Calcium Chaos in Sarcoidosis: A Tale of Severe Hypercalcemia's Diagnostic Challenge.

Sarcoidosis is a systemic inflammatory condition characterized by noncaseating granulomas. Lung involvement is typical, while extrapulmonary manifestations, notably lymphadenopathy, are observed in a significant proportion of cases. The etiology involves complex interactions among immune cells and mediators, resulting in granuloma formation capable of independently producing 1,25-dihydroxyvitamin D, leading to unregulated hypercalcemia and hypercalciuria. Diagnosis can be challenging, especially when hypercalcemia is the initial symptom. The presence of noncaseating granulomas on biopsy is characteristic of sarcoidosis. We present a case of severe hypercalcemia in a 53-year-old woman, initially suggestive of primary hyperparathyroidism due to non-suppressed intact parathyroid hormone (PTH) levels and unilateral intrathyroidal tracer uptake on a technetium 99m sestamibi parathyroid scan. The patient presented with hypertension, acute kidney injury, and severe hypercalcemia. Initial assessment, including a parathyroid scan, hinted at primary hyperparathyroidism. However, further evaluation, including chest computed tomography (CT) and endobronchial biopsy, revealed sarcoidosis with noncaseating granulomas. Prednisone therapy led to normalization of serum calcium and creatinine levels. The case underscores the complexities in diagnosing sarcoidosis, especially when presenting with severe hypercalcemia. Despite non-suppressed PTH and suggestive imaging, the final diagnosis relied on endobronchial biopsy findings. The study highlights the limitations of conventional diagnostic markers, emphasizing the need for a comprehensive and individualized approach.

Altered Expression of Vitamin D Metabolism Genes and Circulating MicroRNAs in PBMCs of Patients with Type 1 Diabetes: Their Association with Vitamin D Status and Ongoing Islet Autoimmunity.

BACKGROUND: The immunomodulatory role of 1,25-Dihydroxy vitamin D3 (1,25(OH)2D3) is exerted through its interaction with the vitamin D receptor (VDR) present on pancreatic and immune cells. While a deficiency in vitamin D has been linked to Type 1 Diabetes Mellitus (T1DM), the exact molecular mechanism driving this down-regulation in T1DM is yet to be fully understood. This study aimed to decipher differences in the expression of genes associated with vitamin D metabolism in T1DM patients and to ascertain if there is a correlation between serum 1,25(OH)2D3 levels and the expression of these genes. We also sought to understand the influence of specific microRNAs (miRNAs) on the expression of vitamin D metabolism genes in peripheral blood mononuclear cells (PBMCs) of T1DM patients. Furthermore, the study delved into the potential implications of altered vitamin D metabolism genes and miRNAs on autoimmune processes. METHODS: Utilizing real-time PCR, we assessed the expression profiles of genes encoding for 1-hydroxylases (CYP27B1) and 24-hydroxylases (CYP24A1), as well as related miRNAs, in PBMCs from 30 T1DM patients and 23 healthy controls. ELISA tests facilitated the measurement of 1,25(OH)2D3, GAD65, and IA-2 levels. RESULTS: Our findings showcased downregulated CYP27B1 mRNA levels, while CYP24A1 expression remained stable compared to healthy subjects (CYP27B1, p = 0.0005; CYP24A1, p = 0.205, respectively). In T1DM patients, the levels of has-miR-216b-5p were found to be increased, while the levels of has-miR-21-5p were decreased in comparison to the control group. Notably, no correlation was identified between the expression of CYP27B1 in T1DM patients and the levels of has-miR-216b-5p, has-miR-21-5p, and 1,25(OH)2D3. A significant negative correlation was identified between CYP27B1 mRNA levels in PBMCs of T1DM and IA2, but not with GAD65. CONCLUSIONS: The study highlights there were reduced levels of both CYP27B1 mRNA and has-miR-21-5p, along with elevated levels of has-miR-216b-5p in the PBMCs of T1DM. However, the absence of a correlation between the expression of CYP27B1, levels of has-miR-216b-5p, and the status of 1,25(OH)2D3 suggests the possible existence of other regulatory mechanisms. Additionally, the inverse relationship between IA2 autoantibodies and CYP27B1 expression in T1DM patients indicates a potential connection between this gene and the autoimmune processes inherent in T1DM.

Vitamin D3 protects against respiratory syncytial virus-induced barrier dysfunction in airway epithelial cells via PKA signaling pathway.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection in infants and young children globally and is responsible for hospitalization and mortality in the elderly population. Virus-induced airway epithelial barrier damage is a critical step during RSV infection, and emerging studies suggest that RSV disrupts the tight junctions (TJs) and adherens junctions (AJs) between epithelial cells, increasing the permeability of the airway epithelial barrier. The lack of commercially available vaccines and effective antiviral drugs for RSV emphasizes the need for new management strategies. Vitamin D3 is a promising intervention for viral infection due to its critical role in modulating innate immune responses. However, there is limited evidence on the effect of vitamin D3 on RSV pathogenies. Here, we investigated the impact of vitamin D3 on RSV-induced epithelial barrier dysfunction and the underlying mechanisms. We found that pre-incubation with 1,25(OH)2D3, the active form of vitamin D3, alleviated RSV-induced epithelial barrier disruption in a dose-dependent manner without affecting viability in 16HBE cells. 1,25(OH)2D3 induced minor changes in the protein expression level of TJ/AJ proteins in RSV-infected cells. We observed increased CREB phosphorylation at Ser133 during 1,25(OH)2D3 exposure, indicating that vitamin D3 triggered protein kinase A (PKA) activity in 16HBE. PKA inhibitors modified the restoration of barrier function by 1,25(OH)2D3 in RSV-infected cells, implying that PKA signaling is responsible for the protective effects of vitamin D3 against RSV-induced barrier dysfunction in airway epithelial cells. Our findings suggest vitamin D3 as a prophylactic intervention to protect the respiratory epithelium during RSV infections.

In Vitro Generation of Human Tolerogenic Monocyte-Derived Dendritic Cells.

Human monocyte-derived dendritic cells (moDC) are commonly used as a research tool to investigate interactions between antigen-presenting cells and T cells. Generation of these cells involves the isolation of CD14 positive monocytes from peripheral blood and their in vitro differentiation into immature moDC by the cytokines GM-CSF and IL-4. Their functional characteristics can then be manipulated by maturing these cells with a cocktail of agents, which can be tailored to induce either immune activating or tolerogenic properties. Here, we describe a protocol for the generation of moDC with stable tolerogenic function, referred to as tolerogenic dendritic cells. These cells have been developed as an immunotherapeutic tool for the treatment of autoimmune disease but have also proven useful to dissect mechanisms of T cell tolerance induction in vitro.

VDR and PDIA3 Are Essential for Activation of Calcium Signaling and Membrane Response to 1,25(OH)2D3 in Squamous Cell Carcinoma Cells.

The genomic activity of 1,25(OH)2D3 is mediated by vitamin D receptor (VDR), whilst non-genomic is associated with protein disulfide isomerase family A member 3 (PDIA3). Interestingly, our recent studies documented that PDIA3 is also involved, directly or indirectly, in the modulation of genomic response to 1,25(OH)2D3. Moreover, PDIA3 was also shown to regulate cellular bioenergetics, possibly through the modulation of STAT signaling. Here, the role of VDR and PDIA3 proteins in membrane response to 1,25(OH)2D3 and calcium signaling was investigated in squamous cell carcinoma A431 cell line with or without the deletion of VDR and PDIA3 genes. Calcium influx was assayed by Fura-2AM or Fluo-4AM, while calcium-regulated element (NFAT) activation was measured using a dual luciferase assay. Further, the levels of proteins involved in membrane response to 1,25(OH)2D3 in A431 cell lines were analyzed via Western blot analysis. The deletion of either PDIA3 or VDR resulted in the decreased baseline levels of Ca2+ and its responsiveness to 1,25(OH)2D3; however, the effect was more pronounced in A431∆PDIA3. Furthermore, the knockout of either of these genes disrupted 1,25(OH)2D3-elicited membrane signaling. The data presented here indicated that the VDR is essential for the activation of calcium/calmodulin-dependent protein kinase II alpha (CAMK2A), while PDIA3 is required for 1,25(OH)2D3-induced calcium mobilization in A431 cells. Taken together, those results suggest that both VDR and PDIA3 are essential for non-genomic response to this powerful secosteroid.

Kidney xenotransplantation: status quo and development trend of physiological research / 器官移植

Organ transplantation is the most effective treatment for all categories of end-stage organ diseases. To resolve the shortage of donors in organ transplantation, widespread attention has been diverted to xenotransplantation. At present, clinicians mainly highlight the problems related to xenotransplantation rejection and viral infection. The physiology of xenotransplantation has been rarely studied. Kidney performs endocrine function by producing erythropoietin (EPO), renin and activating vitamin D. Although these pathways are usually well preserved in allogeneic transplantation, species-specific differences, especially those between pigs and non-human primates, may still affect the physiological function of transplant organs. In this article, the changes of EPO, renin-angiotensin-aldosterone system (RAAS) and active vitamin D3 of pig and human after xenotransplantation were illustrated, aiming to provide reference for subclinical research of xenotransplantation.

IFI44 is an immune evasion biomarker for SARS-CoV-2 and Staphylococcus aureus infection in patients with RA.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) caused a global pandemic of severe coronavirus disease 2019 (COVID-19). Staphylococcus aureus is one of the most common pathogenic bacteria in humans, rheumatoid arthritis (RA) is among the most prevalent autoimmune conditions. RA is a significant risk factor for SARS-CoV-2 and S. aureus infections, although the mechanism of RA and SARS-CoV-2 infection in conjunction with S. aureus infection has not been elucidated. The purpose of this study is to investigate the biomarkers and disease targets between RA and SARS-CoV-2 and S. aureus infections using bioinformatics analysis, to search for the molecular mechanisms of SARS-CoV-2 and S. aureus immune escape and potential drug targets in the RA population, and to provide new directions for further analysis and targeted development of clinical treatments. Methods: The RA dataset (GSE93272) and the S. aureus bacteremia (SAB) dataset (GSE33341) were used to obtain differentially expressed gene sets, respectively, and the common differentially expressed genes (DEGs) were determined through the intersection. Functional enrichment analysis utilizing GO, KEGG, and ClueGO methods. The PPI network was created utilizing the STRING database, and the top 10 hub genes were identified and further examined for functional enrichment using Metascape and GeneMANIA. The top 10 hub genes were intersected with the SARS-CoV-2 gene pool to identify five hub genes shared by RA, COVID-19, and SAB, and functional enrichment analysis was conducted using Metascape and GeneMANIA. Using the NetworkAnalyst platform, TF-hub gene and miRNA-hub gene networks were built for these five hub genes. The hub gene was verified utilizing GSE17755, GSE55235, and GSE13670, and its effectiveness was assessed utilizing ROC curves. CIBERSORT was applied to examine immune cell infiltration and the link between the hub gene and immune cells. Results: A total of 199 DEGs were extracted from the GSE93272 and GSE33341 datasets. KEGG analysis of enrichment pathways were NLR signaling pathway, cell membrane DNA sensing pathway, oxidative phosphorylation, and viral infection. Positive/negative regulation of the immune system, regulation of the interferon-I (IFN-I; IFN-α/ß) pathway, and associated pathways of the immunological response to viruses were enriched in GO and ClueGO analyses. PPI network and Cytoscape platform identified the top 10 hub genes: RSAD2, IFIT3, GBP1, RTP4, IFI44, OAS1, IFI44L, ISG15, HERC5, and IFIT5. The pathways are mainly enriched in response to viral and bacterial infection, IFN signaling, and 1,25-dihydroxy vitamin D3. IFI44, OAS1, IFI44L, ISG15, and HERC5 are the five hub genes shared by RA, COVID-19, and SAB. The pathways are primarily enriched for response to viral and bacterial infections. The TF-hub gene network and miRNA-hub gene network identified YY1 as a key TF and hsa-mir-1-3p and hsa-mir-146a-5p as two important miRNAs related to IFI44. IFI44 was identified as a hub gene by validating GSE17755, GSE55235, and GSE13670. Immune cell infiltration analysis showed a strong positive correlation between activated dendritic cells and IFI44 expression. Conclusions: IFI144 was discovered as a shared biomarker and disease target for RA, COVID-19, and SAB by this study. IFI44 negatively regulates the IFN signaling pathway to promote viral replication and bacterial proliferation and is an important molecular target for SARS-CoV-2 and S. aureus immune escape in RA. Dendritic cells play an important role in this process. 1,25-Dihydroxy vitamin D3 may be an important therapeutic agent in treating RA with SARS-CoV-2 and S. aureus infections.

Functions of 1,25-dihydroxy vitamin D3, vitamin D3 receptor and interleukin-22 involved in pathogenesis of gout arthritis through altering metabolic pattern and inflammatory responses.

BACKGROUND: Gouty arthritis (GA) is a common type of inflammatory arthritis. Recent studies demonstrated that 1,25-dihydroxy vitamin D3 (1,25(OH) 2 VD3) and vitamin D3 receptor (VD-R) play a protective role in acute inflammation, but interleukin-22(IL-22) promotes inflammation, especially for arthritis. However, our understanding of the responses of 1,25(OH) 2VD3 and IL-22 to gout was still unclear. Presently, in-depth metabolomics, bioinformatics and clinical characteristics analyses were performed to elucidate the pathogenesis and valuable clinical indicators of gouty arthritis. METHODS: Peripheral venous blood was taken for investigation. The levels of IL-22 and 1,25(OH)2VD3 were determined in patient's plasma via ELISA, and the mRNA levels of IL-22 and VD-R were measured via qRT-PCR. The interaction network of VD-R and IL22 were constructed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), and the biological function of the related proteins were analyzed by Clusterprofiler Metabolomics were performed to decipher the metabolic variations of GA. RESULTS: The levels of VD-R and 1,25(OH) 2 VD3 were identified to be low. What,s more, GA patients were reported to have high expression of IL-22. And IL-22 levels positively correlated with C-reactiveprotein (CRP) serum levels in the bivariate correlation analysis, whereas the level of 1,25(OH) 2VD3 negatively correlated with that of CRP. GO and KEGG analyses revealed that IL-22 and 1,25(OH) 2 VD3 were involved in stress immunity and inflammatory responses. These pathways are known to play a role in GA pathogenesis. Meanwhile, the metabolic profiles of GA serum showed that the increase in various amino acids and uric acid are involved in GA pathogenesis. Importantly, VD-R and IL22 closely correlated with the level of key metabolites uric acid, whose increase promoted the occurrence of GA. CONCLUSION: GA patients have low levels of VD-R and 1,25(OH) 2 VD3, and high levels of IL-22 together with various amino acids and uric acid. The levels of IL-22 and 1,25(OH) 2VD3 were positively and negatively correlated with C-reactive protein (CRP) serum levels, respectively. Both IL-22 and 1,25(OH) 2 VD3 functioned in GA-related immune and inflammatory responses, and closely correlated with the level of GA-related uric acid. Overall, IL-22, VD-R and 1,25(OH) 2 VD3 play functionally important roles in inflammatory responses and are relevant to gout pathogenesis.

Immunomodulatory effect of vitamin D on immune response to dengue virus infection.

Dengue, an acute febrile illness which in some cases requires hospitalization and occasionally a fatal disease, caused by dengue virus is a potential threat to the public health systems throughout the world. Approved antivirals are not available for treating dengue. Immunomodulators, that can reduce inflammation which if not treated properly results in vascular leakage, are being attempted as therapeutics against severe dengue. Vitamin D, an immunomodulatory hormone, with both antiviral and immunomodulatory effects, is an appropriate choice for investigation as a potential drug against dengue. Investigations of vitamin D levels by many studies have suggested vitamin D levels as a potential marker for predicting severe dengue. In-vitro studies have shown that 1, 25 dihydroxy vitamin D3 (1,25(OH)2D3), active form of vitamin D, can reduce the expression of dengue virus entry receptors, restrict the viral replication and can modulate the expression of inflammatory cytokines in dengue virus infected cells. The results from in-vitro studies also have cautioned that insufficient levels of vitamin D supplementation might increase the virus replication. Available evidence suggests vitamin D based therapeutics against dengue and provides ray of light for treating dengue patients but, the available evidence needs to be supported by beneficial outcomes in clinical trials.

Hypercalcemia of Malignancy Attributed to Cosecretion of PTH and PTHRP in Lung Adenocarcinoma.

INTRODUCTION: Hypercalcemia of malignancy (HCM) portends a very poor prognosis, and no established guidelines exist regarding its management. Most instances of HCM are due to local osteolysis or secretion of parathyroid hormone related-peptide, while less than 1% of all cases are due to ectopic secretion of parathyroid hormone. CASE REPORT: We present an unusual case of HCM due to proposed cosecretion of both parathyroid hormone and parathyroid hormone-related protein in a 36-year-old man with a poorly differentiated lung adenocarcinoma. The patient's hypercalcemia was refractory to conventional measures, including intravenous bisphosphonate therapy (zoledronic acid), and was improved with administration of denosumab. CONCLUSION: This is the youngest and first case of hypercalcemia of malignancy attributed to cosecretion of PTH and PTHrP from an adenocarcinoma. In refractory cases of HCM, denosumab is a potential option when other conventional measures are unsuccessful.

Non-classical effects of vitamin D: Non-bone effects of vitamin D.

Our understanding of vitamin D has improved considerably in recent years. The role of vitamin D in preventing osteoporotic fractures is now well-established. However, an important controversy has emerged in the last decade concerning the effects of the active form of vitamin D (1,25-dihydroxy-vitamin D) on tissues other than bone (non-classical effects). The demonstration that the vitamin D receptor (VDR) is ubiquitously, expressed combined with increasing observational data supporting a relationship between the level of 25-hydroxy-vitamin D in the serum and chronic metabolic disorders, cardiovascular disease and neoplasms, have led to its redefinition as a steroid hormone and the proposal of its use in preventing and/or treating those diseases. This article is an update on the different non-bone or non-classical effects of "vitamin-hormone D", and its potential preventive or therapeutic role in certain diseases, however, this review is not exhaustive. The different modalities of substitution or supplementation proposed in France by the Groupe de Recherche et d'Information sur les Ostéoporoses (GRIO) are also summarised.

Action of 1,25(OH)2D3 on Human Asthmatic Bronchial Fibroblasts: Implications for Airway Remodeling in Asthma.

BACKGROUND: Airway fibroblasts are major contributors to the histopathological feature of airway remodeling in asthma by their implication in the cell invasiveness and profibrogenic secretory phenotype observed in subepithelial fibrosis. 1,25 Dihydroxy vitamin D3 (1,25(OH)2D3) is an important therapeutic agent that blocks many features of airway remodeling induced by profibrogenic mediators, such as transforming growth factor beta 1 (TGF-ß1) or T helper type 1 inflammatory cytokines. OBJECTIVE: We hypothesized that 1,25(OH)2D3 opposes the TGF-ß1 or tumor necrosis factor alpha (TNF-α)-Interleukin 1 beta (IL-1ß) stimulation on airway fibroblast profibrogenic secretory phenotype observed in severe asthmatic patients. Our aim was to investigate the anti-fibrogenic effect of 1,25(OH)2D3 in TGF-ß1 or TNF-α-IL-1ß-stimulated human bronchial fibroblast cells (HBFCs) from severe asthmatic compared with non-asthmatic subjects. PATIENTS AND METHODS: All experiments were performed on primary HBFCs from asthmatic (DHBFCs, n=4) and non-asthmatic subjects (NHBFCs, n=4). mRNA expression and protein quantification of key fibrogenic markers were analyzed by RT-qPCR and ELISA, comparing HBFCs from asthmatic and non-asthmatic subjects. Vitamin D receptor (VDR) mRNA expression and its functionality in HBFCs were assessed by RT-qPCR. HBFCs proliferation was assessed by flow cytometry using BrdU-FITC/7AAD bivariate staining, while HBFCs apoptosis by Annexin V-FITC/7AAD. RESULTS: VDR is constitutively expressed in HBFCs and the addition of 1,25(OH)2D3 significantly increased mRNA expression of CYP24A1 (a direct VDRs' target gene) in both HBFCs groups. DHBFCs cultured in the presence of TGF-ß1 or TNF-α-IL-1ß showed increased mRNA expression and protein secretion of fibrogenic markers when compared to NHBFCs. Additionally, we observed decreased mRNA expression of FN 1, LUM, BGN, MMP2, COL5A1, TIMP1 and CC-chemokines (CCL2, CCL5, CCL11) in response to 1,25(OH)2D3 addition to the TGF-ß1 or TNF-α-IL-1ß-stimulated HBFCs. Cell culture media obtained from TGF-ß1 or TNF-α-IL-1ß-stimulated DHBFCs showed decreased protein secretion (fibronectin 1, lumican, MCP1, RANTES and eotaxin-1) in response to 1,25(OH)2D3 when compared to NHBFCs. 1,25(OH)2D3 inhibited proliferation in TGF-ß1-stimulated HBFCs through G0/G1 cell cycle arrest and these effects were not correlated with the induction of apoptosis. CONCLUSION: DHBFCs under TGF-ß1 or TNF-α-IL-1ß stimulation showed higher fibrogenic capacity when compared to NHBFCs. 1,25(OH)2D3 significantly blocked these effects and highlight 1,25(OH)2D3 as a possible therapeutic target for severe asthma.

Therapeutic effect of 1,25(OH)2-VitaminD3 on fibrosis and angiogenesis of peritoneum induced by chlorhexidine.

The biological activity of vitamin D, which mediated by the vitamin D receptor, is widespread throughout the body. The present study aimed to define whether 1,25-dihydroxy vitamin D3 (1,25-(OH)2D3) can protect against the progression of peritoneum fibrosis (PF) through its impact on the expression of connective tissue growth factor (CTGF) and heat shock protein 47 (HSP47) in vivo and in vitro. The male Sprague-Dawley (SD) rats of PF were induced by daily intraperitoneally injection of chlorhexidine gluconate (CG) for 4 wks. PF Rats were also treated with calcitriol (i.p. 6 ng/100g*d) from initiation of the CG. In calcitriol rats, the ultrafiltration and the ratio of dialysate urea nitrogen to blood urea nitrogen were improved (P < 0.05), pathological changes and peritoneal thickness were milder than that of the PF group. Calcitriol ameliorated high glucose-induced HSP47 expression in peritoneal mesothelial cells via CTGF down-regulation both at the mRNA level and protein level. Furthermore, calcitriol prevented angiogenic mediators of fibrosis by reduced the expression of CD34 and vascular endothelial growth factor (VEGF). The present study demonstrated that 1,25-(OH)2D3 intervention had a partially protective effect on peritoneum fibrosis, which might inhibit CTGF/HSP47 and CD34/VEGF in the peritoneum tissues.

A Dimorphic Diagnosis of a Pleomorphic Disease: An Unusual Cause of Hypercalcemia.

BACKGROUND: Histoplasmosis is a rare cause of 1, 25-dihydroxy vitamin-D-mediated hypercalcemia. In this study, we report 2 cases of hypercalcemia secondary to histoplasmosis seen at Mayo Clinic, Rochester and a review of cases reported in the literature. METHODS: We conducted a PubMed search using the keywords "hypercalcemia" and "histoplasmosis." Fourteen cases of hypercalcemia secondary to histoplasmosis were reported between 1977 and 2020. We identified an additional 2 patients from our institution. RESULTS: We reviewed a total of 16 cases. The median age at presentation was 58.5 years (interquartile range, 41.5-68.75 years), and 13 of 16 patients (81.2%) were men. Serum parathyroid hormone level was available in 13 of 16 (81.25%) patients, of whom 11 patients (84.6%) had a low level, 1 patient (7.6%) had a normal level, and 1 patient (7.6%) had an elevated level. 1, 25-dihydroxy vitamin D level was reported in 9 of 16 (56.25%) patients. Of these, 5 patients (55.5%) had levels within normal limits, and 4 patients (44.4%) had levels above normal. Serum angiotensin-converting enzyme level was evaluated in 4 of 16 patients (25%), and it was elevated in all 4 (100%) cases. Four patients received corticosteroids before a diagnosis of histoplasmosis was made, which resulted in rapidly progressive disease and death in 2 patients. CONCLUSIONS: In patients with granulomatous disorder and hypercalcemia, it is crucial to rule out infectious etiologies before initiating steroids. Histoplasmosis can cause nonparathyroid hormone-mediated hypercalcemia and, if not suspected, may have catastrophic implications.

1,25-Dihydroxy vitamin D3 inhibits the Ras-MEK-ERK pathway and regulates proliferation and apoptosis of papillary thyroid carcinoma.

OBJECTIVE: To explore the effects of 1,25-dihydroxy vitamin D3 [1,25-(OH)2D3] on the proliferation and apoptosis of papillary thyroid carcinoma and to investigate its possible mechanism. MATERIALS AND METHODS: The papillary thyroid carcinoma cell line TPC-1 was cultured, and the cells were divided into control group, the 1,25-(OH)2D3 group, and the 1,25-(OH)2D3 + ML-098 (Ras agonist) group. Cell proliferation was observed by MTT. The colony formation viability of cells was detected by the plate cloning assay. Cell migration was observed by the scratch assay. Apoptosis was detected by flow cytometry. The expression of Ki67 and Caspase-3, and the activity of Ras-MEK-ERK pathway were detected by western blot. RESULTS: Compared with the Control group, the proliferation, colony formation and migration ability of cells in the drug group were significantly decreased. The number of apoptotic cells was significantly increased, the expression of Ki67 protein was decreased, and the expression of Caspase-3 protein was upregulated. The phosphorylation levels of Ras, p-ERK1/2, and p-MEK were decreased. Compared with the drug group, the cloning and migration biological activity of cells in the 1,25-(OH)2D3 + ML-098 group was significantly enhanced (p < 0.05). The number of apoptotic cells was decreased, while the Ki67 protein level was increased. In addition, the Caspase-3 protein level was decreased, and the Ras-MEK-ERK level was also enhanced. Furthermore, the antitumor activity of 1,25-(OH)2D3 was reversed by the Ras agonist ML-098. CONCLUSION: 1,25-(OH)2D3 can inhibit the activity and promote apoptosis of the papillary thyroid carcinoma cell line TPC-1, and its mechanism may be related to the inhibition of the Ras-MEK-ERK pathway activity, thus affecting the proliferation and expression of apoptosis-related proteins.

Short Pretreatment with Calcitriol Is Far Superior to Continuous Treatment in Stimulating Proliferation and Osteogenic Differentiation of Human Adipose Stem Cells.

OBJECTIVE: This study investigated whether short stimulation (30 minutes) of human adipose stem cells (hASCs) with 1,25-dihydroxyvitamin D3 (calcitriol or 1,25-(OH)2VitD3), fitting within the surgical procedure time frame, suffices to induce osteogenic differentiation, and compared this with continuous treatment with 1,25-(OH)2VitD3. MATERIALS AND METHODS: In this experimental study, hASCs were pretreated with/without 10 nM calcitriol for 30 minutes, seeded on biphasic calcium phosphate (BCP), and cultured for 3 weeks with/without 1,25-(OH)2VitD3. Cell attachment was determined 30 minutes after cell seeding. AlamarBlue assay, alkaline phosphatase (ALP) assay, ALP staining, real-time polymerase chain reaction (PCR), and protein assay were used to evaluate the effect of short calcitriol pretreatment on proliferation and osteogenic differentiation of hASCs up to 3 weeks. RESULTS: Pretreatment with 1,25-(OH)2VitD3 enhanced the attachment of hASCs to BCP by 1.5-fold compared to nontreated cells and increased the proliferation by 3.5-fold at day 14, and 2.6-fold at day 21. In contrast, continuous treatment increased the proliferation by 1.7-fold only at day 14. After 2 weeks, ALP activity was increased by 18.5-fold when hASCs were pretreated with 1,25-(OH)2VitD3 for 30 minutes but increased only 2.6-fold when compared with its continuous counterpart. Moreover, after 14 days, pretreatment resulted in significant upregulation of the osteogenic markers RUNX2 and SPARC by 3.6-fold and 2.2-fold, respectively, while this was not observed upon continuous treatment. Finally, 30 minutes pretreatment of hASCs with 1,25-(OH)2VitD3 increased VEGF189 expression, which may contribute to the process of angiogenesis. CONCLUSION: This study is the first research showing that 30 minutes pretreatment of hASCs with 1,25-(OH)2VitD3, not only enhanced cell attachment to the scaffold at seeding time, but also promoted the proliferation and osteogenic differentiation of hASCs more strongly than continuous treatment, suggesting that short pre-treatment with 1,25-(OH)2VitD3 is a promising approach for the regeneration of bones in a one-step surgical procedure.

Novel CYP24A1 Mutation in a Young Male Patient with Nephrolithiasis: Case Report.

BACKGROUND/AIMS: The CYP24A1 gene encodes the vitamin D 24-hydroxylase enzyme, which hydroxylates active forms of vitamin D into inactive forms. Biallelic mutations in the CYP24A1 gene can lead to elevated levels of active vitamin D metabolites and, consequently, to hypercalcemia, hypercalciuria, nephrocalcinosis, and nephrolithiasis; however, monoallelic mutations have been associated only with milder phenotypes. In the present manuscript, we report the case of a young male patient who presented hypercalcemia and nephrolithiasis, suppressed parathormone, and elevated 1,25 dihydroxy vitamin D levels. METHODS: Biochemical analyses were performed on Cobas 8000, F. Hoffmann-La Roche AG, Basel, Switzerland. The proband was initially evaluated for occult malignancies by body imaging, serum electrophoresis, and tumor markers, which did not reveal any pathology. DNA samples of the proband and his sibling were then examined using Sanger sequencing. RESULTS: Genetic analysis revealed 2 compound heterozygous CYP24A1 mutations (p.L148P and p.R223*). The novel nonsense CYP24A1 mutation, p.R223*, was also found heterozygously in other family members with a medical history of nephrolithiasis. CONCLUSIONS: The identification of this gene mutation causing hypercalcemia, hypercalciuria, and renal stones allows the specific management of endogenous vitamin D production.

Phosphaturia in kidney stone formers: Still an enigma.

Calcium kidney stones are common worldwide. Most are idiopathic and composed of calcium oxalate. Calcium phosphate is present in around 80% and may initiate stone formation. Stone production is multifactorial with a polygenic genetic contribution. Phosphaturia is found frequently among stone formers but until recently received scant attention. This review examines possible mechanisms for the phosphaturia and its relevance to stone formation from a wide angle. There is a striking lack of clinical data. Phosphaturia is associated, but not correlated, with hypercalciuria, increased 1,25 dihydroxy-vitamin D [1,25 (OH)2D], and sometimes evidence of disturbances in proximal renal tubular function. Phosphate reabsorption in the proximal renal tubules requires tightly regulated interaction of many proteins. Paracellular flow through intercellular tight junctions is the major route of phosphate absorption from the intestine and can be reduced therapeutically in hyperphosphatemic patients. In monogenic defects stones develop when phosphaturia is associated with hypercalciuria, generally explained by increased 1,25 (OH)2D production in response to hypophosphatemia. Calcification does not occur in disorders with increased FGF23 when phosphaturia occurs in isolation and 1,25 (OH)2D is suppressed. Candidate gene studies have identified mutations in the phosphate transporters, but in few individuals. One genome-wide study identified a polymorphism of the phosphate transporter gene SLC34A4 associated with stones. Others did not find mutations obviously linked to phosphate reabsorption. Future genetic studies should have a wide trawl and should focus initially on groups of patients with clearly defined phenotypes. The global data should be pooled.

Effect of 1,25 dihydroxy vitamin D3 supplementation on pain relief in early rheumatoid arthritis.

BACKGROUND: To assess effect of 1,25 dihydroxy vitamin D3 supplementation on pain relief in early rheumatoid arthritis (RA). MATERIALS AND METHODS: An open-labeled randomized trial was conducted comparing 60,000 IU 1,25 dihydroxy vitamin D3 + calcium (1000 mg/day) combination [Group A] versus calcium (1000 mg/day) only [Group B], as supplement to existing treatment regimen in early RA. Primary outcome included (i) minimum time required for onset of pain relief (Tm) assessed through patients' visual analog scale (VAS); (ii) % change in VAS score from onset of pain relief to end of 8 weeks. Secondary outcome included change in disease activity score (DAS-28). RESULTS: At the end of 8-weeks, Group A reported 50% higher median pain relief scores (80% vs. 30%; P < 0.001) and DAS-28 scores (2.9 ± 0.6 vs. 3.1 ± 0.4; P = 0.012) compared to Group B; however, Tm remained comparable (19 ± 2 vs. 20 ± 2 days; P = 0.419). Occurrence of hypovitaminosis-D was lower (23.3%) compared to Indian prevalence rates and was a risk factor for developing active disease (Odds Ratio (OR) = 7.52 [95% Confidence Interval (CI) 2.67-21.16], P < 0.0001). Vitamin D deficiency was significantly (P < 0.001) more common in female gender, active disease, and shorter mean disease duration. Vitamin D levels were inversely correlated to disease activity as assessed by DAS-28 (r = -0.604; P < 0.001). CONCLUSIONS: Vitamin-D deficiency is a risk factor for developing active disease in RA. Weekly supplementation of 60,000 IU of 1,25 dihydroxy vitamin D3 in early RA results in greater pain relief. The number needed to treat for this additional pain relief was 2. IDENTIFIER: CTRI/2018/01/011532 (www.ctri.nic.in).