A preliminary study to evaluate the impact of pharmaceutical care services on clinical outcome and medication adherence in type 2 diabetes mellitus patients from Ethiopian perspective.

Background: The role of clinical pharmacist in hospital settings of Ethiopia is still new and infant. Objective: To evaluate the impact of pharmaceutical care on clinical outcome and medication adherence in type 2 diabetes mellitus (T2DM) patients. Methods: A single cantered, pre-post interventional study design was carried out by enrolling 100 uncontrolled T2DM patients from March 1-August 30, 2020. The intervention package included assessment of pharmacological and non-pharmacological needs, counselling patients in person at the clinic, and providing educational materials. Results: Of the 100 patients initially enrolled, 87(87%) completed the follow-up and included in the final data analysis. The intervention showed a decrease in average FBG, systolic blood pressure (SBP), low density lipoprotein cholesterol (LDL-C) by 47.3 mg/dL, 22.6mmHg and 31.4mg/dL, while high density lipoprotein cholesterol (HDL-C) and estimated glomerular filtration rate (eGFR) exhibited significant increase by 13.4 mg/dL and 11.5 ml/min/1.73m2 respectively (p<0.0001). In addition, diastolic blood pressure, lipid values, kidney function parameters, and liver function parameters showed significant decrease in post intervention compared to pre-intervention (p<0.05). Medication adherence of the patients increased significantly at 6-month follow-up (p<0.001). Conclusion: These results also suggest the benefits of integrating clinical pharmacist services in multidisciplinary healthcare teams and diabetes management in Ethiopia.

Gangliosides drive the tumor infiltration and function of myeloid-derived suppressor cells.

Although it is now widely appreciated that antitumor immunity is critical to impede tumor growth and progression, there remain significant gaps in knowledge about the mechanisms used by tumors to escape immune control. In tumor cells, we hypothesized that one mechanism of immune escape used by tumors involves the synthesis and extracellular shedding of gangliosides, a class of biologically active cell surface glycosphingolipids with known immunosuppressive properties. In this study, we report that tumor cells engineered to be ganglioside deficient exhibit impaired tumorigenicity, supporting a link between ganglioside-dependent immune escape and tumor outgrowth. Notably, we documented a dramatic reduction in the numbers and function of tumor-infiltrating myeloid-derived suppressor cells (MDSC) in ganglioside-deficient tumors, in contrast with the large MDSC infiltrates seen in ganglioside-rich littermate control tumors. Transient ganglioside reconstitution of the tumor cell inoculum was sufficient to increase MDSC infiltration, supporting a direct connection between ganglioside production by tumor cells and the recruitment of immunosuppressive MDSC into the tumor microenvironment. Our results reveal a novel mechanism of immune escape that supports tumor growth, with broad implications given that many human tumors produce and shed high levels of gangliosides.

Tumor gangliosides accelerate murine tumor angiogenesis.

Tumor cells shed gangliosides and populate their microenvironment with these biologically active membrane glycosphingolipids. In vitro, ganglioside enrichment amplifies receptor tyrosine kinase signaling and activation of vascular endothelial cells. However, a long-standing question is whether in the actual microenvironment of a neoplasm, in vivo, tumor cell ganglioside shedding stimulates angiogenesis. Here we tested the hypothesis that tumor gangliosides have a critical proangiogenic role in vivo using novel murine tumor cells, GM3synthase/GM2synthase double knockout (DKO) cells, genetically completely incapable of ganglioside synthesis and impaired in tumor growth versus wild-type (WT) ganglioside-rich cells. We studied angiogenesis during tumor formation by these ganglioside-depleted cells, quantifying vessel formation, angiogenic factor production/release, and consequences of reconstitution with purified WT gangliosides. DKO cells formed virtually avascular tumors, much smaller than ganglioside-rich WT tumors and displaying a striking paucity of blood vessels, despite levels of VEGF and other angiogenic factors that were similar to those of WT cells. Transient enrichment of the ganglioside milieu of the DKO cell inoculum by adding purified WT gangliosides partially restored angiogenesis and tumor growth. We conclude that tumor gangliosides trigger robust angiogenesis important for tumor growth. Our findings suggest strategies to eliminate their synthesis and shedding by tumor cells should be pursued.

Ganglioside inhibition of CD8+ T cell cytotoxicity: interference with lytic granule trafficking and exocytosis.

Granule exocytosis-mediated cytotoxicity by CD8(+) CTL plays a crucial role in adaptive immunity to tumors and to intracellular pathogens. This T cell effector function has been shown to be defective in various murine tumor models and in human cancer. However, factors and their mechanisms that cause inhibition of CD8(+) T cell lytic function in tumor-bearing hosts remain to be fully defined. We postulate that gangliosides, highly expressed on tumor cell membranes, actively shed into the tumor microenvironment, and having well-established immunosuppressive properties, may be such a factor. We exposed primary mouse CD8(+) CTL to gangliosides derived from three sources (tumors and normal brain). This significantly inhibited cytotoxicity-mediated by granule exocytosis, that is, cytotoxicity of alloantigen-specific and polyclonal CD8(+) CTL in vitro. These molecules did not interfere with the interaction of CD8(+) T cells with their cognate targets. Rather, they inhibited lytic granule release in response both to TCR engagement and to stimuli that induce granule release in a nonpolarized manner. At the subcellular level, confocal microscopic imaging identified inhibition of polarization of lytic granules to the immunological synapse upon target cell recognition. Thus, tumor-shed gangliosides suppress lytic activity of CD8(+) T cells by a novel mechanism, that is, inhibition of trafficking of lytic granules in response to TCR engagement, as well as by interfering with the process of granule exocytosis in CD8(+) T cells.

Loss of Arnt (Hif1ß) in mouse epidermis triggers dermal angiogenesis, blood vessel dilation and clotting defects.

Targeted ablation of Aryl hydrocarbon receptor nuclear translocator (Arnt) in the mouse epidermis results in severe abnormalities in dermal vasculature reminiscent of petechia induced in human skin by anticoagulants or certain genetic disorders. Lack of Arnt leads to downregulation of Egln3/Phd3 hydroxylase and concomitant hypoxia-independent stabilization of hypoxia-induced factor 1α (Hif1α) along with compensatory induction of Arnt2. Ectopic induction of Arnt2 results in its heterodimerization with stabilized Hif1α and is associated with activation of genes coding for secreted proteins implicated in control of angiogenesis, coagulation, vasodilation and blood vessel permeability such as S100a8/S100a9, S100a10, Serpine1, Defb3, Socs3, Cxcl1 and Thbd. Since ARNT and ARNT2 heterodimers with HIF1α are known to have different (yet overlapping) downstream targets our findings suggest that loss of Arnt in the epidermis activates an aberrant paracrine regulatory pathway responsible for dermal vascular phenotype in K14-Arnt KO mice. This assumption is supported by a significant decline of von Willebrand factor in dermal vasculature of these mice where Arnt level remains normal. Given the essential role of ARNT in the adaptive response to environmental stress and striking similarity between skin vascular phenotype in K14-Arnt KO mice and specific vascular features of tumour stroma and psoriatic skin, we believe that further characterization of Arnt-dependent epidermal-dermal signalling may provide insight into the role of macro- and micro-environmental factors in control of skin vasculature and in pathogenesis of environmentally modulated skin disorders.

Peptides mimicking GD2 ganglioside elicit cellular, humoral and tumor-protective immune responses in mice.

INTRODUCTION: Because of its restricted distribution in normal tissues and its high expression on tumors of neuroectodermal origin, GD2 ganglioside is an excellent target for active specific immunotherapy. However, GD2 usually elicits low-titered IgM and no IgG or cellular immune responses, limiting its usefulness as a vaccine for cancer patients. We have previously shown that anti-idiotypic monoclonal antibody mimics of GD2 can induce antigen-specific humoral and cellular immunity in mice, but inhibition of tumor growth by the mimics could not be detected. METHODS AND RESULTS: Here, we isolated two peptides from phage display peptide libraries by panning with GD2-specific mAb ME361. The peptides inhibited binding of the mAb to GD2. When coupled to keyhole limpet hemocyanin (KLH) or presented as multiantigenic peptides in QS21 adjuvant, the peptides induced in mice antibodies binding specifically to GD2 and delayed-type hypersensitive lymphocytes reactive specifically with GD2-positive D142.34 mouse melanoma cells. Induction of delayed-type hypersensitivity (DTH) reaction was dependent on CD4-positive lymphocytes. The immunity elicited by the peptides significantly inhibited growth of GD2-positive melanoma cells in mice. CONCLUSION: Our study suggests that immunization with peptides mimicking GD2 ganglioside inhibits tumor growth through antibody and/or CD4-positive T cell-mediated mechanisms. Cytolytic T lymphocytes most likely do not play a role. Our results provide the basis for structural analysis of carbohydrate mimicry by peptides.

Synovial MMP-3 and TIMP-1 levels and their correlation with cytokine expression in canine rheumatoid arthritis.

The purpose of this study was to investigate whether synovial fluid levels of matrix metalloprotease-3 (MMP-3) and tissue inhibitor of metalloprotease-1 (TIMP-1) are specifically elevated in canine rheumatoid arthritis (CRA) compared to osteoarthritic joint disorders and if these markers are correlated with a specific pattern of cytokine mRNA expression. Synovial fluid samples of 17 dogs with CRA were analysed for MMP-3 and TIMP-1 by two canine sandwich ELISA (enzyme-linked immunosorbent assay) systems. The synovial mRNA content of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), transforming growth factor-beta (TGF-beta), and interferon-gamma (IFN-gamma) was determined by RT-PCR (reverse transcription-polymerase chain reaction). Dogs with osteoarthritis (n = 50) caused by anterior cruciate ligament rupture (ACLR) were used as controls. A significant rise of MMP-3 was found in the synovial fluid of joints with CRA that could not be balanced by sufficient amounts of TIMP-1. The 30-fold surplus of MMP-3 over TIMP-1 was strongly correlated with the synovial mRNA content of IL-1, IL-12 and TGF-beta. Our results point to the potential use of the synovial levels of MMP-3 as a marker for RA in dogs.