Fetal Calf Research Articles

Development and Validation of a Low-Cost, High-Fidelity Simulation Model for Robotic Internal Mammary Artery Harvest Using the da Vinci Xi Robot.

We created and validated a low-cost simulation model for robotic internal mammary artery (IMA) takedown. The simulation model utilized a calf fetus thorax cavity stented open internally and secured to a table. The simulation model was validated at a 2-day robotic cardiac surgery workshop. Each participant harvested one IMA using the da Vinci Xi robot (Intuitive Surgical, Sunnyvale, CA, USA). We compared participant self-reported confidence at robotic IMA harvest before and after using the simulator. Our novel thorax-securing strategy resulted in a stable structure and allowed access to both IMAs from the same 3 ports. The cost to set up the first simulation model was $176 and $133 for every subsequent model. Fifty participants used the simulation model: 42 cardiothoracic surgery attendings and 8 fellows or residents. The feedback form response rate was 78% (n = 39). On the Likert scale, participants rated realism of the calf model to simulate robotic IMA harvesting (0 = not realistic, 10 = highly realistic) with a median of 8 out of 10 (interquartile range [IQR] 7 to 9). Participant confidence (0 = not at all confident, 10 = very confident) in robotic IMA harvesting before and after using the simulator increased (P = 0.001) from a median of 5 (IQR 1 to 7) to 9 (IQR 7 to 10). This robotic IMA harvest simulation model is affordable, realistic, and improved participant confidence in robotic IMA harvest. It may provide a valuable training tool for surgeons learning robotic coronary bypass surgery and allows for training frequency necessary to pass basic learning curves.

Human peripheral blood age-associated (CD11c+Tbet+) B cells: No association with age.

Human peripheral blood age-associated (CD11c+Tbet+) B cells: No association with age.

Physical Characteristics of SurgiMend Meshed Biological ADM in Immediate Prepectoral Implant Breast Reconstruction.

Summary:With increasing acceptance of prepectoral implant breast reconstruction, there has been a requirement for biological acellular dermal matrices with conformational properties, high tensile strength, and rapid integration. SurgiMendPRS Meshed is a biological acellular dermal matrix derived from fetal calf with these specific characteristics for prepectoral implant breast reconstruction. The aim of this study was to test the performance of this mesh by recreating its surgical use ex-vivo using a variety of implants in an effort to define its physical properties. The mesh is usually attached with a number of interrupted sutures to the implant periphery, the variable being at the inferior border, where it can be attached as a snug fit at the level of the inframammary crease (“tent” technique) or sewn behind the implant, cradling the lower pole (“hammock” technique). The results show mesh elasticity to stretch with increasing implant weight. When used as a “hammock,” greater stretch was demonstrated compared with the “tent” technique, allowing greater degrees of ptosis to be achieved. The suture points demonstrated lines of tension that progress evenly over the anterior implant surface. The mesh performed better when used at maximum stretch, but should not be forcibly stretched over an implant as the lines of stress show uneven distribution of lines of tension. These data provide a structural basis on optimum clinical use of this acellular dermal matrix in prepectoral implant breast reconstruction.

Characterization of the Microbiota Associated With 12-Week-Old Bovine Fetuses Exposed to Divergent in utero Nutrition.

A recent study reported the existence of a diverse microbiota in 5-to-7-month-old calf fetuses, suggesting that colonization of the bovine gut with so-called “pioneer” microbiota may begin during mid-gestation. In the present study, we investigated 1) the presence of microbiota in bovine fetuses at early gestation (12 weeks), and 2) whether the fetal microbiota is influenced by the maternal rate of gain or dietary supplementation with vitamins and minerals (VTM) during early gestation. Amniotic and allantoic fluids, and intestinal and placental (cotyledon) tissue samples obtained from fetuses (n = 33) on day 83 of gestation were processed for the assessment of fetal microbiota using 16S rRNA gene sequencing. The sequencing results revealed that a diverse and complex microbial community was present in each of these fetal compartments evaluated. Allantoic and amniotic fluids, and fetal intestinal and placenta microbiota each had distinctly different (0.047 ≥ R2 ≥ 0.019, P ≤ 0.031) microbial community structures. Allantoic fluid had a greater (P < 0.05) microbial richness (number of OTUs) (Mean 122) compared to amniotic fluid (84), intestine (63), and placenta (66). Microbial diversity (Shannon index) was similar for the intestinal and placental samples, and both were less diverse compared with fetal fluid microbiota (P < 0.05). Thirty-nine different archaeal and bacterial phyla were detected across all fetal samples, with Proteobacteria (55%), Firmicutes (16.2%), Acidobacteriota (13.6%), and Bacteroidota (5%) predominating. Among the 20 most relatively abundant bacterial genera, Acidovorax, Acinetobacter, Brucella, Corynebacterium, Enterococcus, Exiguobacterium, and Stenotrophomonas differed by fetal sample type (P < 0.05). A total of 55 taxa were shared among the four different microbial communities. qPCR of bacteria in the intestine and placenta samples as well as scanning electron microscopy imaging of fetal fluids provided additional evidence for the presence of a microbiota in these samples. Minor effects of maternal rate of gain and VTM supplementation, and their interactions on microbial richness and composition were detected. Overall, the results of this study indicate that colonization with pioneer microbiota may occur during early gestation in bovine fetuses, and that the maternal nutritional regime during gestation may influence the early fetal microbiota.

PSII-B-28 Investigation of the presence of bacterial microbiota in 12-week-old bovine fetuses

Abstract A recent study reported the existence of a diverse microbiota in 5-to-7-month-old calf fetuses, suggesting that colonization of the bovine gut with so-called “pioneer” microbiota may begin during mid-gestation. In the present study, we investigated the microbiota in bovine fetuses at early gestation. Amniotic and allantoic fluids, and intestinal and placental (cotyledon) tissue samples harvested from fetuses (n = 33) on day 83 of gestation were processed for the assessment of fetal microbiota using 16S rRNA gene sequencing. The sequencing results revealed that a diverse and complex microbial community was present in allantoic and amniotic fluids, and fetal intestine and placenta on day 83 of gestation in beef cattle. Microbial community structure was significantly different between allantoic and amniotic fluid, and intestinal and placental microbiota (0.047 ≥ R2 ≥ 0.019, P ≤ 0.031). Allantoic fluid had a greater (P &amp;lt; 0.05) microbial richness (number of OTUs) (122 ± 10) compared to amniotic fluid (84 ± 6), intestine (63 ± 7) and placenta (66 ± 6). Microbial diversity (Shannon index) was similar for the intestinal and placental samples, and both were less diverse compared to the fetal fluid microbiota (P &amp;lt; 0.05). At the phylum level, 39 different archaeal and bacterial phyla were detected across all fetal samples, with Proteobacteria (55%), Firmicutes (16.2%), Actinobacteria (13.6%) and Bacteroidetes (5%) predominating. Among the 20 most relatively abundant bacterial genera, Acidovorax, Acinetobacter, Brucella, Corynebacterium, Enterococcus, Exiguobacterium and Stenotrophomonas differed by fetal sample type (P &amp;lt; 0.05). A total of 55 taxa were shared among the four different microbial communities. qPCR of bacteria in the intestine and placenta samples as well as scanning electron microscopy imaging of fetal fluids provided additional evidence for the presence of a microbiota in these samples. Overall, the results of this study indicate that colonization with pioneer microbiota may occur during early gestation in bovine fetuses.

Comparative characteristics of monocytes isolation conditions by adhesion method for in vitro experiments

Introduction . Monocytes and macrophages are cells that play an important role in the function of the innate immune system and may be involved in the pathological process in various diseases. For this reason, in vitro studies of the functional activity of these cells are of great scientific interest. The literature indicates a large number of methods used to isolate primary monocytes but the approaches are often controversial. Aim . To perform a comparative analysis of the influence of various conditions on monocytes adhesion efficiency to determine the most optimal combinations of factors providing the highest yield and purity of the obtained cells. Materials and methods . Peripheral venous blood samples were obtained from 6 healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated by standard centrifugation on Ficoll density gradient (1.077 g/ml). After the centrifugation, PBMC were plated in wells of a 24-well plate, coated with collagen or uncoated, and incubated for 2 or 24 hours in RPMI-1640 medium containing 10% fetal calf (FCS) or autologous human serum (AHS). The number of attached monocytes was assessed by epifluorescence microscopy after staining with Hoechst 33342 with automatic counting of nuclei in ten fields of view. Results . It has been established that incubation of cells for 2 hours in wells without coating in the presence of 10% FCS allows obtaining the largest number of monocytes with median purity of 80.9 (66.5-89.1)%. The number of monocytes obtained by adhesion in collagen-coated wells tended to be lower, regardless of the type of serum used. In our experiment, the adhesion capacity of monocytes was reduced when cells were plated in uncoated wells with 10% AHS. Extended to 24 hours incubation time was accompanied by a significant decrease in the number of attached monocytes in most cases. Conclusion . Incubation of PBMC for 2 hours on uncoated tissue culture plastic in the presence of 10% FCS provides the highest yield and purity of monocytes in comparison with other tested conditions.

Culture and storage of pig embryos

Studies have consistently demonstrated that 4-cell pig embryos can be cultured to the blastocyst stage in a simple salt solution containing bovine serum albumin (BSA). Pig embryos appear to be detrimentally affected by lower levels of lactate and pyruvate than are mouse embryos, but in general their in-vitro requirements are similar. Results from embryos cultured between the 4-cell and blastocyst stages are consistent enough to allow the use of culture for the storage and shipment of pig embryos. However, results obtained after culture and transfer indicate a reduction in viability similar to that observed for cultured cattle and mice embryos. Embryos collected earlier than the 4-cell stage have been difficult to support in vitro beyond one or two cleavage divisions. Pig blastocysts may benefit from serum in their in-vitro environment: lamb, fetal calf and human serum supported continued development but pig serum was detrimental. There are conflicting reports on the efficacy of a more complete medium (Minimum Essential Medium) on the growth of pig embryos. Embryo culture will almost certainly contribute to the application of biotechnology to embryos by providing more appropriate environments for in-vitro manipulations and also, possibly, by providing a system for the early selection of desirable embryonic genotypes.

Optimization of cultivation parameters for bovine respiratory syncytial virus strain Vologda/2019

There are currently many controversial issues in the study of bovine respiratory syncytial infection. In this regard, it is relevant to study the biological properties of the virus, optimize the methods of its cultivation and select the most technologically advanced methods of designing diagnostic and prevention tools for this disease. The aim of this work was to select sensitive cell systems and to optimize the cultivation parameters in selected cell cultures. The Vologda/2019 strain of the bovine respiratory syncytial infection virus isolated from biological material obtained from a calf with respiratory symptoms was used in the experiment. The strain was adapted to the continuous cell culture derived from bovine turbinate tissue (BT) and deposited in the State collection of microorganism strains at FGBI “ARRIAH”. It was established that the continuous cell lines of fetal bovine trachea (FBT) and calf kidney (RBT) are the most sensitive cell systems for the reproduction of the bovine respiratory syncytial virus strain Vologda/2019, the maximum accumulation of the virus was observed in these cell cultures. The cytopathic activity of the virus in the FBT cell culture ranged from 4.78 ± 0.18 to 5.50 ± 0.16 lg TCID 50 /cm 3 , and in the RBT cell culture – from 4.00 ± 0.23 to 4.75 ± 0.20 lg TCID 50 /cm 3 at days 4–5 of cultivation. It was determined that in case of multiplicity of inoculation of FBT and RBT cell cultures with the virus at 0.1 lg TCD 50 /cell and the use of 2% glutamine in the maintenance nutrient medium, as well as 2% horse or cattle blood serum, it is possible to obtain virus material with high cytopathic activity.

CULTURAL PROPERTIES OF BOVINE RESPIRATORY SYNCYTIAL VIRUS STRAIN AB1908

Cattle respiratory diseases are some of the most spread pathologies that can cause economic damage, resulting from fi nancial losses and costs of treatment and diagnostics. One of the major factors contributing to respiratory pathology development is bovine respiratory syncytial infection. The analysis of serological testing, performed by the FGBI “ARRIAH” Reference Laboratory for Cattle Diseases in 2017–2018, showed that respiratory syncytial virus seroprevalence in animals of dairy farms is 60%. Herewith, it was noted that the most susceptible animals to this infection are calves under one year of age. The eff ectiveness of bovine respiratory syncytial infection control measures depends on timely diagnosis; that is why reliable and accurate diagnostic tools are needed, including optimal techniques of virus isolation from pathological material. For successful virus isolation from clinical samples, it is necessary to adhere strictly to optimal parameters of this agent cultivation. This paper presents data on study of bovine respiratory syncytial virus strain AB 1908 cultural properties. The tests performed showed that a continuous bovine turbinate (BT) cell line, continuous bovine fetal trachea (FBT) cell line and continuous bovine calf kidney (RBT) cell line are sensitive for cultivation of this agent and can be used to prepare viral suspension, needed for further research. Virus titre in BT cell culture was 4.33 ± 0.16 – 4.66 ± 0.12 lg TCID50/ cm3, in RBT cell culture – 4.33 ± 0.33 – 4.7 ± 0.36 lg TCID50/cm3 and in FBT cell culture – 4.13 ± 0.20 – 4.78 ± 0.17 lg TCID50/cm3. The following virus cultivation optimal parameters were also determined during this study: the age of the culture for virus inoculation should be 1–2 days and multiplicity of inoculation should be 0.1 TCID50/cell.

Dairy cow reproduction under the influence of heat stress.

Dairy farming is vulnerable to global warming and climate change. Improving and maintaining conception rates (CRs) have a paramount importance for the profitability of any dairy enterprise. There is an antagonistic relationship between fertility and milk yield, and intensive selection for milk yield has severely deteriorated reproductive efficiency. Irrespective of geography and husbandry, modern dairy cows experience heat stress (HS) effects leading to fertility declines, but it worsens in tropical climates. The threshold of HS experience among modern dairy cow has lowered, leading to decreased thermal comfort zone. Studies show that this threshold is lower for fertility than for lactation. HS abatement and robustness response to lactation yield lead to negative energy balance, and cow's reproductive requirements remain unfulfilled. The adverse effects of HS commence from developing oocyte throughout later stages and its fertilization competence; the oestrus cycle and oestrus behaviour; the embryo development and implantation; on uterine environment; and even extend towards foetal calf. Even cows can become acyclic under the influence of HS. These harmful effects of HS arise due to hyperthermia, oxidative stress and physiological modifications in the body of dairy cows. Proper assessment of HS and efficient cooling of dairy animals irrespective of their stage of life at farm is the immediate strategy to reduce fertility declines. Other long- and short-term mitigation strategies to reduce fertility declines during HS include feeding care, reducing disease and mastitis rates, using semen from cooled bulls, timed artificial inseminations (AI), allied hormonal interventions and use of embryo transfer technology. Ultimate long-term solution should be well-planned breeding for fertility improvement and HS tolerance.

Fetal bovine serum (FBS): Past – present – future

The supplementation of culture medium with fetal bovine serum (FBS, also referred to as "fetal calf serum") is still common practice in cell culture applications. Due to a number of disadvantages in terms of quality and reproducibility of in vitro data, animal welfare concerns, and in light of recent cases of fraudulent marketing, the search for alternatives and the development of serum-free medium formulations has gained global attention. Here, we report on the 3rd Workshop on FBS, Serum Alternatives and Serum-free Media, where regulatory aspects, the serum dilemma, alternatives to FBS, case-studies of serum-free in vitro applications, and the establishment of serum-free databases were discussed. The whole process of obtaining blood from a living calf fetus to using the FBS produced from it for scientific purposes is de facto not yet legally regulated despite the existing EU-Directive 2010/63/EU on the use of animals for scientific purposes. Together with the above-mentioned challenges, several strategies have been developed to reduce or replace FBS in cell culture media in terms of the 3Rs (Refinement, Reduction, Replacement). Most recently, releasates of activated human donor thrombocytes (human platelet lysates) have been shown to be one of the most promising serum alternatives when chemically-defined media are not yet an option. Additionally, new developments in cell-based assay techniques, advanced organ-on-chip and microphysiological systems are covered in this report. Chemically-defined serum-free media are shown to be the ultimate goal for the majority of culture systems, and examples are discussed.

The role of herpesviruses (BoHV-1 and BoHV-4) and pestiviruses (BVDV and BDV) in ruminant abortion cases in western Turkey.

The economic impact of abortions in ruminant breeders is one of the biggest problems in livestock. Of the infectious agents, viruses, especially herpesviruses and pestiviruses, are the most important causative agents of abortion in ruminants. In the present study, the role of herpesviruses (bovine herpesvirus-1 (BoHV-1), bovine herpesvirus-4 (BoHV-4)) and pestiviruses (bovine viral diarrhea virus (BVDV), border disease virus (BDV)) was investigated in cases of ruminant abortion between 2007 and 2015 in western Turkey. Out of 81 aborted fetal samples (60 calves, 19 lambs, and 2 kids), 42 were positive, which included 31 calves, 9 lambs, and 2 goats; 39 aborted fetal samples were negative for the pestivirus antigen ELISA. BoHV-1 antigen ELISA was positive in 3 cases which included 2 calves and 1 lamb; the remainder 78 cases were negative. Pestivirus and BoHV-1 were positive in 51.85 and 3.70%, respectively, of the samples. According to PCR analysis, BoHV-4 was not encountered in any of the tested samples. In one of the calf fetus samples, both BVDV and BoHV-1 were positive; in one of the lamb fetus samples, BoHV-1 was positive. There was a much higher level of pestivirus antigen than the other viral agents evaluated in the study (p < 0.0001). The results of this study indicate that pestiviruses are a common viral cause of ruminant abortions in the examined area.

Hyaluronan synthase-2 upregulation protects smpd3-deficient fibroblasts against cell death induced by nutrient deprivation, but not against apoptosis evoked by oxidized LDL

The neutral type 2 sphingomyelinase (nSMase2) hydrolyzes sphingomyelin and generates ceramide, a major bioactive sphingolipid mediator, involved in growth arrest and apoptosis. The role of nSMase2 in apoptosis is debated, and apparently contradictory results have been observed on fibroblasts isolated from nSMase2-deficient fragilitas ossium (homozygous fro/fro) mice. These mice exhibit a severe neonatal dysplasia, a lack of long bone mineralization and delayed apoptosis patterns of hypertrophic chondrocytes in the growth plate. We hypothesized that apoptosis induced by nutrient deprivation, which mimics the environmental modifications of the growth plate, requires nSMase2 activation. In this study, we have compared the resistance of fro/fro fibroblasts to different death inducers (oxidized LDL, hydrogen peroxide and nutrient starvation). The data show that nSMase2-deficient fro/fro cells resist to apoptosis evoked by nutrient starvation (fetal calf serum/glucose/pyruvate-free DMEM), whereas wt fibroblasts die after 48h incubation in this medium. In contrast, oxidized LDL and hydrogen peroxide are similarly toxic to fro/fro and wt fibroblasts, indicating that nSMase2 is not involved in the mechanism of toxicity evoked by these agents. Interestingly, wt fibroblasts treated with the SMase inhibitor GW4869 were more resistant to starvation-induced apoptosis.The resistance of fro/fro cells to starvation-induced apoptosis is associated with an increased expression of hyaluronan synthase 2 (HAS2) mRNAs and protein, which is inhibited by ceramide. In wt fibroblasts, this HAS2 rise and its protective effect did not occur, but exogenously added HA exhibited a protective effect against starvation-induced apoptosis.The protective mechanism of HAS2 involves an increased expression of the heat-shock protein Hsp72, a chaperone with antiapoptotic activity. Taken together, these results highlight the role of nSMase2 in apoptosis evoked by nutrient starvation that could contribute to the delayed apoptosis of hypertrophic chondrocytes in the growth plate, and emphasize the antiapoptotic properties of HAS2.

Loss of CD62L Expression in CD4+FoxP3+ Regulatory T Cells Following Freeze-and-Thaw Prevents Protection Against GVHD in Murine Models

Backgrounds: CD4+FoxP3+ regulatory T cells (Treg) have been shown in pre-clinical murine models and human clinical trials to protect against acute graft-versus-host (GVHD) disease following hematopoietic cell transplantation (HCT). In murine models, Treg expression of CD62L is required for in vivo protection of GVHD (Strober 2005/Blazer 2004). Certain aspects of Treg isolation and administration could affect CD62L expression, such as the use of G-CSF mobilization or freeze-and-thaw. We hypothesized that freeze-thaw of Tregs could reduce CD62L expression and prevent Tregs from protecting against GVHD. Furthermore, we hypothesized that the loss of CD62L expression is mediated at least in part by metalloproteinases (MP).Methods and Results: We used established protocols of GVHD, in which lethal dose of fractionated total body irradiation was followed by murine bone marrow transplantation across major histocompatibility barriers. We sort-purified CD4+Foxp3+ Tregs based on CD25+(high) expression and measured baseline expression of CD62L. Cells were then gradually frozen in fetal calf serum/10%DMSO, and finally stored in liquid nitrogen for at least 7 days. After thawing Tregs showed an average of 58% of the baseline CD62L expression (CD62L expression fresh vs. frozen p=0.0009, n=5). Binding of frozen Tregs to plate bound L-Selectin ligand Mucosal addressin cell adhesion molecule-1 (MAdCAM-1) in vitrowas significantly reduced as compared to fresh Tregs (p=0.0003). To test the potential effect of freeze and thaw in vivo, we used an established murine model in which Tregs are given prior to conventional T cells (Tcon) to protect against GHVD. At the time of transplant conditioned BALB/c donors received 5x10^6 whole bone marrow and 5x10^5 CD4+CD25(high) Tregs (day 0), followed by 1x10^6 Tcon (day +2). Frozen/thawed Tregs and fresh Tregs respectively were given at the same time point at the same quantity of viable cells. Using luciferase-expressing cells (luc+) and BLI imaging we could show that homing of frozen luc+Tregs into pLN at day+5 was impaired as compared to fresh Tregs (p=0.0005). Relative weight loss as an indicator for GVHD was significantly higher and survival was significantly reduced in mice receiving frozen Treg in comparison to mice receiving fresh Treg. To confirm the importance of CD62L expression on Treg function we blocked CD62L in vivo by incubating fresh Tregs with monoclonal blocking antibody Mel14 (120ug/ml) for 1 hour prior to injection. A significantly higher proliferation of luc+ Tcon was observed in mice injected with Mel14-treated Tregs as compared to isotype control-treated Tregs. To test the potential role of metalloproteinases in cleaving CD62L following freeze and thaw, we added various metalloproteinase inhibitors (MPI) during the freezing process. We found that the addition of at least one MPI GM6001 (50nM) can diminish the loss of CD62L.Conclusion: Augmenting or manipulating Tregs of the donor graft is a promising area of cellular therapy that could be used to prevent GVHD in HCT and in potentially many other clinical contexts. We have found that the process of freeze and thaw reduces CD62L expression on Tregs and reduces protection against GVHD. Further, MPs may play role in the loss of CD62L expression and the use of MPIs for frozen products might preserve Treg function. DisclosuresNo relevant conflicts of interest to declare.

Isolation and characterization of mesenchymal stem cells derived from fetal bovine liver

Bovine liver-derived mesenchymal stem cells (bLMSCs) were isolated from the liver tissue of 4-6 months old fetal calf, and then characterized by immunofluorescence and RT-PCR. We found that primary bLMSCs could be subcultured to 44 passages, the total culture time in vitro was 192 days. The results of surface antigen detection showed that bBMSCs expressed CD29, CD44, CD73, CD90, CD106 and CD166 but not expressed endothelial cells and hematopoietic cells specific marker CD34, CD45 and BLA-DR. The results of growth kinetics, colony-forming cell assay and cell cycle analysis indicated that the fetal bovine LMSCs had good proliferation ability in vitro. The cells from passages 7 were successfully induced to differentiate into osteoblasts, adipocytes and chondrocytes. The results indicate the potential for multi-lineage differentiation of bLMSCs that may represent an ideal candidate for cellular transplantation therapy.

Reply to ‘TLR-2 ligand lipomannan from Mycobacterium tuberculosis does not stimulate inflammatory cytokines in dendritic cells’

We thank Hedge et al.[1] for sharing their data obtained with monocyte-derived dendritic cells stimulated with Mycobacterium tuberculosis (MTB)-derived lipomannan. We agree that future studies investigating Toll-like receptor (TLR)2-mediated responses should use a TLR2 ligand derived from the mycobacteria species of interest when one is available. For example, inactivated or whole cell lysate of pathogenic MTB containing all types of TLR2 ligands can be used to investigate immune restoration disease (IRD) associated with MTB infections. We used lipomannan from Mycobacterium smegmatis to demonstrate elevated responses in primary isolate cells from HIV coinfected patients experiencing an IRD associated with MTB [2]. Hedge et al.[1] assert that the responses to lipomannan derived from M. smegmatis would not have been seen had we used lipomannan from MTB. Data in Figure 1 of their letter showed no effect of lipomannan from MTB on cytokine production or the maturation of dendritic cell obtained by 6-day culture of blood monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-4. However, they did not provide data from a positive control (which could be another TLR-2 ligand, such as peptidoglycan or lipotechoic acid from gram-positive bacteria) or M. smegmatis-derived lipomannan. If lipomannan from M. smegmatis also failed to induce tumour necrosis factor (TNF)α, IL-6, IL-1β, IL-12p70, IL-10 or dendritic cell maturation in their system, then their criticism would be invalid. Without a positive control, we question the validity of their assay to measure TLR2-mediated responses of myeloid dendritic cell (mDC). It is important to note that we stimulated peripheral blood mononuclear cells with lipomannan in 10% fetal calf serum/RPMI-1640 medium without any cytokine supplement (e.g. GM-CSF or IL-4) for just 24 h. Concentrations of TNFα, IL-10 and IL-12p40 were then measured in culture supernatant by ELISA. We concluded that these cytokines were probably made by monocytes and/or mDC as we established that these two cell populations expressed much higher levels of TLR2 than other cells. Hedge et al.[1] do not describe TLR2 expression on dendritic cell derived with their protocol. The absence of a response to lipomannan from MTB could be explained by a lack of TLR2 expression. It has been shown that monocyte-derived dendritic cell expressed TLR2 messenger RNA but have lower expression of surface TLR2 receptors compared to freshly isolated monocytes or mDC [3]. Our culture conditions are also different as T-cells were present during stimulation. TLR signals also affect adaptive responses via antigen presenting cell-dependent or cell-independent mechanisms, as reviewed previously [4]. Activated T-cells express TLR2 [5]. Hence, responses mediated by TLR2 ligands may be higher in the presence of T-cells. The structure and immune stimulatory functions of some TLR2 ligands from pathogenic strains of mycobacteria (e.g. MTB) differ from those from nonpathogenic mycobacteria (e.g. M. smegmatis) [6,7]. The critical TLR2 ligands on the mycobacterial cell wall are lipomannan and lipoarabinomannan (LAM). Several studies have investigated responses to lipomannan or LAM by monocyte/macrophage. Lipomannan from pathogenic mycobacteria (e.g. MTB, Mycobacterium kansasii and Mycobacterium chelonae) was able to induce IL-12 production in human macrophage cell line (THP1) and mouse bone marrow-derived macrophages. However, LAM from these pathogenic mycobacteria inhibited IL-12 production in the same cells [8]. Similar results were observed using lipomannan and LAM from Mycobacterium bovis Bacillus Calmette Guerin[9]. Stimulation of THP-1 cells with lipomannan from M. kansasii and M. chelonae induced production of TNFα and IL-8 via TLR2 [6]. TLR2-mediated responses of macrophages to lipomannan were shown to be regulated by mitogen-activated protein kinase pathways [10]. These data suggest that lipomannan from pathogenic (e.g. MTB) and nonpathogenic (e.g. M. smegmatis) mycobacteria is generally proinflammatory. This validates our decision to use lipomannan from M. smegmatis in our study, as it was readily available from a commercial source. The virulence of MTB and other pathogenic mycobacteria was associated with downregulation of TLR2-dependant immune responses by LAM, but not lipomannan. Several studies suggest that the balance of proinflammatory vs. anti-inflammatory responses against mycobacteria infection is probably influenced by the ratio of lipomannan : LAM [11,12]. Future studies should consider the possibility that the presence of lipomannan and/or LAM in lung-associated lymph nodes or in the circulation of HIV patients coinfected with MTB at the commencement of antiretroviral therapy may be a risk factor for MTB-associated IRD. Acknowledgements Conflicts of interest There are no conflicts of interest.

Radiolabeling of DOTATOC with the long-lived positron emitter 44Sc

The positron-emitting radionuclide 44Sc with a half-life of 3.97h and a β+ branching of 94.3% is of potential interest for clinical PET. As so far it is available from a 44Ti/44Sc generator in Mainz, where long-lived 44Ti decays to no-carrier-added (nca) 44Sc. The 44Sc is a trivalent metal cation and should be suitable for complexation with many well established bifunctional chelators conjugated to peptides or other molecular targeting vectors. Thus, the aim of this work was to investigate the potential of 44Sc for labeling of DOTA-conjugated peptides. DOTA-D-Phe1-Tyr3-octreotide (DOTATOC) was used as a model molecule to study and optimize labeling procedure. Reaction parameters such as buffer conditions, concentration of peptide, pH range, reaction temperature and time were optimized. Addition of 21nmol of DOTATOC to 44Sc in ammonium acetate buffer pH 4.0 provided labeling yields >98% within 25min of heating in an oil-bath at 95°C. This time can be reduced to 3min only by applying microwave supported heating. 44Sc-DOTATOC was found to be stable in 0.9% NaCl, PBS pH 7.4, fetal calf and human serums, and also in the presence of competing metal cations (Fe3+, Ca2+, Cu2+, Mg2+), as well as other ligand competitors, like EDTA and DTPA, even after almost 25h incubation at 37°C. Present study shows that nca 44Sc forms stable complexes with the macrocyclic ligand DOTA and that 44Sc-DOTATOC and analog targeting vectors may be synthesized for further preclinical and clinical investigations.

Effects of different concentrations of sevoflurane on adhesion and expression of CD24 and CD44v6 in human lung cancer cell line A549

Objective To investigate the effects of different concentrations of sevoflurane on adhesion and expression of CD24 and CD44v6 in human lung cancer cell line A549.Methods Human lung cancer cell line A549 was obtained from Shanghai Cell Biology Medical Research Institute,Chinese Academy of Sciences and cultured in RPMI1640 culture medium containing 10％ fetal calf serum.The cells were inoculated in 24 well culture plate.After being cultured for 24 h,the cells were randomly divided into 4 groups:control group (group C) and 3 sevoflurane groups exposed to 1.7 ％,3.4 ％ and 5.1％ sevoflurane for 2,4 and 6 h respectively ( groups S1,S2,S3 ).The cells were cultured for another 48 h.Cell adhesion rate was detected by adhesion test and the expression of CD24 and CD44v6 mRNA and protein was determined by RT-PCR and flow cytometry.Results Sevoflurane significantly inhibited the cell adhesion rate and down-regulated CD24 and CD44v6 expression in a concentration and duration of exposure-dependent manner.Conclusion Sevoflurane can inhibit cell adhesion through down-regulation of CD24 and CD44v6 expression. Key words: Anesthetics,inhalation; Cell adhesion; Antigens,CD24; Antigens,CD44

An unusual case of cyclopic and arhinia monster in Mehsana buffalo

Introduction treatment, the placenta was expelled out at night without any assistance. Cyclopia is a rare form of holoprosencephaly The morphological examination of removed and is a congenital disorder (birth defect) characterized dead female fetus revealed that the fetus having a by the failure of the embryonic prosencephalon to single orbit with fused two eye balls (instead of one in properly divide the orbits of the eye into two cavities. typical cyclopia) with two separate corneas. Upper Although, most commonly seen in pig and sheep [1], it eyelid and eyelashes were present but both were is also reported in goat [2] and cattle [3]. Similarly, absent in lower area of central eye. Absence of upper congenital arhinia is an extremely rare anomaly jaw leads to protrusion of tongue from base outside the consisting of an absence of external nasal structures mouth while lower jaw was long and normal and nasal passages. The present case study puts on a (brachygnathism). Muzzle, nostrils and typical nose record of dystocia attributable to a unusual cyclopia were absent (arhinia) and that area was covered with with arhinia fetal monster calf having a typical skin above the central eye. That means the nose and monkey face and its successful per vaginum delivery mouth fail to form, or the nose grows from the roof of through obstetrical maneuvers in Mehsana buffalo. the mouth obstructing airflow, resulting in suffocation Case history and clinical observations which might lead to the death of fetus. Location of fused eyes in single orbit at forehead and absence of A four year old, apparently helathy primiparous, upper jaw and nose made the face of calf just like Mehsana buffalo at full term presented with a history monkey face. The left ear was normal where as the of straining for the last seven hours and ruptured water right one was deformed and hairs were present all over bags. Gynaeco-clinical observation, revealed a dead the body. The skin over the forehead was open fetus in anterior longitudinal presentation and left exposing a musculature mass and there was dorso-iliac position with both the forelimb flexed from rudimentary visible fused eyes which also found in shoulder, where as head was totally deviated downward non-descript cow by Gupta and Anand [3]. Ozcan et so that only ear was palpated (Nap presentation). al., [4] reported a atypical cyclopia in a brown swiss Treatment and Discussion cross calf with most significant malformation such as the presence of a median orbita-like opening that did The dystocial condition was corrected by not contain an eyeball and other defects included obstetrical maneuvers and fetus was brought to normal prosencephalic aplasia, brachygnathia superior and presentation and posture. The delivery of dead female arhinia. Similar type of arhinia and cyclopia in a fetus was carried out by forced traction applied on both German Fleckvieh calf were also reported by Schulze forelimbs and lower jaw after proper lubrication. and Disti [5]. Khasatiya [6] also reported a Thereafter, buffalo was administered with Inj. cebocephalus (cyclopia) monster in a cross-bred cow Dextrose normal saline 5% (2 Litre, I/V), Inj. Calcium and noted that epitheliogenesis imperfecta over the borogluconate (450 ml, I/V), Inj. Strpreptopenicillin forehead and body, having rudimentary and separate (5gm, I/M), Inj. Meloxicam (20 ml, I/M) and Inj. eye balls and atypical deformed ears and jaw. Genetic Dexamethasone (7.5 ml, I/M). Following above Vet. World, 2012, Vol.5(7):429-430 CLINICAL

Endothelial Cells from Embryonic Stem Cells in a Chemically Defined Medium

Endothelial cells (ECs) are desired for their therapeutic potential in a variety of areas including gene therapy, cardiac regeneration, development of tissue-engineered vascular grafts, and prevascularized tissue transplants. Pluripotent embryonic stem cells (ESCs) can be induced to differentiate into ECs in vitro using embryoid bodies, monolayer cultures, or by genetic manipulation and immortalization. However, obtaining homogeneous cultures of proliferating ESC-derived ECs without genetic manipulation is a challenging undertaking and often requires optimization of protocols and rigorous purification techniques. Moreover, current differentiation methods that use medium containing fetal calf or bovine serum components introduce additional challenges because of our limited ability to control the differentiation signals and batch-to-batch variations of serum. We have explored the development of new medium formulations for deriving ECs from murine embryonic stem cells (mESCs) using only chemically defined reagents. We present 2 different medium formulations along with the detailed methodologies, including the optimization of extracellular matrix-derived substrates known to play a role in cell attachment and proliferation as well as cell differentiation. Characterization of the ESC-derived ECs indicate that (1) chemically defined medium formulations reproducibly generate superior ECs compared with previous serum-containing formulations, (2) fibronectin, and not collagen type-IV, is the optimal substrate for EC induction in our chemically defined medium formulations, (3) without additional activation of Notch-signaling, ESC-ECs develop predominantly into venous ECs, and (4) using these medium formulations, a second rigorous selection step is not required to generate proliferating ECs from ESCs, but it does enhance the final purity of the ECs.