Proliferating macrophages in human tumours show characteristics of monocytes responding to myelopoietic growth factors.

Macrophages play essential roles in maintaining tissue homeostasis and immune defence. However, their extensive infiltration into tumours has been linked to adverse outcomes in multiple human cancers. Within the tumour microenvironment (TME), tumour-associated macrophages (TAMs) promote tumour growth and metastasis, making them prime targets for cancer immunotherapy. Recent single-cell analysis suggest that proliferating TAMs accumulate in human cancers, yet their origins and differentiation pathways remain uncertain. Here, we show that a subpopulation of CD163+ TAMs proliferates in situ within the TME of melanoma, lung cancer, and breast cancer. Consistent with their potential role in suppressing anti-tumour activities of T cells, CD163+ TAMs express a range of potent immunosuppressive molecules, including PD-L1, PD-L2, IL-10, and TGF-ß. Other phenotypic markers strongly suggested that these cells originate from CD14+ CCR2+ monocytes, a cell population believed to have minimal capacity for proliferation. However, we demonstrate in vitro that certain myelopoietic cytokines commonly available within the TME induce robust proliferation of human monocytes, especially the combination of interleukin 3 (IL-3) and Macrophage Colony-Stimulating Factor 1 (M-CSF). Monocytic cells cultured with these cytokines efficiently modulate T cell proliferation, and their molecular phenotype recapitulates that of CD163+ TAMs. IL-3-driven proliferation of monocytic cells can be completely blocked by IL-4, associated with the induction of CDKN1A, alongside the upregulation of transcription factors linked to dendritic cell function, such as BATF3 and IRF4. Taken together, our work suggests several novel therapeutic routes to reducing immunosuppressive TAMs in human tumours, from blocking chemokine-mediated recruitment of monocytes to blocking their proliferation.

Safety, pharmacodynamics, and antiviral activity of selgantolimod in viremic patients with chronic hepatitis B virus infection.

Background & Aims: Novel finite therapies for chronic hepatitis B (CHB) are needed, since lifelong treatment is usually required with current available oral antivirals. This phase II study (NCT03615066) evaluated the safety, pharmacodynamics, and antiviral activity of selgantolimod (a Toll-like receptor 8 agonist [TLR8]) with tenofovir alafenamide (TAF). Methods: Viremic patients with CHB not receiving treatment were stratified by HBeAg status and randomized 2:2:1 to TAF 25 mg/day with selgantolimod 3 mg orally once weekly (QW), selgantolimod 1.5 mg QW, or placebo. Combination therapy continued until week (W)24, followed by TAF monotherapy until W48; patients then discontinued TAF and were followed until W96 (treatment-free follow-up [TFFU] period). The primary efficacy endpoint was the proportion with ≥1 log10 IU/ml HBsAg decline at W24. Results: Sixty-seven patients received study drug; 27 were followed during TFFU. Nausea, headache, vomiting, fatigue, and dizziness were the most common adverse events. Most adverse events were grade 1. Alanine aminotransferase flares were not observed up to W48. Four patients experienced alanine aminotransferase and hepatitis flares during TFFU; all had HBV DNA increases. Selgantolimod increased serum cytokines and chemokines and redistributed several circulating immune cell subsets. No patients achieved the primary efficacy endpoint. Mean HBsAg changes were -0.12, -0.16, and -0.12 log10 IU/ml in the selgantolimod 3 mg, selgantolimod 1.5 mg, and placebo groups, respectively, at W48; HBV DNA declined in all groups by ≥2 log10 IU/ml as early as W2, with all groups rebounding to baseline during TFFU. No HBsAg or HBeAg loss or seroconversion was observed throughout TFFU. Conclusions: Selgantolimod up to 3 mg was safe and well tolerated. Pharmacodynamics and antiviral activity in viremic patients support continued study of selgantolimod in combination CHB therapies. Impact and implications: Novel therapeutics for chronic HBV infection are needed to achieve a functional cure. In this study, we confirmed the safety and tolerability of selgantolimod (formerly GS-9688, a TLR8) when administered with tenofovir alafenamide over 24 weeks in viremic patients with chronic HBV infection. Overall, declines in HBsAg levels with selgantolimod treatment were modest; subgroup analysis indicated that patients with alanine aminotransferase levels greater than the upper limit of normal had significantly greater declines compared to those with normal alanine aminotransferase levels (-0.20 vs. -0.03 log10 IU/ml; p <0.001). These findings suggest a potential differential response to selgantolimod based on patients' baseline HBV-specific immune response, which should be considered in future investigations characterizing the underlying mechanisms of selgantolimod treatment and in HBV cure studies using similar immunomodulatory pathways. Clinical trial number: NCT03615066 be found at https://www.gileadclinicaltrials.com/transparency-policy/.

Characterization of adult human skeletal cells in different tissues reveals a CD90+CD34+ periosteal stem/progenitor population.

The periosteum plays a crucial role in bone healing and is an important source of skeletal stem and progenitor cells. Recent studies in mice indicate that diverse populations of skeletal progenitors contribute to growth, homeostasis and healing. Information about the in vivo identity and diversity of skeletal stem and progenitor cells in different compartments of the adult human skeleton is limited. In this study, we compared non-hematopoietic populations in matched tissues from the femoral head and neck of 21 human participants using spectral flow cytometry of freshly isolated cells. High-dimensional clustering analysis indicated significant differences in marker distribution between periosteum, articular cartilage, endosteum and bone marrow populations, and identified populations that were highly enriched or unique to specific tissues. Periosteum-enriched markers included CD90 and CD34. Articular cartilage, which has very poor regenerative potential, showed enrichment of multiple markers, including the PDPN+CD73+CD164+CD146- population previously reported to represent human skeletal stem cells. We further characterized periosteal populations by combining CD90 with other strongly expressed markers. CD90+CD34+ cells sorted directly from periosteum showed significant colony-forming unit fibroblasts (CFU-F) enrichment, rapid expansion, and consistent multi-lineage differentiation of clonal populations in vitro. In situ, CD90+CD34+ cells include a perivascular population in the outer layer of the periosteum and non-perivascular cells closer to the bone surface. CD90+ cells are also highly enriched for CFU-F in bone marrow and endosteum, but not articular cartilage. In conclusion, our study indicates considerable diversity in the non-hematopoietic cell populations in different tissue compartments within the adult human skeleton, and suggests that periosteal progenitor cells reside within the CD90+CD34+ population.

Human placental vascular and perivascular cell heterogeneity differs between first trimester and term, and in pregnancies affected by foetal growth restriction.

Growth-restricted placentae have a reduced vascular network, impairing exchange of nutrients and oxygen. However, little is known about the differentiation events and cell types that underpin normal/abnormal placental vascular formation and function. Here, we used 23-colour flow cytometry to characterize placental vascular/perivascular populations between first trimester and term, and in foetal growth restriction (FGR). First-trimester endothelial cells had an immature phenotype (CD144+/lowCD36-CD146low), while term endothelial cells expressed mature endothelial markers (CD36+CD146+). At term, a distinct population of CD31low endothelial cells co-expressed mesenchymal markers (CD90, CD26), indicating a capacity for endothelial to mesenchymal transition (EndMT). In FGR, compared with normal pregnancies, endothelial cells constituted 3-fold fewer villous core cells (P < 0.05), contributing to an increased perivascular: endothelial cell ratio (2.6-fold, P < 0.05). This suggests that abnormal EndMT may play a role in FGR. First-trimester endothelial cells underwent EndMT in culture, losing endothelial (CD31, CD34, CD144) and gaining mesenchymal (CD90, CD26) marker expression. Together this highlights how differences in villous core cell heterogeneity and phenotype may contribute to FGR pathophysiology across gestation.

Safety and efficacy of the oral TLR8 agonist selgantolimod in individuals with chronic hepatitis B under viral suppression.

BACKGROUND & AIMS: Selgantolimod (GS-9688) is a Toll-like receptor 8 (TLR8) agonist that suppresses HBV in vitro. In a phase II study, we evaluated the safety and efficacy of weekly selgantolimod treatment in virally suppressed individuals with chronic HBV taking oral antiviral treatment. METHODS: Forty-eight patients were randomized into two cohorts (hepatitis B e antigen [HBeAg]-positive and -negative [n = 24 each]) to receive oral selgantolimod 3 mg, 1.5 mg, or placebo (2:2:1) once weekly for 24 weeks while maintaining oral antivirals. The primary efficacy endpoint was the percentage of patients with a ≥1 log10 IU/ml decline in hepatitis B surface antigen (HBsAg) from baseline to week 24. Post-treatment, patients continued on oral antivirals for 24 weeks. RESULTS: The primary endpoint was reached by one participant, who was HBeAg-negative and received selgantolimod 1.5 mg. In contrast with placebo-treated patients (n = 9), only selgantolimod-treated patients (n = 39 total) had HBsAg declines greater than 0.1 log10 IU/ml at weeks 24 (18%, 7/39) and 48 (26%, 10/39), HBsAg loss (5%, 2/39 through 48 weeks), or HBeAg loss (16%, 3/19 through 48 weeks). The most common adverse events in selgantolimod-treated groups were nausea (46%), upper respiratory tract infection (23%), and vomiting (23%). Gastrointestinal disorders were mostly mild and transient. Selgantolimod induced transient dose-dependent increases in serum cytokines, including IL-12p40, IFN-γ, and IL-1RA, as well as rapid redistribution of some circulating immune cell subsets. CONCLUSION: Oral selgantolimod up to 3 mg once weekly for 24 weeks was generally safe and well tolerated and led to serologic changes associated with progression to durable cure in two individuals by week 48. GOV IDENTIFIER: NCT03491553. IMPACT AND IMPLICATIONS: The only robust criterion for stopping treatment in chronic hepatitis B is loss of hepatitis B surface antigen (known as functional cure), which is rare during nucleos(t)ide analogue therapy. It is likely that novel antiviral and immunomodulatory therapies will be needed to achieve finite functional cure. Selgantolimod is an oral Toll-like receptor 8 agonist that has shown antiviral activity in vitro as well as safety in a phase I clinical trial with weekly dosing. In this phase II study, selgantolimod therapy was associated with transient increases in serum cytokines, rapid redistribution of circulating immune cell subsets, modest reductions in HBsAg and HBeAg levels, and occasional loss of HBsAg (5%) and HBeAg (16%) among participants with chronic hepatitis B on nucleos(t)ide analogue therapy with viral suppression. Our results support continued development of selgantolimod as a component of a future hepatitis B cure regimen.

Critical role of diagnostic SARS-CoV-2 T cell assays for immunodeficient patients.

After almost 3 years of intense study, the immunological basis of COVID-19 is better understood. Patients who suffer severe disease have a chaotic, destructive immune response. Many patients with severe COVID-19 produce high titres of non-neutralising antibodies, which are unable to sterilise the infection. In contrast, there is increasing evidence that a rapid, balanced cellular immune response is required to eliminate the virus and mitigate disease severity. In the longer term, memory T cell responses, following infection or vaccination, play a critical role in protection against SARS-CoV-2.Given the pivotal role of cellular immunity in the response to COVID-19, diagnostic T cell assays for SARS-CoV-2 may be of particular value for immunodeficient patients. A diagnostic SARS-CoV-2 T cell assay would be of utility for immunocompromised patients who are unable to produce antibodies or have passively acquired antibodies from subcutaneous or intravenous immunoglobulin (SCIG/IVIG) replacement. In many antibody-deficient patients, cellular responses are preserved. SARS-CoV-2 T cell assays may identify breakthrough infections if reverse transcriptase quantitative PCR (RT-qPCR) or rapid antigen tests (RATs) are not undertaken during the window of viral shedding. In addition to utility in patients with immunodeficiency, memory T cell responses could also identify chronically symptomatic patients with long COVID-19 who were infected early in the pandemic. These individuals may have been infected before the availability of reliable RT-qPCR and RAT tests and their antibodies may have waned. T cell responses to SARS-CoV-2 have greater durability than antibodies and can also distinguish patients with infection from vaccinated individuals.

Full spectrum flow cytometry reveals mesenchymal heterogeneity in first trimester placentae and phenotypic convergence in culture, providing insight into the origins of placental mesenchymal stromal cells.

Single-cell technologies (RNA-sequencing, flow cytometry) are critical tools to reveal how cell heterogeneity impacts developmental pathways. The placenta is a fetal exchange organ, containing a heterogeneous mix of mesenchymal cells (fibroblasts, myofibroblasts, perivascular, and progenitor cells). Placental mesenchymal stromal cells (pMSC) are also routinely isolated, for therapeutic and research purposes. However, our understanding of the diverse phenotypes of placental mesenchymal lineages, and their relationships remain unclear. We designed a 23-colour flow cytometry panel to assess mesenchymal heterogeneity in first-trimester human placentae. Four distinct mesenchymal subsets were identified; CD73+CD90+ mesenchymal cells, CD146+CD271+ perivascular cells, podoplanin+CD36+ stromal cells, and CD26+CD90+ myofibroblasts. CD73+CD90+ and podoplanin + CD36+ cells expressed markers consistent with cultured pMSCs, and were explored further. Despite their distinct ex-vivo phenotype, in culture CD73+CD90+ cells and podoplanin+CD36+ cells underwent phenotypic convergence, losing CD271 or CD36 expression respectively, and homogenously exhibiting a basic MSC phenotype (CD73+CD90+CD31-CD144-CD45-). However, some markers (CD26, CD146) were not impacted, or differentially impacted by culture in different populations. Comparisons of cultured phenotypes to pMSCs further suggested cultured pMSCs originate from podoplanin+CD36+ cells. This highlights the importance of detailed cell phenotyping to optimise therapeutic capacity, and ensure use of relevant cells in functional assays.

Recovering or Persisting: The Immunopathological Features of SARS-CoV-2 Infection in Children.

Background. The profile of cellular immunological responses of children across the spectrum of COVID-19, ranging from acute SARS-CoV-2 infection to full recovery or Long COVID, has not yet been fully investigated. Methods. We examined and compared cytokines in sera and cell subsets in peripheral blood mononuclear cells (B and regulatory T lymphocytes) collected from four distinct groups of children, distributed as follows: younger than 18 years of age with either acute SARS-CoV-2 infection (n = 49); fully recovered from COVID-19 (n = 32); with persistent symptoms (Long COVID, n = 51); and healthy controls (n = 9). Results. In the later stages after SARS-CoV-2 infection, the cohorts of children, both with recovered and persistent symptoms, showed skewed T and B subsets, with remarkable differences when compared with children at the onset of the infection and with controls. The frequencies of IgD+CD27− naïve B cells, IgD+IgM+ and CD27−IgM+CD38dim B cells were higher in children with recent infection than in those with an older history of disease (p < 0.0001 for all); similarly, the total and natural Tregs compartments were more represented in children at onset when compared with Long COVID (p < 0.0001 and p = 0.0005, respectively). Despite the heterogeneity, partially due to age, sex and infection incidence, the susceptibility of certain children to develop persistent symptoms after infection appeared to be associated with the imbalance of the adaptive immune response. Following up and comparing recovered versus Long COVID patients, we analyzed the role of circulating naïve and switched B and regulatory T lymphocytes in counteracting the evolution of the symptomatology emerged, finding an interesting correlation between the amount and ability to reconstitute the natural Tregs component with the persistence of symptoms (linear regression, p = 0.0026). Conclusions. In this study, we suggest that children affected by Long COVID may have a compromised ability to switch from the innate to the adaptive immune response, as supported by our data showing a contraction of naïve and switched B cell compartment and an unstable balance of regulatory T lymphocytes occurring in these children. However, further prospective immunological studies are needed to better clarify which factors (epigenetic, diet, environment, etc.) are involved in the impairment of the immunological mechanisms in the Long COVID patients.

Severe COVID-19 is a T cell immune dysregulatory disorder triggered by SARS-CoV-2.

INTRODUCTION: COVID-19 has had a calamitous impact on the global community. Apart from at least 6 M deaths, hundreds of millions have been infected and a much greater number have been plunged into poverty. Vaccines have been effective but financial and logistical challenges have hampered their rapid global deployment. Vaccine disparities have allowed the emergence of new SARS-CoV-2 variants including delta and omicron, perpetuating the pandemic. AREAS COVERED: The immunological response to SARS-CoV-2 is now better understood. Many of the clinical manifestations of severe disease are a consequence of immune dysregulation triggered by the virus. This may explain the lack of efficacy of antiviral treatments, such as convalescent plasma infusions, given later in the disease. EXPERT OPINION: T cells play a crucial role in both the outcome of COVID-19 as well as the protective response to vaccines. Vaccines do not prevent infection but reduce the risk of a chaotic and destructive cellular immune response to the virus. Severe COVID-19 should be considered a virus-induced secondary immune dysregulatory disorder of cellular immunity, with broad host susceptibility. This perspective of COVID-19 will lead to better diagnostic tests, vaccines, and therapeutic strategies in the future.

The (apparent) antibody paradox in COVID-19.

INTRODUCTION: The immunological response to COVID-19 is only partly understood. It is increasingly clear that the virus triggers an inappropriate host inflammatory reaction in patients experiencing severe disease. AREAS COVERED: The role of antibodies in COVID-19 remains to be fully defined. There is evidence for both protection and harm in different clinical syndromes triggered by SARS-CoV-2. Many patients dying from COVID-19 had both high titers of antibodies to SARS-CoV-2 and elevated viral loads. The uncertain protective role of humoral immunity is mirrored by the lack of benefit of therapeutic convalescent plasma infusions in COVID-19. In contrast, there is increasing evidence that a vigorous T-cell response is protective. Delayed or low avidity T cell reactions were seen in patients suffering severe COVID-19. EXPERT OPINION: These observations suggest T cell responses to SARS-CoV-2 are the dominant long-term protective mechanism following either infection or vaccination. The magnitude and quality of the antibody response is likely to reflect underlying T cell immunity to SARS-CoV-2. Much of what has been learned about COVID-19 will need to be revised following the recent rapid emergence and dominance of the omicron variant of SARS-CoV-2.

Differences in human placental mesenchymal stromal cells may impair vascular function in FGR.

Placentae from pregnancies with foetal growth restriction (FGR) exhibit poor oxygen and nutrient exchange, in part due to impaired placental vascular development. Placental mesenchymal stromal cells (pMSCs) reside in a perivascular niche, where they may influence blood vessel formation/function. However, the role of pMSCs in vascular dysfunction in FGR is unclear. To elucidate the mechanisms by which pMSCs may impact placental vascularisation we compared the transcriptomes of human pMSCs isolated from FGR (<5th centile) (n = 7) and gestation-matched control placentae (n = 9) using Affymetrix microarrays. At the transcriptome level, there were no statistically significant differences between normal and FGR pMSCs; however, several genes linked to vascular function exhibited notable fold changes, and thus the dataset was used as a hypothesis-generating tool for possible dysfunction in FGR. Genes/proteins of interest were followed up by real-time PCR, western blot and immunohistochemistry. Gene expression of ADAMTS1 and FBLN2 (fibulin-2) were significantly upregulated, whilst HAS2 (hyaluronan synthase-2) was significantly downregulated, in pMSCs from FGR placentae (n = 8) relative to controls (n = 7, P < 0.05 for all). At the protein level, significant differences in the level of fibulin-2 and hyaluronan synthase-2, but not ADAMTS1, were confirmed between pMSCs from FGR and control pregnancies by Western blot. All three proteins demonstrated perivascular expression in third-trimester placentae. Fibulin-2 maintains vessel elasticity, and its increased expression in FGR pMSCs could help explain the increased distensibility of FGR blood vessels. ADAMTS1 and hyaluronan synthase-2 regulate angiogenesis, and their differential expression by FGR pMSCs may contribute to the impaired angiogenesis in these placentae.

Cellular interactions with polystyrene nanoplastics-The role of particle size and protein corona.

Plastic waste is ubiquitously spread across the world and its smaller analogs-microplastics and nanoplastics-raise particular health concerns. While biological impacts of microplastics and nanoplastics have been actively studied, the chemical and biological bases for the adverse effects are sought after. This work explores contributory factors by combining results from in vitro and model mammalian membrane experimentation to assess the outcome of cell/nanoplastic interactions in molecular detail, inspecting the individual contribution of nanoplastics and different types of protein coronae. The in vitro study showed mild cytotoxicity and cellular uptake of polystyrene (PS) nanoplastics, with no clear trend based on nanoplastic size (20 and 200 nm) or surface charge. In contrast, a nanoplastic size-dependency on bilayer disruption was observed in the model system. This suggests that membrane disruption resulting from direct interaction with PS nanoplastics has little correlation with cytotoxicity. Furthermore, the level of bilayer disruption was found to be limited to the hydrophilic headgroup, indicating that transmembrane diffusion was an unlikely pathway for cellular uptake-endocytosis is the viable mechanism. In rare cases, small PS nanoplastics (20 nm) were found in the vicinity of chromosomes without a nuclear membrane surrounding them; however, this was not observed for larger PS nanoplastics (200 nm). We hypothesize that the nanoplastics can interact with chromosomes prior to nuclear membrane formation. Overall, precoating PS particles with protein coronae reduced the cytotoxicity, irrespective of the corona type. When comparing the two types, the extent of reduction was more apparent with soft than hard corona.

Common Variable Immunodeficiency Disorders, T-Cell Responses to SARS-CoV-2 Vaccines, and the Risk of Chronic COVID-19.

COVID-19 has had a calamitous effect on the global community. Despite intense study, the immunologic response to the infection is only partially understood. In addition to older age and ethnicity, patients with comorbidities including obesity, diabetes, hypertension, coronary artery disease, malignancy, renal, and pulmonary disease may experience severe outcomes. Some patients with primary immunodeficiency (PID) and secondary immunodeficiency also appear to be at increased risk from COVID-19. In addition to vulnerability to SARS-CoV-2, patients with PIDs often have chronic pulmonary disease and may not respond to vaccines, which exacerbates their long-term risk. Patients with common variable immunodeficiency disorders, the most frequent symptomatic PID in adults and children, have a spectrum of B- and T-cell defects. It may be possible to stratify their risk for severe COVID-19 based on age, ethnicity, the severity of the T-cell defect, and the presence of other comorbidities. Patients with common variable immunodeficiency disorders and other immunodeficiencies are at risk for Chronic COVID-19, a dangerous stalemate between a suboptimal immune response and SARS-CoV-2. Intra-host viral evolution could result in the rapid emergence of vaccine-resistant mutants and variants of high consequence; it is a public health emergency. Vaccination and prevention of Chronic COVID-19 in immunodeficient patients is therefore of the utmost priority. Having a reliable diagnostic assay for T-cell immunity to SARS-CoV-2 is critical for evaluating responses to vaccines in these patients. New treatments for SARS-CoV-2 such as NZACE2-Patari are likely to be particularly beneficial for immunodeficient patients, especially those who fail to mount a robust T-cell response to COVID-19 vaccines.

Perspective: diagnostic laboratories should urgently develop T cell assays for SARS-CoV-2 infection.

Introduction: Diagnostic tests play a critical role in the management of Sars-CoV-2, the virus responsible for COVID-19. There are two groups of tests, which are in widespread use to identify patients who have contracted the virus. The commonly used reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test becomes negative once viral shedding ceases by approximately 2-3weeks. Antibody tests directed to viral antigens become positive after the second week of infection. IgG antibody responses to the virus are muted in children, pregnant females, and those with mild symptoms. IgA and IgM antibodies rapidly wane, although IgG antibodies directed to the receptor-binding domain (RBD) of the spike (S) glycoprotein are more durable. Current data show variability in the sensitivity of commercial and in-house antibody tests to SARS-CoV-2.Areas covered: The role of T cells in acute illness is uncertain, but long-term protection against the virus may rely on memory T cell responses. Measuring memory T cell responses is important for retrospective confirmation of cases, who may have been infected early in the pandemic before reliable RT-qPCR tests were available and whose SARS-CoV-2 antibodies may have become undetectable. Relevant peer-reviewed published references from PubMed are included up to 15 March 2021.Expert opinion: After surveying the literature, the authors present the case for urgent development of diagnostic T cell assays for SARS-CoV-2 by accredited laboratories.

Influence of culture media on the derivation and phenotype of fetal-derived placental mesenchymal stem/stromal cells across gestation.

INTRODUCTION: Derivation of pure fetal placental mesenchymal stem/stromal cells (pMSCs) is key to understand their role in placental development. However, isolated pMSCs are often contaminated by maternal-derived decidual MSCs (dMSCs). EGM-2 medium promotes the derivation of term fetal pMSCs, but the extent of first-trimester maternal pMSC contamination remains unclear. Culture media can also affect MSC phenotype. Here, we examined the effects of culture media on maternal pMSC contamination and fetal pMSC phenotype across gestation. METHODS: pMSCs were derived from first-trimester or term placentae in advanced-DMEM/F12 medium or EGM-2 medium. Proportions of maternal (XX) and fetal (XY) cells in male pMSC cultures were determined by fluorescence in-situ hybridization. pMSC phenotype was analysed by flow cytometry, immunohistochemistry and Alamar blue proliferation assays. RESULTS: When derived in advanced-DMEM/F12, all first trimester pMSC isolates exhibited maternal contamination (>72% XX cells, n = 5), whilst 7/9 term pMSC isolates were >98% fetal. When derived in EGM-2, all first trimester (n = 4) and term (n = 9) pMSC isolates contained 95-100% fetal cells. Fetal pMSCs in EGM-2 proliferated 2-fold (first-trimester) or 4-fold (term) faster than those in advanced-DMEM/F12 (p < 0.05, n = 3). Fetal pMSCs in both media expressed the generic MSC marker profile (CD90+, CD105+, CD73+, CD31-, CD34-, CD144-). However, pMSCs transferred from EGM-2 to advanced-DMEM/F12 increased expression of smooth muscle cell markers calponin and α-smooth muscle actin, and decreased expression of the vascular cell marker VEGFR2 (n = 3). CONCLUSIONS: Deriving first-trimester pMSC in EGM-2 dramatically reduces maternal dMSC contamination. Media affects fetal pMSC phenotype, and careful consideration should be given to application specific culture conditions.

Determinants of translation efficiency in the evolutionarily-divergent protist Trichomonas vaginalis.

BACKGROUND: Trichomonas vaginalis, the causative agent of a prevalent urogenital infection in humans, is an evolutionarily divergent protozoan. Protein-coding genes in T. vaginalis are largely controlled by two core promoter elements, producing mRNAs with short 5' UTRs. The specific mechanisms adopted by T. vaginalis to fine-tune the translation efficiency (TE) of mRNAs remain largely unknown. RESULTS: Using both computational and experimental approaches, this study investigated two key factors influencing TE in T. vaginalis: codon usage and mRNA secondary structure. Statistical dependence between TE and codon adaptation index (CAI) highlighted the impact of codon usage on mRNA translation in T. vaginalis. A genome-wide interrogation revealed that low structural complexity at the 5' end of mRNA followed closely by a highly structured downstream region correlates with TE variation in this organism. To validate these findings, a synthetic library of 15 synonymous iLOV genes was created, representing five mRNA folding profiles and three codon usage profiles. Fluorescence signals produced by the expression of these synonymous iLOV genes in T. vaginalis were consistent with and validated our in silico predictions. CONCLUSIONS: This study demonstrates the role of codon usage bias and mRNA secondary structure in TE of T. vaginalis mRNAs, contributing to a better understanding of the factors that influence, and possibly regulate, gene expression in this human pathogen.

Distinctive Subpopulations of Stromal Cells Are Present in Human Lymph Nodes Infiltrated with Melanoma.

Metastasis of human tumors to lymph nodes (LN) is a universally negative prognostic factor. LN stromal cells (SC) play a crucial role in enabling T-cell responses, and because tumor metastases modulate their structure and function, this interaction may suppress immune responses to tumor antigens. The SC subpopulations that respond to infiltration of malignant cells into human LNs have not been defined. Here, we identify distinctive subpopulations of CD90+ SCs present in melanoma-infiltrated LNs and compare them with their counterparts in normal LNs. The first population (CD90+ podoplanin+ CD105+ CD146+ CD271+ VCAM-1+ ICAM-1+ α-SMA+) corresponds to fibroblastic reticular cells that express various T-cell modulating cytokines, chemokines, and adhesion molecules. The second (CD90+ CD34+ CD105+ CD271+) represents a novel population of CD34+ SCs embedded in collagenous structures, such as the capsule and trabeculae, that predominantly produce extracellular matrix. We also demonstrated that these two SC subpopulations are distinct from two subsets of human LN pericytes, CD90+ CD146+ CD36+ NG2- pericytes in the walls of high endothelial venules and other small vessels, and CD90+ CD146+ NG2+ CD36- pericytes in the walls of larger vessels. Distinguishing between these CD90+ SC subpopulations in human LNs allows for further study of their respective impact on T-cell responses to tumor antigens and clinical outcomes.

microRNAs in Ex Vivo Human Adipose Tissue Derived Mesenchymal Stromal Cells (ASC) Undergo Rapid Culture-Induced Changes in Expression, Including miR-378 which Promotes Adipogenesis.

There is clinical interest in using human adipose tissue-derived mesenchymal stromal cells (ASC) to treat a range of inflammatory and regenerative conditions. Aspects of ASC biology, including their regenerative potential and paracrine effect, are likely to be modulated, in part, by microRNAs, small RNA molecules that are embedded as regulators of gene-expression in most biological pathways. However, the effect of standard isolation and expansion protocols on microRNA expression in ASC is not well explored. Here, by using an untouched and enriched population of primary human ASC, we demonstrate that there are rapid and significant changes in microRNA expression when ASC are subjected to standard isolation and expansion methods. Functional studies focusing on miR-378 indicate that these changes in expression may have an impact on phenotype and function. Specifically, we found that increased levels of miR-378 significantly promoted adipogenesis in late passage ASC. These results are informative to maximizing the potential of ASC for use in various clinical applications, and they have implications for targeting microRNAs as a therapeutic strategy for obesity or metabolic disease.