Unique growth pattern of human mammary epithelial cells induced by polymeric nanoparticles.

Due to their unique properties, engineered nanoparticles (NPs) have found broad use in industry, technology, and medicine, including as a vehicle for drug delivery. However, the understanding of NPs' interaction with different types of mammalian cells lags significantly behind their increasing adoption in drug delivery. In this study, we show unique responses of human epithelial breast cells when exposed to polymeric Eudragit® RS NPs (ENPs) for 1-3 days. Cells displayed dose-dependent increases in metabolic activity and growth, but lower proliferation rates, than control cells, as evidenced in tetrazolium salt (WST-1) and 5-bromo-2'-deoxyuridine (BrdU) assays, respectively. Those effects did not affect cell death or mitochondrial fragmentation. We attribute the increase in metabolic activity and growth of cells culture with ENPs to three factors: (1) high affinity of proteins present in the serum for ENPs, (2) adhesion of ENPs to cells, and (3) activation of proliferation and growth pathways. The proteins and genes responsible for stimulating cell adhesion and growth were identified by mass spectrometry and Microarray analyses. We demonstrate a novel property of ENPs, which act to increase cell metabolic activity and growth and organize epithelial cells in the epithelium as determined by Microarray analysis

Evidence for the Mitochondrial Lactate Oxidation Complex in Rat Neurons: Demonstration of an Essential Component of Brain Lactate Shuttles

To evaluate the presence of components of a putative Intracellular Lactate Shuttle (ILS) in neurons, we attempted to determine if monocarboxylate (e.g. lactate) transporter isoforms (MCT1 and -2) and lactate dehydrogenase (LDH) are coexpressed in neuronal mitochondria of rat brains. Immunohistochemical analyses of rat brain cross-sections showed MCT1, MCT2, and LDH to colocalize with the mitochondrial inner membrane marker cytochrome oxidase (COX) in cortical, hippocampal, and thalamic neurons. Immunoblotting after immunoprecipitation (IP) of mitochondria from brain homogenates supported the histochemical observations by demonstrating that COX coprecipitated MCT1, MCT2, and LDH. Additionally, using primary cultures from rat cortex and hippocampus as well as immunohistochemistry and immunocoprecipitation techniques, we demonstrated that MCT2 and LDH are coexpressed in mitochondria of cultured neurons. These findings can be interpreted to mean that, as in skeletal muscle, neurons contain a mitochondrial lactate oxidation complex (mLOC) that has the potential to facilitate both intracellular and cell-cell lactate shuttles in brain

Increased HIV-1 transcriptional activity and infectious burden in peripheral blood and gut-associated CD4+ T cells expressing CD30

HIV-1-infected cells persist indefinitely despite the use of combination antiretroviral therapy (ART), and novel therapeutic strategies to target and purge residual infected cells in individuals on ART are urgently needed. Here, we demonstrate that CD4+ T cell-associated HIV-1 RNA is often highly enriched in cells expressing CD30, and that cells expressing this marker considerably contribute to the total pool of transcriptionally active CD4+ lymphocytes in individuals on suppressive ART. Using in situ RNA hybridization studies, we show co-localization of CD30 with HIV-1 transcriptional activity in gut-associated lymphoid tissues. We also demonstrate that ex vivo treatment with brentuximab vedotin, an antibody-drug conjugate (ADC) that targets CD30, significantly reduces the total amount of HIV-1 DNA in peripheral blood mononuclear cells obtained from infected, ART-suppressed individuals. Finally, we observed that an HIV-1-infected individual, who received repeated brentuximab vedotin infusions for lymphoma, had no detectable virus in peripheral blood mononuclear cells. Overall, CD30 may be a marker of residual, transcriptionally active HIV-1 infected cells in the setting of suppressive ART. Given that CD30 is only expressed on a small number of total mononuclear cells, it is a potential therapeutic target of persistent HIV-1 infection

Global investments in pandemic preparedness and COVID-19: development assistance and domestic spending on health between 1990 and 2026

Background The COVID-19 pandemic highlighted gaps in health surveillance systems, disease prevention, and treatment globally. Among the many factors that might have led to these gaps is the issue of the financing of national health systems, especially in low-income and middle-income countries (LMICs), as well as a robust global system for pandemic preparedness. We aimed to provide a comparative assessment of global health spending at the onset of the pandemic; characterise the amount of development assistance for pandemic preparedness and response disbursed in the first 2 years of the COVID-19 pandemic; and examine expectations for future health spending and put into context the expected need for investment in pandemic preparedness. Methods In this analysis of global health spending between 1990 and 2021, and prediction from 2021 to 2026, we estimated four sources of health spending: development assistance for health (DAH), government spending, out-of-pocket spending, and prepaid private spending across 204 countries and territories. We used the Organisation for Economic Co-operation and Development (OECD)'s Creditor Reporting System (CRS) and the WHO Global Health Expenditure Database (GHED) to estimate spending. We estimated development assistance for general health, COVID-19 response, and pandemic preparedness and response using a keyword search. Health spending estimates were combined with estimates of resources needed for pandemic prevention and preparedness to analyse future health spending patterns, relative to need. Findings In 2019, at the onset of the COVID-19 pandemic, US$9·2 trillion (95$7·3 trillion (95% UI 7·2–7·4) in 2019; 293·7 times the $24·8 billion (95$43·1 billion in development assistance was provided to maintain or improve health. The pandemic led to an unprecedented increase in development assistance targeted towards health; in 2020 and 2021, $1·8 billion in DAH contributions was provided towards pandemic preparedness in LMICs, and$37·8 billion was provided for the health-related COVID-19 response. Although the support for pandemic preparedness is 12·2% of the recommended target by the High-Level Independent Panel (HLIP), the support provided for the health-related COVID-19 response is 252·2% of the recommended target. Additionally, projected spending estimates suggest that between 2022 and 2026, governments in 17 (95% UI 11–21) of the 137 LMICs will observe an increase in national government health spending equivalent to an addition of 1% of GDP, as recommended by the HLIP. Interpretation There was an unprecedented scale-up in DAH in 2020 and 2021. We have a unique opportunity at this time to sustain funding for crucial global health functions, including pandemic preparedness. However, historical patterns of underfunding of pandemic preparedness suggest that deliberate effort must be made to ensure funding is maintained

Lactate Metabolism and the Response to Nanoparticle Exposure in Normal and Cancerous Breast Cells

To understand lactate metabolism and the response of normal and cancerous breast cells to nanoparticle exposure, we performed two studies using breast cancer cell lines MCF-7 and MDA-MB-231, and a normal breast cell line HMEC 184. In my first study I examined the expression and the localization of the lactate shuttle proteins monocarboxylate transporter (MCT) and lactate dehydrogenase (LDH) isoforms in two breast cancer cell lines MCF-7, MDA-MB-231, compared to the normal breast cell line HMEC 184. I hypothesized that there are changes in the localization and expression of MCTs and LDH isoforms in cancerous breast cells when compared to normal breast cells, and that these changes are associated with the Warburg Effect and correspond to the oxidative capacity of the cancerous cells. My data show that MCT (1, 2, and 4), and LDH isoforms (A and B) are expressed in both normal and cancerous breast cell lines, except that MDA-MB-231 did not express MCT1. MCT1 was highly expressed in normal cells when compared to cancer cell lines. MCT4 was highly expressed in MDA-MB-231, and MCT2 was highly expressed in MCF-7. LDH was highly expressed in both cancerous cell lines compared to the normal cell line, and MCF-7 expressed mainly LDH5 (LDHB), while MDA-MB-231 and HMEC 184 expressed mainly LDH1 (LDHA). Using confocal laser scanning microscopy, I found that MCT2, MCT4, and LDH are localized in mitochondria in addition to their localization in the plasma membrane and cytosol, whereas MCT1 is mainly localized in the plasma membrane. This localization was the same in cancerous and normal cell lines. The changes in the expression of MCT and LDH isoforms corresponded to the metabolic status of each cell line. Both cell lines MCF-7 and MDA-MB-231 produced higher amounts of lactate than the HMEC 184 cell line, but have less endogenous and maximum respiration than the HMEC 184 cell line. In conclusion, I reported changes in the expression of MCT and LDH in breast cancer cells with no change in their localization. These changes corresponded to the breast cancer cells' oxidative capacity. My data support the existence of the previously reported lactate shuttle in cancer, and add a new explanation of its function. My next project examined the effect of co-polymer nanoparticles, Eudragit® RS 100 (ENPs), increasingly being used to coat and deliver drugs including chemotherapy agents, on the metabolic activity and proliferation of the human epithelial breast cells (HMEC 184, MCF-7, MDA-MB-231). I reported novel responses of human epithelial breast cells when exposed to Eudragit nanoparticles. I showed that cells displayed dose-dependent increases in metabolic activity and growth, but lower proliferation rates, than control cells, as evidenced in tetrazolium salt (WST-1) and 5-bromo-2'-deoxyuridine (BrdU) assays. Using mass spectrometry and micrroarry analyses I found that the mechanism for this behaviour stems from the ability of Eudragit nanoparticles to bind to certain proteins in culture media and to bring them closer to the surface of cells. Those proteins are involved in cell adhesion, growth, differentiation, and migration. The effect of nanoparticle treatment in increasing cancer and normal human epithelial breast cell metabolic activity and growth has not been reported previously, and this project highlighted the need for further research to address the potentially counter-productive effects of using nanoparticles in cancer chemotherapy

Evidence for the mitochondrial lactate oxidation complex in rat neurons: demonstration of an essential component of brain lactate shuttles.

To evaluate the presence of components of a putative Intracellular Lactate Shuttle (ILS) in neurons, we attempted to determine if monocarboxylate (e.g. lactate) transporter isoforms (MCT1 and -2) and lactate dehydrogenase (LDH) are coexpressed in neuronal mitochondria of rat brains. Immunohistochemical analyses of rat brain cross-sections showed MCT1, MCT2, and LDH to colocalize with the mitochondrial inner membrane marker cytochrome oxidase (COX) in cortical, hippocampal, and thalamic neurons. Immunoblotting after immunoprecipitation (IP) of mitochondria from brain homogenates supported the histochemical observations by demonstrating that COX coprecipitated MCT1, MCT2, and LDH. Additionally, using primary cultures from rat cortex and hippocampus as well as immunohistochemistry and immunocoprecipitation techniques, we demonstrated that MCT2 and LDH are coexpressed in mitochondria of cultured neurons. These findings can be interpreted to mean that, as in skeletal muscle, neurons contain a mitochondrial lactate oxidation complex (mLOC) that has the potential to facilitate both intracellular and cell-cell lactate shuttles in brain