Yoshihiko Watarai

Early increased chemokine expression and production in murine allogeneic skin grafts is mediated by natural killer cells.

BACKGROUND Increased expression of chemokine mRNA is observed in allogeneic but not syngeneic skin grafts 3-4 days after transplantation. The recipient cells mediating this early inflammatory response in allografts remain unidentified. METHODS Isogeneic and allogeneic skin grafts were transplanted to euthymic and athymic nude mice. mRNA expression and protein production of macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and the murine homolog of Gro(alpha), i.e. KC, from graft homogenates retrieved 3-4 days posttransplantation was tested by Northern blot hybridization and ELISA. To deplete NK cells, recipients were treated with antiasialo GM1 (ASGM1) antisera or with anti-NK1.1 mAb before transplantation. RESULTS Expression of KC, MIP-1alpha, and MIP-1beta mRNA was equivalent in C57BL/6 allogeneic skin grafts and BALB/c isografts at day 2 posttransplant. At day 3 posttransplant, chemokine mRNA levels decreased in isografts but were maintained at high levels in the allografts. Increased early chemokine mRNA was also observed in C57BL/6, but not BALB/c++ grafts on BALB/c athymi(nu/nu) recipients. Treatment of allograft recipients with ASGM1 or with anti-NK1.1 antibody eliminated NK cells from the spleen and allograft infiltrating cell populations and decreased early chemokine mRNA levels in allografts 60-70%. Analyses of allograft homogenates indicated increased levels of KC, MIP-1alpha, and MIP-1beta protein at day 4 posttransplant that were decreased in recipients depleted of NK cells. Early chemokine mRNA levels were equivalent in isogeneic and semiallogeneic F1 grafts. CONCLUSIONS Early chemokine mRNA expression and protein production in allogeneic skin grafts is amplified by recipient natural killer (NK) cells. These results indicate a novel function for infiltrating NK cells in mediating early increased intra-allograft chemokine production and inflammation during the initiation of acute rejection.

Laparoscopic ureteroureterostomy for retrocaval ureter

Abstract Two cases of retrocaval ureter are reported that were successfully treated by a laparoscopic approach. Case 1 was a 20‐year‐old woman who presented with symptoms of a right ureter stone. Case 2 was a 23‐year‐old woman who had suffered from recurrent right flank pain with gross hematuria. A transperitoneal approach was used for case 1, and a retroperitoneal approach was used in case 2. Both were successfully treated with laparoscopic ureteroureterostomy using an intracorporeal suture technique. Laparoscopic surgery should be the first choice for retrocaval ureter not only because of the minimal invasiveness but also because of the cosmetic advantage compared to conventional open surgery. Further technical and instrumental advances are essential for intracorporeal suturing.

Intraallograft Chemokine RNA and Protein During Rejection of MHC-Matched/Multiple Minor Histocompatibility-Disparate Skin Grafts

Chemokines direct leukocyte recruitment into sites of tissue inflammation and may facilitate recruitment of leukocytes into allografts following transplantation. Although the expression of chemokines during rejection of MHC-disparate allografts has been examined, chemokine expression in MHC-matched/multiple minor histocompatibility Ag-disparate allografts has not been tested. The intraallograft RNA expression of several C-X-C and C-C chemokines was tested during rejection of full thickness skin grafts from B10.D2 donors on control Ig-, anti-CD4 mAb-, and anti-CD8 mAb-treated BALB/c recipients. In all recipients, two patterns of intragraft chemokine expression were observed during rejection of these grafts: 1) macrophage-inflammatory protein-1α, macrophage-inflammatory protein-1β, GRO-α (KC), JE, and IFN-γ-inducible protein (IP-10) were expressed at equivalent levels in allo- and isografts for 2–4 days posttransplant and then returned to low or undetectable levels; and 2) IP-10 and monokine induced by IFN-γ (Mig) were expressed in the allografts 3 days before rejection was completed, suggesting a possible role in recruiting primed T cells into the allograft. Three days before completion of rejection, intraallograft IP-10 protein was restricted to the epidermis, whereas Mig was located in the lower dermis and associated with the intense infiltration of mononuclear cells. Treatment of B10.D2 recipients with rabbit antiserum to Mig, but not to IP-10, delayed rejection of the allografts 3–4 days. The results suggest that Mig mediates optimal recruitment of T cells into MHC-matched/multiple minor histocompatibility Ag-disparate allografts during rejection.

Clinical significance of regulatory T-cell-related gene expression in peripheral blood after renal transplantation.

Background. Regulatory T cells (Tregs) have been suggested to be deeply associated with immune tolerance and long-term graft survival in transplantation. Some recipients with stable graft function (ST) could possibly minimize immunosuppression during the maintenance period. However, effective assays for assessing the suitability of patients have yet to be established. The purpose of this study was to elucidate the clinical relevance of Treg-related gene expression such as forkhead box P3 (Foxp3) in peripheral blood after renal transplantation. Methods. Several key molecules related to the function of immune cells such as Treg, including Foxp3, transforming growth factor-&bgr;, cytotoxic T-lymphocyte antigen-4, chemokine receptor 7, toll-like receptor 4, granzyme B, T-bet, GATA3, RORC, &agr;1,2-mannosidase, and proteasome subunit &bgr; 10 were examined in the peripheral blood of 272 renal transplant recipients by quantitative real-time reverse-transcriptase polymerase chain reaction. The expression levels were compared between recipients with chronic rejection and ST. Results. Foxp3 messenger RNA (mRNA) levels were reduced immediately after transplantation and gradually recovered. Pretransplantation levels were closely correlated with 1 year posttransplantation levels. Recipients with chronic rejection had significantly lower levels of Foxp3, chemokine receptor 7, and granzyme B mRNA, and higher levels of toll-like receptor 4 and proteasome subunit &bgr; 10 mRNA compared with those with ST, although Foxp3 was the most relevant marker. Conclusion. There is a possibility that monitoring mRNA expression levels of Treg-related molecules in peripheral blood might offer useful information on patient selection and early detection of rejection when immunosuppression minimization strategy is implemented in renal transplantation.

Comparative study on signal transduction in endothelial cells after anti-a/b and human leukocyte antigen antibody reaction: implication of accommodation.

Background. Recent development of immunosuppressive therapy has provided a platform for clinical human leukocyte antigen (HLA)- and ABO-incompatible kidney transplantation. However, the prognosis seems to be different between the two. Accommodation, the condition of no injury even in the presence of antidonor antibody, is one of the key factors for successful transplantation with antidonor antibody. The purpose of this study was to compare signal transduction between anti-A/B and anti-HLA antibody reaction and to elucidate the mechanisms underlying accommodation. Methods. Blood type A- or B-transferase gene was transfected into human EA.hy926 endothelial cells. After cell sorting, A- or B-expressing cells at high levels were obtained. The effects of anti-HLA and anti-A/B antibody binding on complement-mediated cytotoxicity and signal transduction were examined. Results. Preincubation with anti-HLA antibodies only at low levels (<10% of saturation level) or anti-A/B antibodies at high levels (even at near saturation levels) for 24 hr resulted in resistance to complement-mediated cytotoxicity. Anti-A/B antibody ligation inactivated ERK1/2 pathway and increased complement regulatory proteins such as CD55 and CD59, whereas anti-HLA ligation activated PI3K/AKT pathway and increased cytoprotective genes such as hemeoxygenase-1 and ferritin H. Conclusion. Complement inhibition by upregulation of CD55 and CD59 through ERK1/2 inactivation might play a substantial role in accommodation after ABO-incompatible transplantation, which could also explain the intriguing finding of C4d deposition in the graft without rejection.

Neither pre-transplant rituximab nor splenectomy affects de novo HLA antibody production after renal transplantation

The long-term effect of rituximab and splenectomy on de novo HLA antibody production and chronic antibody-mediated rejection after renal transplantation is uncertain. In order to gain insight on this, we studied 92 ABO-incompatible and 228 ABO-identical/compatible consecutive renal transplant patients and determined their de novo HLA antibody production and graft outcome. Patients with pretransplant donor-specific antibodies had been excluded. ABO-incompatible transplants included 30 recipients treated with rituximab, 51 by splenectomy, or 11 with neither, due to low anti-A or -B antibody titer. Graft survival in ABO-identical/compatible patients (97.7% at 5 years) was significantly higher than in ABO-incompatible (87.0% at 5 years), rituximab (96.7% at 3 years), or splenectomy (85.7% at 5 years) patients. Only four patients had clinical chronic antibody-mediated rejection (two each identical/compatible and incompatible). There was no significant difference in prevalence of de novo HLA antibody, including donor-specific and nondonor-specific antibodies among ABO-identical/compatible patients (13.9%), patients receiving rituximab (14.3%) or splenectomy (13.2%), or among those receiving cyclosporine, tacrolimus, mycophenolate mofetil, mizoribine, and everolimus. Renal function remained stable in most recipients with de novo HLA antibody. Thus, neither pretransplant splenectomy nor rituximab treatment has an inhibitory effect on de novo HLA antibody production during medium-term follow-up. Further study on long-term effects is needed.

Measurement of Prostate Specific Antigen and γ-Seminoprotein Ratio: A New Means of Distinguishing Benign Prostatic Hyperplasia and Prostate Cancer

We measured a ratio of serum prostate specific antigen (PSA) and gamma-seminoprotein concentrations (referred to as the PSA/gamma-seminoprotein ratio) and evaluated its usefulness for the diagnosis of prostate cancer. Between April 1988 and October 1992, 214 men underwent prostatic biopsy and/or transurethral resection of the prostate, and the disease was diagnosed pathologically. Of 214 patients 127 were diagnosed as having benign prostatic hyperplasia, prostatitis or a normal prostate (no cancer), while 87 had prostate cancer. Of 61 patients with a serum PSA level greater than 10 ng./ml. 50 (82.0%) had prostate cancer, compared to 31 of 84 (36.9%) with a serum PSA level of 3.0 to 10 ng./ml. Of 113 patients with a serum gamma-seminoprotein level greater than 4.0 ng./ml. 52 (46.0%) had prostate cancer. The mean plus or minus standard deviation of the PSA/gamma-seminoprotein ratio for 127 patients without cancer was 0.942 +/- 0.564, while that for 87 prostate cancer patients was 12.840 +/- 45.327 (Wilcoxon p < 0.0001). The mean plus or minus standard deviation of the PSA/gamma-seminoprotein ratios for 37 prostate cancer patients with a PSA level of 10 ng./ml. or less and for 50 prostate cancer patients with a PSA level of more than 10 ng./ml. were 2.044 +/- 0.767 and 20.829 +/- 58.757, respectively. Even the mean PSA/gamma-seminoprotein ratio for prostate cancer patients with a PSA level of 10 ng./ml. or less was significantly greater than that for patients without cancer (Wilcoxon p < 0.0001). The sensitivities for PSA (cutoff value 3.0 ng./ml.), gamma-seminoprotein (cutoff value 4.0 ng./ml.) and PSA/gamma-seminoprotein ratio (cutoff value 1.45) were 93.1%, 59.8% and 92.0%, respectively, and the specificities were 49.6%, 52.0% and 91.3%, respectively. Of 91 patients with a PSA/gamma-seminoprotein ratio of 1.45 or more 80 (87.9%) had prostate cancer, while 116 of 123 (94.3%) with a PSA/gamma-seminoprotein ratio of less than 1.45, had no cancer. These results suggest that PSA/gamma-seminoprotein ratio yields the same sensitivity as PSA and more specificity than PSA levels, offering significant advantage over PSA in detecting prostate cancer. The mean plus or minus standard deviations of PSA/gamma-seminoprotein ratios for stages A, B, C and D prostate cancer were 1.847 +/- 0.786 (11 patients), 2.740 +/- 1.536 (30), 7.626 +/- 9.140 (12) and 27.149 +/- 70.500 (34), respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

De Novo Anti-HLA DSA Characteristics and Subclinical Antibody-Mediated Kidney Allograft Injury.

Background It is unclear whether all donor-specific antibodies (DSA) can cause chronic antibody-mediated rejection (AMR). Subclinical stage before manifestation of renal dysfunction may be a critical period for reversing AMR. The aim of our study was to identify factors related to the development of subclinical AMR and to clarify the characteristics of de novo DSA. Methods Eight hundred ninety-nine renal transplants were screened for HLA antibody. De novo DSA were detected in 95 patients. Forty-three patients without renal dysfunction who underwent renal biopsies were enrolled in this study. Eighteen patients (41.9%) were diagnosed with biopsy-proven subclinical AMR and treated with plasmapheresis and rituximab-based therapy, whereas 25 showed no findings of AMR. Results Significant subclinical AMR-related factors were younger recipients, history of acute T cell–mediated rejection and DSA class II, especially DR-associated DSA. Mean fluorescence intensity (MFI) values of DR-DSA were significantly higher, whereas DQ-DSA was not different between subclinical AMR and no AMR. The &Dgr;MFI (>50%), DSA-MFI values greater than 3000, and C1q binding DSA were also significant subclinical AMR-related factors (P < 0.05). Among 18 patients treated for subclinical AMR, 8 patients (44.4%) obtained over 50% reduction of DSA-MFI and/or improvement or no deterioration of pathological findings. In contrast, 25 patients without subclinical AMR did not show renal dysfunction clinically. Moreover, all of the 8 patients with rebiopsy after 2 years continued to demonstrate no AMR. Conclusions About 40% of patients with de novo DSA demonstrated biopsy-proven subclinical AMR, leading to progressive graft injury. To validate the intervention and treatment for de novo DSA-positive patients without renal dysfunction, further study is necessary.

Significant association between chronic antibody-mediated rejection and donor-specific antibodies against HLA-DRB rather than DQB in renal transplantation

De novo production of antidonor HLA antibody has been reported to be associated with chronic antibody-mediated rejection (CAMR). However, some donor-specific antibodies (DSA) do not seem to cause graft injury. Identification of the DSA responsible for CAMR and establishment of effective screening method for early detection of CAMR are therefore essential. All sera from 586 maintenance renal transplant recipients were examined for HLA antibody using ELISA and Luminex-based assay. Positive sera were divided into high (>20% of positive control), moderate (10-20%), and low (2-10%). Donor specificities were analyzed using single antigen beads. ELISA detected only about half of high HLA antibodies (class I: n = 19, class II: n = 46) measured by Luminex-based assay. DSA against class I and class II were identified in 42% and 87% of high antibodies, respectively, including 78% against DQB and 44% against DRB. Renal dysfunction due to CAMR was closely related to high/moderate DRB DSA (n = 11), but not low DRB DSA (n = 9) nor high/moderate/low DQB DSA alone (n = 20). It was speculated that DRB DSA would be more detrimental to the graft, while DQB DSA were readily detectable in blood circulation. Further study, including detailed pathologic analysis of graft biopsy and long-term follow-up, is necessary.

Surgery for giant high-flow renal arteriovenous fistula: experience in one institution

To present five cases of renal arteriovenous fistula (RAVF) seen at our institution, with an emphasis on surgical treatment.