Sensitization to low dose 5-fluorouracil. Subsequent enhancement of its systemic antitumor effect in the rat.

This report describes a novel method of immunochemotherapy; the active immunization to the drug 5-fluorouracil (5-FU) with enhanced antitumor activity resulting from its subsequent systemic administration. Two metastasizing carcinomas in the Fischer strain (F344) rat have been used: a chemically induced bladder carcinoma (FBCa) and a spontaneous mammary adenocarcinoma (MACa). Both tumors grow rapidly and result in 100% mortality within 10 wk of implantation. Neither tumor is sensitive to systemic 5-FU alone. Intradermal sensitization to 5-FU before FBCa tumor implantation, followed by 5-FU administered systemically, resulted in significant tumor regression and improvement in survival with eradication of all tumor and cure in 20% of animals. A similar antitumor effect was observed with the MACa. A comparable drug effect was observed when methotrexate sensitization was given before FBCa implantation followed by systemic MTX. Specificity to the sensitizing drug was demonstrated by the lack of effect of sensitization with either 5-FU or MTX unless followed by systemic therapy with the requisite sensitizing agent. Sensitization to 5-FU has also been assessed after FBCa implantation followed by resection of the local tumor. Resection was performed after distant tumor metastases had occurred, and was followed by systemic 5-FU therapy. Whereas tumor resection alone failed to cure any animal, sensitization to 5-FU increased cure rate fourfold over animals receiving systemic 5-FU alone. Antibody to 5-FU in the sera of sensitized animals has been suggested by an immunoenzymatic staining technique and its specificity confirmed in a radioimmunoassay. It is postulated that a combination of the systemic agent and the antibody elicited to it by sensitization produces the significant antitumor effect observed. The antitumor effect observed with this new approach to immunochemotherapy warrants further experimental and clinical study.

Reversal of cyclosporine-induced mortality with a synthetic polymeric immunostimulant in a murine model of fecal peritonitis.

We have been investigating the effects of a synthetic immunostimulative polymer known as copovithane (Cpv). This agent appears to enhance humoral immunity in untreated and cyclosporine-immunosuppressed mice and is nontoxic in rodents and man. The purpose of this study was to determine whether cyclosporine (CsA) is deleterious to survival in a murine cecal ligation, puncture, and excision (CLPE) model of fecal peritonitis, and--if so--whether this effect could be ameliorated by Cpv without interfering with skin allograft acceptance. Cpv significantly prolongs survival in the CLPE model; the optimal dose for this effect was found to be 100 mg/kg. CsA was found to have a significant and deleterious effect on survival at several dosage levels when administrated 48 and 24 hr before cecal ligation, and immediately before and 16 hr after cecal ligation. Using a dose of CsA sufficient for skin allograft acceptance and the same schedule of administration outlined above, Cpv 100 mg/kg was administered 48 hr prior to cecal ligation. Mice treated with CsA plus Cpv had significantly longer survival than mice treated with CsA alone; furthermore, the survival of CsA-plus-Cpv-treated animals was not significantly different from that of saline-treated controls. Acceptance and survival of H-2 incompatible skin allografts in mice treated with CsA were not affected by Cpv 100 mg/kg/week. We conclude that CsA-induced mortality in the CLPE model can be abrogated by Cpv without adversely affecting skin allograft survival. It may eventually be possible to reduce the incidence of septic complications in clinical allotransplantation by prophylactically administering Cpv to patients on CsA immunosuppression.

Modulation of the immune response to teratocarcinoma in mice sensitized by sperm antigens.

Mouse primitive teratocarcinoma cells share a common surface antigen with morulae, preimplantation embryo cells and murine and human spermatozoa. 129/Sv mice were immunized with either spermatozoa and subsequently inoculated with various doses of teratocarcinoma 6050. A significant inhibition or acceleration of tumor growth was observed when compared with controls immunized with compatible fibroblasts. These effects were sex-dependent, both the incidence and tumor growth being suppressed in sperm-immunized males. The opposite effects were observed in sperm-presensitized females. Immune sera obtained from both male and female 129/Sv mice exhibited a high binding activity to human spermatozoa when tested in a cellular radioimmunoassay. Thus, immunization with sperm antigens provides immunotherapeutic and/or enhancing effects in male and female 129/Sv mice, respectively.

Impaired antibody production in blunt trauma. Possible role for T cell dysfunction.

This study investigates mechanisms of impaired humoral immune response in a well-defined population of blunt trauma patients (n = 18, Injury Severity Score greater than or equal to 20). Spontaneous and pokeweed mitogen-induced polyclonal immunoglobulin production were assessed in cultures of peripheral blood mononuclear cells. The proliferative response to alloantigen and mitogen was assessed in parallel by the mixed lymphocyte reaction and pokeweed mitogen-induced blastogenesis, respectively. Pokeweed mitogen-induced IgG and IgM production was significantly reduced in trauma patients compared with controls. This effect was not reversed by depletion of adherent cells or by the addition of indomethacin. Exogenous interleukin 2 was also ineffective. However, the addition of normal T cells or supernatants from isoantigen-stimulated cultures of these cells to patient B cell-enriched cultures significantly enhanced (by 1.4- to 5.1-fold) the antibody response to pokeweed mitogen. Thus, suppression of humoral antibody response in blunt trauma patients may be due to failure of T-cell mediated help, resulting in insufficient secretion or activity of cytokines required for adequate B cell activation, proliferation, or differentiation into immunoglobulin-secreting cells.

Modulation of biological responses of normal human mononuclear cells by antiestrogens.

The effect of three non-steroidal antiestrogens, Tamoxifen, Toremifene and ICI 164, 384, on various aspects of the immune response was studied in cultures of normal peripheral mononuclear blood cells. The drugs differ somewhat in their effect on the functions tested (pokeweed mitogen-induced immunoglobulin synthesis and cell proliferation, mixed lymphocyte reaction, Interleukin 2 (IL 2) synthesis, IL 2 receptor expression and Tumor Necrosis Factor (TNF alpha) synthesis). However, all three are immunosuppressive. On the other hand, ICI 164, 384 and Tamoxifen were stimulatory for TNF alpha production by adherent cells which may prove as an additional feature in antiestrogen treatment.

Chorionic gonadotropin-induced cell proliferation and polyclonal immunoglobulin synthesis in human mononuclear cells.

Some fetal and placental proteins may play a role in stimulating normal and malignant cell proliferation. We studied the effect of human chorionic gonadotropin (hCG) on the proliferative response of mitogen or alloantigen-activated, human peripheral blood mononuclear cells (PBM). The effect of hCG on polyclonal immunoglobulin synthesis was determined in pokeweed mitogen-activated cultures of PBM. hCG had a statistically discernible, augmenting effect on thymidine uptake in lectin-stimulated mononuclear cell cultures. This effect appeared to be dose-dependent, with an optimal range at 50-150 ng/hCG/ml. Polyclonal IgG and IgM synthesis was also significantly increased, both in PWM-stimulated and PWM-free cultures of PBM. Parallel studies with a rapidly growing EB-virus transformed lymphoblastoid line showed no hCG effect. In contrast to previous reports on the immunosuppressive action of hCG, we conclude that hCG functions, in our experimental conditions, as a mitogen and a stimulator of polyclonal immunoglobulin synthesis.

Regulation of IgM production in thermally injured patients.

This report examines the capacity of autologous and exogenous interleukin-2 (IL2) to regulate and/or induce immunoglobulin M (IgM) production in these patients. Pokeweed mitogen (PWM)-induced lymphocyte proliferation and PWM- and IL2-induced IgM secretion were monitored in vitro during the postburn period (10 to over 60 days) in 40 patients aged 16-72 years, with burns 20-90 per cent TBSA. PWM-induced IgM secretion fluctuated considerably during this period. Twelve of 40 patients demonstrated no IgM production and a significant (P less than 0.001-0.05) proportion of them had profoundly suppressed levels. Of the survivors, restoration of IgM secretion to normal levels was achieved in only 60 per cent at time of discharge. Even more consistently suppressed was exogenous IL2-driven production of IgM. In contrast, PWM-induced lymphoproliferation was normal in over 70 per cent of the patients. Thus, the T-cell-dependent antibody response was suppressed for long periods of time, possibly from some deficiency in IL2-regulated secretion or reception of helper T-cell-derived factors necessary for B cell differentiation into Ig-secreting cells.

Soluble interleukin 2-receptor alpha secretion is related to altered interleukin 2 production in thermally injured patients.

This study examines the relationship between the capacity for interleukin-2 (IL2) production and the magnitude of the in vitro and in vivo secretion of IL2R alpha in 43 patients with major burns (30-90 per cent total body surface area). Throughout the postburn period a significant (P less than 0.001-0.05) proportion of patients studied demonstrated increasingly high levels of serum IL2 ranging from 2 to over 500 U/mL. Serum IL2R alpha also increased, reaching its highest levels at 15-40 days postburn, while serum IL2 gradually declined. In this period in vitro IL2 production and IL2R alpha secretion in patient's cultures were significantly reduced compared to control. However, in parallel cultures supplemented with exogenous IL2, IL2R alpha levels could be significantly increased (2.5 fold). IL2R alpha levels also approached normal in peripheral blood mononuclear cell cultures from recovering patients whose in vitro IL2 production had improved. These observations suggest that in the burn patient altered synthesis and/or secretion of the soluble form of IL2R alpha may be related to IL2 content. Above physiological levels of IL2R alpha and its ligand in postburn serum also indicate that thermal injury induces strong in vivo activation of the lymphoid system.

Induction of the rat in vitro plaque-forming response to sheep erythrocytes by a soluble factor(s) derived from concanavalin A-activated rat T lymphocytes.

Macrophages suppress the in vitro PFC response to sheep erythrocytes by rat spleen cells. We report the activity of a soluble factor released by rat splenic T-lymphocytes following a short incubation (60 min) with Con A (100 micrograms/ml). Culture supernatants added to rat spleen cells in the presence of SRBC significantly increased the PFC response at day 5. The effect was time and dose dependent. The Con A supernatant was effective in increasing PFC response if added to splenocytes during the first 16 h of the culture. The increased response if added to splenocytes during the first 16 h of the culture. The increased response was dose dependent giving maximum PFC at 1%. Depletion of macrophages from the spleen cell population produced a significant increase of the in vitro PFC response, but minimized the enhancing activity of the Con A supernatants. These studies suggest that an activated T-cell product can increase the antibody response to SRBC by completing the helper signal.

Elimination of 51Cr-labelled target cells as a method of in vivo assessment of immune reactions.

This report describes the use of an in vivo 51Cr release assay which is able to monitor various immune reactions in the rat. This assay is able to quantitate the immune response to tumour and also to assess the relative efficacy of different immunostimulants in this tumour model. These data correlate well with survival studies in tumour-bearing rats. This assay can also measure the sensitizing effect of antigens from animals differing at both the major and minor histocompatibility locus using various routes of sensitization and various donor tissues. Allograft survival can also be correlated to in vivo 51Cr release. This method is both reproducible and specific. The in vivo 51Cr release assay is a useful and versatile means of monitoring the development of antitumour responses and the degree of sensitization to histocompatibility antigens in a variety of situations in the rat. It may therefore prove useful as a rapid method for screening new antitumour and immunosuppressive regimens to be used in various studies.

Thermal injury-associated neopterin production: regulation by interleukin-2.

Pteridin neopterin production by monocytes/macrophages has been linked to the biologic activity of immune activation- and/or infection-related cytokines. In patients with thermal injuries who succumb to infections, serum levels of both interleukin-2 (IL-2) and neopterin are significantly increased. However, the relationship between these two markers of immune activation remains unclear. This study examines the role of IL-2 in the biosynthesis of neopterin after major burn. Up to 4 weeks after burn, the levels of plasma neopterin and endotoxin were elevated in all patients studied (N = 9, 30% to > 90% total body surface area). Intact (unsupplemented) peripheral blood mononuclear cell (PBMC) cultures from patients with sepsis secreted high levels of neopterin spontaneously. The spontaneous release of neopterin was significantly decreased (p < 0.05) after supplementation with exogenous IL-2. The reverse was observed in peripheral blood mononuclear cell cultures from infection-free or control groups where relatively low neopterin secretion was markedly augmented in the presence of IL-2. The effect of IL-2 in patient cultures was unrelated to the activity of endogenous interferon gamma, because the production of this cytokine was profoundly reduced. However, IL-2-induced alterations in neopterin secretion paralleled those in the production of tumor necrosis factor alpha. This suggests that after thermal injury, biologic responses of neopterin-secreting peripheral blood mononuclear cells are directly or indirectly regulated by IL-2.

Serum interleukin-2 receptor as a possible mediator of immunosuppression after burn injury.

Concentrations of the interleukin-2 receptor are significantly elevated in serum after burn injury. To examine the immunoregulatory potential of this molecule, suppressive activity of sera from patients with major burns (n = 16; 40% to 80% total body surface area) was assessed before and after immunoaffinity adsorption with interleukin-2. The preadsorption level of interleukin-2 receptor in the pooled serum after burn injury was 6250 U/ml. This serum demonstrated a strong suppressive activity, inhibiting expression of cellular interleukin-2 receptor and proliferative responses of normal human lymphocytes to alloantigen and exogenous interleukin-2 by 60% to 90%. Adsorption of pooled serum after burn injury with interleukin-2 lowered the level of interleukin-2 receptor to 1800 U/ml and reduced its immunosuppressive activity. The percentage of interleukin-2 receptor-bearing cells, and cell proliferative responses, increased by 50% to 70% compared with sham adsorbed pooled serum after burn injury. Thus serum interleukin-2 receptor after burn injury may represent a specific mediator for downregulation of interleukin-2-dependent responses.

Induced immunoglobulin secretion by T-cell-replacing products from blunt trauma patients.

The capacity to induce immunoglobulin (Ig) secretion by soluble T-cell-replacing (TCR) factors derived from alloantigen-stimulated T lymphocytes of blunt trauma patients (n = 15, mean ISS = 24) was examined in Staphylococcus aureus (SAC)-activated normal B-cell cultures. The majority of the patients studied demonstrated a profound suppression of the T-cell-dependent, pokeweed-mitogen-induced Ig production. However, the activity to induce Ig secretion associated with TCRs from the same patients was not reduced compared with that of TCRs from normal subjects. IgM synthesis was normal and IgG secretion induced by TCRs was within the control range (in 6 of 15 patients) or significantly higher (p less than 0.05) than that in the remaining patients. Both patient-derived and control TCRs failed to induce Ig synthesis in cultures of resting B cells and had comparable mitogenic effects on normal SAC-activated and phytohemagglutinin A-activated B and T lymphocytes, respectively. Thus, the intrinsic ability of T lymphocytes to produce B-cell helper factors appears to be unaffected following blunt trauma. Suppression of the T-cell-regulated Ig secretion in traumatized patients may therefore reflect an altered B lymphocyte response to such factors.

Elevated release of tumor necrosis factor-alpha and interferon-gamma by bronchoalveolar leukocytes from patients with bronchial asthma.

Bronchoalveolar lavage (BAL) leukocyte-secreted cytokines are considered to be important mediators of the inflammatory and allergic reactions in the lung. This study examines quantitative changes in the level of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) production in BAL cell cultures derived from patients (n = 11) with bronchial asthma. The secretion of TNF alpha and IFN gamma was determined in intact (unstimulated) and phytohemagglutinin/phorbol myristate acetate (PHA + PMA)-stimulated BAL leukocyte cultures and compared with that in control cultures. In all patients studied, the background and PHA + PMA-induced secretion of TNF alpha and IFN gamma was significantly (p < 0.001) higher than that in parallel control cultures. In contrast to BAL cell preparations, the capacity of TNF alpha and IFN gamma secretion by patients' peripheral blood mononuclear cells (PBMC) did not differ from that of control subjects. High spontaneous release of TNF alpha and IFN gamma by patients' BAL leukocytes, but not PBMC, suggest that in the pathophysiology of bronchial asthma, these cytokines may act as local pathogenic agents in the lung.

Detection of the circulating antibodies to teratocarcinomadefined antigens in patients with testicular tumours.

Sera from twenty-three patients with primary or metastatic testicular tumours of germinal origin were tested for antibodies against teratocarcinoma-associated antigen(s), using an indirect immunofluorescence technique. Human and 129/Sv mouse sperm and mouse teratocarcinoma cell line 402 AX were used as target cells. A total of fifteen sera were identified as positive, six of them when tested against both sperm and tumour cells. Tail staining has been the most prevalent pattern of fluorescence on both human and murine spermatozoa. These observations suggest that antibody to a common teratoma-defined antigen(s) was detected in sera of patients with testicular tumours.

Mechanisms of altered in vitro antibody response in the rat by a small synthetic polymer.

NED 137, a low molecular weight synthetic polymer, enhances the rat humoral immune response to cellular antigens in vivo. The effect of NED 137 on the in vitro antibody response to sheep erythrocytes was studied in normal and macrophage-depleted rat spleen cultures. The polymer stimulated the IgM antibody response of normal splenocytes, and its immunopotentiating activity was increased in macrophage-depleted spleen cell preparations. NED 137 enhanced both the primary and the secondary plaque-forming cell (PFC) response of rat splenocytes in a dose- and time-dependent manner. This stimulatory effect of the polymer was dependent on the presence of antigen and demonstrated antigen specificity. The PFC and mitogenic responses of both normal and macrophage-depleted rat splenocytes were dramatically inhibited in cultures prepared from animals pretreated with NED 137 before culture initiation. The antigen dependency, specificity and potentiation of a secondary response suggest that NED 137 acts through the triggering of the lymphoid system but not as a "conventional" B-cell polyclonal activator.

Tumor necrosis factor alpha regulation of immunoglobulin secretion in trauma patients.

Major trauma-related immune dysfunction is observed at the time of augmented release of immunopathologic mediators. In the present study, T cell-dependent immunoglobulin (Ig) synthesis in peripheral blood mononuclear cell (PBMC) cultures from blunt trauma patients (N = 12, injury severity score (ISS) 27-50), was reduced by 30- > 90%. This coincided with significantly (P < 0.001-0.01) elevated secretion of the biologically active tumor necrosis factor alpha (TNF alpha). Modulation of the TNF alpha activity by anti-TNF alpha antibody (anti-TNF alpha Ab) led to dose-dependent alterations in IgG synthesis. IgG production increased (up to 300%) in cultures treated with 0.5-2 micrograms/ml of the antibody, where low levels of TNF alpha activity often persisted. However, immunoglobulin synthesis was eradicated in preparations exposed to higher concentrations (10 micrograms/ml) of anti-TNF alpha Ab and devoid of TNF alpha biological activity. The treatment with anti-TNF alpha Ab had no effect on mitogen- or alloantigen-induced PBMC proliferation. Thus, in severely traumatized patients, biological activities of endogenous TNF alpha may include modulation of T cell-dependent B lymphocyte function. Immunoregulatory potential of TNF alpha should, therefore, be considered in therapeutic strategies to abrogate its activity.

Immunosuppression follows systemic T lymphocyte activation in the burn patient.

A general consensus that thermal injury affects T lymphocyte function adversely is supported particularly by the observation that burned patients' lymphocytes secrete reduced levels of biologically active IL-2 in vitro. In the same patients, however, high serum concentrations of the low-affinity IL-2 receptor (IL2R alpha), a product of an IL-2-activated gene, have been observed. In this study a significant proportion of patients also demonstrated over-physiological levels (from 2 to 500 U/ml) of serum IL-2 ascertained by immunoassay. Increases in serum IL-2 content correlated significantly (P less than 0.02) with those of serum IL-2R alpha during the first week post-burn. Later, serum IL-2R alpha levels continued to increase up to 30 days while IL-2 eventually declined. Thus, augmented secretion of IL-2R alpha appears related to the high serum IL-2 content. Therefore refractoriness to further immune stimulation may be due to early activation of the lymphoid system, rather than to an intrinsic incapacity of T lymphocytes for generating sequential responses.

Mechanism of enhanced humoral response in rodents by a synthetic polymer.

We previously reported that administration of a low molecular weight (MW=800) synthetic polymer, NED 137, significantly increases humoral and cellular immune responses in the rat. The effect of NED 137 on the murine humoral response to T-dependent (TD) and T-independent (TI) antigens was studied in C57 BL/6, CBA/J and Balb/c mice. The TD antigens (SRBC, DNP-DA with adjuvant) or TI antigen (DNP-Ficoll) were administered simultaneously with NED 137. The polymer significantly increased the direct PFC response to all antigens tested in normal mice. However, it could not restore the PFC response to SRBC in athymic (nu/nu) mice. The effect of NED 137 on accessory cells was studied by the assessment of the in vitro response to SRBC in normal and macrophage-depleted rat spleen cultures. The polymer stimulated both, the primary and secondary IgM response and its immunopotentiating activity was the greatest in macrophage-depleted spleen cell preparations. The lack of effect of NED 137 in systems devoid of functional T cells, dependency on and specificity for a sensitizing antigen and its ability to stimulate a secondary response suggest that this polymer does not act as a "conventional" B-cell polyclonal activator.