Biomedical subjects
Publications and source records attributed to R B Colvin.
DNA ploidy of paraffin-embedded tissue from 11 patients with epithelioid sarcoma and 20 control specimens (granuloma annulare and sarcoid) was studied. DNA aneuploidy was found in 64% of the epithelioid sarcomas and in none of the control samples. Aneuploidy did not correlate with necrosis, vascular invasion, or mitotic rate. There was also no association of aneuploidy with adverse outcome.
The role of cell adhesion molecules (CAM) LFA1, ICAM-1, LFA3, VLA1, VLA4, CD29, CD44, and CD56 in tumor-infiltrating lymphocyte (TIL) and natural killer cell (NK)-mediated killing of target cells was studied. Melanoma cell lines and autologous TIL were derived from seven patients with metastatic melanoma, and cytotoxicity assays were done in the presence and absence of monoclonal antibodies (MoAb) to CAM expressed on melanoma cells or TIL. The melanoma cell lines analyzed were all positive for CD29 and LFA3 expression, negative for LFA1 expression, but showed variable expression of ICAM-1, VLA1, VLA4, CD44, and CD56. The effects of anti-CAM antibodies on TIL-mediated melanoma killing fell into three categories: (1) consistent inhibition of TIL-mediated killing was observed when melanoma cells were pretreated with anti-ICAM1 and anti-LFA-3 MoAb or when TIL were pretreated with anti-LFA1; (2) no effect was observed when melanoma cells were pretreated with anti-CD56; or (3) a discreet, but significant, inhibition was observed when target cells were pretreated with anti-CD29, anti-VLA1, anti-VLA4, and anti-CD44. Cytotoxicity was significantly enhanced by pretreatment of target cells with gamma-interferon (gamma-IFN), although gamma-IFN did not augment surface expression of the CAM studied. The NK-mediated killing of K562 cells was blocked by anti-LFA1, anti-CD18, and anti-ICAM, and partially inhibited by anti-CD44 MoAb. Together, these results suggest that several accessory CAM may play a role in regulating cellular cytotoxicity. Because cytotoxicity generally correlated with the level of expression of CAM in melanoma cells, weak CAM surface expression may provide a means for melanomas to escape immune surveillance.
Paraffin-embedded tissue from 38 patients with early- and advanced-stage juvenile granulosa cell tumor (JGCT) of the ovary was analyzed by flow cytometry to investigate whether the DNA content is related to the histologic features, stage, or clinical outcome. Thirty-three cases were suitable for analysis: twenty-seven Stage Ia, two Stage Ic, and four Stage III. Eighteen (55%) tumors were DNA diploid and fifteen (45%) tumors were DNA aneuploid with a mean S-phase fraction (SPF) of 13.6% and a range of DNA indices from 1.0 to 2.2. Neither the DNA ploidy nor the SPF was associated with the stage of the disease. An analysis of the relation between DNA content and histopathologic features revealed that aneuploidy was associated with high mitotic rates and to a lesser extent with high-grade nuclear atypia. DNA aneuploidy was not associated with aggressive behavior of Stage Ia JGCTs. However, among the four patients with Stage III tumors, the two with diploid, low-SPF tumors were alive and well, whereas the two with aneuploid, high-SPF tumors developed recurrences or died. These data suggest that further studies on the prognostic significance of flow cytometric analysis of DNA content in advanced-stage JGCTs are warranted.
The TCR and CD8 complexes of CD8+ T cells bind to different regions of MHC class I molecules and both play important roles in the response of the CD8+ T cells to Ag/MHC on APCs. In this report, we mimicked common MHC binding with an anti-CD3:anti-CD8 (CD3,8) BSMAB to isolate the effect of CD3: CD8 pairing, compared this with the effect of CD3: CD3 pairing by the parental bivalent anti-CD3 MAB, and with monovalent anti-CD3 binding by an anti-CD3: anti-CD4 (CD3,4) BSMAB. CD3: CD8 pairing induced an increase in cytosolic free [Ca2+] 1.5 to 3.0-fold greater than the increase induced by CD3: CD3 pairing whereas monovalent CD3 binding induced only 20%-30% of the increase. Postbinding receptor migration studies suggested that microaggregation increased from monovalent CD3 binding to CD3: CD3 pairing to CD3: CD8 pairing. Further studies revealed that progressively higher concentrations of antibodies were needed from CD3,8 to CD3,3 to CD3,4 to initiate the same degree of DNA synthesis. These results demonstrated that Ti/CD3 and CD8 can indeed be bridged by a single molecule. A model of direct CD8: CD3 synergism was raised as a possible explanation for the enhanced activation induced by CD3: CD8 pairing. The observed parallel between all three parameters and the number of TCRs that can be directly linked by the Abs raised a nonmutually exclusive model whereby CD3 binding induces activated TCR intermediaries (aTCRi) that progressively synergize with other adjacent aTCRis. In this model, this dominant inter-aTCRi synergism may be enhanced by the di- and multimeric CD8 alpha chains serving as aTCRi-aggregation foci.
Tumor deoxyribonucleic acid (DNA) ploidy was evaluated as an objective parameter that may correlate better with the responsiveness of bladder cancer to chemotherapy plus radiotherapy than do clinical features or histopathological subtypes. A total of 40 patients with localized muscle-invading bladder cancer (clinical stages T2 to T4) underwent prospective treatment on a potential bladder preserving protocol. Tumors of 37 of the 40 patients were analyzed by DNA flow cytometry of multiple paraffin embedded specimens. Transurethral resection, neoadjuvant chemotherapy and 40 Gy. radiotherapy plus cisplatin were followed by urological reevaluation of the tumor (a complete response required a negative biopsy and a negative urine cytology study). A total of 7 noncomplete response patients underwent immediate radical cystectomy whereas the full bladder sparing treatment (radiotherapy to 64.80 Gy. plus cisplatin) was given to 23 complete response patients and 7 noncomplete response patients who were unsuited for surgery. Of the tumors 22 (59%) were purely aneuploid and 10 (27%) were purely diploid. Five tumors contained aneuploid and diploid patterns in different tumor specimens (partly diploid). Current status with a 30-month median followup in surviving patients includes an 82% overall survival rate in the aneuploid group versus 47% in the diploid/partly diploid group. Of all the patients 68% are free of invasive tumor: 82% in the aneuploid group versus 47% in the diploid/partly diploid group. By multivariate analysis pure aneuploidy was significantly (p = 0.05) correlated with freedom from invasive tumor in the bladder (either as persistence or as recurrence) and approached significance (p = 0.08) in correlation with overall patient survival. A longer observation time will be required to confirm this unexpectedly good outcome for patients with pure aneuploid tumors. We hypothesize that pretreatment DNA ploidy status may become a clinically useful prognostic factor in selecting patients for successful treatment with transurethral surgery and neoadjuvant chemotherapy plus radiotherapy.
Significant differences in cellular responses were found among allograft recipients treated with various OKT4A mAb protocols. Recipients of multiple infusion low-dose and 2-bolus OKT4A immunosuppressive regimens regularly showed potent donor-specific cytotoxic CD8+ and CD4+ intragraft T cells and donor-reactive PBMC in MLC tests. In contrast, PBMC isolated from recipients of high-dose OKT4A therapy generally showed very weak or no response to donor-antigens during the later posttransplant periods. Furthermore, an absence of IL2-responsive intragraft cells was found to correlate with stable graft function in these recipients. We conclude that OKT4A mAb, in high doses, can block allosensitization and induce donor-specific nonresponsiveness in vivo. An OKT4A-based therapy, therefore, may have the potential of inducing long-lasting donor-specific immunosuppression, or even tolerance.
CD3,4 (anti-CD3:anti-CD4) bispecific monoclonal antibodies (BSMAB) cause a profound decrease in CD4+ T cells and a marked proliferation of CD8+ T cells in peripheral blood mononuclear cells in vitro. CD3,8 (anti-CD3:anti-CD8) BSMAB causes a reciprocal decrease in CD8+ T cells and a proliferation of CD4+ T cells. The major effector of CD4+ T cell cytolysis in the presence of CD3,4 resides in the CD8+ T cell population. In contrast, both the CD4+ and CD8+ T cells are effective mediators of cytolysis of the CD8+ T cells in the presence of the CD3,8. The likely underlying mechanism in each case is bridging of the CD4 and CD8 of the target cells to the CD3 complexes of the effector cells by antibodies, mimicking the natural encounter between a cytolytic T cell and its target. Proliferation studies indicated that CD3,4 and CD3,8 each can induce proliferation of both CD4+ and CD8+ T cells in the presence of accessory cells. These results suggest that the major selection of the BSMABs occurs via selective destruction of one T cell subset with concurrent stimulation of the remaining CD3+ population. Potential applications of the selective destruction and proliferation include study and manipulation of the T cell subsets in HIV infections, tumor infiltrating lymphocytes, autoimmune diseases, and graft rejection.
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Fibronectins (FN) in guinea pig lymphoid cell culture supernatants have been studied using a panel of polyclonal and monoclonal anti-FN antibodies to clarify their relationship with macrophage agglutination factor (MAggF), an inflammatory lymphokine sharing many properties with this family of high molecular weight glycoproteins. MAggF contained cellular FN epitopes, and was reversibly bound by antibodies specific for cellular FN. Enzyme-linked immunoassay and inhibition of MAggF activity by monoclonal anti-plasma FN antibodies revealed immunoreactive FN in guinea pig lymphoid cell culture supernatants to share three epitopes with plasma FN and to lack a fourth epitope present in plasma FN. Immunoreactive FN in gelatin-affinity purified lymph node cell culture supernatants was polydisperse; MAggF activity (Mr 410 kD) was associated with only 13% of total immunoreactive FN. Although a low molecular weight FN fragment (Mr 67 kD) was associated with MAggF activity in salt-fractionated peritoneal exudate culture supernatants, it was not possible to generate MAggF activity by limited proteolysis of MAggF-inactive, high molecular weight FN in lymph node cell culture supernatants. We conclude that MAggF is a cellular FN containing a number of epitopes in common with plasma FN and suggest it may be a unique species of cellular FN produced by T lymphocytes involved in initiating delayed hypersensitivity reactions.
Monoclonal antibodies were used to determine the relationships between epitopes on the p55 chain of the IL-2 receptor and high-affinity IL-2 binding. Five monoclonal antibodies to the human P55 chain of the IL-2 receptor were induced by immunizing mice with murine L cells that were transfected with human p55 cDNA. Since the p55 chain is the only human antigen expressed on these cells, all antihuman MABs thus generated were directed against this molecule. These antibodies were used to map epitopes on the p55 chain and determine their relationship to high-affinity IL-2 binding. Extensive flow cytometric studies with these MABs and a large panel of other anti-p55 MABs revealed three major patterns of competition. Type I MABs compete with anti-Tac extensively but not with antibodies of other groups. Type II MABs do not block anti-Tac but do block 7E11. Type III MABs do not block either type I or type II antibodies. 125I-IL2 competition studies under high-affinity conditions revealed that types I and II MABs inhibit IL-2 binding. Type III MABs can be resolved into two subgroups, one that inhibits IL-2 binding and one that does not. Together these data suggest that there are at least four distinct immunogenic epitopes on the human p55 chain, with three epitopes related to IL-2 binding. The competitive component evident by a change in Kd on the Scatchard plots suggests that all three epitopes are close to or part of the IL-2-binding site of the p55 chain. The noncompetitive component, as evidenced by the lower number of high-affinity IL-2 receptors induced by these antibodies, suggests that the same epitopes are also close to the site(s) of interaction between the p55 and p70 chains to form the high-affinity receptor. These studies indicated that the IL-2-binding site and site of interaction between the p55 and p70 chains are close together or identical. Modulation studies revealed that one type II antibody (7E11) modulates the p55 chain in the absence of IL2 and the p70 chain, thus revealing that modulation of the p55 chain can occur by an active process, and not merely passively comodulate by the p70 chain upon IL-2 binding. Modulation of the p55 chain alone has no proliferative effect on IL-2-responsive T lymphoblasts. Potentially this antibody-dependent modulation may be used to deliver toxin to activated lymphocytes.
Induration is a characteristic feature of delayed-type hypersensitivity skin reactions and is the usual measure of their intensity. The precise basis of induration has not been established, although activation of the clotting system with consequent fibrin deposition has been clearly implicated. In this study, two subjects with congenital afibrinogenemia, a genetic defect in fibrinogen synthesis, were skin tested with standard microbial antigens: streptokinase-streptodornase, monilia, mumps, and tuberculin purified protein derivative. One positive delayed reaction from each subject was biopsied at 40-48 h and compared with 23 biopsies of similar skin tests in normal volunteers. The eight skin tests in the afibrinogenic subjects lacked induration, although the erythema was similar in size (10-34 mm in diameter), intensity, and time-course to those in normals. Biopsies from the two strongest reactions from the afibrinogenemic subjects showed a typical perivascular mononuclear infiltrate. No more than traces of fibrin/fibrinogen were detected by immunofluorescence, in striking contrast to the abundant fibrin/fibrinogen deposition in 23 positive, indurated reactions in normal subjects. These findings indicate that fibrinogen itself is essential for the development of induration in delayed-type skin reactions in man. As judged by 1-mum sections and fluorescence, this is probably a result of the formation of an extravascular fibrin gel.
Renal allografts were performed between and among animals from three herds of miniature swine that were selectively inbred to homozygosity at the major histocompatibility complex, MSLA. The results suggest several genetic factors which influence the survival of renal allografts in these animals. As expected, the major histocompatibility complex (MHC) was of dominant importance, and all MSLA-mismatched grafts were rejected promptly (12 +/- 3.7 days). Some MSLA-matched grafts were also rejected (30 +/- 15.0 days), indicating that non-MSLA loci also determine antigens which can lead to kidney rejection. Other MSLA-matched grafts were accepted indefinitely. At least one immune response gene that determined ability to reject kidneys across non-MSLA differences seemed to be segregating in our swine population. Animals that had accepted MSLA-matched renal grafts for extended periods demonstrated markedly prolonged survival of subsequent donor skin grafts compared to skin graft survival across the same non-MSLA difference in normal animals. This finding suggests that failure to reject kidneys across non-MSLA differences indicates systemic tolerance, and that there may be a relationship between the induction of such tolerance and the proposed immune response gene controlling rejection.
Renal allografts were performed in miniature swine that were identical at their major histocompatibility locus and were presensitized by skin grafts from their prospective renal donors. All of these renal grafts were rejected in a hyperacute or markedly accelerated manner compared to the survival of comparable grafts in nonsensitized animals. Studies directed at the mechanism of this rejection revealed no circulating recipient antidonor antibodies by several serological assays. In contrast, mixed lymphocyte cultures (MLCs) and cell-mediated lympholysis (CML) assays demonstrated marked recipient antidonor lymphocyte reactivity that appeared after skin grafts, diminished during the tenure of the renal graft in the host circulation, and reappeared after removal of the rejected kidney. These results suggest that cellular immune mechanisms may plan a role in the accelerated rejection of major histocompatibility complex (MHC)-identical renal allografts.
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Kidney transplants between strains of mice which are incompatible at either the K or the D end of the H-2 complex usually function for prolonged periods supporting the lives of nephrectomized recipients. This occurs with no recipient treatment. With multiple H-2 and non-H-2 determined incompatibilities, transplants may be rejected but more slowly than skin grafts. In the strain combination studied most extensively in these experiments (B10.D2 to B6AF(1)) in which the incompatibility was confined to the K end of the H-2 region, about 70 percent of recipients survived for many weeks with normal blood urea nitrogen levels. Skin grafts between untreated members of these strains were rejected promptly (mean survival time of 13.5 +/- 1.1 days) as were kidney transplants to recipients of prior skin grafts. Donor strain skin grafts to recipients of kidney transplants after kidney transplantation enjoyed greatly prolonged survival whereas skin grafts from a third party (A.SW) were rejected normally. If kidney tissue was transferred in the form of free grafts without primary vascular union, it was rejected promptly leaving its recipient highly immunized. Cellular and humoral immunity to donor antigens declined over the first few weeks after transplantation, and the spleens of long-term recipients contained no "killer cells." Recipient lymphoid cells could mount active graft versus host reactions to donor strain antigens on transfer to neonatal mice. Nevertheless, they were distinctly less able to respond specifically by the production of killer cells to donor strain antigens after sensitization in vitro. No evidence that this defect was associated with the presence of suppressor cells was forthcoming from several types of in vivo and in vitro tests.