Tyrphostins reduce chemotherapy-induced intestinal injury in mice: assessment by a biochemical assay.

Intestinal injury that results from chemotherapy belongs to the major factors of dose-limitation in tumour therapy. The tyrphostins AG1714 and AG1801 reduce cisplatin and 5-FU-induced small intestinal mucosal damage, using a quantitative biochemical assay. The assay is based on the determination of the enzymatic activity of gamma-glutamyl transpeptidase, a marker of the brush border epithelium of the small intestine.

CD4+ T lymphocytes as a primary cellular target for BAT mAb stimulation.

BAT is a monoclonal antibody (mAb) produced against membranes of a human Burkitt lymphoma cell line (Daudi) that was selected for its ability to stimulate lymphocyte proliferation. BAT manifests anti-tumor properties in mice bearing a variety of murine tumors. BAT also induced regression of human tumors inoculated into SCID mice that had been engrafted with human lymphocytes. The anti-tumor activity of BAT was related to its immune stimulatory properties. Previous data indicated that T lymphocytes and NK cells mediate in vivo the anti-tumor activity. In order to define the primary target cell for BAT stimulatory activity, the in vitro stimulatory effect of BAT on purified lymphocyte subpopulations was investigated. Human CD4(+), CD8(+) T cells and CD56(+) NK cells were purified and their in vitro response to BAT was investigated. Results indicate that BAT selectively stimulated CD4(+) cells as assessed by proliferation and secretion of IFN-gamma. FACS analysis has also revealed a selective increase in BAT antigen on CD4(+) T cells that were cultured with BAT antibody. The effector cells that mediate BAT-induced tumor eradication may, however, be distinct from those that serve as the primary cellular target of the antibody. Cytokines such as IFN-gamma that are produced by CD4(+) cells may be involved in activation of additional cell types that may be involved in tumor destruction.

Activation of lymphocytes by BAT and anti CTLA-4: comparison of binding to T and B cells.

In this study we compare the binding and immune stimulatory properties of BAT and anti CTLA-4 monoclonal antibodies (mAbs). Both antibodies were previously shown to manifest effective immune responses against tumor cells. We have described that BAT antibody, produced against Daudi, a B lymphoblastoid cell line, binds and activates T cells. In this paper we demonstrate that anti CTLA-4, produced against the T-cell activation determinant CTLA-4, binds also to B lymphoblastoid cell lines like Daudi and Raji. Both antibodies do not bind resting B cells. BAT binds resting T lymphocytes as well as activated T lymphocytes, whereas anti CTLA-4 binds only activated T cells. Competitive binding experiments indicate that the binding sites of BAT and anti CTLA-4 on activated T cells are distinct. We have studied the in vitro stimulatory effect of BAT and anti CTLA-4 on lymphocytes cultured with or without tumor cells. In contrast to BAT that increased the proliferation of lymphocytes that have been cultured with tumor cells, anti CTLA-4 did not synergize with tumor cells to enhance lymphocyte proliferation.

Antibodies to neuroblastoma cell line proteins in patients with schizophrenia.

The presence of antibodies against neural antigens was investigated in the serum of patients with schizophrenia, major depression and normal controls. Different immunological abnormalities, humoral and cellular, were reported in schizophrenia and major depression. The pathogenesis of schizophrenia is multifactorial. An autoimmune mechanism was suggested as a possible factor. We tested the serum of 26 patients with schizophrenia, eight patients with major depression and 22 normal controls. The serum samples were tested for antibody binding to protein extracts of IMR-32 neuroblastma cell line using Western blot analysis. Immunoglobulins of eight patients with schizophrenia (30.71%) reacted with a protein of 80-85 kDa. Serum samples from subjects of other groups did not react with this protein. Sera of all patients with major depression but one, and all normal controls reacted with HSP 60 kDa to different extent. This is an apparent discrepancy with the findings of Kilidireas et al. [Kilidireas, K., Latov, N., Strauss, D.H., Gorig, A.D., Hashim, G.A., Gorman, J.M., Sadig, S.A., 1992. Antibodies to the human 60 kDa heat shock protein in patients with schizophrenia. Lancet 340, 569-572.] who demonstrated the presence of antibodies against HSP 60 kDa in 44% of patients with schizophrenia tested and 8% of normal subjects. HSP 60 kDa is an antigen of many pathogens and antibodies against it might be a result of an infection and cannot be a good indicator for an autoimmune process. The presence of antibodies against a protein of 80-85 kDa should be investigated as a possible specific indicator.

Tyrphostin 4-nitrobenzylidene malononitrile reduces chemotherapy toxicity without impairing efficacy.

In mice, 4-nitrobenzylidene malononitrile (AG1714), which belongs to the tyrphostin family, reduced toxicity induced by doxorubicin and cisplatin without impairing their antitumor efficacy. AG1714 reduced mortality induced by doxorubicin and cisplatin. It prevented, in a dose-dependent manner, cisplatin-induced nephrotoxicity as assessed by measurement of serum creatinine and blood urea nitrogen levels. The protective effect of AG1714 was most pronounced on its administration 2 h before cisplatin. AG1714 also prevented doxorubicin-induced myelosuppression as assessed by the scoring of bone marrow nucleated cells and colony-forming units. Cisplatin-induced small intestinal injury was also protected by AG1714 as assessed by histopathological analysis. In vitro, AG1714 reduced cisplatin-induced apoptosis in a murine fibroblastic cell line (A9) and did not affect doxorubicin-induced apoptosis of B-16 melanoma cells. In contrast to its protective effect against mortality and injury of normal tissues induced by chemotherapy, AG1714 did not impair its antitumor activity and in some tumor models enhanced it. This was evident by using the murine tumors B-16 melanoma, Lewis lung carcinoma, and methylcholanthrene-induced fibrosarcoma and the human tumors SK-28 melanoma and human ovary carcinoma xenografts in nude mice. Experiments in which low and high doses of cisplatin and doxorubicin were administered to tumor-bearing mice demonstrated that AG1714 reduced mortality of high-dose chemotherapy and increased its therapeutic index. AG1714 could provide a novel, useful tool to improve chemotherapy by allowing dose intensification.

A lymphocyte-activating monoclonal antibody induces regression of human tumors in severe combined immunodeficient mice.

Monoclonal antibodies were raised against Daudi B-lymphoblastoid cell line membranes. An mAb (BAT) was selected for its ability to stimulate human and murine lymphocyte proliferation. BAT induced cytotoxicity in human and murine lymphocytes against natural killer cell-sensitive and -resistant tumor cell lines. A single intravenous administration of BAT to mice that had been inoculated with various murine tumors (e.g., B16 melanoma, 3LL carcinoma, and methylcholanthrene fibrosarcoma) resulted in striking antitumor effects as manifested by complete tumor regression and prolonged survival of the treated mice. BAT exhibited a diminished but significant antitumor effect in athymic nude mice, which are deficient in T lymphocytes, and in beige mice, which are deficient in NK cells. Furthermore, selective depletion of T or NK cells in mice reduced the response to the antitumor effect of BAT. These data indicate a dual role for T and NK cells in mediating the antitumor activity of BAT. We report here on the antitumor activity of BAT mAb on human tumor xenografts in mice. BAT demonstrated an antitumor effect in nude mice bearing human colon carcinoma (HT29) xenografts. It failed, however, to inhibit established lung metastases in severe combined immunodeficient (SCID) mice that had been inoculated (i.v.) with SK28 human melanoma. Engraftment of human lymphocytes into SCID mice bearing human melanoma xenografts rendered them responsive to the antitumor effect of BAT. The efficacy of BAT in the regression of human tumors by activation of human lymphocytes indicates its potential clinical use.

Tyrphostin AG 556 improves survival and reduces multiorgan failure in canine Escherichia coli peritonitis.

Tyrosine kinase-dependent cell signaling is postulated to be a pivotal control point in inflammatory responses initiated by bacterial products and TNF. Using a canine model of gram-negative septic shock, we investigated the effect of tyrosine kinase inhibitors (tyrphostins) on survival. Animals were infected intraperitoneally with Escherichia coli 0111: B4, and then, in a randomized, blinded fashion, were treated immediately with one of two tyrphostins, AG 556 (n = 40) or AG 126 (n = 10), or with control (n = 50), and followed for 28 d or until death. All animals received supplemental oxygen, fluids, and antibiotics. Tyrphostin AG 556 improved survival times when compared to controls (P = 0.05). During the first 48 h after infection, AG 556 also improved mean arterial pressure, left ventricular ejection fraction, cardiac output, oxygen delivery, and alveolar-arterial oxygen gradient compared to controls (all P < or = 0.05). These improvements in organ injury were significantly predictive of survival. Treatment with AG 556 had no effect on clearance of endotoxin or bacteria from the blood (both P = NS); however, AG 556 did significantly lower serum TNF levels (P = 0.03). These data are consistent with the conclusion that AG 556 prevented cytokine-induced multiorgan failure and death during septic shock by inhibiting cell-signaling pathways without impairing host defenses as determined by clearance of bacteria and endotoxin.

Immune stimulatory and anti-tumor properties of anti-CD3 and BAT monoclonal antibodies: a comparative study.

A novel monoclonal antibody raised to Daudi cell membranes was found to exhibit immune stimulatory and anti-tumor properties. The activity of this antibody (BAT) which also binds T cells was compared to that of anti-CD3. Anti-CD3 reacts with the T cell receptor complex, induces cell proliferation, and cytolytic activity in vitro and also manifests in vivo anti-tumor effect against murine tumors. Comparison of the two antibodies demonstrates similar induction in vitro of splenocyte proliferation and cytolytic activity. Both BAT and anti-CD3 antibodies manifest anti-tumor activity in mice bearing B16 melanoma. They differ however in the timing of antibody administration post-tumor inoculation which is most effective in eliciting the anti-tumor effect. Whereas BAT is most effective when administered 10 to 14 days post-tumor inoculation, anti-CD3 is effective at an early time. Data also indicate that BAT synergises with tumor cells in eliciting cell proliferation in vitro. In contrast, this effect could not be demonstrated with anti-CD3. The properties of BAT may be of advantage in its potential clinical use.

Late administration of a lipophilic tyrosine kinase inhibitor prevents lipopolysaccharide and Escherichia coli-induced lethal toxicity.

Septic shock induced by gram-negative bacteria results primarily from excessive stimulation by lipopolysaccharide (LPS) of macrophages to produce tumor necrosis factor (TNF)-alpha and interleukin (IL)-1. The cellular effects of LPS, TNF-alpha, and IL-1 are mediated via tyrosine phosphorylation pathways. A recent report indicated that selective inhibitors of tyrosine kinases, tyrphostins of the AG126 family, protect mice against LPS-induced lethal toxicity in mice. Protection was most effective when the tyrphostin was injected before the LPS. In the present study, tyrphostin AG556, which is more lipophilic than those of the AG126 family, was effective in preventing LPS-induced lethal toxicity when administered 2 h after LPS. AG556 also prevented viable Escherichia coli-induced lethal toxicity when given 2 h before and, to a lesser extent, 2 h after the bacterial inoculation. AG556 may block a critical step downstream of the signaling pathway induced by LPS after TNF-alpha production.

p21ras as a common signaling target of reactive free radicals and cellular redox stress.

Reactive free radicals have been implicated in mediating signal transduction by a variety of stimuli. We have investigated the role of p21ras in mediating free radical signaling. Our studies revealed that signaling by oxidative agents which modulate cellular redox status, such as H2O2, hemin, Hg2+, and nitric oxide was prevented in cells in which p21ras activity was blocked either through expression of a dominant negative mutant or by treating with a farnesyltransferase inhibitor, as assessed by NF-kappa B binding activity. Furthermore, the NF-kappa B response to these oxidative stress stimuli was found to be enhanced when cells from the human T cell line, Jurkat, were pretreated with L-buthionine-(S,R)-sulfoximine, an inhibitor of glutathione synthesis. We directly assayed p21ras and mitogen-activated protein kinase activities in Jurkat cells and found both of these signaling molecules to be activated in cells treated with the redox modulating agents. Blocking glutathione synthesis made cells 10- to 100-fold more sensitive to these agents. Finally, using recombinant p21ras in vitro, we found that redox modulators directly promoted guanine nucleotide exchange on p21ras. This study suggests that direct activation of p21ras may be a central mechanism by which a variety of redox stress stimuli transmit their signal to the nucleus.

Activation of human lymphocytes by a monoclonal antibody to B lymphoblastoid cells; molecular mass and distribution of binding protein.

A novel monoclonal antibody (BAT) to the B-lymphoblastoid cell line activates murine lymphocytes and exhibits a striking antitumor activity in mice. In order to evaluate the potential use of this antibody against human cancer, we have investigated its immuno-stimulatory properties on human peripheral blood lymphocytes (PBL). Our findings demonstrate that BAT mAb induces proliferation and cytotoxicity in human PBL against natural-killer-cell-sensitive and natural-killer-cell-resistant tumor cell lines. Interleukin-2 at a low concentration synergizes with BAT mAb in eliciting these effects. BAT mAb binds to human peripheral T cells as revealed by a double-labelling technique using anti-CD3 and BAT mAb. The molecular mass of the antigen recognized by BAT mAb was 48-50 kDa under reducing and non-reducing conditions. This study provides a basis for future experiments to evaluate the use of BAT mAb in the immunotherapy of cancer.

A diagnostic assay for the Wiskott-Aldrich syndrome and its variant forms.

BACKGROUND: The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by severe thrombocytopenia, eczema, and impaired immunity. While the diagnosis is usually straightforward, the syndrome may be expressed in an attenuated form, a phenotype which is difficult to distinguish from other types of congenital thrombocytopenia. Although a molecular-based assay for diagnosis of the spectrum of WAS patients has not been available, recent data indicate that WAS is associated with a specific profile of impaired mitogen responsiveness and suggest that detection of this abnormality may provide a diagnostic marker for all forms of the disease. To address this issue, we have studied patients with classical and atypical WAS for their lymphocyte proliferative responses to four T cell mitogenic stimuli and compared their response patterns to those detected in unaffected children. METHODS: Clinical histories and informed consent were obtained from 23 patients with either classical or putative (ie, atypical) WAS, 16 subjects with other disorders, and 12 healthy children. Peripheral blood mononuclear cells (PBMCs) collected from patients and controls were resuspended in culture medium, stimulated with the T cell mitogens phytohemagglutinin (PHA), concanavalin A (Con A), neuraminidase/galactose oxidase (NAGO), or periodate, and cultured for 60 h in 0.2 mL aliquots. Following a 20 h pulse with 3H-thymidine, cultures were harvested and the 3H-thymidine uptake was evaluated by liquid scintillation counting. RESULTS: The most striking observation involved response to periodate. While lymphocytes from all healthy control children proliferated in response to periodate treatment, cells from both classical as well as atypical WAS patients consistently failed to proliferate in response to this mitogen. By contrast, lymphocyte proliferative responses to PHA, Con A, and NAGO were detected in all patients and controls, although responses generally were lower in cells from classical WAS patients compared to other children. In two WAS patients, bone marrow transplantation and clinical improvement were associated with a change from no periodate response (pre-transplant) to periodate responsiveness (post-transplant). In contrast to the WAS patients, cells from patients with other hematologic and primary immune deficiency diseases responded uniformly to all four mitogens, including periodate. CONCLUSIONS: The data presented here indicate that T cells from patients with either classical or attenuated WAS fail to undergo proliferation in response to periodate, an agent that induced extensive T cell mitogenesis of cells from all healthy controls as well as patients with diseases other than WAS. As the WAS patients' cells did proliferate in response to treatment with other T cell mitogens, it appears that periodate induced T cell proliferation is selectively impaired in WAS and that detection of this defect may be of value in the distinction of both classical and attenuated WAS from other thrombocytopenic conditions.

Nitric oxide-stimulated guanine nucleotide exchange on p21ras.

The protooncogene p21ras, a monomeric G protein family member, plays a critical role in converting extracellular signals into intracellular biochemical events. Here, we report that nitric oxide (NO) activates p21ras in human T cells as evidenced by an increase in GTP-bound p21ras. In vitro studies using pure recombinant p21ras demonstrate that the activation is direct and reversible. Circular dichroism analysis reveals that NO induces a profound conformational change in p21ras in association with GDP/GTP exchange. The mechanism of activation is due to S-nitrosylation of a critical cysteine residue which stimulates guanine nucleotide exchange. Furthermore, we demonstrate that p21ras is essential for NO-induced downstream signaling, such as NF-kappa B activation, and that endogenous NO can activate p21ras in the same cell. These studies identify p21ras as a target of the same cell. These studies identify p21ras as a target of NO in T cells and suggest that NO activates p21ras by an action which mimics that of guanine nucleotide exchange factors.

A monoclonal antibody against a human B lymphoblastoid cell line induces tumor regression in mice.

We have developed a monoclonal antibody (BAT) to Daudi B lymphoblastoid cell line membranes. The antibody was selected for its ability to stimulate lymphocyte proliferation. Splenocytes of BALB/c or C57BL mice given i.v. injections of 10 micrograms/mouse of BAT exhibited increased proliferation and cytotoxic activity. A single i.v. administration of BAT monoclonal antibody 2 weeks after B16 melanoma cell inoculation resulted in a striking antitumor effect as manifested by the elimination of lung metastases and prolonged survival of the treated mice. BAT monoclonal antibody was also effective in the regression of tumors in mice bearing 3LL (Lewis lung carcinoma) and MCA-105 (fibrosarcoma). Transfer of 10(7)-10(8) splenocytes from mice that had been given injections of BAT to B16- or 3LL-inoculated recipients led to a reduction of lung metastases. Splenocytes from B16-inoculated mice that were cured by BAT were more effective than those from mice treated with BAT alone against recipients bearing either B16 or 3LL tumors. The antitumor activity of BAT is related to its immunostimulatory properties.

Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors.

Septic shock results from excessive stimulation of the host immune system, especially macrophages, by lipopolysaccharide (LPS), or endotoxin, which resides on the outer membrane of bacteria. Protein tyrosine kinase inhibitors of the tyrphostin AG 126 family protect mice against LPS-induced lethal toxicity. The protection correlates with the ability of these agents to block LPS-induced production of tumor necrosis factor alpha (TNF-alpha) and nitric oxide in macrophages as well as LPS-induced production of TNF-alpha in vivo. Furthermore, this inhibitory effect correlated with the potency of AG 126 to block LPS-induced tyrosine phosphorylation of a p42MAPK protein substrate in the murine macrophage.

Novobiocin modulates colchicine sensitivity in parental and multidrug-resistant B16 melanoma cells.

The effect of the antibiotic agent novobiocin on the sensitivity of melanoma cells to colchicine and vinblastine was examined in drug-sensitive and drug-resistant B16 melanoma cells. A cell line COL/R was selected for colchicine resistance. The COL/R cell line (resistant to 80 ng/ml colchicine) was found to possess the multidrug-resistant (MDR) phenotype. The cells were shown to be cross-resistant to vinblastine and Adriamycin and to overexpress P glycoprotein. P glycoprotein activity was assessed by using the rhodamine 123 accumulation test. Rhodamine accumulation was markedly decreased in COL/R cells as compared to the parental B16 cells. Verapamil reversed drug resistance and increased rhodamine accumulation in COL/R cells. Novobiocin in combination with colchicine or vinblastine synergistically inhibited the proliferation of parental B16 cells. In COL/R cells, novobiocin markedly decreased colchicine resistance and increased rhodamine accumulation. These data show that novobiocin increases the sensitivity of both parental and MDR melanoma cells to microtubule-disrupting cytotoxic drugs.

Butyrate-induced differentiation in leukemic myeloid cells - in-vitro and in-vivo studies.

A study of the effects of three differentiating agents, butyric acid, retinoic acid and cytosine arabinoside on proliferation and differentiation of primary cultures, obtained from sixteen patients with myelo-proliferative disorder was conducted. The results showed that BA was an effective inhibitor of cell proliferation and inducer of cytodifferentiation. An acute non-lymphoblastic leukemia patient was treated with sodium butyrate. A temporary increase in differentiation-associated parameters were noted. However, the effects of SB were short-lived. The lack of clinical response led to the development of a BA prodrug pivaloyloxymethylbutyrate (AN-9). This prodrug was more potent in vitro than BA in the induction of cytodifferentiation and inhibition of cell proliferation.

Nitric oxide signaling: a possible role for G proteins.

We have previously reported various inductive effects of nitric oxide on human PBMC. We describe a novel and potentially important mechanism of nitric oxide signaling-through direct activation of guanine nucleotide-binding proteins (G proteins). We have found that nitric oxide treatment of membranes isolated from fresh human PBMC enhances the ability of these membranes to hydrolyze [gamma-32P]GTP and bind [gamma-35S]GTP. In addition, treatment of whole cells with nitric oxide yielded membranes with enhanced GTPase activity. Furthermore, the GTPase activity of pure, recombinant Gs alpha, Gi alpha 1, and p21ras was greatly enhanced by nitric oxide. In support of the existence of this pathway in whole cells, we found that the G protein inhibitor, GDP-beta-S, blocked NF-kappa B translocation induced by nitric oxide or LPS in permeabilized cells. In addition, nitric oxide greatly reduced the pertussis toxin-mediated ADP-ribosylation of 45- and 41-kDa proteins in membranes of these cells. Because G proteins play a central role in many diverse signaling systems, activation by an endogenous and inducible oxidant may represent a novel signaling pathway.

Immune stimulatory and anti-tumour properties of haemin.

IL-2 induces tumour regression in some patients with metastatic disease, but the dose of IL-2 is limited by severe toxicity. Agents that increase the expression of IL-2 receptors in the effector cells could be used to improve the effectiveness of IL-2 in mediating its anti-tumour effect. We have reported that haemin increased the expression of IL-2 receptors in human peripheral blood mononuclear cells (PBMC) and synergized with IL-2 in the induction of mitogenicity, cytotoxicity and cytokine production. We now report on haemin-induced immune stimulation and tumour regression in mice. Haemin-induced mitogenicity in mouse splenocytes was potentiated up to two-fold by IL-2. The combination of haemin and IL-2 was also effective in inducing cytotoxicity for natural killer (NK)-resistant target cells. Maximal induction of cytotoxicity was attained at an optimal concentration of haemin of 10 microM. Higher concentrations were less effective. Splenocytes isolated from mice that had been treated in vivo with haemin and IL-2 incorporated twice the amount of 3H-thymidine compared with splenocytes from mice treated with either haemin or IL-2 alone. Cytotoxicity of splenocytes for NK-resistant target cells was not increased following in vivo administration of haemin and IL-2 when fresh splenocytes were tested. Cytotoxicity was enhanced, however, up to five-fold following 48 h in vitro incubation with IL-2. Administration of haemin and IL-2 resulted in a significant decrease (40%) of established hepatic metastases in mice. Either IL-2 or haemin alone at the dose used were ineffective. The anti-tumour effect of haemin and IL-2 was enhanced (63% decrease in metastases) by administration of the thiol compound, N-acetylcysteine. Since haemin can safely be administered to patients, it may represent a new class of biologic response modifiers that could enhance IL-2-mediated anti-tumour effects.

Amino acid alcohols: growth inhibition and induction of differentiated features in melanoma cells.

The effects of a series of D- and L-amino acid alcohols on the proliferation and phenotypic expression of B16 mouse melanoma cells were evaluated. B16 melanoma cells were incubated for different time intervals in the presence of D- or L-phenylalaninol (PHE), D- or L-alaninol (AL), D- or L-leucinol (LE), L-histidinol (HIS), L-tyrosinol (TYR) and L-methioninol (MET). All agents, including the D or L configuration, induced an anti-proliferative effect, although of considerably different magnitude. D-PHE was the most active growth inhibitor. The growth inhibitory effects were accompanied by phenotypic alterations, which included morphological changes and enhancement in the activities of NADPH cytochrome c reductase and tau-glutamyl transpeptidase. These phenotypic alterations correlated with the growth inhibitory effects of the different agents and seem to reflect a higher differentiated state.