Immunochemical studies on the antigenic determinants required to elicit delayed and immediate hypersensitivity reactions.

Hartley and strain 2 guinea pigs were sensitized to chemically defined alpha-DNP(Lys)(n)-BuAm, alpha-DNP(Lys)(n)-, and (Lys)(n)-BuAm peptides and skin tested with individual members of these homologous series, related peptides and hapten-substituted proteins. The immediate skin response (Arthus) could be elicited with hapten-substituted tetra-, penta-, or hexamers, whereas both immediate and delayed skin responses could be provoked by the octamer or nonamer. The hapten is an integral part of the determinant for both immediate and delayed skin reactivity, since poly-L-lysine was unable to elicit either delayed or immediate reactions in sensitized animals. Arthus type cross-reactions occurred only when the sensitizing and test antigen shared a common haptenic determinant. In contrast to this, in this system, delayed type cross-reactions occurred only when the test antigen and the sensitizing antigen contained both a large oligo-L-lysine carrier as well as the same haptenic determinant. These observations imply that the mediation of the delayed response requires a larger determinant than is necessary to elicit the immediate response. The role of high affinity antibody as the mediator of the delayed response is discussed in terms of the size of the antigenic determinants required to elicit this response. It was found that the ability to elicit the delayed response paralleled the immunogenic capacity of these peptides, whereas the immediate response could be elicited by nonimmunogenic peptides. This finding suggests that the delayed response may require the continued biosynthesis of antibody and may be analogous to a local in vivo secondary response.

Immunopotentiating and immunotherapeutic effects of thymic hormones and factors with special emphasis on thymic humoral factor THF-gamma2.

The essential role played by the thymus in the development of the immune response was well documented in many publications. These findings prompted a long series of studies devised to define the factors produced and secreted by thymus cells, which are involved in the development and nature of immunological responsiveness. First experiments done with crude thymus extracts were followed by isolation of purified products and finally by chemical characterization and synthesis of immunologically active thymus-derived peptides. In this article we review the various thymic hormones and factors described, that is, thymosin fractions 5, the thymosins, prothymosin alpha, thymulin (FTS-Zn), thymopoietin, thymostimulin (TP-1), Thymic humoral factor (THF), and THF-gamma2. Studies demonstrating the activity of the various thymic factors in increasing the immunocompetence potential in both in vitro and in vivo conditions are discussed. The immunostimulatory potential of thymic factors was also investigated in experimental models where beneficial therapeutic effects were sought in a situation of immunological malfunction. The last part of the review is dedicated to clinical trials with thymic factors that revealed improvement in the immunocompetence potential in cases of immunodeficiencies, viral infections, and cancer and its correlation with therapeutic effectiveness. It seems that more research is required in order to better define conditions for the use of thymic factors in immunotherapy.

Involvement of inflammatory mediators in macrophage antitumor activity.

This review describes the potential role of macrophages in defense against cancer cells and the regulatory involvement of inflammatory mediators in this role. Interactions between macrophage-derived cytokines (tumor necrosis factor alpha, interleukin-1, IL-6) and their interrelationships with eicosanoids (mainly the cyclooxygenase product prostaglandin E2 and some lipoxygenase metabolites) represent a network that controls the expression of antitumor activity of macrophages either in a cell-to-cell contact system between the effector and the target tumor cell or as cell-free soluble products. Attention is given to the influence of tumor burden on production of cytokines and eicosanoids by macrophages and to the production of these mediators by tumor cells. Emphasis is placed on the roles of TNF-alpha and PGE2 in links between inflammatory and antitumor functions of macrophages. Finally, the perspectives and still existing problems in clinical implications of macrophage-derived cytokines are discussed in terms of a conceivable macrophage-directed immunotherapy of cancer.

An improved quantitative plaque assay for lymphocytes bearing antigen-specific cell receptors.

An improved method for the determination of the number of lymphoid cells bearing antigen-specific receptors is described. The method is based on the use of hapten-coupled bacteriophage (dinitrophenyl-T4) and detection of lytic plaques formed by the action of DNP-T4 on a target E. coli strain. The method is highly specific (up to 90% specific binding) and can be adapted for use with other antigenic determinants chemically attached to an active bacteriophage.

Immunomodulating anticancer alkylating drugs: targets and mechanisms of activity.

CY and L-PAM potentiated specific anti-tumor response in addition to their killing effect. The immunomodulating effect of a low dose of either CY or L-PAM was expressed in mice bearing large s.c. MOPC-315 plasmacytoma tumors. Cured mice were resistant to a challenge dose of the syngeneic tumor and their spleens contained specific cytotoxic T cells. Induction of specific anti-tumor response by a low dose of alkylating drugs was due to expression of "latent anti-tumor" capability. This fitted with the conception that "suppressed concomitant immunity" occurring in tumor-bearing animals can be activated. The immunomodulating activity of alkylating drugs was related to enhancement of T-cell functions:impairment of suppressor T-cell activity,enhancement of effector T-cell activity and increase in production of cytokines at the tumor site. The target tumor killing activity of a low dose alkylating drug was dissociated from its immunomodulating activity by treating mice bearing a tumor resistant to an alkylating drug. A low dose of CY had an immunomodulating effect in human cancer such as reduction of ConA-induced suppressor cell activity in melanoma, some improvement in addition to use of melanoma vaccine, and potentiation of DTH in cancer patients. The immunomodulating effect of alkylating drugs suggest that their use might be beneficial not only for killing tumor cells but also for promoting specific anti-tumor immune response.

Interactions between inflammatory mediators in expression of antitumor cytostatic activity of macrophages.

Antitumor properties and participation in inflammatory events are important characteristics of activated macrophages. We show here that both antitumor cytostatic function of macrophages and participation of these cells at inflammatory sites are controlled by two main groups of mediators: cytokines (IL-1, TNF alpha) and eicosanoids (prostanoids and leukotrienes). These two groups of mediators represent a complex system of mutual interactions in regulation of their production and activities. Multiple sets of experiments with murine macrophages are discussed in favor of the views that PGE2 and lipoxygenase products oppose each other's actions, and that the regulating role of PGE2 in the secretions of cytokines are of pivotal importance in antitumor cytostasis of macrophages in vitro. Such observations can be extended to a situation ex vivo, showing that human macrophages harvested from inflammatory sites have markedly augmented cytostatic expression. It thus appears that the antitumor cytostatic function of macrophages is related to the production of inflammatory mediators by these cells. Accordingly, it might be that occurrence of inflammation in tumor-bearing individuals plays a role in the promotion of antitumor activity of macrophages.

Effect of melphalan administration on the activity of natural killer and natural cytotoxic spleen cells of normal and tumor-bearing mice.

The effect of melphalan (L-PAM; L-phenylalanine mustard) administration on natural killer (NK) and natural cytotoxic (NC) activities of spleen cells from normal noninoculated BALB/c mice and mice bearing large MOPC-315 plasmacytoma tumors was investigated. Injection of L-PAM (7.5 or 15.0 mg/kg) had no effect on NK and NC activities of spleen cells from normal mice whereas stimulation by PHA was markedly suppressed. NK activity was markedly suppressed in spleens of tumor-bearing mice whereas NC activity was not affected. Therapy with L-PAM of tumor-bearing mice led to transient recovery of NK activity which was possibly related to the effect of L-PAM on macrophage suppressive activity on NK cells. It is concluded that NK and NC cell activities and the response to PHA stimulation are selectively affected by L-PAM administration.

Prostaglandin E2 inhibits the release of tumor necrosis factor-alpha, rather than interleukin 1 beta, from human macrophages.

We have reported previously that macrophages obtained from renal patients on continuous ambulatory peritoneal dialysis (CAPD) during an episode of infectious peritonitis display a decrease in intracellular cAMP levels and in spontaneous in vitro release of PGE2 and PGI2. Such macrophages also release large quantities of IL-1 beta and TNF alpha when stimulated in vitro by LPS. In view of the interregulatory effects between PGE2 and macrophage cytokines (IL-1 beta and TNF alpha) in their production, we examined in the present work to what extent the LPS-induced release of either IL-1 beta or TNF alpha in vitro from CAPD-originated peritoneal macrophages is affected by graded doses of exogenous PGE2 (range 0-1000 ng/ml) and by the cyclooxygenase inhibitor indomethacin (INDO) (10(-6) M). IL-1 beta and TNF alpha were determined using an enzyme-linked immunoabsorbent assay and an immunoradiometric assay, respectively. We found that PGE2 invariably induced a dose-dependent decrease in TNF alpha release. In peritoneal macrophages collected during an infection-free period, TNF alpha release decreased from 3225 pg/ml (controls) to 353 pg/ml at 1000 ng/ml of PGE2, and in peritoneal macrophages collected during an episode of infectious peritonitis, it decreased from 4100 pg/ml (controls) to 545 pg/ml at 100 ng/ml of PGE2. However, PGE2 failed to influence the secretion of IL-1 beta. INDO induced an approx. two-fold increase in TNF alpha release, but had no effect on IL-1 beta release. These findings indicate that exogenous and endogenous PGE2 controls the release of TNF alpha rather than IL-1 beta from LPS-stimulated peritoneal macrophages.

Activity of human peritoneal macrophages against a human tumor: role of tumor necrosis factor-alpha, PGE2 and nitrite, in vitro studies.

Human peritoneal macrophages collected from renal patients on continuous ambulatory peritoneal dialysis (CAPD) during inflammation-free periods were induced to express antitumor activity in vitro when cultured in the presence of bacterial lipopolysaccharide (LPS) and even more activity when they were kept in the presence of LPS + IND (indomethacin). The antitumor activity was expressed against a human tumor-cell line, RC43, either in a cell-to-cell contact set-up between the macrophages and the RC43 target cells or when the tumor cells were exposed to supernatants of the cultured macrophages. The antitumor activity of macrophages was correlated to a marked increase in production of tumor necrosis factor-alpha (TNF alpha), not correlated to an increase in nitrite production and inversely correlated to the production of PGE2. The RC43 tumor cells were susceptible to recombinant human TNF alpha, recombinant human IL-1 beta, sodium nitrite and the leukotriene LTB4. The results obtained suggest that activated human macrophages might represent a useful tool for cancer immunotherapy.

Serum levels of tumor necrosis factor determine the fatal or non-fatal course of endotoxic shock.

The role of tumor necrosis factor alpha (TNF alpha) in endotoxin-induced shock was investigated in pigs receiving 5 micrograms kg-1 of Escherichia coli endotoxin (LPS) during 60 min of continuous infusion into the superior mesenteric artery. LPS concentration in aortic plasma, as determined by a chromogenic Limulus amoebocyte lysate (LAL) test, reached a peak of approximately 1000 ng l-1 during LPS infusion, and declined rapidly after discontinuation of the infusion. Serum TNF levels were determined by a bioassay using the L929 murine transformed fibroblast line. Eight of the 17 animals infused with LPS died within 30 min after beginning LPS administration, while the other 9 pigs survived beyond the experimental observation period of 3 h, although they were in a state of shock. No difference in LPS concentration was found between the survivors and the non-survivors. However, the serum TNF levels in non-survivors were significantly higher than in survivors when measured at 30 min after beginning LPS administration. In survivors, the peak increase in serum TNF levels was measured at 60 min after the beginning of LPS injection and returned rapidly to the baseline values. Although the role of TNF inducing rapid death seems to be dominant, the hemodynamic, hematology and blood chemistry disturbances seen during shock continued in survivors long after the return of TNF to baseline levels. These findings indicate that besides TNF other mediators are also involved in the LPS infusion-induced shock.

Correlation between PGE2 production and suppressor activity of alveolar macrophages from patients with interstitial lung diseases.

The suppressive activity of alveolar macrophages (AM) obtained from bronchoalveolar lavage (BAL), on PHA stimulation of autologous peripheral blood lymphocytes (APL) was evaluated. The effect on lymphocyte stimulation was evaluated by coculturing the AM and APL cells at a ratio of 1:1. PGE2 released by AM during the culture period was measured by a radioimmune assay. The patients included in the study were 11 cases with interstitial lung disease (ILD), 8 cases of lung cancer (CA), and 5 controls (CO). Addition of AM of patients from the CA group resulted in slight suppression of lymphocyte stimulation in 4 cases, slight enhancement in 3 cases and no effect in one case. AM from the CO group induced slight suppression in 4 out of 5 cases. AM from all 11 ILD cases exerted a significant high suppressive activity (65.6% +/- 18.2 - P less than 0.001 by comparison with the CO and CA groups). In ILD cases, a dichotomous pattern was found in regard to relation between high suppressive activity of AM and release of PGE2: in idiopathic pulmonary fibrosis (IPF) patients, high suppressive activity of AM (70.4% +/- 15.4) correlated well with elevated secretion of PGE2: 3.58 +/- 0.26 ng/ml/10(5) cells (P less than 0.02 compared to CO). AM from sarcoidosis patients suppressed PHA stimulation by 61.6% +/- 19.3 but secreted only 0.357 +/- 0.26 ng/ml/10(5) cells of PGE2 (P less than 0.02 compared with the idiopathic pulmonary fibrosis group). Therefore, it seems that other factors, in addition to PGE2, might be involved in the suppressive activity of AM from interstitial lung diseases.

Sequential release of tumour necrosis factor, platelet activating factor and eicosanoids during endotoxin shock in anaesthetized pigs: protective effects of indomethacin.

1. The effects of indomethacin were investigated on haemodynamics, haematological and blood glucose values, and the release of tumour necrosis factor (TNF), platelet activating factor (PAF) and eicosanoids in anaesthetized pigs receiving 5 micrograms kg-1 E. coli lipopolysaccharide (LPS) over 60 min into the superior mesenteric artery. The animals were observed for an additional period of 2 h after the termination of LPS infusion. 2. Eight of the 17 animals infused with LPS and not treated with indomethacin died within 30 min after the beginning of LPS infusion (non-survivors), while the other 9 survived the experimental period of 3 h though in a state of shock (survivors). 3. No alterations were observed in plasma concentrations of PAF and eicosanoids (thromboxane B2 (TXB2), 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha) and leukotriene B4 (LTB4] in non-survivors. However, a marked increase was detected in TNF release. A significant, though transient, increase in concentrations of PAF, TNF and eicosanoids occurred in the survivors. The peak in the concentrations of PAF and TXB2 preceded the maximum in TNF values in survivors. 4. Another group of 7 LPS-infused pigs was treated with indomethacin (2 mg kg-1, i.v. bolus 60 min before the start of LPS infusion, followed by a continuous infusion of 3 mg kg-1 h-1). This treatment prevented death and shock despite the high concentrations of circulating TNF and PAF. Concentrations of cyclo-oxygenase enzyme products were reduced, whereas LTB4 release was not affected. The effect of indomethacin on haemodynamic changes occurred earlier than on cyclo-oxygenase products.5. In another group of 6 pigs indomethacin (2mg kg- 1, i.v.) was given 20-25 min after the start of LPS infusion at which time mean arterial blood pressure (MABP) had decreased below 40mmHg indicating imminent death. This indomethacin treatment immediately reversed the hypotension, restored the organ perfusion, delayed the haemoconcentration and thrombocytopenia and prevented death. However, TNF and PAF concentrations remained elevated. Concentrations of cyclo-oxygenase products studied were reduced by the end of the observation period, whereas LTB4 production was unaffected.6. The decrease in MABP induced by exogenous PAF was temporarily prevented by indomethacin.7. These data indicate that the beneficial effect of indomethacin in LPS-induced septic shock is related to cyclo-oxygenase inhibition as well as to a direct vasoconstrictor property of the drug.

Macrophage cytokines render WEHI-3B tumor cells susceptible to cytostasis by prostaglandins.

The growth of the murine myelomonocytic leukemia tumor, WEHI-3B, has been shown to be inhibited by a two-step treatment: first, incubation for one hour with either interleukin-1 (human recombinant IL-1 alpha or tumor necrosis factor (human recombinant TNF-alpha); second, subsequent exposure to prostaglandins. Preincubation with IL-1 rendered the tumor cells more susceptible to subsequent treatment with either prostaglandin E2 or to the stable synthetic analogue of prostacyclin DC-PGI2. Preincubation with TNF-alpha rendered the tumor cells more susceptible to further treatment with PGE2 but not with DC-PGI2. Preconditioning of the tumour cells with either IL-1 alpha or TNF alpha did not affect cytostasis by subsequent culture of tumor cells in presence of either one of the cytokines. It is concluded that the interactions between macrophage cytokines and prostaglandins in enhancement of antitumor activity might imply first binding or induction of certain modifications in the tumor cells by the cytokines which render the cells more susceptible to exposure to prostaglandins.

Protective effect of diclofenac sodium against endotoxic shock in anaesthetized pigs.

The effect of diclofenac sodium was investigated on haemodynamics, haematologic and blood glucose values as well as the release of eicosanoids, tumor necrosis factor (TNF) and platelet activating factor (PAF) in anaesthetized pigs receiving 5 micrograms.kg-1 Escherichia coli lipopolysaccharide (LPS) over 60 min into the superior mesenteric artery. The animals were observed for an additional period of 2 h after the termination of LPS infusion. 15 of the 31 animals infused with LPS and not treated with diclofenac sodium died within 30 min after the commencement of LPS infusion (non-survivors), while the other 16 survived the experimental period of 3-h, though in a shock state (survivors). No alterations were observed in plasma concentrations of PAF or eicosanoids (TXB2, 6-keto PGF1 alpha and LTB4), but a marked increase was detected in TNF release in the non-survivors. A significant, though transient, increase in concentrations of PAF, TNF and eicosanoids studied characterized the survivors. Another group of 7 LPS-infused pigs was treated with diclofenac sodium (2 mg, kg-1, i.v. bolus 60 min before the start of LPS infusion, followed by a continuous infusion of 1 mg kg-1 h-1) 1 mg/kg-1/h-1. This treatment prevented death and shock despite the high concentrations of TNF and PAF. Concentrations of both cyclooxygenase and 5-lipoxygenase enzymes products were reduced. These data indicated that the beneficial effect of diclofenac sodium in LPS induced shock may be related to the reduced production of eicosanoids.

Leukotriene C4 is an essential 5-lipoxygenase intermediate in A23187-induced macrophage cytostatic activity against P815 tumor cells.

Resident peritoneal macrophages incubated with 3.5 x 10(-7) M Calcium ionophore A23187 in tumor cell growth medium (TGM) release large amounts of leukotriene (LT)E4 and an unidentified 5-lipoxygenase product, whereas A23187-stimulated macrophages produce in serum free medium LTD4, predominately. LTC4 and 3H-LTC4 incubated for 20 min at 37 degree C in serum containing TGM, convert into LTE4 and 3H-LTE4, respectively. Thus, LTC4 released from A23187-stimulated macrophages is an intermediate in TGM which rapidly converts into LTE4, probably because of the presence of gamma-glutamyl transpeptidase and cystenylglycinase in TGM. Macrophages express antitumor cytostatic activity towards P815 cells (49-53%) in a cocultured ratio (macrophage: tumor cell) 2:1 when stimulated with 3.5 x 10(-7) M A23187 in TGM. The 5-lipoxygenase inhibitor AA861 reverses the cytostatic activity by 42-58% and it inhibits also the formation of A23187-induced 5-lipoxygenase products from macrophages. Restoration of 38% macrophage- antitumor cytostatic activity by exogenous LTC4 (10(-8) M) indicates that LTC4 is an essential 5-lipoxygenase intermediate in the pathway of required signals underlying A23187-induced macrophage antitumor cytostatic activity. Macrophages not stimulated by A23187 do not express cytostatic activity in the presence of LTC4. This implies that besides LTC4, increased cytosolic [Ca2+] is required for A23187 induction of macrophage cytostatic activity.

Cancer immunotherapy: hopes and pitfalls: a review.

Cancer immunotherapy (IT) started approximately 100 years ago with attempts to use a prepared immune serum against osteosarcoma. Since then, IT was attempted by use of various immunopotentiating agents like whole bacterial cells, bacterial cell fractions, cytokines and thymic humoral factors. The therapeutic efficiency of IT alone was limited and erratic. Accordingly, combined IT with other procedures were used. This included use of "immunomodulating" anticancer drugs as cyclophosphamide (CY) and melphalan (L-PAM) and use of IT in combination with tumor-reducing procedures like surgery, radiation and chemotherapy. The use of immunomodulating drugs was based on findings showing that CY and L-PAM enhance the ability of the immune system to react against tumor cells, in addition to their antitumor activity. Combined treatments were employed with the aim to reduce tumor-burden and as such, render IT more effective. Other therapeutic procedures consisted on use of specific antitumor antibodies as a vehicle for carrying radioactive lethal amounts to tumor cells, use of macrophages activated against tumor cells, use of prostaglandin antagonists and use of specific antitumor vaccines. The general conclusion is that while IT might have some beneficial therapeutic effect especially in conjunction with other procedures, it might be not sufficient to insure cure but it might increase the survival time and improve the quality of life of cancer patients.

Effectiveness of low-dose melphalan treatment against a nonimmunogenic primary induced mouse plasmacytoma.

The fourth i.p. passage of the plasmacytoma "PR" induced by repeated i.p. injections was used for testing chemotherapy with melphalan. The development of "PR" ascitic tumours was slower and the survival time of inoculated mice was longer than that of MOPC-315 inoculated mice. Moreover, the myeloma protein secreted by the "PR" tumour cells, differed from MOPC-315 secreted myeloma protein in its electrophoretic mobility (fast gamma-2) and its characteristics as IgG immunoglobulin. Chemotherapy by a single injection of melphalan 7.5 mg/kg was effective in preventing the development of both MOPC-315 ascitic tumours and "PR" ascitic tumours. Mice cured from MOPC-315 tumours, however, developed antitumour response as shown by resistance to challenge with a high tumourigenic dose and by development of cytotoxic response in vitro against MOPC-315 tumour cells by spleen cells taken from the cured mice. On the other hand, mice cured from "PR" tumour by melphalan were highly susceptible to challenge and their spleen cells were not able to develop a cytotoxic response in vitro against target "PR" tumour cells.

Secretion of suppressor factors by EBV infected B cell lines.

The infection of human peripheral blood B cells with Epstein-Barr Virus (EBV), induced the production of suppressor factor(s) which were released into the supernatant of the B-cell cultures. The induction of suppressive activity was independent of T-cell presence. The suppression was exhibited both against T-cell activity (MLR and mitogenic stimulation) as well as against B-cell mitogenic stimulation of human or murine B lymphocytes. The suppressive factor(s) was of a low molecular weight (equal or less than 5,000), resistant to trypsin and heating at 80 degrees C and its activity was partially inhibited by neuraminidase treatment. These findings indicate that the suppressive factor(s) is not correlated to immunoglobulin production, is not apparently of a protein nature, and might be of ganglioside or siaylated glycoprotein structure. Our present findings suggest that, in addition to T cells, B cells might also play an immunoregulatory role in the expression of immune response potential.

Induction of macrophage antitumor activity by a mycobacterial fraction. In vivo and in vitro studies.

A Methanol Extraction Residue (MER) of BCG tubercle bacillus induced cytostatic activity in vitro against the murine tumor-cell lines YAC-1 and MOPC-315 in resident murine peritoneal macrophages isolated from BALB/c mice. The induction of antitumor activity was not associated with increase in release of TNF-alpha. Macrophages from mice hyperimmunized with MER (MER 3x) or from mice injected once with MER in aqueous suspension released more PGE2 following stimulation in vitro with LPS. Macrophages cultured with either MER or MER+LPS prolonged the survival time of mice bearing palpable RPC5 plasmacytoma s.c. tumors.

Induction of protective antitumor immune response against a murine plasmacytoma not curable by alkylating-drug treatment.

Immunization with viable tumor cells followed by subsequent administration of glutaraldehyde-treated tumor cells induced a protective antitumor immune response in the host toward the alkylating-drug resistant RPC-5 plasmacytoma. This was proven by resistance to challenge with RPC-5 tumor cells, neutralization in Winn tests, by effectiveness of combined chemotherapy with melphalan plus immunotherapy with spleen cells from RPC-5 immunized mice and in vitro by cytotoxicity tests. The specificity of the immune response was ascertained in vivo by comparison with the response toward MOPC-315 plasmacytoma. However, in vitro cytotoxicity tests revealed the occurrence of shared antigens between the RPC-5 and MOPC-315 tumor cells. It is concluded that the ineffectiveness of alkylating-drug treatment toward the RPC-5 tumor is not due to the inability of this tumor to induce a specific antitumor immune response, and that cross-antigenic relationship as revealed by in vitro cytotoxicity tests does not necessarily reflect cross-protection between various plasmacytomas.