Mononuclear White Blood Cells Research Articles

West Nile virus encephalitis: Clinical characteristics and a comparison to other infectious encephalitides

PurposeTo compare functional outcomes and help differentiate between important causative agents of acute infectious encephalitis in adults, focusing on West Nile virus encephalitis (WNVE). MethodsThe electronic database of Tel Aviv Medical Center was screened for patients admitted during 2010–2020 with acute encephalitis. Additionally, patient laboratory results during the same period were screened for CSF samples positive for common pathogens causing encephalitis. The main patient groups were compared in terms of clinical characteristics and functional outcomes. ResultsOne hundred and five infectious encephalitis patients were identified. WNVE patients (n = 31) and VZV encephalitis (VZVE) patients (n = 31) were older than HSV1 encephalitis (HSV1E) patients (n = 15) (median ages 73, 76, 51, respectively). WNVE patients had a more prominent inflammatory profile. CSF characteristics significantly differed between groups, with an extreme mononuclear white blood cell predominance in VZVE patients (median 98%).Functional outcomes at discharge were significantly worse in WNVE patients (median modified Rankin Scale score 4 at hospital discharge, 2.5 at last follow-up) when compared with HSV1E patients (2.5, 1, respectively) and VZVE patients (1.5, 1, respectively). ConclusionIn odds with previous reports, WNVE and VZVE in this study were far more prevalent than HSV1E. Differences in clinical characteristics could prove clinically useful early in encephalitis, including an association of WNVE with a relatively prominent inflammatory profile (somewhat resembling a bacterial infection) and an extreme mononuclear white blood cell predominance in VZVE. The detrimental outcome of WNVE emphasizes the need to advance research on WNV infection.

Oxidative stress and cytotoxicity of co-formulants of glyphosate-based herbicides in HMWB cells

Abstract Background Glyphosate-based herbicides (GBHs) are widely used worldwide because of their effectiveness and low cost. However, there is a growing concern regarding the potential health risks associated with glyphosate exposure. Glyphosate is always marketed in formulations containing ingredients other than the active substance. These co-formulants may have toxic effects on humans and animals. Although several studies have assessed the health risks related to glyphosate, the toxicological effects of the co-formulants must be analysed in more detail to ensure the health and safety of pesticide users and the general population. This study aimed to characterize and compare the cytotoxic effects of co-formulants with those of the active substance and GBH formulations. Methods Human mononuclear white blood (HMWB) cells were exposed to varying concentrations (0.1 µM, 1µM, 10 µM, 100 µM, 1 mM, 10 mM) of glyphosate, three GBHs (Fozat 480, Roundup, and Glyfos), and two co-formulants for 4- and 20-hours. Cytotoxicity was assessed using the CCK-8 assay, and oxidative stress levels in HMWB cells were evaluated using the superoxide dismutase (SOD) assay. Cell viability and SOD activity were analysed using a spectrophotometer at 460 nm absorbance. Results Glyphosate alone did not significantly affect the cell viability. However, notable cytotoxic effects were observed in response to GBHs and adjuvants at concentrations as low as 100 µM. Furthermore, the Oxidative stress levels in cells treated with GBHs or adjuvants were significantly higher than those treated with glyphosate alone. Conclusions These findings suggest that the cytotoxic effects of GBHs are likely attributed to the adjuvants present in the formulations, which promote oxidative stress. Further studies are necessary to evaluate the health risks associated with using glyphosate-based herbicides, and co-formulants should be considered when assessing their safety. Key messages • The safety of GBHs cannot be solely attributed to glyphosate itself but also to the other components in the formulation. • Toxicity evaluation should take into account not only glyphosate but also the co-formulants present in these herbicides when assessing their safety.

In Vitro Protective Effects of Aqueous and Alcoholic Extracts of Caryophyllus aromaticus on Reducing H2O2-Induced DNA Damage in Human Mononuclear White Blood Cells

In Vitro Protective Effects of Aqueous and Alcoholic Extracts of Caryophyllus aromaticus on Reducing H2O2-Induced DNA Damage in Human Mononuclear White Blood Cells

Oxidative Stress and Cytotoxicity Induced by Co-Formulants of Glyphosate-Based Herbicides in Human Mononuclear White Blood Cells.

The use of genetically modified, glyphosate-resistant crops has led to the widespread application of glyphosate-based herbicides (GBHs), making them one of the most widely used herbicide formulations on the market. To enhance the efficacy of the active ingredient, GBHs used in practice often contain other ingredients marked as inert "adjuvants" or "co-formulants", the toxic properties of which are poorly understood. The objective of this study was to compare the cytotoxic effects of pure glyphosate, three GBHs (Roundup Mega, Fozat 480 and Glyfos) and two co-formulants commonly used in GBHs as assessed via CCK-8 assay, and the extent of their potential oxidative damage as assessed via superoxide dismutase (SOD) assay, in order to reveal the role of adjuvants in the toxicity of the formulations. Our results showed that glyphosate alone did not significantly affect cell viability. In contrast, GBHs and adjuvants induced a pronounced cytotoxic effect from a concentration of 100 μM. SOD activity of cells treated with GBHs or adjuvants was significantly lower compared to cells treated with glyphosate alone. This suggests that the adjuvants in GBHs are responsible for the cytotoxic effects of the formulations through the induction of oxidative stress.

Study of biocompatibility and antitumor cytotoxic activity <i>in vitro</i> of Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys strengthened by equal angular pressing

Introduction. The biological activity of potential biodegradable zinc-based alloys that are promising for oncoorthopedics was studied in this work. The alloys were processed by equal-channel angular pressing, which made it possible to increase their strength due to microstructure refinement and the ability to provide the functionality of osteosynthesis, fixed due to the metal structure developed on their basis.Aim. Investigation of effect of equal-channel angular pressing (ECAP) treatment on strength, ductility, degradation rate, biocompatibility in vitro and cytotoxicity against SKOV-3 tumor cells of the Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys.Materials and methods. The Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys in the initial state and after ECAP were used as objects of study, and blood cells of CBA mice were used as model systems. To assess the hemolytic activity, the samples were incubated with red blood cells for 4 and 24 hours at 37 °C, assessing the relative increase in the level of extracellular hemoglobin compared to the intact control. The cytotoxicity of the alloys was assessed by the change in the level of extracellular lactate dehydrogenase (LDH) activity after 24 hours of incubation with mononuclear white blood cells. The study of antitumor cytotoxic activity was carried out on human ovarian cancer cells of the SKOV-3 line in vitro, assessing their survival after 48 hours of incubation with alloy samples using the LDH test.Results. As a result of the studies, it was concluded that the studied alloys after ECAP treatment retained their biocompatibility, since there were no signs of hemolysis and cytotoxicity with respect to blood cells. However, contact with samples of all studied alloys in vitro induced a significant inhibition of the metabolic activity of the ovarian cancer cell culture in comparison with the control. Incubation with alloys samples leads to a decrease in cellular activity by an average of 49 % and 59 % for Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys, respectively. The addition of calcium to the composition of the alloy Zn – 1 %Mg contributed to the growth of antitumor cytotoxic activity.Conclusion. Thus, based on the results of assessing the hemolytic activity and cytotoxicity of the samples, we can conclude that the studied alloys are biocompatible. It was also found that Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys had a pronounced cytotoxic effect on SKOV-3 tumor cells. The obtained data indicate the prospects for the development of a new type of medical devices based on the studied alloys, promising, in particular, for oncoorthopedics: a metal structure developed on their basis can ensure the strength of osteosynthesis, reduce the risk of local recurrence of oncological disease and does not require a second operation to remove the device.

Adenovirus-Associated Urethritis, a Not So Uncommon Entity. A Review of 91 Cases.

Human adenovirus (HAdV) is likely an underdiagnosed cause of urethritis, although it was already associated with urethritis in descriptions published more than 40 years ago. Differential clinical features of this entity, such as meatitis, conjunctivitis, and a predominance of mononuclear white blood cells in first-void urine and/or urethral smear, can be useful to increase diagnostic suspicion. We retrospectively studied 91 episodes of HAdV-associated urethritis diagnosed for 9 years and 6 months after optimizing efforts to detect the pathogen mainly in patients with features suggestive of this condition. Dysuria was the main symptom (84%), whereas meatitis was observed in 34% of cases. Furthermore, 40% of patients had conjunctivitis. Human adenovirus type D was the most prevalent HAdV (56%), although HAdV-C6, a type not previously associated with urethritis, was observed in 12 patients (13%). Urethritis due to HAdV is not uncommon, and it is important to screen for it to avoid unnecessary treatments, contact tracing studies, and checkups. The use of multiplex polymerase chain reaction assays that include HAdV, for the diagnosis of urethritis, would raise awareness of its role in this entity.

Infectious Encephalitis in Adults: Characteristics, Outcome and Clinical Clues (S25.010)

<h3>Objective:</h3> Clinical characterization and comparison between the major causative agents of acute infectious encephalitis in adults. <h3>Background:</h3> The annual incidence of acute infectious encephalitis in adults is reported between 0.5–16 per 100,000 persons. West Nile virus (WNV) is among the most important causative agents of viral encephalitis. <h3>Design/Methods:</h3> We retrospectively characterize the clinical course of acute infectious encephalitis among adult patients admitted to the Tel-Aviv Medical Center between 2010 and 2020 (n=95), comparing major causative agents and focusing on WNV encephalitis (WNVE, n=31), varicella-zoster virus encephalitis (VZVE, n=30) and herpes simplex 1 virus encephalitis (HSV1E, n=13). <h3>Results:</h3> Patient groups differed in terms of age, with WNVE (median age 73) and VZVE (76) patients significantly older compared with HSV1E patients (51). CSF characteristics differed between groups, with VZVE patients having significant mononuclear white blood cell (WBC) predominance (99%) and HSV1E patients having lower CSF protein (57 mg/dL). Blood inflammation markers also differed significantly, with WBC higher in WNVE and C-reactive protein lower in HSV1E. Functional outcome was the worst in WNVE patients at discharge (median modified Rankin Scale [mRS] score 4) and last follow-up (median mRS score 4), reaching statistical significance when compared with VZVE patients. Differences in functional outcome prevailed when excluding patients with poor functional baseline (mRS score &gt; 2 before presentation). Logistic regression found age as an independent variable associated with worse functional outcome, and age, baseline mRS and WNVE (compared to VZVE) as associated with mortality. <h3>Conclusions:</h3> In odds with previous reports, WNVE and VZVE were far more common than HSV1E. Differences in laboratory characteristics could prove clinically useful early in the presentation of acute encephalitis. In some aspects, WNVE patients had a course resembling a bacterial, rather than viral, CNS infection. The detrimental outcome of WNVE emphasizes the need to promote solutions to WNV infection such as vaccinations and specific drugs. <b>Disclosure:</b> Dr. Maayan Eshed has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Bionaut Labs Ltd. Dr. Levinson has nothing to disclose. Dr. Mina has nothing to disclose. Dr. Ashkenazi has nothing to disclose. Dr. Dekel has nothing to disclose. Dr. Cohen-Poradosu has nothing to disclose. Yifat Alcalay has nothing to disclose. Orna Aizenstein Lehavi has nothing to disclose. Yael Paran has nothing to disclose. Dr. Gadoth has received intellectual property interests from a discovery or technology relating to health care.

Kidney Biopsy Features Most Predictive of Clinical Outcomes in the Spectrum of Minimal Change Disease and Focal Segmental Glomerulosclerosis.

Heterogeneity in disease course and treatment response among patients with MCD/FSGS necessitates a granular evaluation of kidney tissue features. This study aimed to identify histologic and ultrastructural descriptors of structural changes most predictive of clinical outcomes in the Nephrotic Syndrome Study Network (NEPTUNE). Forty-eight histologic (37 glomerular, 9 tubulointerstitial, 2 vascular) and 20 ultrastructural descriptors were quantified by applying the NEPTUNE Digital Pathology Scoring System to NEPTUNE kidney biopsies. Outcomes included time from biopsy to disease progression, first complete remission of proteinuria, and treatment response. Relative importance of pathology and clinical predictors was obtained from random forest models, and predictive discrimination was assessed. Among 224 participants (34% Black, 24% Hispanic), model performance was excellent, with predictive discrimination of 0.9 for disease progression, 0.85 for complete remission, and 0.81 for treatment response. The most predictive descriptors of outcomes included both conventional-e.g., global sclerosis or segmental sclerosis and interstitial fibrosis/tubular atrophy-and novel features, including adhesion, interstitial foam cells, deflation, periglomerular fibrosis, mononuclear white blood cells, endothelial cell abnormalities, microvillous transformation, and acute tubular injury. The most predictive descriptors of clinical outcomes among MCD/FSGS patients reflected structural changes in multiple renal compartments. Reporting these descriptors should be standardized to guide prognostication of proteinuric glomerular diseases.

An in silico analysis of genome-wide expression profiles of the effects of exhaustive exercise identifies heat shock proteins as the key players

Physical exercise induces important system disturbances in the human body in a dose-response manner. Meta-analyses of genome-wide expression studies (GWES) might contribute to identify gene expression patterns and to a better understanding of the molecular mechanisms behind the complexity of adaptations to exercise, under a systems biology approach. Here, we aimed to analyze available data for human GWES that have evaluated the effect of exhaustive exercise in peripheral blood mononuclear cells (PBMC) and white blood cells (WBC). Three primary datasets retrieved from the NCBI Gene Expression Omnibus were meta-analyzed using a random effects model in the NetworkAnalyst software. After identifying nine differentially expressed genes (DEGs), we performed functional enrichment analyses to extract relevant biological information. A protein-protein interactions network on DEGs was built to evaluate the associated regulatory pathways. We found that five upregulated genes were members of the heat shock protein family, one of the top stress-response groups of genes. The enrichment analysis revealed key roles of the DEGs on the cellular adaptations to exercise-induced stress (i.e., temperature stimulus, topologically-incorrect and unfolded proteins). Our comparison analysis of DEG signatures found in blood cells with the expression pattern on muscle skeletal tissue showed some common genes. Thus, novel DEGs that might serve as hormetic mediators to exercise-induced adaptations were identified. Further experimental research is needed to validate these findings.

Micronucleus Formation Induced by Glyphosate and Glyphosate-Based Herbicides in Human Peripheral White Blood Cells.

Glyphosate is the most commonly used herbicide around the world, which led to its accumulation in the environment and consequent ubiquitous human exposure. Glyphosate is marketed in numerous glyphosate-based herbicide formulations (GBHs) that include co-formulants to enhance herbicidal effect of the active ingredient, but are declared as inert substances. However, these other ingredients can have biologic activity on their own and may interact with the glyphosate in synergistic toxicity. In this study, we focused to compare the cytogenetic effect of the active ingredient glyphosate and three marketed GBHs (Roundup Mega, Fozat 480, and Glyfos) by investigating cytotoxicity with fluorescent co-labeling and WST-1 cell viability assay as well as genotoxicity with cytokinesis block micronucleus assay in isolated human mononuclear white blood cells. Glyphosate had no notable cytotoxic activity over the tested concentration range (0–10,000 μM), whereas all the selected GBHs induced significant cell death from 1,000 μM regardless of metabolic activation (S9). Micronucleus (MN) formation induced by glyphosate and its formulations at sub-cytotoxic concentrations (0–100 μM) exhibited a diverse pattern. Glyphosate caused statistically significant increase of MN frequency at the highest concentration (100 μM) after 20-h exposure. Contrarily, Roundup Mega exerted a significant genotoxic effect at 100 μM both after 4- and 20-h exposures; moreover, Glyfos and Fozat 480 also resulted in a statistically significant increase of MN frequency from the concentration of 10 μM after 4-h and 20-h treatment, respectively. The presence of S9 had no effect on MN formation induced by either glyphosate or GBHs. The differences observed in the cytotoxic and genotoxic pattern between the active principle and formulations confirm the previous concept that the presence of co-formulants in the formulations or the interaction of them with the active ingredient is responsible for the increased toxicity of herbicide products, and draw attention to the fact that GBHs are still currently in use, the toxicity of which rivals that of POEA-containing formulations (e.g., Glyfos) already banned in Europe. Hence, it is advisable to subject them to further comprehensive toxicological screening to assess the true health risks of exposed individuals, and to reconsider their free availability to any users.

The effects of the dental methacrylates TEGDMA, Bis-GMA, and UDMA on neutrophils in vitro.

ObjectivesThe prevalent usage of methacrylates in modern dentistry demands good knowledge of their biological impacts. While there have been several studies demonstrating the effects of different methacrylic monomers on mononuclear white blood cells, very little is known about the effects caused by these monomers on neutrophilic granulocytes. The objective of this study was to add novel knowledge about how neutrophils are affected by exposure to triethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), and bisphenol A glycol dimethacrylate (Bis‐GMA) alone or in combinations.Materials and MethodsIsolated neutrophils were cultured in the presence or absence of methacrylates. The IL‐8 release was measured using a DuoSet ELISA development kit. Apoptosis and necrosis were analyzed using flow cytometry. The formation of neutrophil extracellular traps (NETs) was investigated using Sytox green DNA staining combined with microscopically examination of released DNA and myeloperoxidase (MPO).ResultsThe release of IL‐8 was significantly increased after exposure to TEGDMA, Bis‐GMA, UDMA, or TEGDMA in combination with Bis‐GMA or UDMA compared to the unstimulated controls. Exposure to TEGDMA, UDMA, and Bis‐GMA for 24 hr separately or in combination did not affect apoptosis or necrosis of the exposed neutrophils. NET structures were formed by neutrophils after exposure to the different combinations of the methacrylates.ConclusionThe combination of TEGDMA and Bis‐GMA had a synergistic proinflammatory effect on neutrophils by increasing the release of IL‐8 and the formation of NET structures. The changes in the normal functions of neutrophils caused by methacrylate exposure may lead to altered inflammatory response and relate to previously reported adverse immune reactions caused by these substances.

Cellular internalization of targeted and non-targeted delivery systems for contrast agents based on polyamidoamine dendrimers

New high-molecular-weight contrast agents based on polyamidoamine (PAMAM) dendrimers for targeted imaging of malignant tumors characterized by overexpression of human epidermal growth factor receptor (EGFR) and human alpha-fetoprotein receptor (RECAF) were designed. Conjugates of second (G2) and third (G3) generation polyamidoamine dendrimers with 1,4,7,10-tetraazocyclodecane-1,4,7,10-tetraacetic acid (DOTA) were obtained. The quantitative composition of the conjugates was determined by 1HNMR spectroscopy. It was shown that four out of the 16 terminal NH2 groups in G2-DOTA and nine out of the 32 groups in G3-DOTA were modified with DOTA. The morphology, size, and charge of the synthesized macromolecules were characterized by dynamic light scattering and electrophoresis. Gadolinium(III) was loaded into the conjugates and the Gd content was determined by atomic emission spectroscopy. For increasing the selectivity of accumulation in the tumor cells, two recombinant proteins able to bind selectively to EGFR and RECAF, namely, human recombinant epidermal growth factor (rEGF) and human recombinant 3rd domain of alpha-fetoprotein (3dAFPpG), were conjugated with G2 and G3 dendrimers. The conjugates containing vector molecules were mainly accumulated via clathrin-dependent endocytosis, whereas G2-DOTA and G3-DOTA were absorbed via caveolin-dependent endocytosis and macropinocytosis. The dendrimer conjugates with vector molecules were intensely accumulated in A549 cells characterized by high expression of EGFR (Herl) and RECAF, whereas the accumulation of conjugates in the control K562 cells (with low expression of Her1) and in the CD14− population of human unstimulated mononuclear white blood cells was insignificant. The 3dAFPpG-conjugated dendrimers were partly recycled. All synthesized conjugates had a rather low toxicity in the range of 350–450 µmol L−1 (IC50).

Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data

BackgroundMany data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone. MethodsKey parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial “Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)” aimed at reconstruction of alveolar bone. ResultsDespite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings. ConclusionsClinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.

Interlaboratory evaluation of the genotoxic properties of pencycuron, a commonly used phenylurea fungicide

Pencycuron, a phenylurea-type antifungal agent, is used in agriculture worldwide for inhibiting the growth of various fungal pathogens of crops. Pencycuron residues were found in vegetables, soil and drinking water. Accordingly, both occupational and consumer exposure can be expected and may be significant. However, human toxicity studies on its genotoxic, mutagenic or carcinogenic potential are lacking. Therefore, a collaborative study was performed in two laboratories to investigate whether pencycuron exposure can induce DNA damage.The genotoxic effect of 0–100 μg/ml pencycuron in in vitro cultures of human mononuclear white blood cells (MWBCs) and human hepatocytes (HepG2) was detected by cytokinesis-block micronucleus assay and comet assay.The combined results of the two labs showed a dose-dependent DNA damage detected by micronucleus frequency, which reached statistical significance at 100 μg/ml concentration after 21-h exposure in HepG2 cells (p = 0.048). Significant genotoxic effect could also be observed in the comet assay from 50 μg/ml concentration in MWBCs, and at 100 μg/ml concentration in HepG2 cells in one lab. Nevertheless, this finding was not confirmed by the other lab in HepG2 cells, where Fpg-dependent oxidative DNA damage could also not be detected.The results indicate that pencycuron may have DNA-damaging potential as well as point out inter-laboratory variability that calls for further studies to confirm the genotoxicity of this fungicide.

PTPN11 Mutations in JMML Confer Dasatinib Sensitivity in a TNK2-Dependent Manner

Introduction: Juvenile myelomonocytic leukemia (JMML) is a rare disease characterized by an overproduction of granulocytes and monocytes, with cells displaying hypersensitivity to stimulation by the cytokine GM-CSF in culture. The only curative approach for JMML is allogeneic hematopoietic stem cell transplant, and disease recurrence is the leading cause of death in these patients, occurring within two years in 50% of patients. New therapeutic strategies are needed to improve treatment and survival.Methods: In order to better identify therapeutic strategies, our lab has developed ex vivo functional assays with small-molecule inhibitors and RNAi for the profiling of actionable targets in primary patient samples. In these assays, mononuclear cells from patient bone marrow or blood are cultured in the presence of small molecule inhibitor or RNAi libraries and cell viability is measured by colorimetric assay. To investigate targets identified in these assays, gene constructs are transfected into HEK293 T17 cells for immunoblots or transduced into mouse bone marrow cells for methylcellulose-based colony formation assays.Results: Mononuclear white blood cells from a patient with recurrent JMML were found to be sensitive to the multi-family kinase inhibitor dasatinib as well as siRNA targeting the dasatinib target tyrosine kinase non-receptor 2 (TNK2, also known as ACK1), suggesting an over-reliance on TNK2 for cell viability. TNK2 was recently demonstrated by our lab and others to regulate signaling downstream of signaling pathways including EGFR, ER, Src, and CSF3R. Based on the ex vivo functional data for this JMML specimen, the patient was placed on daily dasatinib and achieved marked improvement, which bridged the patient to an additional bone marrow transplant, resulting in disease-free survival for another year.This patient had a mutation in PTPN11 (also known as SHP2), which encodes a protein tyrosine phosphatase that is mutated in approximately 35% of JMML patients. PTPN11 is an auto-inhibited phosphatase that dephosphorylates targets in many signaling pathways including RAS-MAPK, with an overall positive effect on pathway activity and cellular proliferation. In JMML, mutations in residues of the N-terminal SH2 domain of PTPN11 result in a constitutively active form of the protein, which increases proliferation and cell survival and is linked to dysregulation of RAS/MAPK.Results of in vitro experiments demonstrate, for the first time, an interaction of TNK2 with mutant PTPN11. Co-expression of mutant-PTPN11 and TNK2 results in increased phospho-PTPN11 at two sites associated with activation of PTPN11 (Y542 and Y580), and co-immunoprecipitation experiments indicate direct interaction of TNK2 with PTPN11. These phosphorylation events coincide with significantly increased MEK/ERK signaling in the context of TNK2/mutant PTPN11 co-expression relative to expression of either gene alone. Expression of a TNK2 mutant harboring a loss-of-function mutation at a key activating residue (TNK2 Y284F) reduces PTPN11/MAPK activation, whereas expression of a gain-of-function, drug-resistant TNK2 gatekeeper mutant increases PTPN11/MAPK activation when compared to wild-type TNK2. Inhibition of TNK2 with either dasatinib or the TNK2-specific inhibitor AIM100 also reduces PTPN11/MAPK activation. Interestingly, phospho-TNK2 levels are significantly reduced in the presence of mutant-PTPN11, suggesting a feedback mechanism that has yet to be fully elucidated.Functional experiments suggest that mutant-PTPN11 and TNK2 work in a synergistic fashion to increase cell transformation and cytokine hypersensitivity. Mouse bone marrow cells transduced with both mutant PTPN11 and TNK2 have significantly more colonies in methylcellulose colony formation assays than with mutant- PTPN11 alone or with wild-type PTPN11 and TNK2. Treatment of cells in colony formation assays with either dasatinib or AIM100 inhibits colony formation, suggesting a dependence on TNK2 kinase activity for cell transformation.Conclusions: Taken together, the preliminary data suggest that PTPN11 mutations may be a functional marker for dasatinib sensitivity, and that the underlying mechanism for this sensitivity may be through the inhibition of TNK2 activity by dasatinib. Our aim is further validate the role of PTPN11 mutations as a marker for efficacy of dasatinib or other TNK2 inhibitors. DisclosuresNo relevant conflicts of interest to declare.

Parenting, Socioeconomic Status Risk, and Later Young Adult Health: Exploration of Opposing Indirect Effects via DNA Methylation.

A sample of 398 African American youth, residing in rural counties with high poverty and unemployment, were followed from ages 11 to 19. Protective parenting was associated with better health, whereas elevated socioeconomic status (SES) risk was associated with poorer health at age 19. Genome-wide epigenetic variation assessed in young adulthood (age 19), was associated with both SES risk and protective parenting. Three categories of genes were identified whose methylation was associated with parenting, SES risk, and young adult health. Methylation was a significant mediator of the impact of parenting and SES risk on young adult health. Variation in mononuclear white blood cell types was also examined and controlled, showing that it did not account for observed effects of parenting and SES risk on health.

Інтенсивність процесів ліпідної пероксидації у кролів з хімічним опіком рогівки на тлі мерказоліл-індукованого гіпотиреозу

&lt;p&gt;Резюме . У статті проаналізовано результати дослідження інтенсивності процесів ліпідної пероксидації у&lt;br /&gt;крові і внутрішньоочній рідині передньої камери ока кролів з хімічним опіком рогівки, викликаним накладанням&lt;br /&gt;фільтрувального паперу, змоченого 1 N NaOH на тлі попередньо змодельованого мерказоліл-індукованого&lt;br /&gt;гіпотиреозу. Встановлено, що у тварин з хімічним опіком рогівки на тлі гіпотиреозу, порівняно з евтироїдними&lt;br /&gt;тваринами, виявляється суттєве зниження продукування активних форм кисню мононуклеарними лейкоцитами&lt;br /&gt;крові, пригнічення інтенсивності ліпідної пероксидації, що виражається у зменшенні концентрації гідропероксидів&lt;br /&gt;ліпідів, ТБК-реагуючих продуктів та основ Шиффа у крові і внутрішньоочній рідині передньої камери ока. Зроблено&lt;br /&gt;висновок, що за умов супутнього дефіциту гормонів щитоподібної залози має місце пригнічення активності&lt;br /&gt;вільнорадикальних процесів як у місці ураження, так і в організмі в цілому.&lt;br /&gt;Ключові слова : опік рогівки, гіпотиреоз, ліпідна пероксидація.&lt;/p&gt;

Detection of malignant cells in serous body fluids by counting high-fluorescent cells on the Sysmex XN-2000 hematology analyzer.

The body fluid mode of the Sysmex XN-2000 hematology analyzer differentiates cells into mononuclear and polymorphonuclear white blood cells (WBC) and high-fluorescent cells (HFC). The aim of this study was to evaluate the performance of the HFC count for detecting malignant cells in serous body fluids. Two-hundred and thirty serous fluids were analyzed on the Sysmex XN body fluid mode. HFC were measured as relative count (HFC/100 WBC) and absolute count (HFC/μL). All samples were microscopically screened on cytospin slides for the presence of malignant cells. Malignant cells were found by microscopic examination in 49 of 230 samples (21.3%). Malignant samples contained significantly higher percentages (10.2 vs. 2.6/100 WBC) and absolute numbers (65 vs. 10/μL) of HFC than nonmalignant samples (P < 0.001). Areas under the ROC curve for relative and absolute HFC count were 0.69 and 0.77, respectively. A cutoff level of ≥17 HFC/μL showed the best performance to predict malignancy, with 88% sensitivity and 61% specificity. As serous body fluids will be more analyzed on automated analyzers in the future, HFC count can be a useful tool to select samples for microscopic review. Microscopic evaluation should be performed if HFC values are above a certain threshold (e.g. ≥17 HFC/μL) or in case of clinical suspicion of malignancy.

Higher levels of protective parenting are associated with better young adult health: exploration of mediation through epigenetic influences on pro-inflammatory processes

The current investigation was designed to examine the association of parenting during late childhood and early adolescence, a time of rapid physical development, with biological propensity for inflammation. Based on life course theory, it was hypothesized that parenting during this period of rapid growth and development would be associated with biological outcomes and self-reported health assessed in young adulthood. It was expected that association of parenting with health would be mediated either by effects on methylation of a key inflammatory factor, Tumor necrosis factor (TNF), or else by association with a pro-inflammatory shift in the distribution of mononuclear blood cells. Supporting expectations, in a sample of 398 African American youth residing in rural Georgia, followed from age 11 to age 19, parenting at ages 11–13 was associated with youth reports of better health at age 19. We found that parenting was associated with changes in TNF methylation as well as with changes in cell-type composition. However, whereas methylation of TNF was a significant mediator of the association of parenting with young adult health, variation in mononuclear white blood cell types was not a significant mediator of the association of parenting with young adult health. The current research suggests the potential value of examining the health-related effects of parenting in late childhood and early adolescence. Further examination of protection against pro-inflammatory tendencies conferred by parenting appears warranted.

Fructose 1,6-bisphosphatase deficiency: clinical, biochemical and genetic features in French patients.

Fructose-1,6-bisphosphatase (FBPase) deficiency is a very rare autosomal recessive disorder caused by a mutation of the fructose-1,6-bisphosphatase gene(FBP1). Disease is mainly revealed by hypoglycemia and lactic acidosis, both symptoms being characteristic for an enzymatic block in the last steps of the gluconeogenesis. Twelve patients with FBPase deficiency were diagnosed in France in the 2001-2013 period, using a diagnostic system based on a single blood sample which allows simultaneous enzyme activity measurement on mononuclear white blood cells and molecular analysis. Sequencing of exons and intron-exon junctions of FBP1 gene was completed in unsolved cases by a gene dosage assay developed for each exon. For most patients, first metabolic decompensation occurred before two years of age with a similar sequence: the triggering factors were fever, fasting, or decrease of food intake. However, diagnosis was made late at a mean age of 3 years, as mitochondrial defects or glycogen storage diseases were firstly suspected. Enzyme activity in leukocytes was dramatically decreased (<10%). Twelve different mutations were identified in 22 alleles among them seven were novels: one missense mutation c.472C > T, one point deletion c.48del, one point duplication c.865dupA, one deletion-insertion, and two splice mutations (c.427-1del and c.825 + 1G > A). We described the first intragenic deletion in FBP1 (g.97,364,754_97,382,011del) in homozygous state. Our report also confirms that this very rare disease is misdiagnosed, as other energetic defects are firstly suspected.