Interferon-gamma sensitizes osteosarcoma cells to Fas-induced apoptosis by up-regulating Fas receptors and caspase-8

BACKGROUND: Osteosarcoma is the third most frequent neoplasm in adolescents. Although chemotherapy, frequently used in pre- and post-operative settings, has resulted in significant improvement in disease-free survival, some patients show little sensitivity to chemotherapy and alternative therapeutic strategies are needed. Because the Fas ligand/Fas receptor (CD95, APO-1) apoptosis pathway is a potential therapeutic target in osteosarcomas, we examined the effect of IFN-gamma on Fas-induced apoptosis in four osteosarcoma cell lines. PROCEDURE AND RESULTS: As measured by flow cytometry, all cell lines expressed cell surface IFN-gamma receptors, and when cultured for 2 days in the presence of IFN-gamma, all cell lines exhibited a significant increase in expression of Fas receptors. By flow cytometric detection of intracellular fragmented DNA as a marker of apoptosis, all cell lines cultured with either IFN-gamma or anti-Fas antibody (clone CH-11) alone showed only moderate apoptosis, whereas significantly high levels of apoptosis occurred in cells cultured with both IFN-gamma and CH-11. Western blotting analysis also revealed that IFN-gamma caused up-regulation of caspase-8 in all cell lines, but no change in Fas-associated death domain protein (FADD/MORT1) or caspase-3. Both caspase-8 and caspase-3 were activated when apoptosis was induced with both IFN-gamma and CH-11. Addition to cultures of z-IETD-fmk, an inhibitor of caspase-8, significantly blocked this apoptosis. CONCLUSIONS: IFN-gamma sensitizes osteosarcoma cells to Fas-induced apoptosis through up-regulation of Fas receptor and caspase-8. Combined immunotherapy with IFN-gamma and either anti-Fas monoclonal antibody or cytotoxic T cells that bear Fas ligand might be a useful adjunctive therapy for patients with osteosarcoma.     

High production of interleukin‐10 and interferon‐γ in influenza‐associated MERS in the early phase

Mild encephalitis/encephalopathy with a reversible splenial lesion (MERS) occurs in various diseases and pathologies, and the clinical symptoms are not consistent with the impaired region. The mechanism of the region specificity is unclear. We investigated the cytokine profiling in cerebrospinal fluid (CSF) and serum obtained from a child with MERS during influenza infection, and compared them with those of serious another serious type of influenza‐associated encephalopathy. There was no elevation of Interleukin (IL)‐1β, which induces convulsion. The inhibitory cytokines of IL‐10 and IFN‐γ were elevated in the early phase in CSF. Comparing them with other patients, the elevation of the cytokine levels were generally mild. Considering that the prognosis of this MERS case was favorable and high levels of inhibitory cytokines including IL‐10 and IFN‐γ might work to localize the lesion and to prevent sequelae.     

Relation between serum IL‐4, IL‐13 and IFN‐γ levels and recurrence of wheezing episodes in infants with acute bronchiolitis

Lower respiratory tract infections are the most important factors among various causes which trigger wheezing in the first year of life. The factors associated with episodic wheezing in children with acute bronchiolitis are still subjects of research. Infections, environmental factors, immunologic mechanisms are sorted as etiologic risk factors of episodic wheezing. We aimed to investigate the relationship between serum interleukin (IL)‐4, IL‐13 and γ‐interferon (IFN‐γ) levels and recurrence of wheezing episodes in infants with acute bronchiolitis. One hundred twenty infants between 3 and 36 months with acute bronchiolitis enrolled in the study. Personal histories, clinical and laboratory data of infants were recorded. The patients were followed for a year. Venous blood samples were obtained to determine serum IL‐4, IL‐13, and IFN‐γ levels during acute bronchiolitis episode. The number of wheezing episodes was significantly higher in infants with a positive family history of allergy. A statistically significant correlation was determined between serum IL‐13 levels of infants and number of wheezing episodes. High serum IL‐13 levels and a positive history of allergy may have important roles in the recurrence of acute bronchiolitis.     

Low levels of interferon‐γ in nasal fluid accompany raised levels of T‐helper 2 cytokines in children with ongoing allergic rhinitis

The T‐helper 2 (Th2) cytokines interleukin‐(IL‐) 4, IL‐5, IL‐6, IL‐10 and the Th1 cytokine IFN‐γ and their associations with eosinophils, eosinophilic cationic protein (ECP) and immunoglobulin (Ig) E were studied in nasal lavage fluid from 60 school children with allergic seasonal rhinitis and 36 nonatopic healthy controls, before and during the pollen season. Eosinophil differential counts and IgE increased significantly in the patients during the pollen season. The eosinophil differential counts, ECP and IgE were all significantly higher during the season than in specimens simultaneously obtained from the nonatopic controls. Before season, the levels of ECP and IgE, but not eosinophils, were significantly higher in the patients than in the controls. During the season the nasal lavage fluid levels of IFN‐γ were significantly lower and the IL‐4/IFN‐γ quotients significantly higher in the allergic than in the control children. In the allergic children, but not in the controls, the nasal fluid levels of the Th2 cytokines IL‐4, IL‐5 and IL‐10 increased during the season, and together with IL‐6, were correlated with the differential counts of eosinophils, and with the levels of ECP and IgE. These findings are compatible with the hypothesis that a deficient release of the Th1 cytokine IFN‐γ plays an important role in the pathogenesis of allergic inflammation. Regardless of whether the defective IFN‐γ secretion is primary or a consequence of suppression by other cytokines, it will in the atopic subjects enhance the release of Th2 cytokines, which in turn will facilitate the development of allergic inflammation.     

Interleukin‐6, interleukin‐8, interleukin‐11, and interferon‐γ levels in nasopharyngeal aspirates from wheezing children with respiratory syncytial virus or influenza A virus infection

The differences between respiratory syncytial virus (RSV) and influenza A virus (IFAV) in the pathogenesis of wheezing in young children have not been clearly defined. The aim of this study was to assess the contributions of RSV vs IFAV in the pathogenesis of upper airway inflammation in wheezy young children. We compared interleukin (IL)‐6, IL‐8, IL‐11, and interferon‐γ (IFN‐γ) levels in nasopharyngeal aspirates (NPA) from non‐asthmatic children with respiratory virus infections (RSV in 17 children and IFAV in 13 children), asthmatic children with viral infections (RSV in nine children, IFAV in 10 children), and 22 unaffected healthy children (controls). Levels of IL‐11 in NPA from asthmatic children were significantly higher than those from non‐asthmatic children with RSV infection, and RSV infection enhanced the IL‐11 production in NPA significantly compared to IFAV infection. Nasopharyngeal epithelium from children with RSV infection secreted more IL‐6 than that of children with IFAV infection. There was little difference in the IL‐8 and IFN‐γ levels between asthmatic and non‐asthmatic children with RSV or IFAV infection. In conclusion, asthma enhanced IL‐11 production in RSV infection rather than IFAV infection in early childhood. There was a trend towards greater IL‐6 production in RSV infection compared with IFAV infection.     

Simultaneous occurrence of foetal and neonatal alloimmune thrombocytopenia and neonatal neutropenia due to maternal neutrophilic autoantibodies: a case study and review of the literature

Foetal and neonatal alloimmune thrombocytopenia (FNAIT) and neonatal neutropenia caused by maternal autoantibodies against neutrophils are rare disorders. We describe a newborn with severe thrombocytopenia and intracerebral bleeding caused by maternal anti-HPA-3a alloantibodies and mild neutropenia caused by maternal autoantibodies against HNA-1b. This appears to be the first case of simultaneous occurrence of these two conditions. CONCLUSION: This case report and review of the literature demonstrate that anti-HPA-3a antibodies can be overlooked by standard assays.     

Maturation of cytokine‐producing capacity from birth to 1 yr of age

Little is known about the immunologic maturation in the early stages of life. The aim of this study was to investigate maturation of immune system from birth to 1 yr of age and to compare immune functions between mothers and their children. Also the effect of atopy to the immune responses of children was examined. Cord blood samples (n = 228) and peripheral blood samples of children (n = 200) and their mothers (n = 208) 1 yr after birth were collected. Whole blood samples were stimulated for 24 and 48 h with Staphylococcal enterotoxin B (SEB), lipopolysaccharide (LPS) and the combination of phorbol ester and ionomycin (P/I). Production of TNF‐α, IFN‐γ, IL‐5, IL‐8 and IL‐10 was determined using ELISA. Significant mother‐to‐child correlation was detected in cytokine‐producing capacity at the age of 1 yr. TNF‐α (P/I, SEB and LPS stimulation), IFN‐γ (P/I and SEB), IL‐5 (P/I and SEB) and IL‐10 (P/I, SEB and LPS) producing capacity increased from birth to 1 yr of age. In general, stimulated cytokine responses were higher in mothers’ than in children’s blood samples, except in the case of P/I and LPS‐stimulated IL‐8, which were highest at birth. Maternal inhalation atopy was associated with increased cord blood IL‐5 (24 and 48 h) and IL‐10 (48 h) production following P/I stimulation. Also children of food atopic mothers expressed elevated cord blood IL‐10 (48 h, P/I) responses and decreased IFN‐γ/IL‐5 ratio (24 h, P/I). In addition, the production of IFN‐γ (24 and 48 h, P/I) and the IFN‐γ/IL‐5 ratio (24 h and 48 h, P/I) at the age of 1 yr was lower among children with food atopic mothers. In conclusion, our results suggest that both adaptive and innate immune responses increase from birth to 1 yr of age, but are still weak in comparison to adult responses. Cytokine responses of children begin to correlate with those of their mothers during the first year of life. Although only few associations were observed between atopy and cytokine‐producing capacity, our results suggest that children of atopic mothers express T_h2‐polarized cytokine pattern.     

Determination of T‐cell subpopulations and intracellular cytokine production (interleukin‐2, interleukin‐4, and interferon‐γ) by cord blood T‐lymphocytes of neonates from atopic and non‐atopic parents

This report describes the results of a prospective study on immunological markers in cord blood for the prediction of allergic diseases in children. First we evaluated methodological aspects of the flow cytometric technique on cord blood cytokine measurements. Subsequently, the T‐cell subsets and percentage of cytokine‐producing cord blood T‐helper (Th) and T‐suppressor/cytotoxic lymphocytes of neonates from atopic and non‐atopic parents were compared. A group of 33 healthy, full‐term newborn infants of whom 23/33 were at risk for atopy (i.e. having at least one parent with one or more atopic symptoms and positive specific immunoglobulin E [IgE] to at least one common inhalant allergen) was studied. A flow cytometric technique was used to analyze cord blood T‐cell subsets and to determine the percentage of interleukin (IL)‐2‐, IL‐4‐, and interferon‐γ (IFN‐γ)‐producing cord blood Th and T‐suppressor/cytotoxic lymphocytes following stimulation with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin. The percentage of CD3 (T lymphocytes), CD3⁺ CD4⁺ (Th lymphocytes), CD3⁺ CD8⁺ (T‐suppressor/cytotoxic lymphocytes), CD19⁺ (B lymphocytes), CD3⁺ CD4⁺ CD45RO⁺ (memory Th lymphocytes), and CD3⁺ CD4⁺ CD45RA⁺ (naive Th lymphocytes) cells was unrelated to parental atopic status. PMA stimulation augmented the percentage of IL‐2‐ and IFN‐γ‐producing Th and T‐suppressor/cytotoxic lymphocytes, whereas the number of IL‐4‐producing T lymphocytes remained very low or undetectable. No differences in the percentage of IL‐2‐, IL‐4‐ and IFN‐γ‐producing Th and T‐suppressor/cytotoxic lymphocytes were found between neonates from atopic and non‐atopic parents. These results will be re‐evaluated when the atopic status of the children at the age of 1 and 2 years can be assessed.     

Peroxisome proliferator‐activated receptor γ 2 mutation may cause a subset of ulcerative colitis

Aim: Previous studies suggest the homeostasis between acquisition of tolerance to the indigenous microflora and protective immune responses appears to be disrupted in inflammatory bowel disease (IBD). Some experimental studies indicate peroxisome proliferator‐activated receptor γ (PPARγ) has been implicated as a regulator of intestinal inflammatory responses. In addition, the toll‐like receptor (TLR)‐4 can regulate expression of PPARγ in colonic epithelial cells. We attempted to demonstrate whether the functional imbalance between TLRs and PPARγ could lead to the onset and some polymorphisms of those genes could contribute to susceptibility to IBD. Methods: RT‐PCR analysis were performed to detect TLR4 and PPARγ mRNA associated with those of P65 of NFκB, TNFα, MyD88, NOD2/CARD15, TLR‐2,5,9, in the diseased colonic mucosa in ulcerative colitis (UC; n = 13) and Crohn's disease (CD; n = 7) compared with normal controls (n = 18). Consequently, we genotyped UC (n = 29) and CD (n = 10) compared with normal controls (n = 134) for the prevalence of suspicious mutations. Results: In a subset of UC patients who were revealed to carry PPARγ Pro12Ala mutation later, impaired expression of normal PPARγ mRNA was noted in the diseased mucosa accompanied with upregulations of MyD88 TLR‐4, 5, 9, P65 and TNFα in mRNA levels. The prevalence of PPARγ Pro12Ala mutation was more frequently found in UC patients compared with CD patients and normal controls (P < 0.05). Conclusions: These findings suggested that imbalances between TLRs and PPARγ in response to luminal bacteria could lead to colonic inflammation in some UC patients. Alternative explanations will be needed for the onset of the rest of UC and CD.     

Influence of cytokine and intercellular adhesion molecule-1 gene polymorphisms on acute rejection in pediatric renal transplantation

Immune response regulation by cytokines is a key to understanding AGR. The influence of the functional polymorphisms in genes coding for TNF-alpha (-308G > A), IL-10 (-819C > T, and -1082A > G), IFN-gamma [(CA)n], TGF-beta1 (+869T > C), and iCAM-1 (R241G and E469K), in addition to HLA and gender matching on the presentation of AGR in 51 pediatric renal recipients during a 36-month post-transplantation follow-up were analyzed. Also, donors and a control group were genotyped. All groups were within Hardy-Weinberg equilibrium for all polymorphisms except IL-10-819C > T and TNF-alpha (p < 0.005 and p < 0.01, respectively) in recipients. Transplants with gender mismatch showed a higher risk for AGR than those between individuals with gender match (OR, 4.227; p = 0.010). Recipients with a high-production compared with low-production TNF-alpha allele experienced earlier AGR (p = 0.030), and those with high-production alleles of both TNF-alpha and IFN-gamma showed a further increased risk (OR = 11.129, p = 0.024). These findings support the notion that a single genotype cannot by itself explain an event as complex as AGR. The sum or combination of different specific alleles of these genes could better account for the immune response to an allograft.     

Absence of biallelic TCRγ deletion predicts induction failure and poorer outcomes in childhood T-cell acute lymphoblastic leukemia

Background

The absence of biallelic TCRγ deletion (ABD) is a characteristic of early thymocyte precursors before V(D)J recombination. The ABD was reported to predict early treatment failure in T‐cell acute lymphoblastic leukemia (ALL). This study aimed to investigate its prognostic value in Taiwanese patients with T‐cell ALL.

Procedure

Forty‐five children with T‐cell ALL were enrolled from six medical centers in Taiwan. Quantitative DNA polymerase chain reaction (Q‐PCR) was performed to check the status of TCRγ deletion. The threshold for homozygous deletions by Q‐PCR was defined as a fold‐change <0.35.

Results

ABD was found in 20 patients [20:45] who had higher incidences of induction failure than those without ABD (P = 0.03; hazard ratio [HR] = 8.13; 95% confidence interval [95% CI] = 1.23–53.77) after multivariate regression analysis. Patents with ABD also had inferior EFS and OS (P = 0.071 and 0.0196, respectively). Multivariate Cox analysis indicated that the association between ABD and overall survival was independent of age and leukocyte count on presentation (P = 0.036; HR = 4.25; 95% CI = 1.10–16.42).

Conclusions

The absence of TCRγ deletion is a predictor of a poor response to induction chemotherapy for pediatric patients with T‐cell ALL in Taiwan. Providing patients with T‐cell ALL and ABD with alternative regimens may be worthwhile to test in future clinical trials. Pediatr Blood Cancer 2012; 58: 846–851. © 2011 Wiley Periodicals, Inc.