Role of glycogen synthase kinase 3 (GSK-3) and its substrate proteins in the development of cardiomyopathy associated with obesity and insulin resistance

Thesis (MScMedSc)--University of Stellenbosch, 2011.ENGLISH ABSTRACT: INTRODUCTION: Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase that was first discovered as a regulator of glycogen synthase thus playing a role in glycogen synthesis (Embi et al. 1980). GSK-3 has also been shown to down regulate the expression of SERCA-2a (a calcium ATPase pump) thus playing a role in myocardial contractility (Michael et al. 2004). However, SERCA-2a activity is regulated by phospholamban (PLM) and sarcolipin (SLN) (Asahi et al. 2003). GSK-3 is constitutively active in cells and can be acutely inactivated by insulin through phosphorylation by PKB/Akt. However, GSK-3 is known to phosphorylate and inhibit IRS-1 protein, thus disrupting insulin signaling (Eldar-Finkelman et al. 1996). In addition, abnormally high activities of GSK-3 protein has been implicated in several pathological disorders which include type 2 diabetes, neuron degenerative and affective disorders (Eldar-Finkelman et al 2009). This led to the development of new generations of inhibitors with specific clinical implications to treat these diseases (Martinez 2008). GSK-3 inhibition has been shown to improve insulin and blood glucose levels and to be cardioprotective during ischemia/reperfusion (Nikoulina et al. 2002; Kumar et al. 2007). AIMS: To determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether a specific GSK-3 inhibitor can prevent or reverse the cardiovascular pathology found in obese and insulin resistant animals. OBJECTIVES: To correlate the alterations in expression and activation of GSK-3 protein in a well characterised rat model of obesity coupled to insulin resistance with: i) myocardial contractile dysfunction and an inability of hearts to withstand ischemia/reperfusion, ii) the activation and expression of phospholamban and SERCA-2a in the sarcoplasmic reticulum, iii) the activation of intermediates (IRS-1, IRS-2 and PKB/Akt) that lie upstream in the activation pathway of GSK-3 and iv) to determine the effects of inhibition of GSK-3 on the abovementioned parameters. METHODS: Age and weight matched male Wistar rats (controls and diet induced obese (DIO) animals) were used in the present study. Controls were fed normal rat chow, while DIOs were fed a rat chow diet supplemented with sucrose and condensed milk, for 8 or 16 weeks. Half of each group of animals were treated with the GSK-3 inhibitor for 4 weeks (from 12 to 16 weeks). After the feeding and treatment period, animals were weighed, sacrificed, hearts removed and freeze clamped immediately or perfused with Krebs-Henseleit buffer and subjected to low flow ischemia (25 min) followed by 30 min reperfusion. Biometric (body weight, intraperitoneal fat, ventricular weight and tibia length) and biochemical (fasting blood glucose and insulin levels) parameters were determined. Expression of GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a and Phospholamban were determined by Western blotting. Ca2+ ATPase activity was determined spectrophotometrically. RESULTS: At both 8 and 16 weeks DIO animals were significantly bigger than control animals and this was associated with increased intraperitoneal fat in DIOs. In DIO animals: IRS-1 was downregulated at 8 weeks and both IRS-1 and IRS-2 as well as PKB/Akt at 16 weeks. There was an increased tendency of GSK-3 expression at both 8 and 16 weeks in DIO animals while SERCA-2a was severely downregulated from 8 weeks onwards and associated with lower Ca2+-ATPase activity. PLM expression was upregulated but its phosphorylation was attenuated. At 16 weeks, baseline heart rate (225 vs 275 in control, P<0.0001, n=6) and rate pressure product (21000 vs 30000 in control, P=0.019, n=6) were significantly lower in hearts from DIO animals. Functional recovery was unchanged but the time to ischemic contracture development was increased (11.6±0.4 control vs 16.2±0.5 min DIO, P<0.01, n=6). Treatment had no effect on total GSK-3 expression. However, GSK-3 phosphorylation was significantly increased in treated controls, while there was no significant difference in DIO animals. However, there was a tendency for an increased GSK-3 phosphorylation in treated DIO animals. GSK-3 inhibitor, improved hypertrophy in DIO animals, while it led to its development in control animals. GSK-3 inhibitor improved IRS-2 expression in both control and DIO animals while it had no effect on IRS-1 and SERCA-2a expression and activity. However, GSK-3 inhibition increased PKB/Akt and phospholamban phosphorylation in DIO animals. CONCLUSION: These findings show that high calorie diet as well as imbalance between energy intake and expenditure lead to the development of obesity and insulin resistance in male Wistar rats. We showed that GSK-3 and its substrate proteins are dysregulated in obesity and insulin resistance. The reduced SERCA-2a expression at baseline may have a negative impact on cardiac function. By treating the animals with GSK-3 inhibitor, we showed that GSK-3 protein may not be responsible for changes seen at baseline. The decreased IRS-1 and SERCA-2a expression may have been caused by a different mechanism other than the actions of GSK-3. However, according to this study, GSK-3 may play a role in regulation of IRS-2 expression but not in IRS-1. Increased PKB/Akt phosphorylation may contribute to the GSK-3 inhibition. In addition, GSK-3 inhibition may reverse cardiac hypertrophy in DIO animals, thus acting as a negative regulator of hypertrophy.AFRIKAANSE OPSOMMING: Inleiding: Glikogeen sintase kinase-3 (GSK-3), 'n serien/threonien proteïen kinase, is oorspronklik ontdek as 'n rolspeler in glikogeen sintese, aangesien dit 'n reguleerder van glikogeen sintase is (Embi et al.1980). Intussen is dit ook bevind dat GSK-3 die uitdrukking van SERCA-2a ('n kalsium ATPase pomp) kan afreguleer en dus sodoende 'n rol speel in miokardiale kontraktiliteit (Michael et al. 2004). Die aktiwiteit van SERCA-2a kan egter ook gereguleer word deur fosfolamban (PLM) en sarkolipin (Asahi et al. 2003). GSK-3 is deurgaans aktief, maar kan tydelik geïnaktiveer word onder kondisies van insulien stimulasie deur PKB/Akt gemedieerde fosforilering. Aan die ander kant is dit bekend dat GSK-3 die IRS-1 proteïen kan fosforileer om dus sodoende insulien sein-transduksie af te reguleer (Eldar-Finkelman et al. 1996). Daarmee saam is abnormaal hoë vlakke van GSK-3 aktiwiteit geassosieer met verskeie patologiese versteurings, insluitend tipe 2 diabetes, neuron degeneratiewe en affektiewe versteurings (Eldar-Finkelman et al. 2009). Daar is dus nuwe generasies GSK-3 inhibitore ontwikkel met die kliniese potensiaal om hierdie patologieë te behandel (Martinez 2008). Dit is al bevind dat GSK-3 inhibisie geassosieer kan word met beide die normalisering van plasma insulien- en glukose vlakke, asook kardiobeskerming in die konteks van iskemie/herperfusie (Nikoulina et al. 2002; Kumar et al. 2007). Doelwitte: Om te bepaal of GSK-3 proteïen en sy substraat proteïene gedisreguleer is onder kondisies van obesiteit en insulien weerstandigheid, asook om vas te stel of 'n spesifieke GSK-3 inhibitor die kardiovaskulêre patologie wat gevind word in obese en insulien weerstandige diere kan verhoed of omkeer. Mikpunte: Om veranderinge in uitdrukking en aktiwiteit van GSK-3 proteïen in 'n goed gekarakteriseerde rotmodel van obesiteit, gekoppel aan insulien weerstandigheid, te korreleer met die volgende: i) miokardiale kontraktiele disfunksie en onvermoë om kardiale iskemie/herperfusie besering te weerstaan, ii) aktivering en uitdrukking van PLM en SERCA-2a in die sarkoplasmiese retikulum, iii) die aktivering van intermediêres wat proksimaal geleë is in die insulienseintransduksiepad van GSK-3 (IRS-1, IRS-2 en PKB/Akt) en iv) om die effek van behandeling met 'n spesifieke inhibitor van GSK-3 op die bogenoemde punte te bepaal. Metodes: Ouderdoms- en gewigsgepaarde manlike Wistar rotte (kontrole en dieet geïnduseerde obees (DIO) diere) is in die studie gebruik. Kontrole diere was normale rotkos gevoer, terwyl die DIO diere op 'n dieet van rotkos aangevul met sukrose en kondensmelk geplaas is vir 'n periode van 8 of 16 weke. Helfte van die diere van elke groep is behandel met die GSK-3 inhibitor vir 4 weke (vanaf week 12 tot 16). Na afloop van die voer- en behandelingsperiode is die diere geweeg, doodgemaak en die harte verwyder om dan of onmiddelik gevriesklamp te word, of retrograad geperfuseer te word met Krebs-Hensleit buffer. Ex vivo geperfuseerde harte is dan blootgestel aan 25 minute lae vloei iskemie gevolg deur 30 minute herperfusie. Biometriese (liggaamsgewig, intraperitoneale vet, ventrikulêre gewig en tibia lengte) en biochemiese (vastende bloedglukose en -insulien vlakke) parameters is telkens bepaal. Western klad tegnieke is gebruik om die uitdrukking en fosforilering van GSK-3, PKB/Akt, IRS-1, IRS-2, SERCA-2a en PLM te bepaal. Ca2+-ATPase aktiwiteit is spektrofotometries bepaal. Resultate: Na beide 8 en 16 weke was die DIO diere beduidend swaarder as die kontrole diere. Hierdie gewigstoename was geassosieer met meer intraperitoneale vet in die DIO diere. Verder, in die DIO diere was IRS-1 afgereguleer na 8 weke, terwyl beide IRS-1 en IRS-2 asook PKB/Akt afgereguleer was na 16 weke. GSK-3 uitdrukking het 'n neiging getoon om toe te neem na beide 8 en 16 weke in die DIO diere, terwyl SERCA-2a beduidend afgereguleer was reeds vanaf 8 weke, geassosieer met laer Ca2+-ATPase aktiwiteit. PLM uitdrukking het toegeneem en die fosforilering daarvan was verlaag. Op 16 weke was die basale harttempo (225 vs 275 in die kontrole groep, P<0.0001, n=6) en tempo druk produk (21000 vs 30000 in die kontrole groep, P=0.019, n=6) betekenisvol laer in die DIO diere. Funksionele herstel het onveranderd gebly, alhoewel die tyd tot iskemiese kontraktuur toegeneem het in die DIO groep (kontrole: 11.6±0.4 min vs DIO: 16.2±0.5 min, P<0.01, n=6). Toediening van die inhibitor het geen effek op totale GSK-3 uitdrukking gehad nie. Fosforilering van GSK-3 was egter wel beduidend verhoog in die behandelde kontrole diere, terwyl daar geen verskille in die DIO groep was nie. Die fosforilering van GSK-3 het wel geneig na 'n toename in die behandelde DIO diere. Die GSK-3 inhibitor het kontrasterende effekte op hipertrofie gehad: dit het dit omgekeer in die DIO groep, maar veroorsaak in die kontrole diere. Daarmee saam het die inhibitor die uitdrukking van IRS-2 in beide DIO en kontrole diere gestimuleer, maar geen effek op IRS-1 en SERCA-2a uitdrukking en aktiwiteit gehad nie. GSK-3 inhibisie het wel PKB/Akt en PLM fosforilering in die DIO diere verhoog. Gevolgtrekking: Hierdie bevindinge toon dat 'n hoë kalorie dieet, tesame met 'n wanbalans tussen energie inname en verbruiking, lei tot die ontwikkeling van obesiteit en insulien weerstand in manlike Wistar rotte. Die studie het ook getoon dat GSK-3 en sy substraat proteïene wel gedisreguleer is in obesiteit en insulien weerstandigheid. Die verlaagde basale uitdrukking van SERCA-2a mag dalk 'n negatiewe impak hê op kardiale funksie. Behandeling van die diere met 'n GSK-3 inhibitor het getoon dat GSK-3 moontlik nie verantwoordelik is vir die basislyn veranderinge nie. Die afname in IRS-1 en SERCA-2a uitdrukking kan moontlik toegeskryf word aan ander meganismes buiten die effekte van GSK-3. Hierdie studie toon wel dat GSK-3 moontlik 'n rol speel in die regulering van die uitdrukking van IRS-2, maar nie IRS-1 nie. Verhoogde PKB/Akt fosforilering mag dalk bydra tot die inhibisie van GSK-3. Daarmee saam blyk dit dat GSK-3 inhibisie hipertrofie kan omkeer in DIO diere, om dan sodoende op te tree as 'n negatiewe reguleerder van hipertrofie, maar in normale kontrole diere, hipertrofie in die hand werk.South African Medical Research CouncilUniversity of Stellenbosch, Dept. of medical Physiolog

The use of nonsteroidal anti-inflammatory drugs by COVID-19 patients – in a nutshell

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a large scale of morbidities and mortalities worldwide, posing a significant threat to global health. COVID-19 has been challenging due to a lack of established treatment guidelines. Nonsteroidal anti-inflammatory drugs (NSAIDs) comprise of a heterogeneous group of compounds used for the symptomatic relief of fever, pain and inflammation. NSAIDs exert their effects by inhibiting prostaglandins' biosynthesis, resulting in anti-inflammatory, analgesic, and antipyretic effects. They may be beneficial in reducing inflammation and prevent fatal cytokine storms in COVID-19. However, the use of NSAIDs by COVID-19 patients has been controversial, with some reports recommending their use, while others contraindicated them. This may be due to the heterogeneous nature of COVID-19 including different strains or cases. There seem to be COVID-19 cases where NSAIDs should not be used; however, there is no evidence that NSAIDs should be avoided in all COVID-19 patients.http://www.sapj.co.zaPharmacolog

An overview of central nervous system tumours

Central nervous system (CNS) tumours refer to tumours that occur in the tissues of the brain and/or spinal cord. These tumours arise as a result of abnormal growth of cells and may begin in different parts of the brain or spinal cord. There are many types of CNS tumours, which are further divided into subtypes. Despite decades of research conducted, CNS tumours remain among the deadliest of all cancers. It is most often challenging to treat these tumours, due to the risks involved, and biological characteristics associated with them. The classification, grading, and characterisation of CNS tumour plays a pivotal role in the management thereof. The current review provides an overview of CNS tumours, classification, grading and treatment, as well as their characterisation with specific focus on gliomas, ependymomas, oligodendrogliomas, meningiomas, medulloblastomas, schwannomas, gangliogliomas, and craniopharyngiomas.https://scimedjournal.org/index.php/SMJPharmacolog

An appraisal of the regulatory policies governing the use of herbal traditional medicine

The regulation of herbal traditional medicine (HTM) is of much importance as it ensures the safety, quality and efficacy thereof. However, there are variations in the regulation of HTM worldwide with some countries being more supportive of HTM than others. This literature review aimed to evaluate and compare the regulatory policies governing the use of HTM in developed and developing countries as well as to determine the regulatory challenges faced by regulatory authorities and governments across the world. The countries investigated in this study were Germany, the United States of America, Japan, South Africa, China and India. Variations were evident between countries, however, Germany and Japan were found to be more advanced with regards to the regulation of HTM. Germany and Japan had stricter regulatory policies and lesser safety concerns. South Africa and the United States of America appear to have inadequate or ineffective HTM regulatory systems which was seen by the countries’ limited or lack of regulations and additional safety concerns. The findings showed the difference in HTM regulation between developed and developing countries were not as large as could be expected. The United States of America (developed country) was found to have poor HTM regulations, while China and India (developing countries) were found to have thorough regulations. The findings also show that both developed and developing countries continue to face challenges with regards to establishing regulations and registration procedures for HTM.http://www.liebertpub.com/tmrdm2022Pharmacolog

The effect of normoxic and hypoxic U-87 glioblastoma paracrine secretion on the modulation of brain endothelial cells

Please read abstract in the article.University of the Western Cape Senate granthttps://www.mdpi.com/journal/cellsdm2022Pharmacolog

Differential effects of normoxic versus hypoxic derived breast cancer paracrine factors on brain endothelial cells

The blood-brain barrier (BBB) is a central nervous system protective barrier formed primarily of endothelial cells that regulate the entry of substances and cells from entering the brain. However, the BBB integrity is disrupted in disease, including cancer, allowing toxic substances, molecules, and circulating cells to enter the brain. This study aimed to determine the mitochondrial changes in brain endothelial cells co-cultured with cancer cells. Method: Brain endothelial cells (bEnd.3) were co-cultivated with various concentrations of breast cancer (MCF7) conditioned media (CM) generated under normoxic (21% O2 ) and hypoxic conditions (5% O2 ). The mitochondrial activities (including; dehydrogenases activity, mitochondrial membrane potential (∆Ψm), and ATP generation) were measured using Polarstar Omega B.M.G-Plate reader. Trans-endothelial electrical resistance (TEER) was evaluated using the EVOM system, followed by quantifying gene expression of the endothelial tight junction (ETJs) using qPCR. Results: bEnd.3 cells had reduced cell viability after 72 h and 96 h exposure to MCF7CM under hypoxic and normoxic conditions

An overview of allergic conjunctivitis

Allergic diseases affect many people across the globe. They significantly impact on the quality of life of the people who are affected, creating personal and economic predicaments. Some of the most commonly diagnosed allergic diseases include atopic dermatitis, rhinitis, allergic conjunctivitis and sinusitis. Allergic conjunctivitis is an allergic disease characterised by the inflammation of the conjunctiva caused by airborne allergens; it presents as itching, excessive lacrimation, discharge and pink eye. Usually it is associated with other allergic conditions such as allergic rhinitis and bronchial asthma. Allergic conjunctivitis is further divided into acute, seasonal allergic conjunctivitis (SAC), and perennial allergic conjunctivitis (PAC). Other conditions, such as eosinophilic oesophagitis, are on the rise and are being diagnosed across all continents except Africa. The diagnosis is primarily clinical. Antihistamines have been the mainstay of therapy for most allergic conditions, except for other conditions that require corticosteroids, or in severe allergic conditions such as anaphylaxis where antihistamines are ineffective as main therapy. It is important to consider first- versus secondgeneration options when treating allergic diseases, also bearing in mind the duration of therapy and any comorbid conditions that a patient might have. This article provides an overview of these conditions and their current management options.http://www.sapj.co.za/index.php/SAPJdm2022Pharmacolog

Barriers to adherence to antiretroviral treatment in a regional hospital in Vredenburg, Western Cape, South Africa

BACKGROUND: South Africa currently runs the largest public antiretroviral treatment (ART) programme in the world, with over 80% of people living with HIV and/or AIDS on ART. However, in order to appreciate the benefits of using ART, patients are subject to uncompromising and long-term commitments of taking at least 95% of their treatment as prescribed. Evidence shows that this level of adherence is seldom achieved because of a multilevel and sometimes interwoven myriad of factors. Objective: We described the challenges faced by patients on ART in Vredenburg with regard to ART adherence. METHODS: A descriptive qualitative research design was used. Eighteen non-adhering patients on ART in the Vredenburg regional hospital were purposefully selected. Using a semistructured interview guide, we conducted in-depth interviews with the study participants in their mother tongue (Afrikaans). The interviews were audio-taped, transcribed verbatim and translated into English. The data were analysed manually using the thematic content analysis method. RESULTS: Stigma, disclosure, unemployment, lack of transport, insufficient feeding, disability grants and alternative forms of therapy were identified as major barriers to adherence, whereas inadequate follow-ups and lack of patient confidentiality came under major criticisms from the patients. CONCLUSION: Interventions to address poverty, stigma, discrimination and disclosure should be integrated with group-based ART adherence models in Vredenburg while further quantitative investigations should be carried out to quantify the extent to which these factors impede adherence in the community.IS

Biomarkers of HIV associated malignancies and of drug interaction between anti-retrovirals (ARVs) and chemotherapy

Thesis (PhD)--Stellenbosch University, 2015.ENGLISH ABSTRACT: INTRODUCTION: Altered immune mechanisms play a critical role in the pathogenesis of Non-Hodgkin lymphoma (NHL), as evidenced by increased rates of NHL among HIV+ patients [De Roos et al., 2012; Mellgren et al., 2012]. AIMS: To determine whether biomarkers of B-, T-cell activation, and inflammation are elevated in HIV+NHL patients; and whether cART influences their expression. METHODS: The expression of CD8+CD38 and FoxP3 were determined by flow cytometry; the serum concentrations of circulating sCD20, sCD23, sCD27, sCD30 and sCD44 were determined by enzyme linked immunosorbent assay (ELISA); and the serum concentrations of circulating IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α were determined by meso-scale discovery (MSD) assay in 141 participants consisting of HIV positive NHL (HIV+NHL), HIV negative NHL (NHL); combination antiretroviral treated HIV+ (HIV+ cART), treatment naive HIV+ (cART-naïve HIV+) patients; and healthy controls. RESULTS: HIV+NHL patients had higher serum concentrations of sCD20 (p<0.0001 and p=0.0359), sCD23 (p=0.0192 and p<0.0001), sCD30 (p=0.0052 and p<0.0001), sCD44 (p=0.0014 and p<0.0001), and IL-4 (p=0.0234 and p=0.03360); and lower expression of FoxP3 (p<0.0001 and p=0.0171) as compared to NHL and HIV+ cART patients. As compared to NHL patients, the serum concentrations of IL-2 (p=0.0115), and TNF-α (p=0.0258) were higher in HIV+NHL patients, while those of IL-1β (p=0.0039) were significantly lower. HIV+NHL patients had higher expression of CD8+CD38 (p=0.0104), serum concentrations of IFN-γ (p=0.0085), and IL-6 (p=0.0265); and lower serum concentrations of IL-12p70 (p=0.0012) than HIV+ cART patients. As compared to controls, NHL had higher concentrationsof all biomarkers investigated except FoxP3 expression. As compared to HIV+ cART and controls, cART-naïve HIV+ patients had higher concentrations of all biomarkers investigated except sCD23 and FoxP3 expression. CONCLUSION: Biomarkers of chronic B- and T-cell activation and inflammation are up-regulated in HIV+NHL and the untreated HIV+ state. cART decreases immune activation and inflammation.AFRIKAANSE OPSOMMING: INLEIDING: Versteurde immuun meganisme speel ‘n kritiese rol in die patogenese van Non-Hodgkin limfoom (NHL), soos aangedui deur verhoogde tempo van NHL onder MIV+ pasiënte [De Roos et al., 2012; Mellgren et al., 2012]. DOELWITTE: Om te bepaal indien biomerkers van B-, T-sel aktivering en inflammasie verhoog is in MIV+NHL pasiënte; en indien kART hul uitdrukking beinvloed. METODE: Die uitdrukking van CD8+CD38 en FoxP3 was bepaal deur vloei sitometrie; die serum konsentrasies van sirkulerende sCD20, sCD27, sCD30 en sCD44 was bepaal deur ensiem gekoppelde immuno sorbant toets (ELISA); en die serum konsentrasies van sirkulerende IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13 en TNF-α bepaal was deur meso-skaal ondekking (MSD) toets in 141 deelnemers bestaande uit MIV positiewe NHL (MIV+NHL); MIV negatiewe NHL (NHL), kombinasie antiretrovirale behandeling MIV+ (MIV+ kART); onbehandelde naïewe MIV+ (kART-naïewe MIV+) pasiente; en gesonde kontroles. RESULTATE: MIV+NHL pasiente het hoë serum konsentrasies van sCD20 (p<0.0001 en p=0.0359), sCD23 (p=0.0192 en p<0.0001), sCD30 (p=0.0052 en p<0.0001), sCD44 (p=0.0014 en p<0.0001), en IL-4 (p=0.0234 en p=0.03360); en verlaagde uitdrukking van FoxP3 (p<0.0001 en p=0.0171) in vergelyking met NHL en MIV+ kART patiente. Vergeleke met NHL pasiente, die serum konsentrasies van IL-2 (p=0.0115), en TNF-α (p=0.0258) was hoër in MIV+NHL pasiente, terwyl die van IL-1β (p=0.0039) beduidend laer was. MIV+NHL pasiente het hoër uitdrukking van CD8+CD38 (p=0.0104), serum konsentrasies van IFN-γ (p=0.0085), en IL-6 (p=0.0265); en laer serum konsentrasies van IL-12p70 (p=0.0012) as MIV+ kART pasiente. Vergeleke met die kontroles, NHL het hoër konsentrasies van al die biomerkers wat geondersoek was behalwe vir FoxP3 uitdrukking. Vergeleke met MIV+ kART en die kontroles, kART-naϊewe MIV+ pasiente het ‘n hoer konsentrasies van al die biomerkers wat ondersoek was behalwe sCD23 en FoxP3 die uitdrukking. GEVOLGTREKKING: Biomerkers van kroniese B- en T-sel aktivering en inflammasie is op-gereguleer in MIV+NHL en die onbehandelde MIV+ toestande. kART het immuun aktivering en inflammasie verminder

Differential effects of normoxic versus hypoxic derived breast cancer paracrine factors on brain endothelial cells

BACKGROUND : The blood-brain barrier (BBB) is a central nervous system protective barrier formed primarily of endothelial cells that regulate the entry of substances and cells from entering the brain. However, the BBB integrity is disrupted in disease, including cancer, allowing toxic substances, molecules, and circulating cells to enter the brain. This study aimed to determine the mitochondrial changes in brain endothelial cells co-cultured with cancer cells. METHOD : Brain endothelial cells (bEnd.3) were co-cultivated with various concentrations of breast cancer (MCF7) conditioned media (CM) generated under normoxic (21% O2) and hypoxic conditions (5% O2). The mitochondrial activities (including; dehydrogenases activity, mitochondrial membrane potential (DYm), and ATP generation) were measured using Polarstar Omega B.M.G-Plate reader. Trans-endothelial electrical resistance (TEER) was evaluated using the EVOM system, followed by quantifying gene expression of the endothelial tight junction (ETJs) using qPCR. RESULTS : bEnd.3 cells had reduced cell viability after 72 h and 96 h exposure to MCF7CM under hypoxic and normoxic conditions. The DYm in bEnd.3 cells were hyperpolarized after exposure to the hypoxic MCF7CM (p < 0.0001). However, the normoxic MCF7CM did not significantly affect the state of DYm in bEnd.3 cells. ATP levels in bEnd.3 co-cultured with hypoxic and normoxic MCF7CM was significantly reduced (p < 0.05). The changes in brain endothelial mitochondrial activity were associated with a decrease in TEER of bEnd.3 monolayer co-cultured with MCF7CM under hypoxia (p = 0.001) and normoxia (p < 0.05). The bEnd.3 cells exposed to MCF7CM significantly increased the gene expression level of ETJs (p < 0.05). CONCLUSIONS : MCF7CM modulate mitochondrial activity in brain endothelial cells, affecting the brain endothelial barrier function.University of the Western Capehttps://www.mdpi.com/journal/biologyam2022Pharmacolog