Combined analysis of cytokine genotype polymorphism and the level of expression with allograft function in African-American renal transplant patients.

Cytokine gene polymorphism and expression levels were evaluated in a group of African-American patients who had undergone renal transplantation. It was hypothesized that possession of specific cytokine alleles might be influential in predisposing the recipient to allograft rejection. Thus, we sought to establish a relationship between cytokine gene polymorphism, the levels of cytokine expression, and the outcome of allograft function. Cytokine genotypes and mRNA transcript levels of IL-2, TNF-alpha, TGF-beta1, IL-10, IL-6 and IFN-gamma were determined using peripheral blood cells. Genomic DNA samples from 77 transplant recipients and 77 controls were tested by a multiplex PCR with specific primers for the above cytokines. The frequency distributions of cytokines were analyzed in respect to the clinical characterization, including delayed graft function (DGF), rejection episodes (REs) and stable graft function (SGF). The mRNA transcript level was tested both at pre- and early post-transplantation (day 1 and day 4) with primers for coding regions of the above cytokines in a RT-PCR assay. The majority of recipients with successful graft function were matched with their donors for only three out of the six HLA alleles. We have shown that the TGF-beta1 T/C G/G high producer and IFN-gamma T/A intermediate producer genotypes were associated with allograft rejection, whereas low IFN-gamma producer and high IL-10 producer genotypes were significantly protective of the allograft. There was some correlation between the TGF-beta1 high producer genotype and DGF, but it was not statistically significant. Overall, 77% of those who experienced REs carried the TGF-beta1 T/C G/G, high producer genotype as compared with 52% who experienced DGF, 39% with SGF (P<0.01, RR=2.0), and 27.3% of controls (P<0.003, RR=2.6). The IFN-gamma T/A intermediate producer genotype was found in 69.2% of patients with REs as compared with 26.8% of patients with SGF (P<0.008, RR=2.85). The IL-10, ATA/ATA low producer genotype was found in 38.5% of recipients with REs and 14.6% of recipients without REs (P<0.04, RR=0.53). Expression levels of mRNA transcript were correlated with genotype data, except for the TGF-beta1 high producer genotype where there was no significant difference between the level of mRNA transcript at pre- and post-transplantation. Low DRbeta1 and high DPbeta1 expression by recipient peripheral blood mononuclear cells before transplantation was associated with more SGF, whereas high DRbeta1 and low DPbeta1 expression at pretransplantation was associated with more REs (DRbeta1, P<0.001 and DPbeta1, P<0.05, respectively). We concluded that, dual analysis of cytokine genotype and expression levels by peripheral cells may be an important clue to understanding the contribution of the recipient's immune response to an allograft pre- and post-transplantation. Identification of peripheral markers diagnostic of rejection could allow advance anticipation of clinical outcome, and might reduce the need for tissue biopsy.

Effect of poverty and other socioeconomic variables on renal allograft survival.

BACKGROUND: Socioeconomic variables including low income and noncompliance impact negatively upon long-term renal allograft survival, especially in African Americans. We sought to determine whether other socioeconomic variables contributed to noncompliance and allograft survival. METHODS: A detailed history of socioeconomic variables was made at the time of renal transplant evaluation in 450 consecutive candidates, 128 of whom (89 African American, 39 Caucasian) have thus far undergone transplantation. Variables evaluated included household income, zip code income, insurance coverage, years of education, literacy, marital status, pretransplantation compliance, and history of substance abuse as well as the usual pre- and posttransplantation demographics. RESULTS: Immunologic graft loss occurred primarily in young African Americans with income below the federal poverty level, whereas nonimmunologic graft loss was distributed across racial, income, and other socioeconomic variables. Immunologic graft loss was also associated with a greater number of HLA mismatches, lower levels of education, and noncompliance with transplant medications and follow-up visits. Recipients with gross illiteracy, however, had excellent graft survival. Pretransplantation substance abuse, but not pretransplantation compliance, was predictive of posttransplantation noncompliance. By multivariate analysis, posttransplantation compliance emerged as the single most important factor predictive of graft survival. CONCLUSIONS: Immunologic graft loss in our population is related to noncompliance with transplant medications, which occurred primarily in recipients with a pretransplantation history of substance abuse and is not related to an inability to pay for medications at the time of graft loss. A change in criteria for acceptance of transplant candidates with a prior history of substance abuse might significantly improve graft survival in this patient population.

Anal carcinoma arising from condyloma acuminata.

Condyloma acuminata is a common anorectal condition that frequently requires surgical evaluation and treatment. We have noted an increased incidence of anal carcinoma in patients with condyloma acuminata. The purpose of this study is to review the incidence of malignant transformation of condyloma in our recent experience. We conducted a 5-year retrospective review of patients with condyloma acuminata treated at a university medical center that serves as a major referral center for the state. From May 1994 through May 1999 257 patients were treated for anal condyloma. During the same time period 74 patients were diagnosed with squamous cell carcinoma of the anus; nine of these patients also had condyloma acuminata (12.2% of patients with anal carcinoma). All nine were immunosuppressed by illness and/or medication. The extent of carcinoma at diagnosis ranged from stage 0 (carcinoma in situ) to stage IVb. Overall 3.5 per cent of patients with condyloma acuminata also had squamous cell carcinoma of the anus. One patient with stage IVb disease died shortly after initial evaluation. Two patients with advanced disease required extensive surgical intervention and had complex postoperative courses. Malignant transformation of condyloma acuminata may be increasing in incidence. This disease progression can be insidious and may be fatal. Screening of high-risk patients might be of value, and more aggressive early management of condyloma may prevent the development of malignancy.

Expression of the placental cytokines tumor necrosis factor alpha, interleukin 1beta, and interleukin 10 is increased in preeclampsia.

OBJECTIVE: We sought to determine whether placental cytokine expression is altered in patients with preeclampsia. STUDY DESIGN: Whole placental tissue was collected at cesarean delivery, and total ribonucleic acid was extracted. Reverse transcriptase-polymerase chain reaction was performed to determine cytokine expression. Product bands were quantitated by scanning densitometry, and results were expressed as a ratio of cytokine/housekeeping gene (cytokine expression index). Statistical analysis was performed by the Student t test and the Mann-Whitney U test. RESULTS: Placentas from 6 patients with preeclampsia and 4 normotensive patients were analyzed. Placental expression of interleukin 1beta and interleukin 10 was greater in preeclamptic women than in normotensive subjects (median interleukin 1beta cytokine expression index, 0.675; range, 0.394-0. 953; vs 0.106; range, 0.084-0.166; P =.011; median interleukin 10 cytokine expression index, 1.042; range, 0.672-1.192; vs 0.126; range, 0.062-0.398; P <.011). Tumor necrosis factor alpha messenger ribonucleic acid was detected in placentas of preeclamptic subjects but not in normotensive control subjects. CONCLUSION: Placentas from preeclamptic patients demonstrated increased expression of interleukin 1beta, interleukin 10, and tumor necrosis factor alpha. This may be in association with placental hypoxia and may contribute to the global endothelial dysfunction observed in preeclampsia.

First-trimester human chorionic villi express both immunoregulatory and inflammatory cytokines: a role for interleukin-10 in regulating the cytokine network of pregnancy.

PROBLEM: T-helper 2 (TH2)-type cytokines [i.e., interleukin (IL)-6, IL-10, and IL-13] and transforming growth factor (TGF)-beta are expressed by the murine decidua and/or placenta and are likely to suppress inflammatory cytokine [i.e., IL-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, IL-1 alpha, and IL-1 beta] production at the maternal-fetal interface. In addition, class I IFNs may protect the fetus from immunologic rejection and viral infections. This study examines the expression of inflammatory/immunoregulatory cytokines and IL-10 production by first-trimester chorionic villi. METHOD OF STUDY: Gestational tissues (n = 5) were obtained following elective terminations performed between 7 and 9 weeks of gestation. Chorionic villous tissues were separated from fetal membranes and decidua, and total RNA was extracted. Cytokine expression was assessed by a reverse transcriptase-polymerase chain reaction technique. Chorionic villi (n = 9; 6-12 weeks gestation) were maintained in organ culture, and human chorionic gonadotropin (hCG) and IL-10 levels were determined by immunoradiometric and enzyme-linked immunosorbent assays, respectively. RESULTS: IFN-gamma and IL-2 were generally not expressed by first-trimester chorionic villi. Low to moderate levels of expression were noted for IL-1 alpha, IL-1 beta, and TNF-alpha. High levels of mRNA were noted for IFN-alpha and IFN-beta, but IFN-tau was not expressed. In all tissues, TGF-beta 1 and IL-13 were either weakly expressed or not expressed. In contrast, moderate to high levels of IL-6 and IL-10 mRNA were detected in each chorionic villous sample. In chorionic villous explants obtained at 6-11 weeks gestation production of hCG and IL-10 was greatest during the first 24 hr ([hCG] = 6961 +/- 815 mIU/mL, [IL-10] = 92 +/- 11 pg/mL) and then declined through 72 hr. CONCLUSIONS: TH1-type cytokines (IL-2, IFN-gamma) are not expressed by first-trimester chorionic villous tissues: This is possibly due to local production of IL-10. In contrast, macrophage-associated cytokines (IL-1 beta and TNF-alpha) are expressed and their regulation may be critical for fetal survival. Finally, class 1 IFNs expressed by early chorionic tissues may protect the fetus from maternal rejection and viral transmission.

Cytokine expression by first-trimester human chorionic villi.

PROBLEM: Communication at the human maternal-fetal interface occurs by an intricate cytokine network. This study examines cytokine expression by normal first-trimester human chorionic villi. METHOD OF STUDY: Tissues were obtained at elective pregnancy terminations (7-9 weeks). Total RNA was isolated from chorionic villi by guanidinium isothiocynate-acid phenol extraction. A reverse transcriptase-polymerase chain reaction technique was used to examine cytokine expression. beta-Actin was used as the housekeeping gene, and mitogen-stimulated lymphocytes served as positive controls. RESULTS: beta-Actin was uniformly expressed by all chorionic villous samples. Interferon (IFN)-alpha and -beta also were highly expressed. Moderate expression was noted for interleukin (IL)-10, IL-6, tumor necrosis factor (TNF)-alpha, and IL-1 beta. In contrast, transforming growth factor-beta 1, IFN-gamma, IL-2, and IL-1 alpha were either weakly expressed or absent in first-trimester villi. CONCLUSIONS: Cytokines may contribute to pregnancy immunotolerance (IFN-alpha, IFN-beta, and IL-10), viral resistance (IFNs), hormone secretion (IL-1 and IL-6), and cellular remodeling (IFN-gamma and TNF-alpha) within the chorionic villous.

Epitope-specific signaling through CD45 on T lymphocytes leads to cAMP synthesis in monocytes after ICAM-1-dependent cellular interaction.

We recently demonstrated that different CD45 monoclonal antibodies (mAb) are able to induce cellular aggregation in human peripheral blood mononuclear cells (PBMC) through LFA-1/ICAM-1 interactions. Such interactions could be down-modulated by protein kinase (PK) A/G inhibitors, but were unaffected by inhibitors of PKC, suggesting the involvement of PKA or PKG in CD45 mAb-induced adhesion. In this study we show that after incubation of PBMC with several (but not all) mAb to CD45, CD45RO and CD45RA, intracellular cAMP, but not cGMP concentrations readily increase, reaching a maximum 30 min after start of activation. As evidenced by several lines of investigation cAMP accumulation was independent of Fc receptor-associated signaling as well as tyrosine phosphatase activity of CD45. In highly pure T lymphocytes, CD45 mAb were unable to induce cAMP synthesis, but readily did so after addition of autologous monocytes. After paraformaldehyde fixation of both quiescent or IFN-gamma/TNF-alpha-preactivated monocytes, cAMP production was no longer detectable, suggesting monocytes as the cell of origin for the increased cAMP synthesis. Further, cAMP accumulation in monocytes occurred after reconstitution to T lymphocytes preincubated with CD45 mAb and extensively washed. Importantly, pretreatment of T lymphocyte/monocyte mixtures with LFA-1 mAb and/or ICAM-1 mAb down-regulated CD45 mAb-induced cAMP synthesis. Finally, we demonstrate that CD45 mAb are not only capable of inducing cAMP production, but also of directly stimulating PKA enzyme activity. Based on the data presented, we propose that CD45 signaling in T lymphocytes subsequently activates cAMP accumulation and PKA activation in monocytes via LFA-1/ICAM-1-dependent cellular interactions.

Correcting prolonged bleeding during renal transplantation with estrogen or plasma.

OBJECTIVE: To determine the efficacy and relative effectiveness of conjugated entrogens (CE) and fresh-frozen plasma (FFP) in normalizing prolonged preoperative bleeding times during renal transplantation. DESIGN: Prospective, randomized trial. SETTING: A university regional referral center for transplantation. PATIENTS: Patients scheduled for renal transplantation with preoperative bleeding times greater than 10 minutes (normal, < 7 minutes) following informed consent were asked to participate in the randomized protocol. Those with bleeding times of 8 to 9.5 minutes were asked, following informed consent, to be a control group receiving neither CE nor FFP. INTERVENTIONS: Following induction of anesthesia and drawing of baseline laboratory tests, patients were administered randomly, using a table of random numbers, either 50 mg of CE or 2 U of FFP. MAIN OUTCOME MEASURES: Bleeding time measurements and other laboratory tests were repeated at the end of surgery as well as at 24 and 48 hours postoperatively. RESULTS: Treatment with CE and FFP decreased the patients' bleeding times from 16.68 +/- 0.8 (SEM) and 17.13 +/- 0.85 minutes to 7.67 +/- 0.79 (P < .001) and 10.50 +/- 1.27 minutes (P < .001), respectively, by the end of surgery. At 24 and 48 hours postoperatively, the CE group had bleeding times of 9.77 +/- 0.99 and 9.81 +/- 1.24 minutes (P < .001 for both), respectively, whereas the FFP group bleeding times were 12.76 +/- 1.57 (P = .003) and 12.14 +/- 1.56 minutes (P = .001), respectively. There were no statistical differences for the control group compared with baseline either at the end of surgery or at 24 hours. CONCLUSIONS: Although both CE and FFP significantly decreased prolonged preoperative bleeding times during renal transplantation, CE might be preferred because of lower risk and cost, as well as a longer duration of action.

Tumor necrosis factor alpha gene expression in human peritoneal macrophages is suppressed by extra-abdominal trauma.

BACKGROUND: Trauma is believed to activate immunocytes but paradoxically also increases the risk of intraperitoneal infection. OBJECTIVE: To investigate these events by evaluating changes in the cytokine control networks of human peritoneal macrophages (PM phi) early after trauma. DESIGN: Case-control study comparing cytokine messenger RNA (mRNA) expression by PM phi from patients with extra-abdominal trauma with that of both peripheral blood mononuclear cells (PBM) and PM phi obtained from healthy individuals. SETTING: Level I trauma center and basic science laboratory in a university hospital center. PATIENTS: Six patients with polytrauma (Injury Severity Score, > or = 15) with clinically negative diagnostic peritoneal lavages performed on routine indications at admission to the emergency department and six healthy age- and sex-matched individuals undergoing inguinal herniorrhaphy under local anesthesia. INTERVENTIONS: Peritoneal macrophages were isolated from diagnostic peritoneal lavages in trauma patients. Identical lavages were performed through the hernia sac in the control group. MEASUREMENTS: Cellular RNA was assayed for tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, IL-6, and IL-10 message by semiquantitative reverse-transcription polymerase chain reaction. RESULTS: Normal PM phi expressed high levels of TNF-alpha mRNA relative to PBM, but expression of the other proinflammatory cytokines was equivalent to that of PBM. Peritoneal macrophage expression of TNF-alpha mRNA was markedly (64-fold) decreased after trauma (P < .001), when PBM expression of IL-10 mRNA was increased (P = .03). CONCLUSIONS: Human PM phi constitutively show high levels of TNF-alpha message expression, and this is down-regulated by polytrauma. This might constitute a functionally "primed" state. If so, TNF-alpha down-regulation might contribute to functional PM phi suppression after systemic injury.

Human peripheral mononuclear cells do not show proinflammatory patterns of cytokine transcription in early trauma: a preliminary report.

Injury has been hypothesized to cause inflammation through systemic release of lipopolysaccharide and pro-inflammatory cytokines, but this has proved difficult to demonstrate in humans. We looked for evidence of an inflammatory pattern of cytokine gene expression by peripheral blood mononuclear cells (PBM) in six polytraumatized patients (ISS = 25 +/- 8) upon ER admission, and in six matched healthy controls. PBM tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-4, IL-6, IL-10, and interferon (IFN)-gamma message was assessed by semi-quantitative reverse-transcription polymerase chain reaction. No increase in expression of any of the pro-inflammatory cytokines (tumor necrosis factor-alpha, IL-1 beta, or IL-6) was found after trauma, and IFN-gamma tended to decrease. Of the immunosuppressive cytokines, IL-10 expression increased 5-fold (p < .05) but no change in IL-4 was discerned. This pattern is fundamentally different from the cytokine expression patterns expected with sepsis or exposure to lipopolysaccharide. These findings are inconsistent with the occurrence of systemic endotoxemia and subsequent global immunocyte activation early after trauma.

Donor specific bone marrow cells suppress lymphocyte reactivity to donor antigens and differentially modulate TH1 and TH2 cytokine gene expression in the responder cell population.

Previous studies have shown that post-transplantation infusion of donor specific bone marrow following a non-specific potent immunosuppressive agent such as antilymphocyte globulin (ALG) can significantly enhance graft survival compared to ALG alone. This enhancement remains variable and is thought to occur through the induction of specific partial tolerance to the renal allograft, but the underlying cellular mechanisms have not been clearly identified. In order to improve the efficacy of this specific immunosuppressive treatment and to study the events leading to enhanced allograft survival, we sought to establish a simple in vitro model based on a mixed lymphocyte reaction (MLR). We show that cellular proliferation seen in a normal MLR can be suppressed by addition of donor specific bone marrow cells (BMC). Significantly, this suppression is not observed with either third party BMC or donor specific peripheral blood mononuclear cells (PBMC). We have defined the optimum conditions of bone marrow infusion regarding number of BMC, their handling and culture, and simple enrichment procedures. Using a semiquantitative polymerase chain reaction assay, we have studied the cytokine gene expression in MLR modulated by donor specific BMC. In an unmodified allogeneic response, the responder cells show increased expression of interleukin-2 (IL-2) gamma-interferon IFN-gamma and receptor (IL-2R) mRNA, and no IL-10 mRNA. When responder cells are cultured with BMC of the stimulator, there is a 256-fold decrease in IL-2 mRNA, and a 64-fold decrease in IFN-gamma and IL-2R mRNA. There is also a 64-fold increase in IL-10 mRNA. This effect is even more marked when the BMC are depleted of CD3+ cells. The kinetics of addition of donor specific BMC to the normal allogeneic MLR culture and specificity of the action of BMC are also elucidated. Our data suggest that the enhancement of graft survival observed with donor BMC may operate through decreased proliferation of reactive T cell clones (due to decreased IL-2/IL-2R) and suppressed monocyte functions (due to decreased IFN-gamma and increased IL-10 gene expression).

Peripheral blood chimerism in renal allograft recipients transfused with donor bone marrow.

Experimental studies have shown that administration of antilymphocyte serum combined with donor bone marrow cells can induce tolerance to allograft tissue. We have initially reported application of these protocols in clinical studies of cadaveric renal allograft recipients who were treated with MALG and donor-specific bone marrow cells. To evaluate the effectiveness of the donor marrow cells in the production of chimerism, a detection method based on 32P-incorporated PCR was established. The 32P PCR was utilized with primers specific for the HLA class II, VNTR (D17S5 and D1S111), and/or Y-chromosome genes to detect the presence of allogeneic chimerism in the recipients. Immediately posttransplant, 26.4% of marrow recipients demonstrated the presence of allogeneic chimerism prior to the marrow transfusion as did 18% in the untransfused controls. In transfused patients, chimerism was detected most frequently during the 1-3-month interval after marrow transfusion (65%), and then diminished to 50-56% at 3-12 months posttransfusion. In the control group the frequency of allogeneic chimerism was gradually decreased and was undetectable in the majority of the patients beyond 3 months posttransplant while marrow-transfused recipients were more likely to have chimeric cells detected consistently beyond 3 months. Rejection episodes were significantly effected by the presence of chimerism in the recipients. Of the transfused patients, 91.3% who demonstrated allogeneic chimerism were rejection-free as compared with 8.7% who experienced at least one rejection episode (P = 0.01). While the presence of allogeneic chimerism in the control group was correlated with rejection-free graft survival, this difference did not reach statistical significance.

IL-2, IL-4, and IFN-gamma gene expression versus secretion in superantigen-activated T cells. Distinct requirement for costimulatory signals through adhesion molecules.

In the complete absence of APCs staphylococcal superantigens induced IL-2, IL-4, IL-5, IFN-gamma, and IL-2R gene transcripts in both highly purified human T cells and FACs sorted CD4+ memory (CD45RA-) T cells. Secretion of IL-2, IL-4, and IFN-gamma, as well as DNA synthesis, on the other hand, required the presence of monocytes. At cytokine gene transcript level, three patterns of expression were noted after superantigen activation of T cells in the presence vs the absence of APC. mRNA levels for IL-2 were markedly up-regulated in the presence of monocytes, IL-4 and IFN-gamma transcripts increased only modestly, and IL-5 and IL-2R mRNA levels were unaffected. Blocking mAbs against LFA-1 and LFA-3 added to staphylococcal enterotoxin B (SEB)-activated cultures of T cells and autologous monocytes, reproducibly decreased both T cell proliferation and genetic expression of IL-2, IL-4, IL-5, and IL-2R, although having little or no effect on IFN-gamma transcripts. Further, under those conditions of blocking, secretion of IL-2 and IL-4 was dramatically decreased, whereas IFN-gamma secretion remained essentially unchanged. In contrast, LFA-1 and LFA-3 mAbs completely abrogated IFN-gamma secretion from PHA-activated T cell-monocyte mixtures, although having no inhibitory effect on T cell proliferation. These results indicate a characteristic and differential involvement of adhesion molecule-mediated signals in superantigen-induced T cell proliferation, differential cytokine gene expression, and cytokine secretion.

Renal retransplantation: the role of race, quadruple immunosuppression, and the flow cytometry cross-match.

To assess the impact of quadruple immunosuppression in black and white recipients of cadaver kidney retransplants, we reviewed data from 178 second or subsequent renal allografts performed at our center between 1985 and 1991. Sixty-six black and 102 white recipients were divided into 3 groups: groups 1 and 2 consisted of patients with a negative complement-dependent cytotoxicity (CDC) T cell cross-match, receiving triple drug therapy (CsA-AZA-prednisone) and quadruple immunosuppressive therapy (quad therapy; Minnesota antilymphoblast globulin-CsA-AZA-prednisone), respectively. Group 3 patients also received quad therapy, but, in addition to a negative CDC cross-match, had a negative T cell flow cytometry cross-match (FCXM). Black and white patients in groups 1 and 2 experienced similar graft survival at 1 year, ranging from 47% to 63% (P = NS). In group 3, 1-year graft survival in whites, but not blacks, improved to 82%, with fewer grafts lost to immunologic causes in the first 90 days after transplant. A parametric analysis of potential risk factors identified a significant effect of better HLA-DR matching (P = 0.0005) on improved graft survival, with previous mismatched antigens (P = 0.04), female donor (P = 0.002), and short duration of previous graft (P = 0.05) as risk factors for graft loss. Race and immunosuppressive protocol did not affect graft survival. In group 3, blacks received fewer well-matched kidneys than whites (P = 0.05), which may have contributed to poorer outcomes for black recipients. Nine of 10 patients undergoing retransplantation with a negative CDC cross-match and a positive T cell FCXM suffered graft loss at a median of 26 days after transplant. Thus, quad therapy did not enhance graft survival for either black or white patients undergoing cadaveric retransplantation. Immunologic considerations, including HLA-DR matching and the FCXM, continue to exert a strong influence on outcomes in these high-risk recipients.

Limiting detection of an amplification signal for HLA-D region and VNTR genes by 32P-PCR.

The limiting detection signal for identification of human genetic markers, such as HLA-D and VNTR genes, was determined using DNA isolated from a series of decreasing numbers of lymphocytes carrying the target marker in the polymerase chain reaction (PCR). The PCR procedure was assembled by incorporating 32P-labeled dCTP in the reaction mixture. Primers specific for detection of MHC Class II genes such as HLA-DR1, -DR2, -DRw52 and -DRw53 were utilized when cells were mismatched by one DR type, and primers for the identification of the region of variable number of tandem repeats (VNTRs) were utilized where cells had the same DR types. The 32P-incorporated amplified DNA was analyzed by polyacrylamide gel electrophoresis followed by exposure to x-ray film. The sensitivity of the test varied for different allelic markers as evaluated by amplification of DNA from each set of a mixture of lymphocytes. The target HLA-DR markers were detectable in a cell ratio of as high as 1:100,000, whereas the VNTR markers were detectable at a 1:1000 cell ratio. The approach described here offers certain advantages: 1) increased sensitivity, 2) quantitative power, 3) reduced assay time, 4) simplified procedure and 5) less expense. This method provides valuable information for studies involving forensic specimens and marrow engraftment after allogenic bone marrow transplantation (BMT) that require discrete representation of one allele relative to another in a heterozygous sample where limited quantities of target DNA are available.

Experience with mycophenolate mofetil (RS61443) in renal transplantation at a single center.

OBJECTIVE: Mycophenolate mofetil (MM) is a new immunosuppressive agent that reversibly inhibits guanine nucleotide synthesis and DNA replication. Its activity is highly selective for T and B lymphocytes. Two open-label multicenter trials of MM in renal transplantation have been performed. This report summarizes the results from one center involved in these two trials. METHODS AND RESULTS: The initial trial of MM was an open-label dose-ranging trial in primary cadaveric renal transplantation. Mycophenolate mofetil was included in the maintenance immunosuppression regimen from the day after transplantation. Of the 21 patients enrolled in this trial, one (5%) was withdrawn for side effects. There was one graft loss due to recurrent renal disease and two patients were withdrawn for difficulty with follow-up. Mean follow-up is 26 months, and patient and graft survival at 2 years are 100 and 95% respectively. The second trial was designed to study the efficacy of mycophenolate in reversing refractory renal allograft rejection. Patients enrolled in the trial had biopsy-proven acute rejection and had previously received at least one course of high-dose corticosteroids and/or OKT3. Of the 26 patients enrolled in this trial, one (4%) was withdrawn for side effects. There were two deaths. Mean follow-up is 20 months, and patient and graft survival at 12 months was 91 and 54%. The incidence of infections in the two groups was 38% and there were no deaths in either group attributable to infection. CONCLUSIONS: The results of these two studies indicate that mycophenolate mofetil could be administered safely to renal allograft recipients for periods up to 2 years. It appears to be effective in reversing acute rejection in a high percentage of patients refractory to other forms of therapy.

A standardized approach to PCR-based semiquantitation of multiple cytokine gene transcripts from small cell samples.

A simple, rapid, reproducible, and nonradioisotopic method for semiquantitative analysis of cytokine mRNAs based on polymerase chain reaction (PCR) is described. RNA isolated from 2.5 million cells has proven sufficient to perform semiquantitative analysis of mRNA for 10 different cytokines. By this approach accurate assessment of mRNA levels for multiple cytokines can be made from as little as 2 ml of blood or about 3 mg of biopsy material. Total cellular RNA is quantitatively recovered by guanidinium isothiocyanate-acid-phenol extraction of a constant number of cells. Further quantitation of RNA is unnecessary. Highly reproducible PCR product formation occurs after specific amplification of aliquots of reverse transcribed test RNA. The photographic image of the ethidium bromide-stained gel accurately reflects the amount of PCR product loaded, both densitometrically and visually. PCR product generation is not affected by the presence of carrier RNA. Thus quantitative recovery of total RNA is possible even from a very small number of cells. Similarly, presence of a large excess of nonspecific RNA from nonexpressing cells does not affect amplification of the specific mRNA under study. A linear relationship between mRNA frequency and PCR product formation is observed over a 256- to 512-fold range. The actual mRNA concentration for each cytokine varies depending on the relative abundance of mRNA for that cytokine relative to total RNA. By performing two amplification cycles (28 and 35) on undiluted and 10-fold diluted RNA samples, the range of detection linearity is extended over a 5000-fold difference in input RNA levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Tapering or discontinuing cyclosporine for financial reasons--a single-center experience.

In patients with primary cadaveric renal transplants and stable allograft function, we assessed the impact of tapering or discontinuing cyclosporine A (CsA) for financial reasons. Forty-two patients whose CsA was discontinued ("no-dose") and 29 patients whose CsA was tapered to 100 to 150 mg/d ("low-dose"; mean, 1.7 mg/kg/d) were examined. Results were compared with 70 age- and race-matched control patients maintained on at least 200 mg/d of CsA (mean, 3.9 mg/kg/d). Follow-up time for all patients averaged 55 +/- 18 months. Late acute rejection episodes occurred more frequently in no-dose than in low-dose (P = 0.017) or control (P = 0.001) patients. In the no-dose group, blacks experienced a greater number of late acute rejections than whites. These late acute rejections often coincided with the discontinuation of CsA and contributed to an increased rate of allograft loss in blacks in the no-dose group compared with black and white controls (P = 0.011). In contrast, no increase in late acute rejection episodes occurred in blacks tapered to low doses of CsA. Black patients who remained on low doses of CsA also exhibited a trend toward allograft survival that was intermediate between that of control and no-dose patients. In those patients who retained functional allografts, mean serum creatinine concentration did not differ between the study groups at the beginning and end of the follow-up period. These findings support continuance of CsA in black primary cadaveric renal transplant patients, even if dosages must be reduced to 100 to 150 mg/d.(ABSTRACT TRUNCATED AT 250 WORDS)