Background: Platelet-rich plasma (PRP) contains pro-angiogenic growth factors including vascular endothelial growth factor (VEGF). Angiogenesis is a necessary component of wound healing in instances of diabetic foot ulcers (DFU). PRP composition varies depending on methods and donor health status. Our group has developed an improved PRP protocol for diabetes treatment. The aims of this study were to examine the levels of the pro-angiogenic factor VEGF in these patient populations with and without diabetes.Methods: PRP was prepared using 24 mL of whole blood from 13 diabetic and 10 non-diabetic patients registered at Klinik Hayandra. Whole blood in sodium citrate tubes were centrifuged at 1,000 rpm for 5 minutes followed by plasma separation. Plasma samples were centrifuged at 3,000 rpm for 5 minutes.Upper platelet-poor plasma layers were discarded, leaving 5 mL of concentrated platelet containing plasma (PRP). Concentrated plasma samples were mixed, aliquoted, stored at −86 ℃, and pooled for platelet count, VEGF, and total protein analyses. Platelet counting was also performed using fresh whole blood and PRP to measure changes following PRP preparation.Results: Diabetic donors had higher whole blood platelet counts than non-diabetic donors, but this difference was not statistically significant. An average increase of more than 250% in platelet number after PRP preparation using our method was noted in both groups. Freezing-thawing samples at −86 ℃ lysed more than 90% of PRP platelets regardless of diabetes status. Diabetic PRP had lower mean total protein and higher VEGF concentrations. Lysed platelets from diabetic donors released more VEGF than those from non-diabetic donors.Conclusions: PRP from diabetic donors had higher VEGF content making autologous PRP application a promising treatment for DFU. However, this should be investigated another appropriate clinical trial.

Pathogenesis of type 2 Diabetes Mellitus (DM) is often associated with chronic low-grade inflammation. This kind of inflammation is characterized by an increased level of pro-inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin (IL)-6 and IL-1β. From an immunological point of view, an inflammatory response is always followed by an anti-inflammatory response as negative feedback to avoid excessive tissue damages. Regulatory T cells are a subset of cluster of differentiation (CD)4+ T cells that have the function to maintain peripheral tolerance and suppress immune response. This review would discuss the impaired function of regulatory T cells in type 2 DM. DM is a group of metabolic diseases characterized by hyperglycemia due to a defect of insulin secretion or a combination of insulin resistance and relative insulin deficiency. Chronic low-grade inflammation has been known as a key factor in the development of insulin resistance. Regulatory T cells (Treg cells) action through contact and non-contact inhibition could suppress inflammatory response in innate and adaptive immune systems. In type 2 DM, the proportion and function of CD4+CD25+Foxp3+ and CD4+CD25+ regulatory T cell decreases due to the reduced number of Treg cells and the Treg cells depletion contributes to metabolic conditions such as insulin resistance. Moreover, Treg cells are more susceptible to apoptosis, the ability of Treg cells to produce anti-inflammatory cytokines such as transforming growth factor β (TGF-β) and IL-10 decreases, and there is an imbalance between the proportion of Th1/Th17 cells and Treg cells. This inadequate anti-inflammatory response gives rise to the chronic low-grade inflammatory condition in type 2 DM.Keywords: type 2 diabetes mellitus, inflammation, regulatory T cell

Type 2 diabetes mellitus (T2DM) is associated with chronic low-grade inflammation, which is marked by the dysregulation of innate and adaptive immune responses. Therefore, reducing inflammation, possibly through an immunoregulatory agent, may play a role in T2DM treatment. Butyrate is the most potent short-chain fatty acid (SCFA), and it exerts anti-inflammatory properties by inhibiting histone deacetylase activity. As an immunoregulatory agent, sodium butyrate can inhibit nuclear factor kB (NF-kB) activation and reduce the production of pro-inflammatory cytokines in immune cells. The aim of the study was to measure the level of plasma butyrate in poorly controlled T2DM and normoglycemic participants and to compare the response of peripheral blood mononuclear cells (PBMCs) to sodium butyrate treatment between the groups by measuring production of the following cytokines: tumor necrosis factor (TNF)-α, interleukin (IL)-6, interferon (IFN)-γ, IL-13, and IL-10. The in vitro study examined the PBMCs of 15 participants with poorly controlled T2DM and 15 normoglycemic participants. PBMCs were cultured with the following stimulations for two days at a temperature of 37°C and 5% CO2: 100 ng/mL lipopolysaccharide (LPS), 1 mM sodium butyrate, or a combination of 100 ng/mL LPS and 1 mM sodium butyrate. Plasma butyrate was measured using gas chromatography-mass spectrometry, and cytokines from culture supernatant were analyzed using magnetic beads multiplex assay. Plasma butyrate levels in participants with poorly controlled T2DM did not significantly differ from those in normoglycemic participants (p = 0.105). Compared to treatment with an LPS-stimulated PBMC culture, treatment with 1 mM sodium butyrate reduced the levels of TNF-α (p < 0.039) and IFN-γ (p < 0.038) in normoglycemic participants. The same general trend was seen in PBMC from participants with poorly controlled T2DM, but higher variability appeared to preclude statistical significance. These data suggest that butyrate may modulate inflammatory cytokine production in human PBMCs, but more research is needed to determine if butyrate is anti-inflammatory in poorly controlled T2DM.

Tumor antigen alpha-fetoprotein (AFP) can promote immune tolerance toward tumor cells by inducing regulatory functions of the immune system. The purpose of this study was to characterize the effects of AFP on dendritic cells (DC) in their antitumor immune response stimulation and subsequent immune tolerance toward tumor cells. Monocytes were cultured in medium with GM-CSF and IL-4 and incubated for 6 days to generate immature DC (imDC). AFP was added into the treatment group at the beginning of the monocyte-derived DC culture. Mature DC (mDC) were generated by an addition of lipopolysaccharide (LPS) into the culture and incubation for another 48 hours. We observed that the addition of AFP in early DC culture was able to decrease the binding of LPS onto imDC surface, which lowered the strength of stimulation and consequently the maturity of DC. As expected, the expression of mDC surface markers, which are known to be crucial in effector cell proliferation and activation such as HLA-DR, CD40, CD80, CD83, and CD86, were confirmed to be reduced on AFP-exposed DC. DC potential in stimulating proliferation of CD4+ T cells was decreased, in line with the reduction of surface markers’ expression. Additionally, an increased secretion of cytokine TGF-β by DC was observed. In summary, AFP inhibited the effector immune responses while increasing the regulatory immune responses in DC. This might lead to tolerance toward antigens and tumor cell survival, such as in cases of hepatocellular carcinoma patients with high levels of AFP.

Endometriosis is a gynecologic disease in women that can cause infertility and chronic pelvic pain with a relatively high recurrence rate. This research was to prove the effects of gallic acid and its derivatives on inflammatory regulation of endometriosis primary cultures in terms of NF-kB mRNA expression and IL-6 secretions. Endometriosis cells are derived from endometriosis tissue of patients undergoing laparoscopy, isolated enzymatically and cultured primarily. The culture cells were treated with gallic acid, heptyl and octyl gallate at doses (25.6 μg/ml, 51.2 μg/ml and 102.4 μg/ml) for 48 h, then induced with 500 ng/ml LPS for 24 h. Inflammatory regulation was assessed from NF-kB mRNA expression with qRT-PCR and IL-6 secretion levels with ELISA. Gallic acid and its derivatives showed a decrease in the relative expression of NF-kB, significantly at dose 102,4 μg/ml. IL-6 although not statistically significant. The result indicated that gallic acid and its derivatives have a potential as anti-inflammatory effect. Gallic acid and its derivative compounds have an effect on decreased relative expression of mRNA NF-kB and IL-6.