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M Kelley

Publications and source records attributed to M Kelley.

At least 73 records · Page 4Linked to original sources
PubMed1996

Immune response to polyvalent melanoma cell vaccine in AJCC stage III melanoma: an immunologic survival model.

BACKGROUND: Our polyvalent, allogeneic melanoma cell vaccine (MCV) induces immunoglobulin M (IgM) and immunoglobulin G (IgG) class antibodies to a 90-kDa glycoprotein melanoma-associated antigen (MAA). Additionally, MCV induces delayed-type hypersensitivity (DTH) responses that we previously correlated with survival. We hypothesized that early DTH responses to MCV and early humoral responses to the 90-kDa MAA expressed on MCV cells may be predictive of overall survival. We tested this hypothesis by monitoring immunologic profiles in 59 patients with melanoma who were receiving MCV after surgical resection of regional lymph node or soft-tissue metastases. METHODS: Blood was drawn before vaccine administration, biweekly for 6 weeks, and then monthly. DTH to MCV was recorded at 0, 2, 4, and 8 weeks of MCV therapy. Mean antibody titers during the first 6-week interval were calculated. Changes in DTH were calculated as the difference between peak and prevaccine values (delta DTH). RESULTS: At a median follow-up of 75.6 months (range 5-138), univariate analysis assigned prognostic significance to gender (p = 0.046), lymph node involvement (p = 0.024), delta DTH (p = 0.044), mean anti-90-kDa MAA IgG (p = 0.0009), and mean anti-90-kDa MAA IgM (p = 0.0014). In multifactorial analysis, only the three immunologic variables significantly impacted survival (p = 0.046, 0.0005, and 0.0053, respectively). A mathematical model based on delta DTH and mean anti-90-kDa MAA IgG and IgM titers closely approximated the observed individual and overall survival rates. CONCLUSIONS: The correlation between overall survival and initial humoral/cellular immune responses to MCV immunotherapy may be useful in selecting patients most likely to benefit from prolonged adjuvant immunotherapy.

Adolescent↗
PubMed1996

Interactions of BMS-181174 and radiation: studies with EMT6 cells in vitro and in solid tumors.

N-7[2-(4-nitrophenyldithio)-ethyl] mitomycin C, (BMS-181174; previously designated as BMY25067) is a mitomycin C analog now in initial clinical trials. The experiments described in this report were performed to assess whether BMS-181174, like mitomycin C and porfiromycin, was selectively toxic to the hypoxic cells in solid tumors and might therefore prove valuable in combination with radiotherapy. In contrast to mitomycin C and porfiromycin, BMS-181174 was more toxic to aerobic EMT6 cells in vitro than to cells made acutely hypoxic. In vitro, BMS-181174 and radiation produced cytotoxicity compatible with either additive or slightly supra-additive cytotoxicity. In vivo, BMS-181174 was effective in killing cells in solid EMT6 tumors. The effects of regimens combining BMS-181174 and radiation in vivo were complex. Combinations of low doses of BMS-181174 plus a large dose of radiation were very effective in killing cells in solid tumors. However, the survival curve plateaued at high doses of BMS-181174, providing evidence for a subpopulation of tumor cells which were resistant to both BMS-181174 and radiation; this was hypothesized to be a hypoxic cell population.

Aerobiosis↗
PubMed1995

Cytotoxicity of BMS-181174. Effects of hypoxia, dicoumarol, and repair deficits.

The mitomycin C (MC) analog BMS-181174 (previously designated as BMY25067) has been shown to be active against a variety of solid tumors in mice. The activity of this compound against tumor cell lines resistant to MC and the different toxicity profiles of BMS-181174 and MC suggested that there may be significant differences in the metabolism and the mechanisms of action of these two compounds. Our studies with a mouse mammary tumor cell line (EMT6), a wild-type Chinese hamster cell line (AA8), and three repair-deficient Chinese hamster cell lines (UV4, UV5, and EM9) supported this concept. BMS-181174 was more toxic to all five cell lines in air than in hypoxia; in contrast, MC is more toxic in hypoxia. Dicoumarol (which increases the cytotoxicity of MC in hypoxia and reduces the cytotoxicity of this drug in air) did not alter the cytotoxicity of BMS-181174. This finding suggests that neither DT-diaphorase nor cytochrome b5 reductase is involved in the activation of BMS-181174. Studies with the repair-deficient cell lines suggest that DNA strand breaks are not important to the cytotoxicity of BMS-181174, and that cross-links and adducts may be the critical lesions; these studies also suggest that the lethal lesions produced by BMS-181174 are the same under aerobic and hypoxic conditions.

Animals↗
PubMed1995

Damnacanthal is a highly potent, selective inhibitor of p56lck tyrosine kinase activity.

Damnacanthal, an anthraquinone isolated from a plant extract, was found to be a potent, selective inhibitor of p56lck tyrosine kinase activity. The structure, potency, and selectivity of damnacanthal were confirmed by independent synthesis and testing. Damnacanthal exhibited an IC50 of 17 nM for inhibition of p56lck autophosphorylation and an IC50 of 620 nM for phosphorylation of an exogenous peptide by p56lck. Damnacanthal had > 100-fold selectivity for p56lck over the serine/threonine kinases, protein kinase A and protein kinase C, and > 40-fold selectivity for p56lck over four receptor tyrosine kinases. It also demonstrated modest (7-20-fold), but highly statistically significant, selectivity for p56lck over the homologous enzymes p60src and p59fyn. Mechanistic studies demonstrated that damnacanthal was competitive with the peptide binding site, but mixed noncompetitive with the ATP site. Although damnacanthal contains a potentially reactive aldehyde moiety, equilibrium dialysis experiments demonstrated that significant amine formation between damnacanthal and amines occurred only at high concentrations of reactants. However, damnacanthal appeared to bind nonspecifically to membrane lipids and was not active in whole cell tyrosine kinase assays. Damnacanthal is the most potent, selective inhibitor of p56lck tyrosine kinase activity described to date and may represent the starting point for the identification of novel, selective inhibitors of p56lck which are active in whole cell as well as in cell-free systems.

Alkaloids↗
PubMed1995

Inhibition of bile acid conjugation by cyclosporin A.

Each of the two steps involved in bile acid conjugation was tested in vitro for its sensitivity to inhibition by cyclosporin A (CsA). Bile acid-CoA: glycine/taurine N-acyltransferase, the enzyme which catalyzes the second step, was tested and found to be insensitive to inhibition by 20 microM CsA. Bile acid:CoA ligase, the enzyme which catalyzes the first step, was found to be inhibited by 25% at 10 microM CsA in the standard assay. The inhibition was competitive vs. bile acid and noncompetitive vs. ATP, and uncompetitive vs. CoA. CsA was also found to interfere with the divalent cation requirement of the enzyme at low concentrations of Mg2+ the maximum inhibition was 70%. The maximum inhibition obtainable at physiologic Mg2+ concentration was 40%. The extent of inhibition was presumably limited by the insolubility of CsA. At concentrations of CsA reached in vivo during drug therapy, CsA can be expected to significantly inhibit bile acid conjugation.

Acyltransferases↗
PubMed1995

Endotoxin receptors (CD14) are found with CD16 (Fc gamma RIII) in an intracellular compartment of neutrophils that contains alkaline phosphatase.

CD14 is a glycosylphosphatidylinositol (GPI)-anchored protein on the surfaces of monocytes and polymorphonuclear leukocytes (PMN) that binds and initiates cellular responses to bacterial LPS. PMN also contain an intracellular pool of CD14 that can be deployed rapidly to the cell surface in response to stimulation with a variety of agonists. To determine which of the well-characterized subcellular compartments of PMN contains CD14, cells were cavitated and fractionated on Percoll gradients. The gradient fractions were assayed for CD14 by ELISA and Western blot and for the marker proteins beta-glucuronidase (azurophil granules), vitamin B12 binding protein (specific granules), alkaline phosphatase (secretory vesicles and plasma membrane), and HLA (plasma membrane). Approximately one-half of the CD14 ran with plasma membrane fractions and one-half with intracellular membranes of light density. Both intracellular and cell surface CD14 were associated tightly with membrane, and both forms showed identical electrophoretic mobility. The intracellular CD14 was clearly not present in azurophil granules or specific granules, but ran precisely with alkaline phosphatase, a marker for secretory vesicles. Parallel studies showed that an additional GPI-linked protein, Fc gamma RIII (CD16), also fractionated precisely with CD14 and alkaline phosphatase. Association of CD14 with secretory vesicles were confirmed by studies on cells stimulated with the formyl peptide fNLLP for 20 min at 37 degrees C before fractionation. This treatment caused translocation of CD14 from intracellular fractions to plasma membrane fractions. No release of the specific granule marker vitamin B12 binding protein was observed under these conditions, whereas two other GPI-anchored proteins, alkaline phosphatase and CD16, moved coincidentally with CD14 to comigrate with the plasma membrane. Time course studies of CD14 and CD16 surface expression confirmed the rapid and coordinate up-regulation of these proteins. Thus, the intracellular compartment containing CD14 and CD16 had the properties of secretory vesicles. These vesicles may represent a specialized membrane domain of PMN enriched in GPI-anchored proteins.

Alkaline Phosphatase↗
PubMed1995

Cryptosporidia--who is at risk?

Cryptosporidium parvum is a coccidian parasite originally described a century ago and, until recently, not considered to be a human pathogen. It has a complex life cycle, including both sexual and asexual reproduction, an auto-infectious cycle, and the ability to complete its development within a single host. The transmission form is a robust, environmentally resistant oocyst, excreted in the stool, which can exist for long periods of time in the environment. Because animals, in particular domesticated livestock, are its primary host, human infection is usually zoonotic. Oocysts often find their way into water supplies, and it resists chlorination and is incompletely filtered from processed drinking water supplies, even when filtration is working optimally. Transmission via ingestion of fecally contaminated swimming pool water, food, fomites, and sexual activities facilitating fecal-oral inoculation have been demonstrated. The major target of C. parvum in the host is the intestinal epithelial cell, resulting in diarrhea, sometimes profuse and persistent, although it may also infect other organs such as the gall bladder and lungs. Pathogenesis involves attachment, probably via a sporozoite lectin, invasion, probably involving apical organelles, replication within a parasitophorous vacuole with the host cell membrane, causing cellular dysfunction. Diagnosis is generally made by visualization of the oocyst form in stool by staining methods, the best of which appears to be auramine and fluorescence microscopy. Those at greatest risk are immunocompromised adults and children, especially those with AIDS, children in day care, travelers to endemic regions, dairy or cattle farm workers of their families or contacts, household contacts of cases or carriers, and possibly owners of infected dogs or cats or their neighbors. There is no specific therapy available, however in the immunocompetent host the illness is self-limited, lasting from a few days to 3 weeks, and long term carriage is uncommon. In the immunocompromised host, infection is prolonged, sometimes asymptomatic, but may result in chronic debilitating diarrhea with dehydration, malabsorption and wasting. Public health measures to reduce contamination of water supplies and vigilant surveillance will reduce the risk to populations. Reducing behaviors favoring fecal-oral transmission, such as certain sexual activities, and scrupulous hygiene in the day care setting would also reduce the likelihood of transmission but not eliminate it. Given our lack of knowledge about Cryptosporidium biology and pathogenesis, high priority should be given to research designed to increase our understanding of the organism and improve the chance of developing useful therapeutic or preventative drugs or strategies.

Adult↗
PubMed1995

The use of combination internal fixation and hybrid external fixation in severe proximal tibia fractures.

Forty-eight patients with 50 severe fractures of the proximal tibia were followed prospectively for 2-4 years (mean 2.7 years) to evaluate the use of limited internal fixation combined with external fixation in the treatment of these injuries. There were 27 men and 21 women ranging in age from 20 to 74 years. Fractures were classified according to the A0 system (Mast J, Ganz R, Jacob R: Planning and reduction technique in fracture surgery. Berlin, Springer-Verlag, 1989), which included 5 A3, 6 C1, 16 C2, and 23 C3 fractures. All patients in this series healed; 48 fractures healed in an average of 12 weeks without subsequent surgery. There were two (4%) nonunions requiring bone graft. The average hospital special surgery knee score was 90 (68-100). Grading criteria for anatomical outcome revealed there were 17 (34%) excellent results, 24 (48%) good results, 6 (12%) fair results, and 3 (6%) poor results. In conclusion, this treatment method is associated with a high percentage of good and excellent results. Combined internal and external fixation combines the advantages of anatomic, stable fixation with less soft-tissue dissection and eliminates the need for large implants.

Adult↗
PubMed1995

Administration of a perfluorochemical emulsion plus carbogen breathing does not alter radiation pneumonitis.

The effects of treatment with a perfluorochemical emulsion plus carbogen on radiation pneumonitis were examined in a rat model system. Rats received thoracic irradiation (15 Gy) and radiation reactions in the lungs were assessed 25 and 35 days later using bronchoalveolar lavage and histologic assessments. The irradiated lungs showed the expected evidence of acute radiation pneumonitis, including protein leaks and also alveolar infiltrates and interstitial infiltrates. Administration of a perfluoro-chemical emulsion (Fluosol; 15 ml/kg) plus carbogen breathing for 30 min before and during irradiation did not enhance the reactions seen in the irradiated lungs.

Animals↗
PubMed1995

Elevated circulating interleukin-7 levels in patients with systemic juvenile rheumatoid arthritis.

OBJECTIVE: Since interleukin-7 (IL-7) has been shown to induce monocyte production of IL-1, IL-6, IL-8, and tumor necrosis factor-alpha, we investigated plasma and synovial fluid levels of IL-7 in patients with juvenile rheumatoid arthritis (JRA). METHODS: IL-7 levels were measured using an ELISA in 60 patients with JRA and in 25 patients with other rheumatic disorders (ORD). RESULTS: In patients with systemic JRA, but not in patients with polyarticular or pauciarticular JRA or with ORD, plasma IL-7 levels were significantly higher (p < 0.0001) than those of healthy controls. IL-7 was undetected in all synovial fluid tested. Among patients with systemic JRA, those with persistent systemic symptoms had IL-7 levels significantly higher (p < 0.01) than those of patients in which systemic symptoms subsided. CONCLUSION: Plasma IL-7 levels are elevated in patients with systemic JRA and are associated with the presence and severity of systemic symptoms.

Adolescent↗
PubMed1995

Cytochrome P-450 2E1 is not the sole catalyst of chlorzoxazone hydroxylation in rat liver microsomes. off.

The contribution of individual cytochrome P-450 isozymes in the hydroxylation of the centrally acting skeletal muscle relaxant chlorzoxazone was determined in rat liver microsomes. The hydroxylation rate of chlorzoxazone was found to be 50% greater in male than female microsomes. Kinetic studies using control male microsomes showed that chlorzoxazone hydroxylation was biphasic with a calculated low Km (33 microM) and high Km (116 microM). Liver microsomes from isoniazid-, beta-naphthoflavone- or dexamethasone-treated male rats produced a Km of 93, 69 and 26 microM, respectively. When chlorzoxazone hydroxylation activity was measured at a high substrate concentration (200 microM), treatment of male rats with isoniazid, acetone, beta-naphthoflavone and dexamethasone produced increases in the activity rate of 124%, 117%, 81% and 32%, respectively. However, when the activity was measured at a low substrate concentration (2 microM), liver microsomes from dexamethasone-treated male and female rats produced 5- and 10-fold induction, respectively. In immunoinhibition studies at 200 microM of chlorzoxazone, antibodies specific for cytochrome P-450 2E1 inhibited the rate of chlorzoxazone hydroxylation in microsomes from control and isoniazid-treated male rats by 68% and 79%, respectively. A monoclonal antibody (C8) against P-450 1A1 inhibited 67% of the activity in microsomes from beta-naphthoflavone-treated male rats but was ineffective inhibiting chlorzoxazone hydroxylation in microsomes from control or dexamethasone-treated male rats. In liver from control female rats, antibodies against cytochrome P-450 2E1 inhibited 80% of chlorzoxazone hydroxylation, whereas it inhibited only 47% of the activity in dexamethasone-treated females.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Metaphyseal dissociation fractures of the proximal tibia. An analysis of treatment and complications.

A study was done of 44 metaphyseal dissociation fractures of the proximal tibia in 42 patients (27 men and 15 women, aged 22 to 77 years; mean, 42 years). Follow-up ranged from 6 months to 4 years. There were 2 study groups: a retrospective group (group 1, 22 fractures) given a variety of treatments ranging from casts to dual plates, and a prospective group (group 2, 22 fractures) treated by combining external fixation and optional minimal internal fixation. There were 12 comminuted fractures in group 1 and 20 in group 2 (P < 0.01). All fractures eventually healed, with an average healing time in group 1 of 3.8 months, and 5.3 months in group 2. There was one delayed union in group 2. Results were graded from poor to excellent, based on pain, range-of-motion, and malunion. There were 6 poor and 4 fair results in group 1, and no poor and 3 fair results in group 2. Complications included 6 deep infections, 5 in group 1 (1 requiring a free-flap procedure); and 1 pin-tract infection resulting in septic arthritis in group 2. There were 7 gastrocnemius flaps required in group 1, and 1 in group 2. The results of this study suggest that patients treated with external fixation had better results with less infection and soft-tissue complications than those treated with conventional internal fixation.

Adult↗
PubMed1994

Determination of the mechanism of reaction for bile acid: CoA ligase.

The reaction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated by kinetic analysis of the reaction as catalysed by guinea pig liver microsomes. The enzyme has an absolute requirement for divalent cation for activity so all kinetic analyses were carried out in excess Mn2+. A trisubstrate kinetic analysis was conducted by varying, one at a time ATP cholate and CoA. Both ATP and cholate gave parallel double reciprocal plots versus CoA, which indicates a ping-pong mechanism with either pyrophosphate or AMP leaving prior to the binding of CoA. Addition of pyrophosphate to the assays changed the parallel plots to intersecting ones; addition of AMP did not. This indicates that pyrophosphate is the first product. The end-product, AMP, was a competitive inhibitor versus ATP, as was cholyl-CoA at saturating concentrations of cholate. Both AMP and cholyl-CoA were uncompetitive inhibitors versus CoA. Based on this information, it was concluded that the reaction follows a bi uni uni bi ping-pong mechanism with ATP binding first, and with the release of the final products, AMP and cholyl-CoA, being random. CoA showed substrate inhibition at high but non-saturating concentrations and this inhibition was competitive versus ATP, which is consistent with the predicted ping-pong mechanism. The ability of cholyl-CoA, but not cholate or CoA, to bind with high affinity to the free enzyme was suggestive of a high affinity of the enzyme for the thioester link.

Adenosine Triphosphate↗
PubMed1994

Dual role of divalent cations in the bile acid:CoA ligase catalyzed reaction.

The role of divalent cations in the bile acid:CoA ligase catalyzed reaction of cholic acid, CoA and ATP to yield cholyl-CoA was investigated using guinea pig liver microsomes as the source of enzyme. EDTA treatment completely eliminated activity indicating an absolute requirement for divalent cation for enzyme activity. Analysis of this requirement revealed that it was twofold. First, the data suggested that ATP which was not complexed with a divalent cation did not appreciably bind to the enzyme and thus a divalent cation complex of ATP is the form of ATP that is the substrate for the enzyme. Further, this was shown to be the basis for the absolute requirement for divalent cation in the reaction. In addition, analysis revealed that there is a secondary site which binds divalent cations with relatively low affinity, and results in a rate enhancement. Binding at this secondary site is estimated to increase the rate by greater than 60%.

Adenosine Triphosphate↗
PubMed1994

Scatter factor modulates the metastatic phenotype of the EMT6 mouse mammary tumor.

EMT6 is a transplantable mouse mammary tumor cell line that has been utilized widely as a model system to study the effects of various treatments on local tumor growth and pulmonary metastasis. In this study, we examined the cellular mechanisms by which scatter factor (SF), a fibroblast-derived cytokine that stimulates epithelial cell motility, may contribute to tumor-cell dissemination, using the EMT6 model system. In vitro, SF stimulated EMT6 cell motility, invasiveness and cell-surface expression of urokinase (an enzyme required for cell migration through tissue). SF differentially stimulated EMT6 cell adhesion to and migration onto surfaces coated with collagen I and laminin. EMT6 cells treated in vitro with SF and injected i.v. into isogeneic BALB/c-Rw mice showed a small but significant increase (1.7-fold) in lung colony formation as compared with control cells. For EMT6 cells in vitro, SF had no effect on DNA synthesis, cell proliferation, cell size distribution, or in vitro colony-forming ability. Thus, the increase in lung colonization may be due to enhanced ability of SF-treated cells to adhere to subendothelial basement membrane or to invade through tissue. Studies of the tissue distribution of SF in BALB/c-Rw mice demonstrated high levels of active factor in the lung. Thus, the presence of endogenous pulmonary SF may have reduced the degree to which SF treatment stimulated EMT6 lung colonization. Significant SF activity was also found in extracts of EMT6 tumors. Cultured EMT6 cells did not produce SF, but did produce high titers of a soluble low-molecular-weight protein activity that is capable of stimulating SF production in human fibroblasts 3- to 5-fold. EMT6 tumor extracts contained high titers of a similar SF-inducing activity. These observations suggest that SF may contribute to the invasive and metastatic phenotype of EMT6 cells via a paracrine mechanism in which tumor cells induce the production of SF in stromal fibroblasts.

Animals↗
PubMed1994

Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14.

CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.

Acute-Phase Proteins↗
PubMed1994

Characterization of the acyl-CoA:amino acid N-acyltransferases from primate liver mitochondria.

The acyl-CoA:amino acid N-acyl-transferases were partially purified from human liver mitochondria. The aralkyl transferase (ArAlk) had glycine conjugating activity toward the following compounds: benzoyl-CoA > butyryl-CoA, salicylyl-CoA > heptanoyl-CoA, indoleacetyl-CoA. Its kinetic properties and responses to salt were very similar to those of bovine ArAlk. Further, its molecular weight was found to be similar to that of the bovine enzyme, in contrast to reports from other laboratories. Thus, it was concluded that the human and bovine ArAlk are not significantly different. The human arylacetyl transferase (AAc) had glutamine conjugating activity toward phenylacetyl-CoA, but only 3-5% as much activity toward indoleacetyl-CoA or 1-naphtylacetyl-CoA, respectively. While this was similar to the bovine AAc, the two forms differed in several respects. First, the human liver AAc was insensitive to salts. Second, glycination of phenylacetyl-CoA by human AAc could only be detected at a high concentration of glycine (50 mM), and the rates were < 2% of the rate of glutamination. In contrast, glycine conjugation predominates with bovine AAc. Kinetic analysis of the glutamination of phenylacetyl-CoA by human AAc revealed a KD for phenylacetyl-CoA of 14 microM and a Km for glutamine of 120 mM. These values indicate that the human AAc is not more efficient at glutamination than the AAc from bovine liver. An AAc was purified from rhesus monkey liver and found to have similar kinetic constants to the human form. This indicates that nonprimate enzymes do not have a defect in glutamine conjugation.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗