Immunotherapy of a carcinogen-induced murine sarcoma with soluble tumor-specific transplantation antigens.

Tumor-specific transplantation antigens (TSTA), extracted from the 3-methylcholanthrene-induced sarcoma MCA-F and partially purified by flat-bed isoelectric focusing, were used to treat syngeneic inbred C3H/HeJ mice bearing supralethal neoplastic challenges. Three weekly injections of 25 micrograms TSTA increased the survival times of hosts inoculated 1 day earlier with ten times the lethal dose of MCA-F cells. In another protocol TSTA injections decreased the incidence and outgrowth of a local metastasis in mice given sc supralethal inoculations and completely resected of established 1-cm tumors. In addition, weekly injections of 25 micrograms MCA-F TSTA decreased the tumor recurrence rate and increased the survival times of hosts with recurrent neoplastic disease by virtue of residual tumor cells following resection of 2-cm masses. The therapeutic effect of TSTA was immunologically specific: Animals cured of local MCA-F recurrences promptly died from primary challenge with the non-cross-reacting sarcoma MCA-D. The results suggest that active specific immunotherapy may represent a useful adjunct to treatment of hosts bearing modest tumor burdens.

Extraction of a murine tumor-specific transplantation antigen with 1-butanol. I. Partial purification by isoelectric focusing.

Treatment of whole viable MCA-F murine sarcoma cells from syngeneic female C3H/HeJ mice with a single-phase solution of 2.5% (vol/vol) 1-butanol yielded a crude membrane protein extract without loss of cell viability. 1-Butanol-extracted cells were capable of in vitro proliferation. Variations in extraction conditions significantly influenced not only protein yield but also viability. The crude butanol extract (CBE) contained a tumor-specific transplantation antigen (TSTA) more potent than that obtained by 3-M KCl extraction of MCA-F cells: a specific activity of 40 TSTA U/mg protein in the former and 2 U/mg in the latter. The antigen was specific and protected host against challenge with MCA-F cells but not the antigenically different MCA-D cells. The growth of MCA-F cells was not reduced by pretreatment with equivalent doses of CBE obtained from MCA-D cells. Partial purification of the antigenic activity by preparative isoelectric focusing revealed that most activity focused in the pH 6.4 fraction, which increased the specific activity to 1,000 TSTA U/mg protein. Thus 1-butanol was an effective agent for the extraction of certain membrane polypeptides, including an immunogenic TSTA, from whole cells.

Immunotherapeutic effects of partially purified tumor-specific transplantation antigens on pulmonary metastases of a 3-methylcholanthrene-induced sarcoma in mice: a preliminary report.

Tumor-specific transplantation antigens (TSTA), which were partially purified by preparative isoelectric focusing of 3-M KCl extracts of a fibrosarcoma MCA-F induced in inbred female C3H/HeJ mice with 3-methylcholanthrene and previously shown to display immunotherapeutic activity against subcutaneous neoplasms, were effective against pulmonary metastases. Weekly sc injections of 25 micrograms fraction 15 decreased the number of pulmonary colonies after iv injection of tumor cells into syngeneic, virgin C3H/HeJ mice. The effect was immunologically specific; the immunoprotective fraction from the heterotypic, antigenically distinct MCA-D tumor did not affect the number of pulmonary MCA-F tumor colonies. Because fraction 15 treatment did not alter the number of extrapulmonary tumor colonies, the survival rates of hosts challenged iv with MCA-F cells were unaffected. The variant cell line MCA-F-4 was selected to detect prolonged host survival as a result of a therapeutic effect against pulmonary metastases. This line was selected for its proclivity for lung colonization and low propensity for growth in extrapulmonary sites. Cross-immunoprotection tests demonstrated that MCA-F-4 shares a TSTA with the parent tumor. Therapeutic administration of fraction 15 prolonged the survival of hosts in two settings: 1) after artificial iv injection of MCA-F-4 cells and 2) as treatment for hosts resected of 1-cm subcutaneous MCA-F-4 primary tumors and at high risk for spontaneous pulmonary metastases. Therefore, fraction 15 displayed therapeutic effects on both artificially induced and spontaneous pulmonary metastases.

Retardation of postsurgical metastases with the use of extracted tumor-specific transplantation antigens and cyclophosphamide.

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.

In vitro migration of lymphocytes through collagen matrix: arrested locomotion in tumor-infiltrating lymphocytes.

Antitumor immunity requires (a) extravasation of lymphocytes from the blood stream to interstitium, (b) locomotion through extracellular matrix to the site of the tumor, (c) effector cell recognition of the tumor target with cell/cell contact and binding of adhesion receptors, (d) T-cell receptor binding to histocompatibility and tumor antigens, and (e) tumor cell lysis. We hypothesize that the tumor microenvironment inhibits lymphocyte locomotion through extracellular matrix as one mechanism by which tumors may avert host defense. Lymphocyte locomotion was investigated in vitro using a three-dimensional collagen gel model. Fresh tumor-infiltrating lymphocytes (TIL) were obtained by enzymatic digestion of melanomas and renal cell carcinoma, and mononuclear cells were isolated by discontinuous Ficoll-Hypaque gradient. The lymphocytes were analyzed for motility from a point of origin between basal and overlay layers of collagen gel. Results showed that TIL migration was almost completely inhibited, compared with migration of normal and cancer patient peripheral blood leukocytes and lymphocytes from lymph nodes. Short-term (24-h) exposure of lymphocytes to cytokines during the assay in the collagen gel matrix had no effect on locomotor ability. Long-term (19, 30, or 35 days) culture of TIL in 200 units/ml of interleukin 2 reinstated locomotor ability. Short-term exposure of any of the lymphocyte populations to interleukin 1-alpha, interleukin 1-beta, interleukin 2, interleukin 3, interleukin 4, alpha-interferon, or gamma-interferon had no effect on migration. Thus, TIL display a uniquely arrested ability to locomote through collagen gel. Inhibition of the locomotion of infiltrating effector cells is possibly a mechanism by which the tumor evades the host immune system.

Effect of continuous administration of interleukin 2 on active specific chemoimmunotherapy with extracted tumor-specific transplantation antigen and cyclophosphamide.

Injection of purified human interleukin 2 (IL-2) directly into the spleen has been shown to potentiate the effect of specific chemoimmunotherapy, using butanol-extracted tumor-specific transplantation antigen (TSTA) and cyclophosphamide (CY) in a C3H/HeJ murine methylcholanthrene-induced fibrosarcoma model. Since IL-2 has a relatively short half-life in serum, continuous infusion of this lymphokine via the intrasplenic (i.s.), i.v., or i.p. routes was administered in an attempt to maintain therapeutic tissue levels. Primary hosts bearing 7-day (4-mm) or 14-day (greater than 10-mm) established s.c. methylcholanthrene F tumors were treated with weekly s.c. doses of 1 micrograms 1-butanol-extracted, isoelectrophoretically purified TSTA, the first of which was combined with a single i.p. injection of 20 mg/kg CY, and/or a 10-day continuous infusion of 120 units IL-2/day by one of the three routes. IL-2 delivered by all routes either by continuous infusion or by bolus injection augmented the chemoimmunotherapeutic efficacy of TSTA/CY against 7-day established tumors. On the other hand, the outcome of 14-day (greater than 10-mm) established tumors depended upon the method and route of administration of IL-2: continuous infusion via the i.v., i.p., or i.s. route prolonged host survival beyond that obtained by bolus administration. Continuous i.s.-IL-2 infusion greatly prolonged, continuous i.p.-IL-2 (120 units/day) slightly extended, and continuous i.v.-IL-2 had no effect on host survival. In a spontaneous pulmonary metastasis model following amputation of a tumor-bearing limb, only the triple regimen of TSTA/CY/i.s.-IL-2 decreased the number of lung colonies and prolonged host survival. Continuous infusion i.s.-IL-2 (120 units/day, 10 days) combined with TSTA/CY induced tumor-specific cytotoxic T-cells, as documented by in vitro 51chromium release cytolytic and in vivo local adoptive transfer assays. Based upon the residual local adoptive transfer assay activity of spleen cells depleted of specific lymphocyte subpopulations using monoclonal antibodies, the immune effectors generated by i.s.-IL-2 plus TSTA/CY bear the Thy 1+, Lyt2+ phenotype and those by i.p. or i.v.-IL-2 plus TSTA/CY, the Thy+, L3T4+ markers. Thus continuous i.s.-IL-2 infusion appears to augment cytotoxic T-cell induction in tumor-bearing hosts undergoing stimulation of helper elements by TSTA and inhibition of suppressor cells by CY.

Intrasplenic administration of interleukin-2 to potentiate specific chemoimmunotherapy in tumor-bearing mice.

Augmentation of specific chemoimmunotherapy by daily, intrasplenic injection of interleukin-2 (IL-2) was assessed in a methylcholanthrene (MCA)-induced fibrosarcoma model in C3H/HeJ mice. Daily access to the spleen was achieved by relocating the organ into the subcutis while leaving its blood supply intact. Following intrasplenic injection of 80 units of human IL-2 into MCA-F tumor-bearing mice for 6 days, spleen cells tested in the local adoptive transfer assay showed specific neutralization of MCA-F, but not the antigenically different MCA-D, tumor. Depletion of the spleen cell population with monoclonal antibodies and complement showed that the responding cell bore the surface markers Thy 1.2 and Lyt 2. Mice bearing established MCA-F tumors underwent a variety of chemoimmunotherapy regimens, including 1 microgram of 1-butanol, extracted isoelectrophoretically purified tumor-specific transplantation antigen, a single i.p. dose of cyclophosphamide (20 mg/kg), and/or either i.p. or intrasplenic injection of 80 units of IL-2. Specific triple chemoimmunotherapy including daily intrasplenic IL-2, but not i.p., administration was superior in the degree of tumor neutralization to all single or double therapy protocols. Furthermore, the combined triple modality inhibited spontaneous lung colonization by clone 9-4, a highly metastatic variant of MCA-F; both the numbers of lung colonies (median, 17; range, 2 to 55, versus median, 3, range, 0 to 42; P less than 0.005) and the incidence were decreased. The combined treatment group displayed 35% of hosts free of lung metastasis, while 100% of the control animals had lung colonies (P less than 0.02). Thus antitumor immunity was augmented in vivo using IL-2 delivered by intrasplenic, but not i.p., injection. Furthermore, chemoimmunotherapy including intrasplenic IL-2 injection potentiated the antitumor immunity achieved with combined tumor-specific transplantation antigen and cyclophosphamide.

Effects of concomitant and sinecomitant immunity on postsurgical metastasis in mice.

The role of concomitant and sinecomitant antitumor resistance in the regulation of metastatic outgrowth was assessed using methylcholanthrene (MCA)-induced tumors in C3H/HeJ mice. Variants of neoplasms MCA-F, MCA-D, and MCA-2A were selected for proclivity for spontaneous lung metastasis and expression of parental tumor-specific transplantation antigens. The incidence of spontaneous lung metastases after resection of a s.c. tumor of clone 9-4, a highly metastatic variant of the MCA-F tumor, was determined by both the size and the duration of neoplastic disease. The coexistence of the primary local tumor retarded lung colonization both from spontaneous and after artificially induced metastases. Greater concomitant immunity leading to a reduced number of artificial metastases after i.v. challenge with clone 9-4 cells was evident in hosts bearing large (1.6 to 1.8 cm) compared to small (0.1 to 0.2 cm) burdens of the nonmetastatic MCA-F (P less than 0.005). Furthermore, i.v. challenge of mice bearing antigenically different tumors revealed that the concomitant inhibition was antigen specific with small tumor burdens, but nonspecific and possibly more efficacious with large tumor burdens. Therefore, concomitant antimetastatic immunity consists of both specific, immune-mediated resistance and nonimmunological mechanisms. Specific concomitant immunity decreases inversely with the progression of the primary, while nonimmunological inhibition of metastasis increases during late stages of primary growth. Abrogation of the strong nonspecific concomitant inhibition by resection of the primary tumor may facilitate lung metastasis. On the other hand, significantly greater inhibition of metastases occurred after resection of 7- or 14-day neoplasms compared to larger tumors (P less than 0.001 or 0.05). Sinecomitant inhibition is antigen specific, probably representing an extension of specific concomitant immunity. These results suggest that adjunctive immunotherapeutic protocols for surgically treated hosts should augment existent specific immunity and promote nonspecific resistance, in order to minimize metastatic outgrowth.

Biochemical characterization of 1-butanol-extracted murine tumor-specific transplantation antigens.

This investigation sought to characterize biochemically the tumor-specific transplantation antigens (TSTA) expressed on the cell surface of a panel of chemically induced fibrosarcomas of C3H/HeJ mice. Results suggest a uniform antigenic framework upon which individual specificities are superimposed. The antigens expressed by the 3-methylcholanthrene-induced fibrosarcomas MCA-D, MCA-F, and MCA-2A fulfill the requirements of a TSTA; namely, immunization of syngeneic hosts with irradiated cells or soluble extracts engenders a tumor-specific immune response such that animals resist challenge with the same, but not another, tumor. Brief incubation of intact tumor cells in single-phase aqueous solutions of 2.5% (v/v) 1-butanol extracts an immunoprotective TSTA, but not alloantigenic activity, from MCA-F cells. This extraction protocol was extended to the two other MCA-induced neoplasms. The butanol-extracted TSTA from the three tumors displayed isoelectric pHs of 6.4 to 6.6 following preparative isoelectric focusing. The tumor-specific immunoprotective activity from all three tumors displayed an apparent molecular weight of 150,000 (150 kDa) during high-performance gel permeation chromatography. The chromatographic properties of the 150 kDa antigens were unaffected by reduction using dithiothreitol, but incubation in acetate buffer, pH 3.0, dissociated the 150 kDa complex into at least two components with molecular weights of 70 to 100 kDa and 20 to 40 kDa. Only the smaller component displayed TSTA activity. The presence of two major components in the 150-kDa antigen was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. TSTA activity was sensitive to digestion with pronase, papain, chymotrypsin, and alpha-mannosidase, but resistant to DNase, RNase, neuraminidase, trypsin, endoglycosidase H, and a mixed-function glycosidase. In addition, the TSTA activity was unaffected by heating. These data demonstrate that MCA carcinogenesis results in the expression of immunologically unique epitopes on biochemically related glycoproteins and suggest a unified mechanism for the generation of TSTA polymorphism.

Immunobiological properties of 1-butanol-extracted cell surface antigens.

Extracts of viable 3-methylcholanthrene-induced murine sarcoma cells (MCA-F and MCA-2A) prepared using single-phase (2.5%) 1-butanol significantly retarded the outgrowth of the homotypic, but not the heterotypic, tumor of syngeneic C3H/HeJ mice. Butanol extracts specifically evoked a delayed hypersensitivity response in tumor-immune syngeneic mice, but not in alloimmune DBA/2J mice. Crude butanol extracts of MCA-F cells did not contain alloantigenic activity, as shown by their inability to block H-2 or Ia-specific antibodies in a complement-dependent cytotoxicity assay. Absorption of these same allospecific reagents with untreated or with butanol-extracted cells indicated that H-2 antigens remain associated with the cell surface during extraction. Thus, butanol appears to release tumor antigens, but not alloantigens, from the cell surface.

Suppressed PHA activation of T lymphocytes in simulated microgravity is restored by direct activation of protein kinase C.

Utilizing clinostatic rotating wall vessel (RWV) bioreactors that simulate aspects of microgravity, we found phytohemagglutinin (PHA) responsiveness to be almost completely diminished. Activation marker expression was significantly reduced in RWV cultures. Furthermore, cytokine secretion profiles suggested that monocytes are not as adversely affected by simulated microgravity as T cells. Reduced cell-cell and cell-substratum interactions may play a role in the loss of PHA responsiveness because placing peripheral blood mononuclear cells (PBMC) within small collagen beads did partially restore PHA responsiveness. However, activation of purified T cells with cross-linked CD2/CD28 and CD3/CD28 antibody pairs was completely suppressed in the RWV, suggesting a defect in signal transduction. Activation of purified T cells with PMA and ionomycin was unaffected by RWV culture. Furthermore, sub-mitogenic doses of PMA alone but not ionomycin alone restored PHA responsiveness of PBMC in RWV culture. Thus our data indicate that during polyclonal activation the signaling pathways upstream of PKC activation are sensitive to simulated microgravity.

Countermeasure for space flight effects on immune system: nutritional nucleotides.

Microgravity and its environment have adverse effects on the immune system. Abnormal immune responses observed in microgravity may pose serious consequences, especially for the recent directions of NASA for long-term space missions to Moon, Mars and deep Space exploration. The study of space flight immunology is limited due to relative inaccessibility, difficulty of performing experiments in space, and inadequate provisions in this area in the United States and Russian space programs (Taylor 1993). Microgravity and stress experienced during space flights results in immune system aberration (Taylor 1993). In ground-based mouse models for some of the microgravity effects on the human body, hindlimb unloading (HU) has been reported to cause abnormal cell proliferation and cytokine production (Armstrong et al., 1993, Chapes et al. 1993). In this report, we document that a nutritional nucleotide supplementation as studied in ground-based microgravity analogs, has potential to serve as a countermeasure for the immune dysfunction observed in space travel.

Locomotion through three-dimensional type I rat tail collagen. A modified mini-assay.

There is an increased application of three-dimensional type I rat tail collagen as an in vitro model for the peritumoral matrix in analysis of lymphocyte migration. The increased demand prompted us to modify the previous methods. We here describe our 'mini'-setup of the collagen model assay, which uses only 1/20 the amount of collagen medium and the number of cells used in the conventional assay. The modified assay was tested for optimal collagen concentration in gel for upward and downward migration, for locomotion from a collagen-gel bead into a collagen overlayer for demonstration of the effect of inhibitors and for differentiation between locomotory properties of lymphocyte subpopulations. The results verify that the mini-assay is an applicable in vitro model, easily read and amenable to limited blood samples such as those obtained from cancer patients, and reflects well known in vivo events.

Evidence that opiate addiction is in part an immune response. Destruction of the immune system by irradiation-altered opiate withdrawal.

Experiments were performed to determine whether physical dependence on opiates (CNS phenomena) can be altered by destruction of the immune system. Irradiation, prior to or after chronic treatment with morphine significantly reduced the opiate-withdrawal syndrome as assessed by naloxone-induced abstinence. This study supports the proposition that addiction to opiates is related, at least in part, to interaction between the central nervous system (CNS) and the immune system.

Differential modification of morphine and methadone dependence by interferon alpha.

Subcutaneous implantation of a pellet of methadone was presented as a novel method for the establishment of physical dependence upon this agent and it was compared to (1) the state of physical dependence induced by multiple injections of methadone, administered over several days, and (2) the dependence established by injections of morphine and the implantation of a morphine pellet. Comparable signs of drug dependence were observed in rats treated with both morphine and methadone following the administration of the opiate antagonist naloxone. The administration of interferon-alpha significantly attenuated the severity of the withdrawal syndrome in dependent rats after chronic exposure to morphine and to a lesser extent after morphine and methadone in combination. In contrast, alpha interferon did not affect 6 of the 7 abstinence signs in animals dependent upon methadone alone. The observations suggest that the states of physical dependence upon morphine and methadone may be separate phenomena that involve different physiological mechanisms. Thus, interferon may be a useful adjunct in the treatment of subjects dependent upon morphine but not in those dependent on methadone.

The brain and the immune system: an intact immune system is essential for the manifestation of withdrawal in opiate addicted rats.

Gamma irradiation (500 rad) is often used to suppress the immune system in mice, rats and man. Recently, it was shown that irradiation prior to chronic morphine treatment, dramatically reduces the severity of naloxone-precipitated withdrawal in morphine-dependent animals. In the present study adoptive transfer of 2-6 x 10(8) splenocytes to irradiated rats prior to chronic morphine treatment restored the severity of all withdrawal signs precipitated by naloxone. In contrast, adoptive transfer of fractionated splenocyte subpopulations only partially restored withdrawal severity; and transfer of irradiated splenocytes, red blood cells or diluted numbers of normal splenocytes did not have any observed restorative effect. These findings suggest that specific cellular activities or factors derived from lymphoid cells are required for the expression of opiate withdrawal.

Effect of splenectomy upon tumor growth: characterization of splenic tumor-enhancing cells in vivo.

The effect of splenectomy upon neoplastic outgrowth was examined prior to and after implantation of methylcholanthrene-induced C3H/HeJ murine tumors. Splenectomy or sham operation performed 6 and 3 days prior to tumor inoculation significantly facilitated tumor growth compared to nonoperated, control mice. Operative procedures 12 days prior to tumor inoculation had no effect on tumor growth rate, suggesting that facilitated tumor growth was related to surgically induced, transient immunosuppression, rather than to the presence or absence of splenic tissue. On the other hand, 3 days after tumor inoculation, sham operations resulted in significant facilitation, but splenectomy yielded retardation of tumor growth. In local adoptive transfer assays, spleen cells from hosts bearing MCA-F tumors for 3 days nonspecifically facilitated the outgrowth of the antigenically noncrossreactive tumors MCA-F, MCA-D and MCA-C. These findings suggest that the spleen is a reservoir of suppressor cells during early stages of tumor growth, since the suppressor activity was not demonstrate 6 or 12 days after tumor inoculation. The nonspecific splenic suppressor cells were radioresistant (700 rads), capable of phagocytizing carbonyl-iron and adherent to plastic dishes. These findings suggest that perioperative immunodepression resulting in facilitated tumor growth is due to transient nonspecific activation of splenic suppressor macrophages.

The influence of hepatocellular function on NK and T cell tumoricidal activity.

Recent studies by this group have demonstrated that hepatocellular integrity is important in the preservation of host cellular immune function. This study evaluated the effect of experimental hepatocellular dysfunction (EHD) on host antineoplastic defense mechanisms. In nonspecific immune studies, we examined the effect of EHD on Wistar Furth (WF) natural killer (NK) cell cytotoxicity; in specific immune studies, we assessed WF C3H/HeJ lymphocytic responsiveness to both T cell mitogen and unmodified syngeneic fibrosarcoma. In concurrent studies, we evaluated the effect of EHD on interleukin-2 (IL-2) synthesis, an important NK and T cell trophic factor. WF rats and C3H/HeJ mice were assigned to three groups: EHD induced by bile duct ligation, sham, and normal control (NC). At day 21 serum bilirubin, WF NK cytotoxicity to YAC-1 tumor cells, WF and C3H/HeJ lymphocytic responsiveness to phytohemagglutinin (PHA) and syngeneic MCA-fibrosarcoma (MCA-F), and WF T-helper IL-2 production were determined in respective groups. Serum total bilirubin was elevated in EHD rats and mice with respect to controls (p less than 0.01). Wistar Furth cytotoxicity to the YAC-1 tumor cells was depressed in EHD animals with respect to sham and NC groups at 12.5:1 (p less than 0.01), 25:1 (p less than 0.05), 50:1 (p less than 0.05), and 100:1 (p less than 0.05) effector/target cell ratios. WF T cell responsiveness to PHA was depressed in EHD with respect to controls (p less than 0.01). C3H/HeJ lymphoproliferative response to MCA-F tumor antigen was also depressed in EHD animals when compared with control groups with the addition of 12.5 X 10(3) (p less than 0.05) and 50 X 10(3) (p less than 0.05) MCA-F cells. These impairments in NK and T cell function in EHD could not be attributed to diminished IL-2 production (EHD vs sham and NC: 112,141 +/- 5232 vs 106,691 +/- 1419 and 120,759 +/- 3248 cpm, respectively). These results demonstrate that hepatocellular failure compromises NK and T cell tumoricidal function, an effect not resultant on diminished T helper IL-2 production.

Modeled microgravity-induced protein kinase C isoform expression in human lymphocytes.

In long-term space travel, the crew is exposed to microgravity and radiation that invoke potential hazards to the immune system. T cell activation is a critical step in the immune response. Receptor-mediated signaling is inhibited in both microgravity and modeled microgravity (MMG) as reflected by diminished DNA synthesis in peripheral blood lymphocytes and their locomotion through gelled type I collagen. Direct activation of protein kinase C (PKC) bypassing cell surface events using the phorbol ester PMA rescues MMG-inhibited lymphocyte activation and locomotion, whereas the calcium ionophore ionomycin had no rescue effect. Thus calcium-independent PKC isoforms may be affected in MMG-induced locomotion inhibition and rescue. Both calcium-dependent isoforms and calcium-independent PKC isoforms were investigated to assess their expression in lymphocytes in 1 g and MMG culture. Human lymphocytes were cultured and harvested at 24, 48, 72, and 96 h, and serial samples were assessed for locomotion by using type I collagen and expression of PKC isoforms. Expression of PKC-alpha, -delta, and -epsilon was assessed by RT-PCR, flow cytometry, and immunoblotting. Results indicated that PKC isoforms delta and epsilon were downregulated by >50% at the transcriptional and translational levels in MMG-cultured lymphocytes compared with 1-g controls. Events upstream of PKC, such as phosphorylation of phospholipase Cgamma in MMG, revealed accumulation of inactive enzyme. Depressed calcium-independent PKC isoforms may be a consequence of an upstream lesion in the signal transduction pathway. The differential response among calcium-dependent and calcium-independent isoforms may actually result from MMG intrusion events earlier than PKC, but after ligand-receptor interaction.

Continuous intrasplenic interleukin-2 combined with antigen-specific chemoimmunotherapy.

A methylcholanthrene-induced fibrosarcoma model in C3H/HeJ mice demonstrated that combined chemoimmunotherapy, including partially purified, 1-butanol-extracted, tumor-specific transplantation antigen (1 microgram), cyclophosphamide (20 mg/kg), and continuous intrasplenic (IS) or intravenous (IV) infusion of purified human interleukin-2 (IL-2) (120 U/d) reduced the outgrowth of 4-mm established tumors, while IL-2 alone only modestly decreased tumor growth. For tumors larger than 1 cm, only the triple regimen with IS IL-2 significantly inhibited tumor growth, whereas IL-2 alone or the triple regimen with IV IL-2 failed to retard tumor growth. Furthermore, the first regimen significantly decreased pulmonary metastases after primary tumor resection. The Lyt-2+ phenotype predominated in the effector population of animals treated with this regimen, while L3T4+ cells predominated in those treated with the triple regimen that included IV IL-2. Thus, continuous IS IL-2 administration potentiates the efficacy of antigen-specific chemoimmunotherapy.