Transcription factor Sp4 is required for hyperalgesic state persistence.

Understanding how painful hypersensitive states develop and persist beyond the initial hours to days is critically important in the effort to devise strategies to prevent and/or reverse chronic painful states. Changes in nociceptor transcription can alter the abundance of nociceptive signaling elements, resulting in longer-term change in nociceptor phenotype. As a result, sensitized nociceptive signaling can be further amplified and nocifensive behaviors sustained for weeks to months. Building on our previous finding that transcription factor Sp4 positively regulates the expression of the pain transducing channel TRPV1 in Dorsal Root Ganglion (DRG) neurons, we sought to determine if Sp4 serves a broader role in the development and persistence of hypersensitive states in mice. We observed that more than 90% of Sp4 staining DRG neurons were small to medium sized, primarily unmyelinated (NF200 neg) and the majority co-expressed nociceptor markers TRPV1 and/or isolectin B4 (IB4). Genetically modified mice (Sp4+/-) with a 50% reduction of Sp4 showed a reduction in DRG TRPV1 mRNA and neuronal responses to the TRPV1 agonist-capsaicin. Importantly, Sp4+/- mice failed to develop persistent inflammatory thermal hyperalgesia, showing a reversal to control values after 6 hours. Despite a reversal of inflammatory thermal hyperalgesia, there was no difference in CFA-induced hindpaw swelling between CFA Sp4+/- and CFA wild type mice. Similarly, Sp4+/- mice failed to develop persistent mechanical hypersensitivity to hind-paw injection of NGF. Although Sp4+/- mice developed hypersensitivity to traumatic nerve injury, Sp4+/- mice failed to develop persistent cold or mechanical hypersensitivity to the platinum-based chemotherapeutic agent oxaliplatin, a non-traumatic model of neuropathic pain. Overall, Sp4+/- mice displayed a remarkable ability to reverse the development of multiple models of persistent inflammatory and neuropathic hypersensitivity. This suggests that Sp4 functions as a critical control point for a network of genes that conspire in the persistence of painful hypersensitive states.

Intrinsic activation of the vitamin D antimicrobial pathway by M. leprae infection is inhibited by type I IFN.

Following infection, virulent mycobacteria persist and grow within the macrophage, suggesting that the intrinsic activation of an innate antimicrobial response is subverted by the intracellular pathogen. For Mycobacterium leprae, the intracellular bacterium that causes leprosy, the addition of exogenous innate or adaptive immune ligands to the infected monocytes/macrophages was required to detect a vitamin D-dependent antimicrobial activity. We investigated whether there is an intrinsic immune response to M. leprae in macrophages that is inhibited by the pathogen. Upon infection of monocytes with M. leprae, there was no upregulation of CYP27B1 nor its enzymatic activity converting the inactive prohormone form of vitamin D (25-hydroxyvitamin D) to the bioactive form (1,25α-dihydroxyvitamin D). Given that M. leprae-induced type I interferon (IFN) inhibited monocyte activation, we blocked the type I IFN receptor (IFNAR), revealing the intrinsic capacity of monocytes to recognize M. leprae and upregulate CYP27B1. Consistent with these in vitro studies, an inverse relationship between expression of CYP27B1 vs. type I IFN downstream gene OAS1 was detected in leprosy patient lesions, leading us to study cytokine-derived macrophages (MΦ) to model cellular responses at the site of disease. Infection of IL-15-derived MΦ, similar to MΦ in lesions from the self-limited form of leprosy, with M. leprae did not inhibit induction of the vitamin D antimicrobial pathway. In contrast, infection of IL-10-derived MΦ, similar to MΦ in lesions from patients with the progressive form of leprosy, resulted in induction of type I IFN and suppression of the vitamin D directed pathway. Importantly, blockade of the type I IFN response in infected IL-10 MΦ decreased M. leprae viability. These results indicate that M. leprae evades the intrinsic capacity of human monocytes/MΦ to activate the vitamin D-mediated antimicrobial pathway via the induction of type I IFN.

Concerted effects of heterogeneous nuclear ribonucleoprotein C1/C2 to control vitamin D-directed gene transcription and RNA splicing in human bone cells.

Traditionally recognized as an RNA splicing regulator, heterogeneous nuclear ribonucleoprotein C1/C2 (hnRNPC1/C2) can also bind to double-stranded DNA and function in trans as a vitamin D response element (VDRE)-binding protein. As such, hnRNPC1/C2 may couple transcription induced by the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D) with subsequent RNA splicing. In MG63 osteoblastic cells, increased expression of the 1,25(OH)2D target gene CYP24A1 involved immunoprecipitation of hnRNPC1/C2 with CYP24A1 chromatin and RNA. Knockdown of hnRNPC1/C2 suppressed expression of CYP24A1, but also increased expression of an exon 10-skipped CYP24A1 splice variant; in a minigene model the latter was attenuated by a functional VDRE in the CYP24A1 promoter. In genome-wide analyses, knockdown of hnRNPC1/C2 resulted in 3500 differentially expressed genes and 2232 differentially spliced genes, with significant commonality between groups. 1,25(OH)2D induced 324 differentially expressed genes, with 187 also observed following hnRNPC1/C2 knockdown, and a further 168 unique to hnRNPC1/C2 knockdown. However, 1,25(OH)2D induced only 10 differentially spliced genes, with no overlap with differentially expressed genes. These data indicate that hnRNPC1/C2 binds to both DNA and RNA and influences both gene expression and RNA splicing, but these actions do not appear to be linked through 1,25(OH)2D-mediated induction of transcription.

Endometriosis inflammation mimicking pseudomyxoma peritonei: A case report.

•Endometriosis may mimic other pathologic processes•Fertility preservation can be considered in young women with atypical endometriosis•Referral to experienced surgeons for maligancy is warranted in atypical endometriosis.

Value of intraoperative neck margin analysis during Whipple for pancreatic adenocarcinoma: a multicenter analysis of 1399 patients.

INTRODUCTION: During pancreaticoduodenectomy (PD) for ductal adenocarcinoma, a frozen section (FS) neck margin is typically assessed, and if positive, additional pancreas is removed to achieve an R0 margin. We analyzed the association of this practice with improved overall survival (OS). METHODS: Patients who underwent PD for pancreatic ductal adenocarcinoma from January 2000 to August 2012 at 8 academic centers were classified by neck margin status as negative (R0) or microscopically positive (R1) on the basis of FS and permanent section (PS). Impact on OS of converting an FS-R1-neck margin to a PS-R0-neck margin by additional resection was assessed. RESULTS: A total of 1399 patients had FS neck margins analyzed. Median OS was 19.7 months. On FS, 152 patients (10.9%) were R1, and an additional 51 patients (3.6%) had false-negative FS-R0 margins. PS-R0-neck was achieved in 1196 patients (85.5%), 131 patients (9.3%) remained PS-R1, and 72 patients (5.1%) were converted from FS-R1-to-PS-R0 by additional resection. Median OS for PS-R0-neck patients was 21.1 months versus 13.7 months for PS-R1-neck patients (P < 0.001) and 11.9 months for FS-R1-to-PS-R0 patients (P < 0.001). Both FS-R1-to-PS-R0 and PS-R1-neck patients had larger tumors (P = 0.001), more perineural invasion (P = 0.02), and more node positivity (P = 0.08) than PS-R0-neck patients. On multivariate analysis controlling for adverse pathologic factors, FS-R1-to-PS-R0 conversion remained associated with significantly worse OS compared with PS-R0-neck patients (hazard ratio: 1.55; P = 0.009). CONCLUSIONS: For patients who undergo pancreaticoduodenectomy for pancreatic ductal adenocarcinoma, additional resection to achieve a negative neck margin after positive frozen section is not associated with improved OS.

IL-32 is a molecular marker of a host defense network in human tuberculosis.

Tuberculosis is a leading cause of infectious disease-related death worldwide; however, only 10% of people infected with Mycobacterium tuberculosis develop disease. Factors that contribute to protection could prove to be promising targets for M. tuberculosis therapies. Analysis of peripheral blood gene expression profiles of active tuberculosis patients has identified correlates of risk for disease or pathogenesis. We sought to identify potential human candidate markers of host defense by studying gene expression profiles of macrophages, cells that, upon infection by M. tuberculosis, can mount an antimicrobial response. Weighted gene coexpression network analysis revealed an association between the cytokine interleukin-32 (IL-32) and the vitamin D antimicrobial pathway in a network of interferon-γ- and IL-15-induced "defense response" genes. IL-32 induced the vitamin D-dependent antimicrobial peptides cathelicidin and DEFB4 and to generate antimicrobial activity in vitro, dependent on the presence of adequate 25-hydroxyvitamin D. In addition, the IL-15-induced defense response macrophage gene network was integrated with ranked pairwise comparisons of gene expression from five different clinical data sets of latent compared with active tuberculosis or healthy controls and a coexpression network derived from gene expression in patients with tuberculosis undergoing chemotherapy. Together, these analyses identified eight common genes, including IL-32, as molecular markers of latent tuberculosis and the IL-15-induced gene network. As maintaining M. tuberculosis in a latent state and preventing transition to active disease may represent a form of host resistance, these results identify IL-32 as one functional marker and potential correlate of protection against active tuberculosis.

Regulation of the extrarenal CYP27B1-hydroxylase.

Provided here is a collective review of research on the extrarenal CYP27B1-hydroxylase that shapes our current and expanding vision of the role this enzyme plays in the intracrinology and paracrinology, as opposed to the traditional endocrinology, of vitamin D to regulate the innate and adaptive immune responses, particularly in human granuloma-forming diseases like tuberculosis. Special emphasis is placed on soluble factors (i.e., cytokines) in the local microenvironment of these human diseases that coordinate amplification and feedback inhibition of the macrophage CYP27B1-hydroxylase. Principal among these factors are Type I and Type II interferons (IFNs); the Type II IFN, IFN-γ, stimulates the production of 1,25-dihydroxyvitamin D (1,25(OH)2D) from 25-hydroxyvitamin D (25OHD) by the granuloma-forming disease-activated macrophage, while the Type I IFNs, IFN-α and IFN-ß, block the hydroxylation reaction. The Type I IFN response is associated with more aggressive disease, while the Type II IFN response, the one that promotes 1,25(OH)2D production by the macrophage, is associated with more confined disease. Tilting the balance in the human immune response toward a confined disease phenotype is enabled by the presence of sufficient extracellular 25OHD to modulate IFN-γ-promoted and substrate 25OH-driven intracellular synthesis of 1,25(OH)2D. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Suppression of iron-regulatory hepcidin by vitamin D.

The antibacterial protein hepcidin regulates the absorption, tissue distribution, and extracellular concentration of iron by suppressing ferroportin-mediated export of cellular iron. In CKD, elevated hepcidin and vitamin D deficiency are associated with anemia. Therefore, we explored a possible role for vitamin D in iron homeostasis. Treatment of cultured hepatocytes or monocytes with prohormone 25-hydroxyvitamin D or active 1,25-dihydroxyvitamin D decreased expression of hepcidin mRNA by 0.5-fold, contrasting the stimulatory effect of 25-hydroxyvitamin D or 1,25-dihydroxyvitamin D on related antibacterial proteins such as cathelicidin. Promoter-reporter and chromatin immunoprecipitation analyses indicated that direct transcriptional suppression of hepcidin gene (HAMP) expression mediated by 1,25-dihydroxyvitamin D binding to the vitamin D receptor caused the decrease in hepcidin mRNA levels. Suppression of HAMP expression was associated with a concomitant increase in expression of the cellular target for hepcidin, ferroportin protein, and decreased expression of the intracellular iron marker ferritin. In a pilot study with healthy volunteers, supplementation with a single oral dose of vitamin D (100,000 IU vitamin D2) increased serum levels of 25D-hydroxyvitamin D from 27±2 ng/ml before supplementation to 44±3 ng/ml after supplementation (P<0.001). This response was associated with a 34% decrease in circulating levels of hepcidin within 24 hours of vitamin D supplementation (P<0.05). These data show that vitamin D is a potent regulator of the hepcidin-ferroportin axis in humans and highlight a potential new strategy for the management of anemia in patients with low vitamin D and/or CKD.

Transcription factors Sp1 and Sp4 regulate TRPV1 gene expression in rat sensory neurons.

BACKGROUND: The capsaicin receptor, transient receptor potential vanilloid type -1 (TRPV1) directs complex roles in signal transduction including the detection of noxious stimuli arising from cellular injury and inflammation. Under pathophysiologic conditions, TRPV1 mRNA and receptor protein expression are elevated in dorsal root ganglion (DRG) neurons for weeks to months and is associated with hyperalgesia. Building on our previous isolation of a promoter system for the rat TRPV1 gene, we investigated the proximal TRPV1 P2-promoter by first identifying candidate Sp1-like transcription factors bound in vivo to the P2-promoter using chromatin immunoprecipitation (ChIP) assay. We then performed deletion analysis of GC-box binding sites, and quantified promoter activity under conditions of Sp1 / Sp4 over-expression versus inhibition/knockdown. mRNA encoding Sp1, Sp4 and TRPV1 were quantified by qRT-PCR under conditions of Sp1/Sp4 over-expression or siRNA mediated knockdown in cultured DRG neurons. RESULTS: Using ChIP analysis of DRG tissue, we demonstrated that Sp1 and Sp4 are bound to the candidate GC-box site region within the endogenous TRPV1 P2-promoter. Deletion of GC-box "a" or "a + b" within the P2- promoter resulted in a complete loss of transcriptional activity indicating that GC-box "a" was the critical site for promoter activation. Co-transfection of Sp1 increased P2-promoter activity in cultured DRG neurons whereas mithramycin-a, an inhibitor of Sp1-like function, dose dependently blocked NGF and Sp1-dependent promoter activity in PC12 cells. Co-transfection of siRNA directed against Sp1 or Sp4 decreased promoter activity in DRG neurons and NGF treated PC12 cells. Finally, electroporation of Sp1 or Sp4 cDNA into cultures of DRG neurons directed an increase in Sp1/Sp4 mRNA and importantly an increase in TRPV1 mRNA. Conversely, combined si-RNA directed knockdown of Sp1/Sp4 resulted in a decrease in TRPV1 mRNA. CONCLUSION: Based on these studies, we now propose a model of TRPV1 expression that is dependent on Sp1-like transcription factors with Sp4 playing a predominant role in activating TRPV1 RNA transcription in DRG neurons. Given that increases of TRPV1 expression have been implicated in a wide range of pathophysiologic states including persistent painful conditions, blockade of Sp1-like transcription factors represents a novel direction in therapeutic strategies.