Low Intensity Laser Irradiation and Growth Factors Influence Differentiation of Adipose Derived Stem Cells into Smooth Muscle Cells in a Coculture Environment over a Period of 72 Hours

Stem cells have the ability to self-renew and differentiate into several specialised cells. Low intensity laser irradiation (LILI) has been shown to have positive effects on cells including adipose derived stem cells (ADSCs). Growth factors such as retinoic acid and transforming growth factor (TGF-1) play significant roles in the differentiation of cells. This study aimed at investigating the role of LILI and growth factors on differentiation of adipose derived stem cells cocultured with smooth muscle cells (SMCs). The study used isolated human adipose derived stem cells and smooth muscle commercial cells (SKUT-1). The cells were cocultured directly in the ratio 1 : 1 using the established methods with and without growth factors (retinoic acid and TGF-1) and then exposed to LILI at a wavelength of 636 nm with 5 J/cm 2 using a diode laser. The cellular proliferation and expression of the both cell type markers were assessed using optical density and flow cytometry at 24 h and 72 h. The study showed that LILI increased the proliferation of cocultured cells. The expression of the smooth muscle cell markers increased in the coculture groups that were exposed to LILI in the presence of growth factors while those of the ADSCs decreased

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance.

Investment in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing in Africa over the past year has led to a major increase in the number of sequences that have been generated and used to track the pandemic on the continent, a number that now exceeds 100,000 genomes. Our results show an increase in the number of African countries that are able to sequence domestically and highlight that local sequencing enables faster turnaround times and more-regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and illuminate the distinct dispersal dynamics of variants of concern-particularly Alpha, Beta, Delta, and Omicron-on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve while the continent faces many emerging and reemerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

The evolving SARS-CoV-2 epidemic in Africa: Insights from rapidly expanding genomic surveillance

INTRODUCTION Investment in Africa over the past year with regard to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) sequencing has led to a massive increase in the number of sequences, which, to date, exceeds 100,000 sequences generated to track the pandemic on the continent. These sequences have profoundly affected how public health officials in Africa have navigated the COVID-19 pandemic. RATIONALE We demonstrate how the first 100,000 SARS-CoV-2 sequences from Africa have helped monitor the epidemic on the continent, how genomic surveillance expanded over the course of the pandemic, and how we adapted our sequencing methods to deal with an evolving virus. Finally, we also examine how viral lineages have spread across the continent in a phylogeographic framework to gain insights into the underlying temporal and spatial transmission dynamics for several variants of concern (VOCs). RESULTS Our results indicate that the number of countries in Africa that can sequence the virus within their own borders is growing and that this is coupled with a shorter turnaround time from the time of sampling to sequence submission. Ongoing evolution necessitated the continual updating of primer sets, and, as a result, eight primer sets were designed in tandem with viral evolution and used to ensure effective sequencing of the virus. The pandemic unfolded through multiple waves of infection that were each driven by distinct genetic lineages, with B.1-like ancestral strains associated with the first pandemic wave of infections in 2020. Successive waves on the continent were fueled by different VOCs, with Alpha and Beta cocirculating in distinct spatial patterns during the second wave and Delta and Omicron affecting the whole continent during the third and fourth waves, respectively. Phylogeographic reconstruction points toward distinct differences in viral importation and exportation patterns associated with the Alpha, Beta, Delta, and Omicron variants and subvariants, when considering both Africa versus the rest of the world and viral dissemination within the continent. Our epidemiological and phylogenetic inferences therefore underscore the heterogeneous nature of the pandemic on the continent and highlight key insights and challenges, for instance, recognizing the limitations of low testing proportions. We also highlight the early warning capacity that genomic surveillance in Africa has had for the rest of the world with the detection of new lineages and variants, the most recent being the characterization of various Omicron subvariants. CONCLUSION Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve. This is important not only to help combat SARS-CoV-2 on the continent but also because it can be used as a platform to help address the many emerging and reemerging infectious disease threats in Africa. In particular, capacity building for local sequencing within countries or within the continent should be prioritized because this is generally associated with shorter turnaround times, providing the most benefit to local public health authorities tasked with pandemic response and mitigation and allowing for the fastest reaction to localized outbreaks. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Differentiation potential of adipose derived stem cells (ADSCs) when co-cultured with smooth muscle cells (SMCs) and the role of low intensity laser irradiation (LILI)

D.Tech. (Biomedical Technology)Stem cells are defined as undifferentiated cells that can proliferate and have the capacity of both self-renewal and differentiation to one or more types of specialised cells (Bishop et al., 2002). The two types of stem cells are embryonic and adult stem cells. Adult stem cells have been isolated from adipose tissue in abundance and with ease (Mvula et al., 2010) and these cells have been differentiated into smooth muscle cells (SMCs) with the enhancement of low intensity laser irradiation and the growth factors (de Villiers et al., 2011). Smooth muscles play an important role in diseases like cancer, hypertension, asthma and others (Rodriguez et al., 2006). Studies have shown that low intensity laser irradiation (LILI) can increase proliferation of cells, cellular attachment, differentiation and production of transforming growth factor-beta 1 (TGF-β1) in cells indicating that in vitro LILI can modulate the activity of cells and tissues (Khadra et al., 2005). Further studies have also discovered that LILI enhances wound healing (Fiszerman and Markmann, 2000). LILI has been successfully used for pain attenuation and to induce wound healing in non-healing defects (Hawkins and Abrahamse, 2005). LILI has been shown to increase viability and proliferation of adipose derived stem cells (ADSCs) (Mvula et al., 2008 and Mvula et al., 2010). Growth factors such as retinoic acids (RA) have been shown to have major influences on cells. They are involved specifically in apoptosis, cell proliferation, differentiation and maturation (Duong and Rochette, 2011; Gudas and Wagner, 2011). Co-culturing is used to achieve several cellular processes including proliferation, differentiation and migration (Kim et al., 2012). When two types of cells are cultured together, they are exposed to a number of complex environmental factors such as cytokines, extracellular matrix components, cell interactions, mechanical stimuli, signalling transcriptional pathways and transcriptional factors such as growth factors. v These factors are able to affect migration, proliferation and differentiation of one cell type into another (Zhang et al., 2012). The aim of this study was to investigate the differentiation potential of ADSCs when co-cultured with (SMCs) and to determine the role of LILI on the co-cultured cells. Short and long term biological effects were monitored on these cells following exposure to LILI and addition of growth factors. The study used commercial and isolated human ADSCs and SMCs (SKUT-1) cells. After growing cells to semiconfluency for ADSCs and confluency for SMCs, they were co-cultured in a ratio of 1:1 using the established methods supplemented with and without growth factors (TGF-β1and RA) and then exposed to LILI. The cellular morphology, viability and proliferation activities of the irradiated cells were then assessed using direct inverted and differential interference contrast microscopy (DIC), trypan blue test, adenosine triphosphate luminescence, optical density analysis, and carboxyfluorescein diacetate succinimdyl ester (CFSE) methods. In particular the expression of the specific markers of both ADSCs, β1 Integrin (CD29) and Thy-1 (CD90) and SMCs, Myosin Heavy Chain (MHC) were investigated through immunoflourescent microscopy and flow cytometric analysis. Up and down regulation of genes involved in the human mesenchymal stem cell array were analysed through Reverse Transcriptase Polymerase Chain Reaction (RTPCR)..

Effect of low level laser irradiation on human adult adipose derived stem cells: an in vitro study

M. Tech.Stem cells are defined as undifferentiated cells that can proliferate indefinitely and have the capacity of both self-renewal and differentiation to one or more types of specialised cells. Traumatic tissue injury and age-related degenerative diseases are a major problem in South Africa and worldwide. Stem cells could be used for tissue engineering and reconstructive surgery. In treating these conditions, the main principle of stem cell therapy is the replacement of damaged and dead cells in injured tissues and organs with new healthy ones expanded in vitro from stem cells (Orlic et al., 2002). These cells can be isolated from adipose tissue in significant numbers and exhibit stable growth and proliferation kinetics in culture and could be differentiated into bone, fat, cartilage and muscle when treated with established lineage-specific factors (Zuk et al., 2002). Low Level Laser Therapy (LLLT) is currently applied in the treatment of numerous diseases and pathological conditions (Gasparyan et al., 2004). LLLT produces positive effects on irradiated cells and tissues such as proliferation of cells, capillary growth and adenosine triphosphate (ATP) activation (Schindl et al., 1998). Low level laser radiation at different intensities has been shown to stimulate as well as to inhibit cellular processes (Moore et al., 2005). Epidermal growth factor (EGF) is a growth factor that plays important roles in the regulation of cell growth, proliferation and differentiation. This study investigated the effect of low level laser radiation alone as well as in combination with EGF on adult adipose derived stem cells (ADSCs) isolated from human adipose tissue. ADSCs were isolated from human adipose tissue through collagenase digestion and cultured in DMEM-F12 containing 10% FBS and antibiotics and incubated at 37°C in a humidified atmosphere of 5% CO2 (Zuk et al., 2001). iii Semi-confluent monolayers of ADSCs were exposed to low level laser at 5 J/cm2 using 636 nm diode laser with a power density of 12.1 mW/cm2 at room temperature in the dark. Cell morphology was monitored at 0, 24 and 48 h using an inverted light inverted microscope. Cell viability was evaluated at 0, 24 and 48 h using the Trypan Blue exclusion test and an adenosine triphosphate (ATP) luminescence assay. bFGF (basic fibroblast growth factor) indirect ELISA and optical density assays were used to monitor cell proliferation at 0, 24 and 48 h post irradiation. In addition the expressions of stem cell markers, β1-integrin and Thy-1, were monitored by immunocytochemical live cell surface labelling and Western blot analysis. Cells were incubated with EGF to enhance proliferation and differentiation and the cell morphology, viability and proliferation were monitored as well as the expressions of stem cell markers, β1-integrin and Thy-1. Morphology of the cells was not altered by irradiating them with 5 J/cm2 using diode laser at 0, 24 and 48 h. Cell viability and proliferation showed an increase at 24 and 48 h post irradiation. At 0 h, there was no significant difference between irradiated and non-irradiated cells in cell viability and proliferation. There was an increase in the expression of β1-integrin and Thy-1 after irradiation as shown by Western blot analysis and immunocytochemical live cell surface labelling. Cell viability and proliferation showed a significant increase at all time points post irradiation with the addition of EGF. There was no noticeable change in cellular morphology at any time point. Low level laser irradiation of human ADSC’s at 636 nm with 5 J/cm2 and 12.1 mW/cm2 increased the viability and proliferation of these cells in vitro. Furthermore, low level laser irradiation appeared to increase the expression of stem cell markers, β1-integrin and Thy-1. In addition, laser irradiation did not alter the morphology of the cultured cells. The addition of EGF to the cells also increased their viability and proliferation as well the expression of the markers, β1-integrin and Thy-1. The study showed that laser irradiation stimulates two important cellular responses namely cell viability and proliferation which indicates that ADSCs may be suitable for tissue engineering and future cell differentiation studies

Differentiation potential of adipose-derived stem cells when cocultured with smooth muscle cells, and the role of low-intensity irradiation

Abstract: The aim of the study was to investigate the differentiation potential of adipose-derived stem cells (ADSCs) when cocultured with smooth muscle cells (SMCs), and to determine the role of low-intensity laser irradiation (LILI). Background data: ADSCs isolated from adipose tissue are isolated with ease and in large amounts. SMCs constitute most parts of the intestinal, urinary, reproductive, and cardiovascular systems. LILI has been found to have positive effects on different cell types, including ADSCs. Methods: The study used ADSCs (Stempro Adipose Derived Stem Cells-R7788-115) and SMCs (SKU-T-1 American Type Culture Collection HTB-114) cell lines. These cell lines were cocultured in a 1:1 ratio with and without growth factors and then exposed to LILI using 636 nm at 5 J/cm2. Results: Cell viability and proliferation increased significantly in the cocultured groups that were exposed to LILI alone, as well as in combination with growth factors. Further, there was a significant decrease in the expression of stem cell markers with a concomitant increase in SMC markers. Conclusions: These results suggest that ADSCs have the ability to differentiate into SMCs when cocultured with SMCs, whereas LILI potentially augments the differentiation potential and need. This further highlights the significant role that LILI has to offer ADSC therapy in regenerative medicine

Six months survival and risk factors for attrition for patients detected with cryptococcal antigenemia through screening in Malawi.

Main objectiveA cohort of adult Malawian people living with HIV (PLHIV) testing positive for cryptococcal antigenemia was observed and followed to determine the outcomes and risk factors for attrition.Methods conceptEligible PLHIV were enrolled at 5 health facilities in Malawi, representing different levels of health care. ART naïve patients, ART defaulters returning to care, and patients with suspected or confirmed ART treatment failure with CD4 ResultsA total of 2146 patients were screened and 112 (5.2%) had cryptococcal antigenemia. Prevalence ranged from 3.8% (Mzuzu Central Hospital) to 25.8% (Jenda Rural Hospital). Of the 112 patients with antigenemia, 33 (29.5%) were diagnosed with concurrent CM at the time of enrollment. Six-month crude survival of all patients with antigenemia (regardless of CM status) ranged from 52.3% (assuming lost-to-follow-up (LTFU) patients died) to 64.9% (if LTFU survived). Patients who were diagnosed with concurrent CM by CSF test had poor survival (27.3-39.4%). Patients with antigenemia who were not diagnosed with concurrent CM had 71.4% (if LTFU died)- 89.8% (if LTFU survived) survival at six months. In adjusted analyses, patients with cryptococcal antigenemia detected after admission to inpatient care (aHR: 2.56, 1.07-6.15) and patients with concurrent CM at the time of positive antigenemia result (aHR: 2.48, 1.04-5.92) had significantly higher hazard of attrition at six months.ConclusionsOverall, our findings indicate a need for routine access to CrAg screening and pre-emptive fluconazole treatment as a way to detect cryptococcal antigenemia and prevent CM in outpatient and inpatient settings. Rapid access to diagnosis and treatment for cryptococcal meningitis (CM) with gold-standard antifungals is needed to improve survival of patients with advanced HIV in Malawi

Draft genomes of <i>Aeromonas caviae</i> from patients with cholera-like illness during the 2022-2023 cholera outbreak in Malawi.

Aeromonas caviae is an increasingly recognized etiological agent of acute gastroenteritis. Here, we report five draft genomes of A. caviae isolated from suspected cholera cases during the 2022-2023 cholera outbreak in Malawi

Genomic insights into the 2022–2023Vibrio cholerae outbreak in Malawi

AbstractMalawi experienced its deadliest Vibrio cholerae (Vc) outbreak following devastating cyclones, with &gt;58,000 cases and &gt;1700 deaths reported between March 2022 and May 2023. Here, we use population genomics to investigate the attributes and origin of the Malawi 2022–2023 Vc outbreak isolates. Our results demonstrate the predominance of ST69 clone, also known as the seventh cholera pandemic El Tor (7PET) lineage, expressing O1 Ogawa (~ 80%) serotype followed by Inaba (~ 16%) and sporadic non-O1/non-7PET serogroups (~ 4%). Phylogenetic reconstruction revealed that the Malawi outbreak strains correspond to a recent importation from Asia into Africa (sublineage AFR15). These isolates harboured known antimicrobial resistance and virulence elements, notably the ICEGEN/ICEVchHai1/ICEVchind5 SXT/R391-like integrative conjugative elements and a CTXφ prophage with the ctxB7 genotype compared to historical Malawian Vc isolates. These data suggest that the devastating cyclones coupled with the recent importation of 7PET serogroup O1 strains, may explain the magnitude of the 2022–2023 cholera outbreak in Malawi.</jats:p