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K Zucker

Publications and source records attributed to K Zucker.

At least 37 records · Page 2Linked to original sources
PubMed1996

Longitudinal induced IL-2 mRNA monitoring in renal transplant patients immunosuppressed with cyclosporine and in unmodified canine renal transplant rejection.

Immune monitoring of transplant patients to define optimal immunosuppression continues to be important, as rejection occurs despite adjustment of dosaging of CsA or even FK506 to achieve "therapeutic-range" blood levels. Because CsA is known to inhibit upregulation of IL-2 mRNA transcription, we prospectively sequentially measured (induced) IL-2 mRNA in PHA-stimulated PBMC cultures from transplant recipients of kidneys from living-related donors (n = 15) using a quantitative PCR assay, with a potential 24-hour turnaround time, to define immunologic events in real time. Reproducible individual patient sensitivity or refractoriness to CsA was determined pretransplant, by adding a range of CsA concentrations to the PBMC cultures and constructing induced IL-2 mRNA regression inhibition curves. However, this was not predictive of rejection episodes, but did correlate well with individual differences in IL-2 mRNA levels posttransplant, despite similar maintenance trough blood concentrations of CsA between patients. In this prospective study, seven patients experienced rejection episodes despite therapeutic CsA trough levels. Three of these, plus one not receiving CsA therapy, who happened to be prospectively tested at the time that rejection was clinically diagnosed, had a decrease in induced IL-2 mRNA before treatment was instituted. As a correlation to this observation in patients, induced IL-2 mRNA levels in unmodified rejection were sequentially measured in PBMC cultures in autologous vs allogeneic canine renal transplants and IL-2, IL-10, TNF-alpha, and IFN-gamma mRNA were also measured in kidney biopsies. Sequential PHA lymphoproliferation assays of [3H] thymidine incorporation on patient and dog PBMC cultures were also performed. Similar to the observations in patients, unmodified rejection in the canine renal allograft model also was accompanied by a decline of PHA-induced IL-2 mRNA in PBMCs as the serum creatinine concentrations became elevated. In the dog kidney biopsies at later phases of rejection, IL-10 mRNA levels were also significantly elevated (p = 0.032).

Animals↗
PubMed1995

De novo membranoproliferative glomerulonephritis in hepatitis C virus-infected renal allograft recipients.

Hepatitis C virus (HCV) is the leading cause of non-A, non-B hepatitis among renal allograft recipients. We sought to identify and describe a proteinuric renal disease occurring in our HCV-infected renal transplant patients. Patients with proteinuria exceeding 1 g/day were identified from a cohort of 98 HCV-infected kidney recipients. Qualitative and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and restriction fragment-length polymorphism of the amplified RT-PCR product was performed to detect circulating HCV RNA, viral titer, and strain type, respectively. An immune complex nephritis (ICN) of the membranoproliferative pattern (MPGN) was found on five of eight biopsies. Two patients infected with the Hutch strain-type developed nephrotic-range proteinuria within three months posttransplant while the remaining three MPGN patients had been transplanted greater than 5 years prior to the onset of proteinuria. Testing for rheumatoid factors, cryoglobulins, hypocomplementemia, and circulating immune complexes failed to show a consistent pattern. Sucrose density gradient (SDG) equilibrium centrifugation was used to determine the buoyant-density of HCV virions from control (HCV-infected nonproteinuric recipients; n = 5) and nephrotic patients (n = 5). Whereas HCV virions from the control patients had a low buoyant density on sucrose gradients, a substantial percentage of the circulating HCV RNA from the MPGN patients was present in the high-density fractions in association with IgM and IgG. Treatment of the pooled high-density layers with NP40 followed by recentrifugation resulted in a shift of the HCV RNA to the medium-density layers. In conclusion, MPGN developed in five HCV-infected kidney recipients despite pharmacologic immunosuppression. Both the physicochemical properties of the HCV virions on SDG and their association with IgG and IgM in the high-density layers provide indirect evidence for the presence of circulating complexes of anti-HCV antibody and HCV antigen(s).

Adult↗
PubMed1995

Transmission of hepatitis C virus by kidney transplantation: impact of perfusion techniques and course of viremia post transplant.

Hepatitis C virus (HCV) infection is the leading cause of post-transplant non-A, non-B hepatitis. Although many end-stage renal disease patients present for transplantation already infected with HCV, some recipients acquire the infection by transmission from the donor organ. We have detected serological evidence for HCV infection in 6.8% of our organ donors using second-generation anti-HCV assays. Approximately one-third of the patients who received an organ from a HCV carrier donor developed chronic transaminasemia and 8 of 14 (56%) patients converted from HCV RNA negative to positive in the posttransplant period. Demonstration of the course of viremia and transaminasemia is presented for 2 patients in whom transmission of HCV occurred. Using pulsatile machine perfusion, we were able to demonstrate that a standard perfusion of 20 h reduced the viral load in the kidney by 75%, additional flushes and a subsequent perfusion reduced the total viral titer by more than 99%. Thus, although transmission of HCV does occur with solid-organ transplantation, differences in the incidence of transmission between centers may be related to techniques of organ preservation.

Hepacivirus↗
PubMed1995

Cloning and expression of canine interleukin-10.

We describe here the cloning of canine IL-10 cDNA (GenBank accession No. U33843) and the expression of recombinant IL-10 in a dog kidney cell line (DK6247) and Chinese hamster ovary cells (CHO). Canine IL-10 exhibits strong sequence homology to the known sequences of human, mouse, rat, and bovine genes at nucleotide and amino acid levels. The IL-10 gene, when introduced into DK and CHO cell lines, produces recombinant IL-10 that causes an inhibitory effect on allogeneic MHC-driven lymphoproliferative responses.

Amino Acid Sequence↗
PubMed1994

Depletion of hepatitis C virus from procured kidneys using pulsatile perfusion preservation.

The safety of the use of kidneys procured from a hepatitis C virus (HCV)-positive cadaver donors in transplantation has recently been the subject of controversy. One factor that is important in determining the transmission of the virus and/or viral liver disease is the total viral inoculum to which the renal allograft recipient is exposed as a result of the transplant. We have studied the effect of a standard pulsatile renal preservation procedure and variations of it on the number of viral copies in organs from HCV-positive donors. An HCV-RNA quantitative reverse transcription-polymerase chain reaction (RT-PCR) method was utilized with a recombinant competitive inhibitor substrate added after cDNA synthesis with the PCR primers within the relatively non-polymorphic 5' untranslated region of the HCV genome. Additionally, strain specificity was found to be detectable using a modification of the technique of restriction fragment-length polymorphism (RFLP-PCR), so that virus from the organ donor could be specifically identified and quantified in the recipient. It was observed that standard preservation procedures using pulsatile perfusion were able to eliminate 75% of the virus from the organ in 20 hr. By modifying this procedure to include additional wash steps and a second pulsatile perfusion, greater than 99% of the virus could be eliminated from the kidney. In a related study, we used quantitative PCR to study requirements for filtration of the virus using HCV-positive serum. It was found that a high-flow-rate ultrafilter with a molecular weight cut-off (MWCO) of 300,000 daltons placed in series to the preservation apparatus was very efficient in eliminating the virus from perfusion solution in less than 2 hr. It can therefore be proposed that with the use of these molecular techniques, pulsatile perfusion coupled with additional viral depletion steps (dilution, and/or filtration) may allow the practical reduction of HCV transmission risk in recipient follow-up studies. The means are thereby presented for similar manipulation of other known or, as yet, unknown transmissible agents.

Base Sequence↗
PubMed1994

The impact of hepatitis C virus infection on renal allograft recipients.

A second generation hepatitis C virus recombinant immunoblot assay (RIBA) was used to screen stored perioperative serum from 641 renal allograft recipients. One hundred and nine (17%) were anti-HCV positive at the time of transplant. RIBA positivity was found to be an independent predictor of post-transplant liver disease in a logistic regression model (P < 0.05). Moreover, RIBA positive patients were at greater risk for infectious events (P = 0.03) and rejection episodes (P = 0.002). The cumulative dose of antilymphoblast globulin administered as induction therapy was an independent predictor of post-transplant liver disease in a dose response relationship. Qualitative PCR showed that 74% of the perioperative RIBA positive patients had detectable HCV RNA in a current serum sample. Further, quantitative HCV RNA analysis with a competitive template PCR and HCV strain identification by restriction fragment length polymorphism demonstrated a large range of HCV RNA copies/ml of serum and three different HCV strains (BK, Hutch and HCV-1). Neither quantity of HCV RNA nor strain type correlated with abnormal transaminases post-transplant. As yet, there has not been an effect of anti-HCV status on actuarial patient and graft survival. This study suggests that anti-HCV is not a contraindication to renal transplantation; however, we would recommend that the pre-transplant evaluation of the anti-HCV positive patient include a liver biopsy to properly stage the disease. Close post-transplant follow-up is required in view of the increased risk for infection and rejection.

Adult↗
PubMed1994

Characterization of anti-canine cytokine monoclonal antibodies specific for IFN-gamma: effect of anti-IFN-gamma on renal transplant rejection.

Two murine monoclonal antibodies specific for IFN-gamma, ADI-1, and ADI-23 (both IgG1 kappa), were generated in BALB/c mice. The ADI-1 exhibited a higher avidity for canine rIFN-gamma than for nIFN-gamma and human rIFN-gamma. In contrast, the ADI-23 showed equal avidity for the three IFN-gamma preparations. The anti-canine IFN-gamma mAb did not bind to mouse and rat rIFN-gamma. The ADI-1, and ADI-23 mAb were also tested for binding to human rTFN-alpha and, contrary to our expectations, it was found that ADI-23 showed significant binding to human rTFN-alpha and rIFN-gamma, in contrast to ADI-1. Both anti-canine IFN-gamma mAb stained 48-h PHA-induced dog lymphoblasts. A two-site mAb ELISA was developed, which was linear in the range of 7-500 ng of canine rIFN-gamma, which indicated that the two mAb detected non-overlapping epitopes on the canine rIFN-gamma molecule. We studied the effect of ADI-1 on the prolongation of canine renal allografts. Recipients of kidney allografts, that were treated with ADI-1 by continuous arterial infusion, were prolonged to 22 and 25 days, compared to 9 and 13 days for animals given the IgG1 isotype control.

Animals↗
PubMed1994

Hepatitis C in liver transplantation: preliminary study of prognostic factors.

At the University of Miami liver transplantation for chronic liver disease in HCV-positive patients has shown good results, with a 92% patients survival rate (follow up 8 to 57 months, median 21). None the less, we found that a large number of patients are expected to develop serious histological graft damage and may need retransplantation, which may place a further strain on the already scarce donor resources. We have conducted a preliminary investigation on the importance of parameters which may correlate with the prognosis of HCV grafts. We found no impact of HLA match or typing. An interesting hypothesis, which deserves further investigation, is that some HCV strains could be more virulent than others and play a role as an independent risk factor. We have identified six strains among our patients and the BK serotype shows a trend to be associated with a worse outcome. We have found that patients developing and maintaining higher liver enzyme levels (ALT and GGT) after transplant and those with higher levels of viremia may be at risk to develop serious damage to their grafts.

Adolescent↗
PubMed1994

Cloning and expression of the cDNA for canine tumor necrosis factor-alpha in E. coli.

We have utilized the reverse transcription polymerase chain reaction (RT-PCR) to clone the protein coding region of canine tumor necrosis factor (TNF-alpha) cDNA. The gene displays 90% sequence homology to the corresponding human TNF-alpha cDNA. The predicted initial translation product is 233 amino acids and shows 88% homology to the human counterpart, and 92% homology with the human putative mature TNF-alpha protein. The canine TNF-alpha clone was used to engineer bacteria to express large amounts of the mature form of recombinant protein. A monoclonal antibody against human TNF-alpha cross-reacted with canine rTNF-alpha using Western blot and ELISA analysis. The purified canine rTNF-alpha had a cytotoxic effect on WEHI 164 clone 13 cells as well as increasing the cell surface expression of major histocompatibility class II antigens on canine kidney cortical cell line (MDCK) in vitro. The availability of canine rTNF-alpha will allow further studies on its role in immunoregulatory mechanisms in the canine transplantation model, both by itself and in conjunction with the already available canine specific recombinant interferon-gamma.

Amino Acid Sequence↗
PubMed1993

Molecular monitoring of the immunosuppressive effects of cyclosporine in renal transplant patients by using a quantitative polymerase chain reaction.

We have developed a rapid (24 hours) quantitative PCR assay to measure the direct effect of cyclosporine on IL-2 mRNA production by activated PBMC cultures from renal transplant patients. The PBMCs were purified from normal laboratory volunteers (group A, n = 26), CsA-treated renal transplant patients with good renal function, tested between 3 and 8 weeks (group B, n = 14) or between 2 and 8 years (group C, n = 15) after surgery, and stimulated with PHA in media supplemented with either patient serum or pooled commercially obtained AB serum. The mRNA was then isolated and, using semiquantitative PCR or quantitative PCR with a competitive inhibitor, the relative levels or exact levels of IL-2 mRNA (in attomoles) could be measured. A 3-day confirmatory lymphoproliferation assay of [3H]thymidine incorporation was also performed on the samples. Kinetic analysis of the data from group A showed that the peak level of IL-2 transcription into mRNA occurred at 6 hours after mitogen stimulation. Increasing in vitro concentrations of CsA in this group resulted in lower IL-2 mRNA levels and a shift in the peak time to 12-24 hours. In the transplant recipients, there was no correlation between individual CsA blood levels and proliferation responses. However, some correlation was found between CsA blood levels and IL-2 mRNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1993

Differential effects of IFN-gamma on kidney cell expression of MHC class II molecules, kidney cell associated molecules and their stimulatory capacity in mixed lymphocyte kidney cell culture.

Mixed cell co-cultures of lymphocytes responding to kidney cells (MLKC), islets of Langerhans (MLIC) and mixed lymphocyte culture (MLC), were used to clarify mechanisms in allogeneic and autoimmune (tissue-associated) antigen presentation. Fresh kidney cortical tubular cells (KC), Madin-Darby canine kidney (MDCK), and dog kidney (6247) (DK) cell lines were used in MLKC reactions, and islets of Langerhans were used in the MLIC as putative antigen presenting cells (APC). The stimulating cells were treated with purified or recombinant dog interferon-gamma (IFN-gamma), and the detection of class II MHC molecule expression was assessed by a moneclonal antibody (mAb) (B1F6). Transcription of MHC class II mRNA and IFN-gamma mRNA was measured by semiquantitative polymerase chain reaction, and detection of a kidney cell (tissue-associated) antigen molecule was assessed by the mAb I1F6 that recognizes 72 and 150 kDa tubular cell protein(s) (KT1). The MDCK cell line constitutively expressed low levels of MHC class II molecules and KT1. The steady-state level of the MHC class II mRNA transcription was virtually unaltered by treatment with IFN-gamma (400 units) for 48 hours; however, the MHC cell surface protein expression was enhanced. The KC and DK cell lines constitutively expressed KT1, but not MHC class II molecules; these cells required a minimum of 4000 units, and a 62-hour incubation with IFN-gamma was needed to upregulate both surface MHC class II molecules and the transcription of corresponding specific mRNA. In the MLKC reaction both the MDCK and DK cell lines, as well as fresh KC cells, could serve as lymphocyte activators. This could be amplified by exogenous IFN-gamma. The removal of APC from the responding T cell population did not reduce the IFN-gamma effect. This indicates that IFN-gamma treatment allows for the expression of all of the co-stimulating factors and/or adhesion molecules necessary for these cells to serve as (surrogate) APC (direct as opposed to indirect antigen presentation). The requirements for purified IFN-gamma to increase this amplification was greater in the MLKC reactions with kidney cells than in the MLC reactions. The mAbs anti-IFN-gamma and I1F6 differed in their ability to inhibit lymphocyte proliferation depending on the different cell types involved. The I1F6 inhibited the MDCK and DK cell-driven MLKC (in the absence of exogenous IFN-gamma).(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1993

Production and characterization of recombinant canine interferon-gamma from Escherichia coli.

We have used the recently cloned cDNA for canine interferon-gamma (IFN-gamma) to engineer bacteria to produce large amounts of the recombinant cytokine. The resulting protein can be recognized by monoclonal and polyclonal antibodies largely species specific for canine IFN-gamma. The purified recombinant IFN-gamma (rIFN-gamma) also had biological activity in vitro in three assay systems: (i) vesicular stomatitis virus plaque inhibition, (ii) class II major histocompatibility complex antigen upregulation on canine kidney parenchymal cells, and (iii) amplification of in vitro tissue-associated lymphoproliferation, all known to be effected by native IFN-gamma (nIFN-gamma). The availability of large amounts of active canine rIFN-gamma will be an important tool in studies of the role of this cytokine in the widely used experimental canine organ transplant model and also will be of diagnostic and therapeutic veterinary interest.

Amino Acid Sequence↗
PubMed1992

Cross-species reactivity of the anti-idiotype anti-OKT3 cascade between mice and humans.

The administration of murine mAb specific for the CD3 epsilon subunit of the TCR complex (OKT3) has been demonstrated to engender in humans an anti-OKT3 idiotypic cascade. This study used murine-derived anti-OKT3 (Ab2) as a bioreagent to determine whether this Ab2 and polyclonal anti-(anti-OKT3) (Ab3) generated in some human kidney transplant patients are idiotypically connected. Two anti-OKT3 mAbs G-880 (IgG1) and M-12 (IgM) were derived by immunizing BALB/c mice with the OKT3-secreting hybridoma. The two mAbs exhibited specificity for OKT3 F(ab)'2 idiotypic determinants. Both mAbs were tested for their ability to inhibit OKT3 induced mitogenesis and to block FITC-OKT3 binding to cell surface CD3 epsilon chain. The M-12 mAb inhibited OKT3-induced mitogenesis and blocked (approximately 60%) the binding of OKT3 to peripheral blood (PBL) T-cell CD3 epsilon chain in flow cytometry. In contrast, the G-880 mAb did not inhibit mitogenesis and only weakly blocked OKT3 binding to CD3 epsilon chain (approximately 12%). Sera of kidney transplant recipients who received OKT3 antirejection therapy and who developed antiidiotypic anti-OKT3 antibodies could be divided into two subgroups exhibiting anti-OKT3 activity: (a) those who had similar specificity as M-12 and failed to enhance the M-12 inhibition of OKT3 binding to PBL T-cell CD3 epsilon chain when added as a third component (n = 3), and (b) those with anti-OKT3 antibodies with idiotype specificity dissimilar to M-12 and who were able to increase the (maximum 60%) inhibition obtained with M-12 in the OKT3 to T-cell CD3-binding assay (n = 4). From these observations, we conclude that M-12 had the characteristics of an Ab2 beta and G-880 that of an Ab2 alpha. Additionally, there was an idiotypic connectivity of mouse-derived M-12 anti-OKT3 (Ab2) and OKT3-engendered human polyclonal anti-(anti-OKT3) (Ab3), in that three of seven patients examined had human serum IgG antibodies that specifically recognized M-12 idiotypic determinants as demonstrated in ELISA.

Animals↗
PubMed1992

Cloning of the cDNA for canine interferon-gamma.

We have taken advantage of conserved regions of cDNA sequences for interferon-gamma (IFN-gamma) from other species to design polymerase chain reaction (PCR) primers capable of amplifying the protein-coding region of the canine mRNA. We report here the cDNA cloning of this region from dog lymphocytes and the cDNA sequence. The predicted amino acid sequence is also reported and compared to the known sequences of these other species. The molecular clone for the canine IFN-gamma will allow direct study of this important cytokine's role in graft rejection in the widely used experimental canine transplant model.

Amino Acid Sequence↗
PubMed1990

The histone H3/H4.N1 complex supplemented with histone H2A-H2B dimers and DNA topoisomerase I forms nucleosomes on circular DNA under physiological conditions.

We have fractionated the whole cell extract of Xenopus oocytes (oocyte S-150) and isolated the endogenous components required for DNA supercoiling and nucleosome formation. Histone H2B and the three oocyte-specific H2A proteins were purified as free histones. Histones H3 and H4 were purified 100-fold in a complex with the acidic protein N1. In the presence of DNA topoisomerase I or II, histone H3/H4.N1 complexes supercoil DNA in a reaction that is inhibited by Mg2+, and this inhibition is relieved by NTPs. The supercoiling reaction induced by H3/H4.N1 complexes is enhanced by free histone H2A-H2B dimers, which by themselves do not supercoil DNA. Nuclease digestions and protein analyses indicate that H3/H4.N1 complexes form subnucleosomal particles containing histones H3 and H4. Nucleosomes containing 146-base pair DNA and the four histones are formed when histones H2A and H2B complement the reaction.

Animals↗
PubMed1990

The transcription complex of the 5 S RNA gene, but not transcription factor IIIA alone, prevents nucleosomal repression of transcription.

Assembly of nucleosomes on a 5 S DNA plasmid with histone H3.H4-N1 complex and histone H2A-H2B dimers causes a marked, 30-300-fold repression of 5 S RNA transcription. This repression is a time-dependent process that parallels the process of nucleosome formation. At physiological histone levels, DNA plasmids carrying nucleosomes with only histones H3 and H4 are transcriptionally permissive. The histone H3-H4 chromatin becomes transcriptionally nonpermissive when histone H2A-H2B dimers complement the nucleosome assembly reaction. H3-H4-H2A-H2B nucleosomes, but not H3-H4 nucleosomes, displace a DNA-bound transcription factor IIIA. In contrast, a preassembled 5 S RNA transcription complex is refractory to inactivation by nucleosomes.

Animals↗