Concurrent Immunosuppression Research Articles

Sirolimus promotes tolerance for donor and recipient antigens after MHC class II disparate bone marrow transplantation in rats

Mixed chimerism after allogeneic bone marrow transplantation (BMT) promotes immunologic tolerance. Graft-vs-host disease (GvHD) can occur when immunosuppressive control of the graft fails. Here we evaluate the influence of concurrent immunosuppression after irradiation-based induction therapy on development of tolerance and GvHD. Conditioning was performed by different doses of total body irradiation (TBI) in a major histocompatibility complex (MHC) class II disparate rat BMT model. Animals received subsequent immunosuppression with either cyclosporine A (CsA) or sirolimus. Nonresponsiveness toward donor and recipient antigens was demonstrated by development of mixed chimerism and/or GvHD. Administration of 10 Gy of TBI prior to BMT alone was associated with severe GvHD. Induction therapy with 8 Gy of TBI alone led to graft rejection in the long-term. Two weeks of immunosuppression with CsA after 8 Gy of TBI resulted in transient chimerism, but was finally associated with a combination of fatal GvHD and graft rejection. Six gray of TBI with CsA treatment for 14 or 28 days caused only mild GvHD, but did not lead to stable chimerism. In contrast, treatment with sirolimus was associated with stable chimerism after 8 Gy of TBI (14-day course) and 6 Gy of TBI (28-day course) accompanied by a low incidence of GvHD. In contrast to CsA, sirolimus facilitates development of tolerance after MHC class II disparate BMT and irradiation-based conditioning, with a low risk of GvHD. Therefore, sirolimus has promising characteristics for inclusion in immunosuppressive protocols.

Effect of Plasmapheresis for Acute Humoral Rejection After Heart Transplantation

Objective The objective of this study was to report our experience of treating acute humoral rejection with plasmapheresis in heart transplant (HT) recipients. Patients and Methods From May 1996 to December 2005, 238 HTs were performed using therapy with cyclosporine or tacrolimus, azathioprine or mycophenolate mofetil, and prednisolone as well as induction treatment with rabbit anti-human thymocyte globulin. Endomyocardial biopsy for rejection surveillance was performed weekly for the first month, monthly for 3 months, yearly after the first year, and whenever rejection was suspected. Immunofluorescence studies with IgG, IgM, C3, C4, C1q, and HLA-DR were performed routinely on the first month biopsy. After a 2-year trial, immunofluorescence studies were not performed routinely, because no significant findings were observed; thus they were performed only when clinical deterioration, unstable hemodynamic status, or suspicion of rejection occurred on routine echocardiographic examinations. Plasmapheresis with fresh frozen plasma exchanging twice the blood volume of the patients was performed for 5 days. Rescue immunosuppression with methylprednisolone (1 g/d) was delivered for 3 days and the immunosuppressants changed, but no intravenous immunoglobulin was prescribed. Results Twelve patients suffered biopsy-proven acute humoral rejection at 3 days to 32 months after HT (mean, 9.4 months). Immunofluorescence studies showed positive HLA-DR in 7 patients; IgG in 4 patients; IgM in 1 patient; C3 in 4 patients; C4 in 1 patient; and C1q in 1 patient. One patient who was 3 months after HT showed only C1q positive but was treated with extracorporeal membrane oxygenation and intra-aortic balloon pumping support and died 1-month after plasmapheresis. Another patient who deteriorated on the 3rd postoperative day and died 3 days after plasmapheresis was considered to have vascular rejection by interstitial edema, vacuolated endothelial cells and no pathognomonic clinical features, although there was no positive immunofluorescence result. All other subjects were discharged from the hospital, although 3 required mechanical support during plasmapheresis. Hypotension with hypocalcemia was frequently noted during plasmapheresis. The 1-year survival rate was 75% ± 11%, and 5-year survival rate, 51% ± 15%. Conclusion Plasmapheresis with concurrent rescue immunosuppression was an effective treatment for acute humoral rejection in HT even with unstable hemodynamics.

Immune Suppression as the Etiology of Failure To Detect Anti-HBc Antibodies in Patients with Chronic Hepatitis B Virus Infection

A lack of anti-HBc antibodies during chronic hepatitis B virus infection is a serological pattern that is rarely observed. In our series of 39 patients with such a confirmed profile, mutations within the precore/core gene were rarely found and the lack of antibody detection was mostly explained by concurrent immunosuppression and the low sensitivities of the serological assays.

Neoral Versus Prograf in Simultaneous Pancreas-Kidney Transplantation With Portal Venous Drainage: Three-Year Results of a Single-Center, Open-Label, Prospective, Randomized Pilot Study

Background The preferential use of tacrolimus (Prograf) over cyclosporine microemulsion (Neoral) in simultaneous pancreas-kidney transplantation (SPKTx) is mainly based on historical, retrospective studies. We herein report the 3-year results of a single-center, prospective, randomized comparison of the two calcineurin inhibitors in the setting of mycophenolate mofetil (MMF)-based immunosuppression and portal drainage of pancreas allografts. Methods Between May 2001 and August 2004, 47 SPKTx recipients who were stratified by recipient sex, were alternatively assigned to treatment with Neoral ( n = 22) or Prograf ( n = 25). Concurrent immunosuppression included induction treatment with basiliximab and maintenance with MMF and steroids. Results After a median follow-up of 24.0 months, all patients remained in the study arm into which they were initially enrolled. No pancreas rejection episode was observed. One acute kidney rejection was recorded in the Neoral arm (4.5%) as compared with 7 (28.0%) including one steroid-resistant episode, in the Prograf arm ( P = .03). The cumulative incidence of adverse events was 31.8% ( n = 7) in the Neoral arm compared with 92.0% ( n = 23) in the Prograf arm ( P < .0001). One patient died in each study arm. Patient, pancreas, and kidney survivals overlapped at 1- and 3-years posttransplant, namely all 95.4% for the Neoral arm compared with 95.8%, 91.8%, and 95.8%, respectively, for the Prograf arm ( P > .05). Conclusions We conclude that in MMF-based immunosuppression there is no convincing evidence that Prograf should be preferred to Neoral in SPKTx.

AIDS-related cancer

<h2>Abstract</h2> This contribution provides a current and comprehensive summary regarding AIDS-related malignancies and their management. Management of AIDS-related malignancies remains complex. Cancers are more aggressive, presenting at a later stage and at unusual sites. Opportunistic infection and concurrent immunosuppression complicate management. In addition to prolonging survival through immune reconstitution in persons with HIV, highly active antiretroviral therapy (HAART) has resulted in a reduction in incidence of Kaposi's sarcoma and certain types of non-Hodgkin's lymphoma. Furthermore, HAART is of therapeutic value in Kaposi's sarcoma.

Pharmacokinetics of Mycophenolic Acid in Kidney Transplant Patients Receiving Sirolimus Versus Cyclosporine

Introduction Mycophenolic acid (MPA) pharmacokinetics exhibit large variability in transplant recipients and may be altered due to concurrent immunosuppressants. Little is known about the influence of sirolimus (SRL) on MPA pharmacokinetics in kidney transplant patients. Methods We studied the areas under concentration-time curves (AUC) for MPA in 15 patients receiving immunosuppression combining SRL with mycophenolate mofetil (MMF). The pharmacokinetic measurements were performed in all patients using three MMF dosing regimens (0.5 g twice a day, 0.75 g twice a day, 1 g twice a day). Similar blood AUC profiles were also sampled from 12 patients treated with a fixed dose of MMF 1 g twice a day and cyclosporine (CsA). MPA was measured using HPLC; the AUC 0–12 of MPA was determined by the trapezoidal method using four sampling time points: C 0, C 1, C 3, C 5. Results While patients on SRL were receiving 0.75 g MMF twice a day, mean AUC 0–12 and C 0 values of MPA were comparable to those of patients receiving CsA and 1 g MMF twice a day (54.1 ± 17.6 and 3 ± 1.87 vs 51.7 ± 16.7 mg · h/L and 2.76 ± 1.57 mg/L, respectively). On the other hand, 0.5 g MMF twice a day with SRL therapy resulted in AUC 0–12 and C 0 values of MPA of 32.3 ± 12.6 mg · h/L and 2.32 ± 1.72 mg/L, respectively, whereas, 1 g MMF twice a day with SRL resulted in AUC 0–12 and C 0 values of MPA of 70.9 ± 19.3 mg · h/L and 4.7 ± 2.44 mg/L, respectively. Conclusions These findings demonstrate that MPA exposure in the presence of SRL is higher than that with CsA. It appears that the MMF dose should be reduced to 0.75 g twice a day in patients receiving SRL to obtain AUC 0–12 of MPA levels comparable to that in patients treated with CsA and MMF 1 g twice a day.

Sirolimus

This review seeks to apply a decision-making algorithm to establish whether clinical pharmacokinetic monitoring (CPM) of sirolimus (rapamycin) in solid organ transplantation is indicated in specific patient populations. The need for CPM of sirolimus, although a regulatory requirement in Europe, has not yet been firmly established in North America and other parts of the world. Sirolimus has demonstrated immunosuppressive efficacy in renal, pancreatic islet cell, liver and heart transplant recipients. The pharmacological response of immunosuppressive therapy with sirolimus cannot be readily evaluated; however, a relationship between trough blood sirolimus concentrations, area under the plasma concentration-time curve (AUC) and the incidence of rejection has been proposed. Furthermore, sirolimus can be measured in whole blood by several assays--high-performance liquid chromatography with detection by tandem mass spectrometry, or with ultraviolet detection, radioreceptor assay or microparticle enzyme immunoassay. Both experimental animal and clinical data suggest that adverse events and their associated severity are correlated with blood concentrations. To prevent rejection and minimise toxicity, a therapeutic range of 4-12 microg/L (measured via chromatographic assays) is recommended when sirolimus is used in conjunction with ciclosporin. If ciclosporin therapy is discontinued, a target trough range of 12-20 microg/L is recommended. Sirolimus pharmacokinetics display large inter- and intrapatient variability, which may change in specific patient populations due to disease states or concurrent immunosuppressants or other interacting drugs. Due to the long half-life of sirolimus, dosage adjustments would ideally be based on trough levels obtained more than 5-7 days after initiation of therapy or dosage change. Once the initial dose titration is complete, monitoring sirolimus trough concentrations weekly for the first month and every 2 weeks for the second month appears to be appropriate. After the first 2 months of dose titration, routine CPM of sirolimus is not necessary in all patients, but may be warranted to achieve target concentrations in certain populations of patients, but the frequency of further monitoring remains to be determined and should be individualised.

PHARMACOKINETICS OF MYCOPHENOLIC ACID IN KIDNEY TRANSPLANT PATIENTS RECEIVING SIROLIMUS VERSUS CYCLOSPORINE

O217 Aims: Mycophenolic acid (MPA) pharmacokinetics exhibit large variability in transplant recipients and may be altered due to concurrent immunosuppressants. It has been recognized that cyclosporine (CsA) decreases the exposure (AUC) to MPA. Conversely, little is known about the influence of sirolimus (SRL) on the AUC of MPA in kidney transplant patients. This trial was performed to assess the optimal dose of MMF during co-administration with SRL, basing on the well-known MPA-therapeutic range targeted in organ transplantation (30-60 mg. hour/L) Methods: We performed AUC measurements for MPA in 15 patients (M/F: 11/4, mean age: 48+/-15 y) receiving immunosuppression combining SRL with mycophynolate mofetil (MMF) and in 12 patients (M/F: 10/2, mean age: 47+/- 9 y) receiving CsA with MMF. For each patient in SRL group, MPA measurements were performed at three MMF dosing regimens (0.5g b.i.d., 0.75g b.i.d., 1g b.i.d.), blood samples were obtained with 2-week interval after MMF regimen modification. Whereas, in CsA group MPA measurements were achieved with 1g b.i.d.. MPA was measured by high-performance liquid chromatography (HPLC), and AUC-MPA was determined by trapezoidal method using 4 sampling time points: C0, C1, C3, C5. SRL doses were unchanged over the whole study course. All patients were Caucasians, transplanted at least since 6 months, and had stable graft function for no less than 3 months (mean serum creatinine: 147+/-61μmol/L in CsA group Vs 141+/-39 μmol/L in SRL group). Results: Mean CsA and SRL trough levels were 99.6+/-51.5 ng/mL and 9.1+/-2.4 ng/mL respectively. Table 1 shows AUC and C0 values of MPA in CsA and SRL groups.FigureIn the overall patients C0-MPA correlates moderately with AUC-MPA (r = 0.45; P<0.0001). Conclusions: These findings demonstrate that administering MMF 0.75 g b.i.d. in patients treated by SRL will result AUC-MPA values equivalent to those treated by CsA and MMF 1g b.i.d.. The use of MMF 1g b.i.d. combining SRL may cause unnecessary overexposure to the drug.

LONG-TERM RESULTS IN SOLID ORGAN TRANSPLANT RECIPIENTS CONVERTED FROM CALCINEURIN-INHIBITORS TO SIROLIMUS

O294 Aims: Maintenance immunosuppressive regimens without calcineurin-inhibitors (CI) have been advocated for minimizing renal and non-renal toxicities and maximizing long-term renal function. Although CI discontinuation and conversion to sirolimus (SRL) has been proposed by several centers, controversy still exist for the indications, the optimal time, the inclusion and exclusion criteria, the desired SRL levels, and the ideal immunosuppressive agents to used with SRL. Methods: We report herein our long-term results (Nov 1999-Feb 2004) in which CI was abruptly discontinued and substituted with a regimen consisting of SRL (target level 10-12 ng/mL), mycophenolate mofetil (MMF) and prednisone (PRED) in a large cohort (n=139) of transplant recipients [82 kidney (KTX), 28 kidney-pancreas (SPK), 8 pancreas alone (PTA), 21 liver (OLTX)]. Indications for conversions were chronic allograft nephropathy, CI nephrotoxicity, thrombotic microangiopathy (TMA) with or without systemic involvement (HUS) and glucose intolerance. PTA and OLTX required CI discontinuation because of renal dysfunction (n=25) and posttransplant diabetes (n=4). Exclusions criteria were: serum creatinine >3.5 mg/dL or glomerular filtration rate <30 ml/min, cholesterol and/or triglicerides >300 mg/ml) and rejection episodes in the previous 3 months. Results: At follow-up, 6 patients (5 KTX and 1 SPK) died from cardiovascular events; 11 patients lost the kidney allograft (1 rejection, 2 non-compliance, 2 polyoma virus, 6 recurrent glomerulonephritis). Renal function improved or stabilized not only in KTX and SPK but also in PTA and OLTX. Patients with systemic HUS requiring dialysis and plasmapheresis and patients with biopsy-proven TMA lesions had complete recovery of renal allograft function. Pancreas graft failed in 3 patients (1 chronic rejection, 2 non-compliance). Blood glucose control improved in 76% patients leading to either insulin-independence or reduction in the need for oral hypoglycemic agents. The combination of SRL and MMF induced reversible leucopenia, but rarely was WBC < 3500 mm3. Because of the immunological stability, PRED was discontinued or reduced in most patient (mean dose 2 mg/day at follow-up). Infectious complications included herpes zoster (2), herpes simplex (2), and pneumonia (4). There were no cytomegalovirus infection and Epstein Barr virus reactivations. No malignancies were observed. Reasons for SRL discontinuation were pneumonitis (1), skin rash (3), mouth ulcers (2), gastrointestinal intolerance (2), joint pain (1), intractable hyperlipidemia (2). Conclusions: This study reports the longest follow-up (> 3 years) and the largest number of transplant recipients (n=139) converted to SRL. An abrupt CI discontinuation and conversion to SRL achieved stable patient and graft survival, stable or improved renal function with minimal bone marrow suppression and manageable lipid abnormalities. Our data suggest that it is possible to obtain adequate immunosuppression with low dose of SRL. The reduction of the concurrent immunosuppressants might have an impact on the low incidence of metabolic and infectious complications.

Disseminated cutaneous leishmaniasis after visceral disease in a patient with AIDS

Leishmaniasis is emerging as a common and serious opportunistic disease for patients with HIV infection. Almost all cases of HIV-Leishmania coinfection have been described in Mediterranean countries and they occur with various clinical presentations, ranging from typical visceral forms to asymptomatic or atypical cases, including cutaneous and mucocutaneous leishmaniasis. Pentavalent antimony compounds have been the mainstays of antileishmanial therapy for half a century and new lipid formulations of amphotericin B seem reliable, but the most effective treatment remains unknown. We describe a patient who was HIV infected and an intravenous drug user, with an unusual disseminated cutaneous leishmaniasis, after an initial visceral disease and after a 13-month maintenance treatment with liposomal amphotericin. The severe concurrent immunosuppression probably played an essential role in leading to this atypical cutaneous form, characterized by diffuse, nonulcerated, nonscabby maculopapular lesions.

Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation with and without donor-specific cell augmentation

Background Immunomodulatory strategies such as donor-specific bone marrow or blood transfusions have been used to promote engraftment after intestinal transplants. We previously showed that delivery of donor antigen via the portal vein can effectively reduce the rate of intestinal graft rejection. The purpose of our current study was to investigate the impact of donor-specific cell augmentation (blood versus bone marrow) via the portal vein on cytokine expression in intestinal grafts versus native livers. Materials and methods We performed heterotopic small intestinal transplants between male Brown-Norway (donor) and female Lewis (recipient) rats. We studied 10 groups according to [1] the type of donor-specific cell augmentation and [2] the use and dose of immunosuppressive therapy. For cell augmentation, donor-specific blood or bone marrow was transfused via the donor portal vein immediately before graft implantation. For immunosuppression, tacrolimus was used post-transplant at a high or low dose. Control rats received neither immunosuppression nor cell augmentation. Tissue samples for histological assessment were obtained at designated time points. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements (IL-1 α, IL-1 β, IL-2, IL-4, IL-5, IL-6, IFN-γ, TNF-α, and TNF-β). Chimerism levels were determined using Q-PCR analysis. Results Without concurrent immunosuppression, neither portal donor-specific blood nor bone marrow transfusion reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood ( versus bone marrow) transfusion. Irrespective of the type of donor-specific cell augmentation, severe rejection caused strong cytokine expression in the grafts of IL-1 α, IL-1 β, IFN-γ, and TNF-α; in the native livers, mainly of TNF-α (with IFN-γ showing hardly any increase). In general, rejection caused stronger cytokine expression in the grafts than in the native livers. Mild rejection correlated well with strong intragraft expression of IL-6, TNF-α, and TNF-β (early rejection markers); severe rejection with IL-1 α, IL-1 β, IFN-γ, and TNF-α (late rejection markers). In addition to cell augmentation per se, the type of cell augmentation also had an impact on cytokine expression in both grafts and native livers. Cell-augmented ( versus tacrolimus-treated) rats showed hardly any differences in intragraft cytokine expression, but the expression of almost all cytokines was significantly stronger in the native livers. With immunosuppression, bone marrow infusion increased intragraft cytokine expression of IL-1 α, IL-1 β, IFN-γ, and TNF α, as well as liver cytokine expression of IL-1 β, compared to blood transfusion. This finding reflected the more advanced rejection stages in the bone marrow infused group; different types of donor-specific cell augmentation had similar effects on liver cytokine expression. In the absence of myoablative therapy, chimerism levels were low, in both cell-augmented and non-cell-augmented groups. Conclusions Rejection and donor-specific cell augmentation independently causes differences in intragraft versus native liver cytokine expression after intestinal transplants. Portal donor-specific blood transfusion, as compared with donor-specific bone marrow infusion, lowered the incidence of rejection and diminished intragraft cytokine up-regulation.

Intravenous hydrocortisone premedication reduces antibodies to infliximab in Crohn's disease: A randomized controlled trial

Background & Aims: We assessed the relationship between antibodies to infliximab (ATI) and the loss of response postinfliximab, infusion reactions and, in a randomized trial, investigated whether intravenous hydrocortisone premedication can reduce ATI. Methods: Initially, we prospectively evaluated clinical response, adverse events, and ATI levels in 53 consecutive patients with Crohn's disease who received 199 infliximab (5 mg/kg) infusions. Subsequently, 80 patients with Crohn's disease were randomized to intravenous hydrocortisone 200 mg or placebo immediately before their first and subsequent infliximab infusions. The primary endpoint was reduction in median ATI levels at week 16. Analysis was by intention to treat. Results: Nineteen of our initial 53 patients (36%) developed ATI, including all 7 patients with serious infusion reactions (median ATI level, 19.6 μg/mL). Eleven of 15 patients (73%) who lost their initial response were ATI positive compared with none of 21 continuous responders, (8.9 vs. 0.7 μg/mL, P < 0.0001). Administering a second infusion within 8 weeks of the first (OR, 0.13; 95% CI, 0.03-0.5; P = 0.0007) or concurrent immunosuppressants (OR, 0.19; 95% CI, 0.04-1.03; P = 0.007) significantly reduced ATI formation. In the placebo-controlled trial, ATI levels were lower at week 16 among hydrocortisone-treated patients (1.6 vs. 3.4 μg/mL, P = 0.02), and 26% of hydrocortisone-treated patients developed ATI compared with 42% of placebo-treated patients, P = 0.06. Conclusions: Loss of initial response and infusion reactions post-infliximab is strongly related to ATI formation and level. Administering a second infusion within 8 weeks of the first and concurrent immunosuppressant therapy significantly reduce ATI formation. Intravenous hydrocortisone premedication significantly reduces ATI levels but does not eliminate ATI formation or infusion reactions.

Mycophenolate Mofetil for Solid Organ Transplantation: Does the Evidence Support the Need for Clinical Pharmacokinetic Monitoring?

The need for clinical pharmacokinetic monitoring (CPM) of the immunosuppressant mycophenolate mofetil (MMF) has been debated. Using a previously developed algorithm, the authors reviewed the evidence to support or refute the utility of CPM of MMF. First, MMF has proven efficacy for prevention of organ rejection in renal and cardiac transplant populations. In addition, the pharmacologically active form of MMF, mycophenolic acid (MPA), can be measured readily in plasma, and relationships between the incidence of rejection and MPA predose concentrations and MPA area under the curve (AUC) have been reported. A lower limit of the therapeutic range (MPA predose concentrations >1.55 microg/mL, as measured by enzyme multiplied immunoassay technique [EMIT], or MPA AUC >30 or 40 microg. h/mL, as measured by high-performance liquid chromatography [HPLC]) has been suggested to prevent rejection in renal allograft patients. Similarly, in cardiac transplant patients, decreased incidences of organ rejection have been reported in patients with MPA concentrations >2 or 3 microg/mL (using EMIT) and total AUC values >42.8 microg. h/mL (using HPLC). However, the relationship between pharmacokinetic parameters and adverse events in renal and cardiac transplant patients remains unclear. Due to the nature of antirejection therapy, the pharmacologic response of MMF is not readily assessable, and therapy is life-long. MPA pharmacokinetics exhibit large inter- and intrapatient variability and may be altered in specific patient populations due to changes in protein binding, concomitant disease states, or interactions with concurrent immunosuppressants. Therefore, on the basis of current evidence, CPM can provide more information regarding efficacy of MMF than clinical judgment alone in select patient populations. However, further randomized, prospective trials are required to clarify unresolved issues. Specifically, an upper limit of the therapeutic range, above which the risk of side effects is increased, needs to be elucidated for MMF therapy. Other future directions for research include determining a practical limited sampling strategy for MPA AUC; clarifying the relationship between free MPA concentrations, efficacy, and toxicity; and defining the pharmacodynamic relationship between activity of inosine monophosphate dehydrogenase (the enzyme inhibited by MPA) and risk of rejection or adverse effects.

Use of an adenoviral vector to express soluble donor-major histocompatibility complex molecules capable of suppressing the immune response in rat transplant recipients

There is an accumulating body of evidence indicating that soluble major histocompatibility complex (sMHC) molecules have donor-specific immunosuppressive properties. One classic, but still unproven, theory is that production of sMHC by liver transplants induces a potent immunosuppressive effect. To mimic this possible effect, we have developed a replication deficient adenovirus (Ad-RQ) to express high levels of donor sMHC class I molecules (sRT1.A a) in the liver. Ad-RQ produced sRT1.A a was measured by enzyme-linked immunosorbant assay (ELISA) after in vitro infection of Lewis (RT1 l) hepatocytes, and in vivo following intravenous virus injection into Lewis rats. Results indicated high sRT1.A a expression in Lewis hepatocyte cultures and, in vivo, high expression was also demonstrated and maintained for at least 1 week. A strong immunosuppressive potential of sMHC in vivo was revealed by prolongation of cardiac (ACI, RT1 a) heart allograft survival in high-responder Lewis rat recipients treated with Ad-RQ alone. Furthermore, limiting dilution cytotoxic T-lymphocyte precursor (CTLp) analysis of lymphocytes from Ad-RQ-treated Lewis recipients receiving an ACI heart transplant indicated a marked decrease in antidonor CTLp frequency. In conclusion, our results demonstrate that viral vectors can be used effectively to express high levels of sMHC molecules, and their immunosuppressive effect, without concurrent immunosuppression, is sufficiently potent to prolong heart transplant survival.

Administration of adenoviral vectors induces gangrene in acutely ischemic rat hindlimbs: Role of capsid protein-induced inflammation

Purpose: The initial purpose of this study was to determine the effects of intravascular adenoviral vector-mediated gene transfer of endothelial nitric oxide synthase (AdeNOS) on experimental hindlimb ischemia in the rat. Unexpectedly, administration of AdeNOS immediately after induction of acute limb ischemia led to limb gangrene. We subsequently sought to define the molecular mechanisms responsible for this unusual effect and to devise adenoviral gene transfer strategies to prevent the development of gangrene in acutely ischemic limbs. Methods: Phosphate-buffered saline or adenoviral vectors containing the bovine endothelial nitric oxide synthase gene (AdeNOS) or no transgene (Ad-E1) were injected intra-arterially into the hindlimb of a rat under vascular isolation immediately after surgical induction of severe ischemia. Hematoxylin and eosin staining was performed on muscle sections to evaluate inflammation. A separate group of animals was injected with an adenovirus containing a nontranscribable genome, treated with cyclosporine, or received delayed administration of the adenoviral vector. Gene expression after delayed adenoviral gene transfer was assessed with immunohistochemistry, Western blotting, and nitric oxide synthase (NOS) activity assay. Results: Both AdeNOS and Ad-E1 caused gangrene of the entire hindlimb within 12 days in a dose-dependent manner, at a threshold concentration of 1 × 109 plaque-forming unit/mL. Adenoviral delivery was associated with more inflammation and edema compared with phosphate-buffered saline histologically. Inactivation of adenoviral DNA transcription did not affect induction of gangrene. However, gangrene was prevented by concurrent immunosuppression with cyclosporine or delayed administration of the vector. Delayed administration allowed adenoviral gene expression as determined by immunohistochemistry, NOS protein levels, and an assay of NOS enzyme activity. Conclusion: Intra-arterial administration of adenoviral vectors, under vascular isolation, immediately after induction of acute ischemia causes inflammation and subsequent limb gangrene. The inflammatory response is unrelated to the expression of the recombinant transgene or the adenoviral genome and is likely due to the adenoviral capsid proteins. However, administration of cyclosporine or delayed injection of the adenoviral vector is a method that can be used for adenoviral mediated gene transfer in limb ischemia. (J Vasc Surg 2001;34:489-96.)

Humanization and molecular modeling of the anti-CD4 monoclonal antibody, OKT4A.

OKT4A, a murine mAb that recognizes an epitope on the CD4 receptor, is a potent immunosuppressive agent in vitro and in a variety of nonhuman primate models of graft rejection and autoimmune disease. Initial human cardiac transplant trials suggest that OKT4A does not cause either cytokine release syndrome or CD4+ cell depletion, but does induce a human anti-mouse Ab (HAMA) response despite strong concurrent immunosuppression. To further investigate the potential of OKT4A as an immunomodulator, it was necessary to decrease its immunogenicity. Therefore, we developed a humanized version of this Ab (gOKT4A-4), which has the same binding affinity and in vitro immunosuppressive properties of OKT4A, but retains only three murine sequence-derived amino acid residues outside of the complementarity-determining regions (CDRs). Detailed computer modeling of both OKT4A and gOKT4A-4 provided a computational rationale for the changes necessary to regain activity after humanization. This has also provided a plausible representation of the Ag binding site. Preliminary clinical results with gOKT4A-4 suggest that we have eliminated the immunogenicity observed in the parent murine Ab.

The differentiation of delayed hemolytic and delayed serologic transfusion reactions: incidence and predictors of hemolysis.

After differentiation of the entities of clinically detectable delayed hemolytic (DHTR) and delayed serologic transfusion reactions (DSTR), previous investigators calculated a DHTR:DSTR incidence ratio of 18:72 from a retrospective review of patients with serologic evidence of DHTR or DSTR. There are no published data on factors that may influence the occurrence of DHTR versus DSTR in a given patient. Retrospective review was conducted of 292 patients at the Mayo Clinic who, between 1980 and 1992, received a clinical diagnosis of DHTR or DSTR concurrently with a serologic diagnosis. Red cell alloantibody specificity, the activity of the patient's reticuloendothelial system, and concurrent immunosuppression were evaluated as potential predictors of the occurrence of DHTR versus DSTR in different patients. The incidence of DHTR or DSTR was 1 in 1899 allogeneic red cell units transfused, with a DHTR:DSTR ratio of 36:64. Alloantibody specificity was the only variable that affected the occurrence of DHTR versus DSTR at the clinical level, with the anti-Jka and anti-Fya specificities, as well as multiple coexisting specificities, significantly associated with detectable hemolysis (p < 0.05). Clinically detectable DHTRs are found to occur more commonly than previously believed when the clinical and serologic diagnoses are made concurrently and appropriate work-ups for hemolysis are ordered. The association of certain alloantibody specificities with detectable DHTRs may have implications for clinical transfusion practice.

Randomised trial comparing tacrolimus (FK506) and cyclosporin in prevention of liver allograft rejection

Summary Studies in the USA and Japan have shown that tacrolimus (FK506) is a potent immunosuppressant. To compare the efficacy and safety of tacrolimus-based and conventional cyclosporin-based immunosuppressive regimens we recruited 545 liver transplant recipients from eight European centres into a randomised open trial. Analysis of the data at 12 months post-transplant showed that tacrolimus was associated with a significant reduction in acute, refractory acute, and chronic rejection episodes. The rates were, for acute rejection, tacrolimus 40·5% vs 49·8% cyclosporin (p=0·040; absolute difference 9·3% [95% Cl 0·9-17·8%]). For refractory acute and chronic rejections the comparisons were 0·8% vs 5·3% (p=0·005) and 1·5% vs 5·3% (p=0·032). These results were seen despite significantly lower corticosteroid usage. The incidence of infection was also lower in patients receiving tacrolimus. Patient and graft survival rates were not significantly different (tacrolimus 82·9% and 77·5%; cyclosporin 77 5% and 72·6°6). Safety data were comparable—the most serious events being renal impairment, disturbances of glucose metabolism, and neurological complications—but these events were more common in the tacrolimus group. In this trial tacrolimus had advantages over cyclosporin in respect of lower rejection rates, even though less concurrent immunosuppression was administered.

Direct detection of circulating hepatitis C virus RNA using probes from the 5' untranslated region.

Diagnostic testing for hepatitis C virus (HCV) infection currently is based on the presence of anti-HCV antibodies or a positive HCV RNA polymerase chain reaction (PCR) test. Although HCV RNA PCR is a sensitive and specific technique, widespread application is limited. Moreover, HCV RNA PCR is subject to false-positive reactions through contamination and is inherently difficult to standardize and quantitate. To overcome limitations of HCV RNA PCR, we produced both cDNA and riboprobes from a 241 nucleotide sequence of the 5' untranslated region of the HCV genome for slot hybridization. Hybridization was absent using normal human serum, horse serum, or hepatic cellular RNA from noninfected liver. Hybridization occurred predominantly with positive-stranded HCV RNA and was abolished by pretreatment with RNase A. Slot hybridization was performed on serum samples from 60 patients with chronic HCV infection and a positive HCV RNA PCR and 20 patients with liver diseases unrelated to HCV who had a negative HCV RNA PCR. Slot hybridization with cDNA and riboprobes showed concordance with HCV RNA PCR of 95 and 98.3%, respectively. There were no false-positive reactions in controls. The sensitivity of riboprobe hybridization was comparable to that of one stage HCV RNA PCR using 5' untranslated region primers. Riboprobe hybridization with the HCV H strain standard was positive in the dilution corresponding to 10(-6) chimpanzee infectious doses50/ml. The density of the hybridization signals correlated significantly with the mass of an RNA standard extracted from the liver of a patient with HCV infection. The relative quantities of HCV RNA in the sera of selected patients varied and were not correlated with the duration of disease or the histopathological stage. The highest relative quantities were associated with concurrent immunosuppression. We conclude that slot hybridization is a sensitive, specific alternative to HCV RNA PCR that can be directly quantitated using appropriate HCV RNA standards.

Fournier’s Gangrene in Hypersensitivity Vasculitis

Fournier’s gangrene in IgE positive hypersensitivity vasculitis demands concurrent high dose immunosuppression, radical surgery and intensive treatment of septic sequelae. Even extended autologous skin grafting is possible with immunosuppression.A case of fulminant necrotizing infection of the genitalia, perineum and abdominal wall during the acute generalization phase of IgE positive hypersensitivity vasculitis is described. Apart from the radical surgical approach, medical treatment demands a different strategy than in classical Fournier’s gangrene. The favorable outcome in a 21-year-old man after several months of intensive care could be realized only by close cooperation of internal specialists, urologists and plastic surgeons. J. Urol., 142: 823–825, 1989)