Percutaneous Ablation, Osteoplasty, Reinforcement, and Internal Fixation for Pain and Ambulatory Function in Periacetabular Osteolytic Malignancies.

Background Osteolytic neoplasms to periacetabular bone frequently cause pain and fractures. Immediate recovery is integral to lifesaving ambulatory oncologic care and maintaining quality of life. Yet, open acetabular reconstructive surgeries are associated with numerous complications that delay cancer treatments. Purpose To determine the effectiveness for short- and long-term pain and ambulatory function following percutaneous ablation, osteoplasty, reinforcement, and internal fixation (AORIF) for periacetabular osteolytic neoplasm. Materials and Methods This retrospective observational study evaluated clinical data from 50 patients (mean age, 65 years ± 14 [SD]; 25 men, 25 women) with osteolytic periacetabular metastases or myeloma. The primary outcome of combined pain and ambulatory function index score (range, 1 [bedbound] through 10 [normal ambulation]) was assessed before and after AORIF at 2 weeks and then every 3 months up to 40 months (overall median follow-up, 11 months [IQR, 4-14 months]). Secondary outcomes included Eastern Cooperative Oncology Group (ECOG) score, infection, transfusion, 30-day readmission, mortality, and conversion hip arthroplasty. Serial radiographs and CT images were obtained to assess the hip joint integrity. The paired t test or Wilcoxon signed-rank test and Kaplan-Meier analysis were used to analyze data. Results Mean combined pain and ambulatory function index scores improved from 4.5 ± 2.4 to 7.8 ± 2.1 (P < .001) and median ECOG scores from 3 (IQR, 2-4) to 1 (IQR, 1-2) (P < .001) at the first 2 weeks after AORIF. Of 22 nonambulatory patients, 19 became ambulatory on their first post-AORIF visit. Pain and functional improvement were retained beyond 1 year, up to 40 months after AORIF in surviving patients. No hardware failures, surgical site infections, readmissions, or delays in care were identified following AORIF. Of 12 patients with protrusio acetabuli, one patient required a conversion hemiarthroplasty at 24 months. Conclusion The ablation, osteoplasty, reinforcement, and internal fixation, or AORIF, technique was effective for short- and long-term improvement of pain and ambulatory function in patients with periacetabular osteolytic neoplasm. © RSNA, 2023.

Endoscopic Versus Open Carpal Tunnel Surgery: Risk Factors and Rates of Revision Surgery.

PURPOSE: The purpose of our study was to compare the 1-year revision surgery rates and outcomes of open versus endoscopic carpal tunnel release. Our hypothesis was that, compared to open release, endoscopic carpal tunnel release was an independent risk factor for revision surgery within 1-year. METHODS: This was a retrospective cohort study of 4338 patients undergoing isolated endoscopic or open carpal tunnel release. Demographic data, medical comorbidities, surgical approach, need for revision surgery, hand dominance, history of prior injection, and Patient Reported Outcomes Measurement Information System upper extremity (UE), pain interference (PI) and physical function scores were analyzed. Multivariable analysis was used to identify the risk factors for revision surgery within one year of the index procedure. RESULTS: In total, 3280 patients (76%) underwent open and 1058 (24%) underwent endoscopic carpal tunnel release. Within one year of the index procedure, 45 patients required revision carpal tunnel release. The average time to revision was 143 days. The rate of revision carpal tunnel release in the open group was 0.71% compared to 2.08% in the endoscopic group. Multivariable analysis demonstrated that endoscopic surgery, male sex, cubital tunnel syndrome, tobacco use, and diabetes were associated independently with revision surgery. CONCLUSIONS: In this study, we found that endoscopic carpal tunnel release was associated independently with a 2.96 times greater likelihood of requiring revision carpal tunnel release within one year, compared to open carpal tunnel release. Male sex, concurrent cubital tunnel syndrome, tobacco use, and diabetes also were associated independently with greater risk of needing revision carpal tunnel release within one year. TYPE OF STUDY/LEVEL OF EVIDENCE: Prognostic II.

1,25-Dihydroxyvitamin D regulates expression of the tryptophan hydroxylase 2 and leptin genes: implication for behavioral influences of vitamin D.

To investigate vitamin D-related control of brain-expressed genes, candidate vitamin D responsive elements (VDREs) at -7/-10 kb in human tryptophan hydroxylase (TPH)2 were probed. Both VDREs bound the vitamin D receptor (VDR)-retinoid X receptor (RXR) complex and drove reporter gene transcription in response to 1,25-dihydroxyvitamin D3 (1,25D). Brain TPH2 mRNA, encoding the rate-limiting enzyme in serotonin synthesis, was induced 2.2-fold by 10 nM 1,25D in human U87 glioblastoma cells and 47.8-fold in rat serotonergic RN46A-B14 cells. 1,25D regulation of leptin (Lep), encoding a serotoninlike satiety factor, was also examined. In mouse adipocytes, 1,25D repressed leptin mRNA levels by at least 84%, whereas 1,25D induced leptin mRNA 15.1-fold in human glioblastoma cells. Chromatin immunoprecipitation sequencing analysis of the mouse Lep gene in response to 1,25D revealed a cluster of regulatory sites (cis-regulatory module; CRM) at -28 kb that 1,25D-dependently docked VDR, RXR, C/EBPß, and RUNX2. This CRM harbored 3 VDREs and single C/EBPß and RUNX2 sites. Therefore, the expression of human TPH2 and mouse Lep are governed by 1,25D, potentially via respective VDREs located at -7/-10 kb and -28 kb. These results imply that vitamin D affects brain serotonin concentrations, which may be relevant to psychiatric disorders, such as autism, and may control leptin levels and affect eating behavior.

Emerging Minimally Invasive Percutaneous Procedures for Periacetabular Osteolytic Metastases.

➤: Periacetabular osteolytic skeletal metastases are frequently associated with pain and impaired ambulatory function. Minimally invasive techniques allow for the restoration of ambulation without interrupting critical systemic cancer therapy. ➤: The open surgical management of massive periacetabular osteolytic lesions, such as by curettage, internal fixation, or complex total hip reconstruction, is associated with blood loss, hospitalization, rehabilitation, and complications such as infection or delayed wound-healing. ➤: Minimally invasive percutaneous procedures have become increasingly popular for the management of periacetabular osteolytic metastases by interventional oncologists and orthopaedic surgeons before complex open surgical procedures are considered. ➤: Minimally invasive procedures may include various methods of cancer ablation and reinforcement techniques. Minimally invasive procedures may entail cancer ablation, polymethylmethacrylate (PMMA) cement reinforcement, balloon osteoplasty, percutaneous screw fixation, or combinations of the aforementioned techniques (e.g., ablation-osteoplasty-reinforcement-internal fixation [AORIF]).

Parathyroid hormone therapy improves MRSA-infected fracture healing in a murine diabetic model.

Introduction: Diabetes mellitus (DM) impairs fracture healing and is associated with susceptibility to infection, which further inhibits fracture healing. While intermittent parathyroid hormone (1-34) (iPTH) effectively improves fracture healing, it is unknown whether infection-associated impaired fracture healing can be rescued with PTH (teriparatide). Methods: A chronic diet-induced type 2 diabetic mouse model was used to yield mice with decreased glucose tolerance and increased blood glucose levels compared to lean-fed controls. Methicillin-resistant Staphylococcus aureus (MRSA) was inoculated in a surgical tibia fracture model to simulate infected fracture, after which mice were treated with a combination of antibiotics and adjunctive teriparatide treatment. Fracture healing was assessed by Radiographic Union Scale in Tibial Fractures (RUST), micro-computed tomography (µCT), biomechanical testing, and histology. Results: RUST score was significantly poorer in diabetic mice compared to their lean nondiabetic counterparts. There were concomitant reductions in micro-computed tomography (µCT) parameters of callus architecture including bone volume/total volume, trabecular thickness, and total mineral density in type 2 diabetes mellitus (T2DM) mice. Biomechanicaltesting of fractured femora demonstrated diminished torsional rigidity, stiffness, and toughness to max torque. Adjuvant teriparatide treatment with systemic antibiotic therapy improved numerous parameters of bone microarchitecture bone volume, increased connectivity density, and increased trabecular number in both the lean and T2DM group. Despite the observation that poor fracture healing in T2DM mice was further impaired by MRSA infection, adjuvant iPTH treatment significantly improved fracture healing compared to antibiotic treatment alone in infected T2DM fractures. Discussion: Our results suggest that teriparatide may constitute a viable adjuvant therapeutic agent to improve bony union and bone microarchitecture to prevent the development of septic nonunion under diabetic conditions.

Bone Mass Changes Following Percutaneous Radiofrequency Ablation, Osteoplasty, Reinforcement, and Internal Fixation of Periacetabular Osteolytic Metastases.

BACKGROUND: The success of orthopedic interventions for periacetabular osteolytic metastases depends on the progression or regression of cancer-induced bone loss. PURPOSE: To characterize relative bone mass changes following percutaneous radiofrequency ablation, osteoplasty, cement reinforcement, and internal screw fixation (AORIF). METHODS: Of 70 patients who underwent AORIF at a single institution, 21 patients (22 periacetabular sites; average follow-up of 18.5 ± 12.3 months) had high-resolution pelvic bone CT scans, with at least one scan within 3 months following their operation (baseline) and a comparative scan at least 6 months post-operatively. In total, 73 CT scans were measured for bone mass changes using Hounsfield Units (HU). A region of interest was defined for the periacetabular area in the coronal, axial, and sagittal reformation planes for all CT scans. For 6-month and 1-year scans, the coronal and sagittal HU were combined to create a weight-bearing HU (wbHU). Three-dimensional volumetric analysis was performed on the baseline and longest available CT scans. Cohort survival was compared to predicted PathFx 3.0 survival. RESULTS: HU increased from baseline post-operative (1.2 ± 1.1 months) to most recent follow-up (20.2 ± 12.1 months) on coronal (124.0 ± 112.3), axial (140.3 ± 153.0), and sagittal (151.9 ± 162.4), p < 0.05. Grayscale volumetric measurements increased by 173.4 ± 166.4 (p < 0.05). AORIF median survival was 27.7 months (6.0 months PathFx3.0 predicted; p < 0.05). At 12 months, patients with >10% increase in wbHU demonstrated superior median survival of 36.5 months (vs. 26.4 months, p < 0.05). CONCLUSION: Percutaneous stabilization leads to improvements in bone mass and may allow for delays in extensive open reconstruction procedures.

Treating 'Septic' With Enhanced Antibiotics and 'Arthritis' by Mitigation of Excessive Inflammation.

Bacterial infection within the synovial joint, commonly known as septic arthritis, remains a clinical challenge as it presents two concurrent therapeutic goals of reducing bacterial burden and preservation of articular cartilage from destructive host inflammation. We hypothesized that mitigation of MRSA-induced inflammatory signaling could diminish destruction of articular cartilage in the setting of septic arthritis when used in conjunction with antibiotics. Herein, we provide evidence which supports a new therapeutic notion that concurrent antimicrobial therapy to address the 'septic' component of the disease with inflammation mitigation to manage the destructive 'arthritis' component. We established a murine model to mimic septic knee arthritis, as well as a variety of other inflammatory joint conditions. This murine septic arthritis model, in conjunction with in vitro and ex-vivo models, was utilized to characterize the inflammatory profile seen in active septic arthritis, as well as post-antibiotic treatment, via transcriptomic and histologic studies. Finally, we provided the clinical rationale for a novel therapeutic strategy combining enhanced antibiotic treatment with rifampin and adjuvant immunomodulation to inhibit post-infectious, excess chondrolysis and osteolysis. We identified that septic arthritis secondary to MRSA infection in our murine model led to increased articular cartilage damage compared to various types of inflammatory arthritis. The activation of the pERK1/2 signaling pathway, which is implicated with the mounting of an immune response and generation of inflammation, was increased in intracellular MRSA-infected synovial tissue and persisted despite antibiotic treatment. Trametinib, an inhibitor of ERK signaling through suppression of MEK1/2, alleviated the inflammation produced by the addition of intra-articular, heat-killed MRSA. Further, when combined with vancomycin and rifampin, mitigation of inflammation by pERK1/2 targeting improved outcomes for MRSA septic arthritis by conferring chondroprotection to articular cartilage and diminishing inflammatory osteolysis within bone. Our results support a new therapeutic notion that cell/biofilm-penetrating antibiotics alongside adjuvant mitigation of excessive intra-articular inflammation accomplish distinct therapeutic goals: reduction of bacterial burden and preservation of articular cartilage integrity.

Enhancement of Impaired MRSA-Infected Fracture Healing by Combinatorial Antibiotics and Modulation of Sustained Inflammation.

Fracture healing is impaired in the setting of infection, which begets protracted inflammation. The most problematic causative agent of musculoskeletal infection is methicillin-resistant Staphylococcus aureus (MRSA). We hypothesized that modulation of excessive inflammation combined with cell-penetrating antibiotic treatments facilitates fracture healing in a murine MRSA-infected femoral fracture model. Sterile and MRSA-contaminated open transverse femoral osteotomies were induced in 10-week-old male C57BL/6 mice and fixed via intramedullary nailing. In the initial therapeutic cohort, empty, vancomycin (V), rifampin (R), vancomycin-rifampin (VR), or vancomycin-rifampin-trametinib (VRT) hydrogels were applied to the fracture site intraoperatively. Rifampin was included because of its ability to penetrate eukaryotic cells to target intracellular bacteria. Unbiased screening demonstrated ERK activation was upregulated in the setting of MRSA infection. As such, the FDA-approved mitogen-activated protein kinase kinase (MEK)1-pERK1/2 inhibitor trametinib was evaluated as an adjunctive therapeutic agent to selectively mitigate excessive inflammation after infected fracture. Two additional cohorts were created mimicking immediate and delayed postoperative antibiotic administration. Systemic vancomycin or VR was administered for 2 weeks, followed by 2 weeks of VRT hydrogel or oral trametinib therapy. Hematologic, histological, and cytokine analyses were performed using serum and tissue isolates obtained at distinct postoperative intervals. Radiography and micro-computed tomography (µCT) were employed to assess fracture healing. Pro-inflammatory cytokine levels remained elevated in MRSA-infected mice with antibiotic treatment alone, but increasingly normalized with trametinib therapy. Impaired callus formation and malunion were consistently observed in the MRSA-infected groups and was partially salvaged with systemic antibiotic treatment alone. Mice that received VR alongside adjuvant MEK1-pERK1/2 inhibition displayed the greatest restoration of bone and osseous union. A combinatorial approach involving adjuvant cell-penetrating antibiotic treatments alongside mitigation of excessive inflammation enhanced healing of infected fractures. © 2022 American Society for Bone and Mineral Research (ASBMR).

Identification of putative transcriptomic biomarkers in irritable bowel syndrome (IBS): Differential gene expression and regulation of TPH1 and SERT by vitamin D.

Irritable bowel syndrome (IBS) is one of the most common gastrointestinal disorders and affects approximately 4% of the global population. The diagnosis of IBS can be made based on symptoms using the validated Rome criteria and ruling out commonly occurring organic diseases. Although biomarkers exist for "IBS mimickers" such as celiac disease and inflammatory bowel disease (IBD), no such test exists for IBS. DNA microarrays of colonic tissue have been used to identify disease-associated variants in other gastrointestinal (GI) disorders. In this study, our objective was to identify biomarkers and unique gene expression patterns that may define the pathological state of IBS. Mucosal tissue samples were collected from the sigmoid colon of 29 participants (11 IBS and 18 healthy controls). DNA microarray analysis was used to assess gene expression profiling. Extraction and purification of RNA were then performed and used to synthesize cDNA. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) was employed to identify differentially expressed genes in patients diagnosed with IBS compared to healthy, non-IBS patient-derived cDNA. Additional testing probed vitamin D-mediated regulation of select genes associated with serotonergic metabolism. DNA microarray analyses led to the identification of 858 differentially expressed genes that may characterize the IBS pathological state. After screening a series of genes using a combination of gene ontological analysis and RT-qPCR, this spectrum of potential IBS biomarkers was narrowed to 23 genes, some of which are regulated by vitamin D. Seven putative IBS biomarkers, including genes involved in serotonin metabolism, were identified. This work further supports the hypothesis that IBS pathophysiology is evident within the human transcriptome and that vitamin D modulates differential expression of genes in IBS patients. This suggests that IBS pathophysiology may also involve vitamin D deficiency and/or an irregularity in serotonin metabolism.

Concurrent targeting of glycolysis in bacteria and host cell inflammation in septic arthritis.

Intracellular infiltration of bacteria into host cells complicates medical and surgical treatment of bacterial joint infections. Unlike soft tissue infections, septic arthritis and infection-associated inflammation destroy cartilage that does not regenerate once damaged. Herein, we show that glycolytic pathways are shared by methicillin-resistant Staphylococcus aureus (MRSA) proliferation and host inflammatory machinery in septic arthritis. MRSA readily penetrates host cells and induces proinflammatory cascades that persist after conventional antibiotic treatment. The glycolysis-targeting drug dimethyl fumarate (DMF) showed both bacteriostatic and anti-inflammatory effects by hindering the proliferation of intracellular MRSA and dampening excessive intraarticular inflammation. Combinatorial treatment with DMF and vancomycin further reduced the proliferation and re-emergence of intracellular MRSA. Combinatorial adjuvant administration of DMF with antibiotics alleviated clinical symptoms of septic arthritis by suppressing bacterial burden and curbing inflammation to protect cartilage and bone. Our results provide mechanistic insight into the regulation of glycolysis in the context of infection and host inflammation toward development of a novel therapeutic paradigm to ameliorate joint bioburden and destruction in septic arthritis.

Dual therapeutic targeting of intra-articular inflammation and intracellular bacteria enhances chondroprotection in septic arthritis.

Bacterial infections involving joints and vital organs represent a challenging clinical problem because of the two concurrent therapeutic goals of bacterial eradication and tissue preservation. In the case of septic arthritis, permanent destruction of articular cartilage by intense host inflammation is commonly seen even after successful treatment of bacterial infection. Here, we provide scientific evidence of a novel treatment modality that can protect articular cartilage and enhanced eradication of causative bacteria in septic arthritis. Locally delivered cell-penetrating antibiotics such as rifampicin effectively eradicate intracellular reservoirs of methicillin-resistant Staphylococcus aureus within joint cells. Furthermore, mitigation of intra-articular inflammation by targeting the NLRP3 (nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3) inflammasome protects articular cartilage from damage in a murine model of knee septic arthritis. Together, concurrent mitigation of intra-articular inflammation and local adjuvant targeting of intracellular bacteria represents a promising new therapeutic strategy for septic arthritis.

Locally delivered adjuvant biofilm-penetrating antibiotics rescue impaired endochondral fracture healing caused by MRSA infection.

Infection is a devastating complication following an open fracture. We investigated whether local rifampin-loaded hydrogel can combat infection and improve healing in a murine model of methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis. A transverse fracture was made at the tibia midshaft of C57BL/6J mice aged 10-12 weeks and stabilized with an intramedullary pin. A total of 1 × 106 colony-forming units (CFU) of MRSA was inoculated. A collagen-based hydrogel containing low-dose (60 µg) and high-dose (300 µg) rifampin was applied before closure. Postoperative treatment response was assessed through bacterial CFU counts from tissue and hardware, tibial radiographs and microcomputed tomography (µCT), immunohistochemistry, and histological analyses. All untreated MRSA-infected fractures progressed to nonunion by 28 days with profuse MRSA colonization. Infected fractures demonstrated decreased soft callus formation on safranin O stain compared to controls. Areas of dense interleukin-1ß stain were associated with poor callus formation. High-dose rifampin hydrogels reduced the average MRSA load in tissue (p < 0.0001) and implants (p = 0.041). Low-dose rifampin hydrogels reduced tissue bacterial load by 50% (p = 0.021). Among sterile models, 88% achieved union compared to 0% of those infected. Mean radiographic union scale in tibia scores improved from 6 to 8.7 with high-dose rifampin hydrogel (p = 0.024) and to 10 with combination local/systemic rifampin therapy (p < 0.0001). µCT demonstrated reactive bone formation in MRSA infection. Histology demonstrated restored fracture healing with bacterial elimination. Rifampin-loaded hydrogels suppressed osteomyelitis, prevented implant colonization, and improved healing. Systemic rifampin was more effective at eliminating infection and improving fracture healing. Further investigation into rifampin-loaded hydrogels is required to correlate these findings with clinical efficacy.

Inflammatory conversion of quiescent osteoblasts by metastatic breast cancer cells through pERK1/2 aggravates cancer-induced bone destruction.

Disruption of bone homeostasis caused by metastatic osteolytic breast cancer cells increases inflammatory osteolysis and decreases bone formation, thereby predisposing patients to pathological fracture and cancer growth. Alteration of osteoblast function induces skeletal diseases due to the disruption of bone homeostasis. We observed increased activation of pERK1/2 in osteolytic breast cancer cells and osteoblasts in human pathological specimens with aggressive osteolytic breast cancer metastases. We confirmed that osteolytic breast cancers with high expression of pERK1/2 disrupt bone homeostasis via osteoblastic ERK1/2 activation at the bone-breast cancer interface. The process of inflammatory osteolysis modulates ERK1/2 activation in osteoblasts and breast cancer cells through dominant-negative MEK1 expression and constitutively active MEK1 expression to promote cancer growth within bone. Trametinib, an FDA-approved MEK inhibitor, not only reduced breast cancer-induced bone destruction but also dramatically reduced cancer growth in bone by inhibiting the inflammatory skeletal microenvironment. Taken together, these findings suggest that ERK1/2 activation in both breast cancer cells and osteoblasts is required for osteolytic breast cancer-induced inflammatory osteolysis and that ERK1/2 pathway inhibitors may represent a promising adjuvant therapy for patients with aggressive osteolytic breast cancers by altering the shared cancer and bone microenvironment.

Perioperative Outcomes in 17,947 Patients Undergoing 2-Level Anterior Cervical Discectomy and Fusion Versus 1-Level Anterior Cervical Corpectomy for Treatment of Cervical Degenerative Conditions: A Propensity Score Matched National Surgical Quality Improvement Program Analysis.

OBJECTIVE: To compare the perioperative morbidity of 2-level anterior cervical discectomy and fusion (ACDF) with that of 1-level anterior cervical corpectomy and fusion (ACCF) for the treatment of cervical degenerative conditions. METHODS: A retrospective study of the 2005-2016 National Surgical Quality Improvement Program database for patients undergoing 2-level ACDF and 1-level ACCF was performed. Patient data included: age, sex, body mass index (BMI), functional status, and American Society of Anesthesiologists (ASA) physical status (PS) classification. Hospital data included: operative time and length of hospital stay (LOS). Thirty-day outcome data included: any, serious, and minor adverse events, return to the operating room, readmission, and mortality. After propensity matching for age, sex, ASA PS classification, functional status, and BMI, multivariate logistic regression analysis was used to compare outcomes between the 2 propensity-matched subcohorts. Finally, multivariate logistic regression that additionally controlled for operative time was performed to compare the 2 propensity-matched subcohorts. RESULTS: A total of 17,497 cases were identified, with 90.20% undergoing 2-level ACDF and 9.80% undergoing 1-level ACCF. Patients undergoing 2-level ACDF were younger, more likely to be female, had higher functional status, and had shorter operative time and LOS (p < 0.001). After propensity score matching, cases undergoing 1-level ACCF had a statistically significant higher rate of serious adverse events (p = 0.005). This difference was no longer significant after controlling for operative time. CONCLUSION: While there was noted to be additional morbidity in 1-level ACCF cases relative to 2-level ACDF cases, the lack of difference once controlling for the surgical time supports using the procedure that best accomplishes the surgical objectives.

Rotational Tibio-Pelvic Constrained Hip Arthroplasty: A Surgical Technique: A Case Report.

CASE: A 25-year-old man presented with chronic bone and soft tissue infection of the right thigh following resection and radiation of epithelioid sarcoma. Multiple revisions and debridement procedures had failed to control the infection and left him unable to ambulate. We describe a modified Van Nes rotationplasty using a constrained, prosthetic hip between the tibia and pelvis following femur resection. With 18 months of follow-up, the patient was able to walk with a prosthetic device without evidence of recurrent infection. CONCLUSIONS: We report this rotationplasty as a potential approach to avoid hip disarticulation in cases requiring extensive debridement for incurable infection.

Gene Expression Profiling and Assessment of Vitamin D and Serotonin Pathway Variations in Patients With Irritable Bowel Syndrome.

BACKGROUND/AIMS: Irritable bowel syndrome (IBS) is a multifaceted disorder that afflicts millions of individuals worldwide. IBS is currently diagnosed based on the presence/duration of symptoms and systematic exclusion of other conditions. A more direct manner to identify IBS is needed to reduce healthcare costs and the time required for accurate diagnosis. The overarching objective of this work is to identify gene expression-based biological signatures and biomarkers of IBS. METHODS: Gene transcripts from 24 tissue biopsy samples were hybridized to microarrays for gene expression profiling. A combination of multiple statistical analyses was utilized to narrow the raw microarray data to the top 200 differentially expressed genes between IBS versus control subjects. In addition, quantitative polymerase chain reaction was employed for validation of the DNA microarray data. Gene ontology/pathway enrichment analysis was performed to investigate gene expression patterns in biochemical pathways. Finally, since vitamin D has been shown to modulate serotonin production in some models, the relationship between serum vitamin D and IBS was investigated via 25-hydroxyvitamin D (25[OH]D) chemiluminescence immunoassay. RESULTS: A total of 858 genetic features were identified with differential expression levels between IBS and asymptomatic populations. Gene ontology enrichment analysis revealed the serotonergic pathway as most prevalent among the differentially expressed genes. Further analysis via real-time polymerase chain reaction suggested that IBS patient-derived RNA exhibited lower levels of tryptophan hydroxylase-1 expression, the enzyme that catalyzes the rate-limiting step in serotonin biosynthesis. Finally, mean values for 25(OH)D were lower in IBS patients relative to non-IBS controls. CONCLUSIONS: Values for serum 25(OH)D concentrations exhibited a trend towards lower vitamin D levels within the IBS cohort. In addition, the expression of select IBS genetic biomarkers, including tryptophan hydroxylase 1, was modulated by vitamin D. Strikingly, the direction of gene regulation elicited by vitamin D in colonic cells is "opposite" to the gene expression profile observed in IBS patients, suggesting that vitamin D may help "reverse" the pathological direction of biomarker gene expression in IBS. Thus, our results intimate that IBS pathogenesis and pathophysiology may involve dysregulated serotonin production and/or vitamin D insufficiency.

SIRT1 enzymatically potentiates 1,25-dihydroxyvitamin D3 signaling via vitamin D receptor deacetylation.

The hormonal metabolite of vitamin D, 1,25-dihydroxyvitamin D3 (1,25D), binds to the vitamin D receptor (VDR) and promotes heterodimerization of VDR with a retinoid-X-receptor (RXR) to genomically regulate diverse cellular processes. Herein, it is revealed for the first time that VDR is post-translationally acetylated, and that VDR immunoprecipitated from human embryonic kidney (HEK293) cells displays a dramatic decrease in acetylated receptor in the presence of 1,25D-ligand, sirtuin-1 (SIRT1) deacetylase, or the resveratrol activator of SIRT1. To elucidate the functional significance of VDR deacetylation, vitamin-d-responsive-element (VDRE)-based transcriptional assays were performed to determine if deacetylase overexpression affects VDR/VDRE-driven transcription. In HEK293 kidney and TE85 bone cells, co-transfection of low amounts (1-5ng) of a SIRT1-expression vector elicits a reproducible and statistically significant enhancement (1.3- to 2.6-fold) in transcription mediated by VDREs from the CYP3A4 and cyp24a1 genes, where the magnitude of response to 1,25D-ligand is 6- to 30-fold. Inhibition of SIRT1 via EX-527, or utilization of a SIRT1 loss-of-function mutant (H363Y), resulted in abrogation of SIRT1-mediated VDR potentiation. Studies with a novel, non-acetylatable VDR mutant (K413R) showed that the mutant VDR possesses enhanced responsiveness to 1,25D, in conjunction with reduced, but still significant, sensitivity to exogenous SIRT1, indicating that acetylation of lysine 413 is relevant, but that other acetylated residues in VDR contribute to modulation of its activity. We conclude that the acetylation of VDR comprises a negative feedback loop that attenuates 1,25D-VDR signaling. This regulatory loop is reversed by SIRT1-catalyzed deacetylation of VDR to amplify VDR signaling and 1,25D actions.