Mimicking the cellular environment does not cause monocyte-derived macrophages to become phenotypically similar to Kupffer cells.

Resident macrophages of various mammalian organs are characterized by several distinctive features in their gene expression profile and phenotype, including involvement in the regulation of organ functions, as well as reduced sensitivity to proinflammatory activation factors. The reasons for the formation of such a specific phenotype remain the subject of intensive research. Some papers emphasize the role of the origin of organ macrophages. Other studies indicate that monocytes that develop in the red bone marrow are also able to form resident macrophages with a phenotype characteristic of a particular organ, but this requires appropriate microenvironmental conditions. In this article, we studied the possibility of differentiation of monocyte-derived macrophages into cells with a Kupffer-like phenotype under the influence of the main stromal components of Kupffer cells macrophage niche: Ito cells, liver sinusoid endotheliocytes and hepatocyte growth factor (HGF). It was found that Kupffer cells are characterized by several features, including increased expression of transcription factors Spic and Id3, as well as MUP family genes, Clusterin and Ngp genes. In addition, Kupffer cells were characterized by a higher proliferative activity. The expression of marker genes of Kupffer cells (i.e. Id3, Spic, Marco and Timd4) increased in monocyte-derived macrophages during coculture with Ito cells, liver sinusoid endothelial cells, macrophage colony-stimulating factor and HGF cells only by 3 days. However, the expression level of these genes was always higher in Kupffer cells. In addition, a complete coincidence of the expressed gene profile in monocyte-derived macrophages and Kupffer cells did not occur even after 3 days of culturing.

De novo start-loss variant in HIRA in patient with DiGeorge-like syndrome.

A case of a newborn with tetralogy of Fallot, corpus callosum hypoplasia, and phenotypic features similar to DiGeorge syndrome. Chromosomal microarray analysis did not reveal any alterations. Whole exome sequencing and Sanger sequencing identified a de novo variant in the HIRA gene resulting in the loss of the start codon.

Treatment of COVID-19 patients with a SARS-CoV-2-specific siRNA-peptide dendrimer formulation.

BACKGROUND: Severe acute respiratory syndrome corona virus (SARS-CoV-2) infection frequently causes severe and prolonged disease but only few specific treatments are available. We aimed to investigate safety and efficacy of a SARS-CoV-2-specific siRNA-peptide dendrimer formulation MIR 19® (siR-7-EM/KK-46) targeting a conserved sequence in known SARS-CoV-2 variants for treatment of COVID-19. METHODS: We conducted an open-label, randomized, controlled multicenter phase II trial (NCT05184127) evaluating safety and efficacy of inhaled siR-7-EM/KK-46 (3.7 mg and 11.1 mg/day: low and high dose, respectively) in comparison with standard etiotropic drug treatment (control group) in patients hospitalized with moderate COVID-19 (N = 52 for each group). The primary endpoint was the time to clinical improvement according to predefined criteria within 14 days of randomization. RESULTS: Patients from the low-dose group achieved the primary endpoint defined by simultaneous achievement of relief of fever, normalization of respiratory rate, reduction of coughing, and oxygen saturation of >95% for 48 h significantly earlier (median 6 days; 95% confidence interval [CI]: 5-7, HR 1.75, p = .0005) than patients from the control group (8 days; 95% CI: 7-10). No significant clinical efficacy was observed for the high-dose group. Adverse events were reported in 26 (50.00%), 25 (48.08%), and 28 (53.85%) patients from the low-, high-dose and control group, respectively. None of them were associated with siR-7-EM/KK-46. CONCLUSIONS: siR-7-EM/KK-46, a SARS-CoV-2-specific siRNA-peptide dendrimer formulation is safe, well tolerated and significantly reduces time to clinical improvement in patients hospitalized with moderate COVID-19 compared to standard therapy in a randomized controlled trial.

Procoagulant Activity in Amniotic Fluid Is Associated with Fetal-Derived Extracellular Vesicles.

Procoagulant activity in amniotic fluid (AF) is positively correlated with phosphatidylserine (PS) and tissue factor (TF)-expressing(+) extracellular vesicles (EVs). However, it is unknown if pathological fetal conditions may affect the composition, phenotype, and procoagulant potency of EVs in AF. We sought to evaluate EV-dependent procoagulant activity in AF from pregnant people with fetuses with or without diagnosed chromosomal mutations. AF samples were collected by transabdominal amniocentesis and assessed for common karyotype defects (total n = 11, 7 healthy and 4 abnormal karyotypes). The procoagulant activity of AF was tested using a fibrin generation assay with normal pooled plasma and plasmas deficient in factors XII, XI, IX, X, V, and VII. EV number and phenotype were determined by flow cytometry with anti-CD24 and anti-TF antibodies. We report that factor-VII-, X-, or V-deficient plasmas did not form fibrin clots in the presence of AF. Clotting time was significantly attenuated in AF samples with chromosomal mutations. In addition, CD24+, TF+, and CD24+ TF+ EV counts were significantly lower in this group. Finally, we found a significant correlation between EV counts and the clotting time induced by AF. In conclusion, we show that AF samples with chromosomal mutations had fewer fetal-derived CD24-bearing and TF-bearing EVs, which resulted in diminished procoagulant potency. This suggests that fetal-derived EVs are the predominant source of procoagulant activity in AF.

Does the uterine microbiota affect the reproductive outcomes in women with recurrent implantation failures?

BACKGROUND: Inefficiency of in vitro fertilization (IVF) programs can be caused by implantation failures. The uterine microbiota can influence the implantation process. However, it still remains unclear whether opportunistic microorganisms detected in the endometrium have a negative impact on the implantation success. The aim of our study was to evaluate the influence of the uterine microbiota on the embryo implantation success in patients undergoing assisted reproductive technologies. METHODS: The study included 130 women diagnosed with infertility. The patients were divided into three groups: group I included women with the first IVF attempt (n = 39); group II included patients with recurrent implantation failure following embryo transfer with ovarian stimulation (n = 27); group III consisted of women with recurrent implantation failure following frozen-thawed embryo transfer (n = 64). We performed microbiological examination of the embryo transfer catheter which was removed from the uterine cavity after embryo transfer; cervical discharge of all the patients was studied as well. Thirty patients were selected for metagenomic sequencing. RESULTS: The study showed that the uterine cavity is not free of microorganisms. A total of 44 species of microorganisms were detected: 26 species of opportunistic organisms and 18 species of commensals (14 species of lactobacilli and 4 species of bifidobacteria). Obligate anaerobic microorganisms and Gardnerella vaginalis were detected more frequently in group I compared to group III (strict anaerobes-15.4 and 1.6%; G. vaginalis-12.8 and 1.6%, respectively) (p < 0.05). However, this fact did not have a negative influence on the pregnancy rate: it was 51.3% in group I, it was 29.6% and 35.9% in women with recurrent implantation failures, respectively. CONCLUSION: Opportunistic microorganisms which were revealed in low or moderate titers (103-105 CFU/ml) in the uterine cavity and cervical canal did not affect the pregnancy rate in the women in the study groups. The microflora of the uterine cavity and cervical canal differed in qualitative composition in 87.9% of patients, therefore, we can suggest that the uterine cavity may form its own microbiota. The microbiota of the uterine cavity is characterized by fewer species diversity compared to the microbiota of the cervical canal.

CYP2C9 and VKORC1 genotyping for the quality of long-standing warfarin treatment in Russian patients.

A total of 263 warfarin naive patients with indications to long-term anticoagulation were included in prospective multicenter study and randomized into Pharmacogenetics and Standard dosing groups. The loading warfarin dose in Pharmacogenetics group was calculated by Gage algorithm and corrected starting on day 5 of treatment according to INR. In Standard dosing group warfarin initial dose was 5 mg and starting on day 3 of treatment it was titrated according to INR. Pharmacogenetics dosing in comparison with prescription of starting dose of 5 mg decreased major bleedings (0 vs. 6, p = 0.031), time to target INR (11 [9-14] vs. 17 [15-24] days, p = 0.046), and frequency of INR fluctuations ≥4.0 (11% vs. 30.9%, p = 0.002). The advantages of the pharmacogenetics dosing were mainly achieved due to the patients with increased warfarin sensitivity.

Preventive Triple Gene Therapy Reduces the Negative Consequences of Ischemia-Induced Brain Injury after Modelling Stroke in a Rat.

Currently, the main fundamental and clinical interest for stroke therapy is focused on developing a neuroprotective treatment of a penumbra region within the therapeutic window. The development of treatments for ischemic stroke in at-risk patients is of particular interest. Preventive gene therapy may significantly reduce the negative consequences of ischemia-induced brain injury. In the present study, we suggest the approach of preventive gene therapy for stroke. Adenoviral vectors carrying genes encoding vascular endothelial growth factor (VEGF), glial cell-derived neurotrophic factor (GDNF) and neural cell adhesion molecule (NCAM) or gene engineered umbilical cord blood mononuclear cells (UCB-MC) overexpressing recombinant VEGF, GDNF, and NCAM were intrathecally injected before distal occlusion of the middle cerebral artery in rats. Post-ischemic brain recovery was investigated 21 days after stroke modelling. Morphometric and immunofluorescent analysis revealed a reduction of infarction volume accompanied with a lower number of apoptotic cells and decreased expression of Hsp70 in the peri-infarct region in gene-treated animals. The lower immunopositive areas for astrocytes and microglial cells markers, higher number of oligodendrocytes and increased expression of synaptic proteins suggest the inhibition of astrogliosis, supporting the corresponding myelination and functional recovery of neurons in animals receiving preventive gene therapy. In this study, for the first time, we provide evidence of the beneficial effects of preventive triple gene therapy by an adenoviral- or UCB-MC-mediated intrathecal simultaneous delivery combination of vegf165, gdnf, and ncam1 on the preservation and recovery of the brain in rats with subsequent modelling of stroke.

In silico size selection is effective in reducing false positive NIPS cases of monosomy X that are due to maternal mosaic monosomy X.

OBJECTIVES: The aim of this study was to establish maternal contribution to false positive noninvasive prenatal DNA screening (NIPS) results and develop the method to distinguish maternal and fetal origin of high-risk monosomy X NIPS calls including mosaic maternal cases. METHOD: A total of 906 women carrying singleton pregnancies have been recruited. Maternal plasma DNA semiconductor massive parallel sequencing was performed to detect common aneuploidies. For the case of high monosomy X risk call, analysis method to distinguish fetal and maternal monosomy X has been additionally applied. RESULTS: According to NIPS results, 18 patients had a high risk of fetal monosomy X. In 11 (61%) cases, fetal aneuploidy was confirmed by karyotyping. Other 7 cases were false positives. In 3 out of 7 cases, additional analysis based on in silico size selection was allowed to assume maternal monosomy X. In these cases, fluorescence in situ hybridization analysis confirmed mosaic monosomy X in maternal blood cells. CONCLUSION: The prevalence of mosaic monosomy X karyotype is 0.3% (3/906)-10 times higher than published before. Additional in silico size-selection and data analysis increases PPV for monosomy X from 61% to 73% for studied population.

Targeting TLR-4 with a novel pharmaceutical grade plant derived agonist, Immunomax®, as a therapeutic strategy for metastatic breast cancer.

BACKGROUND: Previously we demonstrated that the resection of primary 4T1 tumors only slightly prolongs mouse survival, but importantly, creates a "window of opportunity" with attenuated suppressor cell and increased activated T cell populations. This suggests that additional activation of the immune system by immunostimulatory agents during this period may enhance anti-tumor immunity and potentially eradicate micro-metastatic disease in this stringent model. We hypothesized that the immunostimulator Immunomax®, which is comprised of a plant-derived polysaccharide, is non-toxic in humans and stimulates immune defense during the infectious diseases treatment, may have also anti-tumor activity and be beneficial in the adjuvant setting when endogenous anti-tumor responses are present and during the "window of opportunity" in post-resection metastatic breast cancer model. Here we provide the initial report that Immunomax® demonstrates the capacity to eliminate micro-metastatic disease in the post-resection, 4T1 mouse model of breast cancer. METHODS: The efficacy of Immunomax® was evaluated by analyzing survival rate and the number of spontaneous clonogenic tumor cells in the lung homogenates of mice. The frequencies of activated NK, CD4(+) and CD8(+) cells as well as myeloid-derived suppressor cells and Treg cells were evaluated using flow cytometry. Highly purified mouse and human dendritic and NK cells were sorted and the effect of Immunomax® on activation status of these cells was assessed by flow cytometry. The property of Immunomax® as TLR-4 agonist was determined by NF-κB/SEAP reporter gene assay, WB, RT-PCR. RESULTS: Immunomax® injections significantly prolonged overall survival and cured 31% of mice. This immunostimulator activates DCs via the TLR-4, which in turn stimulates tumoricidal NK cells and in vitro, completely inhibits growth of 4T1 cells. Incubation of PBMC from healthy donors with Immunomax® activates NK cells via activation of plasmacytoid DC leading significantly higher efficacy in killing of human NK-target cells K562 compared with non-treated cells. CONCLUSION: This is the first demonstration that Immunomax® is a TLR-4 agonist and the first report of a documented role for this pharmaceutical grade immunostimulator in augmenting anti-tumor activity, suggesting that incorporation of Immunomax® into developing breast cancer therapeutic strategies may be beneficial and with less potential toxicity than checkpoint inhibitors.

Expanding phenotype of MED13-associated syndrome presenting novel de novo missense variant in a patient with multiple congenital anomalies.

BACKGROUND: Whole exome sequencing allows rapid identification of causative single nucleotide variants and short insertions/deletions in children with congenital anomalies and/or intellectual disability, which aids in accurate diagnosis, prognosis, appropriate therapeutic interventions, and family counselling. Recently, de novo variants in the MED13 gene were described in patients with an intellectual developmental disorder that included global developmental delay, mild congenital heart anomalies, and hearing and vision problems in some patients. RESULTS: Here we describe an infant who carried a de novo p.Pro835Ser missense variant in the MED13 gene, according to whole exome trio sequencing. He presented with congenital heart anomalies, dysmorphic features, hydrocephalic changes, hypoplastic corpus callosum, bilateral optic nerve atrophy, optic chiasm atrophy, brain stem atrophy, and overall a more severe condition compared to previously described patients. CONCLUSIONS: Therefore, we propose to expand the MED13-associated phenotype to include severe complications that could end up with multiple organ failure and neonatal death.

The spleen as a possible source of serine protease inhibitors and migrating monocytes required for liver regeneration after 70% resection in mice.

Introduction: The role of the immune system in liver repair is fundamentally complex and most likely involves the spleen. The close connection between the two organs via the portal vein enables delivery of splenic cytokines and living cells to the liver. This study evaluates expression of inflammation-related genes and assesses the dynamics of monocyte-macrophage and lymphocyte populations of the spleen during the recovery from 70% hepatectomy in mice. Methods: The study used the established mouse model of 70% liver volume resection. The animals were sacrificed 24 h, 72 h or 7 days post-intervention and splenic tissues were collected for analysis: Clariom™ S transcriptomic assay, immunohistochemistry for proliferation marker Ki-67 and macrophage markers, and flow cytometry for lymphocyte and macrophage markers. Results: The loss and regeneration of 70% liver volume affected the cytological architecture and gene expression profiles of the spleen. The tests revealed significant reduction in cell counts for Ki-67+ cells and CD115+ macrophages on day 1, Ly6C + cells on days 1, 3 and 7, and CD3+CD8+ cytotoxic lymphocytes on day 7. The transcriptomic analysis revealed significant activation of protease inhibitor genes Serpina3n, Stfa2 and Stfa2l1 and decreased expression of cell cycle regulatory genes on day 1, mirrored by inverse dynamics observed on day 7. Discussion and conclusion: Splenic homeostasis is significantly affected by massive loss in liver volume. High levels of protease inhibitors indicated by increased expression of corresponding genes on day 1 may play an anti-inflammatory role upon reaching the regenerating liver via the portal vein. Leukocyte populations of the spleen react by a slow-down in proliferation. A transient decrease in the local CD115+ and Ly6C+ cell counts may indicate migration of splenic monocytes-macrophages to the liver.

Novel Approaches to Possible Targeted Therapies and Prophylaxis of Uterine Fibroids.

Uterine leiomyomas are the most common benign tumors in women of childbearing age. They may lead to problems of conception or complications during the gestational period. The methods of treatment include surgical (myomectomy and hysterectomy, embolization of arteries) and therapeutic treatment (ulipristal acetate, leuprolide acetate, cetrorelix, goserelin, mifepristone). Both approaches are efficient but incompatible with pregnancy planning. Therefore, there is a call for medical practice to develop therapeutical means of preventing leiomyoma onset in patients planning on becoming pregnant. Based on the analysis of GWAS data on the search for mononucleotide polymorphisms associated with the risk of leiomyoma, in meta-transcriptomic and meta-methylomic studies, target proteins have been proposed. Prospective therapeutic treatments of leiomyoma may be based on chemical compounds, humanized recombinant antibodies, vaccines based on markers of the uterine leiomyoma cells that are absent in the adult organism, or DNA and RNA preparations. Three different nosological forms of the disease associated with driver mutations in the MED12, HMGA2, and FH genes should be considered when developing or prescribing drugs. For example, synthetic inhibitors and vaccines based on matrix metalloproteinases MMP11 and MMP16 are expected to be effective only for the prevention of the occurrence of MED12-dependent nodules.

Molecular diagnosis of tuberous sclerosis complex in fetuses and infants: an institutional case series.

OBJECTIVE: We describe the clinical and genetic characteristics of fetuses and infants diagnosed with tuberous sclerosis complex (TSC) in our centre, prenatally or neonatally, for a better understanding of the benefits of early screening. METHODS: In this retrospective study, we analysed the data on one fetus and nine infants with a definitive TSC diagnosis by genetic criteria (five patients carrying TSC1 variants and 5 patients carrying TSC2 variants). We explored the differences between phenotypes of patients carrying TSC1 and TSC2 pathogenic variants. RESULTS: The most common initial presenting features of TSC were cardiac rhabdomyomas (CRs) that were observed in nine out of ten patients. The most common postnatal features, besides CR, were presented with subependymal nodules-in five patients, and hypomelanotic macules-in four patients. In total, 10 variants causing TSC were detected in this study, including 5 novel variants. We demonstrated that patients with TSC2 variants had earlier onset and more severe clinical manifestations compared with patients carrying TSC1 variants. CONCLUSION: Early diagnosis of TSC improves genetic counselling and perinatal management.

Clinical Evaluation of Nasopharyngeal, Oropharyngeal, Nasal Swabs, and Saliva for the Detection of SARS-CoV-2 by Direct RT-PCR.

Nasopharyngeal swab (NPS) and oropharyngeal swab (OPS) are the most widely used upper respiratory tract specimens for diagnosis of SARS-CoV-2 using RT-qPCR. In contrast, nasal swab (NS) and saliva (SS), recently recommended by the WHO, are rarely used, and their test accuracy is limited. The method for direct RT-PCR detection of SARS-CoV-2 does not require an RNA extraction and is faster and easier than standard RT-PCR tests with RNA extraction. This study aimed to compare the diagnostic performance of upper respiratory tract samples for SARS-CoV-2 detection using the direct RT-PCR without preliminary heat inactivation. Here we report the application and validation of direct RT-PCR SARS-CoV-2 RNA on 165 clinical specimens of NPS/OP, and 36 samples of NS/NPS and 37 saliva samples (for the latter with prior deproteinization). The overall sensitivity estimates were 95.9%, 94.2%, 88.9%, and 94.6% for NPS/OPS/NS/SS samples, respectively, and the specificity was 100% against standard RT-PCR with RNA extraction. Overall, NS and SS testing by direct RT-PCR had sufficient sensitivity to detect SARS-CoV-2. They can be acceptable alternative to NPS/OPS for rapid detection of SARS-CoV-2 infections in future.

Familial Predisposition to Leiomyomata: Searching for Protective Genetic Factors.

In order to determine genetic loci associated with decreasing risk of uterine leiomyomata (UL), a genome-wide association study (GWAS) was performed. We analyzed a group of patients with a family history of UL and a control group consisting of patients without uterine fibroids and a family predisposition to this pathology. Six significant single nucleotide polymorphisms were selected for PCR-genotyping of a large data set of patients with UL. All investigated loci (rs3020434, rs11742635, rs124577644, rs12637801, rs2861221, and rs17677069) demonstrated the lower frequency of minor alleles within a group of women with UL, especially in a subgroup consisting of patients with UL and a familial history of leiomyomata. We also found that the minor allele frequencies of these SNPs in our control group were higher than those across the Caucasian population in all. Based on the obtained data, an evaluation of the common risk of UL was performed. Further work will pave the way to create a specific SNP-panel and allow us to estimate a genotype-based leiomyoma incidence risk. Subsequent studies of genetic variability in a group of patients with a familial predisposition to UL will allow us to make the prediction of the development and course of the disease more individualized, as well as to give our patients personalized recommendations about individual reproductive strategies.

Distinct gene expression patterns for CD14++ and CD16++ monocytes in preeclampsia.

Preeclampsia (PE) is a serious gestational complication affecting the life of a mother and child. The immunophenotype and gene expression profile of isolated blood monocyte subpopulations of pregnant women with PE have not been studied before. In this work, we assessed changes in CD14++ and CD16++ monocyte subpopulations in PE and physiological pregnancy (n = 33). Immunophenotyping, immunomagnetic sorting of monocytes and analysis of the transcriptional profile of their genes were carried out. The percentage of classical monocytes was significantly lower, while the intermediate fraction of monocytes was significantly higher in late-onset PE compared to control. Transcriptome analysis of late-onset PE classical CD14++ monocytes revealed significant activation of inflammation mediated by chemokine and cytokine signalling pathways; apoptosis; regulation of transcription from RNA polymerase II promoter in response to stress and others. The most suppressed signalling pathways were associated with T cell activation and selection. In CD16++ monocytes of late-onset PE cases, positive regulation of cell-cell adhesion, integrin signalling pathway, blood coagulation cascade were the most activated ones. The inflammation mediated by chemokine and cytokine signalling pathway and p53 pathway were the most down-regulated in CD16++ monocytes. The obtained results indicate profound changes occurring to two most polar monocyte subpopulations in PE and their different roles in the pathogenesis of this disease.

Vertical Transmission of SARS-CoV-2 in Second Trimester Associated with Severe Neonatal Pathology.

The effects of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection in women on the gestation course and the health of the fetus, particularly in the first and second trimesters, remain very poorly explored. This report describes a case in which the normal development of pregnancy was complicated immediately after the patient had experienced Coronavirus disease 2019 (COVID-19) at the 21st week of gestation. Specific conditions included critical blood flow in the fetal umbilical artery, fetal growth restriction (1st percentile), right ventricular hypertrophy, hydropericardium, echo-characteristics of hypoxic-ischemic brain injury (leukomalacia in periventricular area) and intraventricular hemorrhage at the 25th week of gestation. Premature male neonate delivered at the 26th week of gestation died after 1 day 18 h due to asystole. The results of independent polymerase chain reaction (PCR), mass spectrometry and immunohistochemistry analyses of placenta tissue, umbilical cord blood and child blood jointly indicated vertical transmission of SARS-CoV-2 from mother to the fetus, which we conclude to be the major cause for the development of maternal vascular malperfusion in the studied case.

Impact TMPRSS2-ERG Molecular Subtype on Prostate Cancer Recurrence.

Currently, seven molecular subtypes of prostate cancer (PCa) are known, the most common of which being the subtype characterized by the presence of the TMPRSS2-ERG fusion transcript. While there is a considerable amount of work devoted to the influence of this transcript on the prognosis of the disease, data on its role in the progression and prognosis of PCa remain controversial. The present study is devoted to the analysis of the association between the TMPRSS2-ERG transcript and the biochemical recurrence of PCa. The study included two cohorts: the RNA-Seq sample of Russian patients with PCa (n = 72) and the TCGA-PRAD data (n = 203). The results of the analysis of the association between the TMPRSS2-ERG transcript and biochemical recurrence were contradictory. The differential expression analysis (biochemical recurrence cases versus biochemical recurrence-free) and the gene set enrichment analysis revealed a list of genes involved in major cellular pathways. The GNL3, QSOX2, SSPO, and SYS1 genes were selected as predictors of the potential prognostic model (AUC = 1.000 for a cohort of Russian patients with PCa and AUC = 0.779 for a TCGA-PRAD cohort).