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The leukocyte aggregation test (LAT) detects the in vitro adhesion of sensitized, but not nonimmune, recipient leukocytes onto donor kidney cell monolayers. The test specifically detects cell-mediated homograft immunity up to 15 days prior to the appearance of clinical signs or alteration of chemical indexes. The presence of a positive reaction always signified incipient homograft rejection, which was usually controlled by intravenously administered, high-dose methylprednisolone sodium succinate (Solu-Medrol) therapy. There was no instance in which methyl-prednisolone treatment effectively reversed rejection in the presence of a negative leukocyte aggregation test. One common form of homograft rejection may be characterized by positive LAT results, a cellular infiltrate on the renal biopsy specimen, and sensitivity to methylprednisolone therapy.

Active immunotherapy with materials extracted from a murine methylcholanthrene-induced sarcoma using single phase solutions of 2.5% 1-butanol evoked host resistance against supralethal burdens of neoplastic cells deposited subcutaneously or delivered hematogenously. After curative resection of progressing 1 cm tumors, postsurgical immunity against subsequent subcutaneous neoplastic challenges was potentiated by weekly injections of crude butanol extracts. In a conventional therapy model, hosts bearing progressing 4 mm subcutaneous tumors displayed neoplastic regression following chemoimmunotherapy with cyclosphosphamide and tumor extracts. Therapeutic effects were demonstrated not only against subcutaneous neoplasms, but also against hematogenously disseminated pulmonary and extrapulmonary metastases introduced by intravenous inoculation of sarcoma cells. These findings suggest that tumor extracts may afford potent therapeutic adjuvants for patients bearing a small body tumor burden but at high risk of disease recurrence.

Because of the nephrotoxic action of trimethoprimsulfamethoxazole (TMP-SMX) in cyclosporine (CsA)-treated patients, combined with the (CsA)-treated patients, combined with the possibility of selecting resistant gram-negative or Nocardia asteroides organisms, a monitoring tool to detect early Pneumocystis carinii (PC) infection permitting a selective treatment approach is highly desirable. A review of 401 consecutive renal transplants revealed 26 cases (18 suspected and 8 histologically proved) of PC infection in 21 cadaver and 5 living-related renal recipients. The diagnosis was confirmed in 8/18 patients who were invasively studied by open-lung biopsy (1/2), bronchoscopy with transbronchial biopsy (4/9), bronchoscopy with brushing (1/2), bronchoscopy with bronchoalveolar lavage (2/5), and transpleural needle biopsy (0/1)-yielding a confirmed incidence of 2% (8/401). All positive invasive studies had been performed prior to or within 24 hr of the inception of TMP-SMX therapy. Nine of ten negative invasive studies were performed after more than 24 hr of treatment. The mean time from transplantation to the onset of clinical symptoms was 2.5 +/- 1.5 months. The infection rate would be 6.5%, assuming all 18 suspected cases would be PC-positive if studied pretreatment. In order to assess the efficacy of a variety of serologic methods of PC detection, qualitative counter-immunoelectrophoresis (CIE) for P carinii antigen (PC-Ag), IgG antibody reactive with PC (enzyme-linked immunosorbent assay [ELISA]), and a latex particle agglutination test (LPA) were performed on 279 sera; 85 sera from the 26 suspected or proved cases, 100 sera from normal age-matched controls, and 94 sera from 78 asymptomatic allograft recipients followed as outpatients. In the eight histologically proven cases, CIE was positive in only 3/8 and turned positive late in the clinical course. LPA was positive in all histologically proved cases; however, it was also positive in 60% of asymptomatic renal recipients. In cases that developed clinical disease, LPA increased in titer weeks to months prior to the onset of symptoms. Additionally, LPA titers decreased or stabilized during successful TMP-SMX therapy, providing an early therapeutic index. Measurement of anti-PC IgG was not useful per se, as it was elevated in both controls and documented PC infection. The combination of very low antibody titer (less than or equal to 1:16) with a positive or increasing LPA PC-Ag titer appeared to be disease-predictive.(ABSTRACT TRUNCATED AT 400 WORDS)

The fate of hosts after tumor resection depends upon the level of their sinecomitant immunity, which can destroy residual neoplastic cells. In addition, active specific immunotherapy can stimulate the resistance of mice incompletely resected of large tumors and therefore destined to develop recurrent disease. The studies presented herein, assessing host resistance by in vivo tumor challenge and an in vitro growth inhibition assay (GIA) measuring the effect of splenic lymphocytes on 3H-thymidine incorporation into monolayers of sarcoma cells, revealed an antagonistic relationship between sinecomitant immunity and tumor antigen immunotherapy. Treatment of hosts bearing high levels of endogenous sinecomitant immunity with Fraction 15 pI 5.95 partially purified from 3 M KCl extracts by preparative isoelectric focusing, decreased their resistance to tumor challenge and the capacity of their spleen cells to perform in the in vitro GIA. When sinecomitant immunity was not demonstrable, therapy with tumor antigen induced host resistance. If therapy was delayed until sinecomitant immunity had naturally waned, hosts displayed improved resistance toward secondary challenge. These findings suggest that not only does antigen therapy effect tumor resistance by a mechanism independent of sinecomitant immunity, but also these two mechanisms are mutually antagonistic. Therefore, the design of active specific immunotherapy protocols utilizing tumor antigens may depend on the level of the host endogenous sinecomitant resistance.

In a 3-methylcholanthrene [(MCA) CAS: 56-49-5]-induced tumor model of C3H/HeJ mice excision of a growing primary tumor decreased concomitant immunity and facilitated experimental lung metastases. Administration of tumor-specific transplantation antigens extracted from viable MCA-F cells with the use of single-phase (2.5%) 1-butanol [crude butanol extract (CBE)] augmented immunity after resection of the primary MCA-F tumor. Two weeks after footpad inoculation of 2 X 10(5) MCA-F cells, the tumor-bearing limbs were amputated and the mice were challenged subsequently with 5 X 10(4) cells of clone-9-4, a metastatic variant of MCA-F, via the tail vein. Whereas treatment with either 50 micrograms CBE sc or 20 mg cyclophosphamide (CY)/kg ip failed to retard lung colonization, combination therapy with the two agents reduced the incidence of lung colonies by 69.8% (26.5 vs. 8; P less than .001) compared with the incidence in the surgery-alone group and by 55.5% (18 vs. 8; P less than .001) compared with the incidence in the group treated with surgery and CY. Furthermore, the combined effects of CBE and CY were immunologically specific: The combined therapy with the non-cross-reactive MCA-D-CBE did not protect against iv challenge with clone-9-4. Treatment with antigenic extracts induced a 21.4% (4.2 vs. 3.3%) decrease in the ratio of Lyt 1+:Lyt 2+ cells in the spleens of tumor-resected mice, which suggested restoration to normal levels. Therefore, in the combined regimen, antigen may induce specific activation of helper lymphocytes, while CY inhibits activation of suppressor cells.

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.

Combination therapy with one dose of 3 M KCl extracted donor-soluble antigen (Ag) and a short course of cyclosporine (CsA) has proven to prolong the survival of kidney allografts by enhancing specific T-suppressor populations. This regimen is tested in rat islet allografts in this study (Lewis to ACI). A 3-day perioperative course of 10 mg/kg/day CsA on Days -1, 0, and 1 did not prolong graft survival (MST = 10.7 +/- 2.5 days vs 9.4 +/- 1.2 days in controls). When this course of CsA therapy was combined with a single dose of donor antigen on Day -1, the survival time was prolonged slightly but significantly (MST = 14.0 +/- 5.8 days). Three cycles of a 3-day course of CsA therapy at 7-day intervals, a total of nine doses of 10 mg/kg/day CsA, were effective in delaying rejection of islet allografts (MST = 26.4 +/- 30.3). Moreover, combined therapy with donor antigen and three cycles of a 3-day course of CsA prolonged the survival of islet allografts (MST = 57.7 +/- 51.4 days) with 50% of recipients still normoglycemic at 60 days after transplantation. These findings indicate that the combination therapy of donor antigen with a short course of CsA has a powerful effect to prevent the rejection of islet allografts, as shown in kidney allografts, in rats.

Methods which dissociate intramolecular noncovalent bonds have been used to prepare soluble derivatives of cell-surface antigens. Applications of these techniques to human colon carcinoma are underway. Continuous tissue-culture strains derived from primary lesions were developed and shown to be composed of malignant epithelial elements. Parallel data on the preparation and activity of soluble materials in a murine model methylcholanthrene system reveal that although cultured cells are a satisfactory source for antigen extraction, they are poor targets of the immune response. The development of methods to quantitate the biologic activity of colon-specific, soluble materials may provide indicator systems to define the antigenic determinants, to permit purification, and to serve as assays of the efficacy of immunotherapy.

To assess the impact of cytomegalovirus (CMV) infection in D+R- patients treated with cyclosporine (CsA)-prednisone immunosuppression, we compared the incidence of CMV infection, severity of disease, and the 1, 2, and 3-year actual graft and patient survival rates of CMV-infected D+R- patients with R+ patients from a group of 516 renal allograft recipients at our center. CMV infection occurred more frequently in 27/56 D+R- patients (48%) versus 111/376 R+ patients (29%) (P less than 0.01). The incidence of CMV was also significantly greater in D+R- versus R- patients receiving CAD grafts (59% vs. 32%, P less than 0.01) and first transplants (47% vs. 30%, P less than 0.05). There were no significant differences in CMV disease severity between the aggregate D+R- and R+ patient groups and when subgroups of these patients receiving cadaveric donor (CAD), living-related donor (LRD), first, or retransplant allografts were compared. The actual 1, 2, and 3-year graft survival rates for D+R- patients (68%, 58%, 68%) were not significantly different from rates in R+ patients (83%, 77%, 63%) with CMV infection. When the 1, 2, and 3-year actual graft survival rates in subgroups of D+R- and R+ patients were compared in CAD, LRD, and first and retransplants, there were no significant differences. The actual 1, 2, and 3-year patient survival rates were not significantly different between D+R- (89%, 92%, 100%) and R+ patients (94%, 91%, 86%) with CMV infection, nor were they different when CMV infected D+R- and R+ patients with CAD, LRD, first or retransplant grafts were compared. These data do not support the policy of denying a seropositive kidney to a seronegative recipient, since the severity of CMV disease and the impact of CMV infection is not significantly different comparing D+R- and R+ patients receiving CsA-prednisone immunosuppression.

Two nonspecific immunological assays were combined with radionuclide scanning to monitor 113 patients thrice weekly following allotransplantation. The nonspecific immune assays included measurement of the percentage of active T rosette-forming cells (% A-T RFCs) and spontaneous blastogenesis (SB). An increase in SB and decrease in % A-T RFCs (greater than 1 sd of normal controls) constituted an immune event. The immune parameters were correlated with thrice weekly radionuclide studies which were computer analyzed for glomerular and tubular function. Alteration of the immunological and radionuclide parameters significantly correlated (P less than 0.001) with 90 rejection episodes displayed by 72 nonantithymocyte globulin (ATG)-treated renal allograft recipients during the first 30 postoperative days. In the absence of clinically defined rejection, changes in immune parameters correlated with (1) decline of radionuclide parameters and (2) alterations in weight, temperature, creatinine clearance, and serum creatinine, suggesting subclinical events. As a result of the effects of ATG on lymphocytes, a similar comparison could not be made for 41 other patients treated with this immunosuppressive drug. The incidence of false positive tests was 12.7%. Thus, the combination of two nonspecific immune parameters, % A-T RFCs and SB, with computerized analysis of radionuclide scans may afford a reliable index to diagnose early rejection or impaired function of renal allografts.

The immunosuppressive effect of the combination of a three day course of cyclosporine with one i.v. injection of 3M KCL-extracted donor antigen or donor blood transfusion was tested across the strong histocompatibility barrier causing rejection within 8 days of kidney transplants from Buffalo (Buf, RT1b) to Wistar-Furth (WFu, RT1u) inbred rats. Administration of 10 mg/kg/day cyclosporine alone for three days (-1, 0, and 1) slightly prolonged graft survival time from 7 to 11 days. The combination of cyclosporine with donor Buf 3M KCl antigen or with a Buf blood transfurion administered one day prior to transplantation caused greater prolongation of graft survival--19 and 25 days, respectively. Neither third-party BN soluble antigen nor BN blood transfustions acted synergistically with cyclosporine to prolong Buf graft survival. Increasing doses of donor-soluble antigen up to an optimal dose of 5 mg proportionately prolonged graft survival; however, administration of 10 mg antigen was less effective than 5 mg. On the other hand, administration of 1 ml of donor blood achieved the maximal effect. Lymphocytes harvested ten days after transplantation from recipients that had received combined therapy with cyclosporine and donor 3M KCl antigen not only displayed specific unresponsiveness to donor stimulator cells in mixed lymphocyte culture, but also specifically suppressed the proliferative response of syngeneic, virgin WFu responder cells to allogeneic donor Buf but not to third-party BN cells. Furthermore, suppressor cell activity was suggested by diminished responses in an in vivo local adoptive mixed lymphocyte culture assay and by prolongation of Buf kidney survival following systemic adoptive transfer. These findings suggest that immunosuppression with cyclosporine permits induction of specific suppressor cells by 3M KCl donor antigen, resulting in specific unresponsiveness to allografts.

Antigen-specific suppressor T cells are induced by donor histocompatibility antigen extracted from spleen cells with 3M KCl combined with cyclosporine (Ag-CsA). A single i.v. injection of 5 mg 3M-KCl-extracted donor Buffalo (Buf, RT1b) antigen (Ag) combined with a three day course of CsA prolonged renal allograft survival in Wistar-Furth (WFu, RT1u) hosts to a greater extent (MST 26.5 days) than CsA alone (MST 11.8 days). Peripheral blood lymphocytes (PBL) or spleen cells harvested from Ag-CsA-treated recipients ten days after transplantation inhibited the mixed lymphocyte reaction (MLR) between normal responder WFu cells and irradiated Buf cells (55.6% and 64.4% suppression, respectively, P less than 0.025), but not third-party Brown-Norway (BN, RT1n) stimulator cells (13.6% and -18.3% suppression, respectively, NS). The suppressor effect was not mediated by cytolytic cells; there was neither primary nor secondary cytolytic activity against 51Cr-labeled Con-A blastoid Buf cells. The suppressor cells were neither adherent to plastic dishes nor to nylon-wool columns. PBL irradiated with 800 rads, but not 1500 rads, suppressed the MLR. A single injection of cyclophosphamide (CY, 25 mg/kg) seven days after transplantation abrogated the suppression induced by Ag-CsA treatment. Moreover, PBL from Ag-CsA recipients failed to suppress the MLR, if depleted either of all T cells by treatment with monoclonal antibody (Mab) W3/13 HLK (pan T cells; % suppression -15.8), or of cytotoxic/suppressor cells with Mab OX-8 (-19.3% suppression) together with rabbit antimouse immunoglobulin and complement. On the other hand, PBL treated with the Mab W3/25 (helper) showed suppressor cell activity (+56.4%, P less than 0.001) similar to untreated cells (62.4%, P less than 0.001). Moreover, adoptive transfer of suppressor T cells purified from pooled lymphocytes by rosetting using Mab significantly prolonged the survival of donor-specific, but not third-party, test grafts in naive secondary hosts. Thus, these studies demonstrated antigen-specific suppressor T cells mediate the long-term unresponsiveness induced by the Ag-CsA regimen.

Blood transfusion therapy confers an immunosuppressive effect on organ transplant recipients. The nonspecific immunosuppressive effect of third-party transfusions is useful for CAD transplantation, while donor transfusions improve the outcome of LRD grafts. Although there is evidence that erythrocytes, modified lymphocytes and/or platelets mediate some effects, the optimal blood product is unknown. Similarly, the mechanism of the effect, be it related to induction of a suppressive immune response, prophylaxis against CMV, active viral infection or a side effect of blood such as iron overload, remains an important issue for investigation. Dissection of the immunosuppressive effect of transfusions may afford insights into host resistance to allografts and new methods to achieve transplantation tolerance.

Active specific immunotherapy, harnessing the strength and specificity of the host immune response to destroy neoplastic cells, may offer an ideal surgical adjuvant treatment modality for human colon cancer. Unfortunately, achievement of this goal has been obscured by 1) the effect of excess residual disease to interfere with the host's destructive response, 2) the weak nature of tumor resistance, 3) the potential adverse effect of concomitant treatments such as chemotherapy, and 4) the present limitation of poorly defined immunogens to induce, as well as insensitive assay systems to detect, host sensitizaion. Recent immunologic and chemical research revealing distinctive surface membrane structures on colon cancer cells suggests that a controlled trial of irradiated, autochtonous cell vaccines (without mycobacterial adjuvants) may provide a new therapeutic tool for Dukes B2 and C stages of human colon cancer.

A human colon epithelial tumor cell line (LS174T) recultured in vitro following passage through hamsters displayed differences in its cell doubling time and synthesis of carcinoembryonic antigen when compared with the cells grown solely in vitro. These animal-passaged cells more closely resembled the parent tumor cell line (LS180) derived from the primary tumor than LS174T, the trypsinized variant of LS180. Analysis of lactate dehydrogenase isoenzymes indicated that the tumor cells recovered from the hamsters were free of xenogeneic host tissue. Furthermore, LS174T grafted to athymic (nude) mice grew as a mucinous adenocarcinoma microscopically resembling the original tumor. The altered growth potential of LS174T was also demonstrated on confluent feeder monolayers of normal cells and by uninhibited multiplication in vitro. These results suggest that, at least in this one case, short-term passage of long-term cultured cells into xenogeneic hosts may effect a phenotypic reversion such that the cells regain properties observed in the primary tumor and the initial in vitro explant.

The effectiveness of local versus systemic low-dose CsA (2 mg/kg/day) therapy delivered by osmotic pump for a 14-day continuous infusion was examined in the rat model. Systemic subtherapeutic CsA treatment of WFu recipients either by oral gavage or intravenously using an osmotic pump resulted in quick rejection of BUF heart allografts within a median survival time (MST) of 8 days in comparison with untreated controls (MST = 7 days). In contrast, direct local subtherapeutic CsA delivery to BUF heart allografts produced significantly (P less than 0.01) prolonged heart allograft survivals up to MST of 40 days. Splenic T cells, isolated on days 10 to 12 from locally immunosuppressed WFu recipients, revealed a nonspecifically reduced proliferative response toward alloantigens. Coculture experiments demonstrate that these T cells have the capacity to inhibit normal T cell proliferative responses in a nonspecific fashion either by their suppressor function or more likely by carrying CsA to the culture plate. In contrast, T cells isolated from WFu recipients three weeks after transplantation and tested in vitro demonstrated the presence in alloantigen specific T suppressor cells that coincided with a decreased frequency of alloantigen-specific T cytotoxic cells and may explain the extended heart allograft survival beyond the time of CsA delivery. CsA therapy delivered directly to the graft resulted in high CsA levels within the heart graft (1108 ng/0.1 g) but subtherapeutic levels in other tissues. These results demonstrate that local drug delivery is effective in inhibiting the rejection process within the graft itself, as manifested by prolonged heart allograft survival. Further, subtherapeutic CsA therapy facilitates development of Ts cells that may be responsible for the survival of heart allografts beyond the CsA delivery time.

The impact of the presence of the allograft on the induction of unresponsiveness by the immunosuppressive combination of cyclosporine (CsA) and 3M KCl-extracted histocompatibility Antigen (Ag) was assessed by comparing the outcome of renal transplants when the regimen was used pretransplantation versus peri- and posttransplantation. WFu rat hosts, which had been pretreated with either one (-11, -10, -9) or three (-25, -24, -23, -18, -17, -16, -11, -10, -9) cycles of CsA and BUF Ag (-11 or -25, -18, -11) prior to implantation of BUF renal allografts, failed to display the prolonged graft survival achieved with this regimen administered in the peri- (-1, 0, +1) and immediate posttransplant (+7, 8, 9, 14, 15, 16) period. However, OX-8 positive, putative T-suppressor (Ts) cells in the spleens of pretreated hosts were able to transfer slightly prolonged BUF allograft survival to virgin, secondary syngeneic hosts. The OX-8-positive cells induced by pretreatment were apparently inhibited from prolonging BUF allografts in primary hosts by cellular elements vulnerable to splenectomy, total-body irradiation, or a three-day peritransplant course of CsA therapy. Therefore the presence of the renal graft at the time of peri and immediate posttransplant administration of Ag-CsA facilitates the induction of unresponsiveness, possibly due to continued release of histocompatibility antigen to stimulate, and/or to providing an important peripheral environment promoting differentiation and maturation of, Ts cells.

A tumor-derived suppressor factor ( TDSF ) has been isolated from 3 M KCl extracts of a chemically induced fibrosarcoma of C3H/HeJ mice by preparative isoelectric focusing. Incubation of TDSF with normal spleen cells induces suppressor cells that enhance tumor growth and inhibit DTH to the chemical sensitizer 2,4-dinitro-1-chlorobenzene (DNCB). Similar suppressogenic activity has been detected in extracts of the 10T1/2 fibroblast line, an ultraviolet-induced fibrosarcoma of C3H/HeN mice, the C57B1/6J Lewis lung carcinoma, and four human colonic adenocarcinoma. TDSF activity was not found in extracts of syngeneic muscle or spleen cells. Chemical characterization of TDSF from the murine fibrosarcoma MCA-F revealed sensitivity to treatment with heat and RNase, partial sensitivity to treatment with trypsin, but resistance to treatment with DNase, pronase, and neuraminidase. TDSF has an apparent molecular weight of greater than 300 kDa by high-performance gel permeation chromatography. Acidic soluble factors isolated from murine and human tumors induce suppressor cells to inhibit cell-mediated immunity in an intact host.

A series of human colonic epithelial cell lines have been cultured from a single patient: LS-180 the original adenocarcinoma, LS-174T a trypsinized variant, and normal colonic tissue. The malignant cells, 20 to 40, mum in diameter and oval to polygonal, exhibited characteristics of normal colonic mucosal cells, namely, abundant microvilli prominent in secretory cells, and the presence of intracytoplasmic mucin vacuoles. The cultured adenocarcinoma cells, but not normal, demonstrated neoplastic properties by producing high levels of carcinoembryonic antigen (CEA) and by the ability to be propagated in hamster cheek pouches and in immunodeprived mice. The CEA production by the newly established line LS-180 released 900 times more CEA per cell into the culture medium and bore 30 times more cell-associated material than the established line, HT-29. These cell lines may permit detection of distinctive chemical, physiological, pharmacologic, and immunologic characteristics of neoplastic colonic cells.

From July 1982 to August 1986, 137 patients received heart allografts at our transplant (Tx) center. Recipients were treated postoperatively with cyclosporine (CsA) and prednisone (Pred), with a minority of patients receiving CsA, Pred, and azathioprine (Aza) as immunosuppression. The impact of pre-Tx immune factors on survival was evaluated, including HLA A, B, and DR mismatches (MM), blood transfusions (BT), immune responder status, crossmatch results, and donor and recipient AIDS-virus (human immunodeficiency virus, HIV-1) status. The overall patient survivals were 75%, 68%, and 62% at one, two, and three years respectively. Pre-Tx, 15/137 (11%) recipient sera and 5/137 (3.6%) donor sera were HIV-1 reactive in both enzyme immunoassay (EIA) and Western blot antibody assays. Two of the 5 recipients of HIV-1 (+) donor allografts are alive 11 and 29 months post-Tx, whereas the other 3 recipients died at 1, 31, and 36 months post-Tx from diseases unrelated to AIDS. All 5 were pre-Tx HIV-1 nonreactive. The survivals for the 15 recipients who tested pre-Tx HIV-1 (+) were 87%, 87%, and 69% at 1, 2, and 3 years, respectively, comparable to the overall group survivals. Pre-Tx strong and weak immune responders had comparable 12-month survivals of 73% and 80%, respectively. Six patients displayed a positive pre-Tx donor crossmatch, two were attributed to autoantibody, and 4 were attributed to donor T cell reactivity. Five of the six patients presently survive 14, 16, 30, 36, and 44 months post-Tx. Recipients treated pre-Tx with 1-4 BTs displayed significantly better 12-month survival (81% vs. 69%, P less than 0.05) and fewer rejections (1.3 +/- 0.9 vs. 1.9 +/- 1.0, P less than 0.05) than untransfused recipients. Recipients of a 0-1 vs. 2 DR donor antigen-mismatch experienced fewer rejections (1.3 +/- 1.0 vs. 1.8 +/- 1.1, P less than 0.05). Evaluation of the combined influence of HLA DR as well as pre-Tx BTs suggested a significantly improved survival (80% vs. 61%, P less than 0.05) and fewer rejection episodes (1.4 +/- 0.9 vs. 2.0 +/- 1.1, P less than 0.05) for 29 well-matched, transfused (0-1 DR MM and 1-4 BT) compared with 43 poorly matched, untransfused (2 DR MM and 0-BT) heart allograft recipients. Moreover, the benefit of DR matching was only observed in untransfused, but not transfused, cardiac recipients.

Giemsa-banded chromosomes were analyzed at intervals during either 34 or 70 serial subcultivations of two cell lines, LS180 and LS174T, established from one primary human colon adenocarcinoma, and at passage 14 of autochthonous normal bowel cells, NB(LS174T). The cell lines were established and subcultured by either scraping or trypsin treatment of primary cultures; the scraped cell line was designated LS180, and the trypsin-dispersed cell line was named LS174T. Early passages of LS174T cells were composed mainly of 46,XX (38%) and 45,X (34%) karyotypes; LS180 cultures possessed cells with 46,XX (54%), 45,X (7.5%), and 47,XX+D (19.5%) chromosome modes. In both cell lines, the 45,X karyotype predominated in later subcultivations. After the fifth passage, all LS180 cells examined exhibited a translocation from the long arm of the X chromosome to the long arm of the No. 5 chromosome. Cultures from the patient's normal bowel mucosa and peripheral blood leukocytes had normal 46,XX karyotypes. Genetic signature analysis sustantiated the common genetic origin of the cell lines, and we concluded that differences observed between LS180 and LS174T were not due to contamination with other cell lines. LS180 and LS174T represent closely related cell lines differing cytogenetically in a translocation.

Radionuclide studies in a renal-transplant patient with congestive heart failure suggested vascular steal from the renal allograft by a contralateral femoral arteriovenous fistula. These reliable, noninvasive diagnostic procedures have potential use in similar settings to evaluate allograft perfusion and function. Correction by removal of the fistula was demonstrated.

The purpose of this investigation was to examine the ability of single-phase aqueous solutions of 1-butanol to release immunoprotective tumor antigen activity from partially purified plasma membranes of the methylcholanthrene-induced fibrosarcoma, MCA-F. Tumor antigen activity was assessed by s.c. immunization of syngeneic C3H/HeJ mice 10 days prior to supralethal challenge. Although brief incubation of intact MCA-F cells in 2.5% butanol releases potent immunoprotective activity, application of this protocol to plasma membranes did not result in antigen extraction. Modification of the extraction protocol using higher concentrations of butanol and longer extraction times did release measurable tumor antigen activity. However, a significant amount of the membrane-associated activity remained with the insoluble membrane fraction, as demonstrated by the immunoprotective capacity of the extracted membranes. The dramatic difference in the extractability of antigen from intact cells and plasma membranes suggested that membrane architecture may influence antigen release. To investigate this possibility, we extracted with butanol MCA-F cells that had been preincubated in colchicine. Treatment of cells with colchicine significantly potentiated the extraction of tumor antigen activity. Augmentation of antigen yield was also observed when plasma membranes were pretreated with colchicine prior to 2.5% butanol extraction. These results suggest that the tumor-specific transplantation antigen may be directly or indirectly associated with the cytoskeleton underlying the plasma membrane.

The pretransplant cellular immune responsiveness of 90 renal failure patients was correlated with subsequent allograft survival. Patients were subdivided in two bases: whether the pretransplant immune parameter values were above (strong responder) or below (weak responder) the group median, and whether they were responsive or anergic to recall skin test antigens. In a group of 72 cadaveric renal allograft recipients, treated with only Imuran and prednisone, the overall 1-year graft survival was 48%. Pretransplant immunocompetence correlated with graft survival: factors predicting longer allograft survival (P < 0.01) included: percentage of active T rosette-forming cells (A-T RFCs) < 36.5%, anergy to microbial skin test (ST) antigens, in vitro spontaneous blastogenesis (SB) < 14,600 cpm, and response to a panel of five donors in mixed lymphocyte culture (PMLC) < 28,000 cpm. In the two groups, weak and strong responders, the 1-year graft survival rates differed: 63% versus 32% when segregated by the A-T RFC parameter, 63% versus 33% for ST, 57% versus 36% for SB, and 63% versus 35% for PMLC. There were no significant differences in the number of HLA mismatches between the two groups. An additional group of 18 patients was treated with adjuvant immunosuppressive therapy by prophylactic administration of antithymocyte globulin (ATG; Upjohn Co.). Strong, but not weak, responders treated with ATG displayed a significantly improved (P < 0.01) 1-year graft survival over that of the untreated group. Thus, pretransplant immunological assessment may guide the selection of adjuvant immunosuppressive therapy to improve renal allograft survival in strong immune responders at high risk of rejection.

Previous work has demonstrated that cyclosporine inhibits generation of the cytoplasmic activation signal. The present study shows that the drug also diminishes the proliferative response of isolated nuclei upon phytohemagglutinin, OKT3, or mixed lymphocyte culture stimulation. Following in vitro action of normal human peripheral blood lymphocytes, nuclei isolated by homogenization and differential centrifugation were tested for 3H-thymidine incorporation upon stimulation with cytoplasmic fractions derived from homologous or heterologous activators. Nuclei isolated from CsA-treated cells showed significantly reduced responses to activation by cytoplasmic fractions from cells stimulated by all three agents with inhibition of OKT3 greater than MLC greater than PHA activation. This inhibition was less than the effect of CsA to disrupt generation of the activation signal by cytoplasmic fractions. Exogenous addition of Interleukin 2 to PHA-treated cells almost completely overcame the inhibitory effect of CsA on both nuclei and cytoplasm. A direct effect was documented by incubation of isolated nuclei with CsA prior to addition of a cytoplasmic activator and assessment of 3H-thymidine incorporation. The degree of direct nuclear inhibition by CsA was proportionate to the intranuclear drug concentration: cells displaying greater than 25% inhibition had higher intranuclear CsA concentrations (126 +/- 49 ng/ml) than unaffected cells (43 +/- 3 ng/ml) (P less than 0.005). These data suggested direct effects of CsA on nuclear proliferative responses, probably related to intranuclear drug binding and independent of its action to inhibit cytoplasmic transduction of the activation signal.

Three properties of cell surface membranes of normal bladder epithelium and of two malignant urothelial lines transformed in vitro by Dr. Y. Hashimoto and Dr. H. S. Kitagawa were analyzed by means of morphological and chemical tools. Under scan electron microscopy, normal bladder epithelium displayed an appearance devoid of the pleomorphic, abundantly distributed, microvillous structures seen on the neoplastic cells. The molecular weight profiles of proteins dispersed from purified plasma membrane fractions demonstrated quantitative differences in the content of three molecular-weight classes between the native and the transformed cells. More striking differences were seen upon two-dimensional analysis of proteins solubilized from these two cell types, using 3 M KCl. These findings suggest that further investigations of the chemical moieties appearing on the cell surface early after transformation may enhance our understanding of proteins amenable for chemical and/or immunological attack to achieve control of the progression of bladder neoplasia.

CsA-Pred therapy yields equivalently good patient survival for LRD and 2 degrees CAD versus 1 degree CAD transplants. There is a long-term graft survival advantage for LRD versus 1 degree CAD transplants (5 years; 83% vs 58%). 2 degrees CAD transplants have inferior graft survival when compared with 1 degree CAD grafts (one year; 78% vs 67%). Multiple donor factors adversely affecting graft outcome include increased warm and cold ischemia times, pulsatile perfusion, use of pressors or diuretics in the donor, donor age less than 10 years, donor blood transfusions, and kidneys shipped from other centers. Recipient factors adversely affecting graft outcome include retransplantation and CMV infection as well as noncompliance with therapy. HLA-matching and pretransplant blood transfusions have not contributed in a statistically significant way to graft outcome although they may affect the quality of graft function at this center. Immunosuppressive therapy with CsA-Pred must be tailored to the individual patient. Continuous IV CsA infusion in the preoperative period and slow steroid taper impact favorably on graft outcome. The complications of CsA therapy include neuroectodermal toxicity, hepatotoxicity, and most importantly, nephrotoxicity. Other problems unique to CsA-Pred therapy include hypertension, delayed graft thrombosis, and de novo hemolytic uremic syndrome. Hepatotoxicity may eventuate in biliary and pancreatic complications necessitating surgical therapy. The overall incidence of infection and neoplasm remains low with CsA-Pred therapy. The use of therapeutic trough CsA level monitoring, as well as pharmacokinetic and pharmacodynamic analyses may assist in clinical decision making regarding administered doses, dosing interval, and discrimination between rejection and nephrotoxicity.

The fibrosarcoma MCA-SP, which was recently induced with methylcholanthrene (MCA) in C3H/HeJ mice, displays poor immunogenicity in in vivo prophylaxis. A cell variant MCA-SPN1, which bears a tumor-specific transplantation antigen (TSTA) cross-reactive with the parental line MCA-SP, was selected because of its proclivity for axillary lymph-node metastases. Although these lymph-node metastases were resistant to sinecomitant (post-excisional) immunity, they were susceptible to combined active and passive specific chemoimmunotherapy, using tumor-specific, 1-butanol-extracted, preparative isoelectric focusing-purified, TSTA (1 microgram weekly sc injections), cyclophosphamide (CY, a single intraperitoneal 20 mg/kg dose), and adoptive transfer of immune splenic T lymphocytes, which had been re-stimulated in vitro with extracted TSTA and interleukin-2. This triple regimen both reduced the incidence of spontaneous lymph-node metastases, and prolonged the survival of tumor-bearing, as well as tumor-resected hosts. The results from local adoptive transfer assay using T-lymphocyte subpopulations of spleen and lymph nodes in these treated hosts suggested that Lyt 2+ cytotoxic T-lymphocytes (CTL) mediated in vivo tumor-neutralization. Thus TSTA/CY/CTL therapy activates tumoricidal host responses effective against the poorly immunogenic MCA-SP tumor and its lymph-node metastases.