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Biomedical subjects

J Scharff

Publications and source records attributed to J Scharff.

15 recordsLinked to original sources
PubMed2001

Graft survival in a rhesus renal transplant model after immunotoxin-mediated T-cell depletion is enhanced by mycophenolate and steroids.

BACKGROUND: Anti-CD3 immunotoxin (IT), a T-cell-depleting agent, prolongs survival of renal allografts in a rhesus monkey model without the need for long-term immunosuppression. In this study we sought to further prolong allograft survival by giving short-term conventional immunosuppression simultaneous with IT administration. METHODS: MHC class II mismatched, juvenile rhesus monkeys were paired as donor and recipient for renal transplantation. Recipients received two to three daily doses of IT starting on the day of transplantation. Additional immunosuppression was given for no more than 60 days. Graft function was monitored by serum creatinine and renal biopsies. Flow cytometry was used to monitor T-cell recovery. RESULTS: Graft survival time (GST) in animals receiving IT was prolonged compared with controls with 50% of IT-treated monkeys surviving >100 days. Animals treated with IT plus mycophenolate mofetil (MMF) and steroids had significantly enhanced GST (mean GST, 305 days) compared with those treated with IT alone (mean GST, 94 days). In contrast, addition of cyclosporine or 40-O-[2-Hydroxyethyl]rapamycin did not significantly increase graft survival time. A comparison among animals from all treatment groups with short (<100 days) and long (>100 days) GST demonstrated that those with the shorter GST had a higher blood T-cell count 2 weeks after transplantation. Full recovery of CD4+ T cells required longer than 6 months. CONCLUSIONS: A combination with MMF and steroids given for 4 days after renal allograft transplantation significantly increases GST in IT-treated monkeys. We hypothesize that MMF and steroids suppress the initial T-cell activation mediated by IT.

Animals↗
PubMed1997

Depletion of T lymphocytes with immunotoxin retards the progress of experimental allergic encephalomyelitis in rhesus monkeys.

FN18-CRM9 is an anti-rhesus anti-CD3 immunotoxin that can transiently deplete T cells to 1% of initial values in both the blood and lymph node compartments and can induce long-term tolerance to mismatched renal allografts. We have investigated the ability of this immunotoxin to interdict the course of an experimental rhesus T-cell-driven autoimmune disease, experimental allergic encephalomyelitis (EAE) induced by myelin basic protein. Monkeys showing CSF pleocytosis were then treated with FN18-CRM9 alone or in combination with cranial irradiation (325 or 650 cGy). EAE in nontreated control monkeys progressed rapidly. Paralysis occurred 4-6 days after CSF pleocytosis. Paralysis was either delayed or never occurred in treated monkeys, and histopathology revealed few inflammatory plaques that were notable for their low or absent T cell content. While T cells repopulate in the periphery posttreatment, they do not return to the CNS in large numbers, suggesting that the newly repopulated T cells have lost their previously acquired CNS homing capability. Anti-CD3 immunotoxin may be useful in treating clinical T-cell-driven autoimmune diseases such as rheumatoid arthritis and multiple sclerosis.

Animals↗
PubMed1997

FN18-CRM9 immunotoxin promotes tolerance in primate renal allografts.

BACKGROUND: Transplant tolerance, rather than immunity, may be favored in the setting of a lower mature lymphoid mass in the recipient induced by anti-T cell agents. A novel immunosuppressive agent, FN18-CRM9, known to specifically kill T cells with great potency, was evaluated in a transplant model. METHODS: In order to ablate recipient T cells, the immunotoxin FN18-CRM9 was administered to rhesus monkey recipients of MHC-mismatched renal allografts. Donor lymphocytes were injected intrathymically into some animals. RESULTS: All monkeys with T-cell depletion by immunotoxin had prolonged allograft survival, and tolerance confirmed by skin grafting has been confirmed in five of six long-surviving recipients. CONCLUSIONS: In this clinically relevant model, profound but transient T-cell depletion by a single agent substantially promotes tolerance.

Animals↗
PubMed1996

A new reagent for the induction of T-cell depletion, anti-CD3-CRM9.

We have developed a new reagent for inducing in vivo T-cell depletion and have tested this reagent in rhesus monkeys. The reagent is an anti-CD3 epsilon immunotoxin based on a diphtheria toxin binding-site mutant, CRM9. After administration to monkeys, T cells are depleted from both the blood and lymph node compartments to < 1% of their initial values. T-cell depletion is associated with transient immunosuppression, as judged by delayed rejection of RhLA-mismatched skin allografts. T cells are repopulated in both compartments; however, the rate of repopulation is age dependent. The rate is rapid in juvenile animals (12 days) and requires > 30 days in old animals. The correlation between repopulation rate and age suggests that the repopulation is thymus dependent and that the repopulated T cells are probably naive T cells. This reagent should be a valuable tool in studying the role of memory T cells in rhesus models of autoimmune diseases and protocols of tolerance induction after organ transplantation.

Animals↗
PubMed1995

An anti-CD3 single-chain immunotoxin with a truncated diphtheria toxin avoids inhibition by pre-existing antibodies in human blood.

Diphtheria toxin (DT) is often used in the construction of immunotoxins. One potential problem using DT-based immunotoxins is the pre-existing anti-DT antibodies present in human blood due to vaccination. The present study examined the effect of human serum with pre-existing anti-DT antibodies on the toxicity of UCHT1-CRM9, an immunotoxin directed against CD3 molecules on T-lymphocytes. Sera with detectable anti-DT antibodies at 1:100 or greater dilutions inhibited the immunotoxin toxicity. Experiments with radio-labeled UCHT1-CRM9 indicate that anti-DT antibodies partially block its binding to the cell surface as well as inhibit the translocation from the endosome to the cytosol. The inhibitory effect could be adsorbed using a full-length DT mutant or B-subfragment. A C-terminal truncation mutant could not adsorb the inhibitory effect, suggesting that the last 150 amino acids contain the epitope(s) recognized by the inhibitory antibodies. Therefore, an anti-CD3 single-chain immunotoxin, sFv-DT390, was made with a truncated DT. The IC50 of sFv-DT390 was 4.8 x 10(-11) M, 1/16 the potency of the divalent UCHT1-CRM9. More importantly, sFv-DT390 toxicity was only slightly affected by the anti-DT antibodies in human sera.

Antibodies, Bacterial↗
PubMed1992

In vivo T-cell ablation by a holo-immunotoxin directed at human CD3.

We have evaluated the in vivo efficacy of anti-CD3-CRM9, a holo-immunotoxin constructed with a diphtheria toxin binding-site mutant. Eighty percent of established human T-cell subcutaneous tumors in nude mice completely regressed following intraperitoneal injection of immunotoxin at a dose set at half the minimum lethal dose assayed in toxin-sensitive animals. Similar regressions produced by a 137Cs source required a dose in excess of 500 cGy. The high degree of in vivo T-cell ablation produced by this immunotoxin is apparently due to maintenance of the toxin translocation function provided by CRM9 and a necessary intracellular routing function supplied by CD3. This immunotoxin may be useful in treating conditions caused by pathologic oligoclonal T-cell expansion such as graft-versus-host disease, autoimmune diseases, and possibly AIDS.

Animals↗
PubMed1992

Evidence that plasma membrane electrical potential is required for vesicular stomatitis virus infection of MDCK cells: a study using fluorescence measurements through polycarbonate supports.

We used fluorescence microscopy of Madin-Darby Canine Kidney (MDCK) cells grown on polycarbonate filters to study a possible link between plasma membrane electrical potential (delta psi pm) and infectivity of vesicular stomatitis virus (VSV). Complete substitution of K+ for extracellular Na+ blocks VSV infection of MDCK cells as well as baby hamster kidney (BHK) cells. When we independently perfused the apical and basal-lateral surfaces of high resistance monolayers, high K+ inhibited VSV infection of MDCK cells only when applied to the basal-lateral side; high K+ applied apically had no effect on VSV infection. This morphological specificity correlates with a large decrease in delta psi pm of MDCK cells when high K+ buffer is perfused across the basal-lateral surface. Depolarization of the plasma membrane by 130 mM basal K+ causes a sustained increase of cytosol pH in MDCK cells from 7.3 to 7.5 as reported by the fluorescent dye BCECF. Depolarization also causes a transient increase of cytosol Ca2+ from 70 to 300 nM as reported by the dye Fura-2. Neither increase could explain the block of VSV infectivity by plasma membrane depolarization. One alternative hypothesis is that delta psi pm facilitates membrane translocation of viral macromolecules as previously described for colicins, mitochondrial import proteins, and proteins secreted by Escherichia coli.

Animals↗
PubMed1989

Enhancement of immunotoxin efficacy by acid-cleavable cross-linking agents utilizing diphtheria toxin and toxin mutants.

We have utilized a new class of acid-cleavable protein cross-linking reagents in the construction of antibody-diphtheria toxin conjugates (Srinivaschar, K., and Neville, D. M., Jr. (1989) Biochemistry 28, 2501-2509). The potency of anti-CD5 conjugates assayed by inhibition of protein synthesis on CD5 bearing cells (Jurkat) is correlated with cross-linker hydrolytic rates. The maximum increase in potency of the cleavable conjugates over non-cleavable conventional conjugates is 50-fold and is specific for the CD5 uptake route as judged by competition with excess anti-CD5. The potency of conjugates made from diphtheria toxin and the anti-high molecular weight melanoma-associated antigen (HMW-MAA) is enhanced 3-10-fold by a cleavable cross-linker. However the potency of transferrin or anti-CD3 diphtheria toxin conjugates is only minimally enhanced (2-3-fold). Mutant diphtheria toxins, CRM103 and CRM9, previously shown to express less than 1/100 of the wild type in binding affinity were substituted into these conjugates as probes for possible intracellular toxin receptor interactions. Both mutants were equally as toxic to Jurkat target cells exhibiting 1/700 the wild-type potency. CRM9 non-cleavable conjugates were equally as potent as wild-type conjugates for transferrin and anti-CD3-mediated uptake but not for anti-CD5-mediated uptake where toxicity was reduced 60-fold over the wild-type analog. The cleavable cross-linker enhanced the toxicity of anti-CD5-CRM103 and anti-CD5-CRM9 conjugates, but potency was only 1/10 that of the analogous wild-type cleavable conjugate. These data are consistent with a model in which potentiation of toxicity of the anti-CD5 and anti-high molecular weight melanoma-associated antigen conjugates by the cleavable cross-linker occurs from an enhanced intracellular toxin-toxin receptor interaction that ultimately results in increased toxin translocation to the cytosol compartment. In contrast, these data indicate that the anti-CD3 and transferrin uptake systems do not require this interaction in agreement with previous work (Johnson, V.G., Wilson, D., Greenfield, L., and Youle, R. J. (1988) J. Biol. Chem. 263, 1295-1300).

Animals↗
PubMed1989

Antiasthmatic effects of onions. Prevention of platelet-activating factor induced bronchial hyperreactivity to histamine in guinea pigs by diphenylthiosulfinate.

Thiosulfinates are responsible for antiasthmatic and anti-inflammatory properties of onions. We tested the effect of diphenylthiosulfinate on platelet-activating factor (PAF)-induced bronchial hyperreactivity to histamine: According to a randomized crossover protocol, groups of 14 guinea pigs inhaled histamine, were then treated orally with either vehicle or with 10-100 mg/kg diphenylthiosulfinate, inhaled 1 microgram PAF, and thereafter the same histamine dose given prior to PAF. In the control group the histamine response increased threefold; in the treated group the histamine response decreased. The effect of 100 mg diphenylthiosulfinate lasted 12 h. Antihistamine effects were not demonstrable in this test system. We conclude that thiosulfinates inhibit PAF-induced hyperreactivity.

Allium↗
PubMed1988

Energy requirements for diphtheria toxin translocation are coupled to the maintenance of a plasma membrane potential and a proton gradient.

Translocation of diphtheria toxin (DT) or ricin to the cytosol is the rate-limiting step responsible for (pseudo) first-order decline in protein synthesis observed in intoxicated cell populations. The requirements for energy utilization in the translocation of both toxins are examined by perturbing the intoxication during this period of protein synthesis decline. Translocation of either toxin is blocked at 4 degrees C and requires energy. Ricin translocation is tightly coupled to ATP hydrolysis with no involvement of membrane potential. Cell depolarization slows the rate of DT translocation but does not block completely. Elimination of transmembrane pH gradients alone does not affect DT translocation; however, in combination with depolarization, translocation is blocked virtually completely. Energy requirements for DT intoxication are mediated by establishing a plasma membrane potential and a pH gradient across some cellular membrane. It is proposed that a postendocytotic vesicle containing processed DT fuses with the plasma membrane. Either component of the proton motive force across the plasma membrane then drives DT translocation. Ricin apparently utilizes a different energy coupling mechanism at a different intracellular site, thus demonstrating toxin specificity in the translocation mechanism.

Adenosine Triphosphate↗
PubMed1986

First 6 months of Medicaid data.

This is the first in a series of comprehensive Medicaid program reports based on National Medical Care Utilization and Expenditure Survey (NMCUES) data. Preliminary analyses are presented based on data from the first half of 1980, which include the personal characteristics and medical care utilization patterns of noninstitutional Medicaid enrollees and the health insurance coverage of the U.S. noninstitutionalized population. More comprehensive analyses employing full calendar year 1980 data will be available in subsequent reports. The information contained in this report is useful in appraising the impact of eligibility, benefit package, and reimbursement policy on Medicaid enrollee health care utilization at both the Federal and the State Medicaid level. Despite the expectations of the Medicaid program's architects that it would be smaller and less significant than the Medicare program, the Medicaid program has experienced dramatic growth in the number of recipients and total expenditures since its creation. By early 1980, State Medicaid program expenditures accounted for between 10 and 15 percent of individual State general operating funds. The Medicaid program is continuing to grow relative to State budgets. State budgets have been expanding by only about 9 percent per year, while Medicaid budget expenditures have been expanding at approximately twice that rate. These factors are forcing States to devise program changes concerning eligibility, benefits, or reimbursement approaches that will enable them to maintain fiscal stability as expenditures increase and the Federal role changes. These problems present a major challenge to policymakers and administrators at both the Federal and State levels. Solutions to these problems are difficult because of the differences in Medicaid programs, the constantly changing character of the Medicaid programs within States, and a lack of data to describe, monitor, and forecast Medicaid program activities in a consistent fashion. NMCUES was designed with these data problems in mind. Survey Background The goal of the National Medical Care Utilization and Expenditure Survey (NMCUES) is to collect information that will improve our understanding of the way Americans use and pay for health care. In addition to providing reliable statistical descriptions of the types of health services consumed and the amount of dollars expended for health care by the Nation, NMCUES was designed to permit health policy analysts to investigate a broad range of issues concerning the financing and delivery of health services in the United States. NMCUES data reflect health care experiences of the civilian noninstitutionalized population during 1980.(ABSTRACT TRUNCATED AT 400 WORDS)

Ambulatory Care↗
PubMed1983

Six months of Medicaid data: a summary from the National Medical Care Utilization and Expenditure Survey.

This is a summary of the first report in a series of three comprehensive Medicaid program reports based on National Medical Care Utilization and Expenditure Survey data. Preliminary analyses are presented based on data from the first half of 1980 which include the personal characteristics and medical care utilization patterns of noninstitutional Medicaid enrollees and the health insurance coverage of the U.S. noninstitutionalized population. More comprehensive analyses employing full calendar year 1980 data will be available in subsequent reports. The information provided in this summary is useful in appraising the impact of eligibility, benefit package, and reimbursement policy on Medicaid enrollee health care utilization at both the Federal and the State Medicaid level.

Data Collection↗
PubMed1981

Sleep paralysis among medical students.

Sleep paralysis is a sensation of an inability to speak or move other muscles when falling asleep or awakening. Sleep paralysis by itself has been reported as occurring infrequently and many clinicians are uncertain of its significance. In contrast, sleep paralysis in conjunction with sleep attacks has been reported as a concomitant of narcolepsy. To further examine the incidence of sleep paralysis, the responses of 80 first-year medical students, 16.25% had experienced predormital, postdormital, or both types of sleep paralysis. These episodes occurred infrequently--only once or twice for most of these students. Reports of sleep paralysis were not associated with sleep attacks or cataplexy. These results support two previous studies which found that sleep paralysis alone occurs frequently among normals.

Adult↗
PubMedDate not supplied

Genetic construction and characterization of an anti-monkey CD3 single-chain immunotoxin with a truncated diphtheria toxin.

We have previously developed a chemically conjugated anti-rhesus monkey CD3 immunotoxin FN18-CRM9 that can deplete in vivo T cells and induce long term tolerance of mismatched renal allograft in rhesus monkeys. This immunotoxin is a monkey analogue of anti-human CD3 immunotoxin UCHT1-CRM9. In this study, we cloned the light and heavy chain variable regions of anti-monkey CD3 monoclonal antibody FN18 and constructed a single-chain Fv (sFv) by linking variable light and variable heavy regions with a (Gly4Ser)3 linker. The single-chain immunotoxin DT390-FN18sFv was constructed by ligating the sFv to the carboxyl terminus of DT390, a truncated form of diphtheria toxin. The DT390-FN18sFv fusion protein was expressed in Escherichia coli and purified with Ni-RTA affinity and anion exchange columns. Similar to the chemically conjugated immunotoxin FN18-CRM9, DT390-FN18sFv can also specifically inhibit protein synthesis in primary monkey T cells in a dose-dependent manner. DT390-FN18sFv at 10(-7) mol/L or FN18-CRM9 at 10(-8) mol/L is sufficient to reduce protein synthesis of monkey primary T cells to less than 5% of the control. The 50% inhibition dosage (IC50) of FN18-CRM9 is 1 x 10(-10) mol/L, while the IC50 of DT390-FN18sFv is 1 x 10(-8) mol/ L, reflecting the lowered affinity of monovalent Fab' FN18 to its parental divalent antibody. The availability of functional FN18sFv will provide the basis for the construction of divalent anti-CD3 immunotoxins for preclinical studies on the induction of tolerance in organ transplantation and experimental autoimmune diseases.

Adenosine Diphosphate Ribose↗