Potential of Interferon-α in Solid Tumours

Interferon-alpha (IFNalpha) is a pleiotropic cytokine with direct and indirect antitumour effects. These include prolongation of the cell cycle time of malignant cells, inhibition of biosynthetic enzymes and apoptosis, interaction with other cytokines, and immunomodulatory and antiangiogenic effects. The first clinical trials in solid tumours used crude preparations of natural IFNalpha and demonstrated that tumour regressions in solid tumours and haematological malignancies were possible. Since the advent of genetic engineering technology, recombinant (r) IFNalpha has been widely evaluated in solid tumours. This review discusses the use and potential of rIFNalpha in solid tumours; the first part focuses on malignant melanoma and metastatic renal cell carcinoma (RCC). In the adjuvant treatment of malignant melanoma, rIFNalpha has been tested in randomised trials in more than 6000 patients. High-dosage IFNalpha (> or =10MU) prolongs disease-free survival (DFS) but not overall survival (OS). Low-dosage IFNalpha (< or =3MU) has not been shown to prolong DFS or OS, and current data do not support its use outside clinical trials. The latest United Kingdom Co-ordinating Committee on Cancer Research meta-analysis of ten randomised trials that used adjuvant rIFNalpha has shown that there is a benefit in DFS but not OS. No conclusions can be reached for intermediate-dosage IFNalpha (5 to 10MU) until the mature results of the European Organization for Research and Treatment of Cancer (EORTC) study 18952 are available. In RCC, current evidence does not support the use of adjuvant IFNalpha. In metastatic malignant melanoma and RCC, reported response rates to rIFNalpha are approximately 15%. In a minority of responding patients, however, these responses can be long-standing. In metastatic malignant melanoma, IFNalpha combined with other cytotoxic agents with or without interleukin-2 has achieved high response rates but has not improved survival. In metastatic RCC, intermediate dosages of rIFNalpha should be used and therapy should probably be prolonged (>12 months); response depends on prognostic factors such as good performance status, whereas survival is affected by factors such as low tumour burden. Nephrectomy should therefore be considered in patients with good performance status prior to IFNalpha immunotherapy in advanced RCC, even in patients with metastatic disease. The toxicity of high-dosage IFNalpha and the lack of definite benefit on OS with high- or low-dosage IFNalpha do not support its use outside clinical trials. Data from the ongoing US Intergroup studies, the ongoing EORTC 18991 study (long-term therapy with pegylated IFNalpha) and mature data from EORTC 18952 (intermediate-dosage IFNalpha) will help establish the role of IFNalpha as adjuvant therapy in malignant melanoma.     

Tumor necrosis factor-α signaling in macrophages

Tumor necrosis factor-α (TNFα) was cloned over 2 decades ago and its identification in part led to the discovery of a super family of tumor necrosis factors (TNFs) and their receptors. TNFα signals through two transmembrane receptors, TNFR1 and TNFR2, and regulates a number of critical cell functions including cell proliferation, survival, differentiation, and apoptosis. Macrophages are the major producers of TNFα and interestingly are also highly responsive to TNFα. Aberrant TNFα production and TNF receptor signaling have been associated with the pathogenesis of several diseases, including rheumatoid arthritis, Crohn's disease, atherosclerosis, psoriasis, sepsis, diabetes, and obesity. TNFα has been shown to play a pivotal role in orchestrating the cytokine cascade in many inflammatory diseases and because of this role as a "master-regulator" of inflammatory cytokine production, it has been proposed as a therapeutic target for a number of diseases. Indeed anti-TNFα drugs are now licensed for treating certain inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. In this review we discuss the discovery of TNFα and its actions especially in regulating macrophage biology. Given its importance in several human diseases, we also briefly discuss the role of anti-TNFα therapeutics in the treatment of inflammatory diseases.     

Liprin-α is involved in exocytosis and cell spreading in mast cells

The active zone is a specialized region of the presynaptic plasma membrane where the neurotransmitter release occurs by exocytosis. Mast cells also release inflammatory mediators by exocytosis resulting in induction of allergic responses. In our previous reports, we found that active zone proteins, Munc13-1 and ELKS regulates exocytosis of mast cell positively. In this study, we investigated the involvement of liprin-α, another active zone protein, in exocytosis in mast cells. We found that three isoforms of liprin-α, liprin-α1, -α2 and -α3 were expressed. Immunocytochemical experiments revealed that liprin-α1 resided both in the cytoplasm and on the plasma membrane. Upon stimulation with antigen, the area of a cell increased remarkably due to cell spreading and the distribution of liprin-α1 became punctuated. Interestingly, knockdown of liprin-α1 caused decrease in exocytotic release and cell spreading. These results suggest that liprin-α1 facilitates exocytosis and cell spreading, and these events might have correlated each other in mast cells.     

Role of SIRPα in regulation of mucosal immunity in the intestine

Mononuclear phagocytes such as dendritic cells (DCs) and macrophages in the lamina propria (LP) are thought to be important for both induction of inflammatory responses and maintenance of immunologic tolerance in the mammalian intestine. The molecular mechanisms by which these cells regulate intestinal immunity have remained poorly understood, however. Signal regulatory protein α (SIRPα) is a transmembrane protein that is specifically expressed in DCs, macrophages and neutrophils. Here, we show that SIRPα is abundant in CD11c(+) CD11b(+) LP cells of the mouse intestine. Whereas SIRPα did not appear to be important for the steady-state homeostasis of mucosal immunity in the intestine, the flagellin-stimulated production of IL-17 or interferon (IFN)-γ by LP cells of SIRPα mutant (MT) mice that lack the cytoplasmic region of the protein was markedly decreased compared with that observed with wild-type cells. Moreover, the flagellin-induced production of IL-6 by LP cells from SIRPα MT mice was also greatly reduced. SIRPα MT mice were also resistant to the development of colitis induced by IL-10 deficiency. Our data thus suggest that SIRPα expressed on CD11c(+) LP cells is important for the production of IL-17 or IFN-γ in the LP as well as for the development of colitis induced by IL-10 deficiency.     

Interfreron-α Alone versus Interferon-α plus Ribavirin in Patients with Chronic Hepatitis C Not Responding to Previous Interferon-α Treatment

OBJECTIVE: To study the effects of monotherapy with leucocyte interferon-alpha (IFNalpha) versus IFNalpha + ribavirin in patients with chronic hepatitis C who were nonresponders to previous courses of recombinant or lymphoblastoid IFNalpha. DESIGN AND SETTING: This was a nonblind randomised study of outpatients at 3 centres in Palermo, Sicily, Italy. PATIENTS AND PARTICIPANTS: We recruited 72 patients (48 males, 24 females), mean age 48.8 +/- 6.6 years (range 31 to 63 years), with biopsy-proven chronic hepatitis C, predominantly genotype 1b. INTERVENTIONS: 24 patients (group A) received IFNalpha 6MU 3 times weekly for 6 months, and 48 patients (group B) received IFNalpha 6MU 3 times weekly + ribavirin 1200 mg/day for 6 months. ALT levels and adverse effects were monitored monthly, and hepatitis C virus (HCV) RNA levels were measured at study entry, at the end of treatment and after a 6-month follow-up. RESULTS: At baseline all patients were HCV-RNA positive and had ALT levels greater than twice normal. Mean post-treatment serum HCV-RNA levels were below baseline in group A, but the virus was eradicated in only 1 patient; 6 patients had normalised serum ALT levels. In group B at end of treatment, 12 patients were negative for HCV-RNA and serum ALT levels were normal in 18. At follow-up, all group A patients had elevated ALT levels and positive HCV-RNA. In group B, 3 patients were still negative for HCV-RNA and 4 had normal ALT. In 4 patients in group B, therapy was suspended because of anaemia, depression and decrease in neutrophil count; a flu-like syndrome was recorded with no frequency difference between groups. CONCLUSIONS: These results suggest that patients with chronic hepatitis C unresponsive to IFNalpha monotherapy could benefit from combination therapy with IFNalpha + ribavirin.     

Interleukin-32α expression in human colonic subepithelial myofibroblasts

Interleukin (IL)-32 is a recently described proinflammatory cytokine, characterized by induction of nuclear factor (NF)-κB activation. We studied IL-32α expression in human colonic subepithelial myofibroblasts (SEMFs). Colonic SEMFs were isolated from normal human colon tissue. IL-32α protein expression was evaluated by Western blot analyses, and IL-32α mRNA expression was analyzed by real-time PCR. IL-32α mRNA was weakly expressed without a stimulus, and its expression was markedly enhanced by IL-1? and TNF-α. IL-1? and TNF-α enhanced intracellular accumulation of IL-32α protein, but IL-32α was not detected in supernatants. Each cytokine dose- and time-dependently induced IL-32α mRNA expression. An inhibitor of phosphatidylinositol 3-kinase (LY294002) significantly suppressed IL-1?- and TNF-α-induced IL-32α mRNA expression, although MAPK inhibitors had no effect. Akt activation in response to these cytokines was confirmed by Western blotting. Blockade of NF-κB activation by an adenovirus expressing a stable mutant form of IκBα markedly suppressed IL-1?- and TNF-α-induced IL-32α mRNA expression. Human colonic SEMFs expressed IL-32α in response to IL-1? and TNF-α. IL-32α mRNA expression depends on the phosphatidylinositol 3-kinase and the NF-κB system.     

Intravenous Immunoglobulin Plus Interferon-α in Autoimmune Hepatitis C

Background: Hepatitis C virus (HCV) may be associated with a variety of autoimmune phenomena causing a therapeutic dilemma for treatment with interferon-α (IFNα), which stimulates autoimmune symptoms, or with corticosteroids, which may lead to an increasing of viral load. To evaluate the possible role of intravenous immunoglobulins (IVIg) in the response of patients treated with IFNα, we administered IVIg plus IFNα and compared the results with a group of patients treated with IFNα alone. Methods: Forty-two patients affected by chronic hepatitis C with probable autoimmune disease were eligible for this open-label, randomised study. All patients tested positively for anti-nuclear antibodies, anti-smooth muscle antibodies, anti-liver/kidney microsomal antibodies and anti-mitochondrial antibodies. Patients were randomly assigned to one of two groups: group A received IVIg at a dosage of 400 mg/kg each day for 5 days, and then 3 MUI of leucocyte IFNα three times a week, while group B received physiological solution followed by the administration of leucocyte IFNα three times a week at the same dosage for 6 months. Complete biochemical response was defined as a sustained normalisation of alanine aminotransferase levels, and complete virological response was defined as complete clearance of virus throughout the entire 6-month follow-up period. Immunological response was measured in terms of Autoimmune Hepatitis (AIH) score, while histological response was based on a reduction in histological activity index (HAI) score. Results: Compared with patients receiving IFNα alone, a higher percentage of patients who received IFNα plus IVIg showed complete virological and histological responses (p = 0.04). More patients in the combination therapy group achieved biochemical and immunological responses, although the differences between the groups were not statistically significant at all time points. Conclusions: Exogenously added Ig might modulate the immune network at various points. We propose that the immunomodulating action of IVIg acts synergistically with IFNα, achieving a better response to IFN treatment in patients with chronic HCV associated with autoimmunity. Data obtained from this preliminary study indicate a positive prospective for the clinical use of gamma globulins in patients with a high probability of autoimmune disorders associated with HCV infection.     

KIT, PDGFRα and EGFR analysis in nephroblastoma

Nephroblastoma prognosis has dramatically improved, but an unfavourable prognostic subgroup warrants development of novel therapeutic strategies. Selective KIT, PDGFRalpha and epidermal growth factor receptor (EGFR) tyrosine kinase inhibition evolved as powerful targeted therapy for gastrointestinal stromal tumours and non-small-cell lung cancer. To investigate a potential role for tyrosine kinase inhibition, we analyzed 209 nephroblastomas for immunohistochemical KIT and EGFR expression, 63 nephroblastomas for mutations in KIT exons 9, 11, 13, EGFR exons 18, 19, 20 and 21, and all 209 nephroblastomas for PDGFRalpha exons 12, 14 and 18. Twenty-two tumours (10.5%) expressed KIT, 31 (14.8%) EGFR, and 10 (4.8%) both KIT and EGFR, respectively. KIT expression was relatively more common among high-risk tumours (6/27; 22.3%) compared to low-/intermediate-risk tumours (26/181; 14.4%). Nine patients deceased, four of which had high-risk tumours with KIT expression in two of four and EGFR expression in one of four. There were no KIT, PDGFRalpha or EGFR mutations. Our results suggest no significant contribution of KIT, EGFR or PDGFRalpha mutations to nephroblastoma pathogenesis. Despite a trend towards association of immunohistochemical KIT and EGFR expression with poor outcome in high-risk nephroblastomas, statistical analysis did not yield significant correlations in this subgroup. Therefore, it remains open if KIT, PDGFRalpha or EGFR tyrosine kinase inhibition constitute a therapeutic target in nephroblastoma in the absence of KIT, PDGFRalpha or EGFR mutations.     

Placental TNF-α signaling in illness-induced complications of pregnancy

Maternal infections are implicated in a variety of complications during pregnancy, including pregnancy loss, prematurity, and increased risk of neurodevelopmental disorders in the child. Here, we show in mice that even mild innate immune activation by low-dose lipopolysaccharide in early pregnancy causes hemorrhages in the placenta and increases the risk of pregnancy loss. Surviving fetuses exhibit hypoxia in the brain and impaired fetal neurogenesis. Maternal Toll-like receptor 4 signaling is a critical mediator of this process, and its activation is accompanied by elevated proinflammatory cytokines in the placenta. We evaluated the role of tumor necrosis factor-α (TNF-α) signaling and show that TNF receptor 1 (TNFR1) is necessary for the illness-induced placental pathology, accompanying fetal hypoxia, and neuroproliferative defects in the fetal brain. We also show that placental TNFR1 in the absence of maternal TNFR1 is sufficient for placental pathology to develop and that a clinically relevant TNF-α antagonist prevents placental pathology and fetal loss. Our observations suggest that the placenta is highly sensitive to proinflammatory signaling in early pregnancy and that TNF-α is an effective target for preventing illness-related placental defects and related risks to the fetus and fetal brain development.     

Effects interferon-2α on seizures in corasol-induced kindling

Intracerebroventricular injection of recombinant human interferon-2α (100–500 U/rat) decreased the mean effective dose of corasol inducing clonic convulsions in 50% kindled Wistar rats. This effect was more pronounced in delayed periods of kindling and was accompanied by enhancement of epileptiform signs on EEG. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 144, No. 7, pp. 11–14, July, 2007     

TNF-α and sICAM-1 in intracranial aneurismal rupture

Introduction  Subarachnoidal hemorrhage (SAH) occurring after aneurismal rupture produces an inflammatory response in the cerebral circulation. Tumor necrosis factor (TNF)-α is a major cytokine in this process. Adhesion molecules provide information on inflammatory reactions taking place in the walls of blood vessels. Clinical evidence suggests a role of soluble intercellular adhesion molecule (sICAM)-1 in early hemorrhagic events. This study aimed to evaluate the implementation of early TNF-α and sICAM-1 serum measurement for the prognosis of patient outcome after intracranial aneurismal rupture. Materials and Methods  The study consisted of 27 patients with a diagnosis of intracranial aneurysm. SAH was evaluated on admission according to the Fisher scale, patients’ consciousness with the Glasgow Coma Scale, clinical grading with the Hunt and Hess scale, and clinical outcome with the Glasgow Outcome Scale (GOS). Blood samples were drawn within 72 h after arrival at the emergency room. Serum concentrations of TNF-α and sICAM-1 were assayed with the ELISA method. Results  The initial serum TNF-α concentration in the aneurismal patients was low and did not correlate with radiological and clinical scores. The serum sICAM-1 level positively correlated with the severity of bleeding assessed by the Fisher scale and negatively with the patient’s scoring in the GOS. Conclusions  This study demonstrated the absence of a systemic TNF-α-mediated inflammatory response at the onset of subarachnoid hemorrhage. Early measurement of serum sICAM-1 levels offers a potential prognostic value in the assessment of patients’ outcome after brain aneurismal rupture.     

Impact of TNF-α and LTα gene polymorphisms on genetic susceptibility in Indian SLE patients

The promoter polymorphisms of tumour necrosis factor-α (TNF-α) and intronic Lymphotoxin-α (LTα) have been implicated as genetic risk factors for systemic lupus erythematosus (SLE) in various ethnic groups. The aim of this study was to investigate an impact of TNF-α (?308G/A; 238G/A) and LTα (+252A/G) gene polymorphisms in disease susceptibility among Indian 200 SLE patients along with 201 healthy controls. The gene polymorphisms were studied by using direct DNA sequencing and Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) methods. Serum levels were measured by multiplex assay. Allelic frequencies of TNF-α ?308A (OR = 2.3, p = 0.0001, Pc = 0.0003) and LTα +252G (OR = 2.1, p < 0.0001, Pc < 0.001) were significantly higher in SLE patients. Frequency of haplotype-AGG was found to be higher in patients than controls (OR = 12.2, p = 0.0050). Serum levels of TNF-α and LTα also were found to be significantly higher in patients showing variant alleles. TNF-α ?308G/A + A/A genotypes (p < 0.01) and LTα +252 A/G + G/G genotypes (p < 0.02) were significantly associated with renal disorders and haematological manifestations. SLE patients with ?308G/A + A/A genotypes showed higher prevalence of anti-dsDNA antibodies (OR = 3.9, p = 0.0014, Pc = 0.0098) and anti-Sm antibodies (OR = 4.1, p = 0.0002, Pc = 0.0014). The present study suggests TNF-α ?308A and LTα +252G as risk alleles for disease susceptibility associated with higher serum levels of TNF-α and LTα and concomitant discrete clinical features among Indian SLE patients.     

HIF-2α expression is suppressed in SCLC cells, which survive in moderate and severe hypoxia when HIF-1α is repressed

Small cell lung carcinoma (SCLC) is extremely aggressive and frequently metastasizes widely in its early stage. Because tumor hypoxia is related to aggressive tumor behavior and the hypoxic adaptation of SCLC is poorly documented, we stained SCLC tumors arranged in a tissue microarray for hypoxia-inducible factor (HIF)-1α and HIF-2α proteins. We found an overall lack of HIF-2α protein expression, which was confirmed in large tumor sections. HIF-1α protein was strongly expressed in most tumors, frequently adjacent to necrotic regions. In concordance, cultured SCLC but not non-small cell lung carcinoma cells showed no or extremely low levels of HIF-2α mRNA and no HIF-2α protein at hypoxia. HIF-1α was stabilized after 4 hours at hypoxia, and its accumulation increased up to 96 hours. SCLC cells survived well and showed net proliferation and low cell death in modest (1% oxygen) and severe (0.1% oxygen) hypoxia. HIF-1α repression virtually did not influence cell death or viability despite reduced levels of hypoxia-inducible genes, such as BNIP3 and BNIP3L. At 1% oxygen no increased autophagy (LC3B-II activation) or NF-κB signaling were detected, whereas the unfolded protein response was activated at severe hypoxia. Our data indicate that HIFs are not exclusively required for SCLC cell survival at modest or severe hypoxia and that additional, yet uncharacterized, hypoxia-driven adaptation pathways may become activated.