Counteracting diabetes-induced adipose tissue derived-stromal cell senescence.

Adipose tissue stromal cells (ADSCs) are prone to functional decline and senescence during metabolic disturbances. In diabetes mellitus (DM), the pathogenic microenvironment induces oxidative stress causing ADSCs to senesce. The senescence associated secretory phenotype (SASP) in turn drives disease progression. The pathogenesis of DM is thus both a cause and consequence of senescence. Therapeutically preventing the onset of senescence in ADSCs may play a significant role in preventing disease progression and directly impact the onset of comorbidities. The purpose of this study was to establish an in vitro model that mimic the DM micro-environment to use as a screening tool to assess the therapeutic efficacy of preventative and restorative agents. Exposing ADSCs (

Pregnancy and diabetic ketoacidosis: fetal jeopardy and windows of opportunity.

Background: Diabetic ketoacidosis (DKA) during pregnancy poses significant risks to both the mother and fetus, with an increased risk of fetal demise. Although more prevalent in women with Type I diabetes (T1D); those with Type 2 diabetes (T2D) and gestational diabetes mellitus (GDM) can also develop DKA. A lack of information about DKA during pregnancy exists worldwide, including in South Africa. Objective: This study examined the characteristics and outcomes associated with DKA during pregnancy. Methods: The study took place between 1 April 2020 and 1 October 2022. Pregnant women with DKA, admitted to Tygerberg Hospital's Obstetric Critical Care Unit (OCCU) were included. Maternal characteristics, precipitants of DKA, adverse events during treatment, and maternal-fetal outcomes were examined. Results: There were 54 episodes of DKA among 47 women. Most DKA's were mild and occurred in the third trimester. Pregestational diabetes dominated (31/47; 60%), with 47% having T1D and 94% requiring insulin. Seven women (7/47, 15%; T2D:6, T1D:1) had two episodes of DKA during the same pregnancy. Most women (32/47; 68%) were either overweight or obese. Yet, despite the T2D phenotype, biomarkers indicated that auto-immune diabetes was prevalent among women without any prior history of T1D (6/21; 29%). Twelve women (26%) developed gestational hypertension during pregnancy, and 17 (36%) pre-eclampsia. Precipitating causes of DKA included infection (14/54; 26%), insulin disruption (14/54; 26%) and betamethasone administration (10/54; 19%). More than half of the episodes of DKA involved hypokalemia (35/54, 65%) that was associated with fetal death (P=0.042) and hypoglycemia (28/54, 52%). Preterm birth (<37 weeks' gestation) occurred in 85% of women. No maternal deaths were recorded. A high fetal mortality rate (13/47; 28%) that included 11 spontaneous intrauterine deaths and two medical terminations, was observed. Conclusion: Women with DKA have a high risk of fetal mortality as well as undiagnosed auto-immune diabetes. There is a strong link between maternal hypokalemia and fetal loss, suggesting an opportunity to address management gaps in pregnant women with DKA.

Nutritional deficiency in South African adults scheduled for bariatric surgery.

Background: Globally, there is a rising trend in obesity, known to increase morbidity and mortality. Metabolic surgery and adequate weight loss decrease mortality but may worsen pre-existing nutrient deficiencies. Most data on pre-existing nutritional deficiencies in the population undergoing metabolic surgery is from the developed world, where an extensive micronutrient assessment is achievable. In resource-constrained environments, the cost of a comprehensive micronutrient assessment must be weighed against the prevalence of nutritional deficiencies and the potential harm if one or more nutritional deficiencies are missed. Methods: This cross-sectional study investigated the prevalence of micronutrient and vitamin deficiencies in participants scheduled to undergo metabolic surgery in Cape Town, South Africa, a low-middle income country. 157 participants were selected and 154 reported on; who underwent a baseline evaluation from 12 July 2017 to 19 July 2020. Laboratory measurements were conducted, including vitamin B12 (Vit B12), 25-hydroxy vitamin D (25(OH)D), folate, parathyroid hormone (PTH), thyroid-stimulating hormone (TSH), thyroxine (T4), ferritin, glycated haemoglobin (HbA1c), magnesium, phosphate, albumin, iron, and calcium. Results: Participants were predominantly female, aged 45 years (37-51), with a preoperative BMI of 50.4 kg/m2 (44.6-56.5). A total of 64 individuals had Type 2 diabetes mellitus (T2D), with 28 undiagnosed cases at study entry (18% of study population). 25(OH)D deficiency was most prevalent (57%), followed by iron deficiency (44%), and folate deficiency (18%). Other deficiencies (vitamin B12, calcium, magnesium, phosphate) were rarely encountered and affected ≤1% of participants. Folate and 25(OH)D deficiency were related to obesity classification, with a higher prevalence in participants with a BMI ≥40 kg/m2 (p <0.01). Conclusion: A higher prevalence of some micronutrient deficiencies was noted compared with data from similar populations in the developed world. The minimum baseline/preoperative nutrient evaluation in such populations should include 25(OH)D, iron studies, and folate. Additionally, screening for T2D is recommended. Future efforts should seek to collate broader patient data on a national scale and include longitudinal surveillance after surgery. This may provide a more holistic picture of the relationship between obesity, metabolic surgery and micronutrient status inform more appropriate evidence-based care.

Early postpartum HbA1c after hyperglycemia first detected in pregnancy-Imperfect but not without value.

BACKGROUND: South African women of childbearing age are disproportionally affected by obesity and at significant risk of Type 2 Diabetes Mellitus (T2DM). Unless pregnant, they do not readily undergo screening for T2DM. With a local focus on improved antenatal care, hyperglycemia is often first detected in pregnancy (HFDP). This may erroneously be attributed to Gestational Diabetes Mellitus (GDM) in all without considering T2DM. Glucose evaluation following pregnancy is essential for early detection and management of women with T2DM in whom persistent hyperglycemia is to be expected. Conventional testing with an oral glucose tolerance test (OGTT) is cumbersome, prompting investigation for alternate solutions. AIM: To compare the diagnostic performance of HbA1c to the current gold standard OGTT in women with HFDP 4-12 weeks post-delivery. METHODS: Glucose homeostasis was assessed with OGTT and HbA1c in 167 women with HFDP, 4-12 weeks after delivery. Glucose status was based on American Diabetes Association criteria. RESULTS: Glucose homeostasis was assessed at 10 weeks (IQR 7-12) after delivery. Of the 167 participants, 52 (31%) had hyperglycemia, which was comprised of 34 (20%) prediabetes and 18 (11%) T2DM. Twelve women in the prediabetes subgroup had diagnostic fasting plasma glucose (FPG) and 2-hour plasma glucose (2hPG), but in two-thirds of the patients (22/34) only one time point proved diagnostic. The FPGs and the 2hPGs of six women with HbA1c-based T2DM were both within the prediabetes diagnostic range. According to the HbA1c measurements, 85% of 52 participants with gold standard OGTT defined hyperglycemia (prediabetes and T2DM) as well as 15 of 18 women with postpartum persistent T2DM were correctly classified. According to FPG, 15 women with persistent hyperglycemia would have been missed (11 with prediabetes and four with T2DM; 29%). When compared to an OGTT, a single HbA1c of 6.5% (48mmol/mol) postpartum demonstrated a sensitivity of 83% and specificity of 97% for the identification of T2DM. CONCLUSION: HbA1c may improve access to postpartum testing in overburdened clinical settings where the required standards of OGTT cannot be guaranteed. HbA1c is a valuable test to detect women who will benefit most from early intervention but cannot unequivocally replace OGTT.

Immunology of chronic low-grade inflammation: relationship with metabolic function.

Inflammation is part of the body's innate immune response and is an essential process that not only defends against harmful bacteria and pathogens but also plays a key role in the maintenance and repair of tissues. Under pathological conditions, there is bilateral crosstalk between immune regulation and aberrant metabolism resulting in persistent inflammation in the absence of infection. This phenomenon is referred to as sterile metabolic inflammation (metainflammation) and occurs if the initiating stimulus is not removed or if the resolution process is disrupted. Disruption of this tightly regulated immune response and its failure to resolve as is evident in metabolic disorders is not only associated with disease progression but also leads to immune senescence and should not be neglected in the clinical management of patients. This review gives an overview of the mechanisms underlying chronic metabolic inflammation, the aberrant metabolic activation of innate immune cells (neutrophils, macrophages, mast cells, dendritic cells), and its role in disease progression using obesity-diabetes as a prime example. Addressing the underlying subclinical metabolic inflammation in addition to achieving glucose control may contribute significantly towards therapeutic interventions aimed at preventing the onset of co-morbidities in diabetic patients.

Transabdominal cerclage during pregnancy: A retrospective single operator series over a quarter century.

OBJECTIVE: To describe the pregnancy outcomes and complications observed in a series of cases of transabdominal cerclage (TAC), which is reserved for highly selected women with recurrent mid-trimester pregnancy loss, due to cervical insufficiency. METHODS: A retrospective audit covering 25 years (January 1, 1997 to December 31, 2021) was performed at the Obstetric Special Care division, Tygerberg Academic Hospital in Cape Town, South Africa. All 118 pregnancies from 94 procedures, operated and managed by the principal author were included for descriptive analysis. RESULTS: Eighty-four (91.3%) of the 92 first pregnancies after first insertion had successful outcomes. All second and third pregnancies (24/24; 100%) were successful. Eight pregnancies did not achieve viability, two women (2/8) did however achieve a successful pregnancy after a subsequent repeat TAC procedure. For the viable pregnancies (110/118), the median gestational age at delivery was 37 weeks (range 28-39 weeks). The median intraoperative blood loss during cerclage insertion was 100 ml (range 25-750 ml). CONCLUSION: In experienced hands, TAC during pregnancy is a safe and effective operation, when other less invasive procedures have failed.

A regenerative approach to the pharmacological management of hard-to-heal wounds.

A wound is considered hard-to-heal when, despite the appropriate clinical analysis and intervention, the wound area reduces by less than a third at four weeks and complete healing fails to occur within 12 weeks. The most prevalent hard-to-heal wounds are associated with underlying metabolic diseases or vascular insufficiency and include arterial, venous, pressure and diabetic foot ulcers. Their common features include an abnormal immune response and extended inflammatory phase, a subdued proliferation phase due to cellular insufficiencies and finally an almost non-existent remodeling phase. Advances in wound care technology, tested in both pre-clinical models and clinical trials, have paved the way for improved treatment options, focused on regeneration. These interventions have been shown to limit the extent of ongoing inflammatory damage, decrease bacterial load, promote angiogenesis and deposition of granulation tissue, and stimulate keratinocyte migration thereby promoting re-epithelialization in these wounds. The current review discusses these hard-to-heal wounds in the context of their underlying pathology and potential of advanced treatment options, which if applied promptly as a standard of care, could reduce morbidity, promote quality of life, and alleviate the burden on a strained health system.

The paracrine effects of adipocytes on lipid metabolism in doxorubicin-treated triple negative breast cancer cells.

Adipocytes in the breast tumour microenvironment promotes acquired treatment resistance. We used an in vitro adipocyte-conditioned media approach to investigate the direct paracrine effects of adipocyte secretory factors on MDA-MB-231 breast cancer cells treated with doxorubicin to clarify the underlying treatment resistance mechanisms. Cell-viability assays, and Western blots were performed to determine alterations in apoptotic, proliferation and lipid metabolism protein markers. Free fatty acids (FFA) and inflammatory markers in the collected treatment-conditioned media were also quantified. Adipocyte secretory factors increased the cell-viability of doxorubicin-treated cells (p < 0.0001), which did not correspond to apoptosis or proliferation pathways. Adipocyte secretory factors increased the protein expression of hormone-sensitive lipase (p < 0.05) in doxorubicin-treated cells. Adipocyte secretory factors increased the utilization of leptin (p < 0.05) and MCP-1 (p < 0.01) proteins and possibly inhibited release of linoleic acid by doxorubicin-treated cells (treatment-conditioned media FFA profiles). Adipocyte secretory factors induced doxorubicin treatment resistance, by increasing the utilization of inflammatory mediators and inhibiting the release of FFA by doxorubicin-treated cells. This further promotes inflammation and lipid metabolic reprogramming (lipid storage) in the tumour microenvironment, which breast cancer cells use to evade the toxic effects induced by doxorubicin and confers to acquired treatment resistance.

The Effect of N-Acetylcysteine and Ascorbic Acid-2-Phosphate Supplementation on Mesenchymal Stem Cell Function in B6.C-Lepob/J Type 2 Diabetic Mice.

Diabetes is a complex multifactorial disorder associated with hyperglycemia, oxidative stress, and inflammation. The pathological microenvironment impairs mesenchymal stem cell (MSC) viability and dysregulates their proregenerative and immune-modulatory function causing maladaptive tissue damage. Targeting stem cells to protect them against impairment could thus delay the onset of complications and enhance the quality of life in diabetes mellitus patients. The aim of this study was to investigate the efficacy of N-acetylcysteine (NAC) and ascorbic-acid-2-phosphate (AAP) oral supplementation as preventative measure against MSC impairment. Healthy wild-type control (C57BL/6J) (male, n = 24) and obese diabetic (B6.C-Lepob/J) (ob/ob) (male, n = 24) mice received either placebo or antioxidant (NAC/AAP) supplementation for a period of 6 weeks. Metabolic parameters (weight and blood glucose) and the oxidative status (serum total serum antioxidant capacity, malondialdehyde) of animals were assessed. At the end of the 6-week supplementation period, bone marrow MSCs were isolated and their functionality (growth rate, viability, adipogenesis, and osteogenesis) assessed ex vivo. Real time quantitative polymerase chain reaction microarray analysis was also performed to assess the expression of 84 genes related to oxidative stress in MSCs. Despite no change in the metabolic profile, NAC/AAP supplementation improved the antioxidant status of diabetic animals and reduced lipid peroxidation, which is indicative of cellular damage. NAC/AAP also improved the population doubling time of MSCs (first 6-days postisolation) and significantly downregulated the expression of two genes (Nox1 and Rag2) associated with oxidative stress compared to placebo treatment. Taken together, this study has shown reduced oxidative stress and improvements in MSC function following in vivo antioxidant supplementation in healthy control and type 2 diabetic mice.

Histology Scoring System for Murine Cutaneous Wounds.

Monitoring wound progression over time is a critical aspect for studies focused on in-depth molecular analysis or on evaluating the efficacy of potential novel therapies. Histopathological analysis of wound biopsies can provide significant insight into healing dynamics, yet there is no standardized and reproducible scoring system currently available. The purpose of this study was to develop and statistically validate a scoring system based on parameters in each phase of healing that can be easily and accurately assessed using either Hematoxylin & Eosin (H&E) or Masson's Trichrome (MT) staining. These parameters included re-epithelization, epithelial thickness index, keratinization, granulation tissue thickness, remodeling, and the scar elevation index. The initial phase of the study was to (1) optimize and clarify healing parameters to limit investigator bias and variability; (2) compare the consistency of parameters assessed using H&E versus MT staining. During the validation phase of this study, the accuracy and reproducibility of this scoring system was independently iterated upon and validated in four different types of murine skin wound models (Excisional; punch biopsy; pressure ulcers; burn wounds). A total of n = 54 histology sections were randomized, blinded, and assigned to two groups of independent investigators (n = 5 per group) for analysis. The sensitivity of each parameter (ranging between 80% and 95%) is reported with illustrations on the appropriate assessment method using ImageJ software. In the validated scoring system, the lowest score (score:0) is associated with an open/unhealed wound as is evident immediately and within the first day postinjury, whereas the highest score (score:12) is associated with a completely closed and healed wound without excessive scarring. This study defines and describes the minimum recommended criteria for assessing wound healing dynamics using the SPOT skin wound score. The acronym SPOT refers to the academic and scientific institutions that were involved in the development of the scoring system, namely, Stellenbosch University, Polish Academy of Sciences, Obatala Sciences, and the University of Texas Southwestern.

Targeting Stem Cells in Chronic Inflammatory Diseases.

Mesenchymal stem cell (MSC) dysfunction is a serious complication in ageing and age-related inflammatory diseases such as type 2 diabetes mellitus. Inflammation and oxidative stress-induced cellular senescence alter the immunomodulatory ability of MSCs and hamper their pro-regenerative function, which in turn leads to an increase in disease severity, maladaptive tissue damage and the development of comorbidities. Targeting stem/progenitor cells to restore their function and/or protect them against impairment could thus improve healing outcomes and significantly enhance the quality of life for diabetic patients. This review discusses the dysregulation of MSCs' immunomodulatory capacity in the context of diabetes mellitus and focuses on intervention strategies aimed at MSC rejuvenation. Research pertaining to the potential therapeutic use of either pharmacological agents (NFкB antagonists), natural products (phytomedicine) or biological agents (exosomes, probiotics) to improve MSC function is discussed and an overview of the most pertinent methodological considerations given. Based on in vitro studies, numerous anti-inflammatory agents, antioxidants and biological agents show tremendous potential to revitalise MSCs. An integrated systems approach and a thorough understanding of complete disease pathology are however required to identify feasible candidates for in vivo targeting of MSCs.

Dysregulated healing responses in diabetic wounds occur in the early stages postinjury.

Chronic wounds are a serious and debilitating complication of diabetes. A better understanding of the dysregulated healing responses following injury will provide insight into the optimal time frame for therapeutic intervention. In this study, a direct comparison was done between the healing dynamics and the proteome of acute and obese diabetic wounds on days 2 and 7 following injury. Full thickness excisional wounds were induced on obese diabetic (B6.Cg-lepob/J, ob/ob, n = 14) (blood glucose 423.25 ± 127.92 mg/dL) and WT control (C57BL/6J, n = 14) (blood glucose 186.67 ± 24.5 mg/dL) mice. Histological analysis showed no signs of healing in obese DM wounds whereas complete wound closure/re-epithelisation, the formation of granulation tissue and signs of re-vascularisation, was evident in acute wounds on day 7. In obese DM wounds, substance P deficiency and increased MMP-9 activity on day 2 coincided with increased cytokine/chemokine levels within wound fluid. LC-MS/MS identified 906 proteins, of which 23 (Actn3, Itga6, Epb41, Sncg, Nefm, Rsp18, Rsp19, Rpl22, Macroh2a1, Rpn1, Ppib, Snrnp70, Ddx5, Eif3g, Tpt1, FABP5, Cavin1, Stfa1, Stfa3, Cycs, Tkt, Mb, Chmp2a) were differentially expressed in wounded tissue on day 2 (P < 0.05; more than two-fold) and 6 (Cfd, Ptms, Hp, Hmga1, Cbx3, Syap1) (P < 0.05; more than two-fold) on day 7. A large number of dysregulated proteins on day 2 was associated with an inability to progress into the proliferative stage of healing and suggest that early intervention might be pivotal for effective healing outcomes. The proteomic approach highlighted the complexity of obese DM wounds in which the dysregulation involves multiple regulatory pathways and biological processes.

Utility of in-hospital post-delivery fasting plasma glucose to predict postpartum glucose status in women with hyperglycaemia first detected in pregnancy: A prospective cohort study.

BACKGROUND: Women with hyperglycaemia first detected in pregnancy (HFDP), including those with gestational diabetes mellitus (GDM), should undergo a glucose evaluation 4-12 weeks after delivery. Globally, suboptimal postpartum return rates limit the opportunity to intervene in women with sustained hyperglycaemia and pragmatic solutions should be sought to bridge this gap. OBJECTIVE: To assess the utility of postpartum in-hospital glucose evaluation to predict the outcome of the oral glucose tolerance test (OGTT) performed 4-12 weeks after delivery. METHODS: The study was performed prospectively at Tygerberg Hospital, Cape Town, South Africa. Women with HFDP, classified as GDM based on the modified National Institute for Health and Care Excellence criteria, who delivered between November 2018 and June 2019 were included in the study. Fasting plasma glucose (FPG) was performed 24-72 hours after delivery (t1) in the postnatal ward, provided glucose lowering medication was discontinued at delivery. An OGTT 4-12 weeks postpartum (t2) was scheduled for the total cohort. We compared glucose values and glucose categories at t1 and t2 and evaluated antenatal characteristics of women who returned, compared to the group that was lost to follow-up. RESULTS: In-hospital post-delivery glucose assessment (t1) was performed in 115 women. Glucose levels were significantly lower at t1 compared to antenatal diagnostic values (t0) and assessment at t2. Of the fourteen women with hyperglycaemia at t2, none had abnormal fasting glucose concentrations at t1. Women with HFDP who fulfilled criteria for overt diabetes at t0, all (24/115) had normal fasting glucose levels at t1 except for IFG in one (1/24). The antenatal characteristics of women with HFDP who returned at t2, were similar to the women who did not return. CONCLUSION: Based on this study, in-hospital fasting glucose 24-72 hours postpartum cannot replace the OGTT 4-12 weeks postpartum. Pragmatic solutions for low postpartum return rates in women with HFDP should be pursued.

Ex vivo antioxidant preconditioning improves the survival rate of bone marrow stem cells in the presence of wound fluid.

The advancement of autologous mesenchymal stem cell (MSC) therapy for the treatment of non-healing diabetic wounds is hampered by endogenous MSC dysfunction and limited viability of cells post-transplantation into the pathological wound environment. The development of effective strategies to restore the functional capabilities of these impaired MSCs prior to transplantation may be a key to their ultimate success as wound repair mediators. The current study therefore investigated whether antioxidant preconditioning [7.5 mM N-acetylcysteine (NAC) + 0.6 mM ascorbic 2-phosphate (AAP)] could restore the growth rate, migration ability and viability of impaired MSCs and whether this restored state is maintained in the presence of diabetic wound fluid (DWF). Healthy control (source: wild type, C57BL/6J mice) (n = 12) and impaired/diabetic MSCs (source: obese prediabetic, B6.Cg-Lepob/J mice) (n = 12) were isolated from the bone marrow of mice. Treatment groups post-isolation were as follow: (a) No treatment (baseline phenotype): MSCs expanded in standard growth media (SGM) (±8 days) and only exposed to growth media. (b) DWF (baseline response): MSCs expanded in SGM (±8 days) followed by exposure to DWF (24 hours, 48 hours, 96 hours). (c) Antioxidant preconditioning (preconditioned phenotype): MSCs expanded in the presence of NAC/AAP (±8 days). (d) Antioxidant preconditioning + DWF (preconditioned response): MSCs expanded in the presence of NAC/AAP (±8 days) followed by exposure to DWF (24 hours, 48 hours, 96 hours). The results demonstrated that expansion of MSCs (both healthy control and impaired diabetic) in the presence of combined NAC/AAP treatment improved ex vivo MSC viability and protected MSCs in the presence of DWF. Despite improved viability, AAP/NAC could however not rescue the reduced proliferation and migration capacity of impaired diabetic MSCs. The protective effect of NAC/AAP preconditioning against the toxicity of DWF could however be a potential strategy to improve cell number post-transplantation.

Model for Studying the Effects of Chronic Metabolic Disease on Endogenous Bone Marrow Stem Cell Populations.

Disease-associated impairment/dysfunction of stem cell populations is prominent in chronic metabolic and inflammatory diseases, such as type 2 diabetes mellitus (DM) where the multifunctional properties (viability, proliferation, paracrine secretion, multilineage differentiation) of bone marrow resident mesenchymal stem cells (MSCs) can be affected. The growth and viability impairments make it difficult to study the underlying molecular mechanisms related to the dysfunction of these cells in vitro. We have consequently optimized the isolation and culture conditions for impaired/dysfunctional bone marrow MSCs from B6.Cg-Lepob/J obese prediabetic mice. The method described here permits ex vivo investigations into disease-associated functional impairments and the dysregulated molecular mechanisms in these primary MSCs through direct comparisons with their healthy wild-type C57BL6/J control mouse counterparts.

Observations on Glucose Excursions With the Use of a Simple Protocol for Insulin, Following Antenatal Betamethasone Administration.

Aims: Pregnant women with diabetes often require preterm delivery. Antenatal betamethasone reduces perinatal morbidity and mortality, but induces hyperglycemia. The primary objective was to observe glucose excursions and determine the preliminary safety of a protocol for subcutaneous insulin following betamethasone administration in an antenatal ward. Material and Methods: This retrospective study included all women with diabetes who received betamethasone due to anticipated preterm delivery. Glucose excursions were evaluated in the fasting state and 2-h postprandial. Blood glucose values ≥14mmol/L or ≤3.5mmol/L were regarded as unacceptable hyper- and hypoglycemia respectively. Events over the first 96 h were documented. Results: This study spanned 52 months and included fifty-nine women. Eleven episodes of defined hypoglycemia occurred in six women, all receiving insulin therapy, but none after a corrective dose of insulin. No serious hypoglycemic incident was reported. Seventeen women experienced hyperglycemic incidents almost entirely (47/56) within 48 h of betamethasone administration, most often postprandially (34/56) and in 85% of episodes, preceded by pre-prandial values >9 mmol/L (29/34). 14 (82.4%) of these women were receiving background insulin therapy. No case with gestational diabetes encountered defined hyperglycemia. Conclusions: This small study demonstrated preliminary safety of the protocol. Enhanced surveillance is necessary for 72 h after initiation of betamethasone.

Isolation and Characterization of Different Mesenchymal Stem Cell Populations from Rat Femur.

Purified mesenchymal stem cells (MSCs) may be used for a multitude of applications, from the study of biological processes such as cell division and coordinated gene expression to tissue engineering and regenerative medicine. However, although highly similar, MSCs isolated and purified from different tissues may be biologically different in the ability of the cells to respond to environmental cues that instigate and propagate changes in cell fate such as differentiation, proliferation, apoptosis, and senescence. Selecting which MSC subtype to study may therefore profoundly influence the outcome of the investigation. Here we outline the isolation, purification, and differentiation of three different MSC subtypes derived from various depots within rat bone. These include MSCs from bone marrow, compact bone, and the proximal femur. Osteoblastic and adipogenic differentiation exemplify differences between these cells.

Antioxidant Preconditioning Improves the Paracrine Responsiveness of Mouse Bone Marrow Mesenchymal Stem Cells to Diabetic Wound Fluid.

Mesenchymal stem cells (MSCs) are a promising therapeutic tool for the treatment of nonhealing diabetic wounds. The pathological nature of the niche microenvironment limits the use of autologous cell therapy in diabetic patients. Prolonged exposure of endogenous MSCs to a pathological microenvironment in vivo reduces their ability to respond to environmental cues. This study investigated the effectiveness of ex vivo antioxidant treatment [N-acetylcysteine (7.5 mM NAC) and Ascorbic acid 2-phosphate (0.6 mM AAP)] to restore the paracrine function of diabetic MSCs. Healthy control [bone marrow stem cells derived from wild-type mice (SCWT)] (source: wild-type C57BL/6J mice) (n = 12) and impaired/dysfunctional [bone marrow stem cells derived from ob/ob mice (SCob)] (source: obese diabetic, B6.Cg-Lepob/J mice) (n = 12) MSCs were isolated. Ex vivo treatment groups (SCWT vs. SCob) were as follows: (1) no treatment (baseline phenotype), (2) stimulated with diabetic wound fluid (DWF) (baseline response), (3) antioxidant preconditioning (preconditioned phenotype), and (4) antioxidant preconditioned with subsequent stimulation with DWF (preconditioned response). The paracrine responsiveness on both the molecular (mRNA expression of 80 cytokines and receptors, quantitative polymerase chain reaction microarray) and protein (23-plex bead-array Luminex assay) level was assessed. At baseline, 31 genes were overexpressed (> × 2-fold) and 39 genes were underexpressed (> × 2-fold) in SCob versus SCWT. In conditioned media, significant differences (P < 0.05) were detected at baseline for two proinflammatory cytokines [tumor necrosis factor alpha (TNFα) and interferon gamma (IFNγ)], four chemokines [keratinocyte chemoattractant (KC), granulocyte colony-stimulating factor (GCSF), Eotaxin, and macrophage chemoattractant protein (MCP1)], and one anti-inflammatory cytokine [interleukin 10 (IL10)]. Following stimulation with DWF, significant differences (P < 0.05) were detected in the secretion of two chemokines [granulocyte macrophage colony-stimulating factor (GMCSF) and Eotaxin], three proinflammatory cytokines (TNFα, IFNγ, and IL9), and four anti-inflammatory cytokines (IL10, IL4, IL13, and IL3). Antioxidant preconditioning significantly dampened the excessive TNFα response observed in SCob and improved the secretion of IL10. Taken together these data suggest that the combined ex vivo treatment of autologous stem cells with NAC and AAP could potentially be an effective strategy to restore the paracrine function of impaired diabetic MSCs before transplantation.

ADSC-conditioned media elicit an ex vivo anti-inflammatory macrophage response.

Obesity-associated inflammatory mechanisms play a key role in the pathogenesis of metabolic-related diseases. Failure of anti-inflammatory control mechanisms within adipose tissue and peripheral blood mononuclear cells (PBMCs) have been implicated in disease progression. This study investigated the efficacy of allogeneic adipose tissue-derived mesenchymal stem cells conditioned media (ADSC-CM) to counteract persistent inflammation by inducing an anti-inflammatory phenotype and cytokine response within PBMCs derived from patients with and without metabolic syndrome. Forty six (n=46) mixed ancestry females (18 - 45 years) were subdivided into a) healthy lean (HL) (n=10) (BMI < 25 kg/m2), b) overweight/obese (OW/OB) (BMI ≥ 25 kg/m2, < 3 metabolic risk factors) (n=22) and c) metabolic syndrome (MetS) (visceral adiposity , ≥ 3 metabolic risk factors) (n=14) groups. Body composition (DXA scan), metabolic (cholesterol, HDL, LDL, triglycerides, blood glucose) and inflammatory profiles (38-Plex cytokine panel) were determined. PBMCs were isolated from whole blood and treated ex vivo with either i) autologous participant-derived serum ii) ADSCs-CM or iii) a successive treatment regime. The activation status (CD11b+) and intracellular cytokine (IL6, IL10, TNFa) expression were determined in M1 (CD68+CD206-CD163-) and M2 (CD68+CD163+ CD206+) macrophage populations using flow cytometry. ADSC-CM treatment, promoted a M2 macrophage phenotype and induced IL10 expression, this was most pronounced in the OW/OB group. This response is likely mediated by multiple complementing factors within ADSC-CM, yet to be identified. This study is the first to demonstrate the therapeutic potential of ADSC-CM to restore the inflammatory balance in immune compromised obese individuals.