The Impact of Cytomegalovirus Infection and Disease on Rejection Episodes in Renal Allograft Recipients

Cytomegalovirus (CMV) infection and disease are potential risk factors for acute allograft rejection in renal transplant recipients. The present study specifically addresses this issue. From October 1994 to July 1997, 477 consecutive renal allograft recipients (397 first transplants and 80 retransplants) were included in the study. CMV infection (cytomegalovirus pp65 antigen in leukocytes) and disease (infection and clinical symptoms or signs of disease) were examined prospectively for 3 months. No CMV prophylaxis was given, and CMV disease was treated with intravenous (i.v.) ganciclovir. The retransplantation of four patients transplanted twice during the study and 22 patients receiving kidneys from human leucocyte antigen (HLA)-identical siblings were excluded from statistical analysis. Rejections were evaluated clinically [277(61%)] and 173 (38%) also had a biopsy verified rejection. CMV infection occurred in 64% of the patients and 24% experienced CMV disease. In a multiple time-dependent Cox analysis, CMV infection and CMV disease were independent significant predictors for clinical acute rejections, RR = 1.6 (1.1-2.5, p = 0.02) and RR = 2.5 (1.2-5.1, p = 0.01), respectively. Among 173 patients with biopsy verified rejection, 72% of the patients had tubulointerstitial rejection whereas 28% had a vascular rejection. CMV disease, but not CMV infection was a predictor of tubulointerstitial rejection, RR = 3.1 (1.1-9.3, p = 0.04). CMV infection and disease are independent risk factors for clinical acute rejection in kidney allograft recipients. CMV disease is an independent risk factor for biopsy verified acute tubulointerstitial rejection in kidney allograft recipients.     

T cell receptor beta chain (TCR-Vβ) repertoire of circulating CD4+ CD25−, CD4+ CD25low and CD4+ CD25high T cells in patients with long-term renal allograft survival

The mechanisms underlying maintenance of renal allografts in humans under minimal or conventional immunosuppression are poorly understood. There is evidence that CD4⁺ CD25⁺ regulatory T cells and clonal deletion, among other mechanisms of tolerance, could play a key role in clinical allograft survival. Twenty‐four TCR‐Vβ families were assessed in CD4⁺ CD25^?, CD4⁺ CD25^low and CD4⁺ CD25^high T cells from patients with long‐term renal allograft survival (LTS), patients exhibiting chronic rejection (ChrRx), patients on dialysis (Dial) and healthy controls (HC) by flow cytometry. LTS patients presented a higher variability in their TCR‐Vβ repertoire, such decreased percentage of Vβ2⁺, Vβ8a⁺ and Vβ13⁺ in CD4⁺ CD25^low and ^high compared with CD4⁺ CD25^? subset and increased Vβ4 and Vβ7 families in CD4⁺ CD25^high T cells exclusively. Additionally, LTS patients, particularly those that were not receiving calcineurin inhibitors (CNI), had increased percentages of CD4⁺ CD25^high T cells when compared with Dial (P < 0.05) and ChrRx (P < 0.05) patients. Our results suggest that a differential expression of particular TCR‐Vβ families and high levels of circulating CD4⁺ CD25^high T cells in long‐term surviving renal transplant patients could contribute to an active and specific state of immunologic suppression. However, the increase in this T cell subset with regulatory phenotype can be affected by CNI.     

Assessment of Tocilizumab (Anti–Interleukin‐6 Receptor Monoclonal) as a Potential Treatment for Chronic Antibody‐Mediated Rejection and Transplant Glomerulopathy in HLA‐Sensitized Renal Allograft Recipients

Extending the functional integrity of renal allografts is the primary goal of transplant medicine. The development of donor‐specific antibodies (DSAs) posttransplantation leads to chronic active antibody‐mediated rejection (cAMR) and transplant glomerulopathy (TG), resulting in the majority of graft losses that occur in the United States. This reduces the quality and length of life for patients and increases cost. There are no approved treatments for cAMR. Evidence suggests the proinflammatory cytokine interleukin 6 (IL‐6) may play an important role in DSA generation and cAMR. We identified 36 renal transplant patients with cAMR plus DSAs and TG who failed standard of care treatment with IVIg plus rituximab with or without plasma exchange. Patients were offered rescue therapy with the anti–IL‐6 receptor monoclonal tocilizumab with monthly infusions and monitored for DSAs and long‐term outcomes. Tocilizumab‐treated patients demonstrated graft survival and patient survival rates of 80% and 91% at 6 years, respectively. Significant reductions in DSAs and stabilization of renal function were seen at 2 years. No significant adverse events or severe adverse events were seen. Tocilizumab provides good long‐term outcomes for patients with cAMR and TG, especially compared with historical published treatments. Inhibition of the IL‐6–IL‐6 receptor pathway may represent a novel approach to stabilize allograft function and extend patient lives.     

CD8+ T‐Cell Depletion and Rapamycin Synergize with Combined Coreceptor/Stimulation Blockade to Induce Robust Limb Allograft Tolerance in Mice

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti‐CD154 mAb, nondepleting anti‐CD4 combined to either depleting or nondepleting anti‐CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long‐term allograft survival in both combinations. Nevertheless, robust donor‐specific tolerance, defined by the acceptance of a fresh donor‐type skin allograft and simultaneous rejection of third‐party grafts, required initial CD8⁺ T‐cell depletion. Mixed donor‐recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL‐2, IFN‐γ and TNF‐α in MLC with donor antigen while significant alloreactivity persisted against third‐ party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor‐recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8⁺ T‐cell depletion and costimulation/coreceptor blockade.     

安胎养血合剂对大鼠移植肾Fas/FasL基因表达及细胞凋亡的影响

目的：研究中药安胎养血合剂对移植肾肾小管上皮细胞凋亡及Fas/FasL基因表达的影响，探讨其免疫抑制作用的机制。方法：建立大鼠肾移植模型，随机分为3组，每组12对。分别为对照组：（Wistar→Wistar大鼠），给予生理盐水灌胃；生理盐水组：（SD→Wistar大鼠），给予生理盐水灌胃；安胎养血合剂组：（SD→Wistar大鼠），给予安胎养血合剂灌胃。于术后第5d处死动物取肾脏组织，通过TUNEL法检测凋亡指数和RT-PCR检测Fas/Fas-L的mRNA表达。结果：用TUNEL检测移植肾脏凋亡指数，生理盐水组凋亡细胞最多，对照组中凋亡细胞极少见，安胎养血合剂组和生理盐水组肾脏细胞凋亡指数比较差异有显著意义（P＜0.05）。生理盐水组肾小管上皮细胞Fas-L的mRNA表达显著增高，安胎养血合剂组Fas-L的mRNA表达显著减少，与生理盐水组之间比较有显著差异（P＜O.05）。结论：安胎养血合剂可以通过下调移植肾脏肾小管上皮细胞Fas-L的mRNA表达，来减少移植肾的细胞凋亡，起到免疫抑制剂的作用。     

New Immunosuppressive Proteins (KX-2, -4, and -5) Induced by Liver Transplantation

Abstract: The presence in rat serum of three novel immunosuppressive proteins is associated with induction of tolerance after liver grafting. One of these, KX-5 (40 kilodal-ton molecular weight) is present 40–100 days after grafting of DA liver into PVG, a combination in which liver grafts are not rejected; two others, KX-2 (87 kilodaltons) and KX-4 (10 kilodaltons), are present in PVG sera in a retransplantation model. N-terminal amino acid sequencing indicated that none of the proteins had been described previously. Induction of KX-5 was inhibited by cyclosporin A. All three proteins inhibited mixed lymphocyte responses nonspecifically. Administration of a single dose of 300 ^g purified KX-5 to PVG rats receiving heterotopic heart allografts led to prolonged graft acceptance. The results raise the possibility of clinical application of the immunosuppressive properties of liver grafts through administration of soluble proteins.     

Organ transplant specificity of tolerance to skin grafts with heart or kidney grafts plus nondepleting anti-CD4 monoclonal antibody (RIB 5/2) and intravenous donor alloantigen administration

BACKGROUND: CD4+ T cells play an essential role in allograft rejection. Monoclonal anti-rat CD4 antibody, RIB 5/2, has been shown to modulate the CD4 glycoprotein without eliminating recipient T cells. A single dose of monoclonal anti-rat CD4 antibody RIB 5/2 plus donor splenocytes results in donor-specific unresponsiveness to heart and kidney allografts, but not skin allografts. This study examined whether tolerance to the more resistant skin graft could also be achieved with RIB 5/2. METHODS: Buffalo (RT1(b)) recipients were given a single dose (20 mg/kg) of monoclonal antibody RIB 5/2 IP plus IV Lewis (RT1(l)) splenocytes (25 x 10(6)) 21 days before Lewis heart, kidney, or skin grafts. In addition, Lewis skin was grafted either simultaneously with or after long- term Lewis heart or kidney allograft acceptance (>50 days). RESULTS: While IV alloantigen plus RIB 5/2 results in long-term acceptance of both heart and kidney, skin allografts are rejected when transplanted alone. Simultaneous transplantation with a Lewis kidney, but not with a Lewis heart, resulted in long-term Lewis skin graft acceptance. However, recipients tolerant to Lewis kidney or heart alone will not accept subsequent Lewis skin grafts, while recipients of simultaneous Lewis skin and kidney grafts subsequently accept a second Lewis, but not third-party Brown Norway (RT1(n)), skin graft. CONCLUSION: RIB 5/2 plus Lewis donor splenocytes tolerize for donor-specific heart and kidney but not skin grafts. However, Lewis skin grafted simultaneously with a Lewis kidney, but not Lewis heart, is accepted and protects a subsequent donor-specific Lewis skin graft.     

Study of the Association of -667 Aquaporin-2 (AQP-2) A/G Promoter Polymorphism with the Incidence and Clinical Course of Chronic Kidney Disease in Korea

Background. Impaired urinary concentration is uniformly present with advanced disease in chronic renal failure. Aquaporin-2 (AQP-2) is known to be expressed in the renal collecting duct cells and participates in urinary concentration in response to vasopressin. Recently, the study of AQP expression in various forms of chronic kidney disease (CKD) demonstrated a reduction in AQP-2 expression associated with a loss of nephrons and the presence of chronic interstitial fibrosis. No information on aquaporin genetic variations in CKD is available to date. The aim of our study was to evaluate the possible impact of aquaporin-2 genotype on the development and clinical course of CKD. Methods. Blood samples from 259 patients with CKD and 106 ethnicity-, age-, and sex-matched healthy controls were collected, and genomic DNA was extracted. AQP-2 -667 genotype was assessed by PCR, followed by restriction fragment length polymorphism analysis. Results. There were no significant differences in genotype and allele frequencies between the patients and healthy controls (p?=?0.3936, p?=?0.2941, respectively). In all, 79 (30.5%) patients had the AQP-2 -667 wild-type A/A, 123 (47.5%) were heterozygous for the G allele, and 57 (22.0%) patients showed homozygosity. After subclassification of CKD according to underlying disease, no significant differences were observed between those patients and controls (p?=?0.72 for diabetic nephropathy, p?=?0.52 for hypertensive nephropathy, p?=?0.27 for chronic glomerulonephritis, and p?=?0.80 for unknown etiology). Genotype and allele frequencies of the AQP-2 gene polymorphism (rs3759126) of hypertensive patients in pre-ESRD did not show a noticeable difference compared with normal blood pressure patients in pre-ESRD (p?=?0.50). No correlation was found to exist between the AQP-2 gene polymorphism (rs3759126) and serum electrolyte levels in pre-ESRD patients (p?=?0.38 for serum sodium level and p?=?0.44 for serum potassium level). Conclusion. Our data indicate that no association exists between the -667 AQP-2 A/G polymorphism and susceptibility to CKD or its clinical course.     

Humoral Heart Rejection (Severe Allograft Dysfunction with no Signs of Cellular Rejection or Ischemia): Incidence, Management, and the Value of C4d for Diagnosis

Severe allograft dysfunction after heart transplant (HT), without ischemia or evidence of cellular rejection upon endomyocardial biopsy (EMB), is a rare but potentially fatal condition that suggests humoral rejection (HR). Its incidence, and the methods of choice for its diagnosis and management, remain uncertain. We retrospectively studied 445 HT patients (April 1991-December 2003) to determine incidence of HR diagnosed by clinical and conventional histopathological criteria. We used immunofluorescence (IF) techniques to test archived frozen EMB issue for IgM, IgG, C1q, C3, fibrin and C4d. Twelve patients (2.7%) fulfilled the criteria for HR after a mean time post-HT of 21.3 +/- 24.7 months (range: 2-72 months). Patients were treated with high doses of steroids and plasmapheresis, with successful recovery in 11 cases. IF studies using classical markers were mainly negative for the six patients with enough EMB tissue for testing. All six patients showed positivity for C4d during the HR episode but not before or after. Humoral rejection was observed in less than 3% of HT patients. Plasmapheresis treatment was highly effective. Classical IF tests were not useful for diagnosis, but C4d appears to be useful both for confirmation of diagnosis and for monitoring response to treatment.     

肿瘤浸润性淋巴细胞在白细胞介素-2、-4协同下局部过继免疫治疗膀胱癌的实验研究

目的　观察膀胱癌肿瘤浸润性淋巴细胞 (TIL)联合不同细胞因子瘤灶内过继免疫抗癌的效应及对机体全身抗肿瘤免疫机制的影响。方法　建立BTT73 9动物模型 ,分离、培养TIL。采用正交设计实验方法 ,将TIL、白细胞介素 (IL) 2、 4及三因素交互组合悬液分别直接注射至瘤体内 ,定期测量肿瘤体积 ,免疫治疗 2周后检测NK细胞活性、T淋巴细胞转刺激指数 ,观察组织学及超微结构变化。结果　比较对照组 ,治疗 2周时各TIL相关组均不同程度抑制了膀胱肿瘤体积的增长 ,且NK细胞活性及T淋巴细胞转化增殖能力得以提高 (P <0 .0 5 )。TIL/IL 2疗法明显抑制了瘤体的增长 ,免疫治疗 1周后即表现出协同增强效应 (P <0 .0 5 ) ,而NK细胞活性及T淋巴细胞转刺激指数也显著提高 (P <0 .0 5 )。TIL/IL 2 /IL 4组获得了较强的抗癌功效 ,但与TIL/IL 2组差异无显著性 (P >0 .0 5 )。超微结构变化显示出TIL强烈的溶癌现象。结论　TIL在细胞因子特别是IL 2协同下瘤灶内注射的局部免疫疗法 ,具有较强的抗膀胱癌效应 ,并显著提高了机体全身抗瘤免疫功能。     

Detection of Equine Herpesvirus (EHV) ‐1, ‐2, ‐4 and ‐5 in Ethiopian Equids with and without Respiratory Problems and Genetic Characterization of EHV‐2 and EHV‐5 Strains

Infections with equine herpesviruses (EHVs) are widespread in equine populations worldwide. Whereas both EHV‐1 and EHV‐4 produce well‐documented respiratory syndromes in equids, the contribution of EHV‐2 and EHV‐5 to disease of the respiratory tract is still enigmatic. This study describes the detection and genetic characterization of EHVs from equids with and without clinical respiratory disease. Virus‐specific PCRs were used to detect EHV‐1, ‐2, ‐4 and ‐5. From the total of 160 equids with respiratory disease, EHV‐5 was detected at the highest prevalence (23.1%), followed by EHV‐2 (20.0%), EHV‐4 (8.1%) and EHV‐1 (7.5%). Concurrent infections with EHV‐2 and EHV‐5 were recorded from nine (5.2%) diseased horses. Of the total of 111 clinically healthy equids, EHV‐1 and EHV‐4 were never detected whereas EHV‐2 and EHV‐5 were found in 8 (7.2%) and 18 (16.2%) horses, respectively. A significantly higher proportion of EHV‐2‐infected equids was observed in the respiratory disease group (32/160, 20.0%; P = 0.005) compared to those without disease (8/111; 7.2%). EHV‐2‐positive equids were three times more likely to display clinical signs of respiratory disease than EHV‐2‐negative equids (OR 3.22, 95% CI: 1.42–7.28). For EHV‐5, the observed difference was not statistically significant (P = 0.166). The phylogenetic analysis of the gB gene revealed that the Ethiopian EHV‐2 and EHV‐5 strains had a remarkable genetic diversity, with a nucleotide sequence identity among each other that ranged from 94.0 to 99.4% and 95.1 to 100%, respectively. Moreover, the nucleotide sequence identity of EHV‐2 and EHV‐5 with isolates from other countries acquired from GenBank ranged from 92.9 to 99.1% and 95.1 to 99.5%, respectively. Our results suggest that besides EHV‐1 and EHV‐4, EHV‐2 is likely to be an important contributor either to induce or predispose equids to respiratory disease. However, more work is needed to better understand the contribution of EHV‐2 in the establishment of respiratory disease.