Heat-inactivated Serum Research Articles

Evaluation of anticancer activity of Melaleuka Alternifolia. (i. e. tea tree oil) on Breast cancer cell line (MDA MB)- An in-vitro study

Introduction and Aims: According to Globocan 2012, India along with United States and China collectively accounts for almost one third of the global breast cancer burden. India is facing challenging situation due to 11.54% increases in incidence and 13.82% increase in mortality due to breast cancer during 2008–2012. The current treatment modality has issues of drug resistance and side effects. Many anticancer drugs currently used clinically have been isolated from plant species hence, investigation of plant species as a source of experimental therapeutic agents, in treating cancer is currently gaining a lot of importance. One such naturally available plant extract Melaleuka Alternifolia (TTO) which belongs to the family of essential oils is a very good antibacterial, antifungal, antiviral, antiprotozoal and anti-inflammatory agent. But currently there is a lot of importance has been given for its anticancer effect. Hence our aim is to evaluate anticancer activity of Melaleuka Alternifolia on Breast cancer cell line (MDA MB) by MTT assay an in vitro method. Material and Methods: Before the start of the study ethical clearance was obtained from Institutional Review Board. The cytotoxicity checked for Breast cancer cell line (MDA MB) and NIH 3T3 mouse fibroblasts which were used as a control in our current study. These cell lines were procured from NCCS Pune, India. 1. MTT solution preparation (stock solution): 5 mg in 1 ml of PBS. 2. Cell culture : The cell lines were maintained in 96 wells micro titer plate containing MEM media supplemented with 10% heat inactivated fetal calf serum (FCS), containing 5% of mixture of Gentamicin (10ug), Penicillin ( 100 Units/ ml) and Streptomycin (100µg/ml) in presence of 5% CO2 at 37oC for 48-72 hours. 3. Cytotoxicity Assay: In vitro growth inhibition effect of test compound was assessed by calorimetric or spectrophotometric determination of conversion of MTT into Formazan blue by living cells. Results: The results represent the mean of five readings. The IC50 value of tea tree oil for breast cancer cell line (MDA MB) after 48 hrs was 25µg/ml. Spearmans rho’s Correlation showed P value Conclusion: TTO has a promising anticancer property against Breast cancer cell line (MDA MB) with its IC50 value 25µg/ml. Hence this TTO with its greater efficacy related to its anticancer activity can be brought to the level of clinical trials in the coming future. Keywords: Cytotoxicity, MTT assay, MDAMB cell line, Tea tree oil.

Criticality of In-House Preparation of Viral Transport Medium in Times of Shortage During COVID-19 Pandemic.

ObjectiveWith the COVID-19 pandemic, there have been supply challenges necessitating that laboratories must prepare their own viral transport medium (VTM), which provides stability for clinical specimens for diagnostic viral testing.MethodsWithin a veteran affairs medical center clinical laboratory, VTM was prepared with a Hanks Balanced Salt Solution (HBSS) 500 mL bottle with phenol red, sterile heat-inactivated fetal bovine serum (FBS), gentamicin sulfate (50 mg/mL), and amphotericin B (250 μg/mL). An antimicrobial mixture was made of 50 mL each of amphotericin B and gentamicin sulfate. Ten mL of FBS and 2 mL of the antimicrobial mixture were mixed into the HBSS bottle, from which 3 mL aliquots were made. Sterility and efficacy check were assessed. These preparations were conducted at our VAMC’s clinical laboratory to assure adequate VTM supply during the COVID-19 shortage.ResultsThe VTM was successfully prepared in-house, supporting uninterrupted testing for the facility and other affiliated medical facilities/centers and community living centers.ConclusionThis quality assurance/improvement report represents the first published manuscript on feasible VTM preparation exclusively within a clinical microbiology laboratory during the COVID-19 pandemic.

Melanin as a Virulence Factor in Different Species of Genus Paracoccidioides.

Paracoccidioidomycosis (PCM) is a granulomatous systemic mycosis caused by the thermo-dimorphic fungi of the genus Paracoccidioides. Melanin production by fungi can affect their pathogenesis and virulence. This study evaluates the production of melanin by different isolates of genus Paracoccidioides and examines how the presence of this polymer affects yeast cell phagocytosis, as well as laccase enzyme production. The results obtained showed that the isolates of genus Paracoccidioides: P. lutzii (Pb01, Pb66, ED01, Pb1578, and Pb8334), P. restrepiensis (PS3-Pb60855), P. brasiliensis (S1-Pb18), and P. americana (PS2-Pbcão) produce melanin in the presence of L-3,4-dihydroxyphenylalanine (L-DOPA). Phagocytosis assays were carried out with peritoneal macrophages from C57Bl/6 mice that were challenged with Pb18, Pb60855, and Pb01. We observed that melanin interferes with phagocytosis in the presence or absence of complement or heat-inactivated serum. This article confirms that different species of the genus Paracoccidioides produce melanin in different magnitudes and that the polymer functions as a virulence factor.

Enhancement of infectivity of insect cell-derived La Crosse Virus by human serum

Given the dual life cycle of arboviruses in insect and animal hosts and the importance of serum factors as a first line antiviral defense, we have examined the outcome of interactions between the arbovirus La Crosse Virus (LACV) and human serum. To mimic the life cycle between species, we used LACV derived from insect (I-LACV) and human keratinocyte (HaCaT) cells. Incubation of I-LACV with normal human serum did not result in neutralization, but instead stabilized I-LACV virions and enhanced the amount of infectious virus. Enhanced infectivity was also seen with heat-inactivated serum devoid of complement activity and with serum from a range of animals including mouse, ferret, and non-human primates. Depletion of antibodies from serum resulted in loss of enhancement of infectivity and sucrose gradient sedimentation assays showed IgG co-sedimenting with I-LACV particles. In agreement with our results with I-LACV, HaCaT-derived LACV was not neutralized by complement or antibodies in normal human serum. However, in contrast to I-LACV, HaCaT-derived LACV infectivity was stable when incubated alone and treatment with serum did not enhance infectivity. Our results indicate that LACV derived from insect cells differs substantially from virus derived from human cells, with I-LACV being dependent on serum factors to enhance infectivity. These findings suggest that understanding differential composition of insect versus animal cell-derived LACV may form the foundation for potential new antiviral approaches.

Oncofertility case report: live birth 10 years after oocyte in vitro maturation and zygote cryopreservation

PurposeThis study aims to report a case of urgent fertility preservation in an oncological patient with collection of immature oocytes in the absence of ovarian stimulation that, through in vitro maturation (IVM), followed by ICSI and cryopreservation of zygotes resulted, 10 years later, in the live birth of a healthy baby.MethodsIn September 2008, our clinic performed IVM in a 32-year-old woman diagnosed with a ductal invasive carcinoma with positive estradiol receptors, negative progesterone receptors and positive human epidermal growth factor receptor 2. The retrieval of immature oocytes was performed in the absence of ovarian stimulation after a simple mastectomy and prior to any chemotherapy treatment. The compact cumulus-oocyte complexes (COCs) collected were placed in Lag medium for 2 h, followed by incubation in IVM medium, supplemented with heat inactivated patient serum, recombinant FSH, and recombinant LH. After 30 h in culture, cumulus cells were removed, the metaphase II oocytes were microinjected, and the zygotes obtained were cryopreserved. In 2017, the zygotes were thawed and cultured until day 3. One embryo was transferred and the other cryopreserved.ResultsFour compact COCs were collected and subjected to IVM. Two oocytes reached metaphase II and were microinjected. Two zygotes were obtained and were cryopreserved at the two pronuclear stage. Approximately 9 years later, the two zygotes were thawed and cultured until day 3. An embryo with 10 cells was transferred and implanted, resulting in the birth of a healthy baby.ConclusionsIn cases where urgency to start adjuvant therapy requires immediate oocyte collection, IVM may be the only option to obtain fully competent mature oocytes allowing for effective preservation of the reproductive potential.

In-House Viral Transport Medium (VTM) Manufacture in the Time of Shortage, Supply and Crisis of COVID-19 at Veteran Affairs Medical Center (VAMC)

Introduction/ObjectiveWith the outbreak of COVID-19 caused by SARS-CoV-2, there have been challenges in the maintenance of adequate supplies both in terms of PPE and for testing. The shortage of commercial VTM for the transport of specimens for PCR testing has created a situation in which laboratories would need to manufacture their own in-house VTM as commonly used commercial VTM is unavailable. However, there is sparse literature on the emergency manufacture of VTM. Here, we describe the VAMC experience in manufacturing/quality control on its own VTM.ResultsVTM was manufactured by pathology and laboratory medicine using strict aseptic technique with Hanks Balanced Salt Solution (HBSS) 500 ml bottle with phenol red, sterile heat-inactivated fetal bovine serum (FBS) gentamicin sulfate (50 mg/mL) and amphotericin B (250 ug/ml). First, 50 ml of amphotericin B and 50 ml of gentamicin sulfate were mixed. Then 10 ml of FBS was mixed with the HBSS bottle and then 2 ml of the gentamicin/amphotericin B mixture was also mixed into the HBSS bottle. 3 ml aliquots were made from this mixture to constitute individual tubes of VTM for clinical use. Sterility for each batch (after 24-hour incubation at 37o C in the CO2 incubator) was assessed visually and by culture on a blood agar, chocolate agar, and thioglycolate mediums. An efficacy check was performed for each batch by spiking positive and negative controls into the VTM aliquots; RT-PCR for SARS-CoV-2 was executed to verify the medium did not degrade viral RNA and produced expected results for room temperature, refrigerated, and frozen samples. Previously manufactured VTM without phenol red also underwent sterility and efficacy checks.ResultsVTM was successfully manufactured in-house, allowing testing to continue despite the shortage. Sterility and efficacy checks on all lots and bottles from which the VTM aliquots were made passed with no growth detected and efficacy passing with all expected positives and negatives resulting as expected.ConclusionTo the author’s knowledge, this represents the first published abstract on VTM manufacture in this most unprecedented crisis involving COVID-19. In this national emergency with corresponding shortage of testing supplies including commercial VTM, the in-house manufacture of VTM is both feasible and prudent to ensure continuity of testing and quality patient/laboratory care.

The Rigor and Transparency Index Quality Metric for Assessing Biological and Medical Science Methods.

SummaryThe reproducibility crisis is a multifaceted problem involving ingrained practices within the scientific community. Fortunately, some causes are addressed by the author's adherence to rigor and reproducibility criteria, implemented via checklists at various journals. We developed an automated tool (SciScore) that evaluates research articles based on their adherence to key rigor criteria, including NIH criteria and RRIDs, at an unprecedented scale. We show that despite steady improvements, less than half of the scoring criteria, such as blinding or power analysis, are routinely addressed by authors; digging deeper, we examined the influence of specific checklists on average scores. The average score for a journal in a given year was named the Rigor and Transparency Index (RTI), a new journal quality metric. We compared the RTI with the Journal Impact Factor and found there was no correlation. The RTI can potentially serve as a proxy for methodological quality.

Establishment and characterization of a cell line from Ghanaian Aedes aegypti (Diptera: Culicidae) focusing on Aedes-borne flavivirus susceptibility.

Mosquitoes are generally considered one of the most important vectors of arboviruses, with Aedes aegypti regarded as the most important in transmission of yellow fever and dengue viruses. To investigate why there are differences in the incidence of dengue fever and Zika in different geographical areas and an absence of outbreaks in Ghana in spite of an abundance of A. aegypti mosquitoes, we established a continuous cell line from embryonic cells of A. aegypti collected in Ghana and assessed its susceptibility to dengue, yellow fever, and Zika viruses. The new cell line (designated AeAe-GH98), having an adhesive spindle-shaped web-like morphology, was serially subcultured in both VP-12 and Schneider's medium supplemented with 10% heat-inactivated fetal bovine serum. AeAe-GH98 cells were found to have a population doubling time of 1.3d during exponential growth. The mosquito colony used to establish the cell line was confirmed to have originated from Africa using microsatellite assay. In terms of susceptibility to Aedes-borne flaviviruses, AeAe-GH98 cells were found to have different degrees of susceptibility to yellow fever, Zika, and dengue virus infection and propagation. While susceptibility of AeAe-GH98 cells to yellow fever and Zika viruses was comparable with that of C6/36 cells, susceptibility to dengue virus was significantly lower. This cell line will serve as a useful tool for determining molecular factors influencing virus-vector susceptibility in vitro.

Does expression of a human complement-regulatory protein on xenograft cells protect them from systemic complement activation?

BackgroundThere are many causes of systemic complement activation, which may have detrimental effects on a pig xenograft. Transgenic expression of one or more human complement-regulatory proteins (hCRPs), e.g., hCD46, provides some protection to the xenograft, but it is not known whether it protects the xenograft from the effects of systemic complement activation. We used wild-type (WT) pig aortic endothelial cells (pAECs) to activate complement, and determined whether the expression of hCD46 on a1,3galactosyltransferase gene-knockout (GTKO) pAECs protected them from injury. MethodsCFSE-labeled and non-labeled pAECs from a WT, a GTKO, or a GTKO/hCD46 pig were separately incubated with heat-inactivated pooled human serum in vitro. Antibody pre-bonded CFSE-labeled and non-labeled pAECs were mixed, and then incubated with rabbit complement. The complement-dependent cytotoxicity was measured by flow cytometry. ResultsThere was significantly less lysis of GTKO/CD46 pAECs (6%) by 50% human serum compared to that of WT (91%, p<0.001) or GTKO (32%, p<0.01) pAECs. The lysis of GTKO pAECs was significantly increased when mixed with WT pAECs (p<0.05). In contrast, there was no significant change in cytotoxicity of GTKO/CD46 pAECs when mixed with WT pAECs. ConclusionsThe expression of hCD46 protected pAECs from systemic complement activation

8. Modifiable polymer promotes a pro-osteogenic, M2-like macrophage phenotype and osteoblastic differentiation of progenitor cells

8. Modifiable polymer promotes a pro-osteogenic, M2-like macrophage phenotype and osteoblastic differentiation of progenitor cells

Treatment of Plasmodium falciparum merozoites with the protease inhibitor E64 and mechanical filtration increases their susceptibility to complement activation.

Plasmodium falciparum malaria killed 451,000 people in 2017. Merozoites, the stage of the parasite that invades RBCs, are a logical target for vaccine development. Treatment with the protease inhibitor E64 followed by filtration through a 1.2 μm filter is being used to purify merozoites for immunologic assays. However, there have been no studies to determine the effect of these treatments on the susceptibility of merozoites to complement or antibodies. To address this gap, we purified merozoites with or without E64 followed by filtration through either a 1.2 or 2.7 μm filter, or no filtration. Merozoites were then incubated in either 10% fresh or heat-inactivated serum followed by surface staining and flow cytometry with monoclonal antibodies against the complement effector molecules C3b or C5b9. To determine the effect of anti-merozoite antibodies, we incubated merozoites with MAb5.2, a mouse monoclonal antibody that targets the merozoite surface protein 1. We used an amine-reactive fluorescent dye to measure membrane integrity. Treatment with E64 resulted in an insignificant increase in the proportion of merozoites that were C3b positive but in a significant increase in the proportion that were C5b9 positive. Filtration increased the proportion of merozoites that were either C3b or C5b9-positive. The combination of filtration and E64 treatment resulted in marked deposition of C3b and C5b9. MAb5.2 induced greater complement deposition than serum alone or an IgG2b isotype control. The combination of E64 treatment, filtration, and MAb5.2 resulted in very rapid and significant deposition of C5b9. Filtration through the 1.2 μm filter selected a population of merozoites with greater membrane integrity, but their integrity deteriorated rapidly upon exposure to serum. We conclude that E64 treatment and filtration increase the susceptibility of merozoites to complement and antibody. Filtered or E64-treated merozoites are not suitable for immunologic studies that address the efficacy of antibodies in vitro.

In vitro phagocytosis of liquid-stored red blood cells requires serum and can be inhibited with fucoidan and dextran sulphate.

Red-blood-cells (RBCs) undergo structural and metabolic changes with prolonged storage, which ultimately may decrease their survival after transfusion. Although the storage-induced damage to RBCs has been rather well described biochemically, little is known about the mechanisms underlying the recognition and rapid clearance of the damaged cells by macrophages. We, here, used a murine model for cold (+4°C) RBC storage and transfusion. Phagocytosis of human or murine RBCs, liquid stored for 6-8weeks or 10-14days, respectively, was investigated in murine peritoneal macrophages. The effects of storage on murine RBCs resembled that described for stored human RBCs with regard to decreased adenosine triphosphate (ATP) levels, accumulation of microparticles (MPs) during storage, and RBC recovery kinetics after transfusion. Under serum-free conditions, phagocytosis of stored human or murine RBCs in vitro was reduced by 70-75%, as compared with that in the presence of heat-inactivated fetal calf serum (FCS). Human serum promoted phagocytosis of stored human RBCs similar to that seen with FCS. By adding fucoidan or dextran sulphate (blockers of scavenger receptors class A (SR-A)), phagocytosis of human or murine RBCs was reduced by more than 90%. Phagocytosis of stored human RBCs was also sensitive to inhibition by the phosphatidylinositol 3 kinase-inhibitor LY294002, the ERK1/2-inhibitor PD98059, or the p38 MAPK-inhibitor SB203580. RBCs damaged during liquid storage may be recognized by macrophage SR-A and serum-dependent mechanisms. This species-independent recognition mechanism may help to further understand the rapid clearance of stored RBCs shortly after transfusion.

Bacillus anthracis Poly-γ-D-Glutamate Capsule Inhibits Opsonic Phagocytosis by Impeding Complement Activation.

Bacillus anthracis poly-γ-D-glutamic acid (PGA) capsule is an essential virulent factor that helps the bacterial pathogen to escape host immunity. Like other encapsulated bacterial species, the B. anthracis capsule may also inhibit complement-mediated clearance and ensure bacterial survival in the host. Previous reports suggest that B. anthracis spore proteins inhibit complement activation. However, the mechanism through which the B. anthracis capsule imparts a survival advantage to the active bacteria has not been demonstrated till date. Thus, to evaluate the role of the PGA capsule in evading host immunity, we have undertaken the present head-to-head comparative study of the phagocytosis and complement activation of non-encapsulated and encapsulated B. anthracis strains. The encapsulated virulent strain exhibited resistance toward complement-dependent and complement-independent bacterial phagocytosis by human macrophages. The non-encapsulated Sterne strain was highly susceptible to phagocytosis by THP-1 macrophages, after incubation with normal human serum (NHS), heat-inactivated serum, and serum-free media, thus indicating that the capsule inhibited both complement-dependent and complement-independent opsonic phagocytosis. An increased binding of C3b and its subsequent activation to C3c and C3dg, which functionally act as potent opsonins, were observed with the non-encapsulated Sterne strain compared with the encapsulated strain. Other known mediators of complement fixation, IgG, C-reactive protein (CRP), and serum amyloid P component (SAP), also bound more prominently with the non-encapsulated Sterne strain. Studies with complement pathway-specific, component-deficient serum demonstrated that the classical pathway was primarily involved in mediating C3b binding on the non-encapsulated bacteria. Both strains equally bound the complement regulatory proteins C4BP and factor H. Importantly, we demonstrated that the negative charge of the PGA capsule was responsible for the differential binding of the complement proteins between the non-encapsulated and encapsulated strains. At lower pH closer to the isoelectric point of PGA, the neutralization of the negative charge was associated with an increased binding of C3b and IgG with the encapsulated B. anthracis strain. Overall, our data have demonstrated that the B. anthracis capsule inhibits complement fixation and opsonization resulting in reduced phagocytosis by macrophages, thus allowing the bacterial pathogen to evade host immunity.

Effect of Different Media on the Bactericidal Activity of Colistin and on the Synergistic Combination With Azidothymidine Against mcr-1-Positive Colistin-Resistant Escherichia coli.

Antimicrobial susceptibility testing (AST) performed according to defined guidelines is important to identify resistance and to predict the clinical success or failure of specific antibiotic therapy. However, these guidelines do not cover all physiological conditions that can have a tremendous impact on in vivo resistance. In this study, we tested the susceptibility of thirteen mcr-1-positive Escherichia coli strains against colistin, one of the last resort antibiotics for treating multi-drug resistant pathogens, in media recommended for ASTs as well as – physiologically more relevant – in human serum and artificial urine (AU). Minimal inhibitory concentration (MIC) values in heat-inactivated human serum were similar to those in cation-adjusted Mueller-Hinton broth (CAMHB), but reduced in native serum for almost all strains that could grow in this media. In AU MIC values for mcr-1 positive E. coli were increased significantly up to 16-fold compared to that in CAMBH, which did not apply to the colistin-susceptible E. coli strains tested. Although different growth media could affect the MIC of colistin alone, their impact on the synergistic effect of the combination with the antiviral drug azidothymidine was minimal. The higher divalent cation concentration combined with acidic pH values is most likely responsible for the increased MIC values of the mcr-1 harboring E. coli strains tested against colistin in AU compared to that in CAMHB. Antimicrobial susceptibility screening procedures for colistin using CAMHB only could lead to an underestimation of resistance under different physiological conditions. Therefore, not only pharmacokinetic but also pharmacodynamic studies in urine are as important as in serum or plasma.

Performance of monocyte activation test supplemented with human serum compared to fetal bovine serum.

The monocyte activation test (MAT) is used to detect pyrogens in pharmaceutical products and serves as replacement of the rabbit pyrogen test. The peripheral blood mononuclear cell-based MAT assay requires the addition of serum to the medium and is performed with either fetal bovine serum (FBS) or human serum (HS). Since the capacity to detect non-endotoxin pyrogens (NEPs) in a sensitive manner is an important strength of MAT compared to the bacterial endo­toxin test, the performance of the MAT using FBS and HS was compared using endotoxin and several NEPs. The MAT was more sensitive for endotoxin when FBS was used, however for most NEPs the MAT was more sensitive when per­formed in HS. Furthermore, heat-inactivation of FBS affected the performance of the MAT for endotoxin to some extent but not for the NEPs. Interestingly, heat-inactivation of HS led to an almost complete loss of reactivity towards endotoxin, reduced the response towards heat-killed Staphylococcus aureus and peptidoglycan, but had minor or no effects on the responses towards R848, flagellin, and Pam3CSK4. Product testing of a human blood-derived product in MAT using HS was beneficial since endotoxin spike recoveries were improved. This product is therefore currently batch released with the HS-based MAT assay. Overall, to guarantee optimal performance of MAT, heat-inactivated serum should be avoided. The HS-based MAT appears to be the first choice to replace the rabbit pyrogen test, while in some cases the FBS-based MAT may be favored.

Ecotin, a microbial inhibitor of serine proteases, blocks multiple complement dependent and independent microbicidal activities of human serum.

Ecotin is a serine protease inhibitor produced by hundreds of microbial species, including pathogens. Here we show, that ecotin orthologs from Escherichia coli, Yersinia pestis, Pseudomonas aeruginosa and Leishmania major are potent inhibitors of MASP-1 and MASP-2, the two key activator proteases of the complement lectin pathway. Factor D is the key activator protease of another complement activation route, the alternative pathway. We show that ecotin inhibits MASP-3, which is the sole factor D activator in resting human blood. In pathway-specific ELISA tests, we found that all ecotin orthologs are potent lectin pathway inhibitors, and at high concentration, they block the alternative pathway as well. In flow cytometry experiments, we compared the extent of complement-mediated opsonization and lysis of wild-type and ecotin-knockout variants of two E. coli strains carrying different surface lipopolysaccharides. We show, that endogenous ecotin provides significant protections against these microbicidal activities for both bacteria. By using pathway specific complement inhibitors, we detected classical-, lectin- and alternative pathway-driven complement attack from normal serum, with the relative contributions of the activation routes depending on the lipopolysaccharide type. Moreover, in cell proliferation experiments we observed an additional, complement-unrelated antimicrobial activity exerted by heat-inactivated serum. While ecotin-knockout cells are highly vulnerable to these activities, endogenous ecotin of wild-type bacteria provides complete protection against the lectin pathway-related and the complement-unrelated attack, and partial protection against the alternative pathway-related damage. In all, ecotin emerges as a potent, versatile self-defense tool that blocks multiple antimicrobial activities of the serum. These findings suggest that ecotin might be a relevant antimicrobial drug target.

110 Absence of transmission of Mycoplasma bovis via naturally contaminated semen during invitro fertilization

Mycoplasma bovis (Mbo) has been isolated from genital tracts of bulls, and it can survive in processed semen. Experimental studies have shown that Mbo inoculation into the uterus or insemination with Mbo-infected semen can cause bursitis, salpingitis, abortion, and infertility. The control of Mbo is very difficult because of latent carrier animals, increasing resistance to antibiotics, and unavailability of effective vaccines. The aim of this study was to follow the passage of Mbo infection from naturally contaminated semen to transferable embryos during bovine invitro embryo production (IVP). (Unless otherwise stated, all chemicals used were purchased from Sigma-Aldrich.) Two batches of slaughterhouse-derived oocytes were matured in tissue culture medium 199 (TCM-199) with glutamax-I (Gibco™; Invitrogen Corporation) supplemented with 0.25mM sodium pyruvate, 100IUmL−1 penicillin, 100µgmL−1 streptomycin, 2ngmL−1 FSH (Puregon, Organon), 1µgmL−1 β-oestradiol (E-2257), and 10% heat-inactivated fetal bovine serum (FBS; Gibco™) for 24h at 38.5°C in maximal humidity in 5% CO2 in air. Matured oocytes were fertilized for 20h in IVF-TL medium supplemented with 10µgmL−1 of heparin and 2mM of PHE at 38.5°C in maximal humidity in 5% CO2 in air, using spermatozoa per mL as a final concentration. The batches of oocytes were divided between uninfected IVP bull (N=205) and naturally Mbo-infected AI bull (N=690). Zygotes were cultured in G1/G2 media (Vitrolife) supplemented with bovine serum albumin, fatty acid free (4mgmL−1), at 38.5°C in maximal humidity in 5% O2, 5% CO2, and 90% N2. Blastocysts were collected for Mbo cultures on Days 7 and 8 (IVF=Day 0). Samples of washed semen, fertilization medium, cumulus cells, culture medium, all wash media, and transferable embryos (with and without zona pellucidae) were collected for Mbo cultures. Half of the embryos were treated with trypsin according to IETS standards after the collections. The Mbo cultures were performed in accordance with procedures previously described by Bölske (1988 Zentralbl. Bakteriol. Mikrobiol. Hyg. A 69, 331-340), followed by detection with real-time PCR. Infection with Mbo does not seem to have negative effects on fertilization (cleavage rates: 77.1% and 89.0% for IVP and Mbo AI bulls, respectively) or embryo development rates (blastocyst rate: 26.3% and 32.5% for IVP and Mbo AI bulls, respectively). Following Mbo cultures, only washed semen was found to be Mbo positive via real-time PCR. We conclude that M. bovis is not likely transmitted in bovine IVP when using naturally infected semen. We acknowledge Tiina Kortelainen for technical assistance and the Ministry of Agriculture and Forestry for funding.

Alternative pathway of complement activation has a beneficial role against Chandipura virus infection.

The complement system is a critical component of both innate and adaptive immune responses. It has both protective and pathogenic roles in viral infections. There are no studies regarding the role of complement system in Chandipura virus (CHPV) infection. The current study has investigated the role of complement pathways in the in vitro neutralization of CHPV in Vero E6 cells. Using normal human serum (NHS), heat-inactivated serum (HIS), human serum deficient of complement factor, respective reconstituted serum, assays like in vitro neutralization, real-time PCR, and flow cytometry-based tissue culture-based limited dose assay (TC-LDA) were carried out for assessing the activation of different complement pathways. NHS from 9/10 donors showed complement dependent neutralization, reduction in viral load and decrease in percentage of CHPV-positive cells compared to their HIS counterparts. EGTA or EDTA pretreatment experiments indicated that CHPV neutralization proceeds through the alternative pathway of the complement activation. Our data showed a strong dependence on C3 for the in vitro neutralization of CHPV. Disparity in CHPV neutralization levels between factor B-deficient and reconstituted sera could be attributed to amplification loop/“tick-over” mechanism. Assays using C3, C5, and C8 deficient sera indicated that complement-mediated CHPV neutralization and suppression of CHPV infectivity are primarily through C3 and C5, and not dependent on downstream complement factor C8. With no specific anti-viral treatment/vaccine against Chandipura, the current data, elucidating role of human complement system in the neutralization of CHPV, may help in designing effective therapeutics.

Antibiofilm efficacy of the gold compound auranofin on dual species biofilms of Staphylococcus aureus and Candida sp.

Staphylococcus aureus (a bacterial pathogen) and Candida sp. (opportunistic fungi) are two clinically relevant biofilm-forming microbes responsible for a majority of community- and nosocomial-acquired infections. Dual species biofilm formation between S. aureus and Candida sp. extremely enhances the antimicrobial resistance of the micro-organisms and is difficult to treat with antibiotic therapy. Hence, it is crucial to explore new antimicrobial agents. Auranofin (AF) is a mixed ligand gold compound and has recently been repurposed as an antibacterial and antifungal agent. However, the effects of AF against dual species biofilm have remained largely untested. In the present study, by constructing biofilms on microplates and urinary catheter surfaces, AF showed strong planktonic cells and biofilm inhibitory effects against mono- and dual culture models of S. aureus and Candida albicans but only exhibited moderate antibiofilm effects on Candida parapsilosis. Auranofin could be synergistic with subminimal inhibitory concentrations of amphotericin B against S. aureus+C. albicans/C. parapsilosis dual biofilms. Auranofin also showed effective antimicrobial effects on vancomycin-resistant strains. However, the antimicrobial effects of AF were decreased in the presence of heat-inactivated foetal bovine serum. In summary, AF could effectively inhibit S. aureus and C. albicans mono- and dual biofilm formation in vitro. Coexistence between Staphylococcus aureus and Candida sp. in dual biofilms leads to increased resistance to some conventionally used antimicrobials, indicating a need for alternative treatments. This study demonstrates the potential for the Au-containing compound AF in the treatment of dual biofilm infections and encourages further investigation of this treatment for clinical use.

Neurokinin-1 Receptor Antagonism Ameliorates Dry Eye Disease by Inhibiting Antigen-Presenting Cell Maturation and T Helper 17 Cell Activation

Neuroinflammation plays an important role in the pathogenesis of ocular surface disease, including dry eye disease (DED), but little is known about the contribution of substance P (SP) to DED. In this study, we investigated the expression of SP at the ocular surface and evaluated its effect on maturation of antigen-presenting cells (APCs), the key cell component involved in the induction of type 17 helper T-cell (Th17) response in DED. The effect of topical blockade of SP signaling was further investigated using neurokinin-1 receptor (NK1R) inhibitors on APC maturation, Th17 cell activation, and disease severity in a mouse model of DED. The results demonstrate that SP is constitutively expressed at the ocular surface, and trigeminal ganglion neurons are the major source of SP in DED. SP derived from trigeminal ganglion enhanced the expression of major histocompatibility complex class II maturation marker by bone marrow-derived dendritic cells, an effect that is abrogated by blockade of SP signaling using NK1R antagonist spantide. Finally, using a well-established murine model of DED, topical treatment of DED mice with NK1R antagonists CP-99,994 and L-733,060 suppressed APC acquisition of major histocompatibility complex class II, reduced Th17 cell activity, and ameliorated DED severity. These findings are of translational value, as they suggest that antagonizing NK1R-mediated SP signaling may be an effective strategy in suppressing Th17-mediated ocular surface disease.