Kenneth D. Chavin

Everolimus With Reduced Tacrolimus Improves Renal Function in De Novo Liver Transplant Recipients: A Randomized Controlled Trial

In a prospective, multicenter, open‐label study, de novo liver transplant patients were randomized at day 30±5 to (i) everolimus initiation with tacrolimus elimination (TAC Elimination) (ii) everolimus initiation with reduced‐exposure tacrolimus (EVR+Reduced TAC) or (iii) standard‐exposure tacrolimus (TAC Control). Randomization to TAC Elimination was terminated prematurely due to a higher rate of treated biopsy‐proven acute rejection (tBPAR). EVR+Reduced TAC was noninferior to TAC Control for the primary efficacy endpoint (tBPAR, graft loss or death at 12 months posttransplantation): 6.7% versus 9.7% (−3.0%; 95% CI −8.7, 2.6%; p<0.001 for noninferiority [12% margin]). tBPAR occurred in 2.9% of EVR+Reduced TAC patients versus 7.0% of TAC Controls (p = 0.035). The change in adjusted estimated GFR from randomization to month 12 was superior with EVR+Reduced TAC versus TAC Control (difference 8.50 mL/min/1.73 m2, 97.5% CI 3.74, 13.27 mL/min/1.73 m2, p<0.001 for superiority). Drug discontinuation for adverse events occurred in 25.7% of EVR+Reduced TAC and 14.1% of TAC Controls (relative risk 1.82, 95% CI 1.25, 2.66). Relative risk of serious infections between the EVR+Reduced TAC group versus TAC Controls was 1.76 (95% CI 1.03, 3.00). Everolimus facilitates early tacrolimus minimization with comparable efficacy and superior renal function, compared to a standard tacrolimus exposure regimen 12 months after liver transplantation.

Multiple vectors effectively achieve gene transfer in a murine cardiac transplantation model. Immunosuppression with TGF-beta 1 or vIL-10.

The application of gene transfer techniques to organ transplantation offers the potential for modulation of immunity directly within an allograft without systemic side effects. Expression vectors and promoter elements are important determinants of gene transfer and expression. In this study, various vectors (naked plasmid DNA, retroviral vector, herpes simplex viral vector, and adenoviral vector) with various promoters (RSV-LTR, SV40, MuLV-LTR, HCMVie1) were directly compared to demonstrate the successful gene transfer and expression of beta-galactosidase in murine myoblasts in vitro and within murine heterotopic, nonvascularized cardiac isografts or allografts in vivo. Expression of transferred genes was not toxic to cells and strength of expression varied according to the type of vector. Plasmid DNA was expressed in myocytes, retroviral vector was expressed in the graft infiltrating cells, and herpes simplex and adenoviral vectors were expressed in both myocytes and graft-infiltrating cells. Preliminary studies evaluated the ability of these vectors to deliver immunologically important signals. Allografts injected with pSVTGF-beta 1, a plasmid-encoding transforming growth factor beta 1 (TGF-beta 1) under the control of the SV40 promoter, showed significant prolongation of graft survival of 26.3 +/- 2.5 days compared with 12.6 +/- 1.1 days for untreated allografts, and 12.5 +/- 1.5 days for the allografts injected with control plasmid (P < 0.05). Allografts injected with MFG-vIL-10, a retroviral vector encoding viral interleukin-10 under the control of the MuLV-LTR, showed prolongation of graft survival of 36.7 +/- 1.3 days versus 12.6 +/- 1.1 days for the untreated allograft, and 13.5 +/- 2.0 days for the allografts injected with control retroviral vector (P < 0.001). Both vectors were transcriptionally active in vivo and did not appear to have toxic effects. Gene therapy for transplantation can induce transient expression of immunologically relevant molecules within allografts that impede immune activation while avoiding the systemic toxicity of conventional immunosuppression.

Effects of garlic (Allium sativum L.) supplementation on cytochrome P450 2D6 and 3A4 activity in healthy volunteers.

Garlic (Allium sativum L.) is a commonly used food and herbal supplement. The objective of this study was to assess in healthy volunteers (N = 14) the influence of a garlic extract on the activity of cytochrome P450 (CYP) 2D6 and 3A4. Probe substrates dextromethorphan (CYP2D6) and alprazolam (CYP3A4) were administered orally at baseline and again after treatment with garlic extract (3 × 600 mg twice daily) for 14 days. Urinary dextromethorphan/dextrorphan ratios and alprazolam plasma concentrations were determined by HPLC at baseline and after garlic extract treatment. The ratio of dextromethorphan to its metabolite was 0.044 ± 0.48 at baseline and 0.052 ± 0.095 after garlic supplementation. There were no significant differences between the baseline and garlic phases (P ≥ .05). For alprazolam, there were no significant differences in pharmacokinetic parameters at baseline and after garlic extract treatment (all P values ≥ .05; maximum concentration in plasma, 27.3 ± 2.6 ng/mL versus 27.3 ± 4.8 ng/mL; time to reach maximum concentration in plasma, 1.9 ± 1.4 h versus 2.4 ± 1.8 h; area under the time‐versus‐concentration curve, 537 ± 94 h · ng · mL−1 versus 548 ± 159 h · ng · mL−1; half‐life of elimination, 13.7 ± 4.4 h versus 14.5 ± 4.3 h). Our results indicate that garlic extracts are unlikely to alter the disposition of coadministered medications primarily dependent on the CYP2D6 or CYP3A4 pathway of metabolism.

CTLA4Ig prolongs allograft survival while suppressing cell-mediated immunity.

T cell activation is the result of antigen-specific interactions with the TCR/CD3 complex and costimulation via other T cell surface receptors. Prevention of costimulation can result in clonal anergy. CTLA4Ig is a fusion protein that binds with high-affinity to the B7/BB1 ligand and blocks the interaction of this ligand with CD28 and CTLA4. We explored the immunosuppressive effects of CTLA4Ig in a murine nonvascularized heterotopic cardiac transplant model and in a model of cell mediated immunity. CTLA4Ig administered in vivo for two days at the time of transplantation resulted in significant prolongation of allograft survival (55 +/- 2.0 vs. 12.2 +/- 0.5 days for control, P < 0.03). Administration at later times or to previously primed animals produced no prolongation of graft survival. CTLA4Ig administered during in vivo immunization to the hapten TNP suppressed the contact sensitivity response and inhibited the subsequent in vitro generation of secondary TNP-specific CTL. CTLA4Ig administered in vivo had no effect on subsequent primary alloantigen-specific CTL or MLR responses--however, when added to culture the fusion protein inhibited the MLR response by 80%, but not the alloantigen-specific CTL response. CTLA4Ig inhibited CD4+ and CD8+ proliferative and cytokine responses to alloantigen. Flow cytometry showed no changes in distribution of subpopulations of T cells. These results confirm the immunosuppressive activity of CTLA4Ig in vivo in an allograft model and show that both CD4+ and CD8+ T cells are suppressed by CTLA4Ig. The most efficacious time of administration is during priming of the immune response at the time of antigen presentation.

Multiple-dose administration of Ginkgo biloba did not affect cytochrome P-450 2D6 or 3A4 activity in normal volunteers.

Standardized extracts from the Ginkgo biloba tree are purported to exert positive neurocognitive effects and may also be useful in the treatment of a variety of vascular and other disorders. This dietary supplement is among the most commonly used herbal preparations in the world. The objective of this study was to assess in normal volunteers (n = 12) the influence of standardized Ginkgo biloba (GB) on the activity of cytochrome P-450 (CYP) 2D6 and 3A4 normal volunteers phenotyped as CYP2D6 extensive metabolizers. Probe substrates dextromethorphan (CYP2D6 activity) and alprazolam (CYP 3A4 activity) were co-administered orally at baseline, and following treatment with GB (120 mg twice daily) for 14 days. Urinary concentrations of dextromethorphan and dextrorphan were quantified and dextromethorphan metabolic ratios (DMRs) were determined at baseline and after GB treatment. Likewise, plasma samples were collected (0–60 hrs) for alprazolam pharmacokinetics at baseline and after GB treatment to assess effects on CYP 3A4 activity. Validated HPLC methods were used to quantify all compounds and relevant metabolites. No statistically significant differences were found between baseline and post-GB treatment DMRs indicating a lack of effect on CYP2D6. For alprazolam there was a 17% decrease in the area under the plasma concentration versus time curve (AUC); (P <0.05). However, the half-life of elimination was not significantly different after GB administration indicating a lack of hepatic CYP3A4 induction. We conclude that standardized extracts of GB at recommended doses are unlikely to significantly alter the disposition of co-administered medications primarily dependent on the CYP2D6 or CYP3A4 pathways for elimination.

Laparoscopic live donor nephrectomy: technical considerations and allograft vascular length.

renal vein results in loss of approximately 1.0 ‐1.5 cm of vein when compared with the open operation. Additionally, in our series of laparoscopic live donor nephrectomies, right and left renal vein lengths have averaged (6SD) 2.560.7 cm and 4.761.0 cm, respectively (P,0.001). A short, thin right renal vein can make the recipient operation difficult. We have experienced allograft renal vein thrombosis and recipient external iliac vein thrombosis in the peritransplant period with this scenario. Therefore, we advocate extreme caution when using the laparoscopic operation on the right side. In general, when employing the laparoscopic approach, multiple left renal arteries are less problematic than a right kidney. If a right-sided operation is planned, either three-dimensional computed tomography (3-D CT) scanning or magnetic resonance angiography should be performed preoperatively to fully assess the venous anatomy. We have found 3-D CT to be particularly useful in identifying venous anomalies and multiple renal veins. Also, we recommend that the laparoscopic operation be modified. Rather than employing an infra- or periumbilical incision for extraction of the kidney, a small (6 ‐ 8 cm) right upper quadrant transverse incision can be used. This allows for complete dissection of the kidney laparoscopically, avoids a flank incision, and enables the renal vessels to be divided in a similar fashion to the open operation. A Satinsky clamp can be placed across the inferior vena cava to allow for maximal venous length to be obtained. This approach promises to offer the advantages of both the laparoscopic and the open operations.

Renal Function at Two Years in Liver Transplant Patients Receiving Everolimus: Results of a Randomized, Multicenter Study

In a 24‐month prospective, randomized, multicenter, open‐label study, de novo liver transplant patients were randomized at 30 days to everolimus (EVR) + Reduced tacrolimus (TAC; n = 245), TAC Control (n = 243) or TAC Elimination (n = 231). Randomization to TAC Elimination was stopped prematurely due to a significantly higher rate of treated biopsy‐proven acute rejection (tBPAR). The incidence of the primary efficacy endpoint, composite efficacy failure rate of tBPAR, graft loss or death postrandomization was similar with EVR + Reduced TAC (10.3%) or TAC Control (12.5%) at month 24 (difference −2.2%, 97.5% confidence interval [CI] −8.8%, 4.4%). BPAR was less frequent in the EVR + Reduced TAC group (6.1% vs. 13.3% in TAC Control, p = 0.010). Adjusted change in estimated glomerular filtration rate (eGFR) from randomization to month 24 was superior with EVR + Reduced TAC versus TAC Control: difference 6.7 mL/min/1.73 m2 (97.5% CI 1.9, 11.4 mL/min/1.73 m2, p = 0.002). Among patients who remained on treatment, mean (SD) eGFR at month 24 was 77.6 (26.5) mL/min/1.73 m2 in the EVR + Reduced TAC group and 66.1 (19.3) mL/min/1.73 m2 in the TAC Control group (p < 0.001). Study medication was discontinued due to adverse events in 28.6% of EVR + Reduced TAC and 18.2% of TAC Control patients. Early introduction of everolimus with reduced‐exposure tacrolimus at 1 month after liver transplantation provided a significant and clinically relevant benefit for renal function at 2 years posttransplant.

Short-term administration of (-)-epigallocatechin gallate reduces hepatic steatosis and protects against warm hepatic ischemia/reperfusion injury in steatotic mice.

Hepatic steatosis increases the extent of cellular injury incurred during ischemia/reperfusion (I/R) injury. (‐)‐Epigallocatechin gallate (EGCG), the major flavonoid component of green tea (camellia sinensis) is a potent antioxidant that inhibits fatty acid synthase (FAS) in vitro. We investigated the effects of EGCG on hepatic steatosis and markers of cellular damage at baseline and after I/R injury in ob/ob mice. Animals were pretreated with 85 mg/kg EGCG via intraperitoneal (ip) injection for 2 days or oral consumption in the drinking water for 5 days before 15 minutes of warm ischemia and 24 hours of reperfusion. After EGCG administration, total baseline hepatic fat content decreased from baseline. Palmitic acid and linoleic acid levels also were reduced substantially in all ECGC‐treated animals before I/R. Alanine aminotransferase (ALT) levels decreased in all EGCG‐treated animals compared with control animals after I/R. Histologic analysis demonstrated an average decrease of 65% necrosis after EGCG administration. EGCG administration also increased resting hepatic energy stores as determined by an increase in cellular adenosine triphosphate (ATP) with a concomitant decrease in uncoupling protein 2 (UCP2) before I/R. Finally, there was an increased level of glutathione (GSH) in the EGCG‐treated mice compared with the vehicle‐treated mice both at baseline and after I/R. In conclusion, taken together, this study demonstrates that treatment with ECGC by either oral or ip administration, significantly protects the liver after I/R, possibly by reducing hepatic fat content, increasing hepatic energy status, and functioning as an antioxidant. (Liver Transpl 2005;11:298–308.)

Cytokine-Responsive Gene-2/IFN-Inducible Protein-10 Expression in Multiple Models of Liver and Bile Duct Injury Suggests a Role in Tissue Regeneration

IFN-inducible protein-10 (IP-10/CXCL10) is a CXC chemokine that targets both T cells and NK cells. Elevation of IP-10 expression has been demonstrated in a number of human diseases, including chronic cirrhosis and biliary atresia. Cytokine-responsive gene-2 (Crg-2), the murine ortholog of IP-10, was induced following CCl4 treatment of the hepatocyte-like cell line AML-12. Crg-2 expression was noted in vivo in multiple models of hepatic and bile duct injury, including bile duct ligation and CCl4, d-galactosamine, and methylene dianiline toxic liver injuries. Induction of Crg-2 was also examined following two-thirds hepatectomy, a model that minimally injures the remaining liver, but that requires a large hepatic regenerative response. Crg-2 was induced in a biphasic fashion after two-thirds hepatectomy, preceding each known peak of hepatocyte DNA synthesis. Induction of Crg-2 was also observed in the kidney, gut, thymus, and spleen within 1 h of two-thirds hepatectomy. Characteristic of an immediate early gene, pretreatment of mice with the protein synthesis inhibitor cycloheximide before either two-thirds hepatectomy or CCl4 injection led to Crg-2 superinduction. rIP-10 was demonstrated to have hepatocyte growth factor-inducing activity in vitro, but alone had no direct mitogenic effect on hepatocytes. Our data demonstrate that induction of Crg-2 occurs in several distinct models of liver injury and regeneration, and suggest a role for CRG-2/IP-10 in these processes.

LAPAROSCOPIC LIVE DONOR NEPHRECTOMY: The Recipient1

Background Laparoscopic live donor nephrectomy offers advantages to the donor in terms of decreased pain and shorter recuperation. Heretofore no detailed analysis of the recipient of laparoscopically procured kidneys has been performed. The purpose of this study was to determine whether laparoscopic donor nephrectomy had any deleterious effect on the recipient. Methods. A retrospective review was conducted of all live donor renal transplantations performed from January 1995 through April 1998. The control group received kidneys procured via a standard flank approach (Open). Rejection was diagnosed histologically. Creatinine clearance was calculated using the Cockroft-Gault formula. Results. A total of 110 patients received kidneys from laparoscopic (Lap) and 48 from open donors. One-year recipient (100% vs. 97.0%) and graft (93.5% vs. 91.1%) survival rates were similar for the Open and Lap groups, respectively. A similar incidence of vascular thrombosis (3.4% vs. 2.1%, P=NS) and ureteral complications (9.1% vs. 6.3%, P=NS) were seen in the Lap and Open groups, respectively. The incidence of acute rejection for the first month was 30.1% for the Lap group and 31.9% for the Open group (P=NS). The rate of decline of serum creatinine level in the early post-transplantation period was initially greater in the Open group, but by postoperative day 4 no significant difference existed. No difference was observed in allograft function long-term. The median length of hospital stay was 7.0 days for both groups. Conclusions. Laparoscopic live donor nephrectomy does not adversely effect recipient outcome. The previously demonstrated benefits to the donor, and the increased willingness of individuals to undergo live kidney donation, coupled with the acceptable outcomes experienced by recipients of laparoscopically procured kidneys justifies the continued development and adoption of this operation.