Age-Related Loss of Innate Immune Antimicrobial Function of Dermal Fat Is Mediated by Transforming Growth Factor Beta.

Dermal fibroblasts (dFBs) resist infection by locally differentiating into adipocytes and producing cathelicidin antimicrobial peptide in response to Staphylococcus aureus (S. aureus). Here, we show that neonatal skin was enriched with adipogenic dFBs and immature dermal fat that highly expressed cathelicidin. The pool of adipogenic and antimicrobial dFBs declined after birth, leading to an age-dependent loss of dermal fat and a decrease in adipogenesis and cathelidicin production in response to infection. Transforming growth factor beta (TGF-ß), which acted on uncommitted embryonic and adult dFBs and inhibited their adipogenic and antimicrobial function, was identified as a key upstream regulator of this process. Furthermore, inhibition of the TGF-ß receptor restored the adipogenic and antimicrobial function of dFBs in culture and increased resistance of adult mice to S. aureus infection. These results provide insight into changes that occur in the skin innate immune system between the perinatal and adult periods of life.

Obesity alters pathology and treatment response in inflammatory disease.

Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and have led the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system1-7, although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (TH2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent TH17 inflammation. We also observed divergent responses to biologic therapies targeting TH2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo TH response towards a TH2-predominant state and prevent aberrant non-TH2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of TH17 pathology and unlocked therapeutic responsiveness to targeted anti-TH2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity.

Antimicrobial peptide-producing dermal preadipocytes defend against Candida albicans skin infection via the FGFR-MEK-ERK pathway.

Dermal fibroblasts (dFBs) defend against deep bacterial skin infections by differentiating into preadipocytes (pAds) that produce the antimicrobial peptide cathelicidin; this differentiation is known as the dermal reactive adipogenesis response. However, the role of dFBs in fungal infection remains unknown. Here, we found that cathelicidin-producing pAds were present in high numbers in skin lesions from patients with cutaneous Candida granulomas. Second, we showed that dermal Candida albicans (C. albicans) infection in mice robustly triggered the dermal reactive adipogenesis response and induced cathelicidin expression, and inhibition of adipogenesis with pharmacological inhibitors of peroxisome proliferator-activated receptor γ (PPARγ) impaired skin resistance to C. albicans. In vitro, C. albicans products induced cathelicidin expression in pAds, and differentiating pAds markedly suppressed the growth of C. albicans by producing cathelicidin. Finally, we showed that C. albicans induced an antimicrobial response in pAds through the FGFR-MEK-ERK pathway. Together, our data reveal a previously unknown role of dFBs in the defense against skin infection caused by C. albicans.

Antimicrobial Peptide LL37 and MAVS Signaling Drive Interferon-ß Production by Epidermal Keratinocytes during Skin Injury.

Type 1 interferons (IFNs) promote inflammation in the skin but the mechanisms responsible for inducing these cytokines are not well understood. We found that IFN-ß was abundantly produced by epidermal keratinocytes (KCs) in psoriasis and during wound repair. KC IFN-ß production depended on stimulation of mitochondrial antiviral-signaling protein (MAVS) by the antimicrobial peptide LL37 and double stranded-RNA released from necrotic cells. MAVS activated downstream TBK1 (TANK-Binding Kinase 1)-AKT (AKT serine/threonine kinase 1)-IRF3 (interferon regulatory factor 3) signaling cascade leading to IFN-ß production and then promoted maturation of dendritic cells. In mice, the production of epidermal IFN-ß by LL37 required MAVS, and human wounded and/or psoriatic skin showed activation of MAVS-associated IRF3 and induction of MAVS and IFN-ß gene signatures. These findings show that KCs are an important source of IFN-ß and MAVS is critical to this function, and demonstrates how the epidermis triggers unwanted skin inflammation under disease conditions.

Dermal extracellular matrix molecules in skin development, homeostasis, wound regeneration and diseases.

The extracellular matrix (ECM) is a dynamic structure that surrounds and anchors cellular components in tissues. In addition to functioning as a structural scaffold for cellular components, ECMs also regulate diverse biological functions, including cell adhesion, proliferation, differentiation, migration, cell-cell interactions, and intracellular signaling events. Dermal fibroblasts (dFBs), the major cellular source of skin ECM, develop from a common embryonic precursor to the highly heterogeneous subpopulations during development and adulthood. Upon injury, dFBs migrate into wound granulation tissue and transdifferentiate into myofibroblasts, which play a critical role in wound contraction and dermal ECM regeneration and deposition. In this review, we describe the plasticity of dFBs during development and wound healing and how various dFB-derived ECM molecules, including collagen, proteoglycans, glycosaminoglycans, fibrillins and matricellular proteins are expressed and regulated, and in turn how these ECM molecules play a role in regulating the function of dFBs and immune cells. Finally, we describe how dysregulation of ECM matrix is associated the pathogenesis of wound healing related skin diseases, including chronic wounds and keloid.

Coordinated Patient Care via Mobile Phone-Based Telemedicine in Secondary Stroke Prevention: A Propensity Score-Matched Cohort Study.

BACKGROUND: To prevent recurrent stroke, patients need to follow evidence-based practices following discharge; however, adherence to these practices is suboptimal. PURPOSE: To evaluate whether a smartphone mobile application can improve medication adherence and stroke awareness in secondary stroke prevention. METHODS: A retrospective study design was used. Patients with ischemic stroke registered in a database between August 2018 and January 2019 were enrolled. Propensity score matching was used to match patients managed with the mobile application compared with regular practice in a 1:2 ratio. RESULTS: Sixty-five patients were paired with 123 controls. Three-month medication adherence was 93.8% in the application group versus 82.9% in the control group ( P = .036). Patients in the application group were more likely to know stroke warning signs ( P = .003) and when to call an ambulance for stroke symptoms (87.7% vs 72.4%, P = .016). CONCLUSIONS: Using a mobile application may increase medication adherence and stroke awareness in secondary stroke prevention.

Identification of Lineage-Specific Transcription Factors That Prevent Activation of Hepatic Stellate Cells and Promote Fibrosis Resolution.

BACKGROUND & AIMS: Development of liver fibrosis is associated with activation of quiescent hepatic stellate cells (HSCs) into collagen type I-producing myofibroblasts (activated HSCs). Cessation of liver injury often results in fibrosis resolution and inactivation of activated HSCs/myofibroblasts into a quiescent-like state (inactivated HSCs). We aimed to identify molecular features of phenotypes of HSCs from mice and humans. METHODS: We performed studies with LratCre, Ets1-floxed, Nf1-floxed, Pparγ-floxed, Gata6-floxed, Rag2-/-γc-/-, and C57/Bl6 (control) mice. Some mice were given carbon tetrachloride (CCl4) to induce liver fibrosis, with or without a peroxisome proliferator-activated receptor-γ (PPARγ) agonist. Livers from mice were analyzed by immunohistochemistry. Quiescent, activated, and inactivated HSCs were isolated from livers of Col1α1YFP mice and analyzed by chromatin immunoprecipitation and sequencing. Human HSCs were isolated from livers denied for transplantation. We compared changes in gene expression patterns and epigenetic modifications (histone H3 lysine 4 dimethylation and histone H3 lysine 27 acetylation) in primary mouse and human HSCs. Transcription factors were knocked down with small hairpin RNAs in mouse HSCs. RESULTS: Motif enrichment identified E26 transcription-specific transcription factors (ETS) 1, ETS2, GATA4, GATA6, interferon regulatory factor (IRF) 1, and IRF2 transcription factors as regulators of the mouse and human HSC lineage. Small hairpin RNA-knockdown of these transcription factors resulted in increased expression of genes that promote fibrogenesis and inflammation, and loss of HSC phenotype. Disruption of Gata6 or Ets1, or Nf1 or Pparγ (which are regulated by ETS1), increased the severity of CCl4-induced liver fibrosis in mice compared to control mice. Only mice with disruption of Gata6 or Pparγ had defects in fibrosis resolution after CCl4 administration was stopped, associated with persistent activation of HSCs. Administration of a PPARγ agonist accelerated regression of liver fibrosis after CCl4 administration in control mice but not in mice with disruption of Pparγ. CONCLUSIONS: Phenotypes of HSCs from humans and mice are regulated by transcription factors, including ETS1, ETS2, GATA4, GATA6, IRF1, and IRF2. Activated mouse and human HSCs can revert to a quiescent-like, inactivated phenotype. We found GATA6 and PPARγ to be required for inactivation of human HSCs and regression of liver fibrosis in mice.

Retinoids Enhance the Expression of Cathelicidin Antimicrobial Peptide during Reactive Dermal Adipogenesis.

A subset of dermal fibroblasts undergo rapid differentiation into adipocytes in response to infection and acutely produce the cathelicidin antimicrobial peptide gene Camp Vitamin A and other retinoids inhibit adipogenesis yet can show benefit to skin disorders, such as cystic acne, that are exacerbated by bacteria. We observed that retinoids potently increase and sustain the expression of Camp in preadipocytes undergoing adipogenesis despite inhibition of markers of adipogenesis, such as Adipoq, Fabp4, and Rstn Retinoids increase cathelicidin in both mouse and human preadipocytes, but this enhancement of antimicrobial peptide expression did not occur in keratinocytes or a sebocyte cell line. Preadipocytes undergoing adipogenesis more effectively inhibited growth of Staphylococcus aureus when exposed to retinoic acid. Whole transcriptome analysis identified hypoxia-inducible factor 1-α (HIF-1α) as a mechanism through which retinoids mediate this response. These observations uncouple the lipid accumulation element of adipogenesis from the innate immune response and uncover a mechanism, to our knowledge previously unsuspected, that may explain therapeutic benefits of retinoids in some skin disorders.

Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells.

A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor ß superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment.

Ctip2 is a dynamic regulator of epidermal proliferation and differentiation by integrating EGFR and Notch signaling.

Epidermal morphogenesis results from a delicate balance between keratinocyte proliferation and differentiation, and this balance is perturbed upon deletion of transcription factor Ctip2. Here we demonstrate that Ctip2, in a cell autonomous manner, controls keratinocyte proliferation and cytoskeletal organization, and regulates the onset and maintenance of differentiation in keratinocytes in culture. Ctip2 integrates keratinocyte proliferation and the switch to differentiation by directly and positively regulating EGFR transcription in proliferating cells and Notch1 transcription in differentiating cells. In proliferative cells, the EGFR promoter is occupied by Ctip2, whereas Ctip2 is only recruited to the Notch1 promoter under differentiating conditions. Activation of EGFR signaling downregulates Ctip2 at the transcript level, whereas high calcium signaling triggers SUMOylation, ubiquitination and proteasomal degradation of Ctip2 at the protein level. Together, our findings demonstrate a novel mechanism(s) of Ctip2-mediated, coordinated control of epidermal proliferation and terminal differentiation, and identify a pathway of negative feedback regulation of Ctip2 during epidermal development.

Apolipoprotein E gene E2/E2 genotype is a genetic risk factor for vertebral fractures in humans: a large-scale study.

PURPOSE: Although many studies have been performed to evaluate whether or not apolipoprotein E gene (APOE) polymorphisms are differentially associated with bone mineral density (BMD) and fractures, the results have been conflicting. This large-scale study was performed to investigate whether a relationship exists between APOE polymorphisms and risk of fracture. METHODS: A hospital-based case-control study was conducted in 3,000 patients with fractures and 3,000 age- and gender-matched healthy controls. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay was applied to assess the APOE gene polymorphisms. RESULTS: Patients with fractures had a significantly higher frequency of APOE E2/E2 genotype [odds ratio (OR) = 2.02, 95% confidence interval (CI) = 1.30, 3.14; P = 0.002] than healthy controls. When stratifying by fracture type, it was found that patients with vertebral fractures had a significantly higher frequency of APOE E2/E2 genotype (OR = 2.86, 95% CI = 1.73, 4.73; P < 0.001). No significant differences were found in nonvertebral (hip or wrist or other) fractures. CONCLUSIONS: Our study suggests that APOE E2/E2 genotype is a potential genetic risk factor for vertebral fractures in humans.

Coordinated regulation of transcription factor Bcl11b activity in thymocytes by the mitogen-activated protein kinase (MAPK) pathways and protein sumoylation.

The transcriptional regulatory protein Bcl11b is essential for T-cell development. We have discovered a dynamic, MAPK-regulated pathway involving sequential, linked, and reversible post-translational modifications of Bcl11b in thymocytes. MAPK-mediated phosphorylation of Bcl11b was coupled to its rapid desumoylation, which was followed by a subsequent cycle of dephosphorylation and resumoylation. Additionally and notably, we report the first instance of direct identification by mass spectrometry of a site of small ubiquitin-like modifier (SUMO) adduction, Lys-679 of Bcl11b, in a protein isolated from a native, mammalian cell. Sumoylation of Bcl11b resulted in recruitment of the transcriptional co-activator p300 to a Bcl11b-repressed promoter with subsequent induction of transcription. Prolonged treatment of native thymocytes with phorbol 12,13-dibutyrate together with the calcium ionophore A23187 also promoted ubiquitination and proteasomal degradation of Bcl11b, providing a mechanism for signal termination. A Bcl11b phospho-deSUMO switch was identified, the basis of which was phosphorylation-dependent recruitment of the SUMO hydrolase SENP1 to phospho-Bcl11b, coupled to hydrolysis of SUMO-Bcl11b. These results define a regulatory pathway in thymocytes that includes the MAPK pathways and upstream signaling components, Bcl11b and the associated nucleosome remodeling and deacetylation (NuRD) complex, SENP proteins, the Bcl11b protein phosphatase 6, the sumoylation machinery, the histone acetyltransferase p300, and downstream transcriptional machinery. This pathway appears to facilitate derepression of repressed Bcl11b target genes as immature thymocytes initiate differentiation programs, biochemically linking MAPK signaling with the latter stages of T-cell development.

Deciphering the age-dependent changes of pulmonary fibroblasts in mice by single-cell transcriptomics.

Background and objectives: The heterogeneity of pulmonary fibroblasts, a critical aspect of both murine and human models under physiological and pathological conditions, is well-documented. Yet, consensus remains elusive on the subtypes, lineage, biological attributes, signal transduction pathways, and plasticity of these fibroblasts. This ambiguity significantly impedes our understanding of the fibrotic processes that transpire in lung tissue during aging. This study aims to elucidate the transcriptional profiles, differentiation pathways, and potential roles of fibroblasts within aging pulmonary tissue. Methods: We employed single-cell transcriptomic sequencing via the 10x Genomics platform. The downstream data were processed and analyzed using R packages, including Seurat. Trajectory and stemness of differentiation analyses were conducted using the Monocle2 and CytoTRACE R packages, respectively. Cell interactions were deciphered using the CellChat R package, and the formation of collagen and muscle fibers was identified through Masson and Van Geison staining techniques. Results: Our analysis captured a total of 22,826 cells, leading to the identification of fibroblasts and various immune cells. We observed a shift in fibroblasts from lipogenic and immune-competent to fibrotic and myofibroblast-like phenotype during the aging process. In the aged stage, fibroblasts exhibited a diminished capacity to express chemokines for immune cells. Experimental validation confirmed an increase of collagen and muscle fiber in the aged compared to young lung tissues. Furthermore, we showed that TGFß treatment induced a fibrotic, immunodeficient and lipodystrophic transcriptional phenotype in young pulmonary fibroblasts. Conclusion: We present a comprehensive single-cell transcriptomic landscape of lung tissue from aging mice at various stages, revealing the differentiation trajectory of fibroblasts during aging. Our findings underscore the pivotal role of fibroblasts in the regulation of immune cells, and provide insights into why age increases the risk of pulmonary fibrosis.

Modulation of Skin Inflammatory Responses by Aluminum Adjuvant.

Aluminum salt (AS), one of the most commonly used vaccine adjuvants, has immuno-modulatory activity, but how the administration of AS alone may impact the activation of the skin immune system under inflammatory conditions has not been investigated. Here, we studied the therapeutic effect of AS injection on two distinct skin inflammatory mouse models: an imiquimod (IMQ)-induced psoriasis-like model and an MC903 (calcipotriol)-induced atopic dermatitis-like model. We found that injection of a high dose of AS not only suppressed the IMQ-mediated development of T-helper 1 (Th1) and T-helper 17 (Th17) immune responses but also inhibited the IMQ-mediated recruitment and/or activation of neutrophils and macrophages. In contrast, AS injection enhanced MC903-mediated development of the T-helper 2 (Th2) immune response and neutrophil recruitment. Using an in vitro approach, we found that AS treatment inhibited Th1 but promoted Th2 polarization of primary lymphocytes, and inhibited activation of peritoneal macrophages but not bone marrow derived neutrophils. Together, our results suggest that the injection of a high dose of AS may inhibit Th1 and Th17 immune response-driven skin inflammation but promote type 2 immune response-driven skin inflammation. These results may provide a better understanding of how vaccination with an aluminum adjuvant alters the skin immune response to external insults.

Dynamic interplay between IL-1 and WNT pathways in regulating dermal adipocyte lineage cells during skin development and wound regeneration.

Dermal adipocyte lineage cells are highly plastic and can undergo reversible differentiation and dedifferentiation in response to various stimuli. Using single-cell RNA sequencing of developing or wounded mouse skin, we classify dermal fibroblasts (dFBs) into distinct non-adipogenic and adipogenic cell states. Cell differentiation trajectory analyses identify IL-1-NF-κB and WNT-ß-catenin as top signaling pathways that positively and negatively associate with adipogenesis, respectively. Upon wounding, activation of adipocyte progenitors and wound-induced adipogenesis are mediated in part by neutrophils through the IL-1R-NF-κB-CREB signaling axis. In contrast, WNT activation, by WNT ligand and/or ablation of Gsk3, inhibits the adipogenic potential of dFBs but promotes lipolysis and dedifferentiation of mature adipocytes, contributing to myofibroblast formation. Finally, sustained WNT activation and inhibition of adipogenesis is seen in human keloids. These data reveal molecular mechanisms underlying the plasticity of dermal adipocyte lineage cells, defining potential therapeutic targets for defective wound healing and scar formation.

Emerging Roles of Adipose Tissue in the Pathogenesis of Psoriasis and Atopic Dermatitis in Obesity.

Obesity is a growing epidemic worldwide, and it is also considered a major environmental factor contributing to the pathogenesis of inflammatory skin diseases, including psoriasis (PSO) and atopic dermatitis (AD). Moreover, obesity worsens the course and impairs the treatment response of these inflammatory skin diseases. Emerging evidence highlights that hypertrophied adipocytes and infiltrated immune cells secrete a variety of molecules, including fatty acids and adipokines, such as leptin, adiponectin, and a panel of cytokines/chemokines that modulate our immune system. In this review, we describe how adipose hypertrophy leads to a chronic low-grade inflammatory state in obesity and how obesity-related inflammatory factors are involved in the pathogenesis of PSO and/or AD. Finally, we discuss the potential role of antimicrobial peptides, mechanical stress and impairment of epidermal barrier function mediated by fast expansion, and dermal fat in modulating skin inflammation. Together, this review summarizes the current literature on how obesity is associated with the pathogenesis of PSO and AD, highlighting the potentially important but overlooked immunomodulatory role of adipose tissue in the skin.

Effect of Iron-Erythrocyte Metabolism-Related Indexes on Posttraumatic Growth in Patients on Maintenance Hemodialysis (MHD).

Purpose: To investigate the effect of iron-erythrocyte metabolism-related indexes on posttraumatic growth in MHD patients and their caregivers. Patients and Methods: A total of 170 pairs of MHD patients and their caregivers in Shanghai Changhai Hospital were enrolled in this research, which used sociodemographic characteristics, the Posttraumatic Growth Inventory (PTGI), the Perceived Social Support Scale (PSSS), and the Medical Coping Modes Questionnaire (MCMQ). The test data of 141 patients were retrieved from the hospital database. Results: Single-factor analysis showed that the PTGI score of patients with a mean corpuscular erythrocyte volume ≥ 100 fL was 85.4 ± 19.8 and those with a mean corpuscular erythrocyte volume lower than 100 fL were 70.6 ± 24.7; the PTGI scores of patients with reticulocytes >1.5% were 68.8 ± 25.8, and those with reticulocytes <1.5% were 78.4 ± 21.1; the PTGI scores of the caregivers whose serum iron was >10.6 µmol /L were 78.2 ± 21.6, and those with serum iron <10.6 µmol /L were 67.9 ± 22.8. The difference in MCMQ scores between the caregivers with transferrin saturation>50% and with transferrin saturation<20% was 18.9 ± 8.4. For the correlation test of serum iron, reticulocyte and PTGI scores for patients, the Pearson correlation coefficients were 0.239 and -0.193, respectively, and the correlation test between erythrocyte distribution width SD and the score of caregivers MCMQ scale, the Pearson correlation coefficient was 0.225; p for all was< 0.05, with significant differences. There was no significant difference in the scores of different scales for total iron binding capacity (TIBC) at different levels. Conclusion: The indexes related to iron erythrocyte metabolism in MHD patients are correlated with ruminant meditation of patients and their caregivers and promotion of posttraumatic growth. Good nutritional status, adequate hematopoietic material, and normal erythrocyte count and function are also important for them.

Bcl11b represses a mature T-cell gene expression program in immature CD4(+)CD8(+) thymocytes.

Bcl11b is a transcription factor that, within the hematopoietic system, is expressed specifically in T cells. Although Bcl11b is required for T-cell differentiation in newborn Bcl11b-null mice, and for positive selection in the adult thymus of mice bearing a T-cell-targeted deletion, the gene network regulated by Bcl11b in T cells is unclear. We report herein that Bcl11b is a bifunctional transcriptional regulator, which is required for the correct expression of approximately 1000 genes in CD4(+)CD8(+)CD3(lo) double-positive (DP) thymocytes. Bcl11b-deficient DP cells displayed a gene expression program associated with mature CD4(+)CD8(-) and CD4(-)CD8(+) single-positive (SP) thymocytes, including upregulation of key transcriptional regulators, such as Zbtb7b and Runx3. Bcl11b interacted with regulatory regions of many dysregulated genes, suggesting a direct role in the transcriptional regulation of these genes. However, inappropriate expression of lineage-associated genes did not result in enhanced differentiation, as deletion of Bcl11b in DP cells prevented development of SP thymocytes, and that of canonical NKT cells. These data establish Bcl11b as a crucial transcriptional regulator in thymocytes, in which Bcl11b functions to prevent the premature expression of genes fundamental to the SP and NKT cell differentiation programs.