Evidence-based interventions for identifying candidate quality indicators to assess quality of care in diabetic foot clinics: a scoping review.

BACKGROUND: Foot ulcers in people with diabetes are a serious complication requiring a complex management and have a high societal impact. Quality monitoring systems to optimize diabetic foot care exist, but a formal and more evidence-based approach to develop quality indicators (QIs) is lacking. We aimed to identify a set of candidate indicators for diabetic foot care by adopting an evidence-based methodology. METHODS: A systematic search was conducted across four academic databases: PubMed, Embase CINAHL and Cochrane Library. Studies that reported evidence-based interventions related to organization or delivery of diabetic foot care were searched. Data from the eligible studies were summarized and used to formulate process and structure indicators. The evidence for each candidate QI was described in a methodical and transparent manner. The review process was reported according to the "Preferred Reported Items for Systematic reviews and Meta-Analysis" (PRISMA) statements and its extension for scoping reviews. RESULTS: In total, 981 full-text articles were screened, and 322 clinical studies were used to formulate 42 candidate QIs. CONCLUSIONS: An evidence-based approach could be used to select candidate indicators for diabetic foot ulcer care, relating to the following domains: wound healing interventions, peripheral artery disease, offloading, secondary prevention, and interventions related to organization of care. In a further step, the feasibility of the identified set of indicators will be assessed by a multidisciplinary panel of diabetic foot care stakeholders.

A multidisciplinary Delphi consensus to define evidence-based quality indicators for diabetic foot ulcer care.

BACKGROUND: Valid measures to assess quality of care delivered to patients with diabetes suffering from diabetic foot ulcer (DFU) are scarce. This study aimed to achieve consensus on relevant and feasible quality indicators (QIs) among stakeholders involved in DFU care and was conducted as the second part of a Belgian QI selection study that sought to identify QIs for DFU care. METHODS: A stakeholder panel, including caregivers from primary care and specialized disciplines active in diabetic foot care as well as a patient organization representative, was recruited. By using the RAND/UCLA Appropriateness Method, stakeholders were asked to rate a list of 42 candidate evidence-based indicators for appropriateness through a 9-point Likert scale. QIs were classified based on the median ratings and the disagreement index, calculated by the inter-percentile range adjusted for symmetry. RESULTS: At the end of a three-phase process, 17 QIs were judged as appropriate. Among them, five were not previously described, covering the following topics: integration of wound care specialty in the multidisciplinary team, systematic evaluation of the nutritional status of the patient, administration of low-density lipoprotein-cholesterol lowering medication and protocolized care (implementation of care and prevention management protocols). CONCLUSIONS: The identified evidence-based QIs provide an assessment tool to evaluate and monitor quality of care delivered to DFU patients. Future research should focus on their complementarity with the existing QIs and their implementation in clinical practice.

Sex differences in diabetic foot ulcer severity and outcome in Belgium.

BACKGROUND: Sex differences are increasingly recognized to play an important role in the epidemiology, treatment and outcomes of many diseases. This study aims to describe differences between sexes in patient characteristics, ulcer severity and outcome after 6 months in individuals with a diabetic foot ulcer (DFU). METHODS: A total of 1,771 patients with moderate to severe DFU participated in a national prospective, multicenter cohort study. Data were collected on demographics, medical history, current DFU and outcome. For data analysis, a Generalized Estimating Equation model and an adjusted Cox proportional hazards regression were used. RESULTS: The vast majority of patients included were male (72%). Ulcers in men were deeper, more frequently displaying probe to bone, and more frequently deeply infected. Twice as many men presented with systemic infection as women. Men demonstrated a higher prevalence of previous lower limb revascularization, while women presented more frequently with renal insufficiency. Smoking was more common in men than in women. No differences in presentation delay were observed. In the Cox regression analysis, women had a 26% higher chance of healing without major amputation as a first event (hazard ratio 1.258 (95% confidence interval 1.048-1.509)). CONCLUSIONS: Men presented with more severe DFU than women, although no increase in presentation delay was observed. Moreover, female sex was significantly associated with a higher probability of ulcer healing as a first event. Among many possible contributing factors, a worse vascular state associated with a higher rate of (previous) smoking in men stands out.

Bottom-up approach to build a 'precision' risk factor classification for diabetic foot ulcer healing. Proof-of-concept.

AIMS: Diabetic foot ulcers (DFU) have a complex multifactorial pathophysiology. It is crucial to identify essential prognostic variables to streamline therapeutic actions and quality-of-care audits. Although SINBAD and University of Texas (UT), the most frequently used prognostic classification systems, were prospectively validated, not all individual parameters were shown to have consistent associations with healing. In this study, we used a bottom-up approach relying on robust methods to identify independent predictors of DFU healing. METHODS: 1,664 DFU patients were included by 34 Belgian diabetic foot clinics (DFCs). Twenty-one patient- and foot-related characteristics were recorded at presentation. Predictors of healing were identified using multivariable Cox proportional hazard regression. Multivariable models were built using backward regression with multiple imputation of missing values and bootstrapping. RESULTS: Five essential independent variables were identified: presentation delay, history of minor amputation, ulcer location, surface area and ischemia. This 5 variable-model showed a better performance compared to models based on existing classification systems. CONCLUSIONS: A bottom-up approach was used to build a prognostic classification for DFU healing based on large databases. It offers new insights and allows to tailor the classification to certain clinical settings. These 5 parameters could be used as a 'precision classification' for specialized DFCs.

Fluorine MR Imaging Probes Dynamic Migratory Profiles of Perfluorocarbon-Loaded Dendritic Cells After Streptozotocin-Induced Inflammation.

PURPOSE: The pathogenesis of type 1 diabetes (T1D) involves presentation of islet-specific self-antigens by dendritic cells (DCs) to autoreactive T cells, resulting in the destruction of insulin-producing pancreatic beta cells. We aimed to study the dynamic homing of diabetes-prone DCs to the pancreas and nearby organs with and without induction of pancreatic stress in a T1D susceptible model of repeated streptozotocin (STZ) injection. PROCEDURES: In vitro labeling of activated bone marrow-derived DCs (BMDCs) from NOD (Nonobese diabetes) mice was performed using zonyl perfluoro-15-crown-5-ether nanoparticles (ZPFCE-NPs). Internalization of particles was confirmed by confocal microscopy. Two groups of NOD.SCID (nonobese diabetic/severe combined immunodeficiency) mice with (induced by low dose STZ administration) or without pancreatic stress were compared. Diabetogenic BMDCs loaded with BDC2.5 mimotope were pre-labeled with ZPFCE-NPs and adoptively transferred into mice. Longitudinal in vivo fluorine MRI (19F MRI) was performed 24 h, 36 h and 48 h after transfer of BMDCs. For ex vivo quantification of labeled cells, 19F NMR and flow cytometry were performed on dissected tissues to validate in vivo 19F MRI data. RESULTS: In vitro flow cytometry and confocal microscopy confirmed high uptake of nanoparticles in BMDCs during the process of maturation. Migration/homing of activated and ZPFCE-NP- labeled BMDCs to different organs was monitored and quantified longitudinally, showing highest cell density in pancreas at 48-h time-point. Based on 19F MRI, STZ induced mild inflammation in the pancreatic region, as indicated by high accumulation of ZPFCE-NP-labeled BMDCs in the pancreas when compared to the vehicle group. Pancreatic draining lymph nodes showed elevated homing of labeled BMDCs in the vehicle groups in contrast to the STZ group after 72 h. The effect of STZ was confirmed by increased blood glucose levels. CONCLUSION: We showed the potential of 19F MRI for the non-invasive visualization and quantification of migrating immune cells in models for pancreatic inflammation after STZ administration. Without any intrinsic background signal, 19F MRI serves as a highly specific imaging tool to study the migration of diabetic-prone BMDCs in T1D models in vivo. This approach could particularly be of interest for the longitudinal assessment of established or novel anti-inflammatory therapeutic approaches in preclinical models.

Enhanced lung inflammatory response in whole-body compared to nose-only cigarette smoke-exposed mice.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by a progressive and abnormal inflammatory response in the lungs, mainly caused by cigarette smoking. Animal models exposed to cigarette smoke (CS) are used to mimic human COPD but the use of different CS protocols makes it difficult to compare the immunological and structural consequences of using a nose-only or whole-body CS exposure system. We hypothesized that when using a standardized CS exposure protocol based on particle density and CO (carbon monoxide) levels, the whole-body CS exposure system would generate a more severe inflammatory response than the nose-only system, due to possible sensitization by uptake of CS-components through the skin or via grooming. METHODS: In this study focusing on early COPD, mice were exposed twice daily 5 days a week to CS either with a nose-only or whole-body exposure system for 14 weeks to assess lung function, remodeling and inflammation. RESULTS: At sacrifice, serum cotinine levels were significantly higher in the whole-body (5.3 (2.3-6.9) ng/ml) compared to the nose-only ((2.0 (1.8-2.5) ng/ml) exposure system and controls (1.0 (0.9-1.0) ng/ml). Both CS exposure systems induced a similar degree of lung function impairment, while inflammation was more severe in whole body exposure system. Slightly more bronchial epithelial damage, mucus and airspace enlargement were observed with the nose-only exposure system. More lymphocytes were present in the bronchoalveolar lavage (BAL) and lymph nodes of the whole-body exposure system while enhanced IgA and IgG production was found in BAL and to a lesser extent in serum with the nose-only exposure system. CONCLUSION: The current standardized CS-exposure protocol resulted in a higher internal load of serum cotinine in the whole-body exposure system, which was associated with more inflammation. However, both exposure systems resulted in a similar lung function impairment. Data also highlighted differences between the two models in terms of lung inflammation and remodelling, and potential sensitization to CS. Researchers should be aware of these differences when designing their future studies for an early intervention in COPD.

Intestinal Delivery of Proinsulin and IL-10 via Lactococcus lactis Combined With Low-Dose Anti-CD3 Restores Tolerance Outside the Window of Acute Type 1 Diabetes Diagnosis.

A combination treatment (CT) of proinsulin and IL-10 orally delivered via genetically modified Lactococcus lactis bacteria combined with low-dose anti-CD3 (aCD3) therapy successfully restores glucose homeostasis in newly diagnosed non-obese diabetic (NOD) mice. Tolerance is accompanied by the accumulation of Foxp3+ regulatory T cells (Tregs) in the pancreas. To test the potential of this therapy outside the window of acute diabetes diagnosis, we substituted autoimmune diabetic mice, with disease duration varying between 4 and 53 days, with syngeneic islets at the time of therapy initiation. Untreated islet recipients consistently showed disease recurrence after 8.2 ± 0.7 days, while 32% of aCD3-treated and 48% of CT-treated mice remained normoglycemic until 6 weeks after therapy initiation (P < 0.001 vs. untreated controls for both treatments, P < 0.05 CT vs. aCD3 therapy). However, mice that were diabetic for more than 2 weeks before treatment initiation were less efficient at maintaining normoglycemia than those treated within 2 weeks of diabetes diagnosis, particularly in the aCD3-treated group. The complete elimination of endogenous beta cell mass with alloxan at the time of diabetes diagnosis pointed toward the significance of continuous feeding of the islet antigen proinsulin at the time of aCD3 therapy for treatment success. The CT providing proinsulin protected 69% of mice, compared to 33% when an irrelevant antigen (ovalbumin) was combined with aCD3 therapy, or to 27% with aCD3 therapy alone. Sustained tolerance was accompanied with a reduction of IGRP+CD8+ autoreactive T cells and an increase in insulin-reactive (InsB12-20 or InsB13-2) Foxp3+CD4+ Tregs, with a specific accumulation of Foxp3+ Tregs around the insulin-containing islet grafts after CT with proinsulin. The combination of proinsulin and IL-10 via oral Lactococcus lactis with low-dose aCD3 therapy can restore tolerance to beta cells in autoimmune diabetic mice, also when therapy is started outside the window of acute diabetes diagnosis, providing persistence of insulin-containing islets or prolonged beta cell function.

Vitamin D-modulated dendritic cells delay lethal graft-versus-host disease through induction of regulatory T cells.

Graft-versus-host disease (GVHD) is the most lethal complication after allogeneic bone marrow transplantation (allo-BMT). Current approaches to prevent GVHD rely on donor lymphocyte/T cell depletion or general immunosuppression, leading to opportunistic infections and cancer relapse. Tolerogenic dendritic cells can induce regulatory T cells (Tregs) with the ability to suppress inflammation and prevent transplant rejection, making them an attractive cellular therapy to control GVHD. Active vitamin D (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) promotes the generation of tolerogenic dendritic cells (1,25D3-DCs). This study aimed to determine the ability of ex vivo generated 1,25D3-DCs to trigger the expansion of Tregs that are able to control lethal xenogeneic GVHD in humanized NOD/LtSz-PrkdcscidIL2rγtm1Wjl (NSG) mice. We demonstrate that 1,25D3-DCs express lower levels of HLA-DR and costimulatory molecules, such as CD80 and CD86, and produce higher levels of IL-10 and TNF-α and lower amounts of IL-12, compared to vehicle-treated DCs. Moreover, these cells express increased levels of various co-inhibitory molecules such as PD-L1 and ILT-3 and the glycoprotein CD52 that is known to suppress T cell activation. Consequently, 1,25D3-DCs are poor stimulators of alloantigen-primed T cells, but foster the generation of antigen-specific suppressive Tregs. When adoptively transferred in humanized NSG mice, these 1,25D3-DC-induced Tregs delayed GVHD caused by the co-transferred autologous human peripheral blood mononuclear cells (PBMCs). These results indicate that 1,25D3-DC-induced Tregs can inhibit xenogeneic GVHD and maintain their immunomodulatory function under conditions of inflammation.

Vitamin D controls the capacity of human dendritic cells to induce functional regulatory T cells by regulation of glucose metabolism.

Tolerogenic dendritic cells (tolDCs) instruct regulatory T cells (Tregs) to dampen autoimmunity. Active vitamin D3 (1α,25-dihydroxyvitamin D3; 1α,25(OH)2D3) imprints human monocyte-derived DCs with tolerogenic properties by reprogramming their glucose metabolism. Here we identify the glycolytic enzyme 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) as a critical checkpoint and direct transcriptional target of 1α,25(OH)2D3 in determining the tolDC profile. Using tracer metabolomics, we show that PFKFB4 activity is essential for glucose metabolism, especially for glucose oxidation, which is elevated upon 1α,25(OH)2D3 exposure. Pharmacological inhibition of PFKFB4 reversed the 1α,25(OH)2D3-mediated shift in metabolism, DC profile and function, as determined by expression of inhibitory surface markers and secretion of regulatory cytokines and factors. Moreover, PFKFB4 inhibition in 1α,25(OH)2D3-treated DCs blocked their hallmark capacity to induce suppressive Tregs. This work demonstrates that alterations in the bioenergetic metabolism of immune cells are central to the immunomodulatory effects induced by 1α,25(OH)2D3.

Regulation of Immune Function by Vitamin D and Its Use in Diseases of Immunity.

Evidence exists for a role for vitamin D and its active metabolites in modulating immune functions. In animal models, vitamin D deficiency is associated with a higher risk for autoimmunity in genetically predisposed subjects and increases in susceptibility to infections. In addition, high-dose vitamin D can improve immune health, prevent autoimmunity, and improve defense against infections. In humans, evidence exists on associations between vitamin D deficiency and impaired immune function, leading to autoimmunity in genetically predisposed people and increased risk for infections; data on therapeutic immune effects of vitamin D supplementation when vitamin D levels are already sufficient are lacking.

Vitamin D endocrinology on the cross-road between immunity and metabolism.

The effects of vitamin D on the immune function have been recognized for more than a quarter of a century. However, our understanding of the multifactorial nature of the effects of vitamin D at the cellular, molecular and metabolic level in different immune cells of the innate and adaptive immune system has dramatically progressed during the last decades. In this review, we summarize the main metabolic pathways preferentially used in different subsets of macrophages, dendritic cells, T and B cells as well as the immunomodulatory effects of vitamin D on these cells. We will highlight the metabolic reprogramming happening in vitamin D-conditioned tolerogenic dendritic cells. A better knowledge of the dynamics of metabolic states in immune subsets and their possible roles in inflammation and autoimmunity may advance the development of novel immunotherapies. Likewise, the implications of effects of vitamin D on immunometabolism may progress our insights in the nature of immune responses in health and disease.

The phenotype and function of murine bone marrow-derived dendritic cells is not affected by the absence of VDR or its ability to bind 1α,25-dihydroxyvitamin D3.

The nuclear vitamin D receptor (VDR) is generally recognized as a ligand-dependent transcription factor that mediates the actions of its natural ligand, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3) on multiple target genes involved in mineral homeostasis, bone development, as well as immune reactivity. As the VDR is widely distributed in nearly all cells of the body, it implies that the vitamin D endocrine system may regulate many cell types and functions. Experiments in VDR null mice established that the VDR has intrinsically critical roles in skin and keratinocyte biology but not in immune responses. Oppositely, absence of the VDR ligand is linked to susceptibility to autoimmunity, illustrating a potential role for the unliganded VDR in the immune system. This discrepancy stimulated us to further investigate the impact of the VDR on the phenotype and function of myeloid dendritic cells (DCs) generated ex vivo from bone marrow precursors of VDR null (with a truncated VDR) and VDR ΔAF2 mice (with a mutated C-terminal activation factor 2 domain thus rendering ligand-induced gene transcription impossible). Absent or unliganded VDR did not affect bone marrow-derived myeloid DC generation. DCs obtained from VDR null and VDR ΔAF2 bone marrow cells had comparable MHC-II, and costimulatory molecule CD86, CD80 and CD40 expression than DCs from wild-type bone marrow cells. Additionally, an unliganded VDR did not affect the cytokine production nor the antigen-specific T cell stimulatory capacity of bone marrow-derived DCs. In conclusion, we showed that although clear effects of 1α,25-dihydroxyvitamin D3 are described on DC generation, absence of VDR or presence of an unliganded VDR does not affect the profile and function of ex vivo generated bone marrow-derived DCs.

Vitamin D3 Induces Tolerance in Human Dendritic Cells by Activation of Intracellular Metabolic Pathways.

Metabolic switches in various immune cell subsets enforce phenotype and function. In the present study, we demonstrate that the active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), induces human monocyte-derived tolerogenic dendritic cells (DC) by metabolic reprogramming. Microarray analysis demonstrated that 1,25(OH)2D3 upregulated several genes directly related to glucose metabolism, tricarboxylic acid cycle (TCA), and oxidative phosphorylation (OXPHOS). Although OXPHOS was promoted by 1,25(OH)2D3, hypoxia did not change the tolerogenic function of 1,25(OH)2D3-treated DCs. Instead, glucose availability and glycolysis, controlled by the PI3K/Akt/mTOR pathway, dictate the induction and maintenance of the 1,25(OH)2D3-conditioned tolerogenic DC phenotype and function. This metabolic reprogramming is unique for 1,25(OH)2D3, because the tolerogenic DC phenotype induced by other immune modulators did not depend on similar metabolic changes. We put forward that these metabolic insights in tolerogenic DC biology can be used to advance DC-based immunotherapies, influencing DC longevity and their resistance to environmental metabolic stress.

1,25-Dihydroxyvitamin D3 and its analog TX527 promote a stable regulatory T cell phenotype in T cells from type 1 diabetes patients.

The emergence of regulatory T cells (Tregs) as central mediators of peripheral tolerance in the immune system has led to an important area of clinical investigation to target these cells for the treatment of autoimmune diseases such as type 1 diabetes. We have demonstrated earlier that in vitro treatment of T cells from healthy individuals with TX527, a low-calcemic analog of bioactive vitamin D, can promote a CD4+ CD25high CD127low regulatory profile and imprint a migratory signature specific for homing to sites of inflammation. Towards clinical application of vitamin D-induced Tregs in autologous adoptive immunotherapy for type 1 diabetes, we show here that 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and TX527 similarly imprint T cells from type 1 diabetes patients with a CD4+ CD25high CD127low regulatory profile, modulate surface expression of skin- and inflammation-homing receptors, and increase expression of CTLA-4 and OX-40. Also, 1,25(OH)2D3 and TX527 treatment inhibit the production of effector cytokines IFN-γ, IL-9, and IL-17. Importantly, 1,25(OH)2D3 and TX527 promote the induction of IL-10-producing CD4+ CD25high CD127low T cells with a stable phenotype and the functional capacity to suppress proliferation of autologous responder T cells in vitro. These findings warrant additional validation of vitamin D-induced Tregs in view of future autologous adoptive immunotherapy in type 1 diabetes.

1,25-Dihydroxyvitamin D3 promotes tolerogenic dendritic cells with functional migratory properties in NOD mice.

The biologically active form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], is able to promote the generation of tolerogenic mature dendritic cells (mDCs) with an impaired ability to activate autoreactive T cells. These cells could represent a reliable tool for the promotion or restoration of Ag-specific tolerance through vaccination strategies, for example in type 1 diabetes patients. However, successful transfer of 1,25(OH)2D3-treated mDCs (1,25D3-mDCs) depends on the capacity of 1,25(OH)2D3 to imprint a similar tolerogenic profile in cells derived from diabetes-prone donors as from diabetes-resistant donors. In this study, we examined the impact of 1,25(OH)2D3 on the function and phenotype of mDCs originating from healthy (C57BL/6) and diabetes-prone (NOD) mice. We show that 1,25(OH)2D3 is able to imprint a phenotypic tolerogenic profile on DCs derived from both mouse strains. Both NOD- and C57BL/6-derived 1,25D3-mDCs decreased the proliferation and activation of autoreactive T cells in vitro, despite strain differences in the regulation of cytokine/chemokine expression. In addition, 1,25D3-mDCs from diabetes-prone mice expanded CD25(+)Foxp3(+) regulatory T cells and induced intracellular IL-10 production by T cells in vitro. Furthermore, 1,25D3-mDCs exhibited an intact functional migratory capacity in vivo that favors homing to the liver and pancreas of adult NOD mice. More importantly, when cotransferred with activated CD4(+) T cells into NOD.SCID recipients, 1,25D3-mDCs potently dampened the proliferation of autoreactive donor T cells in the pancreatic draining lymph nodes. Altogether, these results argue for the potential of 1,25D3-mDCs to restore Ag-specific immune tolerance and arrest autoimmune disease progression in vivo.