Ana L. Viciana

De novo membranoproliferative glomerulonephritis in hepatitis C virus-infected renal allograft recipients.

Hepatitis C virus (HCV) is the leading cause of non-A, non-B hepatitis among renal allograft recipients. We sought to identify and describe a proteinuric renal disease occurring in our HCV-infected renal transplant patients. Patients with proteinuria exceeding 1 g/day were identified from a cohort of 98 HCV-infected kidney recipients. Qualitative and quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and restriction fragment-length polymorphism of the amplified RT-PCR product was performed to detect circulating HCV RNA, viral titer, and strain type, respectively. An immune complex nephritis (ICN) of the membranoproliferative pattern (MPGN) was found on five of eight biopsies. Two patients infected with the Hutch strain-type developed nephrotic-range proteinuria within three months posttransplant while the remaining three MPGN patients had been transplanted greater than 5 years prior to the onset of proteinuria. Testing for rheumatoid factors, cryoglobulins, hypocomplementemia, and circulating immune complexes failed to show a consistent pattern. Sucrose density gradient (SDG) equilibrium centrifugation was used to determine the buoyant-density of HCV virions from control (HCV-infected nonproteinuric recipients; n = 5) and nephrotic patients (n = 5). Whereas HCV virions from the control patients had a low buoyant density on sucrose gradients, a substantial percentage of the circulating HCV RNA from the MPGN patients was present in the high-density fractions in association with IgM and IgG. Treatment of the pooled high-density layers with NP40 followed by recentrifugation resulted in a shift of the HCV RNA to the medium-density layers. In conclusion, MPGN developed in five HCV-infected kidney recipients despite pharmacologic immunosuppression. Both the physicochemical properties of the HCV virions on SDG and their association with IgG and IgM in the high-density layers provide indirect evidence for the presence of circulating complexes of anti-HCV antibody and HCV antigen(s).

Diagnosis of Intraocular Lymphoma by Flow Cytometry

PURPOSE To evaluate flow cytometry of vitreous cellular specimens as a means of diagnosing intraocular lymphoma and ocular inflammatory disease. METHODS We undertook a retrospective, observational study of hematopoietic cell-surface markers in 20 patients with vitreous cellular infiltration in whom lymphoma was considered in the differential diagnosis. Immunophenotyping of vitreous cells obtained by vitrectomy was performed by flow cytometry using antibodies directed against specific cell-surface antigens, including ones associated with B-lymphocyte and T-lymphocyte lymphomas and activated inflammatory cells. Smears were examined cytologically. Cytofluorography was compared with the cytopathologic diagnosis and with final diagnosis. RESULTS With flow cytometry, a diagnosis of intraocular lymphoma was confirmed in two of four patients with known lymphoma, one of whom had recurrent disease after radiation, and not confirmed in two patients who had had prior treatment with radiation or corticosteroids. In six patients with no prior diagnosis of lymphoma, five were diagnosed with lymphoma on the basis of cytofluorography. Thus, seven (70%) of 10 patients with intraocular lymphoma were diagnosed by cytofluorography compared with three (30%) of 10 with lymphoma diagnosed by cytology. With flow cytometry, 10 patients with uveitis or intraocular infections were distinguishable from patients with lymphoma by lack of a monotypic population and, in some cases, by elevated CD4:CD8 ratios and a high percentage of activated cells. CONCLUSIONS Cytofluorography of vitreous cells is an effective alternative or adjunct to cytology. Information can be gained from specimens that are uninterpretable by routine cytology. The optimal technique for diagnosis may vary among institutions.

Thrombocytopenia after liver transplantation.

BACKGROUND Thrombocytopenia after orthotopic liver transplantation (OLT) is a well recognized and prevalent early postoperative complication. The etiology, as well as the effect of this phenomenon on transplant outcome, however, are vague. The aims of this study are to identify factors contributing to thrombocytopenia and to ascertain whether there is any correlation with early rejection and ultimate survival. METHODS This study examines 541 OLTs (541 grafts in 494 patients) that were transplanted at the University of Miami during the 3-year period from June 1994 to September 1997. The patients with severe postoperative thrombocytopenia (nadir platelet count [PLT] < 20,000/mm3), as well as the whole group of patients, were analyzed. The preoperative PLT, intra-operative platelet transfusion requirements, cross-match, recipient and donor cytomegalovirus (CMV) status, infusion of donor bone marrow cells (DBMC), occurrence of early rejection episodes (in the first posttransplant month), and re-transplantation were factors examined for any association with thrombocytopenia. Total bilirubin (TB) and direct bilirubin (dB), hematocrit, white blood cell count (WBC), aspartate aminotransferase and alanine aminotransferase, determined on the day that platelets reached a nadir (nadir day), were also analyzed. RESULTS In 90.9% of the cases, there was a 56.5%+/-23.5% fall in platelets in the immediate posttransplant period (first 2 weeks), but the mean PLT exceeded preoperative levels during the 3rd and 4th postoperative weeks. The nadir of the drop in the PLT most commonly occurred on posttransplant day 4. For preoperative PLT, platelet transfusions during the operation, re-transplantation, early rejection, cross-match, and recipient CMV status, there was significant statistical correlation with any degree of postoperative thrombocytopenia. Four of these factors, preoperative PLT, intra-operative platelet transfusions, re-transplantation, and early rejection, were found to be independently associated with thrombocytopenia in general. None of them was found to be independently correlated with severe thrombocytopenia. A statistically significant correlation between bilirubin and WBC on the nadir day and the degree of thrombocytopenia was observed. No correlation was found between infusion of DBMC or donor CMV serology and thrombocytopenia. Both the nadir PLT and the percentage of the platelet fall were independent predictive factors (p<0.01 and 0.005, respectively) of patient and graft survival. CONCLUSIONS Thrombocytopenia in the immediate posttransplant period is correlated with low preoperative PLT, massive platelet transfusions, and re-transplantation. These factors reflect a poor preoperative condition. There is also a correlation with allograft dysfunction, rejection, and poorer patient and graft survival. A rise in the mean PLT after the 2nd postoperative week reflects proper graft function.

Thromboxane augmentation of alloreactive T cell function

Thromboxane (Tx) plays a vital role in the dysfunction and ultimate rejection of MHC-disparate renal allografts. In addition to its potent vasoconstrictory properties, in vivo studies have implied that Tx is capable of promoting immune cytotoxic T cell function within transplants. In this study, we have examined the in vitro effect of Tx inhibition on alloreactive immune cells using MHC-disparate mouse strain combinations. Coculture of either Tx-synthetase or Tx-receptor inhibitors modified the response of unprimed mouse lymphoid populations in a primary MLR, implying that Tx inhibition and not endoperoxide shunting was responsible for the modulatory effects seen. For example, B10.S lymphoid cells displayed decreased proliferation to H-2 disparate B10.A cells with Tx inhibitors present during the MLR, at pharmacologically active drug concentrations. Moreover, in vitro addition of TxA2 had an augmentory effect on the response in the primary and secondary MLR. Interleukin 2 production and percentages of T cell populations in the primary MLR were not affected by the presence of these compounds, although CD4 and CD8 expression was often increased in the treated populations. Finally, alloreactive primed effector cells also displayed reduced proliferation to specific alloantigen in a secondary MLR when Tx inhibitors were also present, although responses to IL-2 by T cells were not influenced by thromboxane inhibition. These data imply that thromboxane is an important immunoregulatory mediator capable of potentiating the function of naive and primed alloreactive immune T cell populations crucial to the rejection of the transplant.

Destructive allograft fungal arteritis following simultaneous pancreas-kidney transplantation

Fungal arteritis of the Y graft used to revascularize the whole pancreas graft developed in 2 recipients of simultaneous pancreas-kidney transplant that were performed within 36 hr of each other. The vascular infection became manifest 6-7 days following transplantation. In both patients, the vasculitis culminated in an arterial rupture that required immediate operative intervention. This compromise of the Y grafts contributed to loss of both pancreatic grafts and necessitated vascular reconstruction to reperfuse the lower extremity. To date, both patients continue to experience normal kidney transplant function.

CD26 expression and dipeptidyl peptidase IV activity in an aggressive hepatosplenic T‐cell lymphoma

The transmembrane serine aminopeptidase dipeptidyl peptidase IV (DPP IV) (also known as CD26) participates in several immunological functions and has a binding affinity for several molecules, including collagen, which may be an integral mechanism for T cells to traverse endothelial barriers. Since CD26 is phenotypically expressed in certain T-cell malignancies, this study utilized a novel four-color cytofluorographic procedure to measure DPP IV enzymatic activity concurrently with the expression of other surface markers in an aggressive hepatosplenic T-cell lymphoma. Immunophenotypic analysis by flow cytometry revealed the tumor to be CD2+, CD3+, CD5-, CD7+, TcR-gamma/delta+, CD4-, CD8+/-, CD56+, and CD11c+. The CD26 molecule was also expressed, and DPP IV activity was present, with the maximal activity detectable after 10 min of incubation. These results represent the initial description of enzymatically active CD26 in a T-cell malignancy, and raise the possibility that this molecule may be a participant in the pathogenetic mechanisms utilized by the neoplastic cells.

Cytofluorographic evidence that thymocyte dipeptidyl peptidase IV (CD26) activity is altered with stage of ontogeny and apoptotic status

CD26 is a multifunctional molecule implied to have a variety of roles in the immune response including its activity as a membrane exopeptidase (Dipeptidyl peptidase IV) which cleaves several protein molecules. In order to further define the expression and functional activity of CD26 in the developing thymus, we utilized a nondisruptive, cytofluorogenic assay which allowed simultaneous measurement of DPP IV activity with a fluorochrome-conjugated peptide substrate and surface staining of the T lymphocyte lineage antigens CD4 and CD8. Neonatal and adult murine thymi were examined using the three-color assay and significant differences in DPP IV activity were found among the thymocyte subsets defined by their CD4/CD8 phenotype. Single-positive cells bore higher activity than CD4-/CD8- cells and neonates had higher activity than adults. Thymocytes with characteristics consistent with apoptotic cells expressed higher DPP IV activity. Thus, DPP IV appears to be upregulated both as thymocytes mature and among thymocytes which are undergoing programmed cell death. These results suggest that CD26 is ontogenically controlled during T cell maturation and may play a role in thymic deletion of emerging clones.

T cell lymphoma involving the graft of a multivisceral organ recipient.

Posttransplant lymphoproliferative disorders are typically of B cell origin, whereas T cell lymphomas have been rarely documented. We present a case of a non-Hodgkins T cell lymphoma involving the intestinal graft of a multivisceral transplant patient. The patient was a 7-year-old girl who underwent at age 5 a multivisceral transplant secondary to short gut syndrome. Baseline immunosuppressive therapy consisted of FK506, methylprednisone, and mycophenolate mofetil. At 2 years posttransplant she presented with fever, diarrhea, nausea, and vomiting. Multiple endoscopic biopsies revealed a severe intensity, diffuse and focally nodular lymphocytic infiltrate composed predominantly of small, monomorphic lymphoid cells with scattered plasma cells and abundant eosinophils. Immunohistochemically, the majority of the lymphoid cells expressed the pan T cell marker CD3. Southern blot analysis revealed rearrangement of the T cell receptor beta chain gene, with germline configuration of the heavy immunoglobulin chain gene, confirming a clonal T cell genotype. In situ hybridization for Epstein Barr virus revealed rare positive lymphoid cells, that were negative with CD3 by immunohistochemical staining. A detailed clinico-radiological work-up revealed no other sites of involvement by the lymphomatous process. After the diagnosis of posttransplant lymphoproliferative disorder, immunosuppression was reduced with a subsequent partial improvement in the endoscopic appearance of the graft and a focal decrease in the lymphocytic infiltrate seen in the follow-up biopsies. Repeat gene rearrangement studies demonstrated germline configuration of both the T cell receptor beta chain gene and the heavy chain immunoglobulin. gene. To our knowledge, this represents the first description of a T cell lymphoma affecting the intestinal allograft of a multivisceral transplant patient.

Dipeptidyl peptidase IV (CD26) activity in human alloreactive T cell subsets varies with the stage of differentiation and activation status

Dipeptidyl peptidase IV (DPP IV), also known as CD26, is a transmembrane serine aminopeptidase which has an ontogenically related expression on T cells and participates on several immunological functions. CD26 appears to play an important role in alloimmunity during host T cell activation subsequent to alloantigen encounter and is a way by which effector T cells traverse graft endothelial barriers. In order to help to elucidate the role of the CD26 molecule in alloimmune responses, DPP IV activity and CD26 antigenic expression were assessed during the initial phases of completely MHC-disparate human mixed lymphocyte reactions (MLRs) and in several long-term alloreactive T cell clones. Our methods involved the use of a rhodamine-110-conjugated dipeptide substrate specific for DPP IV in two-colour cytofluorographic analysis that allowed stimultaneous lineage marker evaluation. Polyclonal populations of alloreactive CD4 and CD8 T cells contained DPP IV activity at 1 and 10 min of incubation that was variably elevated from resting T cells with the enzyme activity confined to CD26+ cells. T cell clones derived from MLRs were established with IL-2 supplementation and alloantigen restimulation and had reduced CD62L expression with functional specificity to the stimulating MHC. While CD26 expression remained stable, DPP IV activity was variable in the alloreactive T cell clones, with enzyme function in the latter appearing to coincide with the timing of alloantigen restimulation. These studies demonstrate that DPP IV activity varies among phenotypically distinct alloreactive T cell subsets and appears to be altered with the activation status of the effector cells. These findings raise the potential of a role for CD26/DPP IV in the generation of specific alloimmunity. With this methodology, it may be possible to reveal whether specific alterations in the activity of this molecule in T cell populations promote graft acceptance and to determine the molecular requirements for these changes.

Donor-specific skin transplants activate allodestructive T cells in mice resistant to neonatal H-2 tolerance induction.

Mice of the B10 background that are class II I-E nonexpressing demonstrate a relative resistance to neonatal induction of tolerance of class I alloantigens from I-E-expressing B10 strains. The majority of these injected mice delete donor-responsive and “I-E-reactive” (Vβ11+) cells in the immediate postinoculation period, with many remaining deleted of donor-specific T cells before the application of the test skin graft. Utilizing a hemisplenectomy technique in B10.S (I-E−) mice that received as neonates MHC-disparate (B10.S±B10.A)F1 (I-E+) lymphohematopoietic cells, we determined the proportion of adult mice that demonstrated pretrans-plant donor cell chimerism as well as several functional and phenotypic features ascribed to donor responsiveness. Surprisingly, chimeric cells were present in the bulk of recipients and many also exhibited a deletion of Vβ11+ T cells and a lack of alloreactivity to the donor strain allotype. Since chimeric, MLR−, Vβ11-deleted mice would be predicted to be tolerant to allograft challenge, we hypothesized that the test skin graft applied in adulthood was providing stimulatory signals that overcame this state of immunologic unresponsiveness. To examine this issue, injected mice that had been evaluated before skin grafting were challenged with donor-specific skin and evaluated for the same parameters measured in the pretransplant period. The majority (95%) of these mice subsequently rejected the B10.A skin graft in a range of 7–14 days. After graft rejection, Vβ11= cell levels generally increased and chimeric cells were typically eliminated. Thus, the ability to reject allografts is not predicted by a “nonresponsive” immune phenotype or the presence of chimeric cells before application of the test allograft. In fact, the graft appears to provide an in vivo stimulus to the reduced numbers of host donor-specific T cells that results in the removal of chimeric cells and a breakdown of the tolerant state. We conclude that application of orthotopic skin grafts provides the signal(s) necessary to break class I tolerance induced neonatally in the context of I-E disparity.