Biomedical subjects
Publications and source records attributed to S Orr.
Peptide-1-[N-[2-succinamidylethyl]amino]anthraquinones containing five seven amino acid residues including the KCR motif important in AP-1 protein binding to DNA have been synthesised as potential transcription factor inhibitors. These anthraquinone-peptides showed DNA intercalative binding and inhibition of AP-1 protein binding to its DNA consensus sequence.
Curcumin, the yellow pigment in turmeric, has been shown to prevent malignancies in a variety of tissues in rodents, especially in the intestinal tract. Pharmacological activities of curcumin in cells in situ germane to chemoprevention, such as inhibition of expression of cyclooxygenase-2 (COX-2), require drug concentrations in the 10(-5) - 10(-4) M range. The systemic bioavailability of curcumin is low, so that its pharmacological activity may be mediated, in part, by curcumin metabolites. To investigate this possibility, we compared curcumin metabolism in human and rat hepatocytes in suspension with that in rats in vivo. Analysis by high-performance liquid chromatography with detection at 420 and 280 nm permitted characterization of metabolites with both intact diferoylmethane structure and increased saturation of the heptatrienone chain. Chromatographic inferences were corroborated by mass spectrometry. The major metabolites in suspensions of human or rat hepatocytes were identified as hexahydrocurcumin and hexahydrocurcuminol. In rats, in vivo, curcumin administered i.v. (40 mg/kg) disappeared from the plasma within 1 h of dosing. After p.o. administration (500 mg/kg), parent drug was present in plasma at levels near the detection limit. The major products of curcumin biotransformation identified in rat plasma were curcumin glucuronide and curcumin sulfate whereas hexahydrocurcumin, hexahydrocurcuminol, and hexahydrocurcumin glucuronide were present in small amounts. To test the hypothesis that curcumin metabolites resemble their progenitor in that they can inhibit COX-2 expression, curcumin and four of its metabolites at a concentration of 20 microM were compared in terms of their ability to inhibit phorbol ester-induced prostaglandin E2 (PGE2) production in human colonic epithelial cells. Curcumin reduced PGE2 levels to preinduction levels, whereas tetrahydrocurcumin, previously shown to be a murine metabolite of curcumin, hexahydrocurcumin, and curcumin sulfate, had only weak PGE2 inhibitory activity, and hexahydrocurcuminol was inactive. The results suggest that (a) the major products of curcumin biotransformation by hepatocytes occur only at low abundance in rat plasma after curcumin administration; and (b) metabolism of curcumin by reduction or conjugation generates species with reduced ability to inhibit COX-2 expression. Because the gastrointestinal tract seems to be exposed more prominently to unmetabolized curcumin than any other tissue, the results support the clinical evaluation of curcumin as a colorectal cancer chemopreventive agent.
The prevalence of Pneumocystis carinii pneumonia (PCP) in humans caused by more than a single genotype has been reported to range from 10 to 67%, depending on the method used for detection (3, 19). Most coinfections were associated with primary rather than recurrent disease. To better understand the factors influencing the development of coinfections, the time periods between inoculations and the genotype of the infecting organisms were evaluated in the chronically immunosuppressed-inoculated rat model of PCP. P. carinii f. sp. carinii infecting rats differentiated by karyotypic profiles exhibit the same low level of genetic divergence manifested by organisms infecting humans. P. carinii f. sp. carinii karyotype forms 1, 2, and 6 were inoculated into immunosuppressed rats, individually and in dual combinations, spaced 0, 10, and 20 days apart. Infections comprised of both organism forms resulted from admixtures inoculated at the same time. In contrast, coinfections did not develop in most rats, where a 10- or 20-day gap was inserted between inoculations; only the first organism form inoculated was detected by pulsed-field gel electrophoresis in the resultant infection. Organism burdens were reduced with combinations of forms 1 and 2 spaced 20 days apart but not in rats inoculated with forms 1 and 6. A role for the host response in the elimination of the second population and in reduction of the organism burden was suggested by the lack of direct killing of forms 1 and 2 in an in vitro ATP assay, by reduction of the burden by autoclaved organisms, and by the specific reactions of forms 1 and 2 but not forms 1 and 6. These studies showed that the time between inoculations was critical in establishing coinfections and P. carinii f. sp. carinii karyotype profiles were associated with differences in biological responses. This model provides a useful method for the study of P. carinii coinfections and their transmission in humans.
Dendritic cells may be generated ex vivo from CD34+ progenitor cells or peripheral blood mononuclear cells. Initial reports suggested that monocyte-derived dendritic cells (MoDCs) arise from a proliferating precursor and several groups subsequently reported successful retroviral transduction of these cells, again implying that cell division occurs. As this is of importance in the development of immunotherapy protocols, we investigated whether monocytes proliferate as they differentiate into MoDCs and also their susceptibility to retroviral transduction. During MoDC differentiation, there was a 51 +/- 12% reduction in cell number, 98% of cells were in G0/G1, no DNA synthesis was detectable and the cell cycle regulatory proteins pRb and p130 were in the hypophosphorylated forms observed in non-cycling cells. As expected from these results, MoDCs were refractory to transduction with a GALV1 pseudotyped Moloney murine leukaemia virus (MoMLV)-based retroviral vector. In contrast, generation of DCs from purified CD34 progenitors was accompanied by rapid entry into the cell cycle and a 41.1-fold cell expansion at the end of 14 d culture.
2-(4-aminophenyl)benzothiazole (CJM 126) elicits potent growth inhibition in human-derived breast carcinoma cell lines, including oestrogen receptor-positive (ER+) MCF-7wt cells. Analogues substituted in the 3' position with I (DF 129), CH3 (DF 203), or Cl (DF 229) possess an extended profile of antitumour activity with remarkable selective activity in cell lines derived from solid tumours associated with poor prognosis, e.g. breast, ovarian, renal and colon. Growth inhibition occurs via unknown, possibly novel mechanism(s) of action. Two cell lines have been derived from sensitive MCF-7wt breast cancer cells (IC50 value < 0.001 microM) following long-term exposure to 10 nM or 10 microM CJM 126, MCF-7(10 nM 126) and MCF-7(10 microM 126) respectively, which demonstrate acquired resistance to this agent (IC50 > 30 microM) and cross-resistance to DF 129, DF 203 and DF 229. Sensitivity to tamoxifen, benzo[a]pyrene (BP), mitomyin C, doxorubicin and actinomycin D is retained. Resistance may, in part, be conferred by the constitutively increased expression of bcl-2 and p53 proteins detected in MCF-7(10 nM 126) and MCF-7(10 microM 126 lysates. Significantly decreased depletion of CJM 126 (30 microM) from nutrient medium of MCF-7(10 microM 126) cells was observed with predominantly cytoplasmic drug localization and negligible DNA strand breaks. N-acetyl transferase (NAT)1 and NAT2 proteins were expressed by all three MCF-7 sub-lines, but significantly higher expression of NAT2 was accompanied by enhanced acetylation efficacy in MCF-7(10 nM 126) cells. In contrast, CJM 126 (30 microM) was rapidly depleted from nutrient medium of MCF-7(10 microM 126) culture and accessed nuclei of these cells exerting damage to DNA. The major biotransformation product of CJM 126 in MCF-7(10 microM 126) cells was 2-(4-aminophenyl)-6-hydroxybenzothiazole (6-OH 126). This metabolite possessed no antitumour activity. Accordingly, in this sub-line, low constitutive expression and activity of cytochrome P450 (CYP) 1A1 was detected.
The long-term goal of our laboratory is to understand vertebrate host recognition of microbial pathogens. Although our work is primarily curiosity driven, it is certainly true that understanding how a host recognizes microbial pathogens should have some medical application. Probably more than 50,000 people die each year in the United States of septic shock or the systemic inflammatory response syndrome, and there is no good therapy for this problem. Understanding the molecular basis of its origin should suggest novel therapeutic approaches.
Histopathologic evaluation combined with a period of immunosuppression has been the standard procedure for detection of Pneumocystis carinii in commercial rat colonies. Variation in induction regimens and in the sensitivity of detection methods may result in underreporting of the presence of P. carinii in breeding colonies or delay its detection. In the present study, methylprednisolone and cyclophosphamide were evaluated for the ability to induce P. carinii infection in rats from an enzootically infected commercial barrier colony. The presence of P. carinii was detected by histopathologic methods and by amplification of a targeted region of the P. carinii thymidylate synthase gene by PCR over the 8-week study period. Sera taken from rats prior to either induction regimen were evaluated for the presence of P. carinii-specific antibodies by the immunoblotting technique. Few significant differences in ability to induce organism burden or in histopathology were observed between the two immunosuppressive regimens. However, a dramatic loss of weight over the study period was observed in rats treated with methylprednisolone but not in rats treated with cyclophosphamide. Although histopathologic changes attributable to P. carinii did not appear before 2 weeks with either immunosuppressant, the presence of the organism in these animals was detected by immunoblotting and PCR. Cyst scores and the intensities of the histopathologic lesions increased during the study period, but the number of rats exhibiting evidence of P. carinii infection did not change after week 3. These results suggest that use of the PCR method on postmortem lung tissue of rats without prior induction regimens or identification of anti-P. carinii antibodies in antemortem serum samples is a sufficiently sensitive method for detection of the presence of a P. carinii carrier state in rodent breeding colonies.
An ideal cancer chemotherapeutic prodrug is completely inactive until metabolized by a tumour-specific enzyme, or by an enzyme that is only metabolically competent towards the prodrug under physiological conditions unique to the tumour. Human cancers, including colon, breast, lung, liver, kidney and prostate, are known to express cytochrome P450 (CYP) isoforms including 3A and 1A subfamily members. This raises the possibility that tumour CYP isoforms could be a focus for tumour-specific prodrug activation. Several approaches are reviewed, including identification of prodrugs activated by tumour-specific polymorphic CYPs, use of CYP-gene directed enzyme prodrug therapy and CYPs acting as reductases in hypoxic tumour regions. The last approach is best exemplified by AQ4N, a chemotherapeutic prodrug that is bioreductively activated by CYP3A. This study shows that freshly isolated murine T50/80 mammary carcinoma and RIF-1 fibrosarcoma 4-electron reduces AQ4N to its cytotoxic metabolite, AQ4 (T50/80 Km = 26.7 microM, Vmax = 0.43 microM/mg protein/min; RIF-1 Km = 33.5 microM, Vmax = 0.42 microM/mg protein/min) via AQM, a mono-N-oxide intermediate (T50/80 Km = 37.5 microM; Vmax = 1.4 microM/mg protein/min; RIF-1 Km = 37.5 microM; Vmax = 1.2 microM/mg protein/ min). The prodrug conversion was dependent on NADPH and inhibited by air or carbon monoxide. Cyp3A mRNA and protein were both present in T50/80 carcinoma grown in vivo (RIF-1 not measured). Exposure of isolated tumour cells to anoxia (2 h) immediately after tumour excision increased cyp3A protein 2-3-fold over a 12 h period, after which time the cyp protein levels returned to the level found under aerobic conditions. Conversely, cyp3A mRNA expression showed an initial 3-fold decrease under both oxic and anoxic conditions; this returned to near basal levels after 8-24 h. These results suggest that cyp3A protein is stabilized in the absence of air, despite a decrease in cyp3A mRNA. Such a 'stabilization factor' may decrease cyp3A protein turnover without affecting the translation efficiency of cyp3A mRNA. Confirmation of the CYP activation of AQ4N bioreduction was shown with human lymphoblastoid cell microsomes transfected with CYP3A4, but not those transfected with CYP2B6 or cytochrome P450 reductase. AQ4N is also reduced to AQ4 in NADPH-fortified human renal cell carcinoma (Km = 4 microM, Vmax = 3.5 pmol/mg protein/min) and normal kidney (Km = 4 microM, Vmax = 4.0 pmol/mg protein/min), both previously shown to express CYP3A. Germane to the clinical potential of AQ4N is that although both normal and tumour cells are capable of reducing AQ4N to its cytotoxic species, the process requires low oxygen conditions. Hence, AQ4N metabolism should be restricted to hypoxic tumour cells. The isoform selectivity of AQ4N reduction, in addition to its air sensitivity, indicates that AQ4N haem coordination and subsequent oxygen atom transfer from the active-site-bound AQ4N is the likely mechanism of N-oxide reduction. The apparent increase in CYP3A expression under hypoxia makes this a particularly interesting application of CYPs for tumour-specific prodrug activation.
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We analyzed single drugs and combinations of drugs used clinically in the treatment of opportunistic infections and other conditions for their activities against Pneumocystis carinii pneumonia in immunosuppressed rats. When they were used alone, atovaquone, rifabutin, and dapsone were more active than clarithromycin or trimethoprim. Drug combinations were evaluated for synergistic activity by an analysis of variance model for two-way factorial experiments and a response surface model. Atovaquone combined with trimethoprim trimethoprim and some combinations of dapsone and clarithromycin was synergistic; however, the activities of combinations of atovaquone and rifabutin, atovaquone and clarithromycin, and atovaquone and dapsone were simply additive. Lovastatin, which inhibits 3-hydroxy-methylglutaryl coenzyme A reductase, was inactive whether it was used alone or in combination with other agents. None of the synergistic drug combinations was as effective as trimethoprim-sulfamethoxazole. We conclude that the rat model can be used to test combinations of anti-P. carinii agents for synergistic activity by well-established statistical techniques. While some combinations of clinically used antimicrobial drugs have enhanced anti-P. carinii activity, further studies are needed before clinical trials can be contemplated.
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This article uses Medicaid claims data to examine the adequacy of well-child care provided by different ambulatory care providers for selected children enrolled in Maryland Medicaid. Considerable provider variation was observed. The majority of the nearly one-quarter of the children with no well-child visits during the year appeared to have had no regular primary care source. Results contributed to the development of a managed care program designed to increase care accessibility and continuity and improve provider practices. Advantages and disadvantages of using claims data to investigate this public health issue care discussed.
Our previous study using a deletion mutant indicated that the COOH-terminal (CT) domain of the insulin receptor plays important roles in both catalytic efficiency and stability of the receptor kinase (Yan et al., J. Biol. Chem., 268 [1993] 22444). In this study, we purified the CT domain of 98 amino acids from bacterial cells over-expressing the CT domain and examined its effect on insulin and IGF-I receptor protein kinases. The purified CT domain stimulated the kinase activities of purified insulin receptor-transmembrane/cytoplasmic domain (IRTMTPK) and its CT domain-deletion mutant (IRTMTPK delta CT), 3.3-fold and 2.3-fold, respectively, while it was less effective in stimulating the kinase activity of purified IGF-I receptor transmembrane/cytoplasmic domain (IGFIRTMTPK) (1.4-fold). When the effect of the CT domain on autophosphorylation was examined, a marked increase in autophosphorylation was observed only with IRTMTPK delta CT. These results suggest that the CT domain specifically interacts with the insulin receptor cytoplasmic domain, thereby activating the kinase or autophosphorylation activity.
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Two groups of Vietnam veterans--Post-traumatic stress disorder (PTSD, n = 17) and non-PTSD (n = 80)--were developed from a sample of treatment-seeking alcohol abusers. The subjects completed a Combat Experiences Questionnaire, a PTSD Symptom Scale, the MMPI, and the Multidimensional Personality Questionnaire (MPQ). The MPQ proved to be a useful adjunct to the MMPI in describing the major manifestations of PTSD among alcohol-abusing veterans.
The effect on the gingiva of a variety of relationships of a removable partial denture connector to the gingival margin was investigated in 10 subjects. Acrylic resin baseplates were designed incorporating a variety of relationships of the connector to the gingival margin. The appliances were worn for a 12 hour period daily, for 21 days. At baseline and at days 7, 21 and 49, plaque index, gingival index and probing pocket depth measurements were recorded, and subgingival plaque samples were examined using darkfield microscopy. No increase in plaque accumulation was detected in any area at any time interval. There was a significant increase in gingival index at day 21 in areas where the appliance covered the gingival margin. Small increases in probing pocket depth were recorded at day 21, there being no difference between any of the test areas. Baseline values were re-established by day 49. No significant differences were detected in the percentage of motile organisms or spirochaetes counted in the subgingival plaque samples collected from any area. It was concluded that deterioration in gingival health occurred rapidly following the insertion of a removable appliance, and that coverage of the gingival margin, irrespective of the degree of gingival relief, had a detrimental effect.
In this study we evaluated the in vitro immunologic responses of 11 patients receiving immunotherapy with either interleukin-2 (IL-2, 3 X 10(6) units/m2) or IL-2 plus lymphokine-activated killer cells (LAK) over a 30-day period. Blastogenic responses to mitogens or recall antigens were found to significantly decrease during therapy. Pokeweed mitogen immunoglobulin (Ig) production decreased significantly in 7 patients or showed no change. In vivo skin tests for cell-mediated immunity were also performed and the average number of positive responses before IL-2/LAK therapy decreased to no positive responses (day 29 of therapy). Experiments were performed to determine whether decreased responses were a result of active suppression or a dilution effect by immature/mature cells that cannot respond to mitogen or antigen. Pre-therapy cells were mixed with fresh autologous (anergic) cells from day 29 of therapy in varying ratios. Blastogenesis and Ig production was measured. Our findings demonstrate that the decreased immune response observed during and after therapy is a result not of active immunosuppression but rather of a dilution effect by cells with immune dysfunction. We conclude that: (1) in vitro blastogenesis or Ig production tests using mitogens or antigens for patients undergoing IL-2 immunotherapy have no predictive value, and (2) the immune cells that are present are refractory.
In a previous study we evaluated the in vitro immunologic responses of 14 patients receiving immunotherapy with either interleukin-2 (IL-2; 3 x 10(6) units/m2) or IL-2 plus lymphokine-activated killer (LAK) cells over a 45-day period. Blastogenic responses to mitogens or antigens were found to significantly decrease. Pokeweed mitogen immunoglobulin production decreased or showed no change. Multitest skin test response decreased during and after therapy. We concluded that, although natural killer and LAK activity are enhanced during therapy, in vitro blastogenic or immunoglobulin tests using mitogens or antigens for patients undergoing IL-2 immunotherapy have no predictive values and are depressed. In this study, we provide information that patients while receiving IL-2/interferon-alpha immunotherapy demonstrate as in the previous study in vitro reduced immunologic responses by at least 60%; however, in vivo, they had a normal immunoglobulin response to a tetanus booster. The disparity in results (in vitro versus in vivo) is unexplainable. Further analysis of other in vitro and/or in vivo tests is required to determine the effect IL-2 immunotherapy may have on the immune response status.