Noninvasive diagnosis of renal-allograft rejection by measurement of messenger RNA for perforin and granzyme B in urine.

BACKGROUND: Acute rejection is a serious and frequent complication of renal transplantation, and its diagnosis is contingent on the invasive procedure of allograft biopsy. A noninvasive diagnostic test for rejection could improve the outcome of transplantation. METHODS: We obtained 24 urine specimens from 22 renal-allograft recipients with a biopsy-confirmed episode of acute rejection and 127 samples from 63 recipients without evidence of acute rejection. RNA was isolated from the urinary cells. Messenger RNA (mRNA) encoding the cytotoxic proteins perforin and granzyme B and a constitutively expressed cyclophilin B gene were measured with the use of a competitive, quantitative polymerase chain reaction, and the level of expression was correlated with allograft status. RESULTS: The log-transformed mean (+/-SE) levels of perforin mRNA and granzyme B mRNA, which encode cytotoxic proteins, but not the levels of constitutively expressed cyclophiiin B mRNA, were higher in the urinary cells from the 22 patients with a biopsy-confirmed episode of acute rejection than in the 63 recipients without an episode of acute rejection (perforin, 1.4+/-0.3 vs. -0.6+/-0.2 fg per microgram of total RNA; P<0.001; and granzyme B, 1.2+/-0.3 vs. -0.9+/-0.2 fg per microgram of total RNA; P<0.001). Analysis involving the receiver-operating-characteristic curve demonstrated that acute rejection can be predicted with a sensitivity of 83 percent and a specificity of 83 percent with the use of a cutoff value of 0.9 fg of perforin mRNA per microgram of total RNA, and with a sensitivity of 79 percent and a specificity of 77 percent with the use of a cutoff value of 0.4 fg of granzyme B mRNA per microgram of total RNA. Sequential urine samples were obtained from 37 patients during the first nine days after transplantation; and measurements of the levels of mRNA that encoded cytotoxic proteins identified those in whom acute rejection developed. CONCLUSIONS: Measurement of mRNA encoding cytotoxic proteins in urinary cells offers a noninvasive means of diagnosing acute rejection of renal allografts.

Detection of chlamydial antigenic material in ovarian, prostatic, ectopic pregnancy and semen samples of culture-negative subjects.

PROBLEM: The pathogenesis of long-term sequelae in Chlamydia trachomatis infection is poorly understood. While serology indicates previous chlamydial infection, culture studies are frequently negative. We wanted to know whether in chronic cases the bacterium is absent or persists in a dormant state where it evades detection. METHODS OF STUDY: Using immunoperoxidase (IP) staining and in situ hybridization (ISH), we examined tissues of culture-negative subjects. Ovarian biopsy specimens from 19 culture-negative women with pelvic adhesions and/or tubal infertility were analyzed by both methods. Samples of prostates from 10 culture-negative men undergoing prostatectomy for benign hypertrophy, two sets of semen samples from culture-negative sexual partners of 28 women with PID and/or bacterial vaginosis (BV), and ten endometrium-tube sample-pairs from ectopic pregnancies (EPs) were examined by IP only. RESULTS: Seven of the nineteen ovarian specimens tested positive for Chlamydia antigen or deoxyribonucleic acid (DNA) (36%). Of the 10 hypertrophic prostates examined, 4 (40%) were positive. Of the 28 semen samples examined, 10 (35%) tested positive. Tissue samples of 3 cases of EP were positive by IP. CONCLUSIONS: 1. C. trachomatis antigen and nucleic acid can be frequently demonstrated in asymptomatic, culture-negative men and women with chronic infection. 2. Chlamydia antigens may have an etiologic role in benign prostate hypertrophy and EP. 3. Antigenic material may be sexually transmissible. 4. IP and ISH identify temporarily inactive bacteria that may continue to act as immunostimulants and potentially reactivate as Chlamydia infection.

Thrombotic microangiopathy as a complication of long-term therapy with gemcitabine.

Three patients with pancreatic carcinoma treated with gemcitabine for 1 year developed clinical and laboratory findings compatible with an indolent form of the hemolytic-uremic syndrome. Renal biopsy specimens in two of these patients showed the characteristic features of thrombotic microangiopathy, and a skin biopsy specimen from the third patient, who presented with livedo reticularis, showed intravascular fibrin deposition. Thrombotic microangiopathy may represent a toxic effect of long-term gemcitabine therapy.

Molecular executors of cell death--differential intrarenal expression of Fas ligand, Fas, granzyme B, and perforin during acute and/or chronic rejection of human renal allografts.

Two distinct cytolytic pathways have been characterized: one in which the interaction between the Fas antigen and its ligand results in apoptosis, and another in which the pore forming protein perforin and the serine protease granzyme B contribute to DNA fragmentation and cell death. We investigated intrarenal expression of these molecular executors of cell death in light of the potential participation of cytolytically active cellular elements in the antiallograft repertory. Reverse transcriptase-polymerase chain reaction was used to identify intrarenal expression of Fas antigen, Fas ligand, granzyme B and perforin in eighty human renal allograft biopsies; mRNA display was correlated with the Banff histological diagnosis of renal allografts. Our studies demonstrate that: (1) intrarenal expression of Fas ligand mRNA and of granzyme B mRNA are correlates of acute but not chronic rejection; (2) Fas ligand mRNA is not detectable in allografts in the absence of rejection; (3) intrarenal coexpression of members of each lytic pathway (Fas ligand and Fas, granzyme B, and perforin) and that of both pathways (e.g., Fas ligand and granzyme B) are correlates of acute rejection; and (4) a direct correlation exists between the histological severity of acute rejection and intrarenal coexpression of mRNA encoding Fas ligand, Fas, granzyme B, and perforin. Our studies identify, for the first time, the differential expression of the two major lytic pathways in acute and chronic allograft rejection and suggest that specific therapy directed at the cytotoxic attack molecules might be efficacious in the prevention and/or treatment of acute rejection.

Intragraft TGF-beta 1 mRNA: a correlate of interstitial fibrosis and chronic allograft nephropathy.

Chronic allograft nephropathy is a relentlessly progressive process and a major cause of long-term graft dysfunction and ultimate failure. Interstitial fibrosis, tubular atrophy, and glomerular and vascular lesions characterize this mechanistically unresolved disorder. Given the prominent role of TGF-beta 1 in tissue repair and in fibrosis, we have explored the hypothesis that fibrosis and chronic allograft nephropathy would be distinguished by intragraft TGF-beta 1 mRNA expression. This postulate was tested by mRNA phenotyping of RNA isolated from 127 human renal allograft biopsies. Reverse transcription assisted polymerase chain reaction was used to amplify and identify ingraft gene expression. Our investigation demonstrated a significant correlation between intragraft TGF-beta 1 mRNA display and renal allograft interstitial fibrosis and chronic allograft nephropathy. In contrast, intragraft expression of mRNA encoding immunoregulatory cytokines, IL-2, IFN-gamma, IL-4, IL-10, or cytotoxic attack molecules, granzyme B and perforin was not a correlate of interstitial fibrosis or chronic allograft nephropathy. Our studies identify, for the first time, a significant association between intragraft TGF-beta 1 mRNA expression and renal allograft interstitial fibrosis, and advance a candidate molecular mechanism for chronic allograft nephropathy.

Intragraft expression of IL-10 messenger RNA: a novel correlate of renal allograft rejection.

A major conceptual advance is the formulation that type I cytokines (such as IL-2 and IFN-gamma) enhance cellular immunity and are host-protective, and that type II cytokines (such as IL-4 and IL-10) dampen cellular immunity and facilitate the progression of infection. We have explored the intragraft expression of type I and type II cytokines during human renal allograft rejection. RNA was isolated from 98 allograft biopsies, and reverse transcription-PCR was used to amplify and identify intragraft expression of mRNA encoding IL-2, IFN-gamma, IL-4, or IL-10. Intragraft expression of IL-7 mRNA and TGF-beta 1 mRNA was also investigated. Our investigation demonstrated that: (a) intragraft expression of IL-10 mRNA and IL-2 mRNA are significant correlates of acute rejection; (b) IL-4, IL-7, IFN-gamma and TGF-beta 1 mRNA expression do not correlate with acute rejection; and (c) IL-10 does not prevent in vivo expression of IFN-gamma, IL-2, IL-7, or TGF-beta 1. Our studies identify, for the first time, a significant association between intragraft IL-10 mRNA expression and acute rejection, and suggest that treatment strategies capable of constraining IL-10 expression might be of value in the prevention of acute rejection.

Immunotherapy with anti-CD3 monoclonal antibodies and recombinant interleukin 2: stimulation of molecular programs of cytotoxic killer cells and induction of tumor regression.

Adoptive cellular immunotherapy, infusions of interleukin 2 (IL-2) in conjunction with in vitro-activated killer cells, has brought new hope to patients with cancer. The broad application of this strategy, however, is constrained by the need for repeated leukapheresis and by the labor-intensive process of in vitro activation of cells. Also, current protocols generally use nonphysiological and toxic concentrations of IL-2. Identification of an in vivo stimulant that renders T cells responsive to physiologic concentrations of IL-2 represents a potential improvement over existing approaches. We have determined whether in vivo administration of monoclonal antibodies (mAbs) directed at the T-cell surface protein CD3 induces T-cell responsiveness to IL-2, stimulates cytolytic molecular programs of natural killer cells and cytotoxic T cells, and induces tumor regression. These hypotheses were explored in a murine hepatic MCA-102 fibrosarcoma model. We report that in vivo administration of anti-CD3 mAbs plus IL-2 results in intrahepatic expression of mRNA-encoding perforin, cytotoxic T-cell-specific serine esterase, and tumor necrosis factor alpha. Anti-CD3 mAbs alone or IL-2 alone failed to induce or induced minimal expression of these molecular mediators of cytotoxicity. The anti-CD3 mAbs plus IL-2 regimen also resulted in a significantly smaller number of hepatic metastases and a significantly longer survival time of tumor-bearing mice, compared to treatment with anti-CD3 mAbs alone or IL-2 alone. Our findings suggest that a regimen of anti-CD3 mAbs plus IL-2 is a more effective antitumor regimen compared with anti-CD3 mAbs alone or IL-2 alone and advance an alternative immunotherapy strategy of potential value for the treatment of cancer in humans.

MRL/lpr-->severe combined immunodeficiency mouse allografts produce autoantibodies, acute graft-versus-host disease or a wasting syndrome depending on the source of cells.

MRL/lpr (lpr) mice spontaneously develop a lupus-like illness as well as massive lymphadenopathy. Attempts to transfer autoimmunity by adoptive transfer or radiation bone marrow chimeras have been unsuccessful. Since severe combined immunodeficiency (SCID) mice have been engrafted with human and rat xenografts without apparent graft-versus-host disease (GVHD), we subjected SCID mice to low-dose irradiation and reconstituted the mice with spleen cells from young or old lpr mice or with lpr bone marrow. Fourteen out of twenty (70%) of SCID mice engrafted with spleen cells from old lpr mice produced autoantibodies (anti-DNA and anti-Sm) without evidence of the severe lymphoid atrophy previously described for lpr spleen-->+/+ chimeras. SCID mice engrafted with spleen cells from young lpr mice developed acute GVHD and 5/6 (83%) died within 4 weeks post-transfer. Although 8/11 (73%) of lpr-->SCID bone marrow allografts survived for at least 4 months, these mice developed a wasting disease characterized by lymphoid atrophy and fibrosis without the production of autoantibodies. None of the lpr-->SCID grafts resulted in the transfer of double negative T cells or the lymphoproliferative syndrome characteristic of MRL/lpr mice. These findings indicate that SCID mice can be engrafted with splenocytes from old MRL/lpr mice and that B cells continue to secrete autoantibodies for several months in the SCID recipients. This study also demonstrates that, unlike i.p. transplant of xenogeneic cells, acute GVHD is a consistent feature of i.p. transplants of normal allogeneic mononuclear cells into SCID mice.

Analysis of autoantibody production in SCID-systemic lupus erythematosus (SLE) chimeras.

Mice with SCID disease have previously been successfully engrafted with human peripheral blood mononuclear cells (PBMC) obtained from normal individuals and from patients with various diseases. To determine whether SCID mice engrafted with SLE PBMC produced autoantibodies with specificities similar to those in the SLE donor, and to investigate which variables influence autoantibody production in the SCID recipients, we injected PBMC from 16 SLE patients into SCID mice and tested the recipients for autoantibodies to DNA and to five recombinant autoantigens. Ten out of 16 (68%) lupus and six out of nine (67%) normal grafts were successful as determined by the presence of human IgG greater than or equal to 5 micrograms/ml of SCID serum post-transfer. Autoantibodies to La/SSB, Ro/SSA, and RNP were detected in five out of 10 SCID-SLE recipients by ELISA and immunoblotting up to 22 weeks post-engraftment. The detection of autoantibodies in SCID-SLE mice was more closely related to autoantibody levels in donor sera than to total IgG concentrations in the SCID recipients. Autoantibody activity/mg IgG was similar in the donor and recipient sera. Histological evaluation of eight SCID-SLE mice killed 4-22 weeks post-transfer revealed population of the SCID thymus and spleen with mononuclear cells, but no evidence of lupus nephritis or dermatitis. These findings indicate that SCID mice can be engrafted with PBMC from patients with lupus and that specific autoantibodies are produced up to 5 months post-transfer. Failure to develop glomerulonephritis may be explained by low or absent anti-DNA antibodies or by changes in the cellular composition of the PBMC grafts.

Cytomegalovirus infection of renal allografts. Detection by polymerase chain reaction.

Early laboratory diagnosis of acute cytomegalovirus infection in renal transplant recipients is desirable but often difficult. The polymerase chain reaction (PCR) technique for detecting CMV DNA, although it promises a high sensitivity, risks the possibility of detecting latent CMV infection and leading to false-positive results. To address this issue and the feasibility of applying PCR to renal biopsy specimens, we analyzed 37 renal allografts by PCR. Formalin-fixed or Bouin-fixed paraffin-embedded materials were employed, and primers from the LA (late-antigen) region of CMV were used. Amplified products were detected by gel electrophoresis and ethidium bromide staining, followed by Southern blot analysis. Of 21 nephrectomy samples, three showed CMV-specific amplified products by PCR, but CMV inclusion bodies were detected histologically in only one of the three. Of 16 renal biopsies, three specimens were positive by PCR, with rare viral inclusions histologically identified in only one. All PCR-positive patients had clinically significant CMV disease as evidenced by positive CMV culture and/or seroconversion. In contrast, all CMV-seropositive patients without active viral disease had PCR-negative allografts. We conclude that PCR positivity in the renal allograft strongly correlates with active CMV disease but not latent infection. For the diagnosis of active CMV disease in patients with a renal allograft, PCR provides a means that is more sensitive and objective than histologic examination, more specific than serology, and faster than viral culture.

Immunohistochemistry and gene rearrangement studies in the diagnosis of malignant lymphomas: a comparison of 152 cases.

One hundred fifty-two cases (155 specimens) of lymphoproliferative disorders were studied by immunohistochemistry and gene rearrangement analysis. Ninety-five of 96 B-cell lymphomas (99%) showed genotypic B-cell monoclonality. Of these, five cases had rearranged T-cell receptor (TCR) beta chain gene in addition to immunoglobulin heavy chain (IgH) and kappa light chain (Ig-K), one case had rearranged IgH and TCR-gamma chain but not Ig-K or TCR-beta, and two cases had only Ig-K rearrangement. One exceptional case in the B-cell lymphoma group had unrearranged, germline genotypes. In contrast, only 10 of 19 (53%) phenotypic T-cell lymphomas had rearranged TCR-beta, eight with concurrent TCR-gamma rearrangement. Of the remaining nine cases, six had germline configuration, two had rearranged Ig-K only, and one had both IgH and Ig-K rearrangement. This last case was reclassified as T-cell predominant, B-cell lymphoma. Thirteen of 16 cases of Hodgkin's disease had germline configuration; three cases had rearranged IgH and Ig-K, of which two were lymphocyte predominant with light chain monoclonality and one was a recurrence. Among 21 reactive lesions, 17 had germline configuration and four had rearranged IgH and Ig-K genes. Of these four cases, two were orbital lesions, one was a partially involved lymph node, and one developed a nodular lymphoma 9 months later. Our results indicate that almost all B-cell lymphomas have IgH and/or Ig-K rearrangement. In contrast, peripheral T-cell lymphomas have greater genotypic heterogeneity, and germline patterns for TCR genes are not uncommon. Reactive lesions and Hodgkin's disease tend to retain germline configuration, and any exception is often associated with an unusual clinical setting and/or histology. Genotypic analysis is thus most indicated in B-cell lymphomas with equivocal immunohistochemistry findings, T-cell lymphomas, and atypical cases of Hodgkin's disease and reactive lesions.

Central nervous system lymphoma in the acquired immunodeficiency syndrome.

Primary central nervous system (CNS) lymphomas were studied in fifteen autopsied patients with the acquired immunodeficiency syndrome (AIDS). Using the working formulation for non-Hodgkin's lymphomas, the tumors were classified as large cell (7 patients), mixed large and small cell (6 patients), small cleaved cell (1 patient), and unclassifiable (1 patient). The mixed lymphomas displayed unusual features characterized by a high mitotic rate and the presence of numerous medium-sized cells (5 to 10 mus), not classifiable using the working formulation. Focal T cell and lymphoplasmacytoid B cell infiltrates accompanied lymphoma cells at the periphery of and remote from solid tumor masses in 9 cases. Immunohistochemical analysis of the lymphomas suggested B cell neoplasms. All of these patients had concurrent CNS and systemic cytomegalovirus (CMV) infections. The CNS infections were of both viral (CMV, human immunodeficiency virus (HIV), varicella zoster virus (VZV), progressive multifocal leukoencephalopathy (PML) and non-viral (toxoplasmosis, candidiasis) etiology. In the general AIDS population at our institution, the autopsy incidence of CNS infections and systemic CMV was 63% and 60%, respectively. In contrast, the incidence for both these entities was 0% in otherwise healthy, non-AIDS patients with CNS lymphoma supports the hypothesis that viral infection plays a role in the pathogenesis of CNS lymphoma in the immunocompromised. Polyclonal lymphoplasmacytoid B and T cell infiltrates accompanying lymphoma may produce diagnostic difficulties on surgical biopsy. As these infiltrates were a frequent feature in this study, we caution that their recognition does not argue against the presence of CNS lymphoma.

Retroperitoneal accessory spleen.

We report a lower abdominal mass found to be a retroperitoneal accessory spleen. The vascular supply originated from retroperitoneal vessels independent of the splenic, testicular, and spermatic vessels.

Progressive lymph node histology and its prognostic value in patients with acquired immunodeficiency syndrome and AIDS-related complex.

Seventy-four sequential lymph node biopsies from 30 acquired immunodeficiency syndrome (AIDS)/AIDS-related complex (ARC) patients showed temporal histologic progression from explosive follicular hyperplasia (EFH) to mixed follicular hyperplasia/involution (mixed) to follicular involution (FI) to lymphocyte depletion (LD). This histologic progression correlated with symptoms, development of opportunistic infections (OI), and mortality. At initial biopsy, only 50% of the AIDS/ARC patients with EFH/mixed compared to 100% with FI/LD were symptomatic with weight loss, night sweats, diarrhea, fever, or fatigue. 31% of ARC patients with EFH and 63% with FI developed an OI in a median of 69 months and 5 months, respectively; 86% with LD had a concurrent or previous OI. Ninety percent of ARC patients progressing to FI/LD died; 85% of those persisting with EFH/mixed remained alive 18 to 50 months after initial biopsy. AIDS patients with EFH lived twice as long as those with FI/LD. Progressive histology did not correlate with lymphoma. The number of ARC patients developing Kaposi's sarcoma was too small to draw definitive conclusions.

Lymph node biopsy in patients with human immunodeficiency virus infections.

Twenty-one male homosexuals were followed up by repeated lymph node biopsy for a mean (+/- SEM) follow-up of 99 +/- 18 weeks. Four histologic patterns were seen on biopsy: explosive follicular hyperplasia (EFH), follicular involution (FI), a mixed pattern of EFH with FI in the same node, and lymphocyte depletion. Patients with FI and lymphocyte depletion had mean survival times that were significantly less than those for the subjects with EFH. The percentage of lymph node follicles with suppressor cell clusters (T8) in EFH lymph nodes was significantly higher (43% vs 8%) than in nodes from patients without risk for human immunodeficiency virus infection. Helper/suppressor T-cell ratios in control nodes were 1.6; in EFH nodes, 0.97; and in FI nodes, 0.88. A remarkable 33% of patients in this lymphadenopathy group ultimately developed large-cell (B-cell) lymphoma, suggesting that the follicular stimulation noted histologically played a role in the development of this neoplasm. These data show that there is a progressive destruction of lymph node follicles that correlates with the progression of the disease and that lymph node histologic features may provide important prognostic information.