Search results for: bovine macrophages

Authors: Qiaofeng Guo

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Aims: Obesity is often accompanied by insulin resistance (IR) and whole-body inflammation. Aerobic exercise is an effective treatment to improve insulin resistance and inflammation. However, the anti-inflammatory mechanisms of exercise on epididymal and subcutaneous adipose remain to be elucidated. Here, we compared the macrophage polarization between epididymal and subcutaneous adipose after aerobic exercise. Methods: Male C57BL/6 mice were fed a normal diet group or a high-fat diet group for 12 weeks and performed aerobic training on a treadmill at 55%~65% VO₂ max for eight weeks. Food intake, body weight, and fasting blood glucose levels were monitored weekly. The intraperitoneal glucose tolerance test was to evaluate the insulin resistance model. Fat mass, blood lipid profile, serum IL-1β, TNF-α levels, and CD31/CD206 rates were analysed after the intervention. Results: FBG (P<0.01), AUCIPGTT (P<0.01), and HOMA-IR (P<0.01) increased significantly for a high-fat diet and decreased significantly after the exercise. Eight weeks of aerobic exercise attenuated HFD-induced weight gain and glucose intolerance and improved insulin sensitivity. Serum IL-1β, TNF-α, CD11C/CD206 expression in subcutaneous adipose tissue were not changed before and after exercise, but not in epididymal adipose tissue (P<0.01). Conclusion: Insulin resistance is not accompanied by chronic inflammation and M1 polarization of subcutaneous adipose tissue macrophages in high-fat diet mice. Aerobic exercise effectively improved lipid metabolism and insulin sensitivity, which may be closely associated with reduced M1 polarization of epididymal adipose macrophages.

Keywords: aerobic exercise, insulin resistance, chronic inflammation, adipose, macrophage polarization

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Authors: Aqeela Ashraf, Muhammad Imran, Tahir Yaqub, Muhammad Tayyab, Yung Fu Chang

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For the identification of bovine mastitic pathogen, an economical, rapid and sensitive molecular diagnostic assay is developed by PCR multiplexing of gene and pathogenic species specific DNA sequences. The multiplex PCR assay is developed for detecting nine important bacterial pathogens causing mastitis Worldwide. The bacterial species selected for this study are Streptococcus agalactiae, Streptococcus dysagalactiae, Streptococcus uberis, Staphylococcus aureus, Escherichia coli, Staphylococcus haemolyticus, Staphylococcus chromogenes Mycoplasma bovis and Staphylococcus epidermidis. A single reaction assay was developed and validated by 27 reference strains and further tested on 276 bacterial strains obtained from culturing mastitic milk. The multiplex PCR assay developed here is further evaluated by applying directly on genomic DNA isolated from 200 mastitic milk samples. It is compared with bacterial culturing method and proved to be more sensitive, rapid, economical and can specifically identify 9 bacterial pathogens in a single reaction. It has detected the pathogens in few culture negative mastitic samples. Recognition of disease is the foundation of disease control and prevention. This assay can be very helpful for maintaining the udder health and milk monitoring.

Keywords: multiplex PCR, bacteria, mastitis, milk

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Authors: Yi-Hui Lai, Chih-Hsiang Yang, Li-Chung Hsu

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The inflammasome is a protein complex that modulates caspase-1 activity, resulting in proteolytic cleavage of proinflammatory cytokines such as IL-1β and IL-18, into their bioactive forms. It has been shown that the inflammasomes play a crucial role in the clearance of pathogenic infection and tissue repair. However, dysregulated inflammasome activation contributes to a wide range of human diseases such as cancers and auto-inflammatory diseases. Yet, regulation of NLRP3 inflammasome activation remains largely unknown. We discovered a novel DEAD box protein, whose biological function has not been reported, not only negatively regulates NLRP3 inflammasome activation by interfering NLRP3 inflammasome assembly and cellular localization but also mitigate pyroptosis upon pathogen evasion. The DEAD-box protein is the first DEAD-box protein gets involved in modulation of the inflammasome activation. In our study, we found that caspase-1 activation and mature IL-1β production were largely enhanced upon LPS challenge in the DEAD box-containing protein- deleted THP-1 macrophages and bone marrow-derived macrophages (BMDMs). In addition, this DEAD box-containing protein migrates from the nucleus to the cytoplasm upon LPS stimulation, which is required for its inhibitory role in NLRP3 inflammasome activation. The DEAD box-containing protein specifically interacted with the LRR motif of NLRP3 via its DEAD domain. Furthermore, due to the crucial role of the NLRP3 LRR domain in the recruitment of NLRP3 to mitochondria and binding to its adaptor ASC, we found that the interaction of NLRP3 and ASC was downregulated in the presence of the DEAD box-containing protein. In addition to the mechanical study, we also found that this DEAD box protein protects host cells from inflammasome-triggered cell death in response to broad-ranging pathogens such as Candida albicans, Streptococcus pneumoniae, etc., involved in nosocomial infections and severe fever shock. Collectively, our results suggest that this novel DEAD box molecule might be a key therapeutic strategy for various infectious diseases.

Keywords: inflammasome, inflammation, innate immunity, pyroptosis

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Authors: Holm Zerbe, Laura Macias, Hans-Joachim Schuberth, Wolfram Petzl

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Mastitis is among the most important production diseases in cows. It accounts for large parts of antimicrobial drug use in the dairy industry worldwide. Due to the imminent normative to reduce the use of antimicrobial drugs in livestock, new ways for therapy and prophylaxis of mastitis are needed. Recently epigenetic regulation of inflammation by chromatin modifications has increasingly drawn attention. Currently, some epigenetic modifiers have already been approved for the use in humans, however little is known about their actions in the bovine system. The aim of our study was to investigate whether three selected epigenetic modifiers (Vitamin D3, SAHA and S2101) influence the initial immune response towards mastitis pathogens in bovine udder tissue in vitro. Tissue explants of the teat cistern and udder parenchyma were collected from 21 cows and were incubated for 36 hours in the absence and presence of epigenetic modifiers. Additionally, the tissue was stimulated with heat-inactivated particles of Escherichia coli and Staphylococcus aureus, which are regarded as two of the most important mastitis pathogens. After incubation, the explants were tested by RT-qPCR for transcript abundances of immune-related candidate genes. Gene expression was validated in culture supernatants by an AlphaLISA assay. Furthermore, the culture supernatants were analyzed for their chemotactic capacity through a chemotaxis assay. Statistical analysis of data was performed with the program ‘R’ version 3.2.3. Vitamin D3 had no effect on the immune response of udder tissue in vitro after stimulation with mastitis pathogens. The epigenetic modifiers SAHA and S2101 however significantly blocked the pathogen-induced upregulation of CXCL8, TNFα, S100A9 and LAP (P < 0.05). The regulation of IL10 was not affected by treatment with SAHA and S2101. Transcript abundances for CXCL8 were reflected by IL8 contents and chemotactic activity in culture supernatants. In conclusion, these data show the potential of epigenetic modifiers (SAHA and S2101) to block overshooting inflammation in the udder. Thus epigenetic modifiers may serve in future as immune modulators for the treatment and/or prophylaxis of clinical mastitis. (Funded by Deutsche Forschungsgemeinschaft PE 1495/2-1).

Keywords: mastitis, cattle, epigenetics, immunomodulation

Procedia PDF Downloads 196

Authors: Lien Gysens, Bert Vanmechelen, Maarten Haspeslagh, Piet Maes, Ann Martens

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Bovine papillomavirus (BPV) types 1 and 2 play a central role in the etiology of the most common neoplasm in horses, the equine sarcoid. The unknown mechanism behind the unique variety in a clinical presentation on the one hand and the host-dependent clinical outcome of BPV-1 infection, on the other hand, indicate the involvement of additional factors. Earlier studies have reported the potential functional significance of intratypic sequence variants, along with the existence of sarcoid-sourced BPV variants. Therefore, intratypic sequence variation seems to be an important emerging viral factor. This study aimed to give a broad insight in sarcoid-sourced BPV variation and explore its potential association with disease presentation. In order to do this, a nanopore sequencing approach was successfully optimized for screening a wide spectrum of clinical samples. Specimens of each tumour were initially screened for BPV-1/-2 by quantitative real-time PCR. A custom-designed primer set was used on BPV-positive samples to amplify the complete viral genome in two multiplex PCR reactions, resulting in a set of overlapping amplicons. For phylogenetic analysis, separate alignments were made of all available complete genome sequences for BPV-1/-2. The resulting alignments were used to infer Bayesian phylogenetic trees. We found substantial genetic variation among sarcoid-derived BPV-1, although this variation could not be linked to disease severity. Several of the BPV-1 genomes had multiple major deletions. Remarkably, the majority of the cluster within the region coding for late viral genes. Together with the extensiveness (up to 603 nucleotides) of the described deletions, this suggests an altered function of L1/L2 in disease pathogenesis. By generating a significant amount of complete-length BPV genomes, we succeeded in introducing next-generation sequencing into veterinary research focusing on the equine sarcoid, thus facilitating the first report of both nanopore-based sequencing of complete sarcoid-sourced BPV-1/-2 and the simultaneous nanopore sequencing of multiple complete genomes originating from a single clinical sample.

Keywords: Bovine papillomavirus, equine sarcoid, horse, nanopore sequencing, phylogenetic analysis

Procedia PDF Downloads 146

Authors: Gina Leisching, Ray-Dean Pietersen, Carel Van Heerden, Paul Van Helden, Ian Wiid, Bienyameen Baker

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The distinguishing factors that characterize the host response to infection with virulent Mycobacterium tuberculosis (M.tb) are largely confounding. We present an infection study with two genetically closely related M.tb strains that have vastly different pathogenic characteristics. The early host response to infection with these detergent-free cultured strains was analyzed through RNAseq in an attempt to provide information on the subtleties which may ultimately contribute to the virulent phenotype. Murine bone marrow-derived macrophages (BMDMs) were infected with either a hyper- (R5527) or hypovirulent (R1507) Beijing M. tuberculosis clinical isolate. RNAseq revealed 69 differentially expressed host genes in BMDMs during comparison of these two transcriptomes. Pathway analysis revealed activation of the stress-induced and growth inhibitory Gadd45 signaling pathway in hypervirulent infected BMDMs. Upstream regulators of interferon activation such as and IRF3 and IRF7 were predicted to be upregulated in hypovirulent-infected BMDMs. Additional analysis of the host immune response through ELISA and qPCR included the use of human THP-1 macrophages where a robust proinflammatory response was observed after infection with the hypervirulent strain. RNAseq revealed two early-response genes (IER3 and SAA3) and two host-defence genes (OASL1 and SLPI) that were significantly upregulated by the hypervirulent strain. The role of these genes under M.tb infection conditions are largely unknown but here we provide validation of their presence with use of qPCR and Western blot. Further analysis into their biological role under infection with virulent M.tb is required.

Keywords: host-response, Mycobacterium tuberculosis, RNAseq, virulence

Procedia PDF Downloads 183

Authors: Yasser Y. Lenis, Daniela Garcia Gonzalez, Cristian Solarte Bacca, Diego F. Carrillo González, Amy Jo Montgomery, Dursun Barrios

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Introduction: Artificial insemination in bovines helps to promote genetic improvement and can positively impact the rural economy. The Colombian armed conflict has forced a large portion of the rural population to abandon their territory, affecting their education, family integration, and economics. Justification: The achievement of education in rural populations was one of the Millennium Development Goals (MDGs) made by the United Nations. During the last World Summit on Sustainable Development (WSSD), it was concluded that most of the world’s poor, illiterate and undernourished population lives in rural areas; therefore, access to education is considered one of the most significant challenges for governments in countries with developing economies. Objectives: To study the effects of training in artificial insemination and rural management on the perception of knowledge and the level of knowledge in rural residents affected by the armed conflict in Nariño, Colombia. Methods: The perception of knowledge and the theoretical-practical knowledge of 63 rural residents were evaluated on the topics of bovine agribusiness management, artificial insemination, and genetic improvement through the application of three surveys. 1) evaluated the perceived level of knowledge each rural resident had about each topic using the Likert scale, 2) evaluated the theoretical knowledge before training, and 3) evaluated the theoretical knowledge upon completion of training. Results/discussion: Of the surveyed rural residents, 54% stated that they knew how business management improved the performance of their bovine agribusiness, 54% answered the pre-training knowledge test correctly, while 83% correctly answered the post-training knowledge test. Only 6% of surveyed residents perceived that they had prior knowledge of artificial insemination and reproductive anatomy topics. Before training, 35% of surveyed residents answered correctly on these topics, while upon completion of training, 65% answered correctly. Regarding genetic improvement, 11% of participating rural residents stated that they knew this subject. The correct answers on this topic went from 57% to 89% before and post-training. Conclusion: Rural extension programs contribute to closing knowledge gaps in relation to the use of reproductive biotechnologies and bovine management in rural areas affected by armed conflict.

Keywords: agribusiness, insemination, knowledge, reproduction

Procedia PDF Downloads 139

Authors: Homa Torabizadeh, Mohaddeseh Mikani

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Inulinase from Aspergillus niger was covalently immobilized on magnetic nanoparticles (MNPs/Fe₃O₄) covered with soy protein isolate (SPI/Fe₃O₄) functionalized by bovine serum albumin (BSA) nanoparticles. MNPs are promising enzyme carriers because they separate easily under external magnetic fields and have enhanced immobilized enzyme reusability. As MNPs aggregate simply, surface coating strategy was employed. SPI functionalized by BSA was a suitable candidate for nanomagnetite coating due to its superior biocompatibility and hydrophilicity. Fe₃O₄@SPI-BSA nanoparticles were synthesized as a novel carrier with narrow particle size distribution. Step by step fabrication monitoring of Fe₃O₄@SPI-BSA nanoparticles was performed using field emission scanning electron microscopy and dynamic light scattering. The results illustrated that nanomagnetite with the spherical morphology was well monodispersed with the diameter of about 35 nm. The average size of the SPI-BSA nanoparticles was 80 to 90 nm, and their zeta potential was around −34 mV. Finally, the mean diameter of fabricated Fe₃O₄@SPI-BSA NPs was less than 120 nm. Inulinase enzyme from Aspergillus niger was covalently immobilized through gluteraldehyde on Fe₃O₄@SPI-BSA nanoparticles successfully. Fourier transform infrared spectra and field emission scanning electron microscopy images provided sufficient proof for the enzyme immobilization on the nanoparticles with 80% enzyme loading.

Keywords: high fructose syrup, inulinase immobilization, functionalized magnetic nanoparticles, soy protein isolate

Procedia PDF Downloads 263

Authors: Sukwoong Kang

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Background: The application of light-emitting diode (LED)-dependent photobiomodulation (PBM) in promoting post-tendon injury healing has been recently reported. Despite the establishment of a theoretical basis for ligament restoration through PBM, the lack of any empirical evidence deems this therapeutic strategy contentious. Therefore, the aim of this study was to investigate the potency of LED-based PBM in facilitating tendon healing in a murine model. Methods: Migration kinetics were analyzed at two specific wavelengths: 630 and 880 nm. The Achilles tendon in the hind limbs of Balb/c mice was severed via Achilles tendon transection. Subsequently, the mice were randomized into LED non-irradiation and LED irradiation groups. Mice with intact tendons were employed as healthy controls. The wounds were LED-irradiated for 20 min daily for two days. Histological properties, tendon healing mediators, and inflammatory mediators were screened on day 14. Results: The roundness of the nuclei and fiber structure, indicating the degree of infiltrated inflammatory cells and severity of fiber fragmentation, respectively, were considerably lower in the LED irradiation group than in the LED non-irradiation group. Immunohistochemical analysis depicted an increase in tenocytes (SCX+ cells) and a recovery of wounds with reduced fibrosis (lower collagen 3 and TGF-β1) in the LED irradiation group during healing; conversely, the LED non-irradiation group exhibited tissue fibrosis. The ratio of M2 macrophages to total macrophages was higher in the LED irradiation group than in the injured group. Conclusion: LED-based PBM in the Achilles tendon rupture murine model effectuated a rapid restoration of histological and immunochemical outcomes. The aforementioned findings suggest that LED-based PBM presents remarkable potential as an adjunct therapeutic for tendon healing and warrants further research to standardize various parameters to advance and establish it as a reliable treatment regime.

Keywords: photobiomodulation, light-emitting diode, tendon, regeneration

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Authors: Florian Billing, Soren Segan, Meike Jakobi, Elsa Arefaine, Aliki Jerch, Xin Xiong, Matthias Becker, Thomas Joos, Burkhard Schlosshauer, Ulrich Rothbauer, Nicole Schneiderhan-Marra, Hanna Hartmann, Christopher Shipp

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The immune response plays a major role in implant biocompatibility, but an understanding of how to design biomaterials for specific immune responses is yet to be achieved. We aimed to better understand how changing certain material properties can drive immune responses. To this end, we tested immune response to experimental implant coatings that vary in specific characteristics. A layer-by-layer approach was employed to vary surface charge and wettability. Human-based in vitro models (THP-1 macrophages and primary peripheral blood mononuclear cells (PBMCS)) were used to assess immune responses using multiplex cytokine analysis, flow cytometry (CD molecule expression) and microscopy (cell morphology). We observed dramatic differences in immune response due to specific alterations in coating properties. For example altering the surface charge of coating A from anionic to cationic resulted in the substantial elevation of the pro-inflammatory molecules IL-1beta, IL-6, TNF-alpha and MIP-1beta, while the pro-wound healing factor VEGF was significantly down-regulated. We also observed changes in cell surface marker expression in relation to altered coating properties, such as CD16 on NK Cells and HLA-DR on monocytes. We furthermore observed changes in the morphology of THP-1 macrophages following cultivation on different coatings. A correlation between these morphological changes and the cytokine expression profile is ongoing. Targeted changes in biomaterial properties can produce vast differences in immune response. The properties of the coatings examined here may, therefore, be a method to direct specific biological responses in order to improve implant biocompatibility.

Keywords: biomaterials, coatings, immune system, implants

Procedia PDF Downloads 151

Authors: Nabeeha Hassan Abdel Jalil, Lulwa Saeed Al Badi, Mouza Ghafan Alkhyeli, Khaja Mohteshamuddin, Ahmad Al Aiyan, Mohamed Elfatih Hamad, Robert Barigye

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The present study was done to elucidate the prevalence of enzootic bovine leucosis (EBL) in the UAE, the seroprevalence rates of EBL in dairy herds from the Al Ain area, Abu Dhabi (AD) and indigenous cattle at the Al Ain livestock market (AALM) were assessed. Of the 949 sera tested by ELISA, 657 were from adult Holstein-Friesians from three farms and 292 from indigenous cattle at the AALM. The level of significance between the proportions of seropositive cattle were analyzed by the Marascuilo procedure and questionnaire data on husbandry and biosecurity practices evaluated. Overall, the aggregated farm and AALM data demonstrated a seroprevalence of 25.9%, compared to 37.0% for the study farms, and 1.0% for the indigenous cattle. Additionally, the seroprevalence rates at farms #1, #2 and #3 were 54.7%, 0.0%, and 26.3% respectively. Except for farm #2 and the AALM, statistically significant differences were noted between the proportions of seropositive cattle for farms #1 and #2 (Critical Range or CR=0.0803), farms #1 and #3 (p=0.1069), and farms #2 and #3 (CR=0.0707), farm #1 and the AALM (CR=0.0819), and farm #3 and the AALM (CR=0.0726). Also, the proportions of seropositive animals on farm #1 were 9.8%, 59.8%, 29.3%, and 1.2% in the 12-36, 37-72, 73-108, and 109-144-mo-old age groups respectively compared to 21.5%, 60.8%, 15.2%, and 2.5% in the respective age groups for farm #2. On both farms and the AALM, the 37-72-mo-old age group showed the highest EBL seroprevalence rate while all the 57 cattle on farm #2 were seronegative. Additionally, farms #1 and #3 had 3,130 and 2,828 intensively managed Holstein-Friesian cattle respectively, and all animals were routinely immunized against several diseases except EBL. On both farms #1 and #3, artificial breeding was practiced using semen sourced from the USA, and USA and Canada respectively, all farms routinely quarantined new stock, and farm #1 previously imported dairy cattle from an unspecified country, and farm #3 from the Netherlands, Australia and South Africa. While farm #1 provided no information on animal nutrition, farm #3 cited using hay, concentrates, and ad lib water. To the authors’ best knowledge, this is the first serological evidence of EBL in the UAE and as previously reported, the seroprevalence rates are comparatively higher in the intensively managed dairy herds than in indigenous cattle. As two of the study farms previously sourced cattle and semen from overseas, biosecurity protocols need to be revisited to avoid inadvertent EBL incursion and the possibility of regional transboundary disease spread also needs to be assessed. After the proposed molecular studies have adduced additional data, the relevant UAE animal health authorities may need to develop evidence-based EBL control policies and programs.

Keywords: cattle, enzootic bovine leukosis, seroprevalence, UAE

Procedia PDF Downloads 111

Authors: Hosein Maghsoudi

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Rheumatois arthritis (RA) is progressive inflammatory autoimmune diseases that primarily affect the joints, characterized by synovial hyperplasia and inflammatory cell infiltration, deformed and painful joints, which can lead tissue destruction, functional disability systemic complications, and early dead and socioeconomic costs. The cause of rheumatoid arthritis is unknown, but genetic and environmental factors are contributory and the prognosis is guarded. However, advances in understanding the pathogenesis of the disease have fostered the development of new therapeutics, with improved outcomes. The current treatment strategy, which reflects this progress, is to initiate aggressive therapy soon after diagnosis and to escalate the therapy, guided by an assessment of disease activity, in pursuit of clinical remission. The pathobiology of RA is multifaceted and involves T cells, B cells, fibroblast-like synoviocyte (FLSc) and the complex interaction of many pro-inflammatory cytokine. Novel biologic agents that target tumor necrosis or interlukin (IL)-1 and Il-6, in addition T- and B-cells inhibitors, have resulted in favorable clinical outcomes in patients with RA. Despite this, at least 30% of RA patients are résistance to available therapies, suggesting novel mediators should be identified that can target other disease-specific pathway or cell lineage. Among the inflammatory cell population that might participated in RA pathogenesis, FLSc are crucial in initiaing and driving RA in concert of cartilage and bone by secreting metalloproteinase (MMPs) into the synovial fluid and by direct invasion into extracellular matrix (ECM), further exacerbating joint damage. Invasion of fibroblast-like synoviocytes (FLSc) is critical in the pathogenesis of rheumatoid-arthritis. The metalloproteinase (MMPs) and activator of Toll-like receptor 4 (TLR4)/nuclear factor- κB pthway play a critical role in RA-FLS invasion induced by lipopolysaccharide (LPS). The present study aimed to explore the anti-invasion activity of Glycyrrhizic Acid as a pharmacologically safe phytochemical agent with potent anti-inflammatory properties on IL-1beta and TNF-alpha signalling pathways in Bovine fibroblast-like synoviocyte ex- vitro, on LPS-stimulated bovine FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Results showed that Glycyrrhizic Acid suppressed LPS-stimulated bovine FLS migration and invasion by inhibition MMP-9 expression and activity. In addition our results revealed that Glycyrrhizic Acid inhibited the transcriptional activity of MMP-9 by suppression the nbinding activity of NF- κB in the MMP-9 promoter pathway. The extract of licorice (Glycyrrhiza glabra L.) has been widely used for many centuries in the traditional Chinese medicine as native anti-allergic agent. Glycyrrhizin (GL), a triterpenoidsaponin, extracted from the roots of licorice is the most effective compound for inflammation and allergic diseases in human body. The biological and pharmacological studies revealed that GL possesses many pharmacological effects, such as anti-inflammatory, anti-viral and liver protective effects, and the biological effects, such as induction of cytokines (interferon-γ and IL-12), chemokines as well as extrathymic T and anti-type 2 T cells. GL is known in the traditional Chinese medicine for its anti-inflammatory effect, which is originally described by Finney in 1959. The mechanism of the GL-induced anti-inflammatory effect is based on different pathways of the GL-induced selective inhibition of the prostaglandin E2 production, the CK-II- mediated activation of both GL-binding lipoxygenas (gbLOX; 17) and PLA2, an anti-thrombin action of GL and production of the reactive oxygen species (ROS; GL exerts liver protection properties by inhibiting PLA2 or by the hydroxyl radical trapping action, leading to the lowering of serum alanine and aspartate transaminase levels. The present study was undertaken to examine the possible mechanism of anti-inflammatory properties GL on IL-1beta and TNF-alpha signalling pathways in bovine fibroblast-like synoviocyte ex-vivo, on LPS-stimulated bovine FLS migration and invasion as well as MMP expression and explored the upstream signal transduction. Our results clearly showed that treatment of bovine fibroblast-like synoviocyte with GL suppressed LPS-induced cell migration and invasion. Furthermore, it revealed that GL inhibited the transcription activity of MMP-9 by suppressing the binding activity of NF-κB in the MM-9 promoter. MMP-9 is an important ECM-degrading enzyme and overexpression of MMPs in important of RA-FLSs. LPS can stimulate bovine FLS to secret MMPs, and this induction is regulated at the transcription and translational levels. In this study, LPS treatment of bovine FLS caused an increase in MMP-2 and MMP-9 levels. The increase in MMP-9 expression and secretion was inhibited by ex- vitro. Furthermore, these effects were mimicked by MMP-9 siRNA. These result therefore indicate the the inhibition of LPS-induced bovine FLS invasion by GL occurs primarily by inhibiting MMP-9 expression and activity. Next we analyzed the functional significance of NF-κB transcription of MMP-9 activation in Bovine FLSs. Results from EMSA showed that GL suppressed LPS-induced NF-κB binding to the MMP-9 promotor, as NF-κB regulates transcriptional activation of multiple inflammatory cytokines, we predicted that GL might target NF-κB to suppress MMP-9 transcription by LPS. Myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NF-κB and apoptotic signaling pathways, GL inhibited the expression of TLR4 and MYD88. These results demonstrated that GL suppress LPS-induced MMP-9 expression through the inhibition of the induced TLR4/NFκB signaling pathway. Taken together, our results provide evidence that GL exerts anti-inflammatory effects by inhibition LPS-induced bovine FLSs migration and invasion, and the mechanisms may involve the suppression of TLR4/NFκB –mediated MMP-9 expression. Although further work is needed to clarify the complicated mechanism of GL-induced anti-invasion of bovine FLSs, GL might be used as a further anti-invasion drug with therapeutic efficacy in the treatment of immune-mediated inflammatory disease such as RA.

Keywords: glycyrrhizic acid, bovine fibroblast-like synoviocyte, tlr4/nf-κb, metalloproteinase-9

Procedia PDF Downloads 359

Authors: Jihoon Yang, Jeong II Choi

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Mincle is expressed in macrophages and is members of immunoreceptors induced after exposure to various stimuli and stresses. Mincle receptor activation promotes the production of these substances by increasing the transcription of inflammatory cytokines and chemokines. Cytokines, which play an important role in the initiation and maintenance of such inflammatory pain diseases, have a significant effect on sensory neurons in addition to their enhancement and inhibitory effects on immune and inflammatory cells as mediators of cell interaction. Glial cells in the central nervous system play a critical role in development and maintenance of chronic pain states. Microglia are tissue-resident macrophages in the central nervous system, and belong to a group of mononuclear phagocytes. In the central nervous system, mincle receptor is present in neurons and glial cells of the brain.This study was performed to identify the Mincle receptor in the spinal cord and to investigate the effect of Mincle receptor activation on nociception and the changes of microglia. Materials and Methods: C-type lectins(Mincle) was identified in spinal cord of Male Sprague–Dawley rats. Then, mincle receptor ligand (TDB), via an intrathecal catheter. Mechanical allodynia was measured using von Frey test to evaluate the effect of intrathecal injection of TDB. Result: The present investigation shows that the intrathecal administration of TDB in the rat produces a reliable and quantifiable mechanical hyperalgesia. In addition, The mechanical hyperalgesia after TDB injection gradually developed over time and remained until 10 days. Mincle receptor is identified in the spinal cord, mainly expressed in neuronal cells, but not in microglia or astrocyte. These results suggest that activation of mincle receptor pathway in neurons plays an important role in inducing activation of microglia and inducing mechanical allodynia.

Keywords: mincle, spinal cord, pain, microglia

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Authors: Alazar Nebyou, Sujata Pandit

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Lactic acid bacteria (LAB) are essential ingredients in probiotic foods, intestinal microflora, and dairy products that are capable of coping up with harsh gastrointestinal tract conditions and are available in a variety of environments. The objective of this study is to evaluate the probiotic property of LAB isolated from bovine milk. Milk samples were collected from local dairy farms. Samples were obtained using sterile test tubes and transported to a laboratory in the icebox for further biochemical characterization. Preliminary physiological and biochemical identification of LAB isolates was conducted by growing on MRS agar after ten-fold serial dilution. Seven of the best isolates were selected for the evaluation of the probiotic property. The LAB isolates were checked for resistance to antibiotics and their antimicrobial activity by disc diffusion assay and agar well diffusion assay respectively. Bile salt hydrolase activity of isolates was studied by growing isolates in a BSH medium with bile salt. Cell surface property of isolates was assayed by studying their autoaggregation and coaggregation percentage with S. aerues. All isolates were found BSH positive. In addition, BCM2 and BGM1 were susceptible to all antibiotic disks except BBM1 which was resistant to all antibiotic disks. BCM1 and BGM1 had the highest autoaggregation and coaggregation potential respectively. Since all LAB isolates showed gastrointestinal tolerance and good cell surface property they could be considered as good potential probiotic candidates for treatment and probiotic starter culture preparation.

Keywords: probiotic, aggregation, lactic acid bacteria, antimicrobial activity

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Authors: Robnier Reyes, Andrew J. P. Marques, Joel Ramjist, Chris R. Pasarikovski, Victor X. D. Yang

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Over the past three decades, surgical robotic systems have demonstrated their ability to improve surgical outcomes. The LBR Med is a collaborative robotic arm that is meant to work with a surgeon to streamline surgical workflow. It has 7 degrees of freedom and thus can be easily oriented. Position and torque sensors at each joint allow it to maintain a position accuracy of 150 µm with real-time force and torque feedback, making it ideal for complex surgical procedures. Spinal fusion procedures involve the placement of as many as 20 pedicle screws, requiring a great deal of accuracy due to proximity to the spinal canal and surrounding vessels. Any deviation from intended path can lead to major surgical complications. Assistive surgical robotic systems are meant to serve as collaborative devices easing the workload of the surgeon, thereby improving pedicle screw placement by mitigating fatigue related inaccuracies. Moreover, robotic spinal systems have shown marked improvements over conventional freehanded techniques in both screw placement accuracy and fusion quality and have greatly reduced the need for screw revision, intraoperatively and post-operatively. However, current assistive spinal fusion robots, such as the ROSA Spine, are limited in functionality to positioning surgical instruments. While they offer a small degree of improvement in pedicle screw placement accuracy, they do not alleviate surgeon fatigue, nor do they provide real-time force and torque feedback during screw insertion. We propose a semi-autonomous surgical robot workflow for spinal fusion where the surgeon guides the robot to its initial position and orientation, and the robot drives the pedicle screw accurately into the vertebra. Here, we demonstrate feasibility by inserting pedicle screws into ex-vivo bovine rib bone. The robot monitors position, force and torque with respect to predefined values selected by the surgeon to ensure the highest possible spinal fusion quality. The workflow alleviates the strain on the surgeon by having the robot perform the screw placement while the ability to monitor the process in real-time keeps the surgeon in the system loop. The approach we have taken in terms of level autonomy for the robot reflects its ability to safely collaborate with the surgeon in the operating room without external navigation systems.

Keywords: ex vivo bovine bone, pedicle screw, surgical robot, surgical workflow

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Authors: Temesgen Kassa Getahun, Gezahegn Mamo, Beksisa Urge

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A cross-sectional study was conducted from December 2019 to May 2020 with the aim of determining the seroprevalence of brucellosis in dairy cows and their owners in the central highland of Oromia, Ethiopia. A total of 352 blood samples from dairy cattle, 149 from animal owners, and 17 from farm workers were collected and initially screened using the Rose Bengal Plate test and confirmed by the Complement Fixation test. Overall seroprevalence was 0.6% (95% CI: 0.0016–0.0209) in bovines and 1.2% (95% CI: 0.0032–0.0427) in humans. Market-based stock replacement (OR=16.55, p=0.002), breeding by artificial insemination (OR=7.58, p=0.05), and parturition pen (OR = 11.511, p=0.027) were found to be significantly associated with the seropositivity for Brucella infection in dairy cattle. Human housing (OR=1.8, p=0.002), contact with an aborted fetus (OR=21.19, p=0.017), drinking raw milk from non-aborted (OR=24.99, p=0.012), aborted (OR=5.72, p=0.019) and retained fetal membrane (OR=4.22, p=0.029) cows had a significant influence on human brucellosis. A structured interview question was administered to 284 respondents. Accordingly, most respondents had no knowledge of brucellosis (93.3%), and in contrast, 90% of them consumed raw milk. In conclusion, the present seroprevalence study revealed that brucellosis was low among dairy cattle and exposed individuals in the study areas. However, since there were no control strategies implemented in the study areas, there is a potential risk of transmission of brucellosis in dairy cattle and the exposed human population in the study areas. Implementation of a test and slaughter strategy with compensation to farmers is recommended, while in the case of human brucellosis, continuous social training and implementing one health approach framework must be applied.

Keywords: abortion, bovine brucellosis, human brucellosis, risk factors, seroprevalence

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Authors: Carlos Alfredo Camargo Vila, Fredy Augusto Avellaneda Vargas, Debora Alcida Nabarlatz

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Due to the current need to implement environmentally friendly technologies, the possibility of using renewable raw materials to produce bioproducts such as biofuels, or in this case, to produce biolubricant bases, from residual oils (tallow), originating has been studied of the bovine industry. Therefore, it is hypothesized that through the study and control of the operating variables involved in the reverse transesterification method, a biolubricant base with high performance is obtained on a laboratory scale using animal fats from the bovine industry as raw materials, as an alternative for material recovery and environmental benefit. To implement this process, esterification of the crude tallow oil must be carried out in the first instance, which allows the acidity index to be decreased ( > 1 mg KOH/g oil), this by means of an acid catalysis with sulfuric acid and methanol, molar ratio 7.5:1 methanol: tallow, 1.75% w/w catalyst at 60°C for 150 minutes. Once the conditioning has been completed, the biodiesel is continued to be obtained from the improved sebum, for which an experimental design for the transesterification method is implemented, thus evaluating the effects of the variables involved in the process such as the methanol molar ratio: improved sebum and catalyst percentage (KOH) over methyl ester content (% FAME). Finding that the highest percentage of FAME (92.5%) is given with a 7.5:1 methanol: improved tallow ratio and 0.75% catalyst at 60°C for 120 minutes. And although the% FAME of the biodiesel produced does not make it suitable for commercialization, it does ( > 90%) for its use as a raw material in obtaining biolubricant bases. Finally, once the biodiesel is obtained, an experimental design is carried out to obtain biolubricant bases using the reverse transesterification method, which allows the study of the effects of the biodiesel: TMP (Trimethylolpropane) molar ratio and the percentage of catalyst on viscosity and yield as response variables. As a result, a biolubricant base is obtained that meets the requirements of ISO VG (Classification for industrial lubricants according to ASTM D 2422) 32 (viscosity and viscosity index) for commercial lubricant bases, using a 4:1 biodiesel molar ratio: TMP and 0.51% catalyst at 120°C, at a pressure of 50 mbar for 180 minutes. It is necessary to highlight that the product obtained consists of two phases, a liquid and a solid one, being the first object of study, and leaving the classification and possible application of the second one incognito. Therefore, it is recommended to carry out studies of the greater depth that allows characterizing both phases, as well as improving the method of obtaining by optimizing the variables involved in the process and thus achieving superior results.

Keywords: biolubricant base, bovine tallow, renewable resources, reverse transesterification

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Authors: Martyna Ekiert, Andrzej Mlyniec

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Tendons are the biological structures, which primary function is to transfer force generated by muscles to the bones. Unfortunately, damages of tendons are also one of the most common injuries of the human musculoskeletal system. For the most severe cases of tendon rupture, such as the tear of calcaneus tendon or anterior cruciate ligament of the knee, a surgical procedure is the only possible way of full recovery. Tendons used as biological grafts are usually subjected to the process of deep freezing and subsequent thawing. This, in particular for multiple freezing/thawing cycles, may result in changes of tendon internal structure causing deterioration of mechanical properties of the tissue. Therefore, studies on the influence of freezing on tendons biomechanics, including internal water content in soft tissue, seems to be greatly needed. An experimental study of the influence of freezing on mechanical properties of the tendon was performed on fascicles samples dissected form bovine flexor tendons. The preparation procedure was performed with the presence of 0.9% saline solution in order to prevent an excessive tissue drying. All prepared samples were subjected to the different number of freezing/thawing cycles. For freezing part of the protocol we used -80°C temperature while for slow thawing we used fridge temperature (4°C) combined with equalizing temperatures in the standard state (25°C). After final thawing, the mechanical properties of each sample was examined using cyclic loading test. Our results may contribute for better understanding of negative effects of soft tissues freezing, resulting from abnormal thermal expansion of water. This also may help to determine the limit of freezing/thawing cycles disqualifying tissue for surgical purposes and thus help optimize tissues storage conditions.

Keywords: freezing, soft tissue, tendon, bovine fascicles

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Authors: Mayuva Youngsabanant, Suphaphorn Rabiab, Hatairuk Tungkasen, Nongnuch Gumlungpat, Mayuree Pumipaiboon

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The porcine follicular fluid proteins (pFF) of healthy small size ovarian follicles (1-3 mm in diameters) of Large White pig ovaries were collected by sterile technique. They were used for testing the effect on cell viability and characterization of Vero cell lines using MTT assay. Two hundred microliter of round shape Vero cell lines were culture in 96 well plates with DMEM for 24 h. After that, they were attachment to substrate and some changed into fibroblast shape and spread over the surface after culture for 48 h. Then, Vero cell lines were treated with pFF at concentration of 2, 4, 20, 40, 200, 400, 500, and 600 µg proteins/mL for 24 h. Yields of the best results were analyzed by using one-way ANOVA. MTT assay reviewed an increasing in percentage of viability of Vero cell lines indicated that at concentration of 400-600 µg proteins/mL showed higher percentage of viability (115.64 ± 6.95, 106.91 ± 5.27 and 116.73 ± 20.15) than control group. They were significantly different from the control group (p < 0.05) but lower than the positive control group (DMEM with 10% heat treated fetal bovine serum). Cell lines showed normal character in fibroblast elongate shape after treated with pFF except in high concentration of pFF. This result implies that pFF of small size ovarian follicle at concentration of 400-600 µg proteins/mL could be optimized concentration for using as a supplement in Vero cell line culture medium to promote cell viability instead of growth hormone from fetal bovine serum. This merit could be applied in other cell biotechnology researches. Acknowledgements: This work was funded by a grant from Silpakorn University and Faculty of Science, Silpakorn University, Thailand.

Keywords: cell viability, porcine follicular fluid, MTT assay, Vero cell line

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Authors: Islam Nowisser, Noha Farag, Mohamed El Azizi

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Helicobacter pylori is a highly important human pathogen which inhabits about 50% of the population worldwide. Infection with this bacteria is very hard to treat, with high probability of recurrence. H. pylori causes severe gastric diseases, including peptic ulcer, gastritis, and gastric cancer, which has been linked to chronic inflammation. The infection has been reported to be associated with high levels of pro-inflammatory cytokines, especially IL-1β and TNF-α. The aim of the current study is to investigate the molecular mechanisms by which H. pylori activates NLRP3 inflammasome and its contribution to Il-1 β production in an innate cellular model. H. pylori PMSS1 and G27 standard strains, as well as the PMSS1 isogenic mutant strain PMSS1ΔVacA and G27ΔVacA, G27ΔCagA in addition to clinical isolates obtained from biopsy samples from the antrum and corpus mucosa of chronic gastritis patients, were used to establish infection in RAW-264.7 macrophages. The production levels of TNF-α and IL-1β was assessed using ELISA. Since expression of these cytokines is often regulated by the transcription factor complex, nuclear factor-kB (NF-kB), the activation of NF-κB in H. pylori infected cells was also evaluated by luciferase assay. Genomic DNA was extracted from bacterial cultures of H. pylori clinical isolates as well as the standard strains and their corresponding mutants, where they were evaluated for the cagA pathogenicity island and vacA expression. The correlation between these findings and expression of the cagA Pathogenicity Island and vacA in the bacteria was also investigated. The results showed IL-1β, and TNF-α production significantly increased in raw macrophages following H. pylori infection. The cagA+ and vacA+ H. pylori strains induced significant production of IL-1β compared to cagA- and vacA- strains. The activation pattern of NF-κB was correlated in the isolates to their cagA and vacA expression profiles. A similar finding could not be confirmed for TNF-α production. Our study shows the ability of H. pylori to activate NF-kB and induce significant IL-1β production as a possible mechanism for the augmented inflammatory response seen in subjects infected with cagA+ and vacA+ H. pylori strains that would lead to the progression to more severe form of the disease.

Keywords: Helicobacter pylori, IL-1β, inflammatory cytokines, nuclear factor KB, TNF-α

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Authors: Mammar Khames, Mustapha Oumouna

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Brucellosis is one of the most common zoonoses in the world. It represents a serious threat to human health; the existence of brucellosis in Algeria dates back to the beginning of the 19th century. Its transmission to humans is through coccobacilli of the genus Brucella following direct contact with contaminated animals or indirectly through the consumption of their unpasteurized dairy products. The present investigation covers a retrospective study on human brucellosis in the district of Medea over a period from 2011 to 2021 at the level of two public health establishments. In the first place, it is at the level of the Directorate of Public Health and in the infectious department level at Medea Hospital, and at the level of the directorate of agricultural services in the third place. The results showed that during these eleven years of study, 795 cases were collected from the department of health and population, and 141 cases were collected from the infectious department of the district of Medea. A total of 56 cases of bovine brucellosis were obtained from the directorate of agricultural services of the district of Medea. Human brucellosis affects all age groups with different percentages, but the rate has been higher in the 20-44 age group, with a predominance of men. However, the geographic distribution map of the cases shows that the western part of the district was the most affected. A cohort of 141 cases was hospitalized at the infectious service level of Medea Hospital. They were 89 men and 52 women. The most common age reached is [20-44] years. The majority were of rural origin. Two serological reactions were performed for diagnosis: the buffered antigen test and Wright's serodiagnosis. Bovine brucellosis affects all age groups with different percentages, but the rate was higher in the 2-to-4-year age group, with a predominance of females. From these data, we conclude that brucellosis has a strong spread in the region studied.

Keywords: human brucellosis, serology, Medea, Algeria

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Authors: Samaneh Yavari, Christiane Pferrer, Elisabeth Engelke, Alexander Starke, Juergen Rehage

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Background: Three nociceptive thresholds of thermal, electrical, and mechanical thresholds commonly use to evaluate the local anesthesia in many species, for instance, cow, horse, cat, dog, rabbit, and so on. Due to the lack of investigations to evaluate and/or validate such those nociceptive thresholds, our plan was the comparison of two-foot local anesthesia methods of Intravenous Regional Anesthesia (IVRA) and our modified four-point Nerve Block Anesthesia (NBA). Materials and Methods: Eight healthy nonpregnant nondairy Holstein Frisian cows in a cross-over study design were selected for this study. All cows divided into two different groups to receive two local anesthesia techniques of IVRA and our modified four-point NBA. Three thermal, electrical, and mechanical force and pinpricks were applied to evaluate the quality of local anesthesia methods before and after local anesthesia application. Results: The statistical evaluation demonstrated that our four-point NBA has a qualification to select as a standard foot local anesthesia. However, the recorded results of our study revealed no significant difference between two groups of local anesthesia techniques of IVRA and modified four-point NBA related to quality and duration of anesthesia stimulated by electrical, mechanical and thermal nociceptive stimuli. Conclusion and discussion: All three nociceptive threshold stimuli of electrical, mechanical and heat nociceptive thresholds can be applied to measure and evaluate the efficacy of foot local anesthesia of dairy cows. However, our study revealed no superiority of those three nociceptive methods to evaluate the duration and quality of bovine foot local anesthesia methods. Veterinarians to investigate the duration and quality of their selected anesthesia method can use any of those heat, mechanical, and electrical methods.

Keywords: mechanical, thermal, electrical threshold, IVRA, NBA, hind limb, dairy cow

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Authors: Sofia G. Meirinho, Luis G. Dias, António M. Peres, Lígia R. Rodrigues

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The emerging development of electrochemical aptasen sors has enabled the easy and fast detection of protein biomarkers in standard and real samples. Biomarkers are produced by body organs or tumours and provide a measure of antigens on cell surfaces. When detected in high amounts in blood, they can be suggestive of tumour activity. These biomarkers are more often used to evaluate treatment effects or to assess the potential for metastatic disease in patients with established disease. Osteopontin (OPN) is a protein found in all body fluids and constitutes a possible biomarker because its overexpression has been related with breast cancer evolution and metastasis. Currently, biomarkers are commonly used for the development of diagnostic methods, allowing the detection of the disease in its initial stages. A previously described RNA aptamer was used in the current work to develop a simple and sensitive electrochemical aptasensor with high affinity for human OPN. The RNA aptamer was biotinylated and immobilized on a gold electrode by avidin-biotin interaction. The electrochemical signal generated from the aptamer–target molecule interaction was monitored electrochemically using cyclic voltammetry in the presence of [Fe (CN) 6]−3/− as a redox probe. The signal observed showed a current decrease due to the binding of OPN. The preliminary results showed that this aptasensor enables the detection of OPN in standard solutions, showing good selectivity towards the target in the presence of others interfering proteins such as bovine OPN and bovine serum albumin. The results gathered in the current work suggest that the proposed electrochemical aptasensor is a simple and sensitive detection tool for human OPN and so, may have future applications in cancer disease monitoring.

Keywords: osteopontin, aptamer, aptasensor, screen-printed electrode, cyclic voltammetry

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Authors: Y. Landkocz, C. Lepers, P.J. Martin, B. Fougère, F. Roy Saint-Georges. A. Verdin, F. Cazier, F. Ledoux, D. Courcot, F. Sichel, P. Gosset, P. Shirali, S. Billet

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In 2013, the International Agency for Research on Cancer (IARC) classified air pollution and fine particles as carcinogenic to humans. Causal relationships exist between elevated ambient levels of airborne particles and increase of mortality and morbidity including pulmonary diseases, like lung cancer. However, due to a double complexity of both physicochemical Particulate Matter (PM) properties and tumor mechanistic processes, mechanisms of action remain not fully elucidated. Furthermore, because of several common properties between air pollution PM and tobacco smoke, like the same route of exposure and chemical composition, potential mechanisms of synergy could exist. Therefore, smoking could be an aggravating factor of the particles toxicity. In order to identify some mechanisms of action of particles according to their size, two samples of PM were collected: PM0.03 2.5 and PM0.33 2.5 in the urban-industrial area of Dunkerque. The overall cytotoxicity of the fine particles was determined on human bronchial cells (BEAS-2B). Toxicological study focused then on the metabolic activation of the organic compounds coated onto PM and some genetic and epigenetic changes induced on a co-culture model of BEAS-2B and alveolar macrophages isolated from bronchoalveolar lavages performed in smokers and non-smokers. The results showed (i) the contribution of the ultrafine fraction of atmospheric particles to genotoxic (eg. DNA double-strand breaks) and epigenetic mechanisms (eg. promoter methylation) involved in tumor processes, and (ii) the influence of smoking on the cellular response. Three main conclusions can be discussed. First, our results showed the ability of the particles to induce deleterious effects potentially involved in the stages of initiation and promotion of carcinogenesis. The second conclusion is that smoking affects the nature of the induced genotoxic effects. Finally, the in vitro developed cell model, using bronchial epithelial cells and alveolar macrophages can take into account quite realistically, some of the existing cell interactions existing in the lung.

Keywords: air pollution, fine and ultrafine particles, genotoxic and epigenetic alterations, smoking

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Authors: Naouel Boussoualim, Hayat Trabsa, Imane Krache, Seddik Khennouf, Noureddine Charef, Lekhmici Arrar, Abderrahmane Baghiani

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Purpose: To evaluate the in-vitro inhibition of xanthine oxidase (purified from bovine milk) by extracts of Lycium arabicum, as well as it is in vivo hypouricemic and renal protective effects. Methods: Four extracts of Lycium arabicum, methanol (CrE), chloroform (ChE), ethyl acetate (EaE) and aqueous (AqE) extracts, were screened for their total phenolics and potential inhibitory effects on purified bovine milk xanthine oxidase (XO) activity by measuring the formation of uric acid or superoxide radical. The mode of inhibition was investigated and compared with the standard drugs, allopurinol, quercitin, and catechin. To evaluate their hypouricemic effect, the extracts were administered to potassium oxonate-induced hyperuricemic mice at a dose of 50 mg/kg body weight. Results: The results showed that EaE had the highest content of phenolic compounds and was the most potent inhibitor of uric acid formation (IC50 = 0.017 ± 0.001 mg/mL) and formation of superoxide (IC50 = 0.035 ± 0.001 mg/ml). Lineweaver-Burk analysis showed that CrE and EaE inhibited XO competitively, whereas the inhibitory activities exerted by ChE and AqE were of a mixed type. Intraperetoneal injection of L. arabicum extracts (50 mg/kg) elicited hypouricemic actions in hyperuricemic mice. Hyperuricemic mice presented a serum uric acid concentration of 4.71 ± 0.29 mg/L but this was reduced to 1.78 ± 0.11 mg/L by EaE, which was the most potent hyporuricemic extract. Conclusion: L. arabicum fractions have a strong inhibitory effect on xanthine oxidase and and also have a significantly lowering effect on serum and liver creatinine and urea levels in hyperuricemic mice.

Keywords: lycium arabicum, uric acid, creatinine, superoxide, phenolic compounds, flavonoids, hyperuricemia

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Authors: Swati Gautam, Salman Akhtar, S. P. Jaiswar, Amita Jain

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Background: FGTB is now a major global health problem mostly in developing countries including India. In humans, Mycobacterium Tuberculosis (M.tb) is a causating agent of infection. High index of suspicion is required for early diagnosis due to asymptomatic presentation of FGTB disease. In macrophages Toll Like Receptor-2 (TLR-2) is one which mediated host’s immune response to M.tb. The expression of TLR-2 on macrophages is important to determine the fate of innate immune responses to M.tb. TLR-2 have two work. First its high expression on macrophages worsen the outer of infection and another side, it maintains M.tb to its dormant stage avoids activation of M.tb from latent phase. Single Nucleotide Polymorphism (SNP) of TLR-2 gene plays an important role in susceptibility to TB among different populations and subsequently, in the development of infertility. Methodology: This Case-Control study was done in the Department of Obs and Gynae and Department of Microbiology at King George’s Medical University, U.P, Lucknow, India. Total 300 subjects (150 Cases and 150 Controls) were enrolled in the study. All subjects were enrolled only after fulfilling the given inclusion and exclusion criteria. Inclusion criteria: Age 20-35 years, menstrual-irregularities, positive on Acid-Fast Bacilli (AFB), TB-PCR, (LJ/MGIT) culture in Endometrial Aspiration (EA). Exclusion criteria: Koch’s active, on ATT, PCOS, and Endometriosis fibroid women, positive on Gonococal and Chlamydia. Blood samples were collected in EDTA tubes from cases and healthy control women (HCW) and genomic DNA extraction was carried out by salting-out method. Genotyping of TLR2 genetic variants (Arg753Gln and Arg677Trp) were performed by using single amplification refractory mutation system (ARMS) PCR technique. PCR products were analyzed by electrophoresis on 1.2% agarose gel and visualized by gel-doc. Statistical analysis of the data was performed using the SPSS 16.3 software and computing odds ratio (OR) with 95% CI. Linkage Disequiliribium (LD) analysis was done by SNP stats online software. Results: In TLR-2 (Arg753Gln) polymorphism significant risk of FGTB observed with GG homozygous mutant genotype (OR=13, CI=0.71-237.7, p=0.05), AG heterozygous mutant genotype (OR=13.7, CI=0.76-248.06, p=0.03) however, G allele (OR=1.09, CI=0.78-1.52, p=0.67) individually was not associated with FGTB. In TLR-2 (Arg677Trp) polymorphism a significant risk of FGTB observed with TT homozygous mutant genotype (OR= 0.020, CI=0.001-0.341, p < 0.001), CT heterozygous mutant genotype (OR=0.53, CI=0.33-0.86, p=0.014) and T allele (OR=0.463, CI=0.32-0.66, p < 0.001). TT mutant genotype was only found in FGTB cases and frequency of CT heterozygous more in control group as compared to FGTB group. So, CT genotype worked as protective mutation for FGTB susceptibility group. In haplotype analysis of TLR-2 genetic variants, four possible combinations, i.e. (G-T, A-C, G-C, and A-T) were obtained. The frequency of haplotype A-C was significantly higher in FGTB cases (0.32). Control group did not show A-C haplotype and only found in FGTB cases. Conclusion: In conclusion, study showed a significant association with both genetic variants of TLR-2 of FGTB disease. Moreover, the presence of specific associated genotype/alleles suggest the possibility of disease severity and clinical approach aimed to prevent extensive damage by disease and also helpful for early detection of disease.

Keywords: ARMS, EDTA, FGTB, TLR

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Authors: Shujun Xie, Shirong Zhang, Shenglin Ma

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Background: Chronic inflammation might lead to many malignancies, and inadequate resolution could play a crucial role in tumor invasion, progression, and metastases. A randomised, double-blind, placebo-controlled trial shows that IL-1β inhibition with canakinumab could reduce incident lung cancer and lung cancer mortality in patients with atherosclerosis. The process and secretion of proinflammatory cytokine IL-1β are controlled by the inflammasome. Here we showed the correlation of the innate immune system and afatinib, a tyrosine kinase inhibitor targeting epidermal growth factor receptor (EGFR) in non-small cell lung cancer. Methods: Murine Bone marrow derived macrophages (BMDMs), peritoneal macrophages (PMs) and THP-1 were used to check the effect of afatinib on the activation of NLRP3 inflammasome. The assembly of NLRP3 inflammasome was check by co-immunoprecipitation of NLRP3 and apoptosis-associated speck-like protein containing CARD (ASC), disuccinimidyl suberate (DSS)-cross link of ASC. Lipopolysaccharide (LPS)-induced sepsis and Alum-induced peritonitis were conducted to confirm that afatinib could inhibit the activation of NLRP3 in vivo. Peripheral blood mononuclear cells (PBMCs) from non-small cell lung cancer (NSCLC) patients before or after taking afatinib were used to check that afatinib inhibits inflammation in NSCLC therapy. Results: Our data showed that afatinib could inhibit the secretion of IL-1β in a dose-dependent manner in macrophage. Moreover, afatinib could inhibit the maturation of IL-1β and caspase-1 without affecting the precursors of IL-1β and caspase-1. Next, we found that afatinib could block the assembly of NLRP3 inflammasome and the ASC speck by blocking the interaction of the sensor protein NLRP3 and the adaptor protein ASC. We also found that afatinib was able to alleviate the LPS-induced sepsis in vivo. Conclusion: Our study found that afatinib could inhibit the activation of NLRP3 inflammasome in macrophage, providing new evidence that afatinib could target the innate immune system to control chronic inflammation. These investigations will provide significant experimental evidence in afatinib as therapeutic drug for non-small cell lung cancer or other tumors and NLRP3-related diseases and will explore new targets for afatinib.

Keywords: inflammasome, afatinib, inflammation, tyrosine kinase inhibitor

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Authors: Reshu Saxena, R. K. Tripathi

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HIV-1 transmission and spread involves significant host-virus interaction. Potential targets for prevention of HIV-1 lies at the site of mucosal barriers. Thus a better understanding of how HIV-1 infects target cells at such sites and lead their invasion is required, with prime focus on the host determinants regulating HIV-1 spread. HIV-1 Nef is important for viral infectivity and pathogenicity. It promotes HIV-1 replication, facilitating immune evasion by interacting with various host factors and altering cellular pathways via multiple protein-protein interactions. In this study nef was sequenced from HIV-1 patients, and showed specific mutations revealing sequence variability in nef. To explore the difference in Nef functionality based on sequence variability we have studied the effects of HIV-1 Nef in human SupT1 T cell line and (THP-1) monocyte-macrophage cell lines through proteomics approach. 2D-Gel Electrophoresis in control and Nef-transfected SupT1 cells demonstrated several differentially expressed proteins with significant modulation of alpha-enolase. Through further studies, effects of Nef on alpha-enolase regulation were found to be cell lineage-specific, being stimulatory in macrophages/monocytes, inhibitory in T cells and without effect in HEK-293 cells. Cell migration and invasion studies were employed to determine biological function affected by Nef mediated regulation of alpha-enolase. Cell invasion was enhanced in THP-1 cells but was inhibited in SupT1 cells by wildtype nef. In addition, the modulation of enolase and cell invasion remained unaffected by a unique nef variant. These results indicated that regulation of alpha-enolase expression and invasive property of host cells by Nef is sequence specific, suggesting involvement of a particular motif of Nef. To precisely determine this site, we designed a heptapeptide including the suggested alpha-enolase regulating sequence of nef and a nef mutant with deletion of this site. Macrophages/monocytes being the major cells affected by HIV-1 at mucosal barriers, were particularly investigated by the nef mutant and peptide. Both the nef mutant and heptapeptide led to inhibition of enhanced enolase expression and increased invasiveness in THP-1 cells. Together, these findings suggest a possible mechanism of host invasion by HIV-1 through Nef mediated regulation of alpha-enolase and identifies a potential therapeutic target for HIV-1 entry at mucosal barriers.

Keywords: HIV-1 Nef, nef variants, host-virus interaction, tissue invasion

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Authors: Wiolene Montanari Nordi, Debora Botequio Moretti, Mariana Caroline Pontin, Jessica Pampolini, Raul Machado-Neto

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Gills are organs responsible for respiration and osmoregulation between the fish internal environment and water. Under stress conditions, oxidative response and gill plasticity to attempt to increase gas exchange area are noteworthy, compromising the physiological processes and therefore fish health. Colostrum is a dietary source of nutrients, immunoglobulin, antioxidant and bioactive molecules, essential for immunological protection and development of the gastrointestinal epithelium. The hypothesis of this work is that antioxidant factors present in the colostrum, unprecedentedly tested in gills, can minimize or reduce the alteration of its epithelium structure of juvenile pacu (Piaractus mesopotamicus) subjected to high stocking density. The histological changes in the gills architecture were characterized by the frequency, incidence and severity of the tissue alteration and ionic status. Juvenile (50 kg fish/m3) were fed with pelleted diets containing 0, 10, 20 or 30% of lyophilized bovine colostrum (LBC) inclusion and at 30 experimental days, gill and blood samples were collected in eight fish per treatment. The study revealed differences in the type, frequency and severity (histological alterations index – HAI) of tissue alterations among the treatments, however, no distinct differences in the incidence of alteration (mean alteration value – MAV) were observed. The main histological changes in gill were elevation of the lamellar epithelium, excessive cell proliferation of the filament and lamellar epithelium causing total or partial melting of the lamella, hyperplasia and hypertrophy of lamellar and filament epithelium, uncontrolled thickening of filament and lamellar tissues, mucous and chloride cells presence in the lamella, aneurysms, vascular congestion and presence of parasites. The MAV obtained per treatment were 2.0, 2.5, 1.8 and 2.5 to fish fed diets containing 0, 10, 20 and 30% of LBC inclusion, respectively, classifying the incidence of gill alterations as slightly to moderate. The severity of alteration of individual fish of treatment 0, 10 and 20% LBC ranged values from 5 to 40 (HAI average of 20.1, 17.5 and 17.6, respectively, P > 0.05), and differs from 30% LBC, that ranged from 6 to 129 (HAI mean of 77.2, P < 0.05). The HAI value in the treatments 0, 10 and 20% LBC reveals gill tissue with injuries classified from slightly to moderate, while in 30% LBC moderate to severe, consequence of the onset of necrosis in the tissue of two fish that compromises the normal functioning of the organ. In relation to frequency of gill alterations, evaluated according to absence of alterations (0) to highly frequent (+++), histological alterations were observed in all evaluated fish, with a trend of higher frequency in 0% LBC. The concentration of Na+, Cl-, K+ and Ca2+ did not changed in all treatments (P > 0.05), indicating similar capacity of ion exchange. The concentrations of bovine colostrum used in diets of present study did not impair the alterations observed in the gills of juvenile pacu.

Keywords: histological alterations of gill tissue, ionic status, lyophilized bovine colostrum, optical microscopy

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Authors: Samaneh Yavari, Christiane Pferrer, Elisabeth Engelke, Alexander Starke, Juergen Rehage

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Background: Nowadays, bovine limb interventions, especially any claw surgeries, raises selection of the most qualified and appropriate local anesthesia technique applicable for any superficial or deep interventions of the limbs. Currently, two local anesthesia methods of Intravenous Regional Anesthesia (IVRA), as well as Nerve Blocks, have been routine to apply. However, the lack of studies investigating the quality and duration as well as quantity and onset of full (complete) local anesthesia, is noticeable. Therefore, the aim of our study was comparing the onset and quality of both IVRA and our modified NBA at the hind limb of dairy cows. For this abstract, only the onset of full local anesthesia would be consider. Materials and Methods: For that reason, we used six healthy non pregnant non lactating Holestein Frisian cows in a cross-over study design. Those cows divided into two groups to receive IVRA and our modified four-point NBA. For IVRA, 20 ml procaine without epinephrine was injected into the vein digitalis dorsalis communis III and for our modified four-point NBA, 10-15 ml procaine without epinephrine preneurally to the nerves, superficial and deep peroneal as well as lateral and medial branches of metatarsal nerves. For pain stimulation, electrical stimulator Grass S48 was applied. Results: The results of electrical stimuli revealed the faster onset of full local anesthesia (p < 0.05) by application of our modified NBA in comparison to IVRA about 10 minutes. Conclusion and discussion: Despite of available references showing faster onset of foot local anesthesia of IVRA, our study demonstrated that our modified four point NBA not only can be well known as a standard foot local anesthesia method applicable to desensitize the hind limb of dairy cows, but also, selection of this modified validated local anesthesia method can lead to have a faster start of complete desensitization of distal hind limb that is remarkable in any bovine limb interventions under time constraint.

Keywords: IVRA, four point NBA, dairy cow, hind limb, full onset

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