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It is now well established that parenteral drug abuse is a significant risk factor for contracting human immunodeficiency virus type 1 (HIV-1) infection and subsequently developing AIDS. Earlier studies have shown that morphine can modulate various immune responses and therefore support the premise that morphine is a cofactor in susceptibility to and progression of HIV infection. Dysregulation of interferon (IFN) production, nonspecific apoptosis of T cells, and the immune response to soluble HIV gene products have been associated with potential mechanisms of pathogenesis in HIV disease. The present study was undertaken to examine the immunomodulatory role of morphine on HIV protein-induced lymphocyte proliferative responses, Sendai and Newcastle disease virus-induced alpha IFN (IFN-alpha) and IFN-beta production by lymphocytes and fibroblast cells, respectively, and induction of apoptosis of normal lymphocytes in vitro. Our results demonstrate that HIV protein-induced human lymphocyte proliferative responses were significantly inhibited by morphine in a dose-dependent manner. Furthermore, morphine significantly inhibited both IFN-alpha and IFN-beta production by normal lymphocytes and fibroblasts but induced apoptosis of normal lymphocytes. Inhibition of IFN-alpha production by morphine could be reversed by the opiate receptor antagonist naloxone. This suggests that the immunomodulatory effects of morphine are mediated through the opioid receptor. These studies support a role of morphine as a cofactor in the pathogenesis of HIV infection and describe some of the possible pathologic mechanisms which underlie the immunoregulatory effects of morphine.

The study is aimed to determine the role of luteolin (3',4',5,7-tetrahydroxyflavone), alone and in combination with human interferon-beta (IFN-beta), in modulating the immune response(s) of peripheral blood mononuclear cells (PBMCs) isolated from multiple sclerosis (MS) patients. PBMC proliferation in the presence or absence of these drugs was determined and the production of pro-inflammatory cytokines (IL-1beta, TNF-alpha), and the ratio of cell migration mediator MMP-9, and its inhibitor, TIMP-1 was assessed in the culture supernatants. Luteolin reduced, in a dose-dependent manner, the proliferation of PBMCs, and modulated the levels of IL-1beta and TNF-alpha released by PBMCs in the culture supernatants. Luteolin reduced the MMP-9/TIMP-1 ratio via lowering MMP-9 production. In the majority of cases, luteolin, when combined with IFN-beta, had additive effects in modulating cell proliferation, IL-1beta, TNF-alpha, MMP-9 and TIMP-1.

We have described a nonantibody type inhibitor of interferons (IFN) in the blood of patients with AIDS, advanced neoplastic disorders, and lupus erythematosus in earlier reports (1,2). In the present study we show that the semipurified inhibitor blocks the antiproliferative signal of IFN-alpha in Daudi cells and the membrane potential shifting ability of IFN-alpha is modulated by the interferon inhibitor preparation (IFI).

Synthesis of interferon (IFN) in response to Sendai virus and the development of the resulting antiviral state were studied in human (BG-9) and murine (L-929) fibroblast cell cultures in the presence of the calmodulin antagonists, trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). Compared to control cultures, 16-fold and 8-fold more IFN was formed in human and murine cells, respectively, when 10 microM TFP was present in the medium for 24 h prior to IFN induction with Sendai virus. W-7 did not affect IFN production in human cells, but enhanced it in L-929 cells by 4- to 8-fold. TFP inhibited the antiviral state induced by homologous IFN in the two cell systems, and at 20 microM, there was a 3,000-fold increase in vesicular stomatitis virus (VSV) yield. It also reduced the maintenance of the antiviral state in human cells. In contrast, W-7 had no effect on the development of the antiviral state in either of the two cell systems. Thus, calcium-calmodulin dependent cellular processes are involved in both induction of IFN and its action. The several patterns response to TFP and W-7 may reflect different ligand-binding sites on calmodulin.

Interferon (IFN) resistance is an important factor in the pathophysiology of neoplastic disorders, certain viral infections (e.g., AIDS), and autoimmune diseases (e.g., lupus erythematosus and Wegner's granulomatosis). In addition, in some of these disorders, there is also decreased ability to produce IFNs. The capacity of viruses and neoplastic processes to interfere with the IFN system are thought to represent a “virus-against-host” or “cancer-against-host” defense mechanism. Four resistance factors have been identified: 1) release of free IFN-α/β type 1 receptors into the circulation that, at appropriate concentrations, capture and inactivate IFNs; 2) a new IFN inhibitory protein has been isolated and its chemical structure is under study; 3) prostaglandin E2, which is produced by certain tumor cells, inhibits IFN production; and 4) high levels of cAMP phosphodiesterases present, for example in certain tumor cells, reduces cAMP, an important second messenger in IFN synthesis. Studies are under way to reverse these inhibitory effects and to increase endogenous interferon production.

An inhibitor of interferon antiviral activity, which is absent in healthy HIV-seronegative persons, was detected in the sera of all 29 HIV-seropositive study participants. The relationship of the level of interferon inhibitor to CD4 count and HIV-RNA copy number was statistically significant in distinct models. Levels of interferon inhibitor declined by an average of 41-60% in patients who underwent a change in anti-retroviral therapy. Interferon inhibitor levels appear to decline as CD4 cell count rises and HIV-RNA levels fall. This suggests that interferon inhibitor may have a significant role in the host immune response to HIV infection.

A three-group design of alcoholics, heavy smokers and controls was used to investigate the status of the interferon system, including natural killer cell activity. Group differences were indicated via discriminant function analysis which correctly classified 86% of subjects. Test-retest relations were investigated for 14 subjects following a 30-day alcoholic inpatient program. Whereas significant immune suppression was indicated at the time of detoxification, recovery was evident at the 30-day follow-up. Results are discussed in terms of the significance of alcohol abuse on immune system functioning with consequences for susceptibility to viruses, bacteria and medical illnesses.

Spleen cells from C57BL/6 mice maintained on alcohol containing liquid diet for two weeks were evaluated for different immune functions. On an average, 22% fewer spleen cells were recovered from alcohol-fed mice when compared to cells from control animals. In alcohol-fed mice, the relative frequency of B cells increased, whereas total T cells including CD4 + cells decreased significantly. Alcoholic mice, when challenged with poly(rI) poly(rC), produced significantly less interferon than control mice. In vitro production of interferon alpha and gamma by the spleen cells of alcoholic mice was reduced by 67–90%. No significant differences were seen in the level of natural killer cell activity in spleen cells of control and alcoholic mice. These results suggest that chronic alcohol intake can result in not only changes in the number of immune cells, but more importantly affect their biological functions such as their ability to produce interferons.

Human leukocyte interferon (HL-IF)binds to AGPOLY(A)TM, AGPOLY(U)TM and AGPOLY(I)TM. The bound interferon could be displaced from all three polyribonucleotides by including sodium chloride in the eluant. The nature of interaction of HL-IF with polyribonucleotides is electrostatic and not hydrophobic since its binding was not prevented in the presence of 50% ethylene glycol. The binding of HL-IF on AGPOLY(I)TM is stronger at lower pH since an increase in ionic strength is required to displace it.

Two human cell lines (HT1080 and MRC5) were found to be resistant to the antiproliferative effects of human interferon α. These cells showed also a reduced antiviral response, when virus yield was measured by a plaque assay. The binding of 125 I-labeled α interferon to HT1080 and MRC5 cells was greatly reduced, relative to another human cell line (A549) which responded well to the antiproliferative and antiviral activities of interferon. These results suggested that the poor response of HT1080 and MRC5 cells was due to the presence of fewer interferon receptors than in A459 cells. Some functional receptors, however, appeared to be present in HT1080 and MRC5 cells, since treatment with interferon resulted in a slight reduction in virus yield and in a modest increase in 2′,5′-oligo(A) polymerase activity.

The antiviral activity of exogenously applied HuIFN-alpha was significantly reduced in cells pretreated with non-toxic concentrations of adriamycin. With low concentrations of adriamycin (up to 0.125 micrograms/ml) the effect was reversible in all cells tested. In human foreskin (BG-9) cells, that are more resistant to adriamycin toxicity, higher nontoxic concentrations of adriamycin (0.625 micrograms/ml) caused an irreversible decrease in sensitivity to HuIFN-alpha. Under these conditions, adriamycin pretreatment irreversibly inhibited the rate of cellular protein synthesis in BG-9 cells by about 30%. In contrast, cellular RNA synthesis was inhibited by 80% in 4 h after exposure to the drug, but was restored to almost normal levels (97% of control) at day 4 after removal of the drug. These results suggest that the loss of sensitivity of adriamycin-treated BG-9 cells to HuIFN-alpha may be related to the inhibition of cellular protein synthesis.

Human leukocyte-derived interferon (HuIFN-alpha) can be displaced from controlled-pore glass (CPG) with alkylamines at 0.1-0.3 M concentration. The eluting power of an alkylamine correlates positively with the extent of its alkylation: CH3NH3 + less than (CH3)2NH2 + less than (CH3)3NH+ less than (CH3)4N+. Interferon can also be recovered using an electrolyte and ethylene glycol as cosolvents. Apparently, both hydrophobic and electrostatic forces are involved in the adsorption of HuIFN-alpha to CPG.

Human leukocyte interferon (HL-IF), produced by mixed leukocytes infected with Newcastle disease virus, was resolved into three distinct fractions when chromatographed on concanavalin A-agarose. The major portion (70--75%) of interferon appeared in the breakthrough (BT fraction). The bound interferon (25--30%) was displaced from the column as two peaks: the first was eluted with 0.01 M methyl alpha-D-mannoside, yielding 15-20% of the interferon activity (alpha-MM fraction), and the second by including ethylene glycol (70%) in the eluant, yielding the remaining 5--15% of the interferon (EG fraction). No interferon was retained when HL-IF produced in the presence of glycosylation inhibitors (tunicamycin or 2-deoxy-D-glucose) was chromatographed on concanavalin A-agarose, suggesting that the fraction of interferon retained by this lectin is glycosylated. The three fractions of interferon (BT, alpha-MM, and EG) were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cross-species antiviral activity, and neutralization by specific antisera. The BT fraction contains exclusively the 16 000 molecular weight component of human leukocyte interferon. The majority of the alpha-MM fraction (90%) is the 21 000 molecular weight component. However, the EG fraction contains the 16 000 and 21 000--23 000 molecular weight components in essentially equal proportions. On the basis of cross-species antiviral activity and neutralization by specific antisera, the BT and alpha-MM fractions are leukocyte-type interferon and the EG fraction seems to be primarily of fibroblast type.

Antiviral and antiproliferative activities of three naturally occurring components of leukocyte-derived human interferon (HuIFN-alpha) separated by concanavalin A-agarose affinity chromatography were studied in a variety of neoplastic cell lines. Significant differences were seen with these different components. The results strongly suggest that careful consideration must be given while selecting any one component of HuIFN-alpha for any of the antiviral or antiproliferative studies. There is no clear evidence that glycosylation of HuIFN-alpha has any significant influence on its in vitro antiviral or antiproliferative activities, although apparently glycosylated and non-glycosylated components gave different antiviral and antiproliferative responses in different tumor cells.

The extracellular, acid-soluble cell products (EASCP) from Newcastle disease virus-infected L 929 cells contain both interferon, defined as antiviral activity, and refractoriness inducing principle, defined as an activity that inhibits interferon production. L cells pretreated with EASCP and then infected with Newcastle disease virus give rise to EASCP with decreased amounts of interferon but an increased ratio of refractoriness inducing principle activity to interferon activity in a dose related manner. The antiviral activity of an EASCP preparation is not dependent upon its refractoriness inducing principle level, but is entirely dependent on its interferon content. Our results provide additional evidence that interferon and refractoriness inducing principle are different biological entities and not polymorphic functions of the interferon molecule.

The influence of tumor load, surgical trauma, and bacterial sepsis upon the ability of patient's peripheral leukocytes to produce interferon-alpha (IFN-alpha), the detectable serum IFN levels and circulating serum IFN inactivators were studied. Peripheral blood leukocytes of patients with solid tumors had significantly reduced ability to produce IFN-alpha. Complete resectional surgery resulted in restoration of their ability to produce normal IFN-alpha levels. Circulating IFN levels were detectable in 70% of patients with localized disease while only in 20% of patients with metastatic disease. Interferon-alpha activators were detected in 45% of all patients. Both circulating interferon and IFN-alpha inactivators became undetectable upon tumor resection. Surgical trauma is accompanied by a transient but definite decrease in IFN-alpha production capability. Bacterial sepsis during postoperative days, in patients who successfully recovered, was definitely accompanied by increase in IFN-alpha production capability. Our findings suggest that advanced malignant epithelial tumors have an adverse effect upon the patient's ability to produce interferon and are often accompanied by the presence of circulating serum interferon inactivators. These effects can be reversed by surgical resection of the malignant neoplasm.

Interferon inhibitory activity was found in the plasma of 11 of 14 patients with the acquired immune deficiency syndrome (AIDS). This was not seen in 75 normal persons, including six whose specimens were randomly and blindly interspersed among the patient samples. Plasma from a single patient with the AIDS prodrome (AIDS-related complex), who later demonstrated AIDS, did not contain interferon inhibitors but did contain high levels of interferon. Three patients with AIDS-unrelated Kaposi's sarcoma had neither significant levels of interferon nor interferon inhibitory activity. The existence of interferon inhibitory activity in the plasma has to be taken into account when interferon preparations are administered therapeutically.

Purpose Previous studies had reported that normal individuals do not have measurable levels of interferons in their circulation, whereas high levels have been found in patients in the early stages of AIDS (acquired immunodeficiency syndrome) and in those with AIDS-related complex (ARC). This study was undertaken to compare levels of interferon and interferon inhibitors in plasma samples from patients with AIDS, ARC, Kaposi's sarcoma, or varicella-zoster virus infection, and from control subjects. Patients and methods A total of 206 persons were tested for the presence of interferon and interferon inhibitors in their plasma: 76 with ARC or AIDS, with or without Kaposi's sarcoma or lymphoma; 32 with varicella-zoster infection; 12 with AIDS-unrelated Kaposi's sarcoma; and 86 normal control subjects at high or low risk of AIDS with or without positive antibody levels to human immunodeficiency virus-1. Total interferon activity was measured by bioassay and the subtypes were not separated. Results Of 86 normal control subjects, 85 had no significant levels of interferon or interferon inhibitor. One disease-free homosexual exhibited measurable interferon levels. Patients acutely infected with varicella-zoster virus showed no measurable interferon or inhibitor levels except if they were in a high-risk group for AIDS. Seventy-six patients with ARC or AIDS exhibited measurable circulating interferon levels. Only patients with AIDS had interferon inhibitors in their circulation. Of 12 patients with Kaposi's sarcoma unrelated to AIDS, none had measurable interferon inhibitor levels, but some exhibited measurable interferon levels. Conclusion It is suggested that levels of interferon inhibitor should be considered when interferon is used therapeutically in viral or neoplastic diseases.

The influence of splenectomy upon the growth of B16-F10 malignant melanoma and changes in interferon-synthesizing ability in mice were studied. Surgical stress alone temporarily diminished the ability of mice to respond to interferon induction by poly rIrC. Two weeks following the surgery, mock-splenectomized mice fully regained their interferon synthesis ability. However, this was not true in the case of splenectomized mice. They remained refractory to interferon induction. The removal of the spleen had no obvious effect on the rate of pulmonary metastasis in mice injected with B16-F10 malignant melanoma in relation to the mock-splenectomized or control mice. Mice that were splenectomized and inoculated with B16-F10 melanoma also remained refractory to interferon induction.

The interferon (IFN) system of human cornea cells in culture was studied. IFN produced by these cells by infection with Newcastle disease virus (NDV) was shown, by neutralization studies with specific antisera against human alpha interferon (HuIFN-alpha) and human beta interferon (HuIFN-beta), to contain 90-95% antiviral activity characteristic of the HuIFN-beta and 5-10% that of HuIFN-alpha. The chromatographic behavior of human cornea IFN on Con A-Sepharose and zinc chelate agarose columns was identical to that of HuIFN-beta produced by human foreskin cells. The cornea cells developed marked resistance when exposed to either HuIFN-beta or human gamma interferon (HuIFN-gamma) against vesicular stomatitis virus (VSV), but to a much lesser degree against HSV-1. Both the laboratory-adapted strain and a clinical isolate of HSV-1 were found to be resistant to HuIFN-beta and HuIFN-gamma as compared with VSV. The clinical isolate of HSV-1 was, however, more sensitive to HuIFN-gamma than the laboratory-adapted strain. Furthermore, a combination of HuIFN-beta and HuIFN-gamma did not significantly increase the level of antiviral state induced in cornea cells against HSV-1. These results suggest that in-vitro culture of human cornea cells can be a valuable system to evaluate the potential of chemotherapeutic agents against common ophthalmic viral infections.

Antiviral activity of interferon secreted by human leukocytes into the culture fluid in the presence of tunicamycin (1–2 µg/ml) was significantly decreased, by 50–60 percent, in comparison to that produced in the absence of the antibiotic. No loss in antiviral activity occurred when tunicamycin was added to already harvested interferon preparations. Some physico-chemical and biological properties of human leukocyte interferon synthesized in the presence of tunicamycin (HL-IFT) were apparently altered by comparison with those of control preparations of human leukocyte interferon (HL-IF): HF-IFT had only one molecular weight component of 16,000 daltons in contrast to the two components of HL-IF of 16,000 and 21,000 daltons. HL-IFT also had a higher apparent hydrophobicity and was less efficiently neutralized by an antibody raised against HL-IF. However, some other properties remained unchanged: isoelectric point, pI about 6; affinity for immobilized polyriboinosinic acid and a spectrum of cross-species antiviral activity. These data support the notion that the major component of HL-IF (70 percent, 16,000 daltons) is apparently nonglycosylated whereas the minor component (30 percent, 21,000 daltons) is glycosylated via saccharide-lipid intermediates.

Publisher Summary This chapter presents methodology to show that the molecular size heterogeneity of human leukocyte interferon (HLIF) can be eliminated when interferon is produced in the presence of tunicamycin, a specific glycosylation inhibitor. The evidence suggests that the molecular basis of this heterogeneity may be the relative extent of glycosylation of its molecular components. Tunicamycin is used as a specific inhibitor, to probe the glycosylation status of HLIF. Tunicamycin is shown to alter the properties of HLIF molecules that are released into the production media. The use of tunicamycin as a probe, is particularly revealing, because its inhibitory action suggests the N-glycosidic nature of linkage between the polypeptide and its saccharide moiety. HLIF is known to contain two distinct molecular weight components when subjected to sodium dodecyl sulfate-disc gel electrophoresis: 21,000 and 16,000 dalton components in the approximate ratio of 3:7. If HLIF contains sialic acid, then impairment of its glycosylation by tunicamycin should alter its behavior on isoelectric focusing.

Heat-stable calmodulin immunoreactivity and bioactivity were detected in crude preparations of various types of human, murine and chicken interferons (IFNs). Calmodulin containing HuIFN-α was retained on a trifluorophenothiazine-Sepharose column. The two activities were separated by serial elutions with 50 μM Ca2+ (HuIFN-α) followed by 2 mM EGTA (calmodulin). While maintaining its full antiviral activity, calmodulin free HuIFN-α inhibited enhancement of Ca2+-ATPase activity in vitro by authentic purified eukaryote calmodulin. These results indicate that IFNs are calmodulin-binding proteins and that the secretion of both IFNs and calmodulin occurs from IFN-induced cells.

Publisher Summary This chapter reveals that human leukocyte interferon (HLIF) is composed of several homologous proteins that are coded for by separate genes in the genome. HLIF can be resolved into two molecular weight components by sodium dodecyl sulfate (SDS)-disc gel electrophoresis and, in addition, has been shown to contain fibroblast-type interferon as a minor component. These three molecular components: (1) 21,000-dalton component (Le21), (2) 16,000-dalton component (Le16), and (3) a fibroblast- type minor component (“F” type) cannot be effectively resolved either by gel filtration or by SDS-disc gel electrophoresis. The chapter describes the efficient separation of these three molecular components, in a single step, by chromatography of HLIF on concanavalin Aagarose. NDV is routinely used as an interferon inducer and human serum is present throughout the production period. It is conceivable that the nature or extent of molecular heterogeneity will vary if these production conditions are changed.

It is becoming increasingly evident that changes in various parameters of the interferon system and in the levels of prostaglandins and cyclic nucleotides have a definite role during the health and during the onset, progression and the final outcome of any disease process. In spite of ever increasing excitement over the possible clinical application of interferons, very little is known about the changes and sources of variations in the interferon production capabilities of different individuals. Heterogeneity of human interferons is now well document from the earlier work (1) as well as from more recent work based upon DNA recombinant technology (2–4). However, the clinical significance of such a heterogeneity is not known. Cloned interferons used at present in the treatment of any type of infections are a product of a single gene. It is quite possible that expression of different interferon genes varies considerably in different types of infections and malignancies. If that is the case, it may be necessary to use a custom-tailored interferon preparation for each type of infection. Such an approach at best appears to be several years away. In view of lack of such a knowledge, it seems appropriate that serious attempts should be made towards better understanding of the host’s own interferon system during infection and ways and means of its manipulation for maximal benefit. In this text, some attempts have been made in this direction.