Association of Blood NK Cell Phenotype with the Severity of Liver Fibrosis in Patients with Chronic Viral Hepatitis C with Genotype 1 or 3.

BACKGROUND: NK cells phenotype and functional state in different genotypes of chronic viral hepatitis C (CVHC), depending on liver fibrosis severity, have not been sufficiently studied, which limits the possibilities for the development of pathology therapy. METHODS: The CVHC diagnosis was based on the EASL recommendations (2018). Clinical examination with liver elastometry was performed in 297 patients with genotype 1 and in 231 patients with genotype 3 CVHC. The blood NK cells phenotype was determined by flow cytometry in 74 individuals with genotype 1 and in 69 individuals with genotype 3 CVHC. RESULTS: The frequency of METAVIR liver fibrosis stages F3-F4 was 32.5% in individuals with genotype 3, and 20.5% in individuals with genotype 1 CVHC (p = 0.003). In patients with both genotype 1 and genotype 3 CVHC, a decrease in the total number of blood NK cells, CD56brightCD16+ NK cells and an increase in the proportion of CD56dimCD16+ NK cells, CD94+ and CD38 + CD73+ NK cells were registered in patients with fibrosis stage F3-F4 by METAVIR in comparison with persons with METAVIR fibrosis stage F0-F1. CONCLUSIONS: In patients with both genotype 1 and genotype 3 CVHC, an imbalance in the ratio between cytokine-producing and cytotoxic NK cells and an increase in the content of NK cells that express inhibitory molecules were determined in patients with severe liver fibrosis.

A Key Role of CD8+ T Cells in Controlling of Tuberculosis Infection.

The main role in the control of tuberculosis infection is played by macrophages and Th1 and CD8+ T cells. The study aimed to identify the most diagnostically significant CD8+ T cell subsets in tuberculosis patients. METHODS: Peripheral blood samples from patients with clinical, radiological, and bacteriologically confirmed pulmonary tuberculosis (TB, n = 32) and healthy subjects (HC, n = 31) were collected and analyzed using 10-color flow cytometry. RESULTS: The frequency of the EM4 CD3+CD8+ cells was reduced in the peripheral blood of patients with pulmonary tuberculosis, while the relative and absolute number of EM1 CD3+CD8+ cells increased compared to the control group. CD57 expression was reduced in patients with pulmonary tuberculosis on EM1, EM2, and pE1 CD3+CD8+ cells, whereas the EM3 cells had a high level of CD57 expression. The relative and absolute number of Tc2 (CCR6-CXCR3-) cells in peripheral blood in patients with pulmonary tuberculosis was increased, while the frequency of Tc1 (CCR6-CXCR3+) was decreased, compared to healthy donors. CONCLUSIONS: Patients with pulmonary tuberculosis have an abnormal CD3+CD8+ cell profile and demonstrate their impaired maturation and functional activity.

A Feasibility Study of AlzLife 40 Hz Sensory Therapy in Patients with MCI and Early AD.

Alzheimer's Disease (AD) and Mild Cognitive Impairment (MCI) are debilitating diseases that affect millions of individuals and have notoriously limited treatment options. One emerging therapy, non-invasive 40 Hz sensory therapy delivered through light and sound has previously shown promise in improving cognition in Alzheimer Disease (AD) rodent models. Small studies in humans have proven safe and tolerable, however exploration of feasibility and utility is limited. The purpose of this study is to examine the feasibility of this treatment in a human population through a smart tablet application that emits light and sound waves at 40 Hz to the user over the span of 1 h a day. Confirmation of entrainment of 40 Hz stimulation in the cerebral cortex was performed via EEG. 27 preliminary subjects with subjective cognitive complaints, Mild Cognitive Impairment, or AD were enrolled in the study; 11 participants completed 6 months of therapy. Of those that discontinued treatment, other health issues and difficulties with compliance were the most common causes. Participants were followed with Montreal Cognitive Assessment (MOCA) and Boston Cognitive Assessment (BOCA). For participants with subjective cognitive complaints, 2 of the 4 had improved MOCA score and 1 of 4 had improved BOCA score. For the participant with MCI, his MOCA score improved. For AD participants, 2 out of 6 had improved MOCA score and 3 of the 6 stayed stable, while 3 of 6 BOCA score improved. 4 of 11 participants specifically increased their MOCA scores in the Memory Index section. Of the 8 participants/caregivers able to speak to perceived usefulness of the study, 6 spoke to at least some level of benefit. Of these 6, 2 enrolled with subjective cognitive complaint, 1 had MCI, and 3 had AD. The therapy did not have reported side effects. However, those who did not finish the study experienced issues obtaining and operating a smart tablet independently as well as complying with the therapy. Overall, further exploration of this treatment modalities efficacy is warranted.

Recombinant Human Interleukin-2 Corrects NK Cell Phenotype and Functional Activity in Patients with Post-COVID Syndrome.

Post-COVID syndrome develops in 10-20% of people who have recovered from COVID-19 and it is characterized by impaired function of the nervous, cardiovascular, and immune systems. Previously, it was found that patients who recovered from infection with the SARS-CoV-2 virus had a decrease in the number and functional activity of NK cells. The aim of the study was to assess the effectiveness of recombinant human IL-2 (rhIL-2) administered to correct NK cell phenotype and functional activity in patients with post-COVID syndrome. Patients were examined after 3 months for acute COVID-19 of varying severity. The phenotype of the peripheral blood NK cells was studied by flow cytometry. It was found that disturbances in the cell subset composition in patients with post-COVID syndrome were characterized by low levels of mature (p = 0.001) and cytotoxic NK cells (p = 0.013), with increased release of immature NK cells (p = 0.023). Functional deficiency of NK cells in post-COVID syndrome was characterized by lowered cytotoxic activity due to the decreased count of CD57+ (p = 0.001) and CD8+ (p < 0.001) NK cells. In the treatment of patients with post-COVID syndrome with recombinant IL-2, peripheral blood NK cell count and functional potential were restored. In general, the effectiveness of using rhIL-2 in treatment of post-COVID syndrome has been proven in patients with low levels of NK cells.

seco-Pregnane Glycosides from Australian Caustic Vine (Cynanchum viminale subsp. australe).

Cynanchum viminale subsp. australe, more commonly known as caustic vine, is a leafless succulent that grows in the northern arid zone of Australia. Toxicity toward livestock has been reported for this species, along with use in traditional medicine and its potential anticancer activity. Disclosed herein are novel seco-pregnane aglycones cynavimigenin A (5) and cynaviminoside A (6), together with new pregnane glycosides cynaviminoside B (7) and cynavimigenin B (8). Cynavimigenin B (8) contains an unprecedented 7-oxobicyclo[2.2.1]heptane moiety in the seco-pregnane series, likely arising from a pinacol-type rearrangement. Interestingly, these isolates displayed only limited cytotoxicity in cancer and normal human cell lines, in addition to low activity against acetylcholinesterase and Sarcoptes scabiei bioassays, suggesting that 5-8 are not associated with the reported toxicity of this plant species.

Features of Peripheral Blood Th-Cell Subset Composition and Serum Cytokine Level in Patients with Activity-Driven Ankylosing Spondylitis.

Th cells may exhibit pathological activity depending on the regulatory and functional signals sensed under a wide range of immunopathological conditions, including ankylosing spondylitis (AS). The relationship between Th cells and cytokines is important for diagnoses and for determining treatment. Accordingly, the aim of this study was to investigate the relationship between Th-cell subset composition and serum cytokine profile for patients with activity-driven AS. In our study, patients were divided into two groups according to disease activity: low-activity AS (ASDAS-CRP < 2.1) and high-activity AS (ASDAS-CRP > 2.1). The peripheral blood Th cell subset composition was studied by flow cytometry. Using multiplex analysis, serum cytokine levels were quantified and investigated. It was found that only patients with high-activity AS had reduced central memory (CM) Th1 cells (p = 0.035) but elevated numbers of CM (p = 0.014) and effector memory (EM) Th2 cells (p < 0.001). However, no activity-driven change in the Th17 cell subset composition was observed in AS patients. Moreover, low-AS activity patients had increased numbers of Tfh17 EM cells (p < 0.001), whereas high-AS activity was associated with elevated Tfh2 EM level (p = 0.031). The serum cytokine profiles in AS patients demonstrated that cues stimulating cellular immunity were increased, but patients with high-AS activity reveled increased IL-5 level (p = 0.017). Analyzing the data obtained from AS patients allowed us to conclude that Th cell subset differentiation was mainly affected during the CM stage and characterized the IL-23/IL-17 regulatory axis, whereas increased humoral immunity was observed in the high-AS activity group.

TREC/KREC Levels and T and B Lymphocyte Subpopulations in COVID-19 Patients at Different Stages of the Disease.

BACKGROUND: T and B cell-mediated immunity can be assessed using T cell receptor excision circle (TREC) and Kappa-deleting recombination excision circle (KREC) analysis, respectively, and successful implementation of this method requires evaluation of the correlation between the TREC frequencies and T cell subsets as well as KREC levels and B lymphocyte subsets. The aim of the present study was to evaluate the correlation between the TREC/KREC concentrations and T/B lymphocyte subsets at different stages of COVID-19. METHODS: We examined 33 patients in the acute stage of COVID-19 (including 8 patients with poor outcomes) and 33 COVID-19 survivors. TREC/KREC concentrations were measured using quantitative real-time PCR. T/B lymphocyte subsets were determined using flow cytometry. RESULTS: Blood TREC and KREC levels were found to be significantly lower in the acute stage of COVID-19 compared to control values. Moreover, a zero blood TREC level was a predictor of a poor disease outcome. Reductions in CD3+CD4+CD45RO-CD62L- and CD3+CD8+CD45RO-CD62L- T cell counts (as well as in the main fractions of B1 and B2 B cells) indicated a favorable outcome in COVID-19 patients in the acute stage of the disease. Decreased CD3+CD4+CD45RO-CD62L+ and CD3+CD8+CD45RO-CD62L+ T cell frequencies and increased CD3+CD8+CD45RO-CD62L- cell counts were found to indicate a poor outcome in patients with acute COVID-19. These patients were also found to have increased B1 cell counts while demonstrating no changes in B2 cell counts. The levels of effector T cell subsets an naïve B cells were normal in COVID-19 survivors. The most pronounced correlations between TREC/KREC levels and T/B cell subsets counts were observed in COVID-19 survivors: there were positive correlations with naïve T and B lymphocytes and negative correlations with central and effector memory T cell subsets. CONCLUSIONS: The assessment of correlations between TREC and T cell subsets as well as KREC levels and B cell subset counts in patients with acute COVID-19 and COVID-19 survivors has shown that blood concentrations of TREC and KREC are sensitive indicators of the stage of antigen-independent differentiation of adaptive immunity cells. The results of the TREC and KREC analysis correlated with the stages of COVID-19 and differed depending on the outcome of COVID-19.

Boston cognitive assessment (BOCA) - a comprehensive self-administered smartphone- and computer-based at-home test for longitudinal tracking of cognitive performance.

Longitudinal cognitive testing is essential for developing novel preventive interventions for dementia and Alzheimer's disease; however, the few available tools have significant practice effect and depend on an external evaluator. We developed a self-administered 10-min at-home test intended for longitudinal cognitive monitoring, Boston Cognitive Assessment or BOCA. The goal of this project was to validate BOCA. BOCA uses randomly selected non-repeating tasks to minimize practice effects. BOCA evaluates eight cognitive domains: 1) Memory/Immediate Recall, 2) Combinatorial Language Comprehension/Prefrontal Synthesis, 3) Visuospatial Reasoning/Mental rotation, 4) Executive function/Clock Test, 5) Attention, 6) Mental math, 7) Orientation, and 8) Memory/Delayed Recall. BOCA was administered to patients with cognitive impairment (n = 50) and age- and education-matched controls (n = 50). Test scores were significantly different between patients and controls (p < 0.001) suggesting good discriminative ability. The Cronbach's alpha was 0.87 implying good internal consistency. BOCA demonstrated strong correlation with Montreal Cognitive Assessment (MoCA) (R = 0.90, p < 0.001). The study revealed strong (R = 0.94, p < 0.001) test-retest reliability of the total BOCA score one week after participants' initial administration. The practice effect tested by daily BOCA administration over 10 days was insignificant (ß = 0.03, p = 0.68). The effect of the screen size tested by BOCA administration on a large computer screen and re-administration of the BOCA to the same participant on a smartphone was insignificant (ß = 0.82, p = 0.17; positive ß indicates greater score on a smartphone). BOCA has the potential to reduce the cost and improve the quality of longitudinal cognitive tracking essential for testing novel interventions designed to reduce or reverse cognitive aging. BOCA is available online gratis at www.bocatest.org .

The Boston cognitive assessment: Psychometric foundations of a self-administered measure of global cognition.

Objective: The Boston Cognitive Assessment (BoCA) is a novel, computerized, self-administered assessment of global cognition. This work sought to establish the validity and reliability of the BoCA. Method: Two studies were conducted. The first study used a sample of 43 outpatients from a clinic in eastern Massachusetts to evaluate the content validity and internal consistency of the BoCA. The second study used a sample of 38 patients seen at an outpatient specialty neurological clinic to evaluate the BoCA's test-retest reliability after one week. Results: In the first study, participants without cognitive diagnoses scored significantly higher on both the BoCA and the Telephone Interview for Cognitive Status (TICS) compared to those with mild Neurocognitive Disorders. Correlational analyses revealed moderate correlations between several of the BoCA tasks and measures of related abilities. Exploratory factor analysis of the BoCA tasks revealed one robust factor accounting for a plurality (i.e., 42%) of variance in participant scores. The BoCA demonstrated good internal consistency (α = 0.79) and strong correlations (r = 0.80, p < 0.01) with the TICS. The second study revealed strong (r = 0.89, p < 0.001) test-retest reliability of the total BoCA score one week after participants' initial administration. Conclusions: This work provides evidence of the BoCA's psychometric properties as a self-administered screener of global cognition, and supports its implementation in clinical practice and future studies. Clinical implications, future directions, and limitations are discussed.

Activation of PKC supports the anticancer activity of tigilanol tiglate and related epoxytiglianes.

The long-standing perception of Protein Kinase C (PKC) as a family of oncoproteins has increasingly been challenged by evidence that some PKC isoforms may act as tumor suppressors. To explore the hypothesis that activation, rather than inhibition, of these isoforms is critical for anticancer activity, we isolated and characterized a family of 16 novel phorboids closely-related to tigilanol tiglate (EBC-46), a PKC-activating epoxytigliane showing promising clinical safety and efficacy for intratumoral treatment of cancers. While alkyl branching features of the C12-ester influenced potency, the 6,7-epoxide structural motif and position was critical to PKC activation in vitro. A subset of the 6,7-epoxytiglianes were efficacious against established tumors in mice; which generally correlated with in vitro activation of PKC. Importantly, epoxytiglianes without evidence of PKC activation showed limited antitumor efficacy. Taken together, these findings provide a strong rationale to reassess the role of PKC isoforms in cancer, and suggest in some situations their activation can be a promising strategy for anticancer drug discovery.

EBC-232 and 323: A Structural Conundrum Necessitating Unification of Five In Silico Prediction and Elucidation Methods.

Structurally unique halimanes EBC-232 and EBC-323, isolated from the Australian rainforest plant Croton insularis, proved considerably difficult to elucidate. The two diastereomers, which consist an unusual oxo-6,7-spiro ring system fused to a dihydrofuran, were solved by unification and consultation of five in silico NMR elucidation and prediction methods [i.e., ACDLabs, olefin strain energy (OSE), DP4, DU8+ and TD DFT CD]. Structure elucidation challenges of this nature are prime test case examples for empowering future AI learning in structure elucidation.

Resistance to Acetylsalicylic Acid in Patients with Coronary Heart Disease Is the Result of Metabolic Activity of Platelets.

Sensitivity to acetylsalicylic acid (ASA) is important in the treatment of patients with coronary heart disease (CHD) after coronary artery bypass grafting (CABG). Patients were divided into ASA sensitive (sASA) and ASA resistant (rASA) by the activity of platelet aggregation induced arachidonic acid (ARA) together with ASA. Induced platelet aggregation activity was studied in sASA and rASA patients with CHD before and after CABG. The level of synthesis of primary and secondary reactive oxygen species (ROS) by platelets was determined using chemiluminescent analysis. The activity of NAD- and NADP-dependent dehydrogenases in platelets was determined by the bioluminescent method. It was found that the aggregation activity of platelets depended on the sensitivity of CHD patients to ASA and decreased during postoperative ASA therapy. The most pronounced differences in metabolic parameters of platelets in sASA and rASA patients were detected by Nox2 activity. The synthesis of secondary ROS by platelets of CHD patients did not depend on the sensitivity of patients to ASA but increased during postoperative treatment with ASA. The activity of NAD(P)-dependent dehydrogenases in platelets did not differ in sASA and rASA patients with CHD.

Kalparinol, a Salvialane (Isodaucane) Sesquiterpenoid Derived from Native Australian Dysphania Species That Suggests a Putative Biogenetic Link to Zerumbone.

Dysphania is a genus of plants endemic to the Australian continent, occurring primarily in arid and temperate zones. Despite their prevalence, very little in the way of phytochemical and/or bioactivity investigation of native Dysphania has been performed. Herein reported is the isolation and elucidation of (6E,9E)-zerumbone epoxide and a hitherto unreported isomer, (6Z,9E)-zerumbone epoxide, from D. kalpari. In addition, a novel isodaucane sesquiterepene, kalparinol, was isolated from both D. kalpari and D. rhadinostachya. The coisolation of the humulene and isodaucane skeletons, combined with the lack of any cadalane systems, could suggest an alternate novel biogenetic pathway originating from zerumbone, which is unlike any other proposals for the isodaucene system.

Humulene Diepoxides from the Australian Arid Zone Herb Dysphania: Assignment of Aged Hops Constituents.

Dysphania is an abundant genus of plants, many of which are endemic to the Australian continent, occurring primarily in arid and temperate zones. Despite their prevalence, very few investigations into the phytochemistry of native Dysphania have been undertaken. Described herein, is the isolation and elucidation of two enantiomeric diastereomers of humulene diepoxide C from D. kalpari and D. rhadinostachya, of which unassigned diastereomers of humulene diepoxide C have been previously reported as components in beer brewed from aged hops. In addition, two (+)-humulene diepoxiols (humulene diepoxiol C-I and C-II) were isolated from D. rhadinostachya. Analysis of Chinook hops oil confirmed the presence of both humulene diepoxide C-I and C-II as trace components, and in turn enabled GC-MS peak assignment to the relative stereochemistry. Anticancer assays did not reveal any significant activity for the (+)-humulene diepoxides. Antifungal assays showed good activity against a drug-resistant strain of C. auris, with MIC50 values of 8.53 and 4.91 µm obtained for (+)-humulene diepoxide C-I and C-II, respectively.

Basimarols A, B, and C, Highly Oxygenated Pimarane Diterpenoids from Basilicum polystachyon.

The highly oxygenated pimarane diterpenoids basimarols A, B, and C (3-5) were isolated from the plant species Basilicum polystachyon, which was collected within the Australian arid zone. Structure elucidation was performed using a suite of spectroscopic techniques, including X-ray crystallography. Anticancer and anti-DENV activity of 3-5 was explored, but only limited activity was observed. More extensive antiviral evaluation of stachyonic acid A (1), which was also isolated from B. polystachyon, revealed broad spectrum antiviral activity against West Nile virus (Kunjin strain, WNVKun) and human influenza viruses H1N1 and H3N2.

Stachyonic Acid: A Dengue Virus Inhibitor from Basilicum polystachyon.

Stachyonic acid A, arising from the first in-depth phytochemical investigation of the herb Basilicum polystachyon, was found to display potent inhibitory activity against dengue virus, with limited cytotoxicity. Andrographolide, a known dengue virus inhibitor and closely related labdane-type diterpene, is structurally more complex but displayed poor antiviral activity in the PRNT assay, and increased cytotoxicity in comparison. Furthermore, a Diels-Alder reaction with PTAD identified the active pharmacophore of stachyonic acid to be the conjugated diene.

New Casbanes and the First trans-Cyclopropane seco-Casbane from the Australian Rainforest Plant Croton insularis.

Investigation of the Australian rainforest plant Croton insularis revealed seven new cis-, two new trans-cyclopropane casbanes, and the first trans-cyclopropane seco-casbane. The relative configuration of the cyclopropane moiety for all compounds (EBC-182, 217, 218, 220, 343, 357, 358, 361, 365, 373; EBC=EcoBiotics Compound) was assigned using 13 C NMR data. Comparison of the experimental electronic circular dichroism (ECD) spectra with the theoretical curves, calculated by TD-DFT at the B3LYP/6-31+G**//B3LYP/6-31+G** level, in conjunction with NOE data afforded the absolute configuration. EBC-180, 181 and 220 displayed potent activity against cervical carcinoma (HeLa cells).

miR-204-5p and miR-3065-5p exert antitumor effects on melanoma cells.

MicroRNA (miR)-204-5p was previously identified to be downregulated in melanoma compared with melanocytic nevi. This observation prompted a functional study on miR-204-5p and the newly-identified miR-3065-5p, two miRNAs suggested to be tumor-suppressive oncomiRs. Application of miR-204-5p mimics or inhibitors resulted in a decrease or increase, respectively, in melanoma cell proliferation and colony formation. miR-204-5p mimics hindered invasion, whereas miR-204-5p inhibitors stimulated cancer cell migration. Modulation of miR-3065-5p led to a decrease in melanoma cell proliferation, altered cell cycle distribution and increased expression levels of its target genes HIPK1 and ITGA1, possibly due to functional modifications identified in these cells. miR-204-5p and miR-3065-5p demonstrated antitumor capacities that may need to be taken into account in the development of melanoma treatment approaches.

Furofuran lignans from the Simpson Desert species Eremophila macdonnellii.

The Eremophila plant family, which occurs in the arid zones of Australia, have witnessed extensive investigation, mostly inspired by Aboriginal traditional medicine. A wide and varied biological and phytochemical profile has been reported for over 18 individual species of Australian Eremophila, although E. macdonnellii from the Simpson Desert has not yet been investigated. Isolation and elucidation of one new and six known furofuran lignans are reported. The new lignan, epimethoxypiperitol, displayed moderate anti-cancer activity against the breast cancer cell line (MCF-7).

An improved preparation of phorbol from croton oil.

Background: Croton oil is the only commercial source of the diterpenoid phorbol (1a), the starting material for the semi-synthesis of various diesters extensively used in biomedical research to investigate cell function and to evaluate in vivo anti-inflammatory activity. While efficient chemoselective esterification protocols have been developed for phorbol, its isolation from croton oil is technically complicated, and involves extensive manipulation of very toxic materials like the oil or its native diterpenoid fraction. Results: The preparation of a crude non-irritant phorboid mixture from croton oil was telescoped to only five operational steps, and phorbol could then be purified by gravity column chromatography and crystallization. Evidence is provided that two distinct phorboid chemotypes of croton oil exist, differing in the relative proportion of type-A and type-B esters and showing different stability to deacylation. Conclusion: The isolation of phorbol from croton oil is dangerous because of the toxic properties of the oil, poorly reproducible because of differences in its phorboid profile, and time-consuming because of the capricious final crystallization step. A solution for these issues is provided, suggesting that the poor-reproducibility of croton oil-based anti-inflammatory assays are the result of poor quality and/or inconsistent composition of croton oil.