Viable Leishmania parasites in the absence of an in vitro IFN-γ response in asymptomatic carriers

ABSTRACT Asymptomatic infection (the absence or inapparent signs and symptoms) has been observed in many endemic areas of leishmaniasis, however, little is known about the parasitological and immunological factors associated with this type of infection. This study aimed to identify the in vitro expression of IFN-γ in asymptomatic carriers of viable Leishmania parasites. Asymptomatic infection was identified using the Montenegro skin test in an at-risk population from Yucatan, Mexico. Parasite viability was evinced in the blood by 7SL RNA transcripts amplification. The expression of mRNA IFN-γ was analyzed in peripheral blood mononuclear cells stimulated with soluble Leishmania antigen, using RT-qPCR. Parasite viability was observed in 33.3 % (5/15) of asymptomatic subjects. No differences were found in the expression of IFN-γ between asymptomatic and healthy subjects, and no correlation was found between the presence of viable parasites and the expression of IFN-γ. This study demonstrates the persistence of Leishmania parasites in the absence of an in vitro IFN-γ response in asymptomatic carriers from Mexico.

Type 1–type 2 interferon imbalance in dry eye disease

Purpose: Dry eye disease (DED) is characterized by altered ocular surface proinflammatory and antiinflammatory factors. Interferons (IFNs) are a class of pleiotropic cytokines well known for their antimicrobial, inflammatory, and immunomodulatory roles. Hence, this study investigates the ocular surface expression of different types of IFNs in patients with DED. Methods: The cross?sectional, observational study included patients with DED and normal subjects. Conjunctival impression cytology (CIC) samples were obtained from the study subjects (controls, n = 7; DED, n = 8). The mRNA expression levels of type 1 IFN (IFN?, IFN?), type 2 IFN (IFN?), and type 3 IFN (IFN?1, IFN?2, IFN?3) were measured by quantitative PCR (polymerase chain reaction) in CIC samples. IFN? and IFN? expression under hyperosmotic stress was also studied in human corneal epithelial cells (HCECs) in vitro. Results: The mRNA expression levels of IFN? and IFN? were significantly lower and that of IFN? was significantly higher in DED patients compared to healthy controls. The mRNA levels of IFN?, IFN?, and IFN? were significantly lower compared to IFN? in DED patients. An inverse association between tonicity?responsive enhancer?binding protein (TonEBP; hyperosmotic stress maker) and IFN? or IFN? expression and a positive association between TonEBP and IFN? expression was observed in CIC samples. The expression of IFN? was lower than IFN? in HCECs undergoing hyperosmotic stress compared to HCECs without the stress. Conclusion: The presence of an imbalance between type 1 and type 2 IFNs in DED patients suggests newer pathogenic processes in DED, plausible ocular surface infection susceptibility in DED patients, and potential therapeutic targets in the management of DED

Inmunodeficiencias secundarias relacionadas con la presencia de autoanticuerpos anticitocinas / Secondary immunodeficiencies related to the presence of anti-cytokine autoantibodies

Resumen Los autoanticuerpos anticitocinas (ACAA) han sido reportados como causa importante de inmunodeficiencias secundarias. Altos títulos de autoanticuerpos neutralizantes pueden causar susceptibilidad a diferentes enfermedades infecciosas potencialmente mortales. Por ejemplo, se ha informado que autoanticuerpos neutralizantes contra IFNγ se correlacionan con susceptibilidad a infecciones micobacterianas y patógenos fúngicos intracelulares. Autoanticuerpos contra IL-6 se detectaron en pacientes con abscesos subcutáneos y celulitis estafilocócica recurrente; asimismo, pacientes con criptococosis, nocardiosis y proteinosis alveolar pulmonar fueron positivos a autoanticuerpos contra GM-CSF. También se ha establecido una relación entre los autoanticuerpos contra IL-17 e IL-22 y las infecciones crónicas por Candida en mucosas, que se han identificado en pacientes con poliendocrinopatía autoinmune tipo 1 o timoma. Recientemente se han reportado autoanticuerpos contra interferón tipo I durante el inicio de COVID-19 aguda. Estos ACAA se asemejan a defectos genéticos en citocinas o en sus rutas de señalización. Por ello, pueden considerarse fenocopias de inmunodeficiencias primarias. De esta forma, la detección de ACAA podría ser importante en el diagnóstico, particularmente en pacientes con enfermedades de aparición tardía, para decidir los tratamientos apropiados. Esta revisión presenta una descripción general de la comprensión actual de las inmunodeficiencias secundarias asociadas a ACAA.

Abstract Anti-cytokine autoantibodies (ACAA) have been reported to be an important cause of secondary immunodeficiencies. High titers of neutralizing autoantibodies may cause susceptibility to different life-threatening infectious diseases. For example, neutralizing autoantibodies against IFNγ have been reported to be correlated with susceptibility to mycobacterial infections and intracellular fungal pathogens. Autoantibodies against IL-6 were detected in patients with subcutaneous abscesses and recurrent staphylococcal cellulitis; on the other hand, patients with cryptococcosis, nocardiosis, and pulmonary alveolar proteinosis were positive for autoantibodies to GM-CSF. A relationship has also been established between autoantibodies against IL-17 and IL-22 and chronic mucosal Candida infections, which have been identified in patients with APECED or thymoma. Autoantibodies against type-I IFN have been recently reported during the onset of acute COVID-19. These ACAAs resemble genetic defects in cytokines or their signaling pathways. Therefore, they may be considered to be primary immunodeficiencies phenocopies. Consequently, the detection of ACAA could be important in the diagnosis of patients, particularly in the case of late-onset diseases, in order to decide appropriate treatments. This review presents an overview of current understanding of ACAA-associated secondary immunodeficiencies.

Re-engineering of bicistronic plasmid pGPD/IFN to construct fusion gene co-expressing Glyceraldehyde 3-phosphate dehydrogenase gene (GAPDH) of Edwardsiella tarda and Interferon-gamma (IFN-?) gene of Labeo rohita (Hamilton) and its in vitro functional analysis

Edwardsiella septicemia disease in the cultured Indian major carps is caused by the fish pathogen Edwardsiella tarda and it is preventable by DNA vaccination. Here, we tried to develop a bicistronic DNA vaccine pGPD/IFN expressing the Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene of Edwardsiella tarda and Interferon-gamma (IFN-?) gene of Labeo rohita. The vaccine showed high protective efficiency in our previous studies; however as a limitation of bicistronic construct the expression of gene cloned in second frame (B) is poor. To overcome this limitation we re-engineered the construct and designed a fusion gene co-expressing the GAPDH and IFN-? genes as one frame with an aim to get the optimum expression of both the genes. For this purpose, a fusion insert comprising GAPDH and IFN-? coding sequences was cloned in to pcDNA3.1(+) plasmid vector. The fusion genes' in vitro expression was confirmed in the striped snakehead fish cell line (SSN-1). Successful expression of the re-engineered fusion gene DNA vaccine in the cell line was achieved at 48h post-transfection, which was confirmed by amplifying the expression transcripts of GAPDH and IFN-? genes. Thus, the study concludes that the re-engineered fusion vaccine pcGPD/IFN (pcDNA3.1(+) plasmid having fusion GPD/IFN) is functional and can be effectively utilized to vaccinate rohu (Labeo rohita) as it contains the species-specific immune gene (IFN-?) as an adjuvant

Effect of acute or subchronic stress on T cell response in peripheral blood: Regulatory role of vitamin D

The immune response, orchestrated by helper (Th1, Th2, and Th17) and regulatory (Treg) T cells, is modulated by stress and Vitamin D (Vit-D). Although the immunomodulatory functions of both are known, their specific roles on Th cells have not been fully clarified, yet. On this background, we aimed to investigate the effect of acute or subchronic stress on the distribution of peripheral T lymphocytes, as well as the immunomodulatory role of Vit-D. Young adult male, Swiss-albino mice (30–40g) were allocated to the control, acute stress (AS), subchronic stress (ChS), control+Vit-D, AS+Vit-D, and ChS+Vit-D groups (n=11/group). The combined cold (2-h at 4°C)-immobilization (2-h in a restrainer) stress protocol was employed as one day in AS groups and five consecutive days in ChS groups. Vit-D (2?g/kg ip) was applied every other day, until the end of the protocol. Serum cortisol, Vit-D and cytokine levels (IL-4, IFN-?, and IL-17A) were measured, and lymphocytes from blood samples were subtyped by flow-cytometry. Stress exposure caused differential Th and Treg responses, acute stress shifting the response to Th1, and subchronic stress shifting the response to Th2. Th17 and Treg cells were lower in subchronic stress exposed mice. These changes became comparable to control values in Vit-D treated groups. The T cell response, crucial for immune system function, differs on the basis of stress exposure as such the Vit-D treatment. The tolerogenic profile created by Vit-D should be considered for management of stress-related diseases. Our results may help to provide a better understanding of disease pathogenesis.

Construction of interferon alpha/beta receptor subunit 1 gene knockout Caco-2 cell line based on CRISPR/Cas9 system / 中国生物制品学杂志

@#Objective To knockout interferon alpha/beta receptor subunit 1(IFNAR1) gene in human colorectal adenocarcinoma cells Caco-2 using clustered regularly interspaced short palinmic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system to construct IFNAR1 knockout Caco-2 cell line.Methods The single guide RNA(sgRNA)sequence was designed to specifically recognize the exon region of IFNAR1 gene using CRISPR/Cas9 technology,and the LentiCRISPRv2-IFNAR1-sgRNA recombinant plasmid was constructed.Caco-2 cells were infected with the plasmid packaged by lentivirus and screened by puromycin resistance.The obtained monoclonal cell lines were cultured by limited dilution method,which were verified for the effect of IFNAR1 gene knockout by target gene sequencing and Western blot,and detected for the mRNA levels of CXC chemokine ligand 10(CXCL10)and interferon-stimulatd gene 20(ISG20)in IFNAR1knockout cells by adding exogenous IFNβ.Results Sequencing results of plasmid LentiCRISPRv2-IFNAR1-sgRNA showed that the insertion sites were all located at the sticky end of BsmBⅠenzyme digestion.Two IFNAR1 knockout monoclonal cell lines were obtained.The sequencing results showed that Caco-2-IFNAR1-KO1 had 5 bp deletion in the sixth exon of IFNAR1,and Caco-2-IFNAR1-KO2 had 18 bp deletion and 1 bp insertion in the seventh exon.Compared with wild-type Caco-2 cells,Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells showed no expression of IFNAR1 protein.Compared with no IFNβ stimulation,the mRNA levels of CXCL10 gene(t = 0.566 and 1.268 respectively,P>0.05)and ISG20 gene(t =1.522 and 1.733 respectively,P>0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells stimulated by 50 ng/mL IFNβ showed no significant increase.While compared with those of wild-type Caco-2 cells,the mRNA levels of CXCL10gene(t = 6.763 and 6.777 respectively,P<0.05)and ISG20 gene(t = 5.664 and 5.65 respectively,P<0.05)in Caco-2-IFNAR1-KO1 and Caco-2-IFNAR1-KO2 cells decreased significantly under the stimulation of 50 ng/mL exogenous IFNβ.Conclusion Caco-2 cell line with IFNAR1 knockout was successfully constructed by using CRISPR/Cas9 technology,and the downstream molecules activated by IFNAR(interferon alpha/beta receptor)in this cell line were obviously inhibited,which provided a powerful tool for further exploration of the innate immune response and replication packaging mechanism of Caco-2 cells after virus infection.

Effect of interferon receptor 1 silenced human diploid MRC⁃5 cell line on replication of varicella⁃zoster virus / 中国生物制品学杂志

@#Abstract：Objective To improve the replication level of varicella⁃zoster virus（VZV）in human diploid cell line MRC⁃5 and increase the yield of VZV vaccine by reducing the expression of interferon（IFN）related genes via optimizing the cell line MRC⁃5. Methods Interferon receptor 1（IFNAR1）silenced MRC⁃5 cell line（MRC⁃5IFNAR1⁃）was constructed by CRISPR／Cas9 gene editing technology，which was determined for the relative expression of IFNAR1 mRNA，and for those of mRNA of IFN related genes IFNβ and OAS1 after VZV infection by qRT⁃PCR to evaluate the effect of gene silencing. Gene mutation sequences were further identified by sequencing of the silenced sites. The replication of VZV in MRC⁃5 and MRC⁃5IFNAR1⁃ cell lines was compared 168 h after VZV infection by using qRT⁃PCR and plaque formation unit（PFU）assay， to evaluate the effect of MRC⁃5IFNAR1⁃cell line on VZV replication. Results The growth status of MRC⁃5IFNAR1⁃ cell line wasconsistent with that of MRC ⁃ 5 cells，and the relative expression of IFNAR1 mRNA decreased by 73%；The relative expressions of IFNβ and OAS1 mRNA in MRC⁃5IFNAR1⁃ cell line were 61% and 90% lower than those in MRC⁃ 5 cells respectively after VZV infection；In addition，168 h after VZV infection，the level of DNA replication and the titer of VZV increased by 5. 7 folds and 4 folds respectively. Conclusion The successful establishment of MRC⁃5IFNAR1⁃ cell line may be a potential scheme to increase the yield of vaccines based on human diploid cells，and provided a reference for expanding production of VZV vaccine.

Clinical study of hepatitis B vaccine in achieving hepatitis B surface antibody seroconversion in patients with functional cure

Abstract Hepatitis B Surface Antigen (HBsAg) seroclearance is the highest treatment goal recommended by the current guidelines for hepatitis B. Levels of antibodies to HBsAg (anti-HBs) are strongly associated with HBsAg recurrence, but hepatitis B vaccination may increase the anti-HBs seroconversion rate and reduce recurrence. We conducted a retrospective clinical study to ascertain the effect of this vaccination on the seroconversion rate and levels of protective anti-HBs after HBsAg. In this retrospective study, we distributed a questionnaire through an online survey platform to collect information related to hepatitis B vaccination in patients with functional cure of hepatitis B with Interferon-α (IFNα) therapy. We enrolled 320 patients who achieved functional cure from IFNα therapy. Of these, 219 patients had received hepatitis B vaccination according to their personal preference and drug accessibility after HBsAg seroclearance, whereas the remaining 101 patients did not receive hepatitis B vaccination. The anti-HBs seroconversion rate of 78.1% in the vaccinated group was significantly greater than that in the unvaccinated group (41.6%) (p < 0.001). Stratified comparisons with anti-HBs of ≥ 100 IU/L and ≥ 300 IU/L showed that both proportions in the vaccinated group were greater than those in the unvaccinated group (71.2% vs. 32.7% and 56.2% vs. 17.8%, respectively, all p-values < 0.001). Logistic regression analysis showed that the odds ratio of vaccination was 4.427, which was the strongest influencing factor for anti-HBs, reaching 100 IU/L or higher. Hepatitis B vaccination in patients after HBsAg seroclearance not only increased the anti-HBs seroconversion rate but also significantly increased antibody levels, with good safety, indicating the clinical value of vaccine therapy for patients with functional cure.

Relationship between IL-17, TNF-α, IL-10, IFN-γ, and IL-18 polymorphisms with the outcome of hepatitis B virus infection in the Turkish population

SUMMARY OBJECTIVE: Hepatitis B virus is a global threat that can lead to liver cirrhosis and hepatocellular carcinoma. For the treatment of chronic hepatitis B virus, polymorphisms might be an option for gene treatments. This study aimed to investigate the effects of IL-17, TNF-α, IL-10, IFN-γ, and IL-18 gene polymorphisms on hepatitis B virus infection in the Turkish population. METHODS: The genotypes and allele distribution of 75 patients exposed to hepatitis B virus and 50 healthy control individuals were analyzed. The real-time polymerase chain reaction method was used for identification. RESULTS: A correlation was observed between susceptibility to hepatitis B virus infection and IL-17 Exon 3/3'UTR (rs1974226) C, IL-17 Exon 3 (rs763780) A, IL-18 (-607) (rs1946518) A alleles, and IL-17 Exon 3 (rs763780) AA genotype (p=0.006, p=0.009, p=0.025, and p=0.008, respectively). Furthermore, IL-18 (-137) (rs187238) TT genotype and TNF-α-308 (rs1800629) G and A alleles, were associated with protection against hepatitis B virus infection (p=0.0351 and p=0.032, respectively). CONCLUSION: This study demonstrated that TNF-α (-308), IL-17 (Exon 3/3' UTR), IL-17 (Exon 3), and IL-18 (-607) polymorphisms are associated with hepatitis B virus infection. Therefore, these may serve as potential therapeutic targets for chronic viral hepatitis in the Turkish population.

Innate immune response mediated by interferon during influenza B virus infection / 中国生物制品学杂志

@#ObjectiveTo explore the innate immune response mediated by interferon(IFN) induced by influenza B virus(IBV)infection.MethodsThe activation of IFN signaling pathway and the expression of IFN-stimulated genes were detected by qPCR using Madin Darby canine kidney(MDCK)cells infected with IBV as model. The supernatants of MDCK cells infected with IBV for 36 h and 48 h were collected and mixed with fresh medium to culture MDCK cells infected with IBV. The antiviral effect of endogenous IFN was detected by qPCR. After adding JAK-STAT pathway inhibitor CP,the supernatant of IBV infected MDCK cells was collected and the cells were cultured. The effect of JAK-STAT pathway inhibition on the antiviral effect of endogenous IFN was detected by qPCR.ResultsIBV effectively activated IFN signal pathway and induced the production of cytokines dominated by typeⅠIFN(IFNα,IFNβ)and typeⅢIFN(IFNλ1,IFNλ3).Meanwhile,MDCK cells infected with IBV induced a series of IFN-stimulated genes(ISGs)with broad-spectrum antiviral effect,such as ISG15,CCL5,CXCL10,MX1 and RIG-I. After CP was used to inhibit JAK-STAT pathway,the ability of ISGs production induced by IBV infection in MDCK cells and the corresponding antiviral effect were significantly inhibited.ConclusionMDCK cells infected with IBV effectively activated type Ⅰ and type Ⅲ IFN mediated JAK-STAT signaling pathways,which provided a reference for the further understanding the interaction between IBV and host.

Topological analysis of IFN-induced transmembrane proteins in porcine peripheral blood lymphocytes and changes of transcription level after infection with porcine reproductive and respiratory syndrome virus / 中国生物制品学杂志

@#Objective To analyze the topology of IFN-induced transmembrane(IFITM) protein in porcine peripheral blood lymphocytes(PBMCs) and detect the change of IFITM mRNA transcription in PBMCs after porcine reproductive and respiratory syndrome virus(PRRSV) infection in vitro.Methods PRRSV,porcine circovirus 2(PCV2) and Japanese encephalitis virus(JEV) negative anticoagulant blood of piglets were collected aseptically and isolated for PBMCs.Porcine IFITM CDS sequence was amplified by PCR,sequenced and analyzed for topology.PBMCs were infected with PRRSV in vitro.Cell samples were collected at 12,24,36 and 48 h after infection,detected for PRRSV infection by RT-PCR,and detected for mRNA transcription level changes of IFITM1,IFITM2 and IFITM3 by RT-PCR.Results The porcine PBMCs were successfully isolated and the full-length sequence of IFITM CDS derived from PBMCs was cloned.The porcine IFITM protein might have two topological structures.PBMCs inoculated with PRRSV for 24 h produced obvious cytopathic effect.PRRSV was replicated in PBMCs.The transcription levels of IFITM1,IFITM2 and IFITM3 mRNA in PBMCs were significantly up-regulated at the early stage of PRRSV infection,and reached the peak at 12h after infection,and then gradually decreased;The transcription level of IFITM1 mRNA increased at 36 h after virus infection and then declined rapidly.Conclusion PRRSV infection in vitro significantly up-regulated the transcription level of IFITM mRNA in PBMCs,indicating that IFITM was involved in the antiviral immune response of PBMCs.This study provided a reference for revealing the natural immune response against PRRSV in vivo.

Epidemiological characteristics of allergic rhinitis and its correlation with serum IFN-γ and IL-4 levels / 公共卫生与预防医学

Objective To analyze the epidemiological characteristics of AR and its correlation with serum IFN-γ and IL-4 levels in Bazhong City, and to provide theoretical basis for clinical prevention and treatment of AR. Methods Adopt the method of multistage stratified random survey from January 2019 to January 2020 bazhong 3 armour hospital otolaryngology seeing a doctor , with face to face questionnaire survey form the people generally, AR number of statistics, the main clinical symptoms and related symptoms, AR merger disease situation, according to the illness severity was divided into mild and moderately severe group, Five mL of elbow venous blood was extracted from AR patients, and 22 kinds of allergens were determined by using the allergen-specific IgE antibody detection kit (western blot). Serum IFN-γ and IL-4 levels were determined by enzyme-linked immunosorbent assay. Pearson correlation analysis was performed on serum IFN-γ and IL-4 levels and severity of allergic rhinitis. Results Among 1 243 patients who completed the questionnaire, the prevalence of AR was 275 (22.12%). There was significant difference in the prevalence of AR among different age groups (χ2=6.809 , P2=7.914 , P<0.0) and the prevalence of AR in workers was the highest (26.48%). Among 275 AR patients in Bazhong City, 153 cases (55.64%) had seasonal allergic rhinitis and 122 cases (44.36%) had perennial allergic rhinitis. The main clinical symptoms of AR patients were sneezing in 234 cases (85.09%) and nasal obstruction in 197 cases (71.64%). AR combined with bronchial asthma in 59 cases (21.45%), conjunctivitis in 28 cases (10.18%); the peak of AR incidence was mainly in July. Among 22 allergens, dust mite was the most common allergen in 139 cases (50.55%), followed by penicillin in 56 cases (20.36%). The main inducing factors were pollen 112 cases (40.73%) and cold 98 cases (35.64%). According to the severity of the disease, they were divided into mild group (n=178) and moderate to severe group (n=97). The serum IL-4 level in moderate and severe groups was significantly higher than that in mild group (P<0.05). The serum IFN-γ level in moderate and severe groups was significantly lower than that in mild group (P<0.05). Pearson correlation analysis showed that il-4 level was positively correlated with disease severity (r=0.492, P<0.05). IFN-γ was negatively correlated with the severity of the disease (r=-0.459, P<0.05). Conclusion The prevalence of AR is high in Bazhong city, and the main clinical symptom is sneezing. Among the complications, bronchial asthma is the most common. Colds and weather changes are the main factors causing AR.

Impact of genetic ancestry on the distribution of interferon-λ4 rs12979860 polymorphism in a global population of Buenos Aires, Argentina / Impacto de la ancestría genética en la distribución del polimorfismo de interferón-λ4 rs12979860 en una población global de Buenos Aires, Argentina

ABSTRACT Human interferon-λ4 is a cytokine involved in early stages of antiviral responses. Strikingly, some allelic variants with diminished antiviral activity reduce the susceptibility to viral infections, thus they would have suffered a positive selection pressure throughout the evolutionary history of the genus Homo. An intronic variant within the IFNλ4 locus (rs12979860, T˃C) emerged as one of the main gene determinants of the response to HCV and other viruses. The rs12979860-C allele has a differential frequency in African, European and Native American populations, though South American data are scarce. Here we characterize for the first time the distribution of rs12979860 genotypes in a sample of the global population of Buenos Aires, Argentina, assessing its association with European, Native American and African parental components. The rs12979860 genotypes were determined by PCR-RFLP in DNA samples from donors of a blood banks of Buenos Aires (n=96), whose genetic individual ancestry (European, African or Native American) had been previously determined using molecular markers. The distribution of rs12979860-CC, CT and TT was 29.17%, 50.0% and 20.83%, respectively. A significant increase in the frequency of CC among donors with a strong European contribution and a greater impact of the Native American component among donors carrying the T allele were observed. Native American and European components were associated to the rs12979860 distribution in a sample of the global population of Buenos Aires, while no differences were directly attributable to the African ancestry. Considering interferon´s key role in antiviral responses, our results may contribute to both bioanthropological and immunogenetic studies associated with infectious diseases.

RESUMEN El interferón-λ4 humano es una citoquina involucrada en la respuesta antiviral. Algunas variantes alélicas con menor actividad antiviral, paradójicamente, reducen la susceptibilidad a infecciones virales, por lo que habrían sufrido una presión de selección positiva en la historia evolutiva del gיnero Homo. Una variante dentro del locus de IFNλ4 (rs12979860, T˃C), con distribución diferencial en poblaciones africanas, europeas y nativas americanas, surgió como uno de los principales determinantes genéticos de la respuesta al HCV y otros virus. Aquí caracterizamos por primera vez la distribución de los genotipos de rs12979860 en una muestra de la población cosmopolita de Buenos Aires, Argentina, evaluando el impacto de su ancesrtría. Se determinaron diferentes genotipos de rs12979860 por PCR-RFLP en muestras de ADN de donantes de bancos de sangre de Buenos Aires (n=96), cuya ancestría individual había sido previamente determinada mediante diferentes marcadores moleculares. La distribución global de rs12979860-CC, CT y TT fue 29,17%; 50,0% y 20,83%, respectivamente. Se observó un aumento significativo de la frecuencia del genotipo CC entre individuos con fuerte aporte europeo y un mayor impacto del componente nativo-americano entre portadores del alelo T. Los componentes nativo-americano y europeo se asociaron a la distribución rs12979860 en una muestra poblacional global de Buenos Aires, mientras que no se vieron diferencias directamente asociadas a la ancestría africana. Considerando el papel clave del interferón en la respuesta antiviral, nuestros resultados pueden contribuir a estudios con un enfoque bioantropológico así como a estudios inmunogenéticos asociados a enfermedades infecciosas.

Ficus carica hairy roots: In vitro anti-leishmanial activity against Leishmania major promastigotes and amastigotes

Objective: To investigate the biochemical capacity, and in vitro inhibitory effects of hairy roots from two cultivars of Ficus carica L. (Sabz and Siah) on Leishmania major promastigotes and amastigotes. Methods: In the hairy roots, the activity of antioxidant enzymes compared to normal leaves and roots, and the presence of some phenolic compounds in comparison with fruits were investigated. The IC 50 values of hairy roots in promastigotes was determined by tetrazolium-dye 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and trypan blue assays. By calculating the infectivity index of peripheral blood mononuclear cells (PBMCs), the leishmanicidal activity (IC 50 values) of hairy roots for amastigotes was estimated. The effects of hairy roots (IC 50 values) treatment on the levels of IFN-γ and iNOS expression, intracellular reactive oxygen species, and iNOS protein expression in infected-PBMCs were determined. Results: Based on antioxidant enzyme assays and high performance liquid chromatography analysis, hairy roots exhibited high antioxidant capacity and contained high levels of phenolic compounds. According to the results of tetrazolium-dye 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide and trypan blue assays, the hairy root extracts of both cultivars showed considerable dose-dependent inhibitory effects against Leishmania major promastigotes. Depending on the concentration and exposure time, treatment of infected-PBMCs with hairy root extracts caused the generation of a significant reactive oxygen species, up-regulation of IFN-γ and iNOS genes expression, and high value of iNOS protein compared to controls. Conclusions: The findings of this study suggest that the hairy roots of Ficus carica can be considered as a promising natural source of antileishmanial agents.

Screening and identification of H-2 d-restricted T cell epitopes in fusion and attachment glycoproteins of Nipah virus by ELISPOT / 中华微生物学和免疫学杂志

Objective:To screen and identify H-2 d-restricted T cell epitopes in fusion (F) and attachment (G) glycoproteins of Nipah virus (NiV) in mice. Methods:The complete peptides (single peptide contains 15 amino acids, and 10 amino acids were repeated in the front and back peptides) derived from F and G antigens were mixed into peptide libraries. BALB/c mice were immunized with DNA vaccines expressing NiV F and G proteins alone and in combination. The full sequence peptide libraries of F and G antigens were mixed into peptide pools by matrix design, and spleen cells of immunized mice were collected and analyzed by IFN-γ ELISPOT assay to detect the dominant H-2 d-restricted epitope peptides. Results:Twelve dominant H-2 d-restricted peptides were screened from the F protein-specific peptide library and the 56th peptide produced the strongest reaction. Four dominant peptides were screened from the G protein-specific peptide library and the 72nd peptide produced the strongest reaction. Conclusions:In this study, 12 F antigen-specific and 4 G antigen-specific H-2 d restricted dominant T cell epitopes of NiV were screened and identified by IFN-γ ELISPOT, which could provide reference for immunological analysis of NiV and vaccine research.

Acupuncture combined with western medication for ocular myasthenia gravis: a randomized controlled trial / 中国针灸

OBJECTIVE@#To compare the clinical efficacy between acupuncture combined with western medication and simple western medication for ocular myasthenia gravis (OMG), and to explore its possible mechanism.@*METHODS@#A total of 60 patients of ocular myasthenia gravis were randomized into an acupuncture combined with western medication group (30 cases, 1 case dropped off) and a western medication group (30 cases, 2 cases dropped off). Oral pyridostigmine bromide tablet and prednisone acetate tablet were given in the western medication group. On the basis of the treatment in the western medication group, Tongdu Tiaoqi acupuncture (acupuncture for unblocking the governor vessel and regulating qi ) was applied at Baihui (GV 20), Fengfu (GV 16), Hegu (LI 4), Zusanli (ST 36), etc. in the acupuncture combined with western medication group, once a day, 6 days a week. The treatment was given 8 weeks in both groups. Before and after treatment, the OMG clinical absolute score was observed, electrophysiological indexes of orbicularis oculi (value of mean jitter, percentage of jitter >55 μs and percentage of blocks) were measured by single-fiber electromyography (SFEMG), serum levels of acetylcholine receptor antibody (AChR-Ab), interferon-gamma (IFN-γ) and interleukin-4 (IL-4) were detected by ELISA method.@*RESULTS@#After treatment, the OMG clinical absolute scores, values of mean jitter, percentages of jitter >55 μs, percentages of blocks and serum levels of AChR-Ab, IFN-γ and IL-4 were decreased compared before treatment in both groups (P<0.05), and those in the acupuncture combined with western medication group were lower than the western medication group (P<0.05).@*CONCLUSION@#Acupuncture combined with western medication can effectively improve ptosis, palpebra superior fatigability, eye movement disorder and neuromuscular junction dysfunction in patients with ocular myasthenia gravis, the therapeutic effect is superior to simple western medication. Its mechanism may be related to down-regulating serum levels of AChR-Ab, IFN-γ and IL-4 and promoting the recovery of orbicularis oculi function.

Study on the effect of miR-223-3p regulating transcription factor Rbpj on the differentiation of Th1 and Th17 cells in rats with experimental autoimmune uveitis / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the regulatory effect of miR-223-3p on the expression of transcription factor Rbpj and on the differentiation of Th1 and Th17 cells in experimental autoimmune uveitis(EAU)rats.<p>METHODS: The regulatory role of miR-223-3p in Rbpj gene expression was investigated by a dual luciferase expression reporter system. In the present study, 24 female Lewis rats were randomly divided into EAU model group, normal control(NC)group and blank control(BC)group, and each group included 8 rats. The EAU model group was injected with interphotoreceptor retinoid-binding protein(IRBP)emulsion containing Mycobacterium tuberculin H37RA and complete Freund's adjuvant to induce uveitis, while the NC group was injected with an equal volume of emulsion without IRBP peptide. The rats in the BC group received the same volume of sterile saline solution. At 12d after immunization, the spleen, lymph node and eye tissues in both groups were aseptically isolated, and the expression levels of miR-223-3p and Rbpj RNAs were detected by real-time quantitative PCR(Q-PCR); Meanwhile, the expression levels of Rbpj, IFN-γ and IL-17 proteins were detected by ELISA, and the levels of Th1 and Th17 cell lineages in each tissue from each groups were detected by flow cytometry. <p>RESULTS: The results of dual fluorescein assay indicated that Rbpj was the target gene which regulated by miR-223-3p. At 12d after immunization, compared with NC group, the relative expression levels of miR-223-3p in spleen, lymph node and eye tissues from EAU model rats were 0.33±0.29, 0.11±0.12 and 0.18±0.11, respectively, accompanied by the down-regulated expression, and the differences were statistically significant(all <i>P</i><0.05); Rbpj mRNA levels were 3.00±0.06, 1.52±0.12 and 3.01±0.34, respectively, and were all up-regulated, while the differences were statistically significant(all <i>P</i><0.05). Moreover, the differences in miR-223-3p and Rbpj mRNA levels in spleen, lymph node and eye tissues of rats in the blank control group were not statistically significant compared with those in the NC group(<i>P</i>>0.05); ELISA results revealed that the expression levels of RBPJ, IFN-γ and IL-17 proteins in all tissues from EAU rats at 12d after immunization were significantly higher than those in the NC group( all <i>P</i><0.05), and there was no statistically significant difference in the expression levels of Rbpj, IFN-γ and IL-17 protein in all tissues of rats in the blank control group compared with the NC group(<i>P</i>>0.05); Meanwhile, flow cytometry results showed that the proportions of Th1 and Th17 cell lineages in all tissues from EAU model group were significantly higher than those from the NC group at 12d after immunization, and the differences were statistically significant(all <i>P</i><0.05). Furthermore,there was no significant change in the proportion of Th1 and Th17 cells in each tissue in the BC and NC groups(all <i>P</i> >0.05). <p>CONCLUSION: The miR-223-3p can negatively regulate the expression of the transcription factor Rbpj of Notch signaling pathway. The down-regulated miR-223-3p expression in EAU rats can increase the expression levels of Rbpj gene and protein, and aggravate the differentiation of Th1 and Th17 cells and the expression levels of related molecules IFN-γ and IL-17, which in turn affect the development of uveitis.

The Roles of IFN-γ in oncoimmunology and immunotherapy / 中国肿瘤生物治疗杂志

@#[摘 要] IFN-γ在抗肿瘤过程中发挥了重要作用，近年来人们逐渐认识到了其在肿瘤免疫治疗中的必要性。IFN-γ作用于肿瘤发生发展的全过程，其在抗肿瘤方面的作用机制主要分为免疫机制和非免疫机制。IFN-γ可以直接抑制肿瘤细胞增殖、促进肿瘤细胞凋亡、抑制肿瘤血管生成，协同固有免疫和适应性免疫的NK、NKT细胞、γδT细胞以及CD4+/CD8+T细胞等完成肿瘤杀伤。然而也有报道IFN-γ的促肿瘤作用，主要表现为在长期慢性炎症和肿瘤微环境中重塑免疫微环境促进免疫逃逸。因此，在肿瘤免疫治疗中要注意IFN-γ的双向效应，防止产生定向选择造成肿瘤免疫抑制。

Effect of Astragali Radix-Curcumae Rhizoma compatibility combined with 5-fluorouracil on Th17/Treg balance and tumor-related mRNA and protein expression in orthotopic xenograft model mice of CT26.WT colorectal carcinoma / 中国中药杂志

Astragali Radix-Curcumae Rhizoma(AR-CR) is a combination commonly used in the clinical treatment of tumors. Based on the T helper 17(Th17)/regulatory T cell(Treg) balance, the present study explored the possible mechanism of AR-CR combined with 5-fluorouracil(5-FU) on the tumor growth of orthotopic xenograft model mice of colorectal carcinoma. Ninety male BALB/c mice were randomly divided into nine groups, i.e., a blank group, a model group, a 5-FU group, high-, medium-, and low-dose AR-CR(2∶1) groups, and high-, medium-, and low-dose AR-CR+5-FU groups, with 10 mice in each group. The orthotopic xenograft model of CT26.WT colorectal carcinoma was induced in mice except those in the blank group. Twenty-four hours after the ope-ration, mice in the blank group and the model group received normal saline by gavage(10 mL·kg~(-1), once per day), and those in the 5-FU group received 5-FU by intraperitoneal injection(25 mg·kg~(-1), once every other day). Mice in the AR-CR groups received AR and CR decoctions by gavage(12, 6, and 3 g·kg~(-1), once a day) and those in the combination groups received AR and CR decoctions and 5-FU(doses and administration methods were the same as above). After intervention for three weeks, all mice were sacrificed and tumor tissues were collected. The tumor mass was weighed and the average tumor weight was calculated. The changing trend of Th17/Treg(%) in the CD4~+T lymphocytes of the spleen tissues of the mice in each group was detected. The mRNA expression in the blood and protein expression in the tumor tissues of transforming growth factor-β(TGF-β), tumor necrosis factor-α(TNF-α), interferon-γ(IFN-γ), Smad4, N-cadherin, matrix metalloproteinase-7(MMP-7) were detected. The experimental results revealed that compared with the model group, the groups with drug intervention showed reduced tumor mass(P<0.01), decreased CD4~+IL-17~+ in the spleen tissues to varying degrees(P<0.001), and increased proportion of CD4~+Foxp3~+(P<0.001 or P<0.05), indicating that Th17/Treg maintained dynamic balance, and the effect of the combination groups was predominant. Additionally, the mRNA expression in the blood and protein expression in the tumor tissues of TGF-β, TNF-α, IFN-γ, Smad4, N-cadherin, and MMP-7 declined to varying degrees in a dose-dependent manner(P<0.01 or P<0.001). The AR-CR combined with 5-FU can inhibit the tumor growth of orthotopic xenograft model mice of CT26.WT colorectal carcinoma. The mechanism may be related to maintenance of Th17/Treg dynamic balance in the body and down-regulation of TGF-β, TNF-α, IFN-γ, Smad4, N-cadherin, and MMP-7 expression.

Suppressing fatty acid synthase by type I interferon and chemical inhibitors as a broad spectrum anti-viral strategy against SARS-CoV-2

SARS-CoV-2 is an emerging viral pathogen and a major global public health challenge since December of 2019, with limited effective treatments throughout the pandemic. As part of the innate immune response to viral infection, type I interferons (IFN-I) trigger a signaling cascade that culminates in the activation of hundreds of genes, known as interferon stimulated genes (ISGs), that collectively foster an antiviral state. We report here the identification of a group of type I interferon suppressed genes, including fatty acid synthase (FASN), which are involved in lipid metabolism. Overexpression of FASN or the addition of its downstream product, palmitate, increased viral infection while knockout or knockdown of FASN reduced infection. More importantly, pharmacological inhibitors of FASN effectively blocked infections with a broad range of viruses, including SARS-CoV-2 and its variants of concern. Thus, our studies not only suggest that downregulation of metabolic genes may present an antiviral strategy by type I interferon, but they also introduce the potential for FASN inhibitors to have a therapeutic application in combating emerging infectious diseases such as COVID-19.