Differentiation of mouse embryonic stem cells into cells with spermatogonia-like morphology with chemical intervention-dependent increased gene expression of LIM homeobox 1 (Lhx1).

Spermatogonial stem cells (SSCs) originate from gonocytes that differentiate from primordial germ cells (PGCs). In the developing mouse testis, expression of the gene LIM homeobox 1 (Lhx1) marks the most undifferentiated SSCs, which has not yet been reported for spermatogonia-like cells generated in vitro. Previously, it was shown that a chemical intervention in male mouse embryonic stem (ES) cells in serum culture, including Sirtuin 1 (SIRT1) inhibitor Ex-527, DNA methyltransferase (DNMT) inhibitor RG-108 and electrophilic redox cycling compound tert-butylhydroquinone (tBHQ), was associated with molecular markers of PGC to gonocyte differentiation. Here, we report the in vitro differentiation of male mouse ES cells, cultured under dual chemical inhibition of GSK3β and MEK (2i) with leukemia inhibitory factor (LIF) (2iL) and serum, into cells with spermatogonia-like morphology (CSMs) and population-averaged expression of spermatogonia-specific genes by removal of 2iL and a specific schedule of twice daily partial medium replacement. Combination of this new protocol with the previously reported chemical intervention increased population-averaged gene expression of Lhx1 in the resulting CSMs. Furthermore, we detected single CSMs with strong nuclear LHX1/5 protein signal only in the chemical intervention group. We propose that further investigation of CSMs may provide new insights into male germline development

Case series on granulosa cell tumour in cattle with practical hints on diagnostics and outcome

Granulosa cell tumours are the most common neoplasm of the bovine ovary and present with a wide range of clinical signs. This case series comprises five case reports of ovarian granulosa cell tumours in cattle. The affected animals had different breeds (Red Holstein, Eringer, Swiss Braunvieh, crossbred beef) and ranged in age from 1 year 4 months to 20 years 11 months. The diversity of the cases gives an overview of the diagnostic possibilities as well as the possible treatments and outcomes of the disease. Anti-mullerian hormone (AMH) was measured in one of the affected animals, and was extremely elevated (>150 pmoL/L) compared to reported normal values (<3 pmoL/L). Three animals underwent standing laparotomy with unilateral ovariectomy, and the remaining two animals were slaughtered. One of the cases confirms the previously published reports that malignancy or metastasis is possible in cattle. Another case indicates that successful surgical treatment with subsequent resumption of reproduction is possible. In summary, the initial tentative diagnosis of GCT is made by ultrasound-assisted rectal examination of the genital tract or ovaries and can subsequently be supported by evaluation of AMH levels. The decision to proceed with treatment should be made without delay, as surgical removal of the altered ovary with subsequent resumption of reproductive activity and cessation of undesirable behaviour is possible

Wnt4 and LAP2alpha as pacemakers of Thymic Epithelial Senescence

Age-associated thymic involution has considerable physiological impact by inhibiting de novo T-cell selection. This impaired T-cell production leads to weakened immune responses. Yet the molecular mechanisms of thymic stromal adipose involution are not clear. Age-related alterations also occur in the murine thymus providing an excellent model system. In the present work structural and molecular changes of the murine thymic stroma were investigated during aging. We show that thymic epithelial senescence correlates with significant destruction of epithelial network followed by adipose involution. We also show in purified thymic epithelial cells the age-related down-regulation of Wnt4 (and subsequently FoxN1), and the prominent increase in LAP2α expression. These senescence-related changes of gene expression are strikingly similar to those observed during mesenchymal to pre-adipocyte differentiation of fibroblast cells suggesting similar molecular background in epithelial cells. For molecular level proof-of-principle stable LAP2α and Wnt4-over-expressing thymic epithelial cell lines were established. LAP2α over-expression provoked a surge of PPARγ expression, a transcription factor expressed in pre-adipocytes. In contrast, additional Wnt4 decreased the mRNA level of ADRP, a target gene of PPARγ. Murine embryonic thymic lobes have also been transfected with LAP2α- or Wnt4-encoding lentiviral vectors. As expected LAP2α over-expression increased, while additional Wnt4 secretion suppressed PPARγ expression. Based on these pioneer experiments we propose that decreased Wnt activity and increased LAP2α expression provide the molecular basis during thymic senescence. We suggest that these molecular changes trigger thymic epithelial senescence accompanied by adipose involution. This process may either occur directly where epithelium can trans-differentiate into pre-adipocytes; or indirectly where first epithelial to mesenchymal transition (EMT) occurs followed by subsequent pre-adipocyte differentiation. The latter version fits better with literature data and is supported by the observed histological and molecular level changes

The role of the chemokine receptor CXCR4 in infection with feline immunodeficiency virus

Infection with feline immunodeficiency virus (FIV) leads to the development of a disease state similar to AIDS in man. Recent studies have identified the chemokine receptor CXCR4 as the major receptor for cell culture-adapted strains of FIV, suggesting that FIV and human immunodeficiency virus (HIV) share a common mechanism of infection involving an interaction between the virus and a member of the seven transmembrane domain superfamily of molecules. This article reviews the evidence for the involvement of chemokine receptors in FIV infection and contrasts these findings with similar studies on the primate lentiviruses HIV and SIV (simian immunodeficiency virus)

Purification of Nanoparticles by Size and Shape

Producing monodisperse nanoparticles is essential to ensure consistency in biological experiments and to enable a smooth translation into the clinic. Purification of samples into discrete sizes and shapes may not only improve sample quality, but also provide us with the tools to understand which physical properties of nanoparticles are beneficial for a drug delivery vector. In this study, using polymersomes as a model system, we explore four techniques for purifying pre-formed nanoparticles into discrete fractions based on their size, shape or density. We show that these techniques can successfully separate polymersomes into monodisperse fractions

Thoracic Electrical Impedance Tomography—The 2022 Veterinary Consensus Statement

Electrical impedance tomography (EIT) is a non-invasive real-time non-ionising imaging modality that has many applications. Since the first recorded use in 1978, the technology has become more widely used especially in human adult and neonatal critical care monitoring. Recently, there has been an increase in research on thoracic EIT in veterinary medicine. Real-time imaging of the thorax allows evaluation of ventilation distribution in anesthetised and conscious animals. As the technology becomes recognised in the veterinary community there is a need to standardize approaches to data collection, analysis, interpretation and nomenclature, ensuring comparison and repeatability between researchers and studies. A group of nineteen veterinarians and two biomedical engineers experienced in veterinary EIT were consulted and contributed to the preparation of this statement. The aim of this consensus is to provide an introduction to this imaging modality, to highlight clinical relevance and to include recommendations on how to effectively use thoracic EIT in veterinary species. Based on this, the consensus statement aims to address the need for a streamlined approach to veterinary thoracic EIT and includes: an introduction to the use of EIT in veterinary species, the technical background to creation of the functional images, a consensus from all contributing authors on the practical application and use of the technology, descriptions and interpretation of current available variables including appropriate statistical analysis, nomenclature recommended for consistency and future developments in thoracic EIT. The information provided in this consensus statement may benefit researchers and clinicians working within the field of veterinary thoracic EIT. We endeavor to inform future users of the benefits of this imaging modality and provide opportunities to further explore applications of this technology with regards to perfusion imaging and pathology diagnosis

Statins Disrupt CCR5 and RANTES Expression Levels in CD4(+) T Lymphocytes In Vitro and Preferentially Decrease Infection of R5 Versus X4 HIV-1

BACKGROUND: Statins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1 (HIV-1) through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1 incorporation into viral particles. PRINCIPLE FINDINGS: Here we demonstrate that treatment of an enriched CD4(+) lymphocyte population with lovastatin (Lov), mevastatin (Mev) and simvastatin (activated and non-activated, Sim(A) and Sim(N), respectively) can reduce the cell surface expression of the CC-chemokine receptor CCR5 (P<0.01 for Sim(A) and Lov). The lowered CCR5 expression was associated with down-regulation of CCR5 mRNA expression. The CC-chemokine RANTES protein and mRNA expression levels were slightly increased in CD4(+) enriched lymphocytes treated with statins. Both R5 and X4 HIV-1 were reduced for their infection of statin-treated cells; however, in cultures where statins were removed and where a decrease in CCR5 expression was observed, there was a preferential inhibition of infection with an R5 versus X4 virus. CONCLUSIONS: The results indicate that the modulation of CC-chemokine receptor (CCR5) and CC-chemokine (RANTES) expression levels should be considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1 activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment could be considered as a way to modulate immune induction such as during vaccination protocols

Stromal cell-derived factor 1 (SDF-1) and antenatal human B cell lymphopoiesis: Expression of SDF-1 by mesothelial cells and biliary ductal plate epithelial cells

The chemokine stromal cell-derived factor 1 (SDF-1) stimulates the growth of pre-B cells in vitro, and mice with a disrupted SDF-1 gene have abnormal fetal liver B cell lymphopoiesis. The origin of SDF-1 production has not been determined yet. Using an anti-SDF-1 mAb, we performed immunohistochemical studies in four human embryos and five fetuses to define which cells express the SDF-1 protein at sites of antenatal B cell lymphopoiesis. All mesothelial cells contained SDF-1 at all stages of development, including in the intraembryonic splanchnopleuric mesoderm early into gestation. In fetal lungs and kidneys, SDF-1 was expressed by epithelial cells, and a few B lymphoid precursors, expressing V pre-B chains, were also detected. In the fetal liver, in addition to mesothelial cells, biliary epithelial cells were the only cells to contain SDF-1. Pre-B cells expressing V chains were abundant and exclusively located around the edge of portal spaces, in close contact with biliary ductal plate epithelial cells. They did not colocalize with biliary collecting ducts. Biliary ductal plate epithelial cells and liver B cell lymphopoiesis display a parallel development and disappearance during fetal life. These results indicate that early B cell lymphopoiesis in the splanchnopleura may be triggered by mesothelial cells producing SDF-1. Later into gestation, biliary ductal plate epithelial cells may support B cell lymphopoiesis, thus playing a role similar to that of epithelial cells in the avian bursa of Fabricius, and of thymic epithelial cells for T cell lymphopoiesis

Protocol for a phase 1 homeopathic drug proving trial

<p>Abstract</p> <p>Background</p> <p>This study protocol adapts the traditional homeopathic drug proving methodology to a modern clinical trial design.</p> <p>Method</p> <p>Multi-centre, randomised, double-blind, placebo-controlled phase 1 trial with 30 healthy volunteers. The study consists of a seven day run-in period, a five day intervention period and a 16 day post-intervention observation period. Subjects, investigators and the statisticians are blinded from the allocation to the study arm and from the identity of the homeopathic drug. The intervention is a highly diluted homeopathic drug (potency C12 = 10²⁴), Dose: 5 globules taken 5 times per day over a maximum period of 5 days. The placebo consists of an optically identical carrier substance (sucrose globules). Subjects document the symptoms they experience in a semi-structured online diary. The primary outcome parameter is the number of specific symptoms that characterise the intervention compared to the placebo after a period of three weeks. Secondary outcome parameters are qualitative differences in profiles of characteristic and proving symptoms and the total number of all proving symptoms. The number of symptoms will be quantitatively analysed on an intention-to-treat basis using ANCOVA with the subject's expectation and baseline values as covariates. Content analysis according to Mayring is adapted to suit the homeopathic qualitative analysis procedure.</p> <p>Discussion</p> <p>Homeopathic drug proving trials using the terminology of clinical trials according GCP and fulfilling current requirements for research under the current drug regulations is feasible. However, within the current regulations, homeopathic drug proving trials are classified as phase 1 trials, although their aim is not to explore the safety and pharmacological dynamics of the drug, but rather to find clinical indications according to the theory of homeopathy. To avoid bias, it is necessary that neither the subjects nor the investigators know the identity of the drug. This requires a modification to the informed consent process and blinded study materials. Because it is impossible to distinguish between adverse events and proving symptoms, both must be documented together.</p> <p>Trial registration</p> <p>ClinicalTrials.gov identifier: NCT01061229.</p