Mutation screening of interferon-gamma (IFNγ) as a candidate gene for asthma

Background Reduced levels of interferon gamma (IFNγ) mRNA and protein have been detected in the bronchoalveolar lavage fluid of atopic asthmatics. IFNγ is secreted by TH₁ cells while IL-4 and IL-5 are secreted by TH₂ cells and an imbalance in the TH₁/TH₂ response may be responsible for atopic asthma. The gene for IFN4gM is located on chromosome 12; a region of the genome which has been shown in linkage studies to be associated with asthma. Objective To determine if there are any mutations present in the coding exons and 5’flanking region of the IFNγ gene in atopic asthmatic subjects compared with controls to explain the lower levels of this cytokine as an inherited, rather than acquired, factor in the asthmatic subjects. Methods The four exons and 5’flanking region of the IFNγ gene were amplified by polymerase chain reaction (PCR) from genomic DNA of 265 individuals from a Western Australian and a Venezuelan population. The PCR products were examined by single strand conformational polymorphism and heteroduplex analyses to see if there were any changes in the DNA migration patterns which would suggest the presence of a sequence variation. Results The four exons and the 5’flanking region of the IFNγ gene were amplified from 265 individuals from two populations. Single strand conformational polymorphism and heteroduplex analyses did not reveal any mutations in the regions examined. Conclusions The gene for IFNγ appears to be highly conserved as no sequence variations were detected in 265 individuals. These results suggest that mutations of the IFNγ gene are unlikely to be a significant cause of an inherited asthma diathesis.     

急性病毒性心肌炎与Cx43的相关实验研究

目的:通过对急性病毒性心肌炎组与正常组小鼠通道蛋白43、干扰素-γ及白介素-10水平的比较,探索病毒性心肌炎及其心律失常与通道蛋白43的关系并探讨可能机制。方法:20只雄性4周龄BALB/c小鼠予腹腔接种柯萨奇病毒B组3型建立病毒性心肌炎模型,6只予Eagle s液腹腔注射作为正常组。第14 d处死存活小鼠。ELISA法、实时定量PCR法测通道蛋白43、干扰素-γ和白介素-10改变,免疫组织化学法检测心肌细胞膜上通道蛋白43表达。结果:模型对照组通道蛋白43表达少于正常组。正常组通道蛋白43和白介素-10mRNA表达高于模型对照组(3.890±0.072对1.000±0.127,P<0.05和1.660±0.029对1.000±0.031,P<0.05),模型对照组干扰素-γmRNA高于正常组(1.000±0.061对0.139±0.032,P<0.01)。血清干扰素-γ、白介素-10的改变与其核酸水平的改变基本一致。结论:病毒性心肌炎急性期与心律失常有关的连接蛋白通道蛋白43明显减少,Th1/Th2免疫失衡在其减少的机制中起作用。     

全反式维甲酸联合干扰素-γ对人肝癌细胞株SMMC-7721抗增殖作用的研究

目的探讨全反式维甲酸(all-trans retinoic acid,ATRA)和干扰素-γ(Interferon-gamma,IFN-γ)联合作用对于人肝癌细胞株SMMC-7721的抑制作用。方法用ATRA和IFN-γ处理SMMC-7721细胞后,应用MTT法检测SMMC-7721细胞的增殖抑制率,电镜观察细胞形态的变化,流式细胞仪检测细胞凋亡,蛋白质免疫印迹法(Western blot)检测增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)的表达变化。结果ATRA作用于SMMC-7721后,抑制细胞增殖并且诱导细胞凋亡发生,和IFN-γ联合作用后,这种作用加强;SMMC-7721细胞中PCNA的表达在ATRA作用后减少,ATRA与IFN-γ联合作用后进一步减少。结论IFN-γ可以增强ATRA对于肝癌细胞的抑制及诱导细胞凋亡的作用,并且明显下调了PCNA的表达。     

INTERFERON-GAMMA AND INTERLEUKIN-2 SUPPRESS THE EXPERIMENTAL METASTASIS OF MOUSE RENAL ADENOCARCINOMA

While cytokines such as interferon-alpha (IFN-α), interferon-gamma (IFN-γ) and interleukin-2 (IL-2) are partially effective in the treatment of metastatic renal cell carcinoma, these cytokines have not been investigated for their ability to suppress the development of metastasis. We investigated whether they suppressed experimental pulmonary metastasis by subline-2 of streptozotocin-induced mouse renal adenocarcinoma (MRAC-PM2). IFN-γ (1 × 10⁴ and 1 × 10⁵ U/mouse) and IL-2 (1 × 10⁴ and 1 × 10⁵ U/mouse) suppressed the experimental pulmonary metastasis of MRAC-PM2 in a dose-dependent manner, but 1 × 10⁵ lU/mouse of IFN-α did not. The combination of 1 × 10⁵ U IFN-γ with 1 × 10⁵ U IL-2 had the most powerful inhibitory effect on pulmonary metastasis without any side-effects. In addition, the combination of 1 × 10⁵ U IFN-γ with 1 × 10⁵ U IL-2 had a synergistic inhibitory effect on the growth of subcutaneous tumors. These results indicate that either IFN-γ or IL-2, or their combination, produce a more favorable effect than IFN-α on the metastatic development of renal adenocarcinoma.     

Interferon-γ and urine neopterin in attacks of the hyperimmunoglobulinaemia D and periodic fever syndrome

Abstract. The hyperimmunoglobulinaemia D and periodic fever (hyper-IgD) syndrome is typified by recurrent unpredictable febrile attacks with abdominal pain, joint involvement (arthralgias/arthritis), headache, skin lesions and a polyclonal elevation of serum IgD (> 100 Umr^-1). Interferon-gamma (IFN-γ) is a major proinflammatory cytokine which could play a role in the pathogenesis of the attacks. There is a need for parameters (if possible non-invasive) to monitor disease activity. A potential candidate is neopterin which is released by monocytes/macrophages when stimulated with IFN-γ, excreted unchanged in urine, and appears to be an early and sensitive marker for activation of the immune system. We measured rectal body temperature, serum IFN-γ, and urine neopterin in 10 hyper-IgD patients both during and between attacks. The body temperature rose to a mean of 38–9d?C on the first day of the attack and normalized within 5 days. Serum IFN-γ during the first day of the attack was 2.98 IUmL^-1 and was significantly lower during remissions. The urine neopterin excretion was 268 ± 170 μmolmol^-1 creatinine between attacks and was significantly increased to 638 ± 275 μmolmor^-1 creatinine on the first day of symptoms. Maximal urine neopterin values were reached on the fourth day of the attack (1051 ± 387 μmolmor^-1 creatinine) and excretion gradually declined and attained values below 400 μmolmol^{- '} creatinine after 9 days. There was a good correlation between serum IFN-γ and urine neopterin. The increases in serum IFN-γ and urine neopterin suggest activation of the cellular immunity during the febrile attacks of the hyper-IgD syndrome. Furthermore, the activation of the cellular immune system appears to persist several days after normalization of the body temperature. The significant correlation between IFN-γ; and urine neopterin in the hyper-IgD syndrome accords with experimental data suggesting that IFN-γ is the dominant factor in the release of neopterin. Our study shows that urinary neopterin is a good quantitative and qualitative parameter to monitor disease activity in patients with the hyper-IgD syndrome     

Association of interferon-gamma +874A polymorphism with reduced long-term inflammatory response in haemodialysis patients.

BACKGROUND: We have studied the effects of interferon (IFN)-gamma allelic variations on expression levels of pro- and anti-inflammatory cytokines and on long-term inflammatory status in haemodialysis patients. METHODS: Genotyping was performed in 123 patients for single nucleotide polymorphisms in the first intron of the IFN-gamma gene (+874 T/A). They were prospectively followed for 2 years. Cytokine mRNA levels in whole blood cells (detected by real time (RT)-PCR technique) and serum C-reactive protein (CRP) concentrations were compared in patient groups with different IFN-gamma genotypes. Serum CRP was evaluated every month and inflammatory state was defined as percent of abnormal values (above 5 mg/l) over total determinations. Of the total, 102 patients survived and completed 24+/-1 monthly CRP determinations. The IFN-gamma +/-874 A/A, 'low-producer' genotype was associated with decreased (P<0.05) mRNA levels of IFN-gamma and of interleukin-6 and with a lower (P<0.05) frequency of CRP elevation (37+/-6%) than the +/-874 A/T and T/T, 'intermediate and high-producer' genotypes (59+/-6%, and 60+/-5%, respectively). The mRNA levels of tumor necrosis factor-alpha, IL-10 and of transforming growth factor-beta1 were not different in the three groups of patients. Pooled analysis in deceased (10+/-3 monthly CRP determinations) and survived patients confirmed the results obtained in the patients who completed the follow-up period. CONCLUSIONS: The 'low-producer' IFN-gamma +874 A/A genotype was associated with a preventive effect on long-term CRP elevation in haemodialysis patients possibly mediated by decreased gene expression of IFN-gamma and IL-6.     

Immunologic disturbances in cow's milk allergy, 2: evidence for defective interferon-gamma generation

We have investigated the role of interferon-gamma (IFN-gamma) in the regulation of antigen-specific T-cell function in patients with cow's milk allergy. The study population consisted of 22 patients, aged from 7. 6 to 56. 9 months, who had challenge-proven cow's milk allergy (CMA) manifested with either skin (n=9) or gastrointestinal (n=13) symptoms. In addition, 11 age-matched children and 6 adults, mean (SD) age 31 (7) years, were studied as controls. Patients with challenge-proven CMA were rechallenged to establish whether they had acquired clinical tolerance to cow's milk. The spontaneous and mitogen-induced IFN-gamma and interleukin-4 (IL-4) generation of isolated lymphocytes was evaluated in vitro with commercial ELISA Kits at diagnosis and at reassessment. At diagnosis, the IFN-gamma production was not detectable in patients with CMA as compared with control children. IL-4 production was almost undetectable in all subjects in this study. However, at reassessment the CMA patients who had acquired clinical tolerance to cow's milk (n=16) showed enhanced IFN-gamma production, when compared with that of control children, but still lower when compared with that of healthy adults. Our results indicate that the maturation of IFN-gamma producing T-cells is delayed in CMA, which could lead to a disturbance in the regulation of T-cell function. This defect might be an important etiologic factor for CMA.     

A type 2 (Th2-like) pattern of immune response predominates in the pulmonary interstitium of patients with cryptogenic fibrosing alveolitis (CFA).

CFA is an inflammatory condition of the lungs resulting in scarring, pulmonary failure and death. The etiology of the disease is unknown, but the pathogenesis is believed to involve a persistent immunological reaction to unidentified antigen in the lung resulting in tissue damage. Recent advances in our understanding of the immune system have shown that different patterns of stimulatory cytokines are produced at sites of inflammation by a range of cell types. Patterns of cytokine reproduction by inflammatory cells are recognized to be associated with different patterns of immunological response, and these have been described as type 1 (or Th1-like) and type 2 (or Th2-like) on this basis. We have studied cytokine expression in the intestinal inflammatory cell infiltrate in lung tissue from patients with CFA using mRNA in situ hybridization and immunohistochemistry. Our results show that while there is evidence for both a type 1 (characterized by interferon-gamma (IFN-gamma) and type 2 (characterized by IL-4 and IL-5) response present in CFA, the type 2 (or Th2) pattern of cytokines appears to predominate. This would be consistent with a possible role for the humoral immune response in the pathogenesis of this condition. In addition, recent evidence suggests that IL-4 and IFN-gamma may be important regulatory factors for pulmonary fibroblasts. The relative paucity of IFN-gamma may contribute to the excessive fibroblast activation, deposition of collagen and scar formation that occurs in CFA.     

Multiple sclerosis: occurrence of myelin basic protein peptide-reactive T cells in healthy family members

Genetic factors influence the susceptibility to multiple sclerosis (MS). This disease is accompanied by augmented T cell responses to CNS myelin components such as myelin basic protein. To evaluate the familial occurrence of such T cell autoreactivity, we have studied 12 MS families including 37 healthy first-degree relatives for occurrence of numbers of interferon-gamma (IFN-γ) secreting cells among blood mononuclear after culture in presence of myelin basic protein (MBP), eight synthetic MBP peptides and the control antigen acetylcholine receptor (AChR). There were no differences between MS patients and healthy family members regarding frequencies of autoreactive T cells recognizing MBP, the eight different MBP peptides or AChR. None of the MBP peptides predominated as T cell antigen among the MS patients or their unaffected family members. In some families the highest number of MBP peptide reactive T cells were found among unaffected family members. No correlation was observed between numbers of MBP or MBP peptide reactive T cells in various subjects and their HLA-DR-DQ phenotypes. In conclusion, this study has revealed the presence of MBP and MBP peptide reactive T cells of similar frequencies in MS patients and their healthy family members.