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Abstract Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.

Abstract Introduction Brain abscesses are rare but potentially fatal condition and can be associated with cyanotic congenital heart disease of which 5–18.7% of these patients that develop cerebral abscess commonly have tetralogy of Fallot (TOF). Case presentation We report a case of 3-year-old Muganda male that presented with convulsions, cyanosis and difficulty in breathing. The patient had a combination intervention of medical treatment and surgical drainage of the abscess. Post-operative Computerized tomography scan images and pre-operative brain Computerized tomography scans were compared. The multiple rings enhancing lesions were reduced in number and sizes. The largest measured ring was 44 × 22.5×16mm compared to the previous; 42 × 41×36mm. The mass effect had reduced from 16 mm to 7.5 mm. The periventricular hypodensities persisted. Findings showed radiological improvement with residual abscesses, subacute subdural hematoma and pneumocranium. The patient was treated with intravenous ceftriaxone 1 g OD for six weeks and he showed marked improvement and was discharged home after 3 months. Conclusion A comprehensive strategy involving medications, surgical drainage, and early neurosurgical consultation is vital in treating brain abscesses in uncorrected TOF. Early identification of the pathogen, appropriate antibiotic therapy, and vigilant follow-up through clinical assessments and imaging are crucial, potentially spanning a 4–8-week treatment.

Abstract Background In Uganda, medicinal plants have been utilized to treat a variety of ailments, including cancer. However, there is little information available about the medicinal plants used to treat cancer in the Elgon subregion. As a result, the current study documented the plant species used in the management of cancer in the Elgon sub-region. Methods Data were gathered by observation, self-administered questionnaires, interview guides, and guided field trips. Analyzing descriptive statistics and creating graphs were done using SPSS (version 21.0) and GraphPad Prism® version 9.0.0, respectively. Well-established formulae were used to calculate quantitative indices. The narratives were interpreted using major theories and hypotheses in ethnobotany. Results A total of 50 plant species from 36 families were documented, and herbal knowledge was mainly acquired through inheritance. Fabaceae and Asteraceae comprised more plant species used in herbal preparation. Most plants were collected from forest reserves (63%); herbal therapies were made from herbs (45%); and leaves were primarily decocted (43%). The most frequently used plants were Tylosema fassoglensis, Hydnora abyssinica, Azidarachata indica, Prunus Africana, Kigelia africana, Syzygium cumini, Hydnora africana, Rhoicissus tridentata, Albizia coriaria, and Plectranthus cuanneus. All the most commonly used plants exhibited a high preference ranking (60–86%) and reliability level (74.1–93.9%). Generally, the ICF for all the cancers treated by medicinal plants was close to 1 (0.84–0.95). Conclusions The ten most commonly utilized plants were favored, dependable, and most important for treating all known cancers. As a result, more investigation is required to determine their phytochemistry, toxicity, and effectiveness in both in vivo and in vitro studies. This could be a cornerstone for the pharmaceutical sector to develop new anticancer medications.

Abstract Background Expansive intracranial hematomas (EIH) following traumatic brain injury (TBI) continue to be a public health problem in Uganda. Data is limited regarding the neurosurgical outcomes of TBI patients. This study investigated the neurosurgical outcomes and associated risk factors of EIH among TBI patients at Mulago National Referral Hospital (MNRH). Methods A total of 324 subjects were enrolled using a prospective cohort study. Socio-demographic, risk factors and complications were collected using a study questionnaire. Study participants were followed up for 180 days. Univariate, multivariable, Cox regression analyses, Kaplan Meir survival curves, and log rank tests were sequentially conducted. P-values of

Background: In the East African region, herbal plants are essential in the treatment and control of cancer. Given the diverse ecological and cultural makeup of the regional states, it is likely that different ethnic groups will use the same or different plants for the same or different diseases. However, since 2019, this has not been compiled into a single study. Purpose: The study aimed to compile and record the medicinal plants utilized in East Africa from April 2019 to June 2023 to treat various cancer types. Materials and methods: The study examined 13 original studies that included ethnobotanical research conducted in East Africa. They were retrieved from several internet databases, including Google Scholar, Scopus, PubMed/Medline, Science Direct, and Research for Life. The study retrieved databases on plant families and species, plant parts used, preparation methods and routes of administration, and the country where the ethnobotanical field surveys were conducted. Graphs were produced using the GraphPad Prism 8.125 program (GraphPad Software, Inc., San Diego, CA). Tables and figures were used to present the data, which had been condensed into percentages and frequencies. Results: A total of 105 different plant species from 45 different plant families were identified, including Asteraceae (14), Euphorbiaceae (12), Musaceae (8), and Apocynaceae (7). Uganda registered the highest proportion (46% of the medicinal plants used). The most commonly mentioned medicinal plant species in cancer management was Prunus africana . Herbs (32%), trees and shrubs (28%), and leaves (45%) constituted the majority of herbal remedies. Most herbal remedies were prepared by boiling (decoction) and taken orally (57%). Conclusion: East Africa is home to a wide variety of medicinal plant species that local populations and herbalists, or TMP, frequently use in the treatment of various types of cancer. The most frequently used families are Asteraceae and Euphorbiaceae, with the majority of species being found in Uganda. The most frequently utilized plant species is Prunus africana . Studies on the effectiveness of Prunus africana against other malignancies besides prostate cancer are required.

Atherosclerosis is an inflammatory reaction that develops at specific regions within the artery wall and at specific sites of the arterial tree over a varying time frame in response to a variety of risk factors. The mechanisms that account for the interaction of systemic factors and atherosclerosis-susceptible regions of the arterial tree to mediate this site-specific development of atherosclerosis are not clear. The dynamics of blood flow has a major influence on where in the arterial tree atherosclerosis develops, priming the site for interactions with atherosclerotic risk factors and inducing cellular and molecular participants in atherogenesis. But how this accounts for lesion development at various locations along the vascular tree across differing time frames still requires additional study. Currently, murine models are favored for the experimental study of atherogenesis and provide the most insight into the mechanisms that may contribute to the development of atherosclerosis. Based largely on these studies, in this review, we discuss the role of hemodynamic shear stress, SR-B1, and other factors that may contribute to the site-specific development of atherosclerosis.

Non-communicable diseases (NCDs) are rising rapidly in urbanizing populations in sub-Saharan Africa. Assessment of inflammatory and metabolic characteristics of a urbanizing African population and the comparison with populations outside Africa could provide insight in the pathophysiology of the rapidly increasing epidemic of NCDs, including the role of environmental and dietary changes. Using a proteomic plasma profiling approach comprising 92 inflammation-related molecules, we examined differences in the inflammatory proteome in healthy Tanzanian and healthy Dutch adults. We show that healthy Tanzanians display a pro-inflammatory phenotype compared to Dutch subjects, with enhanced activity of the Wnt/β-catenin signalling pathway and higher concentrations of different metabolic regulators such as 4E-BP1 and fibroblast growth factor 21. Among the Tanzanian volunteers, food-derived metabolites were identified as an important driver of variation in inflammation-related molecules, emphasizing the potential importance of lifestyle changes. These findings endorse the importance of the current dietary transition and the inclusion of underrepresented populations in systems immunology studies.

Abstract Background Agricultural pesticides may exert strong selection pressures on malaria vectors during the aquatic life stages and may contribute to resistance in adult mosquitoes. This could reduce the performance of key vector control interventions such as indoor-residual spraying and insecticide-treated nets. The aim of this study was to investigate effects of agrochemicals on susceptibility and fitness of the malaria vectors across farming areas in Tanzania. Methods An exploratory mixed-methods study was conducted to assess pesticide use in four villages (V1–V4) in south-eastern Tanzania. Anopheles gambiae (s.l.) larvae were collected from agricultural fields in the same villages and their emergent adults examined for insecticide susceptibility, egg-laying and wing lengths (as proxy for body size). These tests were repeated using two groups of laboratory-reared An. arabiensis, one of which was pre-exposed for 48 h to sub-lethal aquatic doses of agricultural pesticides found in the villages. Results Farmers lacked awareness about the linkages between the public health and agriculture sectors but were interested in being more informed. Agrochemical usage was reported as extensive in V1, V2 and V3 but minimal in V4. Similarly, mosquitoes from V1 to V3 but not V4 were resistant to pyrethroids and either pirimiphos-methyl or bendiocarb, or both. Adding the synergist piperonyl butoxide restored potency of the pyrethroids. Pre-exposure of laboratory-reared mosquitoes to pesticides during aquatic stages did not affect insecticide susceptibility in emergent adults of the same filial generation. There was also no effect on fecundity, except after pre-exposure to organophosphates, which were associated with fewer eggs and smaller mosquitoes. Wild mosquitoes were smaller than laboratory-reared ones, but fecundity was similar. Conclusions Safeguarding the potential of insecticide-based interventions requires improved understanding of how agricultural pesticides influence important life cycle processes and transmission potential of mosquito vectors. In this study, susceptibility of mosquitoes to public health insecticides was lower in villages reporting frequent use of pesticides compared to villages with little or no pesticide use. Variations in the fitness parameters, fecundity and wing length marginally reflected the differences in exposure to agrochemicals and should be investigated further. Pesticide use may exert additional life cycle constraints on mosquito vectors, but this likely occurs after multi-generational exposures. Graphical Abstract

PURPOSEClinical breast examination (CBE) is one of the most common methods used for early detection of breast cancer in low- and middle-income countries. CBE alone is limited by lack of specificity and may result in unnecessary diagnostic procedures. We evaluated the feasibility of integrating CBE, fine-needle aspiration biopsy (FNAB), and rapid on-site evaluation (ROSE) in triaging palpable breast masses for specialized cancer care.MATERIALS AND METHODSAn intensive breast cancer screening event was conducted at a national trade fair by a multidisciplinary team of care providers targeting a healthy population in Dar es Salaam, Tanzania. All adults age ≥ 18 years were invited to participate. CBE was performed by oncologists and/or pathologists. FNAB was performed by a pathologist on palpable masses that were then categorized as benign, indeterminate, or suspicious for malignancy or definitively malignant based on ROSE.RESULTSA total of 208 individuals (207 females, one male; median age, 36 years; range, 18-68 years) were screened. Most (90.8%, 189 of 208) had normal findings, whereas 7.2% (15 of 208), 1% (2 of 208), and 1% (2 of 208) had a palpable mass, breast pain, and nipple discharge, respectively. Two participants had lesions too small for palpation-guided biopsy and clinically consistent with fibroadenomas; the participants were counseled, and observation was recommended. FNAB was performed on 13 breast masses, with 9 of 13 (69%) categorized as benign and 4 of 13 (31%) suspicious for malignancy. Final cytopathologic review of referred patients confirmed one case to be breast adenocarcinoma, one was suggestive of fibroadenoma, and two showed inflammations.CONCLUSIONIntegration of CBE with ROSE and FNAB was feasible in a breast cancer screening program in Tanzania. In settings with constrained resources for cancer care, this may be an effective method for triaging patients with breast masses.

The authors profile stool metagenomics and plasma metabolomics in Tanzanian individuals and uncover a gradient of gut microbial profiles, from rural through urban Tanzania towards Western populations. Integration with ex vivo blood microbial stimulations reveals immune responses associated with histidine and arginine pathways, mediated by Bifidobacterium longum and Akkermansia muciniphila.

# Background The burden of non-communicable diseases (NCDs), including cancer, in Africa is rising. Policymakers are charged with formulating evidence-based cancer control plans; however, there is a paucity of data on cancers generated from within Africa. As part of efforts to enhance cancer research training in East Africa, we performed a needs assessment and gap analysis of cancer-related research training resources in Tanzania. # Methods A mixed-methods study to evaluate existing individual, institutional, and national resources supporting cancer research training in Tanzania was conducted. Qualitative data were collected using in-depth interviews while quantitative data were collected using self-administered questionnaires and online surveys. The study also included a desk-review of policy and guidelines related to NCD research and training. Study participants were selected to represent five groups: (i) policymakers; (ii) established researchers; (iii) research support personnel; (iv) faculty members in degree training programs; and (v) post-graduate trainees. # Results Our results identified challenges in four thematic areas. First, there is a need for coordination and monitoring of the cancer research agenda at the national level. Second, both faculty and trainees identified the need for incorporation of rigorous training to improve research competencies. Third, sustained mentoring and institutional investment in development of mentorship resources is critical to empowering early career investigators. Finally, academic institutions can enhance research outputs by providing adequate research infrastructure, prioritizing protected time for research, and recognizing research accomplishments by trainees and faculty. # Conclusions As we look towards establishment of cancer research training programs in East Africa, investment in the development of rigorous research training, mentorship resources, and research infrastructure will be critical to empowering local health professionals to engage in cancer research activities.

Macrophages are core cellular elements of both early and advanced atherosclerosis. They take up modified lipoproteins and become lipid-loaded foam cells and secrete factors that influence other cell types in the artery wall involved in atherogenesis. Apoproteins E, AI, and SAA are all found on HDL which can enter the artery wall. In addition, apoE is synthesized by macrophages. These three apoproteins can promote cholesterol efflux from lipid-loaded macrophages and have other functions that modulate macrophage biology. Mimetic peptides based on the sequence or structure of these apoproteins replicate some of these properties and are potential therapeutic agents for the treatment of atherosclerosis to reduce cardiovascular diseases.

ApoE is a multifunctional protein that is expressed by many cell types that influences many aspects of cardiovascular physiology. In humans, there are three major allelic variants that differentially influence lipoprotein metabolism and risk for the development of atherosclerosis. Apoe-deficient mice and human apoE isoform knockin mice, as well as hypomorphic Apoe mice, have significantly contributed to our understanding of the role of apoE in lipoprotein metabolism, monocyte/macrophage biology, and atherosclerosis. This brief history of these mouse models will highlight their contribution to the understanding of the role of apoE in these processes. These Apoe−/− mice have also been extensively utilized as an atherosensitive platform upon which to assess the impact of modulator genes on the development and regression of atherosclerosis.

Cholesterol efflux capacity associates strongly and negatively with the incidence and prevalence of human CVD. We investigated the relationships of HDL's size and protein cargo with its cholesterol efflux capacity using APOB-depleted serum and HDLs isolated from five inbred mouse strains with different susceptibilities to atherosclerosis. Like humans, mouse HDL carried >70 proteins linked to lipid metabolism, the acute-phase response, proteinase inhibition, and the immune system. HDL's content of specific proteins strongly correlated with its size and cholesterol efflux capacity, suggesting that its protein cargo regulates its function. Cholesterol efflux capacity with macrophages strongly and positively correlated with retinol binding protein 4 (RBP4) and PLTP, but not APOA1. In contrast, ABCA1-specific cholesterol efflux correlated strongly with HDL's content of APOA1, APOC3, and APOD, but not RBP4 and PLTP. Unexpectedly, APOE had a strong negative correlation with ABCA1-specific cholesterol efflux capacity. Moreover, the ABCA1-specific cholesterol efflux capacity of HDL isolated from APOE-deficient mice was significantly greater than that of HDL from wild-type mice. Our observations demonstrate that the HDL-associated APOE regulates HDL's ABCA1-specific cholesterol efflux capacity. These findings may be clinically relevant because HDL's APOE content associates with CVD risk and ABCA1 deficiency promotes unregulated cholesterol accumulation in human macrophages.

Natural antibodies (NAbs) are important regulators of tissue homeostasis and inflammation and are thought to have diverse protective roles in a variety of pathological states. E06 is a T15 idiotype IgM NAb exclusively produced by B-1 cells, which recognizes the phosphocholine (PC) head group in oxidized phospholipids on the surface of apoptotic cells and in oxidized LDL (OxLDL), and the PC present on the cell wall of Streptococcus pneumoniae. Here we report that titers of the E06 NAb are selectively increased several-fold in Cd1d-deficient mice, whereas total IgM and IgM antibodies recognizing other oxidation specific epitopes such as in malondialdehyde-modified LDL (MDA-LDL) and OxLDL were not increased. The high titers of E06 in Cd1d-deficient mice are not due to a global increase in IgM-secreting B-1 cells, but they are specifically due to an expansion of E06-secreting splenic B-1 cells. Thus, CD1d-mediated regulation appeared to be suppressive in nature and specific for E06 IgM-secreting cells. The CD1d-mediated regulation of the E06 NAb generation is a novel mechanism that regulates the production of this specific oxidation epitope recognizing NAb.

BackgroundHypercholesterolemia‐induced decreased availability of nitric oxide (NO) is a major factor in cardiovascular disease. We previously established that cholesterol suppresses endothelial inwardly rectifying K+ (Kir) channels and that Kir2.1 is an upstream mediator of flow‐induced NO production. Therefore, we tested the hypothesis that suppression of Kir2.1 is responsible for hypercholesterolemia‐induced inhibition of flow‐induced NO production and flow‐induced vasodilation (FIV). We also tested the role of Kir2.1 in the development of atherosclerotic lesions. Methods and ResultsKir2.1 currents are significantly suppressed in microvascular endothelial cells exposed to acetylated–low‐density lipoprotein or isolated from apolipoprotein E–deficient (Apoe−/−) mice and rescued by cholesterol depletion. Genetic deficiency of Kir2.1 on the background of hypercholesterolemic Apoe−/−mice, Kir2.1+/−/Apoe−/− exhibit the same blunted FIV and flow‐induced NO response as Apoe−/−or Kir2.1+/− alone, but while FIV in Apoe−/− mice can be rescued by cholesterol depletion, in Kir2.1+/−/Apoe−/− mice cholesterol depletion has no effect on FIV. Endothelial‐specific overexpression of Kir2.1 in arteries from Apoe−/− and Kir2.1+/−/Apoe−/− mice results in full rescue of FIV and NO production in Apoe−/− mice with and without the addition of a high‐fat diet. Conversely, endothelial‐specific expression of dominant‐negative Kir2.1 results in the opposite effect. Kir2.1+/−/Apoe−/−mice also show increased lesion formation, particularly in the atheroresistant area of descending aorta. ConclusionsWe conclude that hypercholesterolemia‐induced reduction in FIV is largely attributable to cholesterol suppression of Kir2.1 function via the loss of flow‐induced NO production, whereas the stages downstream of flow‐induced Kir2.1 activation appear to be mostly intact. Kir2.1 channels also have an atheroprotective role.

apoE is a multi-functional protein expressed in several cell types and in several organs. It is highly expressed in adipose tissue, where it is important for modulating adipocyte lipid flux and gene expression in isolated adipocytes. In order to investigate a potential systemic role for apoE that is produced in adipose tissue, mice were generated with selective suppression of adipose tissue apoE expression and normal circulating apoE levels. These mice had less adipose tissue with smaller adipocytes containing fewer lipids, but no change in adipocyte number compared with control mice. Adipocyte TG synthesis in the presence of apoE-containing VLDL was markedly impaired. Adipocyte caveolin and leptin gene expression were reduced, but adiponectin, PGC-1, and CPT-1 gene expression were increased. Mice with selective suppression of adipose tissue apoE had lower fasting lipid, insulin, and glucose levels, and glucose and insulin tolerance tests were consistent with increased insulin sensitivity. Lipid storage in muscle, heart, and liver was significantly reduced. Adipose tissue macrophage inflammatory activation was markedly diminished with suppression of adipose tissue apoE expression. Our results establish a novel effect of adipose tissue apoE expression, distinct from circulating apoE, on systemic substrate metabolism and adipose tissue inflammatory state.

Macrophage conversion to atherosclerotic foam cells is partly due to the balance of uptake and efflux of cholesterol. Cholesterol efflux from cells by HDL and its apoproteins for subsequent hepatic elimination is known as reverse cholesterol transport. Numerous methods have been developed to measure in vivo macrophage cholesterol efflux. Most methods do not allow for macrophage recovery for analysis of changes in cellular cholesterol status. We describe a novel method for measuring cellular cholesterol balance using the in vivo entrapment of macrophages in alginate, which retains incorporated cells while being permeable to lipoproteins. Recipient mice were injected subcutaneously with CaCl2 forming a bubble into which a macrophage/alginate suspension was injected, entrapping the macrophages. Cells were recovered after 24 h. Cellular free and esterified cholesterol mass were determined enzymatically and normalized to cellular protein. Both normal and cholesterol loaded macrophages undergo measureable changes in cell cholesterol when injected into WT and apoA-I-, LDL-receptor-, or apoE-deficient mice. Cellular cholesterol balance is dependent on initial cellular cholesterol status, macrophage cholesterol transporter expression, and apolipoprotein deficiency. Alginate entrapment allows for the in vivo measurement of macrophage cholesterol homeostasis and is a novel platform for investigating the role of genetics and therapeutic interventions in atherogenesis.

* Context.--Rapid onsite evaluation (ROSE) is critical in determining sample adequacy and triaging cytology samples. Although fine-needle aspiration biopsy (FNAB) is the primary method of initial tissue sampling in Tanzania, ROSE is not practiced. Objective.--To investigate the performance of ROSE in determining cellular adequacy and providing preliminary diagnoses in breast FNAB in a low-resource setting. Design.--Patients with breast masses were recruited prospectively from the FNAB clinic at Muhimbili National Hospital. Each FNAB was evaluated by ROSE for overall specimen adequacy, cellularity, and preliminary diagnosis. The preliminary interpretation was compared to the final cytologic diagnosis and histologic diagnosis, when available. Results.--Fifty FNAB cases were evaluated, and all were adequate for diagnosis on ROSE and final interpretation. Overall percentage of agreement (OPA) between preliminary and final cytologic diagnosis was 84%, positive percentage of agreement (PPA) was 33%, and negative percentage of agreement (NPA) was 100% ([kappa] = 0.4, P Conclusions.--False-positive rates of ROSE diagnoses for breast FNAB are low. While preliminary cytologic diagnoses had a high false-negative rate, final cytologic diagnoses had overall high concordance with histologic diagnoses. Therefore, the role of ROSE for preliminary diagnosis should be considered carefully in low-resource settings, and it may need to be paired with additional interventions to improve pathologic diagnosis. doi: 10.5858/arpa.2022-0398-OA, Breast cancer (BC) is the second leading cause of cancer-related mortality among women in Tanzania, with a majority of patients (63%-84%) presenting with advanced disease. (1-4) Tanzania has a hierarchical [...]

Natural killer T (NKT) cells are a distinct subset of lymphocytes that bridge the innate and adaptive immune response and can be divided into type I invariant NKT cells (iNKT) and type II NKT cells. The objective of this study is to examine the effects of NKT cell on lipid metabolism and the initiation and progression of atherosclerosis in LDL receptor deficient (LDLR−/−) mice. Mice were fed an atherogenic diet for 4 or 8 weeks and plasma lipids, lipoproteins, and atherosclerosis were measured. The selective absence of iNKT cells in Jα18−/−LDLR−/− mice led to an increase in plasma cholesterol levels in female mice. Transgenic Vα14tg/LDLR−/− mice with elevated numbers of iNKT cells had increased late atherosclerosis of the innominate artery, though absence of either iNKT cells or all NKT cells and other CD1d expressing cells had varying effects on atherosclerotic lesion burden in the ascending aortic arch and aortic root. These studies not only highlight the potential modulatory role played by NKT cells in atherosclerosis and lipid metabolism, but also raise the possibility that divergent roles may be played by iNKT and CD1d restricted cells such as type II NKT cells or other CD1d expressing cells.

Obesity is a chronic inflammatory state characterized by infiltration of adipose tissue by immune cell populations, including T lymphocytes. Natural killer T (NKT) cells, a specialized lymphocyte subset recognizing lipid antigens, can be pro- or anti-inflammatory. Their role in adipose inflammation continues to be inconclusive and contradictory. In obesity, the infiltration of tissues by invariant NKT (iNKT) cells is decreased. We therefore hypothesized that an excess iNKT cell complement might improve metabolic abnormalities in obesity. Vα14 transgenic (Vα14tg) mice, with increased iNKT cell numbers, on a LDL receptor-deficient (Ldlr-/-) background and control Ldlr-/- mice were placed on an obesogenic diet for 16 weeks. Vα14tg.Ldlr-/- mice gained 25% more weight and had increased adiposity than littermate controls. Transgenic mice also developed greater dyslipidemia, hyperinsulinemia, insulin resistance, and hepatic triglyceride accumulation. Increased macrophage Mac2 immunostaining and proinflammatory macrophage gene expression suggested worsened adipose inflammation. Concurrently, these mice had increased atherosclerotic lesion area and aortic inflammation. Thus, increasing the complement of iNKT cells surprisingly exacerbated the metabolic, inflammatory, and atherosclerotic features of obesity. These findings suggest that the reduction of iNKT cells normally observed in obesity may represent a physiological attempt to compensate for this inflammatory condition.

Apoprotein E (apoE) is a multifunctional protein. Its best-characterized function is as a ligand for low-density lipoprotein (LDL) receptor family members to mediate the clearance of apoB-containing atherogenic lipoproteins. Among its other functions, apoE is involved in cholesterol efflux, especially from cholesterol-loaded macrophage foam cells and other atherosclerosis-relevant cells, and in reverse cholesterol transport. Reverse cholesterol transport is a mechanism by which excess cellular cholesterol is transported via lipoproteins in the plasma to the liver where it can be excreted from the body in the feces. This process is thought to have a role in the attenuation of atherosclerosis. This review summarizes studies on the role of apoE in cellular cholesterol efflux and reverse cholesterol transport and discusses the identification of apoE mimetic peptides that may promote these pathways.

Inbred strains of mice with differing susceptibilities to atherosclerosis possess widely varying plasma HDL levels. Cholesterol absorption and lipoprotein formation were compared between atherosclerosis-susceptible, low-HDL C57BL6/J mice and atherosclerosis-resistant, high-HDL FVBN/J mice. [3H]cholesterol and triglyceride appeared in the plasma of FVB mice gavaged with cholesterol in olive oil at a much higher rate than in C57 mice. The plasma cholesterol was found almost entirely as HDL-cholesterol in both strains. Inhibition of lipoprotein catabolism with Tyloxapol revealed that the difference in the rate of [3H]cholesterol appearance in the plasma was due entirely to a greater rate of chylomicron secretion from the intestine of the FVB mice. Lipid absorption into the 2nd quarter of the small intestine is greater in the FVB mice and indicates that this region may contain the factors that give rise to the differences in absorption observed between the two mouse strains. Additionally, ad libitum feeding prior to cholesterol gavage accentuates the absorption rate differences compared with fasting. The resultant remodeling of the increased levels of chylomicron in the plasma may contribute to increased plasma HDL. Intestinal gene expression analysis reveals several genes that may play a role in these differences, including microsomal triglyceride transfer protein and ABCG8.

The synthesis of apoE by adipocytes has profound effects on adipose tissue lipid flux and gene expression. Using adipose tissue transplantation from wild-type (WT) to apoE knockout (EKO) mice, we show that adipose tissue also contributes to circulating apoE. Different from circulating apoE produced by bone marrow transplantation (BMT), however, adipose tissue-derived apoE does not correct hyperlipidemia or suppress atherosclerosis. ApoE secreted by macrophages has a more acidic isoform distribution, and it increases binding of reconstituted VLDL particles to hepatocytes and fibroblasts more effectively than apoE secreted by adipocytes. The incremental binding can be entirely accounted for by binding to the LDL receptor. After BMT into EKO hosts, plasma cholesterol and macrophage-derived apoE are largely within IDL/LDL- and HDL-sized particles. After adipose tissue transplantation, most cholesterol and adipocyte apoE remain in VLDL. After BMT, circulating apoE no longer demonstrates predominance of acidic isoforms compared with that circulating after fat transplantation. In conclusion, fat transplantation provides circulating apoE levels similar to those provided by bone marrow transplantation, but it does not suppress hyperlipidemia or atherosclerosis. A potential mechanism contributing to this difference is differential binding to cell surface lipoprotein receptors.

Plasma HDL levels are inversely associated with atherosclerosis. Inbred mouse strains differ in plasma HDL levels and susceptibility to atherosclerosis. Atherosclerosis-susceptible C57BL/6J mice possess plasma HDL levels 2-fold lower than atherosclerosis-resistant FVB/NJ mice. Polymorphisms have been previously identified between the two mouse strains in the major HDL apolipoproteins, ApoA-I and ApoA-II, which may affect their function on HDL. To begin to understand the HDL differences, we here report on a detailed comparison of the lipid-associated functions of the two mouse ApoA-I proteins. We demonstrate that these polymorphisms significantly alter the protein self-association properties, the ability of the proteins to clear lipid micelles from solution, and their binding affinity for mature mouse HDL. The changes in lipid binding do not appear to alter the ability of the protein to promote cholesterol efflux from cells or the formation of nascent HDL from primary hepatocytes. These apolipoprotein polymorphisms do not change the rate at which HDL protein or cholesterol are catabolized in vivo. Although the presence of the polymorphisms in ApoA-I alters important factors in HDL formation, the basis for the differences in the HDL plasma levels observed in the various mouse strains is more complex and requires additional investigation.

Obesity is a chronic inflammatory state characterized by altered levels of adipose tissue immune cell populations. Natural killer T (NKT) cells are CD1d restricted lymphocyte subsets that recognize lipid antigens whose level decreases in obese adipose tissue. However, studies in mice with deficiency or increased levels of NKT cells have yielded contradictory results, so the exact role of these cells in obesity and adipose tissue inflammation is not yet established. We previously showed that Ldlr−/− mice with excess invariant NKT (iNKT) cells demonstrate significant weight gain, adiposity, metabolic abnormalities, and atherosclerosis. The current study evaluates the effects of NKT cell deficiency on obesity, associated metabolic changes, and atherosclerosis in Jα18−/−Ldlr−/− (lacking iNKT cells) and Cd1d−/−Ldlr−/− (lacking invariant and type II NKT cells) mice, and control mice were fed an obesogenic diet (high fat, sucrose, cholesterol) for 16 weeks. Contrary to expectations, Ja18−/−Ldlr−/− mice gained significantly more weight than Ldlr−/− or Cd1d−/−Ldlr−/− mice, developed hypertriglyceridemia, and had worsened adipose tissue inflammation. All the mice developed insulin resistance and hepatic triglyceride accumulation. Ja18−/−Ldlr−/− mice also had increased atherosclerotic lesion area. Our findings suggest that iNKT cells exacerbates the metabolic, inflammatory, and atherosclerotic features of diet-induced obesity. Further work is required to unravel the paradox of an apparently similar effect of iNKT cell surplus and depletion on obesity.

Modifying apolipoprotein (apo) A-I mimetic peptides to include a proline-punctuated α-helical repeat increases their anti-inflammatory properties as well as allows better mimicry of full-length apoA-I function. This study compares the following mimetics, either acetylated or biotinylated (b): 4F (18mer) and 4F-proline-4F (37mer, Pro). b4F interacts with both mouse HDL (moHDL) and LDL in vitro. b4F in vivo plasma clearance kinetics are not affected by mouse HDL level. Administration of biotinylated peptides to mice demonstrates that b4F does not associate with lipoproteins smaller than LDL in vivo, though it does associate with fractions containing free hemoglobin (Hb). In contrast, bPro specifically interacts with HDL. b4F and bPro show opposite binding responses to HDL by surface plasmon resonance. Administration of acetylated Pro to apoE−/− mice significantly decreases plasma serum amyloid A levels, while acetylated 4F does not have this ability. In contrast to previous reports that inferred that 4F associates with HDL in vivo, we systematically examined this potential interaction and demonstrated that b4F does not interact with HDL in vivo but rather elutes with Hb-containing plasma fractions. bPro, however, specifically binds to moHDL in vivo. In addition, the number of amphipathic α-helices and their linker influences the anti-inflammatory effects of apoA-I mimetic peptides in vivo.

Hyperlipidemia is a major risk factor for developing atherosclerosis in humans, and epidemiological studies have correlated specific lipoprotein levels with cardiovascular disease risk. Murine models of atherosclerosis rely on the induction of hyperlipidemia for vascular lesions to form, but the pathogenic contributions attributed to different lipoprotein populations are not well defined. To address this issue, we analyzed over 300 LDL receptor (LDLR) deficient mice that have been fed a high-fat diet and for which a full lipoprotein profile and aortic root atherosclerosis values were assessed. Overall, aortic root atherosclerosis is best predicted by plasma VLDL cholesterol levels with less predictive value derived from either LDL or HDL cholesterol. Triglyceride levels are more atherogenic in female mice, especially immune competent females, and depletion of the adaptive immune system leads to a global reduction in plasma lipid levels and aortic root lesion size yet does not appear to alter the atherogenic potential of individual lipoprotein subspecies. In contrast, HDL-cholesterol is a better predictor of aortic root atherosclerosis in apoE-deficient mice. In summary, this large scale analysis of high-fat diet fed LDLR deficient mice highlight the relationship between different plasma lipid components, especially VLDL-cholesterol, and aortic root atherosclerosis.

We hypothesize that apolipoprotein A-I (apoA-I) mimetic peptides better mimicking the punctuated α-helical repeats of full-length apoA-I are more anti-inflammatory and anti-atherogenic. This study compares a monomeric apoA-I mimetic helix to three different tandem helix peptides in vitro: 4F (18 mer), 4F-proline-4F (37 mer, Pro), 4F-alanine-4F (37 mer, Ala), and 4F-KVEPLRA-4F [the human apoA-I 4/5 interhelical sequence (IHS), 43 mer]. All peptides cleared turbid lipid suspensions, with 4F being most effective. In contrast to lipid clearance, tandem peptides were more effective at remodeling mouse HDL. All four peptides displaced apoA-I and apoE from the HDL, leaving a larger particle containing apoA-II and peptide. Peptide-remodeled HDL particles show no deficit in ABCG1 cholesterol efflux despite the loss of the majority of apoA-I. Tandem peptides show greater ability to efflux cholesterol from lipid-loaded murine macrophages, compared with 4F. Although 4F inhibited oxidation of purified mouse LDL, the Ala tandem peptide increased oxidation. We compared several tandem 4F-based peptides with monomeric 4F in assays that correlated with suggested anti-inflammatory/anti-atherogenic pathways. Tandem 4F-based peptides, which better mimic full-length apoA-I, exceed monomeric 4F in HDL remodeling and cholesterol efflux but not LDL oxidation protection. In addition, apoA-I mimetic peptides may increase reverse cholesterol transport through both ABCA1 as well as ABCG1 pathways.

In this overview to a new thematic series on the immune system and atherogenesis, I provide a very brief summary of current conceptions of atherogenesis, of the innate and adaptive immune systems, and of the participation of the latter in atherogenesis, with particular emphasis on studies of the involvement of the immune system in atherosclerosis reported in the last 2 years. This is followed by a short outline of the eight reviews that will make up this thematic series.The overview is concluded with some caveats that should be considered in the analysis of atherosclerosis in experimental animals.

This study tests the hypothesis that autoantibodies to oxidation epitopes on oxidized LDL (OxLDL) promote the clearance of OxLDL from the plasma. Human LDL (hLDL) was injected into immune-competent apolipoprotein E-deficient (apoE−/−) mice and immune-deficient apoE−/−/recombination-activating gene-deficient mice that lack mature T and B cells and thus antibodies. There was a progressive decrease in human apoB-100 in the plasma in all mice, but the rate of clearance was not greater in the immune-competent mice than in the immune-deficient mice. Interestingly, oxidized phospholipid (OxPL) epitopes as detected by the EO6 antibody on the hLDL increased over time, suggesting de novo oxidation of the LDL or transfer of OxPL to the particles. Because the native LDL was not extensively modified, we also examined the clearance of copper OxLDL. Although the extensively OxLDL was cleared faster than the native LDL, there was no difference in the rate of clearance as a function of immune status. There appeared to be some transfer of OxPL to the endogenous murine LDL.Together, these results suggest that oxidation-specific antibodies do not participate to any great extent in the clearance of OxLDL from plasma. However, it is possible that such antibodies may bind to oxidation epitopes and modulate lesion formation within the vessel wall.

Serum amyloid A (SAA) circulates bound to HDL3 during the acute-phase response (APR), and recent evidence suggests that elevated levels of SAA may be a risk factor for cardiovascular disease. In this study, SAA-HDL was produced in vivo during the APR and without the APR by injection of an adenoviral vector expressing human SAA-1. SAA-HDL was also produced in vitro by incubating mouse HDL with recombinant mouse SAA and by SAA-expressing cultured hepatoma cells. Whether produced in vivo or in vitro, SAA-HDL floated at a density corresponding to that of human HDL3 (d 1.12 g/ml) separate from other apolipoproteins, including apolipoprotein A-I (apoA-I; d 1.10 g/ml) when either apoA-I or apolipoprotein E (apoE) was present. In the absence of both apoA-I and apoE, SAA was found in VLDL and LDL, with low levels in the HDL and the lipid-poor fractions suggesting that other HDL apolipoproteins are incapable of facilitating the formation of SAA-HDL.We conclude that SAA does not exist in plasma as a lipid-free protein. In the presence of HDL-associated apoA-I or apoE, SAA circulates as SAA-HDL with a density corresponding to that of human HDL3. In the absence of both apoA-I and apoE, SAA-HDL is not formed and SAA associates with any available lipoprotein.

Genetic variations of paraoxonase (PON) correlate with HDL cholesterol and apolipoprotein A-I (apoA-I), suggesting antiatherogenic properties. Atherosclerosis occurs naturally in humans and rabbits but not in mice. We compared variations of PON arylesterase activity (PON AEase, phenylacetate substrate) in humans, rabbits, and mice. In humans and rabbits, >95% of PON AEase is HDL associated. In mice, about 30% of PON AEase is lipid poor. In the absence of apoA-I in mice, total PON AEase is reduced and >60% is lipid poor. PON AEase level and distribution is restored in apoA-I−/− mice injected with adenoviruses encoding human apoA-I and in transgenic mice expressing human apoA-I at a steady-state level. Thus, while apoA-I is not required for the HDL association of PON AEase, induced variations in apoA-I correlate with changes in HDL-associated, but not lipid-poor, PON AEase. PON AEase associates only with apoA-I- or apoE-containing HDL but not VLDL. In the absence of both apoA-I and apoE, PON AEase is all-lipid-poor. PON AEase is displaced from HDL by ultracentrifugation and following incubation with serum amyloid A.Variations in the PON distribution between HDL and lipid-poor fractions may have important consequences in its antioxidant activity and in atherogenesis.

Self-medication with antimicrobial agents is a common form of self-care among patients globally with the prevalence and nature differing from country to country. Here we assessed the prevalence and predictors of antimicrobial self-medication in post-conflict northern Uganda. A cross-sectional study was carried out using structured interviews on 892 adult (≥18 years) participants. Information on drug name, prescriber, source, cost, quantity of drug obtained, and drug use was collected. Households were randomly selected using multistage cluster sampling method. One respondent who reported having an illness within three months in each household was recruited. In each household, information was obtained from only one adult individual. Data was analyzed using STATA at 95% level of significance. The study found that a high proportion (75.7%) of the respondents practiced antimicrobial self-medication. Fever, headache, lack of appetite and body weakness were the disease symptoms most treated through self-medication (30.3%). The commonly self-medicated antimicrobials were coartem (27.3%), amoxicillin (21.7%), metronidazole (12.3%), and cotrimoxazole (11.6%). Drug use among respondents was mainly initiated by self-prescription (46.5%) and drug shop attendants (57.6%). On average, participants obtained 13.9±8.8 (95%CI: 12.6-13.8) tablets/capsules of antimicrobial drugs from drug shops and drugs were used for an average of 3.7±2.8 days (95%CI: 3.3-3.5). Over half (68.2%) of the respondents would recommend self-medication to another sick person. A high proportion (76%) of respondents reported that antimicrobial self-medication had associated risks such as wastage of money (42.1%), drug resistance (33.2%), and masking symptoms of underlying disease (15.5%). Predictors of self-medication with antimicrobial agents included gender, drug knowledge, drug leaflets, advice from friends, previous experience, long waiting time, and distance to the health facility. Despite knowledge of associated risks, use of self-medication with antimicrobial drugs in management of disease symptoms is a common practice in post-conflict northern Uganda.

High density lipoprotein (HDL) cholesterol levels are inversely related to cardiovascular disease risk and associated with a reduced risk of type 2 diabetes. Apolipoprotein A-I (apoA-I; major HDL protein) mimetics have been reported to reduce atherosclerosis and decrease adiposity. This study investigated the effect of L4F mimetic peptide and apoA-I overexpression on weight gain, insulin resistance, and atherosclerosis in an LDL receptor deficient (Ldlr-/-) model fed a high fat high sucrose with cholesterol (HFHSC) diet.Studies in differentiated 3T3-L1 adipocytes tested whether L4F could inhibit palmitate-induced adipocyte inflammation. In vivo studies used male Ldlr-/- mice fed a HFHSC diet for 12 weeks and were injected daily with L4F (100 µg/mouse) subcutaneously during the last 8 weeks. Wild-type and apoA-I overexpressing Ldlr-/- mice were fed HFHSC diet for 16 weeks.Neither L4F administration nor apoA-I overexpression affected weight gain, total plasma cholesterol or triglycerides in our studies. While pre-treatment of 3T3-L1 adipocytes with either L4F or HDL abolished palmitate-induced cytokine expression in vitro, L4F treatment did not affect circulating or adipose tissue inflammatory markers in vivo. Neither L4F administration nor apoA-I overexpression affected glucose tolerance. ApoA-I overexpression significantly reduced atherosclerotic lesion size, yet L4F treatment did not affect atherosclerosis.Our results suggest that neither L4F (100 µg/day/mouse) nor apoA-I overexpression affects adiposity or insulin resistance in this model. We also were unable to confirm a reduction in atherosclerosis with L4F in our particular model. Further studies on the effect of apoA-I mimetics on atherosclerosis and insulin resistance in a variety of dietary contexts are warranted.

Serum amyloid A (SAA) is an acute phase protein of unknown function that is involved in systemic amyloidosis and may also be involved in atherogenesis. The precise role of SAA in these processes has not been established. SAA circulates in plasma bound to high density lipoprotein-3 (HDL3). The pathway for the production of SAA-containing HDL is not known. To test whether apolipoprotein (apo)A-I-HDL is required in the production of SAA-HDL, we analyzed the lipopolysaccharide (LPS)-induced changes in apoA-I+/+ and apoA-I–/– mice. In apoA-I+/+ mice, after injection of LPS, remodeling of HDL occurred: total cholesterol increased and apoA-I decreased slightly and shifted to lighter density. Dense (density of HDL3) but large (size of HDL2) SAA-containing particles were formed. Upon fast phase liquid chromatography fractionation of plasma, >90% of SAA eluted with HDL that was enriched in cholesterol and phospholipid and shifted “leftward” to larger particles. Non-denaturing immunoprecipitation with anti-mouse apoA-I precipitated all of the apoA-I but not all of the SAA, confirming the presence of SAA-HDL devoid of apoA-I. In the apoA-I–/– mice, which normally have very low plasma lipid levels, LPS injection resulted in significantly increased total and HDL cholesterol. Greater than 90% of the SAA was lipid associated and was found on dense but large, spherical HDL particles essentially devoid of other apolipoproteins. We conclude that serum amyloid A (SAA) is able to sequester lipid, forming dense but large HDL particles with or without apoA-I or other apolipoproteins. The capacity to isolate lipoprotein particles containing SAA as the predominant or only apolipoprotein provides an important system to further explore the biological function of SAA.—Cabana, V. G., C. A. Reardon, B. Wei, J. R. Lukens, and G. S. Getz. SAA-only HDL formed during the acute phase response in apoA-I+/+ and apoA-I–/– mice. J. Lipid Res. 1999. 40: 1090–1103.