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-Hodgkin's 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.

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.

In situ enzymatic oligonucleotide amplification of hepatitis C virus-RNA in liver biopsy specimens (reverse transcriptase in situ polymerase chain reaction) after orthotopic liver transplantation for hepatitis C-related liver disease.

BACKGROUND: Hepatitis C infection recurs after orthotopic liver transplantation for hepatitis C virus (HCV)-related end-stage liver disease. Overlapping histopathologic features may present difficulties in differentiating recurrent HCV in the allograft from other conditions, especially rejection. METHODS: In this study, we evaluated the presence of HCV-RNA by reverse transcriptase in situ polymerase chain reaction (RT in situ RCR) in hepatic tissue, after orthotopic liver transplantation for HCV-related liver disease. Further, detection of HCV-RNA was correlated with the serum HCV-RNA levels, histopathology, and clinical outcome. RESULTS: Twenty-five patients were part of this study. Seventeen patients were transplanted for HCV cirrhosis and eight for an underlying disease other than HCV. None of the patients in the non-HCV group had in situ RT-PCR detection of HCV-RNA. Positive RT in situ PCR for HCV was found in 9 of 17 HCV patients, and the patients had a clinical course consistent with recurrent hepatitis C disease. Four of these nine patients had an initial histologic diagnosis of rejection. The other eight patients in the HCV group had negative RT in situ PCR, and none of them had a course compatible with recurrent HCV disease, although four patients were histologically diagnosed as having chronic C hepatitis. The mean HCV-RNA level (log/mL) in the patients who had in situ detection of HCV-RNA was 7.01+/-0.26. Although RT-PCR was negative in 8 of 17 HCV patients, the patients were serologically viremic and the mean HCV-RNA level was 6.05+/-0.33 (P=0.03). CONCLUSIONS: Our findings indicate that the HCV in situ RT-PCR assay may be helpful in the differentiation of recurrent hepatitis C disease from rejection. This may further help in the adjustment of immunosuppression.

Human thymocyte dipeptidyl peptidase IV (CD26) activity is altered with stage of ontogeny.

The nonintegrin receptor CD26, also known as dipeptidyl peptidase IV (DPP IV) is a transmembrane 110- to 120-kDa serine aminopeptidase glycoprotein with multiple functions, including cellular trafficking through extracellular matrix, and costimulatory potential during T cell activation, and is an influence upon T cell differentiation during their maturation in the thymus. In order to further define the expression and functional activity of this membrane exopeptidase in human thymus, we utilized a nondisruptive, cytofluorogenic assay which allowed simultaneous measurement of intracellular DPP IV activity using a fluorochrome-conjugated peptide substrate with surface staining of plasma membrane-associated T lymphocyte lineage antigens CD4 and CD8, as well as CD26. Human thymi were examined using the three-color assay, and significant differences in time-dependent DPP IV activity were found among the thymocyte subsets defined by their CD4/CD8 phenotype. In this regard, CD4(-)/CD8(-) thymocytes displayed the lowest DPP IV activity and had higher activity than the smaller-sized CD26(+) cells. Thymocytes containing greater percentages of apoptotic cells expressed lower DPP IV activity than viable cells. Thus, DPP IV appears to be upregulated as thymocytes mature and is reduced among thymocyte populations enriched for cells undergoing programmed cell death, suggesting that CD26-associated enzymatic activity is ontogenically controlled during T cell maturation and may be involved in thymic deletion of emerging clones.

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.

Adenovirus enterocolitis in human small bowel transplants.

This report describes two cases of pediatric small bowel transplant patients who developed diffuse adenovirus enterocolitis of their allografts. Based upon the presenting symptoms for this complication, in both patients a differential diagnosis of allograft rejection versus viral infection was clinically entertained. The clinical condition in both instances rapidly deteriorated and both patients died shortly after the development of the symptoms of fulminant septicemia. Autopsies were performed and histologic examination revealed extensive denudation of the gastrointestinal mucosa with edema and a marked acute and chronic inflammatory infiltrate involving the entire wall of the grafts. Numerous viral intranuclear and intracytoplasmic inclusions were evident and an immunohistochemical stain specific for adenovirus was strongly positive in the infected cells. In addition, while in the first case the adenovirus appeared confined to the GI tract, the second patient displayed numerous viral inclusions in the lung as well as within multiple liver abscesses. At this point, the incidence of adenovirus as a cause of gastroenteritis in small bowel transplant patients remains to be determined. We believe that the importance of recognizing this particular type of viral infection in this group of patients lies primarily in differentiating it from other viral organisms (e.g., CMV) that require a specific antiviral therapy. Moreover, an identification of this entity could help avoid a misdiagnosis of rejection which could lead to an unnecessary increase in immunosuppressive therapy and a possible exacerbation of the underlying condition.

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.

The apical submembrane cytoskeleton participates in the organization of the apical pole in epithelial cells.

In a previous publication (Rodriguez, M.L., M. Brignoni, and P.J.I. Salas. 1994. J. Cell Sci. 107: 3145-3151), we described the existence of a terminal web-like structure in nonbrush border cells, which comprises a specifically apical cytokeratin, presumably cytokeratin 19. In the present study we confirmed the apical distribution of cytokeratin 19 and expanded that observation to other epithelial cells in tissue culture and in vivo. In tissue culture, subconfluent cell stocks under continuous treatment with two different 21-mer phosphorothioate oligodeoxy nucleotides that targeted cytokeratin 19 mRNA enabled us to obtain confluent monolayers with a partial (40-70%) and transitory reduction in this protein. The expression of other cytoskeletal proteins was undisturbed. This downregulation of cytokeratin 19 resulted in (a) decrease in the number of microvilli; (b) disorganization of the apical (but not lateral or basal) filamentous actin and abnormal apical microtubules; and (c) depletion or redistribution of apical membrane proteins as determined by differential apical-basolateral biotinylation. In fact, a subset of detergent-insoluble proteins was not expressed on the cell surface in cells with lower levels of cytokeratin 19. Apical proteins purified in the detergent phase of Triton X-114 (typically integral membrane proteins) and those differentially extracted in Triton X-100 at 37 degrees C or in n-octyl-beta-D-glycoside at 4 degrees C (representative of GPI-anchored proteins), appeared partially redistributed to the basolateral domain. A transmembrane apical protein, sucrase isomaltase, was found mispolarized in a subpopulation of the cells treated with antisense oligonucleotides, while the basolateral polarity of Na+-K+ATPase was not affected. Both sucrase isomaltase and alkaline phosphatase (a GPI-anchored protein) appeared partially depolarized in A19 treated CACO-2 monolayers as determined by differential biotinylation, affinity purification, and immunoblot. These results suggest that an apical submembrane cytoskeleton of intermediate filaments is expressed in a number of epithelia, including those without a brush border, although it may not be universal. In addition, these data indicate that this structure is involved in the organization of the apical region of the cytoplasm and the apical membrane.

Recurrent Crohn's disease in transplanted bowel.

BACKGROUND: Intestinal transplantation is used in patients with short-bowel syndrome after repeated resections for Crohn's disease. We report the apparent clinical recurrence of Crohn's disease in a transplanted intestine. METHODS AND FINDINGS: The patient, a 33-year-old Hispanic woman, underwent small-bowel transplantation in December, 1994. Immunosuppression with tacrolimus, methylprednisolone, bone-marrow infusions, and OKT3 was given. In July, 1995, the patient had recurrent abdominal symptoms. The histological diagnosis of Crohn's disease was established by the independent interpretations of four experienced gastrointestinal histopathologists. INTERPRETATION: The prompt appearance of this autoimmune disorder (within 6 months of transplantation), despite massive immunosuppression may provide important insights into the nature of Crohn's disease and of the recurrence of autoimmune disease during immunosuppression.

DNA flow cytometric measurements in breast cancer: a review of the instrumentation principles and clinical significance.

Breast cancer treatment remains a controversial and challenging issue. Treatment of this disease is often guided by the clinical stage and type of tumor present, as well as information provided by a variety of prognostic indicators which can be employed. DNA analysis by flow cytometry is a relatively new technique that is emerging as a powerful tool to predict the biologic behavior of breast tumors. A majority of studies have demonstrated that the S-phase fraction measured by DNA flow cytometric is an independent predictor of patient outcome. There is less conclusive information regarding the association of DNA ploidy with the clinical behavior of breast malignancies, although, tumor grading often closely parallels this DNA parameter. New flow cytometry methodology involves multiparameter analysis that allows a simultaneous examination of DNA ploidy and cell cycle analysis with surface or internal molecules. This newer approach permits a precise delineation of DNA characteristics in distinct cell populations within a tumor and will likely enhance the utility of this procedure in determining the biologic behavior of the breast tumor.

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.