Interferon responses in schizophrenia and major depressive disorders.

The spontaneous and induced interferon (IFN) production in whole blood cultures was examined in 45 psychiatric inpatients and in 65 normal controls. Among inpatients there were 32 who were chronic schizophrenics (14 women, 18 men) and 13 who were severely depressed (11 women, 2 men). The analysis of the pooled results of assays in the heterogeneous population showed that leukocytes of the psychiatric patients produced significantly lower levels of IFN after stimulation with virus (NDV), lipopolysaccharide (LPS), and IFN spontaneously released without the inducers that control cells. In contrast, there was no difference between the psychiatric patients and controls in IFN response to phytohemagglutinin and phorbol myristate acetate (PHA + PMA). The results apparently confirmed observations made by Moises et al (1985) and Katila et al (1989). We have also tested our hypothesis that the statistics may mask the individual pattern of IFN response related to the specific psychiatric diagnosis, however. In fact, in the group of chronic schizophrenics we have found either high or low responders to all IFN inducers (NDV, PHA + PMA and LPS). Furthermore, the patients with high IFN response had dominant positive symptoms of schizophrenia (delusions, hallucinations, bizarre behavior and thought disorder). Whereas, in the patients with low IFN response the negative symptoms prevailed (asociality or withdrawal, flat affect, attention impairment, abolition or apathy). In plasma samples of schizophrenics, factors were detected that transferred a hypersensitivity to the IFN inducers to normal donor leukocytes. For instance, in leukocytes cultured in the presence of plasma from schizophrenics, there were 71% of high IFN responders after stimulation with NDV, versus 26% of high IFN responders in the presence of plasma from normal controls. We suggest that the factors may belong to the class of opioid peptides, which interact with the production of cytokines including IFNs.

Modulation of cytokine production by a selenoorganic compound (AE-22) in hyperreactive or hyporeactive bronchoalveolar leukocytes of asthmatics or lung cancer patients.

We have found that many synthetic selenoorganic compounds, including ebselen, have immunotropic activity. These studies were designed to assess the effect of the analog of ebselen bis[2-pyridyl (2-carbamoyl) phenyl]diselenide (AE-22) on human leukocytes that may express various activation states. The cells were obtained from bronchoalveolar lavage (BAL) cells of patients with various inflammatory lung diseases. The AE-22-treated BAL cells from patients with bronchial asthma (n = 6) and with small cell lung cancer (SCLC) (n = 6) were compared with these in the peripheral blood leukocytes (PBL) from the same donors. The control group comprised 5 patients who underwent diagnostic examination and were free of any cancer or concomitant diseases. Secretion of TNF-alpha, IL-6, and IFN-gamma was considered as a marker of BAL or PBL cell activation. Different response of the cells and various effects of AE-22 were observed in relation to the origin and functional state of leukocytes. It was established that AE-22 can induce TNF-alpha, IL-6, and IFN-gamma in a dose-dependent manner in BAL cells and PBL isolated from healthy individuals. However, BAL cells were found to be less reactive than PBL as cytokine producers. In contrast, AE-22 had no effect on BAL cells obtained from patients with lung cancer, which were found to be hyporeactive to phytohemagglutinin and bacterial lipopolysaccharide and did not produce TNF-alpha, IL-6, or IFN spontaneously. The spontaneous release of cytokines by BAL cells from bronchial asthma patients, but not by PBL from the same individuals, was significantly (p < 0.01) higher than that from the cultures of healthy control subjects. The high secretion of cytokines by the locally activated BAL cells was significantly (p < 0.01) reduced after administration of AE-22. The results suggest that AE-22 has immunomodulatory activity. AE-22 can downregulate the hyporeactive BAL cells from asthmatics, but it appears to be inactive in BAL cells of cancer patients who can tolerate the cytokine inducers.

A bioassay employing polyethylene glycol for measuring neutralization of interferon by specific antibodies.

A bioassay employing polyethylene glycol (PEG) for measuring the level of interferon-neutralizing antibodies is described. The method is based on incubating a concentrated preparation of interferon with an appropriately diluted anti-interferon serum or globulin for 1 h at 37 degrees C, precipitating the antigen-antibody complexes as well as some free serum protein with sterile PEG at a final concentration of 10% for 18 h at 4 degrees C, separating the sediment by centrifugation, and titrating the residual interferon in the supernate. It is not necessary to remove PEG from the samples since it is not toxic for tissue culture at a final concentration of less than 1%.

Hyporesponsiveness of human alveolar leukocytes to interferon-alpha and interferon-gamma inducers.

Leukocytes were obtained from bronchoalveolar lavages (BAL) of 36 patients including 10 with lung cancer, 15 with inflammatory lung diseases and 11 healthy control patients undergoing diagnostic investigation. The entire alveolar cell population responded weakly to the classic interferon (IFN) inducers: Newcastle disease virus (NDV), phytohemagglutinin (PHA) and lipopolysaccharide (LPS). This refers mainly to normal healthy volunteers. Alveolar leukocytes from patients with inflammatory lung diseases and nonsteroid treated lung cancer responded better to the interferon inducers than did cells from other patients. The IFN-alpha or IFN-gamma response of whole blood leukocytes to the same inducers was 10 to 100-fold higher than that of the alveolar cells. Alveolar macrophages from 6 healthy individuals and 3 patients with inflammatory lung disease were cultured in vitro for 6 days. The IFN response to inducers appears to depend on the origin of the cultured cells. It increased in the initially hyporeactive macrophages from healthy subjects and decreased in the relatively reactive cells from the patients with inflammatory lung diseases. We suggest that the hyporeactivity to IFN induction is a physiological state of the alveolar leukocytes which are a specialized cell population having constant exposure to inhaled agents such as dust, smoke, microorganisms and their by-products. The hyporesponsiveness to IFN induction of the alveolar cells may have an important physiological role in protecting lungs against hyperproduction of cytokines involved in the inflammatory and allergic reactions.

Choline and halogen derivatives of CMA (9-oxo-10-acridine acetic acids) as tools for monitoring the interaction of the interferon inducers via a specific receptor.

New choline and halogen derivatives of CMA (9-oxo-10-acridine acetic acid) were investigated as interferon (IFN) inducers in mice and in the mouse bone marrow-derived macrophage cultures. Two of the choline derivatives, DMCMA and CSCMA, were active IFN inducers presumably because they were hydrolyzed so as to release CMA. The halogen analogues of CMA were inactive or weak IFN inducers in vivo and in vitro. On the contrary, the Br and I derivatives of CMA were potent inhibitors of IFN induction by CMA in vitro. The behavior of the agonists and antagonists of CMA suggests that the induction of interferon may occur indirectly via a specific CMA-receptor complex.

Classification of cytokines according to the receptor code.

A simple classification of cytokines is presented. It is based on voluminous information available from published papers of many outstanding researchers of the cytokines and their receptors and/or summarized by experts in this field. The cytokines are divided into 7 families according to their receptor code. Such arrangement of apparently different cytokines may explain the biological significance of pleiotropy and redundancy. Furthermore, it may stimulate the development of the comprehensive classification of cytokines together with classical hormones, neuromediators, neuromodulators and related substances that participate in complex biological communication system.

Antiviral activity of Wratizolin.

Wratizolin, the new non-steroidal anti-inflammatory drug, was found to inhibit the replication of Herpes simplex, type 1 and 2, viruses (HSV-1, HSV-2) in vitro. The selectivity of the antiviral action is low because the drug disturbs cellular metabolism. Wratizolin at the relatively low concentration (37 micrograms/ml) inactivates partially purified HSV-2 after incubation for 30 min at 37 degrees. The addition of albumin to the reaction mixture inhibits the virucidal activity of the drug. Wratizolin has little if any effect on the production and action of interferon. The drug used topically as 2% solution in dimethylsulfoxide inhibits the evolution of lesions induced in the guinea pig skin by HSV-2. However, the drug did not prevent the replication of HSV-2 in the guinea pig skin.

Effects of Wratizolin on growth and metabolism of cells in vitro.

The new isothiazole derivative--Wratizolin was found to have several properties which are characteristic of the acidic non-steroidal antiinflammatory drugs, among others--indomethacin. The minimal cytotoxic concentration (MCC) of Wratizolin for the chicken or mouse embryonic monolayer tissue cultures maintained in the media without serum is 6.25 microM. In the medium containing from 3 to 10% calf serum MCC for several animal cells was found to be from 12.5 to 62.5 microM. Wratizolin inhibited the synthesis of DNA in the cultured human lymphocytes at the subtoxic concentration only. Wratizolin, in a characteristic way, stimulates the synthesis of lactic acid and increases the consumption of glucose by the chick or mouse fibroblasts. The drug at concentration of 10(-6) to 10(-12) M stimulates the multiplication of mouse L cells. In all above tests Wratizolin is more active than indomethacin.

Combined action of interferons and transforming growth factor beta on the proliferation of human fibroblasts.

The effects of interferons (IFNs) and transforming growth factor-beta (TGF-beta) used alone and in combination on the multiplication of human embryonic diploid fibroblasts were studied. The experimental conditions were standardized and medium containing 2% of a single batch of fetal calf serum, which was almost minimal dose required for good the cell attachment and growth, was used. The observed effects were dose related. IFNs (types alpha, beta, or gamma) at concentration of 1 to 100 U/ml or TGF-beta at concentration of 0.1 to 1.0 ng/ml stimulated the cell multiplication by 20 to 25% when compared to the control cultures incubated without the factors. At higher doses both IFNs (10(2) to 10(5) U/ml) or TGF-beta (1 to 10 ng/ml) inhibited the proliferation of the cells. The antimitogenic action of IFNs and TGF-beta was synergistic. We suggest that IFNs as well as TGF-beta are bifunctional growth regulators although their antimitotic action dominates over their possible growth stimulatory activity. The latter action may be a secondary phenomenon due to interaction of IFNs or TGF-beta with other factors present in cell culture. We have shown that epidermal growth factor (EGF) may increase the growth stimulatory action of low doses of IFN-gamma or TGF-beta.

Human lymphoid target cells for the cytokine-inducing seleno-organic compounds.

Several seleno-organic compounds including ebselen are known as antiinflammatory and antioxidant agents. They also have glutathione peroxidase-like activity and are inhibitors of leukotrienes and prostaglandins. We have recently discovered that these drugs are inducers of cytokines, mainly interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) and mitogenic interleukins in human peripheral blood leukocytes (PBL) but not in the mouse or rat lymphoid cells. We described a production of IFN-gamma and TNF-alpha by various subsets of PBL stimulated with 2-phenyl-1,2-benzisoselenazol-3(2H)-one (ebselen) or bis [2-(N-phenyl-carbamoyl)]phenyl diselenide. IFN-gamma was produced mainly by E-rosette positive lymphocytes. However, the presence of monocytes was required for the optimal production of IFN-gamma. Also soluble mediators released by monocytes enhanced IFN-gamma synthesis. On the other hand, TNF-alpha was produced mainly by the adherent monocytes. Its synthesis was enhanced by the addition of T or B lymphocytes or conditioned medium from the culture of the stimulated lymphocytes. The relative concentrations of the subsets of lymphocytes or monocytes was important for the maximum production of both IFN-gamma and TNF-alpha. High concentration of lymphocytes inhibited the cytokine production.

Application of antisera to interferon in studying oncogenic viruses.

Susceptibility of BALB/c mice to infection with Moloney sarcoma virus (MSV) and to Herpes simplex virus type 2 (HSV-2) was considerably increased by administration of sheep anti-mouse interferon (anti-IF) serum. The regression of the MSV-induced tumors was inhibited when the weanling (three-to-four-week-old) mice were injected with the anti-IF serum. Using the anti-IF serum it has been found that the antagonism between HSV-2 and Rauscher leukemia virus in the mouse is not mediated by interferon. It is suggested that interferon is an important factor controlling growth of virus and/or virus induced tumor cells in the mouse before it develops a strong immunological response.

Platelet-derived growth factor (PDGF) enhances the growth of Moloney sarcoma virus-induced tumors in mice.

The intramuscular (i.m.) administration of bovine or human platelet derived growth factor (PDGF) had a marked potentiating effect on the growth of Moloney sarcoma virus (MSV)-induced tumors in Balb/c mice. PDGF had the tumor-promoting-activity when administered i.m. on the side of inoculation of MSV two days after the virus, and subsequently every second day for 2-3 weeks. The extent of MSV-induced tumor promoting activity of PDGF was age and sex-related. In view of the recently described antagonism in action between growth factors and interferons it is suggested that both families of hormones play an important role in controlling the neoplastic proliferation.

Double infection of BALB/c mice with Rauscher murine leukemia and Herpes simplex virus type 2.

When BALB/c mice were first infected with Rauscher murine leukemia virus (MuLV-R) and then superinfected with Herpes simplex virus type 2 (HSV-2) the inhibition of the evolution of splenomegaly was observed. The effect did not depend on the route of HSV-2 administration. Experiments in which potent anti-interferon serum was administered to double infected mice suggested that the antagonism between MuLV-R and HSV-2 was not mediated by endogenous interferon, HSV-2 was found to replicate in the LPS stimulated spleen cells which are also target cells for MuLV-R; this suggested that the intrinsic interference between both viruses could take place. Mice with Rauscher virus induced disease were found to be more susceptible to infection with Herpesviruses than normal mice.

Hormonal-like modulation of growth of moloney virus-induced tumors by interferon and platelet-derived growth factor.

Continued intramuscular (i.m.) administration of highly purified preparations of mouse interferon (MuIFN) and/or bovine platelet-derived growth factor (PDGF) to the Moloney sarcoma virus (MSV) infected Balb/c mice resulted in a hormonal-like modulation of growth of tumors. Large dose MuIFN treatment (10(5) units per mouse daily, for 14 days) inhibited the MSV-induced tumors. On the other hand, small-dose MuIFN treatment (100 units per mouse daily, for 14 days or 10(4) units per mouse, every second day in 3-4 doses) enhanced the tumor growth. The simultaneous administration of large doses of MuIFN and PDGF significantly diminished the antitumor effect of IFN. The results suggest that the negative and positive interaction between IFN and PDGF takes place in vivo.

Biological evaluation of Wratizolin penetration from ointments to the skin.

The degree of Wratizolin penetration to the skin from ointments prepared in 6 different media was determined. Experiments were carried out in mice and guinea pigs using: a) method determining the decrement of cytotoxic and antibacterial activity of the tested ointment contacted with the skin, b) biological tests to check the presence-of the drug in tissue extracts. Absorption of Wratizolin appeared dependent upon the kind of base ointment. The preparation penetrated well when suspended in polyethylene glycol base ointment (PEG).

New procedure for purification of human platelet-derived growth factor.

A new simple procedure was developed for purification of human platelet-derived growth factor (PDGF). PDGF was isolated and concentrated by acid/ethanol extraction of platelet lysate. Purification of this extract was accomplished by initial ion-exchange chromatography on DEAE-Sephadex and subsequent separation of "cationic" unadsorbed fraction on CM-Sephadex and controlled-pore glass--CPG-10. The product migrated as two biologically active homogeneous fractions, PDGF I (Mr = 34 000) and PDGF II (Mr = 31 000) in analytical gel electrophoresis in the presence of SDS. PDGF had isoelectric point ranging from 9.5-10.6. Multiplication-stimulating activity of PDGF was estimated using the clone of Balb/c 3T3 cells propagated in microplates in Eagle's minimal essential medium supplemented with 2% of platelet poor plasma serum. Purified PDGF was active in stimulating 3T3 cell proliferation at 0.4 ng/ml.

Synthesis of 1,4-diamino-anthraquinone-2-sulfonic acid derivatives coupled to dextran as carriers for interferon.

Ten compounds (II, VI-XIV) of the simpler structure similar to Cibacron Blue were synthesized and bound to dextran with ether binding. Eight obtained high-molecular-weight compounds (D-VII to D-XIV) displayed in biological assays high affinity for mouse or human interferons.

Colostrinine: a proline-rich polypeptide from ovine colostrum is a modest cytokine inducer in human leukocytes.

A proline-rich polypeptide (PRP), now named colostrinine, molecular weight 18,000, was isolated from ovine colostrum and characterized by Janusz, Lisowski et al. The nonapeptide (NP) which is an active fragment of PRP was obtained by chemical synthesis. In mice, PRP has many regulatory effects on the humoral and cellular immune response. The present paper describes PRP as a cytokine inducer. PRP at concentration of 1-100 micrograms/ml was found to induce production of interferon (IFN) and tumor necrosis factor (TNF) in human peripheral blood leukocytes and in whole blood cultures. The effects were dose related. The identified till now cytokines induced by PRP were IFN-gamma and TNF-alpha but many other cytokines may be stimulated also. NP was considerably less active as the cytokine inducer than the natural PRP. Two volunteers given orally once daily for two to three weeks 100 or 200 micrograms PRP in tablets were found to develop the tolerance of IFN induction and had the modified TNF response. Furthermore, the PRP-treated volunteers showed signs of psycho-stimulation. Taken together our observations suggest that ovine PRP is active in humans and may have therapeutic value as an immunostimulant and/or neurotropic cytokine.

Biological activity of sera obtained by hyperimmunizing rabbits with interferon from mouse L cells.

Rabbits immunized with interferon from mouse L cells for long periods of time produced interferon-neutralizing antibodies with titers from 1:80 to 1,2000. The anti-interferon sera also contained antibodies against antigens contaminating the interferon preparations such as albumin, bovine gamma-globulin, chicken albumin, extract from L cells, and Sindbis virus antigens. Some sera also displayed cytotoxic activity against cells of transplantable murine leukemia. These antibodies could be removed by specific absorption. Titers of antibodies neutralizing interferon were not correlated with the titers of antibodies against concomitant antigens. In hyperimmunized rabbits interferon-neutralizing antibodies persisted for long periods of time in spite of interrupting immunization.

Tolpa Torf Preparation (TTP) induces interferon and tumor necrosis factor production in human peripheral blood leukocytes.

Tolpa Torf Preparation (TTP) is a natural immunomodulating drug registered in Poland for use in humans. TTP is a biologically active low molecular weight fraction of an extract from peat containing organic substances, primary bound sugars, amino-acids, uronic and huminic acids and mineral salts. The toxicity of TTP is remarkably low, eg. cytotoxicity (CD50) for human peripheral blood leukocytes (PBL) is 1-9 mg/ml. We have discovered that TTP is an interferon (IFN) and tumor necrosis factor (TNF) inducer in human PBL. The IFN and TNF response of the PBL cultures was dose dependent. The optimal concentration of TTP for IFN or TNF response was 10-100 micrograms/ml. The cytokines stimulated by TTP were IFN-gamma, IFN-alpha and TNF-alpha. Ten commercial batches of TTP have been found to be active as cytokines inducers although variations in their activities were observed. On the other hand, 8 batches of TTP rejected by the producer because of the inadequate immunostimulating activity determined in mice, were found to be significantly less active than the commercial preparations. Over 115 buffy coats from the individual blood donors were used to prepare PBL cultures for this study. Approximately 20% of the PBL cultures were unresponsive to TTP. The IFN and TNF response of PBL to other inducers: phytohemagglutinin (PHA) or lipopolysaccharides (LPS) also varied. Whereas only 7% of PBL could not be stimulated by PHA, as much as 20-50% of PBL failed to produce IFN or TNF when treated with LPS. We suggest that TTP may have clinically useful activities connected with the capacity of stimulation of IFNs and TNF production.