Interferon Type II Research Articles

Construction and screening of human immunodeficiency virus-1 vpr gene RNA interference vector in vitro

Objective To screen the small interfering RNA (siRNA) fragment targeted on human immunodeficiency virus-1 (HIV-1) vpr gene and to investigate the efficacy of siRNA interference. Methods Two oligonucleotide fragments were designed and synthesized targeted on HIV-1 vpr gene, and the expression vector was constructed. Plasmids containing HIV-1 vpr gene were transfected into the human embryonic kidney 293 T (HEK293T) cells. Set up two experimental groups (siRNA56, siRNA160), and a negative control group. Blank HEK293T cells were set as control group. The total RNA and protein were extracted from cells. Real-time polymerase chain reaction and Western blot were used to confirm the efficacy of siRNA targeted on HIV-1 vpr gene at nucleic acids level and protein level. The concentrations of interleukin-17 (IL-17) and interferon-γ (IFN-γ ) were detected by enzyme-linked immunosorbent assay. Data were analyzed by one-way ANOVA. Results The pRNAT U6.1/Neo-vpr-56/160 expression vectors were successfully constructed and confirmed by DNA sequencing in mammalian cells. siRNA56 and siRNA160 could down-regulate the expression of HIV-1 vpr gene by 69.0% and 76.1% at mRNA level, respectively and 76.3% and 86.5% in protein level, respectively. The concentrations of IL-17 in control group, negative control group, siRNA56 and siRNA160 group were (1.936±0.415), (1.815±0.393), (1.935±0.356), and (2.034±0.421) pg/mL, respectively. And the concentrations of IFN-γ in four groups were (1.673±0.234), (1.648±0.332), (2.169±0.362), and (2.301±0.412) pg/mL, respectively. Conclusions The plasmid expressing pRNAT U6.1/Neo-vpr-56/160 is successfully constructed. siRNA targeted on different gene fragment can down-regulate the expression of HIV-1 vpr gene. Key words: Human immunodeficiency virus-1; Genes, vpr; RNA interference; Interleukin-17; Interferon type Ⅱ

In vitro effects of tea polyphenols on the secretion of TNF-α, IFN-γ and IL-2R by peripheral blood CD8＋ T lymphocytes from vitiligo patients

Objective To detect the level of cytokines and their receptors secreted by peripheral blood CD8＋ T lymphocytes from vitiligo patients, and to evaluate the influence of tea polyphenols on their secretion. Methods CD8＋ T lymphocytes were isolated from the peripheral blood of 12 patients with progressive vitiligo, 12 patients with stable vitiligo and 10 healthy controls, and cultured in vitro for 20 days. Then, some CD8＋ T lymphocytes were treated with tea polyphenols of 100 mg/L for two days. Enzyme-linked immunosorbent assay （ELISA） was carried out to determine the levels of tumor necrosis factor （TNF）-a, interferon （IFN）-a and interleukin-2 receptor （IL-2R） in the culture supernatants of CD8＋ T lymphoeytes before and after the treatment with tea polyphenols. Analysis of variance and t test were conducted to assess differences in these parameters among these groups and changes between pretreatment and posttreatment, respectively. Results There was a sequential decrease in the level of TNF-a （（ 191.302±6.077） vs. （175.966± 2.467） vs. （173.664 ± 3.600） ng/L, F = 4.784, P 〈 0.05）, bat a sequential increase in that of IFN-a （ （280.182 ± 36.070） vs. （371.670 ± 24.352） vs. （447.147 ± 8.432） ng/L, F= 9.036, P〈 0.01） and IL-2R （（8.375 ± 0.161） vs. （8.845 ±0.161） vs. （9.345 ± 0.125） ng/L, F = 9.639, P 〈 0.01） in the culture supernatant of CD8＋ T lymphocytes from patients with progressive vitiligo to patients with stable vitiligo and healthy controls. After two days of tea polyphenol treatment, a statistical decrease was observed in the level of TNF-a in the culture supematant of CD8 ＋ T lymphocytes from patients with progressive vitiligo （（ 164.797 ± 1.784） ng/L, P 〈 0.01 ）, patients with stable vitiligo （（166.150 ± 3.576） rig/L, P 〈 0.05） and healthy controls （（155.028 ± 5.759） ng/L, P 〈 0.05）, but no statistical changes were noted for IFN-＇y or IL-2R （all P 〉 0.05） despite a slight elevation in the level of IFN-＇y in the culture supernatant of CD8＋ T lymphocytes from patients with stable vitiligo and in that of IL-2R from all the three groups as well as a mild reduction in that of IFN-Y from patients with progressive vitiligo and healthycontrols. Conclusions The changes of cytokines and their receptors secreted by CD8＋ T lymphocytes may be associated with the induction and progression of vitiligo. Tea polyphenols may treat vitiligo via suppressing the secretion of TNF-a by CD8＋ T lymphocytes. Key words: Vitiligo, CDS-positive T-lymphocytes; Tumor necrosis factor alpha; Interferon type II; Recep-tors, interleukin-2; Tea polyphenols

T-helper type 1 immune response in cutaneous lesions of patients with Penicilliosis marneffei

Objective To observe immunopathological changes and cellular immune response in cutaneous lesions of patients with Penicilliosis marneffei (PSM).Methods Skin specimens were resected from cutaneous lesions of 50 patients with PSM,including 30 cases positive and 20 cases negative for human immunodeficiency virus (HIV),and from the normal skin of 10 healthy human controls.Hematoxylin-eosin staining was performed to observe histopathological changes,and immunohistochemistry to determine the expression level of tumor necrosis factor (TNF)-α,interferon (IFN)-γ interleukin (IL)-2,CD4 and CD8,in these tissue samples.Results Three histological patterns were observed in the cutaneous lesions of PSM,including granulomatous reaction in 9 HIV-positive specimens,suppurative reaction in 10 HIV-positive specimens and 9 HIV-negative specimens,anergic or necrotic reaction in 20 HIV-positive specimens and 2 HIV-negative specimens.Immunostaining was negative for CD4,CD8,TNF-α,IFN-γand IL-2 in the normal control skin.Of the 30 HIV-positive specimens,3 were strongly positive,8 mediately positive,7 positive,and 12 were negative,for CD8.The expression intensity of CD8 was significantly stronger in HIV-positive specimens than in the normal control specimens (P ＜ 0.01),while no significant difference was observed for the expression of the other tested cytokines or molecules between the HIV-positive specimens and normal control specimens (all P ＞ 0.05).The HIV-negative specimens showed enhanced expression of CD4 (2 cases strongly positive,2 moderately positive,9positive,9 negative),IL-2 (1 case moderately positive,8 positive,11 negative),IFN-γ (4 cases moderately positive,7 positive,9 negative) and TNF-α (3 cases strongly positive,2 moderately positive,5 positive,10negative) compared with the normal control specimens (all P ＜ 0.05).Meanwhile,the expression intensity of CD4 and IFN-γ significantly decreased in HIV-positive skin specimens when compared to HIV-negative specimens (both P ＜ 0.05).Enhanced expressions of CD4,IFN-γ and IL-2 were observed in specimens with granulomatous reaction compared with those with anergic or necrotic reaction(all P ＜ 0.05),while the specimens with suppurative reaction showed no statistical differences in these parameters when compared to those with granulomatous reaction and anergic or necrotic reaction (all P ＞ 0.05).Conclusions The histopathological changes in cutaneous lesions are closely associated with the immune status of patients with PSM,and T-helper type 1 immune response may play a pivotal role in the defense against Penicillium marneffei infection. Key words: Penicillium; Tumor necrosis factor-alpha; Interferon type Ⅱ ; Interleukin-2; CD4-positive T-lymphocytes; CD8-positive T-lymphocytes; Pathologic processes

The expression and role of DNA methyltrsansferase 3a in colonic mucosa of patients with ulcerative colitis

Objective To explore the association between DNA methyltrsansferase 3a (DNMT3a) expression and interleukin-4 (IL-4),interferon-gamma (IFN-γ) in mononuclear cells of colonic laminapropriamucosae in ulcerative colitis (UC) patients.Methods From November 2009 to March 2010,sixty colonic mucosa biopsy specimens through colon scopy were collected,specimens of active UC or normal controls were 30 in each group.The expression of DNMT3a,IL-4,and IFN-γ was detected with immunohistochemical method (SABC) in mononuclear cells of colonic laminapropriamucosae,and their relation with different degrees of colonic inflammatory response was analyzed.Results The positive expression rate of DNMT3a,IL-4,IFN-γ in mononuclear cells of colonic laminapropriamucosae in active UC group was significant higher than that in control group and there were statistically significant differences (x2 =16.596,P=0.000;x2 =42.857,P=0.000;x2 =6.667,P=0.024;).The expression of them was higher in sever inflammatory response than that in mild inflammatory response,and its expression intensity increased as inflammatory activity became more severe.There was positive correlation between IL-4,IFN-γ and DNMT3 expression in UC group (r=0.46,P=0.01 ;r=0.559,P=0.001).Conclusions The expression of DNMT3a,IL-4 and IFN-γ is associated with the degree of colonic inflammatory response.DNA methylation maybe involved in Th1/Th2 immune balance regulation in UC pathogenesis. Key words: Colitis, ulcerative; DNA methylation; DNA modification methylases; Interleukin-4; Interferon Type Ⅱ

Comparison of an in-house tuberculosis-specific IFN-γ release assays with T-SPOT TB in latent tuberculosis infection diagnosis among HIV-infected individuals

Objective To evaluate the diagnostic value of two tuberculosis-specific IFN-γ release assays in latent tuberculosis infection among HIV-infected individuals. Methods The levels of tuberculosis antigen-specific IFN-γin 102 HIV patients from AIDS Outpatient Clinic of Shenzhen Third People's Hospital were detected by in-house tuberculosis-specific IFN-γ ELISpot assay and commercial T-SPOT TB kit, and tuberculin skin test (TST) were done at the same time. There were 66 males and 36 females,and the average age was 35. Results Seventeen HIV infected patients were positive in both IFN-γ ELISpot and T-SPOT TB methods, the sensitivity, specificity positive predictive value(PPV), negative predictive value(NPV) and compliance rates of ELISpot were 94. 4% ,94. 0% ,77. 3% ,98. 8% and 94. 1% ,respectively. Three patients were positive in both IFN-γELISpot and T-SPOT TB methods, the sensitivity, specificity, PPV, NPV and compliance rates of TST were 16. 7%, 98. 8%, 75.0%, 84. 7% and 84. 3%, respectively. The average number of spots using three kinds of antigen ESAT-6, Pool A,Pool B obtained were 26. 89 ±5. 77,18. 96 ±4. 75 and 14. 51 ± 3.77, respectively. Only ESAT-6 and Pool B have a statistically significant difference (H=7.557,P = 0.022 9), no significant difference was shown between other groups. There was no significant difference between the positive rate and the CD4+ T cellls number(x2 =0. 860 8 ,P =0. 650 2) ,as the same as the T-SPOT TB (x2 = 1. 396 4, P = 0. 497 5 ). Conclusions The performance of this in-house tuberculosis-specific IFN-γ ELISPot assay was comparable to T-SPOT assay in diagnosis of latent tuberculosis infection, and the sensitivity and specificity of both these two assays were all much higher than TST. They canbe recommended in diagnosing latent tuberculosis infection in HIV infected patients. Key words: HIV infections; Tuberculosis; Interferon type Ⅱ; Immunoenzyme techniques

Low-dose interferon-γ influences differentiation of oligodendrocyte precursor cell through P27kip1

Objective To expore the mechanism of low-dose interfone-γ(IFN-γ) influences on differentiation of oligodendrocyte precursor cell. Methods The cerebral cortex samples were obtained from one day old SD rats to form mixed single cell suspensions. After culturing in full medium for 7 to 10 days, succession and differential velocity adherent technique were performed to acquire oligodendrocyte precursor cell and cultured in serum-free medium. IFN-γ, AG490 and Fludarabine were added during the culture of oligodendrocyte precursor cell and reverse transcription-polymerase chain reaction, Western blot and flow cytometry were performed to evaluate the expression of intracellular P27kip1 and its influence on the differentiation of oligodendrocyte precursor cell. Results (1)The expression of P27kip1 mRNA and protein was lower in IFN-γ group than in control group (t=85. 535, P<0. 05;t= 12. 481, P<0. 05), while the expression of P27kip1 mRNA and protein in IFN-γ+AG490 group and IFN-γ+Fludarabine group were both higher than those in IFN-γ group (P<0. 05). (2) The phosphorylation levels of JAK2/STAT1 in INF-γ group were higher than that in the other three groups (P<0. 05). (3) The percentage of myelin basic protein positive cells was (68. 42 ± 2. 53)% in IFN-γ group, lower than that in control group [(88.21 ± 1.97)%](t=10.682, P < 0.05). Myelin basic protein positive cells in IFN-γ + AG490 group were (57. 63 ±2. 75) %, lower than those in the IFN-γ group. The same figure in IFN-γ+Fludarabine group were (79. 53±4. 15)% , higher than those in IFN-γ group (t = 3.957, P<0.05). Conclusions Low-dose IFN-γ can regulate the expression of intracellular P27kip1 through JAK2/STAT1 signal transduction pathway and Fludarabine may participate in this process and improve the differentiation and maturation of oligodendrocyte precursor cell. Key words: Interferon type Ⅱ; Tyrphostins; Vidarabine; Cyclin-dependent kinase inhibitor p27; Oligodendroglia; Cell differentiation

Effects of BCG-PSN on 2,4-dinitrochlorobenzene-induced atopic dermatitis-like skin lesions in Nc/Nga mice

Objective To determine the effect of bacille Calmette-Guerin-polysaccharide nucleic acid (BCG/PSN)on 2,4-dinitrochlorobenzene(DNCB)-induced atopic dermatitis-like skin lesions in Nc/Nga mice.Methods Fifteen mice were randomly and equally classified into 3 groups,i.e.,control group receiving topical acetone on foot pad and abdomen and intraperitoneal injection of physiological saline,model group receiving topical 5% DNCB solution and intraperitoneal injection of physiological saline,treatment group receiving 5% DNCB solution and intraperitoneal iniection of BCG/PSN,and all drugs were used every other day for 7 weeks.Further more,0.1% DNCB was topically applied on the ear and neck of Nc/Nga mice once a week from week 2 to week 7.The effects of BCG/PSN were evaluated by ear thickness,skin histopathology and immunological parameters.Results Repeated application of DNCB caused the development of eczematous dermatitis in mice.Mice in model group chnieally manifested skin dryness,erythema,edema and erosion with histopathological changes including dermal and epidermal thickening,hyperkeratosis,and inflammatory infiltration.The serum levels of IL-4 and IrE in model group were significantly higher than those in control group[(174.72±12.64)μg/L vs (17.32±3.56)μg/L,(91.49±6.32)ng/L vs (83.95±6.63)ng/L,both P<0.05].Increased serum IL-12 and IFN-γ and decreased serum IgE were observed in treatment group compared with the model group[(122.10±4.64)ng/L vs (20.14±6.15)ng/L(73.89±2.39)ng/L vs (51.53±3.45)ng/L, (84.27±9.35)μg/L vs (174.72±12.64)μg/L, all P<0.05].Conclusion BCG/PSN might be beneficial for the treatment of atopie dermatitis-like skin lesions in Nc/Nga mice by enhancing the secretion of IL-12 and IFN-γ and suppressing the synthesis of IgE. Key words: Dermatitis,atopic; Mice,Nc/Nga; BCG-PSN; Interleukins; Interferon type Ⅱ; Immunoglobulin E

Proliferation of and production of interferon-γ by drug-specific peripheral T cells from pafients with severe drug eruption

Objective To detect the proliferation of and production of interferon-γ by drug-specific peripheral T cells from patients with severe drug eruption.Methods Peripheral blood mononuclear cells (PBMCs) were isolated from 10 patients with severe drug eruption,10 patients with mild or moderate drug eruption and 10 normal human controls,stimulated with causative drugs to obtain drug-specific T cells.Then,both PBMCs and drug-specific T cells were stimulated with causative drugs or unrelated drugs followed by the detection of secretion levels of IFN-γ with ex vivo enzyme-linked immunodotting (ELISpot) assay and cultured ELlSpot assav respectively.Results After stimulation with causative drugs,a higher level of IFN-γ was secreted by PBMCs and drug-specific T cells from patients with severe drug eruption compared with those from normal human controls (both P<0.01).and by drug-specific T cells than by PBMCs (P<0.01).The culture with unrelated drugs could neither induce the generation of drug-specific T cells nor promote the secretion of IFN-γ by PBMCs from the patients.Drug-specific T cells still existed in the peripheral blood of 3 patients within 1 to 3 years after recovery of drug eruption.Conclusions There are drug-specific T cells in peripheral blood of patients with severe drug eruption,and they may persist for a certain period of time after recovery of drug eruption.Ex vivo ELISpot combined with cultured ELISpot may be applied to the identification of causative drugs in vivo. Key words: Drug eruptions; T-lymphocytes; Interferon type Ⅱ

Analysis of Nef-specific interferon-γ secretion responses in HIV-1 B/C recombinant virus infectors

Objective To analyze characteristics of Nef-specific T lymphocyte responses in Chinese HIV-1 recombinant subtype B/C infectors. Methods 19 HIV-1 recombinant subtype B/C infectors infected within 1 year, 40 chronic infectors infected for more than 3 years were enrolled in this cohort study. Elispot assay was used to observe HIV-1 specific T lymphocyte responses in HIV-1 recombinant subtype B/C infectors. Results Nef-specific T lymphocyte responses of interferon-gamma secretion were identified in 15 Chinese HIV-1 recombinant subtype B/C infectors infected within 1 year. The specific T lymphocytes were mainly targeted at four peptides which span from Nef 83 to 135: EVA7081.1, EVAT081.5, EVA7081.6 and EVAT081.48. Responses were identified in 29(75. 2%) infectors with more than 3 years of infection and the specific T lymphocytes were mainly targeted at three peptides which span from Nef 63 to 101 : EVA7081.43, EVA7081.44, EVAT081.45, EVA7081.47, EVA7081.48 and EVA7081.49. The average magnitude of response in infectors with less than 1 year of infection was 284. 13 SFC/106 PBMC. The average magnitude of response in infectors with more than 3 years of infection was 152. 44 SFC/106 PBMC. There was a significant difference between the two groups (U = 91. 000, P = 0. 002). Conclusions HIV-1recombinant subtype B/C infectors at different stages of diseases (less than 1 year and more thank 3 years) can recognize central region of Nef. The magnitude of Nef-specific IFN-γ secretion T lymphocyte responses in this cohort gradually decrease with disease progression. Key words: HIV infections; HIV-1; Nef Gene products,human immunodeficiency virus; T-lymphocytes; Interferon type Ⅱ

Effects of triptolide on interferon-γ signaling in a human keratmocyte cell line HaCaT.

Objective To investigate the effects of triptolide on the expression of a series of proteins associated with interferon-γ (IFN-γ)signaling in HaCaT keratinocytes.Methods After pretreatment with difrerent dosages of triptolide(10-10-10-7 mol/L),HaCaT cells were stimulated by recombinant human IFN-γ(rhIFN-γ,500 U/mL)for various periods followed by the collection of cells.Then,total protein was extracted from these cells and subjected to Western blotting for the detection of expression of interferon-γ receptor α(IFN-γRα),phosphorylated Janus kinase 2(pJAK2)and suppressor of cytokine signaling (SOCS1).Results Triptolide at the concentrations of 10-8 mol/L and 10-7 mol/L significantly inhibited the IFN-γRα expression upregulated by rhIFN-γ(both P＜0.05).The expression of pJAK2 induced bv rhIFN-γ was also suppressed by triptolide at the concentrations of 10-9 moI/L and 10-8 mol/L(both P＜0.05).The inhibition of triptolide on IFN-γRα and pJAK2 expression was dose-dependent and the 50%inhibitory concentrations(IC50 value)were 1.37×10-8 mol/L and 2.83×10-9 mol/L,respectively.On the contrary,triptolide upregulated the expression of SOCS1 stimulated by rhIFN-γ at the concentrations of 10-10,10-9 and 10-8 mol/L(P＜0.05,0.05,0.01,respectively)with the 50%effective dosage(ED50 value)at 3.32 × 10-11 mol/L.Conclusions By inhibiting the expression of IFN-γRα as well as phosphorylation of JAK2 and upregulating the expression of SOCS1,triptolide inhibits the phosphorylation of STAT-1,resulting in the inhibition of genetic transcription of multiple inflammatory factors induced by IFN-γ signaling in HaCaT keratinocytes,and the inhibition probably contributes to the efficacy of triptolide in the treatment of IFN-γ-dependent inflammatory skin disorders,such as psoriasis. Key words: Triptolide; Interferon type Ⅱ; Receptors,interferon; Janus kinase 2:Suppressor of cytokine signaling proteins

Expression and significance of interleukin-18 in lesions of chronic eczema.

Objective To explore the role of IL-18 in the pathogenesis of chronic eczema. Methods Twenty-seven patients with chronic eczema were enrolled into this study along with 12 normal human controls. The severity of eczema was evaluated by eczema area and severity index (EASI) in patients. Skin specimens and vein blood samples were obtained from all the subjects. Reverse-transcription PCR was performed to detect the mRNA expression of IL-18 and IFN-γ in the skin tissue, and enzyme linked immunosorbent assay (ELISA) to measure the protein expression of IL-18 and IFN-γ in the sera of these subjects. Results The mRNA expression level in patients and controls was 1.04±0.29 pg/mL and 0.52±0.15 pg/mL for IL-18, respectively, 0.96±0.34 pg/mL and 0.47±0.12 pg/mL for IFN-γ, respectively; a significant increase was observed in the mRNA expression level of both IFN-γ and IL-18 in the patients than in the controls (both P 0.01), between the patients and controls. No significant correlation was observed betweenthe serum level of IL-18 or IFN-γ and sererity of eczema in the patients (both P>0.01). Conclusions IL-18 may be involved in the pathogenesis of chronic eczema. Also, in local lesions, IL-18 seems to correlate with the induction of production of Th1 type cytokines, such as IFN-γ which could subsequently mediate hypersensitivity response. Key words: Eczema; Interleukin-18; Interferon type Ⅱ

Detection and significance of Th1/Th2 in peripheral blood of patients with colonic cancer

Objective To detect the levels of Thl and Th2 type cytokines of peripheral blood CD+4 T lymphocytes in the patients with colonic cancer and to observe the shift of Th1/h2 in the patients with colonic cancer. To discuss the relationship between Th1/Th2 balance and colonic cancer progression. Methods After collecting the peripheral blood samples of research objects, the levels of IFN-γ and IL-4 were detected by flow cytometrys of Th1 Th2 cytokine marked. The data were analyzed by SPSS11.0. Results The level of IFN-γ of peripheral blood CD+4 T lymphocytes in colonic cancer patients and healthy donors were(11.7±1.86) % and (16.4±6.07)% respectively. The level of IFN-γ in malignant group was significantly lower(P ＜ 0.05). Thl/Th2 (IFN-γ/IL-4)was lower too(P ＜ 0.05). Th1/Th2 (IFN-γ/IL-4)in colonic cancer patients and healthy donors were 4.2±0.94 and 9.7±1.11 respectively. But the level of IL-4 was significantly higher than those in healthy control (P＜0.05). Conclusion Th2 dominates in peripheral blood CD+4 T lymphocytes of colonic cancer patient. Th1/Th2 disequilibrium leads to immune function hypofunction. It may be one of the reasons of colonic cancer recur, rence or colonic cancer metastasis. Key words: Intestinal neoplasms; Cytokines; Interferon type Ⅱ ; Interleukin-4

Relationship of IFN-γ inducible protein 10 with dermatoses

Chemokines and their receptors play an important role in the development of many dermatoses.For example,IFN-γ inducible protein 10(IP-10)is a newly identified chemokine in CXC subfamily which has been studied by numerous researches in recent years.It is secreted by more than 30 activated cells,such as fibroblasts,mononuclear macrophages,endothelial cells and iymphocytes.Functionally,IP-10 can attract neutrophils,facilitate the release ofvarious inflammatory factors and induce Th1 inflammation as a chemokine.This paper describes the structure and expression of IP-10 and its receptors,as well as their relationship with skin disorders. Key words: Interferon type II; Chemokines,CXC; Skin diseases