Deficient IFN alpha production in hairy cell leukemia

Since the application of low doses of IFN-alpha is necessary to maintain remissions in Hairy Cell Leukemia (HCL) it is of interest whether peripheral blood mononuclear cells (MNC) of HCL patients can be induced in vitro to produce IFN-alpha. 9 patients suffering from advanced HCL were included in the study. The diagnoses were confirmed by characteristic findings in peripheral blood and bone marrow biopsies. For IFN treatment we initially used natural IFN-alpha (Bioferon) and switched later to recombinant IFN-alpha2 (Boehringer). MNC of 5 patients before IFN therapy and of 6 patients during IFN therapy (2-47 weeks) were induced by phytohemagglutinin (PHA), Corynebacterium parvum (C.p.), and sendai virus (SV). PHA is known to induce IFN-gamma. Both, C.p. and SV induced IFN-alpha but no IFN-gamma in MNC of healthy controls and of IFN treated breast cancer patients. In HCL patients normal antiviral activities could be induced by PHA. Zero or only low antiviral activities could be induced in MNC from 9 patients tested on 22 occasions. It is concluded that MNC from patients with advanced HCL can be induced to produce IFN-gamma but no IFN-alpha. Since IFN-alpha but not IFN-gamma is produced by monocytes it is likely that reduced numbers of monocytes which were found in our HCL patients before and during IFN treatment account for the described deficiency of IFN-alpha production.     

Phagocytic activity and protein content of human monocytes cultivated in the presence of various homologous interferon preparations

We have studied the effect of different types of interferons (IFN) on phagocytic activity and protein content when present during in vitro cultivation of human blood monocytes. Recombinant IFN-alpha and partially and highly purified leukocyte IFN preparations blocked the increase in phagocytic activity and protein content that occurs during in vitro cultivation of human monocytes. Fibroblast IFN blocked the increase in protein content, but did not significantly alter the phagocytic activity. IFN-gamma slightly enhanced phagocytic activity and protein content, while lymphoblastoid IFN preparations had no effect. The phagocytic activity and protein content of monocytes matured in vitro without IFN and then treated with IFN for 24 h was also tested. Phagocytosis via the non-specific receptors and the protein content was reduced by treatment of these cells with the IFN-alpha preparations. On the other hand Fc-receptor mediated phagocytosis was stimulated by IFN-gamma. Our data indicate that IFN effects on monocytes in culture varies depending on type and possibly subtypes of IFNs, and also on the timing of the treatment.     

Effect of Retreatment with Interferon Alone or Interferon plus Ribavirin on Hepatitis C Virus Quasispecies Diversification in Nonresponder Patients with Chronic Hepatitis C

Alpha interferon (IFN-alpha) treatment is effective on a long-term basis in only 15 to 25% of patients with chronic hepatitis C. The results of recent trials indicate that response rates can be significantly increased when IFN-alpha is given in combination with ribavirin. However, a large number of patients do not respond even to combination therapy. Nonresponsiveness to IFN is characterized by evolution of the hepatitis C virus (HCV) quasispecies. Little is known about the changes occurring within the HCV genomes when nonresponder patients are retreated with IFN or with IFN plus ribavirin. In the present study we have examined the genetic divergence of HCV quasispecies during unsuccessful retreatment with IFN or IFN plus ribavirin. Fifteen nonresponder patients with HCV-1 (4 patients with HCV-1a and 11 patients with HCV-1b) infection were studied while being retreated for 2 months (phase 1) with IFN-alpha (6 MU given three times a week), followed by IFN plus ribavirin or IFN alone for an additional 6 months (phase 2). HCV quasispecies diversification in the E2 hypervariable region-1 (HVR1) and in the putative NS5A IFN sensitivity determining region (ISDR) were analyzed for phase 1 and phase 2 by using the heteroduplex tracking assay and clonal frequency analysis techniques. A major finding of this study was the relatively rapid evolution of the HCV quasispecies observed in both treatment groups during the early phase 1 compared to the late phase 2 of treatment. The rate of quasispecies diversification in HVR1 was significantly higher during phase 1 versus phase 2 both in patients who received IFN plus ribavirin (P = 0.017) and in patients who received IFN alone (P = 0. 05). A trend toward higher rates of quasispecies evolution in the ISDR was also observed during phase 1 in both groups, although the results did not reach statistical significance. However, the NS5A quasispecies appeared to be rather homogeneous and stable in most nonresponder patients, suggesting the presence of a single well-fit major variant, resistant to antiviral treatment, in agreement with published data which have identified an IFN sensitivity determinant region within the NS5A. During the entire 8 months of retreatment, there was no difference in the rate of fixation of mutation between patients who received combination therapy and patients who were treated with IFN alone, suggesting that ribavirin had no major effects on the evolution of the HCV quasispecies after the initial 2 months of IFN therapy.     

Aerosolized interferon-alpha treatment in patients with multi-drug-resistant pulmonary tuberculosis

Multi-drug-resistant tuberculosis (MDR-TB) has emerged as an obstacle to the control of tuberculosis. Recent data however, suggest that interferon-(IFN)-gamma and IFN-alpha may improve disease evolution in subjects affected with pulmonary tuberculosis caused by multi-resistant (IFN-gamma) and sensitive (IFN-alpha) strains. The mechanisms involved are not known, even though it has been reported that IFN-gamma-secreting CD4+ Th cells may possess antitubercular effects. In addition, IFN-alpha can induce IFN-gamma secretion by CD4+ Th cells, and both types of IFN may stimulate macrophage activities. The aim of this study was to explore the possibility that aerosolized IFN-alpha, administered concomitantly with conventional antitubercular chemotherapy, may improve the course of pulmonary tuberculosis. After six months of directly observed therapy (DOT), seven patients who were non-responders to a second line antitubercular therapy were given an IFN-alpha aerosol (3 MU, three times a week) for two months as adjunctive therapy. All strains were resistant to at least two first-line drugs. After IFN-alpha administration, the patients were followed up for a further six months with the same DOT. Sputum samples were collected monthly during the study period, with the exception of the IFN-alpha administration period, when the observations were performed weekly. High resolution computed tomography (HRCT) chest scans were performed before and after IFN-alpha inhalations. The analysis of the results showed that the mean number of Mycobacterium tuberculosis (Mt) had remained statistically unchanged (p = 0.80) during the first 6 months of DOT. During the following 2 months of IFN-alpha administration, 5 patients became negative (p = 0.02). After the end of treatment a progressive increase in Mt number was observed (p = 0. 02). Sputum cultures remained positive for all patients throughout the study period, although a significant decrease (p = 0.02) in the colony number per culture was observed after adjunctive treatment with IFN-alpha. After stopping administration of IFN-alpha, a significant increase (p = 0.03) in the colony number per culture was noted as well as in Mt numbers. HRCT scans were slightly improved in all patients. These preliminary data suggest that aerosolized IFN-alpha may be a promising adjunctive therapy for patients with MDR-TB. Optimal doses and schedules however, require further studies.     

Expression of IFN-alpha and IFN-gamma receptors on normal human small resting T lymphocytes and large granular lymphocytes

The expression of interferon (IFN) receptors was studied on freshly isolated human lymphocytes from normal donors. Highly enriched populations of small resting T lymphocytes and large granular lymphocytes (LGL) were found to constitutively express high-affinity receptors for IFN-alpha and IFN-gamma. Both types of lymphocytes also had lower-affinity IFN-alpha binding sites. T lymphocytes had a mean of 230 IFN-alpha and 520 IFN-gamma high-affinity receptors per cell, whereas LGL had 520 IFN-alpha and 760 IFN-gamma receptors. However, because LGL were larger than the T lymphocytes, the IFN receptor density was similar on the two types of lymphocytes. The affinity of binding was similar on the two types of normal lymphocytes and on the cultured lymphoblastoid cell line Daudi. The number of IFN receptors per cell and the affinities of the IFN-receptor interactions varied little among the normal donors. Both the freshly isolated normal lymphocytes and the cultured cell line Daudi had separate receptors for type I (alpha and beta) and type II (gamma) IFN. Taken together, our data indicate that two types of freshly isolated normal lymphocytes constitutively express IFN receptors that are similar to those present on the lymphoblastoid cell line Daudi derived from a patient with Burkitt's lymphoma.     

Dosing schedule-dependent change in the disruptive effects of interferon-alpha on the circadian clock function

Altered homeostatic regulation, including the disturbance of circadian rhythms, is often observed in patients undergoing interferon (IFN) therapy. We reported previously that IFN-alpha has the ability to modulate the circadian clock function at the molecular level and that the alteration of clock function could be overcome by changing the dosing schedule. In this study, we investigated the influence of IFN-alpha on the intrinsic biological rhythms in mice by comparing two dosing schedules, continuous administration and repetitive injection. Continuous administration of IFN-alpha to mice decreased the rhythm amplitude of locomotor activity, body temperature, leukocyte counts, and plasma corticosterone levels. The treatment also suppressed the oscillation in the expression of clock genes in the liver. On the other hand, modulation effects were scarcely observed in mice treated with repetitive injection of IFN-alpha. These results indicate that treatment with IFN-alpha does not always modulate the circadian clock function. This notion was also supported by in vitro findings that the inhibitory action of IFN-alpha on the expression of clock genes was dependent on its exposure time to cells. The alteration of clock function induced by IFN-alpha could be avoided by optimizing the dosing schedule.     

Safe and effective treatment of spontaneous neoplasms with interleukin 12 electro‐chemo‐gene therapy

Electroporation improves the anti‐tumour efficacy of chemotherapeutic and gene therapies. Combining electroporation‐mediated chemotherapeutics with interleukin 12 (IL‐12) plasmid DNA produces a strong yet safe anti‐tumour effect for treating primary and refractory tumours. A previously published report demonstrated the efficacy of a single cycle of IL‐12 plasmid DNA and bleomycin in canines, and, similarly, this study further demonstrates the safety and efficacy of repeated cycles of chemotherapy plus IL‐12 gene therapy for long‐term management of aggressive tumours. Thirteen canine patients were enrolled in this study and received multiple cycles of electro‐chemo‐gene therapy (ECGT) with IL‐12 pDNA and either bleomycin or gemcitabine. ECGT treatments are very effective for inducing tumour regression via an antitumour immune response in all tested histotypes except for sarcomas, and these treatments can quickly eradicate or debulk large squamous cell carcinomas. The versatility of ECGT allows for response‐based modifications which can overcome treatment resistance for affecting refractory lesions. Importantly, not a single severe adverse event was noted even in animals receiving the highest doses of chemotherapeutics and IL12 pDNA over multiple treatment cycles. This report highlights the safety, efficacy and versatility of this treatment strategy. The data reveal the importance of inducing a strong anti‐tumour response for successfully affecting not only the treated tumours, but also non‐treated metastatic tumours. ECGT with IL12 pDNA plus chemotherapy is an effective strategy for treating multiple types of spontaneous cancers including large, refractory and multiple tumour burdens.     

Neurophysiological effects of recombinant interferon-gamma and -alpha in man

Treatment of malignant tumors with interferon (IFN) is in some patients accompanied by serious neurological side effects. The present study assessed neurophysiological changes in spontaneous EEG activity, visual-evoked cortical potentials (VEPs), and brainstem auditory-evoked potentials (BAEPs) during IFN-gamma or IFN-alpha therapy in 9 patients. In addition, blood pressure, heart rate and body temperature were monitored. In all sessions under IFN, the latency of the P100 component of the VEP was shortened as compared to baseline conditions. IFN also reduced latencies of BAEP components, and diminished amplitudes of the spontaneous EEG activity within the alpha and beta frequency band. These latter effects were somewhat less consistent than those on VEPs. The major neurophysiological changes appeared to be similar for IFN-gamma and IFN-alpha. The results are in accord with an excitatory effect of IFN on central nervous activity. The magnitude of changes excludes a neurotoxicity of IFN-gamma or IFN-alpha at the doses used in this study.     

Biological basis for a proper clinical application of alpha interferons

In recent decades the structure and the function of interferons (IFNs) have been elucidated and it is now clear that IFN is not only a protein but a group of proteins with parallel, but not analogous or identical, biochemical and biological properties. Among them there are at least 12 subtypes of IFN-alpha which are all involved in the control of several cellular functions and are all actively engaged in host defence mechanisms against infections. These acquisitions led to the clinical use of different types of IFN-alpha with appreciable success in several diseases. There are however several possibilities to optimize IFN-alpha treatment which can and must be addressed. For instance, as regards IFN-alpha biology and therapy issues, we need to understand how the different subtypes can generate similar signalling outputs but also govern specific cellular responses and more in general why the body produces so many of these IFN-alphas. This paper highlights the complexity of the IFN-alpha system and tries to summarize what is currently known of the distinctive properties of each individual IFN-alpha subtype. The data presented on the functional distinctions between IFN-alpha subtypes suggest that the commercially available IFN-alpha preparations may differ in some biological, pharmacological and therapeutic aspects and that a wiser and more prudent use of the different IFN-alpha preparations may provide more therapeutic benefits to patients.     

Type I IFN-beta gene therapy suppresses cardiac CD8+ T-cell infiltration during autoimmune myocarditis

Gene therapy using DNA encoding type I IFN subtypes IFNA6, IFNA9 and IFNB suppresses murine cytomegalovirus (MCMV)-myocarditis, a predominantly cell-mediated disease in BALB/c mice. CD8(+) T cells are the principal cell type within the inflamed myocardium. As such, we investigated the effects of IFN subtype treatment on this T-cell subset and other cell types in the cardiac infiltrate. In the acute phase of disease, IFNA6 and IFNA9 treatments significantly reduced the number of CD8(+) T cells within the foci of cellular infiltration in the heart. During the chronic phase, which is primarily autoimmune in nature, IFNB treatment significantly reduced CD8(+) T cells. B-cell and neutrophil numbers in the cardiac infiltrate were also reduced following IFNB immunotherapy. Although early inflammatory responses are important for resolution of virus infection, high numbers of lymphocytes persisting in the myocardium may lead to exacerbation of disease. Our data suggests that type I IFN DNA therapy regulates cardiac cellular infiltration. Thus, treatment with IFN-beta administered prophylactically to high-risk patients in acquiring CMV infection may reduce the development of chronic autoimmune myocarditis.     

Interferon-beta from melanoma cells suppresses the proliferations of melanoma cells in an autocrine manner

Interferon (IFN)-alpha and IFN-beta have been utilized in the treatment of melanoma as a form of cytokine therapy. While previous studies have demonstrated that melanocytes and melanoma cells produce a number of cytokines, it remains unclear whether or not melanocytes and melanoma cells per se produce IFN-alpha or IFN-beta. In the present study, we investigated the expression of IFN-alpha or IFN-beta in human melanocytes and five melanoma cell lines: G-361, C32TG, MMAc, MEWO and VMRC-MELG at both mRNA and protein levels. Both IFN-alpha and IFN-beta mRNA were detected in normal human melanocytes. Likewise, IFN-alpha mRNA was detected in all five melanoma cell lines. However, IFN-beta mRNA was only detected in one melanoma cell line, VMRC-MELG. When melanocytes and melanoma cells were treated with a potent IFN inducer, polyinosinic:polycytidylic acid (poly I:C), the mRNA expression of both IFN-alpha and IFN-beta was significantly upregulated. Poly I:C was not able to induce melanocytes or melanoma cells to produce detectable amounts of IFN-alpha protein, but able to induce a significant amount of IFN-beta in melanocytes and two of the melanoma cell lines: MMAc and VMRC-MELG. Moreover, similar to exogenous IFN-alpha and IFN-beta, poly I:C significantly inhibited the proliferation of all five melanoma cell lines. This suppressive effect was partially blocked by anti-IFN-beta antibody treatment in the IFN-beta-producing melanoma cell lines: MMAc and VMRC-MELG, but not in the non-IFN-beta-producing cell lines: G-361, C32TG and MEWO. Collectively, these studies have demonstrated for the first time that human melanocytes and melanoma cells produce IFN-beta. Furthermore, melanoma cells are capable of suppressing their own proliferation via secretion of endogenous IFN-beta. This finding may have important implications for melanoma therapy.     

Is steroid therapy needed in the treatment of destructive thyrotoxicosis induced by alpha-interferon in chronic hepatitis C?

OBJECTIVE: Treatment with interferon (IFN) of patients affected by chronic hepatitis C (CH-C) may produce alterations in thyroid function, such as hypothyroidism, Graves'-like hyperthyroidism and destructive thyrotoxicosis (DT). IFN-induced DT is characterized by suppressed serum TSH levels, normal or elevated FT4 and FT3 concentrations, with the presence or absence of thyroid peroxidase antibodies and antithyroglobulin antibodies, the absence of thyroid receptor antibodies and radioactive iodine uptake suppressed or <5%. DESIGN: IFN-induced DT is a mild clinical disease, because thyroid-destructive processes last for a short time and involve a small portion of the gland. At present, the therapeutic approach in DT suggests IFN withdrawal and 1-2 months of methylprednisolone treatment. METHODS: In consideration of possible untoward side effects of steroid treatment in patients with CH-C, we studied two groups of patients with CH-C who developed DT after treatments with various preparations of recombinant IFN (with or without ribavirin). Patients sequentially entered the study during a 4-year period, at the time of DT diagnosis, when IFN therapy was discontinued. The first 12 subjects (group A) were treated with 8-16 mg/day methylprednisolone for 30-40 days after IFN withdrawal; in the following 15 patients (group B), IFN withdrawal was not followed by any additional treatment. All patients underwent clinical and laboratory controls of thyroid function at 1, 2, 3 and 6 months after DT diagnosis. RESULTS: The results showed restoration of euthyroidism in both group A and group B patients at 6 months after DT diagnosis, regardless of steroid treatment. CONCLUSIONS: The simple withdrawal of IFN therapy in patients with CH-C, who had developed DT, appears to be effective in the treatment of the thyroid disease. This therapeutic approach should be preferred in order to avoid possible undesired side effects of steroid therapy in patients with CH-C.     

Recent Observations on Mesenchymal Tumours in Adults and Children

Recognition of the types and characteristics of mesenchymal tumours is important, since each type has biological features peculiar to itself that govern its growth and behaviour and it is essential to know these if treatment is to be effectual. Because many differentiated cell types are capable of acting as facultative fibroblasts, histological diagnosis is often in error. It has been found that no matter how bizarre a tumour may appear, if explanted in vitro, the cells will betray their true nature. By this means the various mesenchymal tumour types have been classified, many examples collected and followed up and their biological potentialities recorded. In more recent studies these uncommon tumours in children have been studied and many of them found to be less malignant than in adults. New varieties of mesenchymal tumours have been discovered in recent years, such as pseudosarcomatous fasciitis, elastofibroma dorsi, and bizarre variants of smooth muscle tumour.     

Bromocriptine in management of large pituitary tumours.

Bromocriptine has an accepted place in the management of small pituitary tumours that secrete either prolactin or growth hormone. The treatment of large tumours with extrasellar extensions is more difficult, however: though surgery is the standard treatment, it is often unsuccessful in returning excessive hormone secretion to normal and may cause hypopituitarism. A prospective trial was undertaken to assess the frequency with which changes in pituitary function and size of large tumours occurs. Nineteen patients were studied before and during treatment with bromocriptine (7.5 to 60 ml/day) for three to 22 months, using contrast radiology and a detailed assessment of pituitary function. Eighteen patients had hyperprolactinaemia and two of these also had raised concentrations of growth hormones; one patient had an apparently non-functioning tumour. In 12 patients (63%) tumour size decreased with bromocriptine and no tumour enlarged. Nine patients had visual-field defects, which improved in seven, becoming normal in five. Pituitary function improved in nine patients (47%) becoming entirely normal in three. Bromocriptine should be the treatment of choice in patients with large pituitary tumours with extrasellar extensions, provided close supervision is maintained.     

Use of bacteria in anti-cancer therapies

While a number of valid molecular targets have been discovered for tumours over the past decade, finding an effective way of delivering therapeutic genes specifically to tumours has proved more problematic. A variety of viral and non-viral delivery vehicles have been developed and applied in anti-cancer gene therapies. However, these suffer from either inefficient and/or short-lived gene transfer to target cells, instability in the bloodstream and inadequate tumour targeting. Recently, various types of non-pathogenic obligate anaerobic and facultative anaerobic bacteria have been shown to infiltrate and selectively replicate within solid tumours when delivered systemically. This has prompted the development of cancer gene therapy protocols that use such bacteria as gene delivery vehicles. Here, we review the evidence for the success of these in pre-clinical models and clinical trials, as single modality treatments and in combination with conventional cancer therapies.     

Analysis of the factors motivating HCV-infected patients to accept interferon therapy

ABSTRACT: BACKGROUND: The aims of this study were to analyze factors motivating the acceptance of interferon (IFN) therapy and to clarify the prevalence of oral mucosal diseases in hepatitis C virus (HCV)-infected Japanese patients treated with IFN. FINDINGS: A total of 94 HCV-infected patients who were admitted to our hospital for IFN therapy were asked questions regarding their motivation to accept IFN therapy and were investigated for the presence of oral lichen planus (OLP) before and during IFN treatment. Recommendation and encouragement from other people were the most common factors motivating the acceptance of IFN therapy (49/94, 52.13%). The other motivators were independent decision (30.85%), economic reasons (5.32%), and others. According to multivariate analysis, three factors - sex (male), retreatment after previous IFN therapy, and independent decision to accept IFN therapy - were associated with patients after curative treatment of hepatocellular carcinoma (HCC). The adjusted odds ratios for these three factors were 26.06, 14.17, and 8.72, respectively. The most common oral mucosal lesions included OLP in 11 cases (11.70%). One patient with OLP had postoperative squamous cell carcinoma of the tongue. The rate of sustained virological response (SVR) was 45.45% in cases with OLP and 54.55% in cases without OLP. There were no patients who discontinued IFN therapy because of side effects such as oral mucosal diseases. CONCLUSIONS: We should give full explanation and recommend a course of treatment for a patient to accept IFN therapy. The system to support liver disease as well as oral diseases is also necessary for patient treated for IFN therapy.     

Use of a selectable marker regulated by alpha interferon to obtain mutations in the signaling pathway.

We have selected mutations in genes encoding components of the signaling pathway for alpha interferon (IFN-alpha) by using a specially constructed cell line. The upstream region of the IFN-regulated human gene 6-16 was fused to the Escherichia coli guanine phosphoribosyltransferase (gpt) gene and transfected into hypoxanthine-guanine phosphoribosyltransferase-negative human cells. These cells express gpt only in the presence of IFN-alpha. They grow in medium containing hypoxanthine, aminopterin, and thymidine plus IFN and are killed by 6-thioguanine plus IFN. Two different types of mutants were obtained after treating the cells with mutagens. A recessive mutant, selected in 6-thioguanine plus IFN, was completely resistant to IFN-alpha but responded normally to IFN-gamma and, unexpectedly, partially to IFN-beta. A constitutive mutant, selected in hypoxanthine-aminopterin-thymidine alone, was abnormal in expressing endogenous genes in the absence of IFN. Both types revert infrequently, allowing selection for complementation of the defects by transfection.     

Interferon-alpha inhibits proliferation in human T lymphocytes by abrogation of interleukin 2-induced changes in cell cycle-regulatory proteins.

IFN-alpha exerts prominent regulatory functions on the immune system. One such effect is the inhibition of proliferation of in vitro stimulated T lymphocytes. The exact physiological function of this activity is not known, but it has been implicated in the antiviral effects of IFN, its antitumor action in T-cell malignancies, and the regulation of the in vivo T-cell response. Here, we have investigated the mechanism underlying the IFN-alpha-mediated growth inhibition of normal human PHA- and IL-2-stimulated T lymphocytes by an analysis of how IFN-alpha treatment influences known molecular events that normally accompany the transition from quiescence to proliferation in these cells. IFN-alpha treatment was found to profoundly block S-phase entry of stimulated T lymphocytes. This correlated with a strong inhibition of IL-2-induced changes in G1-regulatory proteins, including the prevented up-regulation of G1 cyclins and cyclin-dependent kinases as well as an abrogation of mitogen-induced reduction of p27Kip1 levels. This latter effect was due to a maintained stability of the p27Kip1 protein in the IFN-alpha-treated cells. In line with these findings, phosphorylation of the pocket proteins was abrogated in IFN-alpha-treated cells. Furthermore, our data indicate that IFN-alpha has selective effects on the pathways that emerge from the IL-2 receptor because IFN-alpha treatment does not block IL-2-induced up-regulation of c-myc or Cdc25A.