An orthotopic syngeneic mouse model of bortezomib-resistant multiple myeloma.

While bortezomib has significant benefits in multiple myeloma (MM) therapy, the disease remains incurable due to the invariable development of bortezomib resistance. This emphasises the need for advanced models for preclinical evaluation of new therapeutic approaches for bortezomib-resistant MM. Here, we describe the development of an orthotopic syngeneic bortezomib-resistant MM mouse model based on the most well-characterised syngeneic MM mouse model derived from spontaneous MM-forming C57BL/KaLwRij mice. Using bortezomib-resistant 5TGM1 cells, we report and characterise a robust syngeneic mouse model of bortezomib-resistant MM that is well suited to the evaluation of new therapeutic approaches for proteasome inhibitor-resistant MM.

Complex-Type N-Glycans Are Associated with Cartilage Degeneration within Different Loading Sites of the Tibial Plateau for Knee Osteoarthritis Patients.

Abnormal N-glycosylation has been shown to play an important role in the pathogenesis of multiple diseases. However, little is known about the relationship between N-glycosylation and knee osteoarthritis (KOA) progression at the tissue level. Thus, the aim of this study was to quantify the cartilage histomorphometric changes in formalin-fixed paraffin-embedded (FFPE) tissue collected from the lateral and medial compartments of the tibial plateau KOA patients (n = 8). Subsequently, N-glycans were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) followed by in situ MS/MS fragmentation. Overall, the Osteoarthritis Research Society International (OARSI) histological grade and cartilage surface fibrillation index were significantly higher, and chondrocyte size in the superficial zone was much larger, for the medial high-loaded cartilage compared to the lateral less-loaded cartilage. Among 92 putative N-glycans observed by MALDI-MSI, 3 complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, and 1 oligomannose-type N-glycan, (Hex)9(HexNAc)2, were significantly higher in intensity in the medial cartilage compared to the lateral cartilage, whereas 2 tetra-antennary fucosylated-type N-glycans, (Hex)3(HexNAc)6(Fuc)2 and (Hex)3(HexNAc)6(Fuc)3, were significantly higher in intensity in the lateral cartilage than the medial cartilage. Our findings indicate that complex-type N-glycans are associated with higher severity of cartilage degeneration and may influence the cellular processes of KOA.

High-Resolution N-Glycan MALDI Mass Spectrometry Imaging of Subchondral Bone Tissue Microarrays in Patients with Knee Osteoarthritis.

N-glycan alterations contribute to the progression of several joint diseases, including knee osteoarthritis (KOA). However, molecular changes in KOA subchondral trabecular bone, when exposed to different joint loading forces, are still unknown. The aim of this study was, therefore, to demonstrate the feasibility to differentiate N-glycan changes in subchondral trabecular bone from four different joint loading forces of the tibial plateau regions (i.e., Lateral Anterior (L-A), Lateral Posterior (L-P), Medial Anterior (M-A), and Medial Posterior (M-P)) in KOA patients (n = 10) using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) at 20 µm spatial resolution. The degree of cartilage degeneration was evaluated histologically, and the subchondral bone tissue microarrays (TMAs) were subsequently manually constructed from formalin-fixed paraffin-embedded (FFPE) KOA osteochondral (i.e., cartilage-subchondral bone) tissues. Overall, the Osteoarthritis Research Society International (OARSI) histological grade was significantly higher and the size of chondrocytes in the superficial zone was much larger for both M-A and M-P compared to L-A and L-P of cartilage (p = 0.006, p = 0.030, p = 0.028, and p = 0.010; respectively). Among the 65 putative N-glycans observed by MALDI-MSI, 2 core fucosylated bi-antennary N-glycans, m/z 1809.64; (Hex)5(HexNAc)4(Fuc)1 and 2100.73; (NeuAc)1(Hex)5(HexNAc)4(Fuc)1, were significantly higher in intensity in M-A compared to L-A of the trabecular bone (p = 0.027, and p = 0.038, respectively). These N-glycans were then further structurally characterized by in situ MS/MS fragmentation post-MALDI-MSI. Our results demonstrate, for the first time, N-glycan alterations can occur at different joint loading forces in the KOA tibial plateau and the feasibility of subchondral bone TMA construction for N-glycan MALDI-MSI.

Acute C-Terminal Crosslinking Telopeptide of Type I Collagen (CTX-1) Suppression with Milk Calcium or Calcium Carbonate Is Independent of Visceral Fat in a Randomized Crossover Study in Lean and Overweight Postmenopausal Women.

BACKGROUND: Postmenopausal women with higher visceral adipose tissue (VAT) present with suppressed bone resorption (lower C-terminal crosslinking telopeptide of type I collagen; CTX-1) and turnover (lower osteocalcin) but whether this blunts the effect of calcium is unknown. OBJECTIVES: The primary outcome of this study was the effect of VAT on changes in CTX-1 after intake of 2 forms of calcium. Secondary outcomes included changes in parathyroid hormone (PTH), serum calcium, phosphorus, and alkaline phosphatase (ALP). METHODS: Randomized open three period crossover trial conducted between 2017 and 2019 at the University of South Australia among 77 lean and overweight postmenopausal women (53-79 y) with BMI <25 kg/m2 and >27 kg/m2, respectively. Participants received a single dose of milk (1000 mg calcium), calcium carbonate tablet (1000 mg calcium), and fruit juice (no calcium) in random order with a 7-d washout period. Blood samples were collected at baseline and hourly for 5 h. Data was analyzed by repeated measures ANOVA of log-transformed data. RESULTS: At baseline, women with higher VAT had significantly lower CTX-1 and higher PTH (44% lower and 30% higher, respectively, between Q4 and Q1, P < 0.0001). VAT had no influence on the acute changes in CTX-1 or PTH with calcium or juice. A suppression of 44% in CTX-1 was seen with calcium carbonate and milk and a suppression of 18% with juice. PTH was suppressed more with calcium carbonate (47%) compared to milk (22%). Milk calcium reduced PTH and CTX-1 at 2 h, whereas calcium carbonate reduced PTH in 1 h. The suppression in CTX-1 was slower with lowest concentrations at 4-5 h. CONCLUSIONS: Intake of 1000 mg calcium from milk or from calcium carbonate is effective in acutely suppressing bone resorption in postmenopausal women irrespective of visceral fat. This trial is registered at http://www.ANZCTR.org.au/ACTRN12617000779370.aspx as ACTRN 12617000779370).

Acute C-Terminal Crosslinking Telopeptide of Type I Collagen (CTX-1) Suppression with Milk Calcium or Calcium Carbonate Is Independent of Visceral Fat in a Randomized Crossover Study in Lean and Overweight Postmenopausal Women.

BACKGROUND: Postmenopausal women with higher visceral adipose tissue (VAT) present with suppressed bone resorption (lower C-terminal crosslinking telopeptide of type I collagen; CTX-1) and turnover (lower osteocalcin) but whether this blunts the effect of calcium is unknown. OBJECTIVES: The primary outcome of this study was the effect of VAT on changes in CTX-1 after intake of 2 forms of calcium. Secondary outcomes included changes in parathyroid hormone (PTH), serum calcium, phosphorus, and alkaline phosphatase (ALP). METHODS: Randomized open three period crossover trial conducted between 2017 and 2019 at the University of South Australia among 77 lean and overweight postmenopausal women (53-79 y) with BMI <25 kg/m2 and >27 kg/m2, respectively. Participants received a single dose of milk (1000 mg calcium), calcium carbonate tablet (1000 mg calcium), and fruit juice (no calcium) in random order with a 7-d washout period. Blood samples were collected at baseline and hourly for 5 h. Data was analyzed by repeated measures ANOVA of log-transformed data. RESULTS: At baseline, women with higher VAT had significantly lower CTX-1 and higher PTH (44% lower and 30% higher, respectively, between Q4 and Q1, P < 0.0001). VAT had no influence on the acute changes in CTX-1 or PTH with calcium or juice. A suppression of 44% in CTX-1 was seen with calcium carbonate and milk and a suppression of 18% with juice. PTH was suppressed more with calcium carbonate (47%) compared to milk (22%). Milk calcium reduced PTH and CTX-1 at 2 h, whereas calcium carbonate reduced PTH in 1 h. The suppression in CTX-1 was slower with lowest concentrations at 4-5 h. CONCLUSIONS: Intake of 1000 mg calcium from milk or from calcium carbonate is effective in acutely suppressing bone resorption in postmenopausal women irrespective of visceral fat. This trial is registered at http://www.ANZCTR.org.au/ACTRN12617000779370.aspx as ACTRN 12617000779370).

Mass spectrometry imaging spatially identifies complex-type N-glycans as putative cartilage degradation markers in human knee osteoarthritis tissue.

N-Glycan alterations contribute to the pathophysiology and progression of various diseases. However, the involvement of N-glycans in knee osteoarthritis (KOA) progression at the tissue level, especially within articular cartilage, is still poorly understood. Thus, the aim of this study was to spatially map and identify KOA-specific N-glycans from formalin-fixed paraffin-embedded (FFPE) osteochondral tissue of the tibial plateau relative to cadaveric control (CTL) tissues. Human FFPE osteochondral tissues from end-stage KOA patients (n=3) and CTL individuals (n=3), aged >55 years old, were analyzed by matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Overall, it was revealed that 22 N-glycans were found in the cartilage region of KOA and CTL tissue. Of those, 15 N-glycans were more prominent in KOA cartilage than CTL cartilage. We then compared sub-regions of KOA and CTL tissues based on the Osteoarthritis Research Society International (OARSI) histopathological grade (1 to 6), where 1 is an intact cartilage surface and 6 is cartilage surface deformation. Interestingly, three specific complex-type N-glycans, (Hex)4(HexNAc)3, (Hex)4(HexNAc)4, and (Hex)5(HexNAc)4, were found to be localized to the superficial fibrillated zone of degraded cartilage (KOA OARSI 2.5-4), compared to adjacent cartilage with less degradation (KOA OARSI 1-2) or relatively healthy cartilage (CTL OARSI 1-2). Our results demonstrate that N-glycans specific to degraded cartilage in KOA patients have been identified at the tissue level for the first time. The presence of these N-glycans could further be evaluated as potential diagnostic and prognostic biomarkers.

A Novel Nanostructured Surface on Titanium Implants Increases Osseointegration in a Sheep Model.

BACKGROUND: A nanostructured titanium surface that promotes antimicrobial activity and osseointegration would provide the opportunity to create medical implants that can prevent orthopaedic infection and improve bone integration. Although nanostructured surfaces can exhibit antimicrobial activity, it is not known whether these surfaces are safe and conducive to osseointegration. QUESTIONS/PURPOSES: Using a sheep animal model, we sought to determine whether the bony integration of medical-grade, titanium, porous-coated implants with a unique nanostructured surface modification (alkaline heat treatment [AHT]) previously shown to kill bacteria was better than that for a clinically accepted control surface of porous-coated titanium covered with hydroxyapatite (PCHA) after 12 weeks in vivo. The null hypothesis was that there would be no difference between implants with respect to the primary outcomes: interfacial shear strength and percent intersection surface (the percentage of implant surface with bone contact, as defined by a micro-CT protocol), and the secondary outcomes: stiffness, peak load, energy to failure, and micro-CT (bone volume/total volume [BV/TV], trabecular thickness [Tb.Th], and trabecular number [Tb.N]) and histomorphometric (bone-implant contact [BIC]) parameters. METHODS: Implants of each material (alkaline heat-treated and hydroxyapatite-coated titanium) were surgically inserted into femoral and tibial metaphyseal cancellous bone (16 per implant type; interference fit) and in tibial cortices at three diaphyseal locations (24 per implant type; line-to-line fit) in eight skeletally mature sheep. At 12 weeks postoperatively, bones were excised to assess osseointegration of AHT and PCHA implants via biomechanical push-through tests, micro-CT, and histomorphometry. Bone composition and remodeling patterns in adult sheep are similar to that of humans, and this model enables comparison of implants with ex vivo outcomes that are not permissible with humans. Comparisons of primary and secondary outcomes were undertaken with linear mixed-effects models that were developed for the cortical and cancellous groups separately and that included a random effect of animals, covariates to adjust for preoperative bodyweight, and implant location (left/right limb, femoral/tibial cancellous, cortical diaphyseal region, and medial/lateral cortex) as appropriate. Significance was set at an alpha of 0.05. RESULTS: The estimated marginal mean interfacial shear strength for cancellous bone, adjusted for covariates, was 1.6 MPa greater for AHT implants (9.3 MPa) than for PCHA implants (7.7 MPa) (95% CI 0.5 to 2.8; p = 0.006). Similarly, the estimated marginal mean interfacial shear strength for cortical bone, adjusted for covariates, was 6.6 MPa greater for AHT implants (25.5 MPa) than for PCHA implants (18.9 MPa) (95% CI 5.0 to 8.1; p < 0.001). No difference in the implant-bone percent intersection surface was detected for cancellous sites (cancellous AHT 55.1% and PCHA 58.7%; adjusted difference of estimated marginal mean -3.6% [95% CI -8.1% to 0.9%]; p = 0.11). In cortical bone, the estimated marginal mean percent intersection surface at the medial site, adjusted for covariates, was 11.8% higher for AHT implants (58.1%) than for PCHA (46.2% [95% CI 7.1% to 16.6%]; p < 0.001) and was not different at the lateral site (AHT 75.8% and PCHA 74.9%; adjusted difference of estimated marginal mean 0.9% [95% CI -3.8% to 5.7%]; p = 0.70). CONCLUSION: These data suggest there is stronger integration of bone on the AHT surface than on the PCHA surface at 12 weeks postimplantation in this sheep model. CLINICAL RELEVANCE: Given that the AHT implants formed a more robust interface with cortical and cancellous bone than the PCHA implants, a clinical noninferiority study using hip stems with identical geometries can now be performed to compare the same surfaces used in this study. The results of this preclinical study provide an ethical baseline to proceed with such a clinical study given the potential of the alkaline heat-treated surface to reduce periprosthetic joint infection and enhance implant osseointegration.

Gelatin-coated indium tin oxide slides improve human cartilage-bone tissue adherence and N-glycan signal intensity for mass spectrometry imaging.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has been successfully used to elucidate the relative abundance and spatial mapping of analytes in situ. Currently, sample preparation workflows for soft formalin-fixed paraffin-embedded (FFPE) tissues, such as brain, liver, kidney, and heart, have been successfully developed. However, hard tissues, such as cartilage-bone, tooth, and whole mouse body, have resulted in the loss of morphology or tissue during the heat-induced epitope retrieval (HIER) step on commercially available conductive indium tin oxide (ITO) slides. Therefore, we have successfully developed a novel and cost-effective sample preparation workflow in which commercial conductive ITO slides are pre-coated with gelatin and chromium potassium sulfate dodecahydrate to improve the adherence of FFPE human osteoarthritic cartilage-bone tissue sections. Gelatin-coated ITO slides also resulted in overall higher N-glycan signal intensity for not only FFPE osteoarthritic cartilage-bone tissue but also for FFPE hard-boiled egg white used as a quality control to assess the quality of sample preparation and MALDI-MSI acquisition. In summary, we present a novel straightforward workflow to improve slide adherence and morphological preservation of FFPE cartilage-bone tissue sections during HIER while improving the signal intensity of N-glycans spatially mapped from the same tissue sections by MALDI-MSI.

Mass Spectrometry Imaging as a Potential Tool to Investigate Human Osteoarthritis at the Tissue Level.

Osteoarthritis (OA) is the most common degenerative joint disease, predicted to increase in incidence year by year due to an ageing population. Due to the biological complexity of the disease, OA remains highly heterogeneous. Although much work has been undertaken in the past few years, underlying molecular mechanisms leading to joint tissue structural deterioration are not fully understood, with only few validated markers for disease diagnosis and progression being available. Discovery and quantitation of various OA-specific biomarkers is still largely focused on the bodily fluids which does not appear to be reliable and sensitive enough. However, with the advancement of spatial proteomic techniques, several novel peptides and proteins, as well as N-glycans, can be identified and localised in a reliable and sensitive manner. To summarise the important findings from OA biomarker studies, papers published between 2000 and 2020 were searched via Google Scholar and PubMed. Medical subject heading (MeSH) terms 'osteoarthritis', 'biomarker', 'synovial fluid', 'serum', 'urine', 'matrix-assisted laser desorption/ionisation', 'mass spectrometry imaging', 'proteomic', 'glycomic', 'cartilage', 'synovium' AND 'subchondral bone' were selectively used. The literature search was restricted to full-text original research articles and written only in English. Two main areas were reviewed for OA biomarker studies: (1) an overview of disease-specific markers detected from different types of OA bio-samples, and (2) an up-to-date summary of the tissue-specific OA studies that have utilised matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI). Overall, these OA biomarkers could provide clinicians with information for better the diagnosis, and prognosis of individual patients, and ultimately help facilitate the development of disease-modifying treatments.

Vitamin D levels in an Australian and New Zealand cohort and the association with pregnancy outcome.

BACKGROUND: Pregnant women are at increased susceptibility to vitamin D deficiency. Hence, there is continuing interest in determining how vitamin D influences pregnancy health. We aimed to compare vitamin D status in two distinct populations of pregnant women in Australia and New Zealand and to investigate the relationship between vitamin D status and pregnancy outcome. This included evaluating possible effect measure modifications according to fetal sex. METHODS: Serum 25-hydroxy vitamin D (25(OH)D) was measured at 15 ± 1 weeks' gestation in 2800 women from Adelaide and Auckland who participated in the multi-centre, prospective cohort SCreening fOr Pregnancy Endpoints (SCOPE) study. RESULTS: Mean serum 25(OH)D in all women was 68.1 ± 27.1 nmol/L and 28% (n = 772) were considered vitamin D deficient (< 50 nmol/L). Serum 25(OH)D was lower in the women recruited in Adelaide when compared to the women recruited in Auckland and remained lower after adjusting for covariates including maternal body mass index and socioeconomic index (Adelaide: 58.4 ± 50.3 vs. Auckland: 70.2 ± 54.5 nmol/L, P < 0.001). A 53% decreased risk for gestational diabetes mellitus (GDM) was observed with high (> 81 nmol/L) "standardised" vitamin D status when compared to moderate-high (63-81 nmol/L, aRR, 0.47; 95% CI: 0.23, 0.96). Marginal sex-specific differences occurred between vitamin D status and GDM: women carrying a female fetus had a 56% decreased risk for GDM in those with low-moderate levels of standardised vitamin D (44-63 nmol/L) compared to moderate-high levels (aRR: 0.44; 95% CI: 0.20, 0.97), whilst in women carrying a male fetus, a 55% decreased risk of GDM was found with high standardised vitamin D when compared to moderately-high vitamin D, but this was not statistically significant (aRR: 0.45; 95% CI: 0.15, 1.38). CONCLUSIONS: High serum 25(OH)D at 15 ± 1 weeks' gestation was shown to be protective against the development of GDM. A possible association between fetal sex, vitamin D status and GDM provides further questions and encourages continual research and discussion into the role of vitamin D in pregnancy, particularly in vitamin D replete populations.

Vitamin D Activity and Metabolism in Bone.

PURPOSE OF REVIEW: In addition to the actions of the endocrine hormone, 1alpha,25-dihydroxyvitamin D (1,25(OH)2D) in stimulating intestinal calcium absorption, the regulation of bone mineral metabolism by 1,25(OH)2D is also considered an important contributor to calcium homeostasis. However, recent evidence suggest that 1,25(OH)2D acting either via endocrine or autocrine pathways plays varied roles in bone, which suggests that vitamin D contributes to the maintenance of bone mineral in addition to its catabolic roles. This review highlights the contrasting evidence for the direct action for vitamin D metabolism and activity in bone. RECENT FINDINGS: Numerous cells within bone express vitamin D receptor (VDR), synthesise and catabolise 1,25(OH)2D via 25-hydroxyvitamin D 1alpha-hydroxylase (CYP27B1), and 25-hydroxyvitamin D 24-hydroxylase (CYP24A1) enzymes, respectively. Recent evidence suggests that all three genes are required to regulate processes of bone resorption, mineralization and fracture repair. The actions of vitamin D in bone appear to negatively or positively regulate bone mineral depending on the physiological and pathological circumstances, suggesting that vitamin D plays pleiotropic roles in bone.

Mechanisms of vitamin D3 metabolite repression of IgE-dependent mast cell activation.

BACKGROUND: Mast cells have gained notoriety based on their detrimental contributions to IgE-mediated allergic disorders. Although mast cells express the vitamin D receptor (VDR), it is not clear to what extent 1α,25-dihydroxyvitamin D3 (1α,25[OH]2D3) or its predominant inactive precursor metabolite in the circulation, 25-hydroxyvitamin D3 (25OHD3), can influence IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo. OBJECTIVE: We sought to assess whether the vitamin D3 metabolites 25OHD3 and 1α,25(OH)2D3 can repress IgE-dependent mast cell activation through mast cell-25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) and mast cell-VDR activity. METHODS: We measured the extent of vitamin D3 suppression of IgE-mediated mast cell degranulation and mediator production in vitro, as well as the vitamin D3-induced curtailment of PCA responses in WBB6F1-Kit(W/W-v) or C57BL/6J-Kit(W-sh/W-sh) mice engrafted with mast cells that did or did not express VDR or CYP27B1. RESULTS: Here we show that mouse and human mast cells can convert 25OHD3 to 1α,25(OH)2D3 through CYP27B1 activity and that both of these vitamin D3 metabolites suppressed IgE-induced mast cell-derived proinflammatory and vasodilatory mediator production in a VDR-dependent manner in vitro. Furthermore, epicutaneously applied vitamin D3 metabolites significantly reduced the magnitude of skin swelling associated with IgE-mediated PCA reactions in vivo; a response that required functional mast cell-VDRs and mast cell-CYP27B1. CONCLUSION: Taken together, our findings provide a mechanistic explanation for the anti-inflammatory effects of vitamin D3 on mast cell function by demonstrating that mast cells can actively metabolize 25OHD3 to dampen IgE-mediated mast cell activation in vitro and in vivo.

The paired-box homeodomain transcription factor Pax6 binds to the upstream region of the TRAP gene promoter and suppresses receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation.

Osteoclast formation is regulated by balancing between the receptor activator of nuclear factor-κB ligand (RANKL) expressed in osteoblasts and extracellular negative regulatory cytokines such as interferon-γ (IFN-γ) and interferon-ß (IFN-ß), which can suppress excessive bone destruction. However, relatively little is known about intrinsic negative regulatory factors in RANKL-mediated osteoclast differentiation. Here, we show the paired-box homeodomain transcription factor Pax6 acts as a negative regulator of RANKL-mediated osteoclast differentiation. Electrophoretic mobility shift and reporter assays found that Pax6 binds endogenously to the proximal region of the tartrate acid phosphatase (TRAP) gene promoter and suppresses nuclear factor of activated T cells c1 (NFATc1)-induced TRAP gene expression. Introduction of Pax6 retrovirally into bone marrow macrophages attenuates RANKL-induced osteoclast formation. Moreover, we found that the Groucho family member co-repressor Grg6 contributes to Pax6-mediated suppression of the TRAP gene expression induced by NFATc1. These results suggest that Pax6 interferes with RANKL-mediated osteoclast differentiation together with Grg6. Our results demonstrate that the Pax6 pathway constitutes a new aspect of the negative regulatory circuit of RANKL-RANK signaling in osteoclastogenesis and that the augmentation of Pax6 might therefore represent a novel target to block pathological bone resorption.

Vitamin D is a potential treatment for the management of gastrointestinal mucositis.

PURPOSE OF THE REVIEW: Gastrointestinal mucositis (GM) is a severe side effect of cancer treatments, negatively impacting the patient's quality of life, and has limited treatment. GM consists of complex biological processes involving apoptosis and inflammation, leading to damage and ulceration of the gastrointestinal system. Recently, vitamin D has been shown to have multiple roles in the gut, including immunomodulation, epithelial barrier regulation and microbiome regulation. Hence, this review aims to put forth vitamin D as a potential therapeutic due to its protective role in the intestine. RECENT FINDINGS: Recent studies have shown that vitamin D can reduce intestinal inflammation by reducing NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation. Vitamin D also targets and maintains the intestinal epithelial barrier via the tight junction protein expression and the inhibition of microbiome translocation. Significant evidence also suggests that vitamin D exerts multiple therapeutic effects through binding to vitamin D receptors (VDRs), and the downregulation of VDR has been associated with the severity of the disease. Additionally, vitamin D deficiency is reported in cancer patients. SUMMARY: There is a dire need for effective treatment for GM, and recent animal and human studies show that vitamin D may be a potential therapy to prevent or treat GM.

Unbiased gene expression analysis of the delayed fracture healing observed in Zucker diabetic fatty rats.

Aims: Impaired fracture repair in patients with type 2 diabetes mellitus (T2DM) is not fully understood. In this study, we aimed to characterize the local changes in gene expression (GE) associated with diabetic fracture. We used an unbiased approach to compare GE in the fracture callus of Zucker diabetic fatty (ZDF) rats relative to wild-type (WT) littermates at three weeks following femoral osteotomy. Methods: Zucker rats, WT and homozygous for leptin receptor mutation (ZDF), were fed a moderately high-fat diet to induce T2DM only in the ZDF animals. At ten weeks of age, open femoral fractures were simulated using a unilateral osteotomy stabilized with an external fixator. At three weeks post-surgery, the fractured femur from each animal was retrieved for analysis. Callus formation and the extent of healing were assessed by radiograph and histology. Bone tissue was processed for total RNA extraction and messenger RNA (mRNA) sequencing (mRNA-Seq). Results: Radiographs and histology demonstrated impaired fracture healing in ZDF rats with incomplete bony bridge formation and an influx of intramedullary inflammatory tissue. In comparison, near-complete bridging between cortices was observed in Sham WT animals. Of 13,160 genes, mRNA-Seq analysis identified 13 that were differentially expressed in ZDF rat callus, using a false discovery rate (FDR) threshold of 10%. Seven genes were upregulated with high confidence (FDR = 0.05) in ZDF fracture callus, most with known roles in inflammation. Conclusion: These findings suggest that elevated or prolonged inflammation contributes to delayed fracture healing in T2DM. The identified genes may be used as biomarkers to monitor and treat delayed fracture healing in diabetic patients.

Antibacterial Nanostructured Surfaces Modulate Protein Adsorption, Inflammatory Responses, and Fibrous Capsule Formation.

The present study interrogates the interaction of highly efficient antibacterial surfaces containing sharp nanostructures with blood proteins and the subsequent immunological consequences, processes that are of key importance for the fate of every implantable biomaterial. Studies with human serum and plasma pointed to significant differences in the composition of the protein corona that formed on control and nanostructured surfaces. Quantitative analysis using liquid chromatography-mass spectrometry demonstrated that the nanostructured surface attracted more vitronectin and less complement proteins compared to the untreated control. In turn, the protein corona composition modulated the adhesion and cytokine expression by immune cells. Monocytes produced lower amounts of pro-inflammatory cytokines and expressed more anti-inflammatory factors on the nanostructured surface. Studies using an in vivo subcutaneous mouse model showed reduced fibrous capsule thickness which could be a consequence of the attenuated inflammatory response. The results from this work suggest that antibacterial surface modification with sharp spike-like nanostructures may not only lead to the reduction of inflammation but also more favorable foreign body response and enhanced healing, processes that are beneficial for most medical devices implanted in patients.

Efficacy and mechanisms of action of vitamin D in experimental polyarthritis.

Vitamin D (vit D) status has been linked to the occurrence and severity of auto-immune and inflammatory diseases. This study evaluates the effects of vit D status on adoptive transfer of adjuvant-induced arthritis (ATA). Rats maintained on diets replete or deficient in vit D3 received arthritogenic thoracic duct cells and were monitored for severity of arthritis. CD45(+) cells obtained by collagenase digestion of hind-paw synovium-rich tissues (SRTs) were analysed to observe the effects of dietary vit D3 on the inflammatory process. Arthritis was more severe in vitamin D-deficient (vit-D(-)) rats compared with vitamin D-replete (vit-D(+)) rats. Resolution was delayed in vit-D(-) rats compared with vit-D(+) rats, or rats fed standard chow. During the acute phase of ATA, numbers of CD45(+) cells were significantly increased in the SRTs of vit-D(-) rats compared with vit-D(+) rats. This increase involved T-cells, polymorphonuclear leukocytes, macrophages, dendritic cells (DCs) and MHC II(hi) cells that resemble activated monocytes. A major difference between the dietary groups was that most DCs at the peak of inflammation in vit-D(-) rats were CD4(-), whereas in convalescent vit-D(+) rats most expressed CD4. Multiple categories of genes expressed by DCs differed between deficient and replete rats, with deficiency being associated with relative upregulation of certain pro-inflammatory genes and replete status being associated with upregulation of genes associated with resolution of inflammation. The findings indicate that ATA is more severe and prolonged in vit-D deficiency, that vit-D deficiency promotes accumulation of CD4(-) DCs in synovium during ATA and that a gene-expression profile is likely to contribute to the observed increased severity and duration of arthritis.

Skeletal response to lentiviral mediated gene therapy in a mouse model of MPS VII.

Mucopolysaccharidosis VII (MPS VII) is an autosomal recessive, lysosomal storage disorder caused by ß-glucuronidase (GUSB) deficiency, resulting in the accumulation of glycosaminoglycans (GAGs), in a variety of cell types. Severe, progressive skeletal pathology, termed dysostosis multiplex, is a prominent clinical feature of MPS VII. We have evaluated a gene therapy protocol for its efficacy in preventing the development and progression of bone pathology in MPS VII mice treated with a lentiviral vector at birth or at 7 weeks. Two weeks after injections, high levels of vector expression were observed in liver, spleen and bone marrow and to a lesser extent in kidney, lung and heart. Widespread clearance of GAG storage was observed in somatic tissues of both groups and some clearance of neuronal storage was observed in mice treated from birth. Micro-CT analysis demonstrated a significant decrease in vertebral and femoral bone mineral volume, trabecular number, bone surface density and cortical bone thickness in both treatment groups. Lumbar and femoral bone lengths were significantly decreased in untreated MPS VII mice, while growth plate heights were increased and these parameters did not change upon treatment. Small improvements in performance in the open field and rotarod behaviour tests were noted. Overall, systemic lentiviral-mediated gene therapy results in a measurable improvement in parameters of bone mass and architecture as well as biochemical and enzymatic correction. Conversely, growth plate chondrocytes were not responsive to treatment, as evidenced by the lack of improvement in vertebral and femoral bone length and growth plate height.

Decreased body weight in young Osterix-Cre transgenic mice results in delayed cortical bone expansion and accrual.

Conditional gene inactivation using the Cre/loxP system has lead to significant advances in our understanding of the function of genes in a wide range of disciplines. It is becoming increasingly apparent in the literature, that Cre transgenic mice may themselves have a phenotype. In the following study we describe the bone phenotype of a commonly used Cre transgenic mouse line to study osteoblasts, the Osx-GFP::Cre (Osx-Cre) mice. Cortical and trabecular bone parameters were determined in the femurs of Osx-Cre mice at 6 and 12 weeks of age by microtomography (µCT). At 6 weeks of age, Osx-Cre mice had reduced body weight by 22% (P < 0.0001) and delayed cortical bone expansion and accrual, characterized by decreases in periosteal circumference by 7% (P < 0.05) and cortical thickness by 11% (P < 0.01), compared to wild type controls. Importantly, the cortical bone phenotype of the skeletally immature Osx-Cre mice at 6 weeks of age could be accounted for by their low body weight. The delayed weight gain and cortical growth of Osx-Cre mice was overcome by 12 weeks of age, with no differences observed between Osx-Cre and wild type controls. In conclusion, Osx-Cre expressing mice display a delayed growth phenotype in the absence of doxycycline treatment, evidenced by decreased cortical bone expansion and accrual at 6 weeks of age, as an indirect result of decreased body weight. While this delay in growth is overcome by adulthood at 12 weeks of age, caution together with appropriate data analysis must be considered when assessing the experimental data from skeletally immature Cre/loxP knockout mice generated using the Osx-Cre mouse line to avoid misinterpretation.

Therapeutic Potential of a Novel Vitamin D3 Oxime Analogue, VD1-6, with CYP24A1 Enzyme Inhibitory Activity and Negligible Vitamin D Receptor Binding.

The regulation of vitamin D3 actions in humans occurs mainly through the Cytochrome P450 24-hydroxylase (CYP24A1) enzyme activity. CYP24A1 hydroxylates both 25-hydroxycholecalciferol (25(OH)D3) and 1,25-dihydroxycholecalciferol (1,25(OH)2D3), which is the first step of vitamin D catabolism. An abnormal status of the upregulation of CYP24A1 occurs in many diseases, including chronic kidney disease (CKD). CYP24A1 upregulation in CKD and diminished activation of vitamin D3 contribute to secondary hyperparathyroidism (SHPT), progressive bone deterioration, and soft tissue and cardiovascular calcification. Previous studies have indicated that CYP24A1 inhibition may be an effective strategy to increase endogenous vitamin D activity and decrease SHPT. This study has designed and synthesized a novel C-24 O-methyloxime analogue of vitamin D3 (VD1-6) to have specific CYP24A1 inhibitory properties. VD1-6 did not bind to the vitamin D receptor (VDR) in concentrations up to 10-7 M, assessed by a VDR binding assay. The absence of VDR binding by VD1-6 was confirmed in human embryonic kidney HEK293T cultures through the lack of CYP24A1 induction. However, in silico docking experiments demonstrated that VD1-6 was predicted to have superior binding to CYP24A1, when compared to that of 1,25(OH)2D3. The inhibition of CYP24A1 by VD1-6 was also evident by the synergistic potentiation of 1,25(OH)2D3-mediated transcription and reduced 1,25(OH)2D3 catabolism over 24 h. A further indication of CYP24A1 inhibition by VD1-6 was the reduced accumulation of the 24,25(OH)D3, the first metabolite of 25(OH)D catabolism by CYP24A1. Our findings suggest the potent CYP24A1 inhibitory properties of VD1-6 and its potential for testing as an alternative therapeutic candidate for treating SHPT.