Mechanical properties of blood exosomes and lipoproteins after the rat whole blood irradiation with X-rays in vitro explored by atomic force microscopy.

Blood is a two-component system with two levels of hierarchy: the macrosystem of blood formed elements and the dispersed system of blood nanoparticles. Biological nanoparticles are the key participants in communication between the irradiated and non-irradiated cells and inducers of the non-targeted effects of ionizing radiation. The work aimed at studying by atomic force microscopy the structural, mechanical, and electrical properties of exosomes and lipoproteins (LDL/VLDL) isolated from rat blood after its exposure to X-rays in vitro. MATERIALS AND METHODS: The whole blood of Wistar rats fed with a high-fat diet was irradiated with X-rays (1 and 100 Gy) in vitro. The structural and mechanical properties (the elastic modulus and nonspecific adhesion force) of exosome and lipoprotein isolates from the blood by ultracentrifugation method were studied using Bruker Bioscope Resolve atomic force microscope in PF QNM mode, their electric properties (the zeta-potential) was measured by electrophoretic mobility. RESULTS: Lipoproteins isolated from non-irradiated blood were softer (Me(LQ; UQ): 7.8(4.9;12.1) MPa) compared to blood nanoparticles of its exosome fraction (34.8(22.6;44.9) MPa) containing both exosomes and non-membrane nanoparticles. X-ray blood irradiation with a dose of 1 Gy significantly weakened the elastic properties of lipoproteins. Exposure of the blood to 100 Gy X-rays made lipoproteins stiffer and their nonspecific adhesive properties stronger. The radiation effects on the mechanical parameters of exosomes and non-membrane nanoparticles in exosome fractions differed. The significant radiation-induced change in electric properties of the studied nanoparticles was detected only for lipoproteins in the blood irradiated with 1 Gy X-rays. The low-dose radiation-induced changes in zeta-potential and increase in lipoprotein size with the appearance of a soft thick surface layer indicate the formation of the modified lipoproteins covered with a corona from macromolecules of irradiated blood. CONCLUSION: Our data obtained using the nanomechanical mapping mode of AFM are the first evidence of the significant radiation-induced changes in the structural and mechanical properties of the dispersed system of blood nanoparticles after the X-ray irradiation of the blood.

Clinical heterogeneity of sleep quality, emotional and behavioral characteristics, and eating habits in adolescents with obesity: A cluster analysis.

This study aims to examine the role of sleep quality, emotional and behavioral characteristics, and eating behavior in adolescents with obesity by means of cluster analysis. One hundred ninety-four adolescents (78 girls, 116 boys) aged 12-17 (mean 14.3 ± 2.7) with obesity (ICD-10 code E 66.0) entered the study. The Adolescent Sleep Wake Scale was used to evaluate sleep quality. The Achenbach Youth Self-Report for Ages 11-18 questionnaire was used to evaluate emotional and behavioral disturbances. The Dutch Eating Behavior Questionnaire was used to assess maladaptive eating. k-Means cluster analysis was used to clarify heterogeneity. Four clusters were identified: the first included anxious, depressed, socially withdrawn adolescents with thought problems and somatic complaints, non-aggressive, obedient, and having mean values on the sleep quality scale, inclined to restrict their food consumption. The second consisted of rule-breaking youngsters with poor sleep quality, reluctant to restrict their food consumption. The third comprised subjects with the highest values on the sleep quality and lowest values on emotional and behavioral problems, aimed at restricting food consumption. The fourth comprised adolescents with obvious signs of emotional disorders, poorest sleep quality, disinhibited behavior, and emotional and external eating. No statistically significant difference was found between the clusters in sex distribution. Patient allocation to the second or fourth cluster was associated with significantly higher body mass index values, as opposed to the reference third cluster.    Conclusion: There is significant variation in teenagers with obesity who have divergent psychological profiles, which should be taken into account. What is Known: • Patients with obesity present with different clinical characteristics. What is New: • Adolescents with obesity may be partitioned into clusters described in terms of emotional, behavioral patterns, including sleep characteristics, and maladaptive eating habits.

Presentation of severe brucellosis in 5-year-old boy - challenges and results.

Brucellosis is highly contagious zoonotic bacterial disease caused by gram-negative genus. It has a wide spectrum of clinical manifestations and due to variety and nonspecificity of clinical signs the diagnostics can be very complicated. We present a clinical case of severe chronic brucellosis in a 5-years old boy with long-term course of disease and multiorgan involvement. A different complication of brucellosis including severe syndrome of inappropriate ADH secretion (SIADH) are discussed. Despite severe course of disease patient achieved significant clinical improvement due to multidisciplinary approach and optimal etiotropic and pathogenetic treatment.

iPSC-Derived Macrophages: The Differentiation Protocol Affects Cell Immune Characteristics and Differentiation Trajectories.

The generation of human macrophages from induced pluripotent stem cells (iMacs) is a rapidly developing approach used to create disease models, screen drugs, study macrophage-pathogen interactions and develop macrophage-based cell therapy. To generate iMacs, different types of protocols have been suggested, all thought to result in the generation of similar iMac populations. However, direct comparison of iMacs generated using different protocols has not been performed. We have compared the productivity, the differentiation trajectories and the characteristics of iMacs generated using two widely used protocols: one based on the formation of embryoid bodies and the induction of myeloid differentiation by only two cytokines, interleukin-3 and macrophage colony-stimulating factor, and the other utilizing multiple exogenous factors for iMac generation. We report inter-protocol differences in the following: (i) protocol productivity; (ii) dynamic changes in the expression of genes related to inflammation and lipid homeostasis following iMac differentiation and (iii) the transcriptomic profiles of terminally differentiated iMacs, including the expression of genes involved in inflammatory response, antigen presentation and lipid homeostasis. The results document the dependence of fine iMac characteristics on the type of differentiation protocol, which is important for further development of the field, including the development of iMac-based cell therapy.

Case Report: COVID-19-Associated ROHHAD-Like Syndrome.

It is known that the SARS-CoV-2 virus may cause neurologic damage. Rapid-onset obesity, hypoventilation, hypothalamus dysfunction, and autonomic dysregulation (ROHHAD) syndrome is a disease of unknown etiology with a progressive course and unclear outcomes. The etiology of ROHHAD syndrome includes genetic, epigenetic, paraneoplastic, and immune-mediated theories, but to our knowledge, viral-associated cases of the disease have not been described yet. Here we present the case of a 4-year-old girl who developed a ROHHAD syndrome-like phenotype after a COVID-19 infection and the results of 5 months of therapy. She had COVID-19 pneumonia, followed by electrolyte disturbances (hypernatremia and hyperchloremia), hypocorticism and hypothyroidism, central hypoventilation-requiring prolonged assisted lung ventilation-bulimia, and progressive obesity with hypertriglyceridemia, dyslipidemia, hyperuricemia, and hyperinsulinemia. The repeated MRI of the brain and hypothalamic-pituitary region with contrast enhancement showed mild post-hypoxic changes. Prader-Willi/Angelman syndrome as well as PHOX2B-associated variants was ruled out. Treatment with non-steroidal anti-inflammatory drugs and monthly courses of intravenous immunoglobulin led to a dramatic improvement. Herein the first description of ROHHAD-like syndrome is timely associated with a previous COVID-19 infection with possible primarily viral or immune-mediated hypothalamic involvement.

Health-related quality of life in 4-to-6-year-old children with type 1 diabetes mellitus estimated by children and their mothers.

Administration of pediatric Health Related Quality of Life (HRQoL) inventories frequently assesses both the child and parent perspectives in young children with type 1 diabetes mellitus (T1DM), but parent-proxy and child self-reports may differ, and little is known on these discrepancies. The aim is to evaluate HRQoL estimated by young children with T1DM and by their mothers, potential discrepancies in the children-maternal estimates and the factors influencing these discrepancies. Thirty-five 4-to-6-year-old children (19 boys) with T1DM admitted to the Pediatric Endocrinology Department were approached with the self-report KINDL questionnaire for children aged 4-6 years (Kiddy-KINDL for children). Their mothers were approached with the parental version (Kiddy-KINDL for parents). Both versions enable measuring child HRQoL in physical, emotional wellbeing, self-esteem, family, friends, everyday functioning, and the disease dimensions, as well as KINDL total on a 0-100 scale. Statistically significant differences were found between children's and maternal estimates on the KINDL total and "Disease" scales, in that the maternal proxy-reports produced lower values. A statistically significant difference between self- and proxy-reports was found for the KINDL "Emotional wellbeing" scale values, and the maternal proxy-reports yielded higher estimates compared with children's self-reports. These associations remained significant after adjustment for major potential confounders. Maternal education, maternal marital status, insulin regimen, and achievement of glycemic control modified the effect of child-maternal discrepancies.Conclusion: Attempts should be made to improve parental understanding of child problems related to his/her disease with due account to individual family social and demographic characteristics. What is Known: • HRQoL in children with T1DM has been advocated as an important complementary outcome to clinical and laboratory markers. • Self-and parental proxy-reports on HRQoL may differ, but little is known on these discrepancies and on the factors influencing them in young children with T1DM. What is New: • Mothers tend to underestimate general and disease-related components of HRQoL but likely to overestimate psychological wellbeing of their ill young children with T1DM. • Maternal education, marital status, insulin regimen, and achievement of glycemic control modify estimations of HRQoL and child-maternal discrepancies.

The coincidence of two rare diseases with opposite metabolic phenotype: a child with congenital hyperinsulinism and Bloom syndrome.

OBJECTIVES: Congenital hyperinsulinism (CHI) is a group of rare genetic disorders characterized by insulin overproduction. CHI causes life-threatening hypoglycemia in neonates and infants. Bloom syndrome is a rare autosomal recessive disorder caused by mutations in the BLM gene resulting in genetic instability and an elevated rate of spontaneous sister chromatid exchanges. It leads to insulin resistance, early-onset diabetes, dyslipidemia, growth delay, immune deficiency and cancer predisposition. Recent studies demonstrate that the BLM gene is highly expressed in pancreatic islet cells and its mutations can alter the expression of other genes which are associated with apoptosis control and cell proliferation. CASE PRESENTATION: A 5-month-old female patient from consanguineous parents presented with drug-resistant CHI and dysmorphic features. Genetic testing revealed a homozygous mutation in the KCNJ11 gene and an additional homozygous mutation in the BLM gene. While 18F-DOPA PET scan images were consistent with a focal CHI form and intraoperative frozen-section histopathology was consistent with diffuse CHI form, postoperative histopathological examination revealed features of an atypical form. CONCLUSIONS: In our case, the patient carries two distinct diseases with opposite metabolic phenotypes.

Differential Morphological Diagnosis of Various Forms of Congenital Hyperinsulinism in Children.

Introduction: Congenital hyperinsulinism (CHI) has diffuse (CHI-D), focal (CHI-F) and atypical (CHI-A) forms. Surgical management depends on preoperative [18F]-DOPA PET/CT and intraoperative morphological differential diagnosis of CHI forms. Objective: to improve differential diagnosis of CHI forms by comparative analysis [18F]-DOPA PET/CT data, as well as cytological, histological and immunohistochemical analysis (CHIA). Materials and Methods: The study included 35 CHI patients aged 3.2 ± 2.0 months; 10 patients who died from congenital heart disease at the age of 3.2 ± 2.9 months (control group). We used PET/CT, CHIA of pancreas with antibodies to ChrA, insulin, Isl1, Nkx2.2, SST, NeuroD1, SSTR2, SSTR5, DR1, DR2, DR5; fluorescence microscopy with NeuroD1/ChrA, Isl1/insulin, insulin/SSTR2, DR2/NeuroD1 cocktails. Results: Intraoperative examination of pancreatic smears showed the presence of large nuclei, on average, in: 14.5 ± 3.5 cells of CHI-F; 8.4 ± 1.1 of CHI-D; and 4.5 ± 0.7 of control group (from 10 fields of view, x400). The percentage of Isl1+ and NeuroD1+endocrinocytes significantly differed from that in the control for all forms of CHI. The percentage of NeuroD1+exocrinocytes was also significantly higher than in the control. The proportion of ChrA+ and DR2+endocrinocytes was higher in CHI-D than in CHI-F, while the proportion of insulin+cells was higher in CHI-A. The number of SST+cells was significantly higher in CHI-D and CHI-F than in CHI-A. Conclusion: For intraoperative differential diagnosis of CHI forms, in addition to frozen sections, quantitative cytological analysis can be used. In quantitative immunohistochemistry, CHI forms differ in the expression of ChrA, insulin, SST and DR2. The development of a NeuroD1 inhibitor would be advisable for targeted therapy of CHI.

In Vivo Antidepressant Efficacy of 3-Substituted Thietane-1,1-dioxide Derivative - A Preliminary Study for Novel Anti-Depression Therapy in Neurological Disorders.

BACKGROUND: Psychosocial stress-induced depressive behavior is linked to the etiology of several neurological diseases viz., PTSD, and neurodegenerative disease like Alzheimer's Disease (AD). The repeated bouts of social stress defeat can be induced using Resident-Intruder- Paradigm (RIP) and Chronic Mild Social Stress (CMSS) animal models to assess the stress-induced depressive behavioral patterns. OBJECTIVES: The aim of this study to examine the anti-depressive efficacy of 3-methoxythietane- 1,1-dioxide (N-14) in RIP models of behavioral alterations. METHODS: In this study, we have used Sprague-Dawley rats in Resident-Intruder-Paradigm (RIP), where intruders interacted with residents Day 0 to Day +5 for 10 minutes to invoke CMSS in intruders and became defeated/submissive rats due to the depressive-like behavioral alterations in social activity, explorations, grooming, defense, aggressive behavior, social interaction, freeze, rearing etc., with residents. Control intact animals are included in group I, group II received N-14 alone; group III received CMSS, and group IV received cotreatment of N14 with CMSS. N-14 (2 mg/kg) was administered intraperitoneally from Day 0 to Day +5 to intact animals and intruder animals under conditions of CMSS. RESULTS: Several behavioral tests viz., forced swim test, open field test, and elevated-plus maze test were used to examine the above behavioral dynamic parameters. The dynamic interaction between Residents and Intruders during the study showed substantial alterations in exploratory activity, aggressiveness, defensive behavior, body weight, and thymus mass in stressed animals. N-14 cotreatment has mitigated sociability, exploratory activity, aggressiveness increased social adaptability and defensive behavior. An extensive rise in active forms of defense and submission latency indicates that N-14 has induced antidepressant activity with a psycho-sedative component of action. CONCLUSION: Serendipitously, we observed the ameliorative capability of N-14 cotreatment to mitigate depressive-behavioral symptoms in intruders.

Plasma kisspeptin levels in boys with hypogonadotropic delayed puberty.

BACKGROUND: In hypogonadotropic forms of delayed puberty (DP), hypophyseal follicle-stimulating (FSH) and luteinizing (LH) hormones, normally released with GnRH stimulation, are detected low. Since kisspeptin (KP) is a strong stimulant of GnRH neurons, it is considered to have a role in DP etiology. It may be hypothesized that abnormal plasma levels of KP are indicative of DP. The study aimed at evaluation and comparison of plasma KP levels in boys of pre-pubertal age, with normal puberty and diagnosed primary hypogonadotropic forms of DP. METHODS: The study comprised 22 boys with verified hypogonadotropic DP (age 14-17 years), 25 boys with normal puberty (age 14-17 years), and 28 pre-pubertal boys (age 6-9 years). Triprorelin stimulation test was performed in DP patients. Plasma KP values were compared between three groups. RESULTS: Statistically significant difference was found for the overall distribution of the plasma KP values across different groups (Kruskal-Wallis H=21.95, P<0.001). The highest values were found in the DP group (median: 45.0 pg/mL). Median values in the pre-pubertal boys and in the normal pubertal adolescents were equal to 13.8 pg/mL. No statistically significant difference was found for plasma KP levels in the DP boys who had either positive or negative response to Triptorelin stimulation test. Plasma KP level exceeding 16.9 pg/mL was a reliable predictor of hypoganadotropic DP (sensitivity 72.7%, specificity 92.0%). CONCLUSIONS: Plasma KP levels are elevated in hypogonadotropic DP cases and may serve as a useful diagnostic tool in evaluating boys with DP.

Comparative performance of QuantiFERON-TB Gold versus skin test with tuberculosis recombinant allergen (Diaskintest) among patients with suspected pulmonary tuberculosis in Russia.

BACKGROUND: The early identification of Mycobacterium tuberculosis infection can prevent tuberculosis (TB) transmission. A skin test with a tuberculosis recombinant allergen (Diaskintest) is a new method for identification that has been implemented in Russia. This study was performed to compare the performances of Diaskintest and QuantiFERON-TB Gold (QFT) in adults and children with suspected TB in Moscow, Russia. METHODS: Adults (n=85) and children (n=96) were tested using Diaskintest and QFT. Concordance and comparative analyses were performed. RESULTS: Diaskintest and QFT were concordant in 84% of adults and 90% of children (overall concordance 87%, κ>0.6, Kc>0.5). The concordance between QFT, Diaskintest, and the final diagnosis was good in adults (86% and 81%, respectively) and moderate in children (77% and 79%, respectively). In adults, QFT had a higher sensitivity for detecting TB than Diaskintest (82% and 68%, respectively); in children, Diaskintest was more sensitive (73% and 65%, respectively). In patients with a confirmed TB diagnosis, negative Diaskintest/QFT results were associated with low disease activity. Combined Diaskintest/QFT results identified TB patients with higher sensitivity and specificity than each test separately. CONCLUSIONS: Diaskintest is a low-cost diagnostic tool that shows a test positivity rate similar to QFT and can be used in combination with QFT as an adjunctive test for TB diagnosis.

Cell Differentiation Degree as a Factor Determining the Role for Different T-Helper Populations in Tuberculosis Protection.

Efficient tuberculosis (TB) control depends on early TB prediction and prevention. Solution to these tasks requires knowledge of TB protection correlates (TB CoPs), i.e., laboratory markers that are mechanistically involved in the protection and which allow to determine how well an individual is protected against TB or how efficient the candidate TB vaccine is. The search for TB CoPs has been largely focused on different T-helper populations, however, the data are controversial, and no reliable CoPs are still known. Here we discuss the role of different T-helper populations in TB protection focusing predominantly on Th17, "non-classical" Th1 (Th1*) and "classical" Th1 (cTh1) populations. We analyze how these populations differ besides their effector activity and suggest the hypothesis that: (i) links the protective potential of Th17, Th1*, and cTh1 to their differentiation degree and plasticity; (ii) implies different roles of these populations in response to vaccination, latent TB infection (LTBI), and active TB. One of the clinically relevant outcomes of this hypothesis is that over-stimulating T cells during vaccination and biasing T cell response toward the preferential generation of Th1 are not beneficial. The review sheds new light on the problem of TB CoPs and will help develop better strategies for TB control.

Vitamin D Insufficiency in Overweight and Obese Children and Adolescents.

Excessive body weight and obesity in childhood and adolescence are becoming more and more important unfavorable factors that entail extremely adverse consequences and require close attention of physicians of any specialty. Along with the high prevalence of obesity and metabolic syndrome in pediatric patients, children and adolescents in the majority of countries are diagnosed with vitamin D deficiency. Among the non-calcaemic effects of vitamin D, a significant role is played by its impact on the hormonal regulation of glucose metabolism and the synthesis of adipokines by fat tissue. The review presents literature data indicative of a close pathogenic relationship between vitamin D insufficiency and impaired tissue insulin sensitivity. It demonstrates the role of vitamin D insufficiency in immune reactions resulting in development of subclinical inflammation in fat tissue infiltrated with macrophages and lymphocytes. It also shows the role of adipokines, immune system cells and pro-inflammatory cytokines produced by them in the pathogenesis of obesity, as well as the function of vitamin D as an endocrine and paracrine regulator of the process of inflammation in adipose tissue. The relationships between the principal adipokines (leptin, adiponectin, resistin) are revealed in the presence of normal vitamin D content and in vitamin D deficiency. The carbohydrate and lipid metabolism parameters in overweight children and adolescents with vitamin D insufficiency are analyzed. A high prevalence of vitamin D insufficiency in overweight and obese children and adolescents (increasing along with the severity of obesity) is demonstrated. The review also presents the current recommendations for the correction of vitamin D insufficiency and underlines the need for higher cholecalciferol doses to achieve serum calcifediol targets in overweight and obese children and adolescents.

Basic clinical and pathogenetic aspects of developing the complications during multiple pregnancies.

OBJECTIVE: Introduction: In order to evaluate the value of the Placenta Growth Factor (PlGF) in the developing the gestational complications during multiple pregnancies, a study of this indicator in serum of 320 pregnant women with multiple pregnancies in the first trimester, as well as 40 pregnant women with single pregnancy, constituted a control group. The aim: of the study is to investigate the effect of the placental growth factors on gestational process during multiple pregnancies. PATIENTS AND METHODS: Materials and methods: A prospective study of maternity pregnancy in 320 females with multiple pregnancies was conducted, which comprised the main group of the subjects and 40 healthy women with unipolar pregnancy. The level of PlGF in serum was determined by solid phase enzyme analysis using monoclonal antibody sets in the first trimester of pregnancy. Indicators of the hemostasis system (vascular thrombocyte and coagulation link) were evaluated according to generally accepted methods. Dopplerometry of placental and fetal blood flow was performed in uterine arteries, arteries and umbilical cord veins, middle cerebral artery of the fetus. RESULTS: Results: Women with multiple pregnancies are at the risk of gestational complications - premature births in 67.8% (p<0.01), feto placental dysfunction, pre eclampsia - in 17.5% (p <0.05) cases. The revealed violations of the vascular thrombocyte and coagulation homeostasis in the first trimester of pregnancy are the main risk factors for early premature abortion. It has been shown that the low level of placental growth factor in serum of pregnant women with multiple pregnancies in the case of premature labor, feto placental dysfunction and pre-eclampsia (111.23 ± 8.4, 203.24 ± 6.4 and 305.86 ± 7.4 pg / ml) compared with the corresponding indicators for single-pregnancy (418.2 ± 10.4 pg / ml) is a prognostic marker for the development of gestational complications. CONCLUSION: Conclusions: Timely medical correction of gestational complications during multiple pregnancies with the use of micronized progesterone, low molecular weight heparins, angio protectants allowed prolonging the pregnancy with mono choric type of placentation by 3.2 weeks (up to 34.2 ± 2.4 weeks), and in the case of dichoric twins - to full-term pregnancy.

The problem of miscarriage in multiple pregnancy.

OBJECTIVE: Introduction: We present the results of the combined method of treatment and prevention of miscarriage in women with a multiple pregnancy and a high risk of the threat of terminating the pregnancy because of using the obstetric unloading pessaries, combined with micronized progesterone. The efficiency of this method of treatment is evidenced by the rapid elimination of clinical symptoms of threatened abortion, accelerating the regression of ultrasound markers, reducing the number of complications in of pregnant women and reducing the time of their stay in hospital. The aim: To evaluate the effectiveness of the handling the obstetric pessary in combination with micronized progesterone at women with multiple pregnancies and a high risk of miscarriage. PATIENTS AND METHODS: Materials and methods: We analyzed 120 cases of multiple pregnancies with signs of miscarriage within the terms from 16 to 28 weeks. The first group of surveyed were 40 pregnant women with twins and signs of miscarriage, who in the scheme of treatment and prophylactic measures were proposed to use the unloading obstetric pessaries in combination with continuous therapy by natural micronized progesterone until 36 weeks of pregnancy. The second group included 40 pregnant women with twins, who were laid seam on the cervix because of cervical incompetence and were applied short-term courses of therapy by gestagens. The control group comprised 40 pregnant women with twins at the age of 16-28 weeks of pregnancy who were conducted the therapy about the threat of miscarriage according to the current clinical protocols (Order of the Ministry of Health of Ukraine No. 624). It was carried out the analysis of the course of pregnancy, childbirth, the postpartum period and the state of neonatal adaptation in the surveyed groups. RESULTS: Results: In the first group, urgent childbirth occurred in 34 (85%) cases, in group II in 29 (72.5%) cases, in control group - 25 (62.5%) cases. Cesarean delivery was performed in 7 (17.5%) of Group I pregnant women, in 9 (22.5%) of Group II patients and in 11 (27.5%) in the pregnant group. When studying the state of neonatal adaptation of newborns in the examined groups, the following results were obtained. The average weight of the newborns in group I was 3245 ± 280 g, in group II 2865 ± 365 g, in the control group - 2975 ± 325 g (p> 0.05). The evaluation of the state of newborns on the Apgar scale, respectively at the 1st and 5th minutes, was respectively: in newborns of the I group, 7.5 ± 1.4 and 8.4 ± 1.3 points, in group II - 7.3 ± 1.6 and 8.2 ± 1.1 points, in the control group - 7.2 ± 1.6 and 8.6 ± 1.2 (р 1-р 2> 0.05). CONCLUSION: Conclusions: Comprehensive prophylaxis of non-pregnancy in multiple pregnancies, combining the use of a traumatic cardiac cervix with the help of unloading obstetric pussies with progesterone preparations, allows prolonging pregnancy, preventing the development of prematurity, contributes to the improvement of perinatal indicators.

Non-invasive approach to diagnosis of pulmonary tuberculosis using microdroplets collected from exhaled air.

In this report we present a proof-of-principle study aimed at developing non-invasive diagnostics for pulmonary TB that are based on analyzing TB biomarkers in exhaled microdroplets of lung fluid (MLFs). Samples were collected on electrospun filters recently developed by the authors, and then tested for the presence of Mycobacterium tuberculosis (Mtb) cells, Mtb DNA, and protein biomarkers (secreted Mtb antigens and antigen-specific antibodies). The latter were detected using rapid ultra-sensitive immunochemistry methods developed in our laboratory. Neither Mtb cells (limit of detection, LOD = 1 cell) nor Mtb DNA (LOD âˆ¼ 10 CFU) were found in the MLF samples exhaled by TB patients. However, immunoglobulin A (IgA) was found in over 90% of samples from TB patients and healthy volunteers. Antigen-specific IgA were detected at higher rates in the patient samples as compared to those from nominally healthy volunteers resulting in a modest discrimination level of 72% sensitivity and 58% specificity. As such, this novel, non-invasive and fast breath diagnostic method shows promise for further development.

Th1, Th17, and Th1Th17 Lymphocytes during Tuberculosis: Th1 Lymphocytes Predominate and Appear as Low-Differentiated CXCR3+CCR6+ Cells in the Blood and Highly Differentiated CXCR3+/-CCR6- Cells in the Lungs.

Th1 lymphocytes are considered the main mediators of protection against tuberculosis (TB); however, their phenotypic characteristics and relationship with Th17 and Th1Th17 populations during TB are poorly understood. We have analyzed Th1, Th17, and Th1Th17 lymphocytes in the blood and pulmonary lesions of TB patients. The populations were identified based on the production of IFN-γ and/or IL-17 and the coexpression of CXCR3 (X3) and CCR6 (R6). In the blood, IL-17+ and IFN-γ+IL-17+ lymphocytes were barely detectable (median, <0.01% of CD4+ lymphocytes), whereas IFN-γ+ lymphocytes predominated (median, 0.45%). Most IFN-γ+ lymphocytes (52%) were X3+R6+, suggesting their "nonclassical" (ex-Th17) nature. In the lungs, IL-17+ and IFN-γ+IL-17+ lymphocytes were more frequent (0.3%, p < 0.005), yet IFN-γ+ cells predominated (11%). Phenotypically, lung CD4+ cells were X3+/loR6- The degree of differentiation of blood effector CD4+ lymphocytes (evaluated based on CD62L/CD27/CD28 coexpression) increased as follows: X3+R6+ < X3+R6- < X3-R6-, with X3-R6- cells being largely terminally differentiated CD62L-CD27-CD28- cells. Lung CD4+ lymphocytes were highly differentiated, recalling blood X3+/-R6- populations. Following in vitro stimulation with anti-CD3/anti-CD28 Abs, X3+R6+CD4+ lymphocytes converted into X3+R6- and X3-R6- cells. The results demonstrate that, during active TB, Th1 lymphocytes predominate in blood and lungs, document differences in X3/R6 expression by blood and lung CD4+ cells, and link the pattern of X3/R6 expression with the degree of cell differentiation. These findings add to the understanding of immune mechanisms operating during TB and are relevant for the development of better strategies to control it.

Severe Tuberculosis in Humans Correlates Best with Neutrophil Abundance and Lymphocyte Deficiency and Does Not Correlate with Antigen-Specific CD4 T-Cell Response.

It is generally thought that Mycobacterium tuberculosis (Mtb)-specific CD4+ Th1 cells producing IFN-γ are essential for protection against tuberculosis (TB). In some studies, protection has recently been associated with polyfunctional subpopulation of Mtb-specific Th1 cells, i.e., with cells able to simultaneously secrete several type 1 cytokines. However, the role for Mtb-specific Th1 cells and their polyfunctional subpopulations during established TB disease is not fully defined. Pulmonary TB is characterized by a great variability of disease manifestations. To address the role for Mtb-specific Th1 responses during TB, we investigated how Th1 and other immune cells correlated with particular TB manifestations, such as the degree of pulmonary destruction, TB extent, the level of bacteria excretion, clinical disease severity, clinical TB forms, and "Timika X-ray score," an integrative parameter of pulmonary TB pathology. In comparison with healthy Mtb-exposed controls, TB patients (TBP) did not exhibit deficiency in Mtb-specific cytokine-producing CD4+ cells circulating in the blood and differed by a polyfunctional profile of these cells, which was biased toward the accumulation of bifunctional TNF-α+IFN-γ+IL-2- lymphocytes. Importantly, however, severity of different TB manifestations was not associated with Mtb-specific cytokine-producing cells or their polyfunctional profile. In contrast, several TB manifestations were strongly correlated with leukocyte numbers, the percent or the absolute number of lymphocytes, segmented or band neutrophils. In multiple alternative statistical analyses, band neutrophils appeared as the strongest positive correlate of pulmonary destruction, bacteria excretion, and "Timika X-ray score." In contrast, clinical TB severity was primarily and inversely correlated with the number of lymphocytes in the blood. The results suggest that: (i) different TB manifestations may be driven by distinct mechanisms; (ii) quantitative parameters and polyfunctional profile of circulating Mtb-specific CD4+ cells play a minor role in determining TB severity; and (iii) general shifts in production/removal of granulocytic and lymphocytic lineages represent an important factor of TB pathogenesis. Mechanisms leading to these shifts and their specific role during TB are yet to be determined but are likely to involve changes in human hematopoietic system.

Investigation of urine proteome of preterm newborns with respiratory pathologies.

A serious problem during intensive care and nursing of premature infants is the invasiveness of many examination methods. Urine is an excellent source of potential biomarkers due to the safety of the collection procedure. The purpose of this study was to determine the features specific for the urine proteome of preterm newborns and their changes under respiratory pathologies of infectious and non-infectious origin. The urine proteome of 37 preterm neonates with respiratory diseases and 10 full-term newborns as a control group were investigated using the LC-MS/MS method. The total number of identified proteins and unique peptides was 813 and 3672 respectively. In order to further specify the defined infant-specific dataset these proteins were compared with urine proteome of healthy adults (11 men and 11 pregnant women) resulting in 94 proteins found only in infants. Pairwise analysis performed for label-free proteomic data revealed 36 proteins which reliably distinguished newborns with respiratory disorders of infectious genesis from those with non-infectious pathologies, including: proteins involved in cell adhesion (CDH-2,-5,-11, NCAM1, TRY1, DSG2), metabolism (LAMP1, AGRN, TPP1, GPX3, APOD, CUBN, IDH1), regulation of enzymatic activity (SERPINA4, VASN, GAPDH), inflammatory and stress response (CD55, CD 93, NGAL, HP, TNFR, LCN2, AGT, S100P, SERPINA1/C1/B1/F1).

Antigen-Specific IFN-γ Responses Correlate with the Activity of M. tuberculosis Infection but Are Not Associated with the Severity of Tuberculosis Disease.

IFN-γ is a key cytokine in antituberculosis (TB) defense. However, how the levels of its secretion affect M. tuberculosis (Mtb) infection is not clear. We have analyzed associations between IFN-γ responses measured in QuantiFERON®-TB Gold In-tube (QFT) assay, TB disease severity, and Mtb infection activity. TB severity was evaluated based on the results of radiological, microbiological, and clinical examinations. Antigen-driven IFN-γ secretion did not correlate with TB severity. Mitogen-induced IFN-γ secretion correlated inversely with the form of pulmonary pathology and the area of affected pulmonary tissue; the levels of spontaneous IFN-γ secretion correlated with patients' age (r = 0.395, p = 0.001). Mtb infection activity was evaluated based on radiological data of lung tissue infiltration, destruction, dissemination or calcification, and condensation. The rate of positive QFT results and the levels of antigen-driven IFN-γ secretion increased in a row: patients with residual TB lesions < patients with low TB activity < patients with high TB activity. Thus, antigen-driven IFN-γ secretion and QFT results did not associate with TB severity but associated with the infection activity. The results suggest that quantitative parameters of IFN-γ secretion play a minor role in determining the course of TB disease but mirror the activity of the infectious process.