Triterpenoids with antiproliferative activities from the twigs and leaves of Melaleuca linariifolia.

One new pentacyclic triterpenoid, urs-12,16-dien-3-one (1), together with twelve known pentacyclic triterpenoids (2‒13), were isolated from the twigs and leaves of Melaleuca linariifolia. Their structures were characterized by their 1D- and 2 D-NMR spectra analysis and mass spectra studies. Furthermore, all isolated compounds were tested the inhibitory effect on proliferation of six human cancer cell lines in vitro, including NCI-H441, NCI-H460, A549, SKOV3, hela, and caki-1 cells. Among them, compounds 3, 5, 7, 9, 12, and 13 exhibited moderate antiproliferative activities with IC50 values ranging from 3.85 to 33.31 µM.

Pathogenesis and Treatment of Cytokine Storm Induced by Infectious Diseases.

Cytokine storm is a phenomenon characterized by strong elevated circulating cytokines that most often occur after an overreactive immune system is activated by an acute systemic infection. A variety of cells participate in cytokine storm induction and progression, with profiles of cytokines released during cytokine storm varying from disease to disease. This review focuses on pathophysiological mechanisms underlying cytokine storm induction and progression induced by pathogenic invasive infectious diseases. Strategies for targeted treatment of various types of infection-induced cytokine storms are described from both host and pathogen perspectives. In summary, current studies indicate that cytokine storm-targeted therapies can effectively alleviate tissue damage while promoting the clearance of invading pathogens. Based on this premise, "multi-omics" immune system profiling should facilitate the development of more effective therapeutic strategies to alleviate cytokine storms caused by various diseases.

Development and applications of a monoclonal antibody against caprine interferon-gamma.

BACKGROUND: Interferon-gamma (IFN-γ) is an important mediator of type I immune response and has antiviral, immunoregulatory and anti-tumor properties, plays a wide range of roles in inflammation and autoimmune diseases. The aim of this study was to obtain monoclonal antibody (mAb) against caprine IFN-γ by immunizing of BALB/c mice with the purified rIFN-γ. RESULTS: Recombinant caprine IFN-γ was expressed in Escherichia coli strain BL21 (DE3) and monoclonal antibodies against caprine IFN-γ were produced by immunizing of BALB/c mice with rIFN-γ. One hybridoma secreting mAb was screened by enzyme-linked immunosorbent assay (ELISA) which was designated as 2C. MAb secreted by this cell line were analyzed through ELISA, western blot and application of the mAb was evaluated by immunofluorescence analysis using goat lip tissues infected with Orf virus. ELISA analysis revealed that mAb 2C can specifically recognize rIFN-γ protein and culture supernatant of goat peripheral blood mononuclear cells (PBMCs) stimulated by concanavalin A (Con A) but cannot recognize the fusion tag protein of pET-32a. Western blot analysis showed that mAb 2C can specifically react with the purified 34.9 kDa rIFN-γ protein but does not react with the fusion tag protein of pET-32a. Immunofluorescence results demonstrated that mAb 2C can detect IFN-γ secreted in histopathological sites of goats infected with Orf virus. CONCLUSIONS: A caprine IFN-γ-specific mAb was successfully developed in this study. Further analyses showed that the mAb can be used to detect IFN-γ expression level during contagious ecthyma in goats.

Preparation of a novel monoclonal antibody against caprine interleukin-17A and its applications in immunofluorescence and immunohistochemistry assays.

BACKGROUND: Interleukin-17 (IL-17), the characteristic cytokine secreted by T helper 17 lymphocytes (Th17 cells), plays a pivotal role in host defense and many inflammatory or autoimmune diseases. The aim of this study was to obtain purified protein caprine IL-17A (cIL-17A) as an antigen for preparing an IL-17A-specific monoclonal antibody (mAb). RESULTS: The coding sequence (CDS) region of cIL-17A was cloned from the peripheral blood mononuclear cells (PBMCs) of dairy goats and then inserted into the expression vector PET 32a and transformed into competent TransB (DE3) cells. Recombinant fusion protein obtained under optimized conditions was used to immunize BALB/c mice for preparing monoclonal antibodies. Finally, the supernatants of two hybridoma cell lines showing positive reaction with the recombinant fusion protein and negative reaction with fusion tags of PET 32a were collected for western blot, immunofluorescence (IF) and immunohistochemistry (IHC) analysis. Our results showed that the maximum amount of soluble protein could be obtained directly in the supernatant when the recombinant expression cells were induced by isopropyl-ß-d-thiogalactoside (IPTG) at a concentration of 0.3 mmol/L at 16 °C for 42 h. Western blot analysis showed that the mAb H8 could recognize the eukaryotically expressed cIL-17A in the supernatant of transfected HEK293T cells. Immunofluorescence and immunohistochemistry assays showed that mAb H8 could strongly recognize both the eukaryotically expressed and natural cIL-17A. CONCLUSIONS: The monoclonal antibody mAb H8 prepared in this study may be a potential tool for the detection of cIL-17A and beneficial for investigating the pathogenesis of various IL-17-associated diseases.

Immunological abnormalities in patients with primary biliary cholangitis.

Primary biliary cholangitis (PBC), an autoimmune liver disease occurring predominantly in women, is characterized by high titers of serum anti-mitochondrial antibodies (AMAs) and progressive intrahepatic cholestasis. The immune system plays a critical role in PBC pathogenesis and a variety of immune cell subsets have been shown to infiltrate the portal tract areas of patients with PBC. Amongst the participating immune cells, CD4 T cells are important cytokine-producing cells that foster an inflammatory microenvironment. Specifically, these cells orchestrate activation of other immune cells, including autoreactive effector CD8 T cells that cause biliary epithelial cell (BEC) injury and B cells that produce large quantities of AMAs. Meanwhile, other immune cells, including dendritic cells (DCs), natural killer (NK) cells, NKT cells, monocytes, and macrophages are also important in PBC pathogenesis. Activation of these cells initiates and perpetuates bile duct damage in PBC patients, leading to intrahepatic cholestasis, hepatic damage, liver fibrosis, and eventually cirrhosis or even liver failure. Taken together, the body of accumulated clinical and experimental evidence has enhanced our understanding of the immunopathogenesis of PBC and suggests that immunotherapy may be a promising treatment option. Herein, we summarize current knowledge regarding immunological abnormalities of PBC patients, with emphasis on underlying pathogenic mechanisms. The differential immune response which occurs over decades of disease activity suggests that different therapies may be needed at different stages of disease.

MOLECULAR CLONING, SEQUENCING, EXPRESSION AND BIOLOGICAL ACTIVITY OF GIANT PANDA (AILUROPODA MELANOLEUCA) INTERFERON-GAMMA.

The giant panda (Ailuropoda melanoleuca) is an endangered species and indigenous to China. Interferon-gamma (IFN-γ) is the only member of type □ IFN and is vital for the regulation of host adapted immunity and inflammatory response. Little is known aboutthe FN-γ gene and its roles in giant panda.In this study, IFN-γ gene of Qinling giant panda was amplified from total blood RNA by RT-CPR, cloned, sequenced and analysed. The open reading frame (ORF) of Qinling giant panda IFN-γ encodes 152 amino acidsand is highly similar to Sichuan giant panda with an identity of 99.3% in cDNA sequence. The IFN-γ cDNA sequence was ligated to the pET32a vector and transformed into E. coli BL21 competent cells. Expression of recombinant IFN-γ protein of Qinling giant panda in E. coli was confirmed by SDS-PAGE and Western blot analysis. Biological activity assay indicated that the recombinant IFN-γ protein at the concentration of 4-10 µg/ml activated the giant panda peripheral blood lymphocytes,while at 12 µg/mlinhibited. the activation of the lymphocytes.These findings provide insights into the evolution of giant panda IFN-γ and information regarding amino acid residues essential for their biological activity.

High prevalence of Eimeria infection in dairy goats in Shaanxi province, northwestern China.

A survey of dairy goats for infection with Eimeria species of coccidia was conducted in the Shaanxi province, northwestern China between December and November 2010, including Saanen and Guanzhong breeds. A total of 584 fecal samples (250 and 334 from Saanen and Guanzhong dairy goats, respectively) in six farms were collected. Eimeria oocysts were seen in 568 (97.3%) fecal samples, with six species, namely Eimeria jolchijevi, Eimeria arloingi, Eimeria alijevi, Eimeria caprina, Eimeria hirci, and Eimeria christenseni. The most prevalent were E. arloingi in Saanen and Guanzhong dairy goats, with an overall prevalence of 83.3% and 84.4%, and the lowest prevalence were E. christenseni (26.9%) and E. hirci (20.7%) for Saanen and Guanzhong Dairy goats, respectively. Two or more Eimeria species were commonly presented in all the age groups; 80.0% and 81.4% of positive Saanen and Guanzhong dairy goats carried more than two species, and 1.6% and 6.5% of two breeds had six species. The results of the present survey suggested that Eimeria infection is wide and severe in the Saanen and Guanzhong dairy goats, which suggested that integrated strategies should be implemented to prevent and control coccidial infection in dairy goats in this province.

Interleukin-17 mediates lung injury by promoting neutrophil accumulation during the development of contagious caprine pleuropneumonia.

Contagious caprine pleuropneumonia (CCPP) is a highly contagious infectious disease of goats caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp). CCPP outbreaks usually result in high morbidity and mortality of the affected goats, making this disease a major cause of economic losses to goat producers globally. However, the pathogenesis of CCPP remains unclear. Here, we show that IL-17-driven neutrophil accumulation is involved in the lung damage in CCPP goats. During CCPP development, intense inflammatory infiltrates could be observed in the injured lungs. Specifically, neutrophils were observed to be present within the alveoli. Increased IL-17 release drove the excessive influx of neutrophils into the lung, as IL-17 effectively stimulated the production of neutrophil chemoattractants from lung epithelial cells following Mccp infection. Our data highlight a critical role of IL-17-driven neutrophil accumulation in the pathogenesis of CCPP and suggest that IL-17 may potentially be a useful immunotherapeutic target for the treatment of CCPP.

Compositional and Functional Comparisons of the Microbiota in the Colostrum and Mature Milk of Dairy Goats.

Goat milk is essential for the initial development of kids by providing a great source of commensal bacteria. In this study, we analyzed the microbiota of the milk of 30 healthy Saanen dairy goats. The 30 samples comprised 15 colostrum and 15 mature milk samples, collected from three different farms of Shaanxi Province. Colostrum samples were collected daily for five days post-delivery and mature milk was collected on the 7th, 10th, 20th, 30th, and 40th days. The result showed that microbial alpha diversity was higher in the mature milk compared with that in the colostrum. Linear discriminant analysis effect size (LEfSe) was performed to detect differentially abundant taxa in colostrum and goat milk. According to taxonomy results, Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes were the predominant bacteria phyla in both colostrum and mature milk. In addition, lactation stage noticeably influenced the composition of milk microbiota. Specifically, Novosphingobium, Brachybacterium, Psychrobacter, Lactobacillus, Yersinia, Roseateles, Rothia, Sanguibacter, Cloacibacterium, Variovorax, Sphingobacterium, and Coxiella were enriched in the colostrum, while Georgenia, Peptostreptococcus, Bacteroidales, Yaniella, Planomicrobium, Cloacibacterium, Azospirillum, Turicibacter, Cupriavidus, Herbaspirillum, Rhodobacteraceae, and Aeromonadales were the dominant genera in the mature milk. The enriched metabolic functions of the goat milk microbiota were predicted by PICRUSt and classified by KEGG pathway. Moreover, the abundances of environmental information processing, cellular processes pathway, genetic information processing pathway, organismal systems pathway, and metabolism pathway were significantly different between microbiota of colostrum and mature milk. Altogether, our study disclosed the significant difference between the microbial communities of colostrum and mature milk and provided grounds for further research in dairy microbiology.

The Role of Monocytes and Macrophages in Autoimmune Diseases: A Comprehensive Review.

Monocytes (Mo) and macrophages (Mϕ) are key components of the innate immune system and are involved in regulation of the initiation, development, and resolution of many inflammatory disorders. In addition, these cells also play important immunoregulatory and tissue-repairing roles to decrease immune reactions and promote tissue regeneration. Several lines of evidence have suggested a causal link between the presence or activation of these cells and the development of autoimmune diseases. In addition, Mo or Mϕ infiltration in diseased tissues is a hallmark of several autoimmune diseases. However, the detailed contributions of these cells, whether they actually initiate disease or perpetuate disease progression, and whether their phenotype and functional alteration are merely epiphenomena are still unclear in many autoimmune diseases. Additionally, little is known about their heterogeneous populations in different autoimmune diseases. Elucidating the relevance of Mo and Mϕ in autoimmune diseases and the associated mechanisms could lead to the identification of more effective therapeutic strategies in the future.

The protective and pathogenic roles of IL-17 in viral infections: friend or foe?

Viral infections cause substantial human morbidity and mortality, and are a significant health burden worldwide. Following a viral infection, the host may initiate complex antiviral immune responses to antagonize viral invasion and replication. However, proinflammatory antiviral immune responses pose a great threat to the host if not properly held in check. Interleukin (IL)-17 is a pleiotropic cytokine participating in a variety of physiological and pathophysiological conditions, including tissue integrity maintenance, cancer progression, autoimmune disease development and, more intriguingly, infectious diseases. Abundant evidence suggests that while IL-17 plays a crucial role in enhancing effective antiviral immune responses, it may also promote and exacerbate virus-induced illnesses. Accumulated experimental and clinical evidence has broadened our understanding of the seemingly paradoxical role of IL-17 in viral infections and suggests that IL-17-targeted immunotherapy may be a promising therapeutic option. Herein, we summarize current knowledge regarding the protective and pathogenic roles of IL-17 in viral infections, with emphasis on underlying mechanisms. The various and critical roles of IL-17 in viral infections necessitate the development of therapeutic strategies that are uniquely tailored to both the infectious agent and the infection environment.

Interactions Between the Gut Microbiota and the Host Innate Immune Response Against Pathogens.

The mammalian intestine is colonized by over a trillion microbes that comprise the "gut microbiota," a microbial community which has co-evolved with the host to form a mutually beneficial relationship. Accumulating evidence indicates that the gut microbiota participates in immune system maturation and also plays a central role in host defense against pathogens. Here we review some of the mechanisms employed by the gut microbiota to boost the innate immune response against pathogens present on epithelial mucosal surfaces. Antimicrobial peptide secretion, inflammasome activation and induction of host IL-22, IL-17, and IL-10 production are the most commonly observed strategies employed by the gut microbiota for host anti-pathogen defense. Taken together, the body of evidence suggests that the host gut microbiota can elicit innate immunity against pathogens.

Methods for Enhancing Clustered Regularly Interspaced Short Palindromic Repeats/Cas9-Mediated Homology-Directed Repair Efficiency.

The evolution of organisms has provided a variety of mechanisms to maintain the integrity of its genome, but as damage occurs, DNA damage repair pathways are necessary to resolve errors. Among them, the DNA double-strand break repair pathway is highly conserved in eukaryotes, including mammals. Nonhomologous DNA end joining and homologous directed repair are two major DNA repair pathways that are synergistic or antagonistic. Clustered regularly interspaced short palindromic repeats genome editing techniques based on the nonhomologous DNA end joining repair pathway have been used to generate highly efficient insertions or deletions of variable-sized genes but are error-prone and inaccurate. By combining the homology-directed repair pathway with clustered regularly interspaced short palindromic repeats cleavage, more precise genome editing via insertion or deletion of the desired fragment can be performed. However, homologous directed repair is not efficient and needs further improvement. Here, we describe several ways to improve the efficiency of homologous directed repair by regulating the cell cycle, expressing key proteins involved in homologous recombination and selecting appropriate donor DNA.

Pathogenicity of blood orf virus isolates in the development of dairy goat contagious pustular dermatitis.

Contagious pustular dermatitis is an exanthematous zoonotic disease caused by the orf virus. Pandemic outbreaks of this disease cause great economic losses, while the pathogenesis of this disease still remains obscure. In this study, blood samples were collected from 628 asymptomatic goats across China for PCR-based virus detection. We detected the orf virus in the blood of asymptomatic goats. Moreover, the orf virus obtained from the blood of infected goats was infectious and induced typical symptoms of contagious pustular dermatitis after inoculation of uninfected dairy goats. In summary, our data provide evidence that asymptomatic animals may be carriers of orf virus. Our findings should contribute to elucidating the details underlying the pathogenesis of contagious pustular dermatitis.

Seroprevalence of Toxoplasma gondii infection in dairy goats in Shaanxi Province, Northwestern China.

BACKGROUND: Toxoplasma gondii is an important zoonotic pathogen causing significant human and animal health problems. Infection in dairy goats not only results in significant reproductive losses, but also represents an important source of human infection due to consumption of infected meat and milk. In the present study we report for the first time seroprevalence of T. gondii infection in Guanzhong and Saanen dairy goats in Shaanxi province, Northwestern China. RESULTS: Sera from 751 dairy goats from 9 farms in 6 counties were examined for T. gondii antibodies with an indirect haemagglutination (IHA) test. Antibodies to T. gondii were detected in 106 (14.1%) serum samples, with antibody titres ranging from 1:64 to 1:1024. Seropositive goats were found in all 9 farms and seroprevalences in Guanzhong (16.3%, 75/461) and Saanen (10.7%, 31/290) dairy goats were not statistically significantly different. All the factors (sex, age and location) reported in the present study affected prevalence of infection, and seroprevalence increased with age, suggesting postnatal acquisition of T. gondii infection. CONCLUSIONS: The results of the present survey indicate that infection by T. gondii is widely prevalent in dairy goats in Shaanxi province, Northwestern China, and this has implications for prevention and control of toxoplasmosis in this province.

[Study on the cloning, in vitro expression and bioactivity of Ailuropoda melanoleuca Qinling subspecies interleukin-2].

AIM: To identify if any difference exisit between the population of Sichuan and Qinling giant panda. express IL-2 protein in BL21(DE3)with biological activity. METHODS: IL-2 was amplified by RT-PCR from adult Qinling giant panda peripheral blood lymphocyte which was induced by ConA, and cloned into prokaryotic expression vector of pET32a-IL-2. The fusion protein was expressed in BL21(DE3)/pET system and identified by SDS-PAGE and Western blot; inoculate rabbit with purified expression products to prepare polyclonal antibody; use lymphocytes proliferation in vitro to detect biological activity. RESULTS: The protein of IL-2 was obtained by recombination expression, molecule weight is 34 000 .The specificity of polyclonal antibody was obtained, in vitro activity test indicated that the recombinant protein IL-2 having an activity of promoting the proliferation of lymphocytes. The effect can be stopped by polyclonal antibody which was prepared before. CONCLUSION: The IL-2 gene of Qinling giant panda was cloned and expressed in E.coli successfully, and the homology of IL-2 gene in these two population is 99.4% and the recombination protein can promote the proliferation of lymphocytes in vitro from giant panda.

[Expression of xenogentic epidermal growth factor receptor ectodomain and its antitumor effect].

AIM: To construct the EGFR protein vaccine and evaluate its antitumor effect. METHODS: The gene of chicken EGFR ectodomain was amplified by PCR, inserted into the pGEX-4T-2 vetor, and then exporessed in E.coli BL21 with glutathione S-transferase in a fusion protein form. The fusion protein was purified by metal affinity chromatography, and refolded by dialysis.Then the mice were immunized with the fusion protein three times. After that, these mice were vaccinated with Lewis cells. At last the growth of tumors was measured and the surm antibody response was measured by ELISA. RESULTS: The ectodomain gene of EGFR was ligated into prokaryotic expression vetor. The expression of the fusion protein was analyzed by SDS-PAGE. The relative moleclar mass of the protein is about M(r); 50,000. After the third immunization, all the mice immunized with the fusion protein showed an antibody response towards the EGFR protein. Compared with the control group, the fusion protein group showed good anti-tumor effects. CONCLUSION: The xenogentic EGFR protein vaccine can overcome the host's immune tolerance problem and induce the production of specific antibodies against.