 cases of normal mesothelium) using the murine monoclonal antibody PC10. We also compared it with mitosis counts expressed as the mitotic volume index (MV index). There were differences between malignant mesothelioma, reactive mesothelium, and normal mesothelium for percentage of PCNA immunoreactive cells (mean +/- SD; 27 +/- 9, 9.5 +/- 5.1, and 3.6 +/- 1.6, respectively) and for their MV index (20.3 +/- 4.5, 9.4 +/- 2.1, and 3.6 +/- 0.6, respectively). The median actuarial survival was 10.1 months for patients with less than 25 per cent PCNA immunoreactive cells, 9.4 months for patients with less than 20 mitoses per mm2 of tumoural tissue, 5.9 months for patients with more than 25 per cent PCNA immunoreactive cells, and 5.3 months for patients with more than 20 mitoses per mm2 of tumoural tissue. Our results suggest that PCNA immunoreactivity is useful in discriminating between neoplastic and non-neoplastic mesothelium and that it may have prognostic value in malignant mesothelioma.

Adult↗
PubMed1993

Immunohistochemical staining of ras oncogene product in neoplastic and non-neoplastic mesothelial tissues: immunoreactivity for N-ras and lack of immunohistochemical staining for Ha-ras and K-ras.

Immunohistochemical staining of 36 malignant mesotheliomas and 45 cases of non-neoplastic mesothelium including 20 specimens with signs of hyperplasia were investigated using murine monoclonal antibodies directed against p21 ras protein, Ha-ras protein, K-ras protein, and N-ras protein. All cases of non-neoplastic mesothelium and the majority of the malignant mesotheliomas (78 per cent) showed cytoplasmic and often submembranous immunoreactivity in more than 50 per cent of the cells with both the pan-ras and N-ras antibody. No immunoreactivity was observed for Ha- and K-ras. There was no statistically significant difference with respect to immunoreactivity between neoplastic and non-neoplastic mesothelium or between the various mesothelioma subtypes.

Adult↗
PubMed1993

Infective aortic endocarditis after percutaneous balloon aortic valvuloplasty.

Infective aortic endocarditis developed in an elderly patient after a percutaneous balloon aortic valvuloplasty. The transesophageal echocardiogram demonstrated a perivalvular abscess. The patient underwent surgical replacement of the infected valve, but later succumbed to renal failure. The development of infective aortic endocarditis should be recognized as a potentially fatal complication of percutaneous balloon aortic valvuloplasty. The important measures in preventing bacteremia during percutaneous balloon aortic valvuloplasty and the appropriate role of operation are discussed.

Aged↗
PubMed1993

Measurement of urinary sugars by HPLC in the estimation of intestinal permeability: evaluation in pediatric clinical practice.

Determinations of the permeability of the intestine to various sugars have been used to assess intestinal mucosal abnormalities, but the widespread application of such tests has been prohibited by the complexity of most classically used techniques. In this study, we evaluated the sensitivity and practicability of using HPLC to estimate intestinal permeability, with mannitol and lactulose as mono- and disaccharides, respectively. The results of the permeability tests were compared with those of intestinal biopsy in 20 children. All patients with an abnormal intestinal biopsy showed a low mannitol recovery, and the patients with allergic symptoms showed a high lactulose recovery. Our preliminary data suggest that HPLC is reliable, gives results comparable with those of other methods, and is a practical way to determine intestinal permeability noninvasively.

Adolescent↗