Montreal cognitive assessment in Brazilian adults with sickle cell disease: The burdens of poor sociocultural background.

Sickle cell disease (SCD) patients are at higher risk of developing silent cerebral infarcts and overt stroke, which may reflect cognitive impairment, functional limitations, and worse quality of life. The cognitive function of Brazilian adult SCD patients (n = 124; 19-70 years; 56 men; 79 SS, 28 SC, 10 S/ß0, 7 S/ß+) was screened through Montreal Cognitive Assessment (MoCA) and correlated the results with possible predictive factors for test performance, including sociocultural, clinical, laboratory data and brain imaging. The Median MoCA score was 23 (8-30); 70% had a 25-or-less score, suggesting some level of cognitive impairment. There were no significant associations between MoCA results and any clinical or laboratory data in SS and SC patients; however, a significant correlation (P = 0.03) with stroke was found in HbS/ß-thalassemic patients. Correlations were further detected according to sociodemographic conditions, such as age (r = -0.316; P < 0.001), age at first job (r = 0.221; P = 0.018), personal (r = 0.23; P = 0.012) and per capita familiar incomes (r = 0.303; P = 0.001), personal (r = 0.61; P = 0), maternal (r = 0.536; P = 0), and paternal educational status (r = 0.441; P = 0). We further sought independent predictors of performance using multivariable regressions and increased education was an independent predictor of better scores in MoCA (0.8099, 95% confidence interval [CI]: 0.509-1.111). Brain imaging analysis showed significant and progressive atrophy in important cerebral areas related to memory, learning, and executive function. These data point to the high prevalence and impact of cognitive decline in adult SCD patients, mirrored in brain atrophic areas. It is also possible to observe the influence of sociodemographic conditions on patients' cognitive performances and the need for creating focused therapeutic plans that address these deficiencies. Moreover, the absence of a significant correlation of MoCA values with stroke in the SS and SC groups may be related to the worst sociocultural and economic conditions of the Brazilian African descent population, in which the impact of low educational stimulation on cognitive function can outweigh even the anatomical damage caused by the disease.

Sickle Cell Disease in Brazil: Current Management.

Sickle cell disease (SCD) comprises inherited red blood cell disorders due to a mutation in the ß-globin gene (c20A > T, pGlu6Val) and is characterized by the presence of abnormal hemoglobin, hemoglobin S, hemolysis, and vaso-occlusion. This mutation, either in a homozygous configuration or in compound states with other ß-globin mutations, leads to polymerization of hemoglobin S in deoxygenated conditions, causing modifications in red blood cell shape, particularly sickling. Vaso-occlusive crisis (VOC) is the hallmark of the disease, but other severe complications may arise from repeated bouts of VOCs. SCD is considered a global health problem, and its incidence has increased in some areas of the world, particularly the Americas and Africa. Management of the disease varies according to the region of the world, mainly due to local resources and socioeconomic status. This review aimed to describe more recent data on SCD regarding available treatment options, especially in Brazil. New treatment options are expected to be available to all patients, particularly crizanlizumab, which is already approved in the country.

Sickle cell disease and increased adverse maternal and perinatal outcomes in different genotypes.

BACKGROUND: Sickle cell disease (SCD) comprises a heterogeneous group of inherited hemolytic disorders that increases the risk of maternal and perinatal complications due to chronic systemic inflammatory response, endothelial damage and vaso-occlusion. The contribution of genotypes to the severity of outcomes during pregnancy is not completely established. METHODS: A retrospective study of medical charts was performed to compare maternal and perinatal outcomes in Hb SS, Hb SC disease and sickle-beta thalassemia (Hb Sß) pregnancies followed at a high-risk antenatal care unit over a 6-year period. A descriptive analysis of morphological findings was performed of the placenta when pathology reports were available. RESULTS: Sixty-two SCD pregnant women [25 Hb SS (40 %), 29 Hb SC (47 %) and 8 Hb Sß (13 %)] were included. Overall, SCD was associated with maternal complications (77 %), preterm birth (30 %), cesarean section (80 %) and a need of blood transfusion. In general there were no statistically significant differences between genotypes. The only significant difference was the hemoglobin level at first antenatal care visit which was lower for the homozygous genotype (7.7 g/dL) compared to Hb SC and Hb Sß (9.7 g/dL and 8.4 g/dL, respectively; p-value = 0.01). Ten of 15 evaluated placentas showed abnormal morphological findings CONCLUSION: SCD, regardless of the underlying genotype, is associated with increased adverse maternal and perinatal outcomes and placental abnormalities associated with maternal vascular malperfusion.

BMP6 and VDR gene polymorphisms are associated with osteonecrosis in a sickle cell anaemia cohort.

The occurrence and severity of osteonecrosis in sickle cell anaemia (SCA) vary due to risk factors, including genetic modifiers. Bone morphogenetic proteins (BMPs), particularly BMP6, and the vitamin D receptor (VDR) play key roles in cartilage and bone metabolism, making them potential contributors to orthopaedic outcomes in SCA. Here, we evaluated the association of polymorphisms in BMP6 (rs3812163, rs270393 and rs449853) and VDR (FokI rs2228570 and Cdx2 rs11568820) genes with osteonecrosis risk in a Brazilian SCA cohort. A total of 177 unrelated SCA patients were selected. The AA genotype of BMP6 rs3812163 was independently associated with a lower osteonecrosis risk (p = 0.015; odds ratio (OR): 0.38; 95% confidence interval (CI): 0.18-0.83) and with the long-term cumulative incidence of osteonecrosis (p = 0.029; hazard ratio: 0.56, 95% CI: 0.34-0.94). The VDR rs2228570 TT genotype was independently associated with a lower osteonecrosis risk (p = 0.039; OR: 0.14; 95% CI: 0.02-0.90). In summary, our results provide evidence that BMP6 rs3812163 and the VDR rs2228570 might be implicated in osteonecrosis pathophysiology in SCA and might help identify individuals at high risk.

Comparative transcriptomic analysis of circulating endothelial cells in sickle cell stroke.

Ischemic stroke (IS) is one of the most impairing complications of sickle cell anemia (SCA), responsible for 20% of mortality in patients. Rheological alterations, adhesive properties of sickle reticulocytes, leukocyte adhesion, inflammation and endothelial dysfunction are related to the vasculopathy observed prior to ischemic events. The role of the vascular endothelium in this complex cascade of mechanisms is emphasized, as well as in the process of ischemia-induced repair and neovascularization. The aim of the present study was to perform a comparative transcriptomic analysis of endothelial colony-forming cells (ECFCs) from SCA patients with and without IS. Next, to gain further insights of the biological relevance of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction network (PPI) construction and in silico prediction of regulatory factors were performed. Among the 2469 DEGs, genes related to cell proliferation (AKT1, E2F1, CDCA5, EGFL7), migration (AKT1, HRAS), angiogenesis (AKT1, EGFL7) and defense response pathways (HRAS, IRF3, TGFB1), important endothelial cell molecular mechanisms in post ischemia repair were identified. Despite the severity of IS in SCA, widely accepted molecular targets are still lacking, especially related to stroke outcome. The comparative analysis of the gene expression profile of ECFCs from IS patients versus controls seems to indicate that there is a persistent angiogenic process even after a long time this complication has occurred. Thus, this is an original study which may lead to new insights into the molecular basis of SCA stroke and contribute to a better understanding of the role of endothelial cells in stroke recovery.

Consensus of the Brazilian Association of Hematology, Hemotherapy and Cellular Therapy (ABHH) and the Brazilian Ministry of Health - General management of blood and blood products on the tests necessary for the release of exceptional medicines for sickle cell disease.

To date, hydroxyurea is the only effective and safe drug that significantly reduces morbidity and mortality of individuals with Sickle cell disease. Twenty years of real-life experience has demonstrated that hydroxyurea reduces pain attacks, vaso-occlusive events, including acute chest syndrome, the number and duration of hospitalizations and the need for transfusion. The therapeutic success of hydroxyurea is directly linked to access to the drug, the dose used and adherence to treatment which, in part, is correlated to the availability of hydroxyurea. This consensus aims to reduce the number of mandatory exams needed to access the drug, prioritizing the requesting physician's report, without affecting patient safety.

Acupuncture as a Complementary Treatment for Leg Ulcers in Sickle-Cell Disease.

Background: Sickle-cell diseases (SCD) are a group of hereditary disorders in which a specific mutation in the gene that encodes the hemoglobin ß chain leads to formation of an anomalous hemoglobin molecule (HbS) with high polymerization power. This leads to sickling of erythrocytes in situations of low oxygen tension, such as in microcirculation, resulting in peripheral microvasculature occlusion, chronic hemolysis, inflammation, and damage to several target organs. Malleolar ulcers are among the most-debilitating complications of the disease, as they are associated with significant pain, secondary infections, and social impact due to their aesthetic impairment. There are no completely satisfactory therapeutic options for this complication; local healing agents, antibiotics, and dressings are used, with high rates of recurrence and complications, such as osteomyelitis and even limb amputation. Case: This case study evaluated the effect of Traditional Chinese Medicine techniques on chronic malleolar ulcers in a 49-year-old male patient. Ten sessions of systemic acupuncture (combinations involving Source, Master, Energetic Action, and Extraordinary Vessels points), auriculotherapy, and dressing with magnets were conducted. Results: Although the primary outcome sought was not reached (decrease in ulcer diameters), this patient had great reduction of local pain, a decrease in limb edema, and important reduction of his inflammatory condition, reflected in his decreasing blood levels of C-reactive protein. Conclusions: These results show that acupuncture should be considered as an important auxiliary treatment for SCD complications.

Calcitonin gene-related peptide and its correlation with prognosis in severe pneumonia among children below 6 years at PICU of Al Zahraa university hospital.

Pneumonia is known to be the biggest cause of death in children younger than five years old. In pulmonary diseases such as pulmonary arterial hypertension, asthma, acute lung injury, and pulmonary fibrosis, calcitonin gene related peptide (CGRP) has been linked to the regulation of inflammation, proliferation, and fibrosis. However, its ability to foretell the emergence of severe pneumonia is questionable. We aimed to determine whether blood levels of CGRP correlate with the outcome of critically ill children. This case-control study included 45 children with severe pneumonia admitted to the pediatric intensive care unit and 45 children with matched age and sex as controls. We investigated the serum level of CGRP as well as routine laboratory investigations of both groups. The CGRP level was lower in the patient group with median of 77 ng/L ranged from 55 to 183 as compared to control group with median of 230 ng/L ranged from 133 to 664 (p≤0.001). Also, CGRP level was significantly higher in the survived group with median of 96.1 ng/L ranged from 55 to 183 than the non-survived group with median of 63.4 ng/L ranged from 55.5 to 120.9 (p=0.022). In conclusion, we found that serum level of CGRP was extremely low in critical and extremely critically ill patients, and thus can be used as a predictor of mortality in children with severe pneumonia.

COVID-19 anti immunoglobulin G antibodies serum levels among health care workers post COVID -19 vaccination: A single center study.

Coronavirus disease 2019 (COVID-19) pandemic has urged the scientific community internationally to find answers in terms of therapeutics and vaccines to control the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The post vaccination immune response differs between individuals especially health care workers who are the first line of defense to combat this disease. Our aim was to measure levels of anti-IgG antibodies titer post COVID-19 vaccination among health care workers in Suez Canal University Hospital. The study included 141 healthcare workers. Of these, 54 were physicians, 80 nurses, 6 health service workers, and one security guard. We used the Roche Elecsys Anti-SARS-CoV-2 assay for serological detection of IgG. Seropositive was found in 96.5% of the participants, and 43.3% of them had evidence of the prior history of COVID-19 infection. The highest titers of IgG in sera were found in the youngest age groups (20 - <35) years with a mean of 335.1 U/ ml. Participants who received the Sinovac vaccine had the highest mean IgG titer, 354.6U/ml; followed by Sinopharm (mean 352.15 U/ml) then Pfizer and Moderna (311.7U/ml) and AstraZeneca vaccine had the least mean level (267.31U/ml). Fatigue was the most significant short side effect occurring with 34% of the participants. In conclusion, there was a significant rising in serum IgG titer post-vaccine, and better antibody response in those previously infected with COVID-19. The post-COVID-19 vaccine serum IgG titers were affected by age, prior history of COVID-19 infection, and type of vaccine while short side effects post-vaccination may be affected by age and type of the vaccine.

Lower expression of NOTCH components in peripheral blood mononuclear cells of allogeneic hematopoietic cell transplant patients

ABSTRACT Introduction: Chronic graft-versus-host disease (cGvHD) not only remains the main cause of late mortality after allogeneic hematopoietic cell transplant, but also has the capacity of causing severe organ impairment in those who survive. The Notch, a highly conserved ligand-receptor pathway, is involved in many immunological processes, including inflammatory and regulatory responses. Recently, mouse models have shown that the blockage of canonical Notch signaling prevents GvHD. Objective and Method: Due to the lack of data on the Notch pathway in human chronic GvHD, we sought to study the expression of NOTCH components in primary samples of patients who received allo-HCT and presented active cGvHD or a long-term clinical tolerance to cGvHD. Results: Our results showed a significantly lower expression of NOTCH components in both groups that received allo-HCT, independently of their cGvHD status, when compared to healthy controls. Conclusion: Moreover, there were no differences in gene expression levels between the active cGvHD and clinically tolerant groups. To our knowledge, this is one of the first studies performed in human primary samples and our data indicate that much remains to be learned regarding NOTCH signaling as a new regulator of GvHD.

PML/RARa leukemia induced murine model for immunotherapy evaluation.

Even though leukemia murine models are valuable tools for new drug therapy studies, most of these models consist of immunocompromised mice, which do not exhibit immune responses. In order to obtain an adequate leukemia model, we established an acute promyelocytic leukemia transplantation-based model (PML/RARa) in immunocompetent BALB/c mice, thus making it possible to study drug-induced cellular immune responses in leukemia. The development of PML/RARa leukemia was confirmed by leukocytosis (76.27 ± 21.8 vs. 3.40 ± 1.06; P < 0.0001), anemia (7.46 ± 1.86 vs. 15.10 ± 0.96; P < 0.0001), and thrombocytopenia (131.85 ± 39.32 vs. 839.50 ± 171.20; P < 0.0001), and the presence of blasts in the peripheral blood of mice (approximately 50% blasts; P < 0.0001), 15 days after the transplants. These findings were corroborated through differential counts, flow cytometry, and in vivo imaging, which indicated increased number of immature cells in the bone marrow (15.75 ± 3.30 vs 6.69 ± 0.55; P < 0.001), peripheral blood (7.88 ± 2.67 vs 1.22 ± 0.89; P < 0.001), and spleen (35.21 ± 4.12 vs 1.35 ± 0.86; P < 0.0001), as well as promyelocytes in the bone marrow (41.23 ± 4.80 vs 5.73 ± 1.50; P < 0.0001), peripheral blood (46.08 ± 7.52 vs 1.10 ± 0.59; P < 0.0001) and spleen (35.31 ± 8.26 vs 2.49 ± 0.29; P < 0.0001) of PML/RARa mice. Compared to basal conditions of untransplanted mice, the PML/RARa mice exhibited frequencies of T lymphocytes CD4 helper = 14.85 ± 2.91 vs 20.77 ± 2.9 in the peripheral blood (P < 0.05); 12.75 ± 1.33 vs 45.90 ± 2.02 in the spleen (P < 0.0001); CD8 cytotoxic = 11.27 ± 3.44 vs 11.05 ± 1.22 in the peripheral blood (P > 0.05); 10.48 ± 1.16 vs 30.02 ± 1.80 in the spleen (P < 0.0001); natural killer (NK) cells = 3.68 ± 1.35 vs 6.84 ± 0.52 in the peripheral blood (P < 0.001); 4.43 ± 0.57 vs 6.40 ± 1.14 in the spleen (P < 0.05); B cells 2.50 ± 0.60 vs 15.20 ± 5.34 in the peripheral blood (P < 0.001); 17.77 ± 4.39 vs 46.90 ± 5.92 in the spleen (P < 0.0001); neutrophils = 5.97% ± 1.88 vs 31.57 ± 9.14 (P < 0.0001); and monocytes = 6.45 ± 2.97 vs 15.85 ± 2.57 (P < 0.001), selected as classical (3.33 ± 3.40 vs 57.80 ± 16.51, P < 0.0001), intermediate (57.42 ± 10.61 vs 21.75 ± 5.90, P < 0.0001), and non-classical monocytes (37.51 ± 10.85 vs 18.08 ± 7.13, P < 0.05) in the peripheral blood; and as classically activated (M1) within in the bone marrow (3.70 ± 0.94 vs 1.88 ± 0.39, P < 0.05) and spleen 15.19 ± 3.32 vs 9.47 ± 1.61, P < 0.05), in addition to alternatively activated (M2) macrophages within the bone marrow (23.06 ± 5.25 vs 1.76 ± 0.74, P < 0.0001) and spleen (46.51 ± 11.18 vs 30.58 ± 2.64, P < 0.05) compartments. All-trans retinoic acid (ATRA) treatment of PML/RARa mice reduced blast (immature cells) in the bone marrow (8.62 ± 1.81 vs 15.76 ± 1.25; P < 0.05) and spleen (8.75 ± 1.31 vs 35.21 ± 1.55; P < 0.0001) with no changes in the peripheral blood (10.13 ± 3.33 vs 7.88 ± 1.01; P > 0.05), as well as reduced promyelocytes in the bone marrow (19.79 ± 4.84 vs 41.23 ± 1.81; P < 0.05), peripheral blood (31.65 ± 3.92 vs 46.09 ± 2.84; P < 0.05) and spleen (24.84 ± 2.03 vs 41.46 ± 2.39; P < 0.001), and increased neutrophils of the peripheral blood (35.48 ± 7.24 vs 7.83 ± 1.40; P < 0.05) which was corroborated by reducing of immature cells and increase of neutrophil in the stained smears from PML/RARa mice, thus confirming that this model can be used in drug development studies. Our results show the effective induction of PML/RARa leukemia in BALB/c mice, thus producing a low-priced and reliable tool for investigating cellular immune responses in leukemia.

The Effects of Green Tea Catechins in Hematological Malignancies.

Green tea catechins are bioactive polyphenol compounds which have attracted significant attention for their diverse biological activities and potential health benefits. Notably, epigallocatechin-3-gallate (EGCG) has emerged as a potent apoptosis inducer through mechanisms involving caspase activation, modulation of Bcl-2 family proteins, disruption of survival signaling pathways and by regulating the redox balance, inducing oxidative stress. Furthermore, emerging evidence suggests that green tea catechins can modulate epigenetic alterations, including DNA methylation and histone modifications. In addition to their apoptotic actions, ROS signaling effects and reversal of epigenetic alterations, green tea catechins have shown promising results in promoting the differentiation of leukemia cells. This review highlights the comprehensive actions of green tea catechins and provides valuable insights from clinical trials investigating the therapeutic potential of green tea catechins in leukemia treatment. Understanding these multifaceted mechanisms and the outcomes of clinical trials may pave the way for the development of innovative strategies and the integration of green tea catechins into clinical practice for improving leukemia patient outcomes.

Filter cake extract from the beet sugar industry as an economic growth medium for the production of Spirulina platensis as a microbial cell factory for protein.

BACKGROUND: Beet filter cake (BFC) is a by-product of sugar beet processing, which is difficult to dispose of and involves severe environmental concerns. Spirulina platensis is a microalga with a high protein content essential for human and animal nutrition. The present study aimed to utilize the beet filter cake extract (BFCE) to produce Spirulina platensis commercially. However, the cultivation of S. platensis on BFCE to produce economically single-cell protein has not been reported previously. RESULTS: The batch experiment revealed the maximum dry weight at Zarrouk's medium (0.4 g/L) followed by 0.34 g/L in the treatment of 75% BFCE. The highest protein content was 50% in Zarrouk's medium, followed by 46.5% in 25% BFCE. However, adding a higher concentration of 100% BFCE led to a protein content of 31.1%. In the adaption experiment, S platensis showed an increase in dry cell weight and protein content from 25 to 75% BFCE (0.69 g/L to 1.12 g/L and 47.0% to 52.54%, respectively) with an insignificant variation compared to Zarrouk's medium (p ≤ 0.05), indicating that S. platensis can be economically produced when cultivated on 75% BFCE The predicated parameters from response surface methodology were NaNO3 (2.5 g/L), NaHCO3 (0.67 g/L), BFCE (33%) and pH = 8, which resulted in biomass yield and protein content (0.56 g/L and 52.5%, respectively) closer to that achieved using the standard Zarrouk's medium (0.6 g/L and 55.11%). Moreover, the total essential amino acid content was slightly higher in the optimized medium (38.73%) than SZM (36.98%). CONCLUSIONS: Therefore, BFCE supplemented medium could be used as a novel low-cost alternative growth medium for producing a single-cell protein with acceptable quantity and quality compared to the standard Zarrouk's medium.

Novel Insights into the Pathophysiology and Treatment of Sickle Cell Disease.

The polymerization of hemoglobin under deoxygenation is the main pathophysiological event in sickle cell diseases, described more than 70 years ago. The last two decades have seen a major increase in knowledge about the cascade of events that follow the polymerization of hemoglobin and the ensuing sickling of red blood cells. Several distinctive therapeutic targets have been discovered as a result, and a few drugs with innovative mechanisms of action are already on the market, while several others are the focus of ongoing trials. The aim of this narrative review is to describe some of the more recent data in the SCD literature regarding pathophysiology and novel treatments.

The antitumor effects of WNT5A against hematological malignancies.

The bone marrow (BM) microenvironment (niche) is abnormally altered in acute myeloid leukemia (AML), leading to deficient secretion of proteins, soluble factors, and cytokines by mesenchymal stromal cells (MSC) that modifies the crosstalk between MSC and hematopoietic cells. We focused on a WNT gene/protein family member, WNT5A, which is downregulated in leukemia and correlated with disease progression and poor prognosis. We demonstrated that WNT5A protein upregulated the WNT non-canonical pathway only in leukemic cells, without modulating normal cell behavior. We also introduced a novel WNT5A-mimicking compound, Foxy-5. Our results showed reduction of crucial biological functions that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy, as well as G0/G1 cell cycle arrest. Additionally, Foxy-5 induced early-stage macrophage cell differentiation, a crucial process during leukemia development. At a molecular level, Foxy-5 led to the downregulation of two overexpressed leukemia pathways, PI3K and MAPK, which resulted in a disarrangement of actin polymerization with consequent impairment of CXCL12-induced chemotaxis. Notably, in a novel tri-dimensional bone marrow-mimicking model, Foxy-5 led to reduced leukemia cell growth and similar results were observed in a xenograft in vivo model. Overall, our findings highlight the pivotal role of WNT5A in leukemia and demonstrate that Foxy-5 acts as a specific antineoplastic agent in leukemia, counterbalancing several leukemic oncogenic processes related to the crosstalk in the bone marrow niche, and represents a promising therapeutic option for AML. WNT5A, a WNT gene/protein family member, is naturally secreted by mesenchymal stromal cells and contributes to the maintenance of the bone marrow microenvironment. WNT5A downregulation is correlated with disease progression and poor prognosis. The treatment with Foxy-5, a WNT5A mimetizing compound, counterbalanced several leukemogenic processes that are upregulated in leukemia cells, including ROS generation, cell proliferation, and autophagy and disruption of PI3K and MAPK signaling pathways.

Does the Mediterranean Diet Have Any Effect on Lipid Profile, Central Obesity and Liver Enzymes in Non-Alcoholic Fatty Liver Disease (NAFLD) Subjects? A Systematic Review and Meta-Analysis of Randomized Control Trials.

The effectiveness of the Mediterranean diet (MD) in non-alcoholic fatty liver disease (NAFLD) subjects has been evaluated in several randomized controlled trials (RCTs). This systematic review and meta-analysis aimed to evaluate the overall effects of MD intervention in a cohort of NAFLD patients targeting specific markers such as central obesity, lipid profile, liver enzymes and fibrosis, and intrahepatic fat (IHF). Google Scholar, PubMed, and Scopus were explored to collect relevant studies from the last 10 years. RCTs with NAFLD subjects were included in this systematic review with a mean intervention duration from 6 weeks to 1 year, and different intervention strategies, mainly including energy restriction MD (normal or low glycaemic index), low-fat MD with increased monounsaturated and polyunsaturated fatty acids, and increased exercise expenditure. The outcomes measured in this meta-analysis were gamma-glutamyl transferase (GGT), alanine aminotransferase (ALT), total cholesterol (TC), waist circumference (WC), and liver fibrosis. Ten randomized controlled trials, which involved a total of 737 adults with NAFLD, were included. According to the results, the MD seems to decrease the liver stiffness (kPa) by -0.42 (CI95% -0.92, 0.09) (p = 0.10) and significantly reduce the TC by -0.46 mg/dl (CI95% -0.55, -0.38) (p = 0.001), while no significant findings were documented for liver enzymes and WC among patients with NAFLD. In conclusion, the MD might reduce indirect and direct outcomes linked with NAFLD severity, such as TC, liver fibrosis, and WC, although it is important to consider the variations across trials. Further RCTs are necessary to corroborate the findings obtained and provide further evidence on the role of the MD in the modulation of other disorders related to NAFLD.

Optimized multiplex immunofluorescence for the characterization of tumor immune microenvironment in neoplastic paraffin-preserved tissues.

The study of neoplastic cells enabled the discovery of important tumor-related biomarkers which resulted in new forms of early diagnosis, therapeutic options, and prognostic markers. Thus, immunofluorescence (IF), a high throughput imaging technology, represents a valuable method that enables the virtual characterization and localization of diverse cell types and targets, preserving tissue architecture and spatial surroundings. IF staining and analysis of formalin-fixed paraffin-embedded (FFPE) tissues are considered a challenge due to several difficulties, such as tissue autofluorescence, non-specific antibody binding, and image acquisition and quality. This study aimed to develop a multiplex-fluorescence staining technique with high-contrast and high-quality multiple-color images to enrich the investigation of important biomarkers. We present a robust optimized multiple-immunofluorescence procedure that reduced sample autofluorescence, enabled the use of simultaneous antibodies on the same sample, and showed super-resolution imaging through precise antigen localization. We illustrated the utility of this powerful method in FFPE neoplastic appendix, lymph node and bone marrow biopsies, and a 3D-coculture system, in which cells are enabled to grow and interact with their surroundings in all 3D dimensions. Our optimized multiple-immunofluorescence method represents a powerful tool for better understanding the complexity of tumor cells, characterizing cell populations and spatial localization, revealing predictive and prognostic biomarkers, and identifying immunologic phenotypes in a single and limited sample. This valuable IF protocol successfully enables tumor microenvironment profiling that could contribute to the study of cellular crosstalk and the niche, and to the identification of predictive biomarkers for neoplasms.

Piezo1 activation augments sickling propensity and the adhesive properties of sickle red blood cells in a calcium-dependent manner.

Haemoglobin S polymerization in the red blood cells (RBCs) of individuals with sickle cell anaemia (SCA) can cause RBC sickling and cellular alterations. Piezo1 is a mechanosensitive protein that modulates intracellular calcium (Ca2+ ) influx, and its activation has been associated with increased RBC surface membrane phosphatidylserine (PS) exposure. Hypothesizing that Piezo1 activation, and ensuing Gárdos channel activity, alter sickle RBC properties, RBCs from patients with SCA were incubated with the Piezo1 agonist, Yoda1 (0.1-10 µM). Oxygen-gradient ektacytometry and membrane potential measurement showed that Piezo1 activation significantly decreased sickle RBC deformability, augmented sickling propensity, and triggered pronounced membrane hyperpolarization, in association with Gárdos channel activation and Ca2+ influx. Yoda1 induced Ca2+ -dependent adhesion of sickle RBCs to laminin, in microfluidic assays, mediated by increased BCAM binding affinity. Furthermore, RBCs from SCA patients that were homo-/heterozygous for the rs59446030 gain-of-function Piezo1 variant demonstrated enhanced sickling under deoxygenation and increased PS exposure. Thus, Piezo1 stimulation decreases sickle RBC deformability, and increases the propensities of these cells to sickle upon deoxygenation and adhere to laminin. Results support a role of Piezo1 in some of the RBC properties that contribute to SCA vaso-occlusion, indicating that Piezo1 may represent a potential therapeutic target molecule for this disease.

Comparative transcriptome analysis of endothelial progenitor cells of HbSS patients with and without proliferative retinopathy.

Among sickle cell anemia (SCA) complications, proliferative sickle cell retinopathy (PSCR) is one of the most important, being responsible for visual impairment in 10-20% of affected eyes. The aim of this study was to identify differentially expressed genes (DEGs) present in pathways that may be implicated in the pathophysiology of PSCR from the transcriptome profile analysis of endothelial progenitor cells. RNA-Seq was used to compare gene expression profile of circulating endothelial colony-forming cells (ECFCs) from HbSS patients with and without PSCR. Furthermore, functional enrichment analysis and protein-protein interaction (PPI) networks were performed to gain further insights into biological functions. The differential expression analysis identified 501 DEGs, when comparing the groups with and without PSCR. Furthermore, functional enrichment analysis showed associations of the DEGs in 200 biological processes. Among these, regulation of mitogen-activated protein (MAP) kinase activity, positive regulation of phosphatidylinositol 3-kinase (PI3K), and positive regulation of Signal Transducer and Activator of Transcription (STAT) receptor signaling pathway were observed. These pathways are associated with angiogenesis, cell migration, adhesion, differentiation, and proliferation, important processes involved in PSCR pathophysiology. Moreover, our results showed an over-expression of VEGFC (vascular endothelial growth factor-C) and FLT1 (Fms-Related Receptor Tyrosine Kinase 1) genes, when comparing HbSS patients with and without PSCR. These results may indicate a possible association between VEGFC and FLT1 receptor, which may activate signaling pathways such as PI3K/AKT and MAPK/ERK and contribute to the mechanisms implicated in neovascularization. Thus, our findings contain preliminary results that may guide future studies in the field, since the molecular mechanisms of PSCR are still poorly understood.