Adult-onset hypophosphatemic osteomalacia as a cause of widespread musculoskeletal pain: A retrospective case series of single center experience.

BACKGROUND: Osteomalacia (OM) is frequently confused with various musculoskeletal or other rheumatic diseases, especially in patients with adult-onset widespread musculoskeletal pain because of its low prevalence and non-specific manifestations. AIM: To facilitate the early diagnosis and etiology-specific treatment of adult-onset hypophosphatemic OM. METHODS: A retrospective review of medical records was performed to screen adult patients who visited a physiatry locomotive medicine clinic (spine and musculoskeletal pain clinic) primarily presenting with widespread musculoskeletal pain at a single tertiary hospital between January 2011 and December 2019. We enrolled patients with hypophosphatemia, high serum bone-specific alkaline phosphatase levels, and at least one imaging finding suggestive of OM. RESULTS: Eight patients with adult-onset hypophosphatemic OM were included. The back was the most common site of pain. Proximal dominant symmetric muscle weakness was observed in more than half of the patients. Bone scintigraphy was the most useful imaging modality for diagnosing OM because radiotracer uptake in OM showed characteristic patterns. Six patients were diagnosed with adefovir (ADV)-induced Fanconi syndrome, and the other two patients were diagnosed with tumor-induced OM and light-chain nephropathy, respectively. After phosphorus and vitamin D supplementation and treatment for the underlying etiologies, improvements in pain, muscle strength, and gait were observed in all patients. CONCLUSION: Mechanical pain characteristics, hypophosphatemia, and distinctive bone scintigraphy patterns are the initial diagnostic indicators of adult-onset hypophosphatemic OM. ADV-induced Fanconi syndrome is the most common etiology of hypophosphatemic OM in hepatitis B virus-endemic countries.

Expression of Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2) by human and rabbit meibomian glands and meibocytes.

PURPOSE: Previously, we showed that Acyl-CoA wax-alcohol acyltransferase 2 (AWAT2), an essential enzyme required for meibum wax ester synthesis, was not expressed by immortalized human meibomian gland epithelial cells (hMGEC) in culture. To begin to understand the mechanisms controlling AWAT2 expression, we have analyzed its expression in human and rabbit meibomian glands and cultured meibocytes. METHODS: Rabbit meibocyte progenitor cells (rMPC) were first grown in Cnt-BM.1 basal medium (Cellntec) supplemented with rhEGF, FGF10, and ROCK inhibitor (Y-27632 dihydrochloride), and then passed at 70-80% confluency with Accutase. Differentiation of rMPC to meibocytes (rMC) was induced by removal of Y-27632 and addition of 1 mM calcium with and without PPARγ agonists. RNA from the tissue, primary, passaged rMPC and differentiated rMC were obtained for AWAT2 qPCR analysis. Proteins and cells were evaluated for western blotting and neutral lipid synthesis, respectively. For comparison, human meibomian glands were separated for RNA and protein analysis. hMGEC was cultured to collect RNA and protein. RESULTS: Rabbit rMPCs were successfully grown, passaged, and differentiated, showing a significant increase in lipid droplet accumulation. AWAT2 RNA was highly expressed in tissue but showed a -16.9 log2 fold decrease in primary and passaged rMPCs and was not induced by differentiation to rMC. By comparison, human meibomian glands showed high expression of AWAT2, and hMGEC expressed non-detectable levels of AWAT2 transcripts or protein. CONCLUSIONS: AWAT2 expression is lost in cultured rMPC and rMC suggesting that cells in culture do not undergo complete meibocyte differentiation and require yet to be identified culture conditions.

Preparation and characterization of 3D human glioblastoma spheroids using an N-octanoyl glycol chitosan hydrogel.

The current 2D culture model systems developed for drug screening are not sufficient to reflect the characteristics of in vivo solid tumors. Therefore, more effective in vitro tumor model systems must be developed for translational studies on therapeutic drug screening and testing. Herein, we report a new ultra-low adhesion (ULA) hydrogel for generating 3D cancer cell spheroids as tumor models in vitro. N-octanoyl glycol chitosan (OGC) was synthesized and coated onto the surface of a typical cell culture dish. Cell spheroids were effectively formed on the OGC-coated surface, and phenotypes of the tumor cells were well maintained during culture. More importantly, U373-MG cells cultured on OGC-coated plates were more resistant to doxorubicin than cells cultured on typical plates. Our OGC-based ULA system may offer a convenient method for 3D cell culture to provide enhanced performance in cancer research, drug screening and toxicology.

Effectiveness of nivolumab versus regorafenib in hepatocellular carcinoma patients who failed sorafenib treatment.

BACKGROUND/AIMS: Several treatment options are currently available for patients with hepatocellular carcinoma (HCC) failing previous sorafenib treatment. We aimed to compare the effectiveness of regorafenib and nivolumab in these patients. METHODS: Consecutive HCC patients who received regorafenib or nivolumab after failure of sorafenib treatment were included. Primary endpoint was overall survival (OS) and secondary endpoints were time to progression, tumor response rate, and adverse events. Inverse probability of treatment weighting (IPTW) using the propensity score was conducted to reduce treatment selection bias. RESULTS: Among 150 study patients, 102 patients received regorafenib and 48 patients received nivolumab. Median OS was 6.9 (95% confidence interval [CI], 3.0-10.8) months for regorafenib and 5.9 (95% CI, 3.7-8.1) months for nivolumab (P=0.77 by log-rank test). In multivariable analysis, nivolumab was associated with prolonged OS (vs. regorafenib: adjusted hazard ratio [aHR], 0.54; 95% CI, 0.30-0.96; P=0.04). Time to progression was not significantly different between groups (nivolumab vs. regorafenib: aHR, 0.82; 95% CI, 0.51-1.30; P=0.48). HRs were maintained after IPTW. Objective response rates were 5.9% and 16.7% in patients treated with regorafenib and nivolumab, respectively (P=0.04). CONCLUSION: After sorafenib failure, the use of nivolumab may be associated with improved OS and better objective response rate as compared to using regorafenib.

A microfluidic gradient device for drug screening with human iPSC-derived motoneurons.

We developed a microfluidic gradient device to utilize as a drug screening system with human induced pluripotent stem cell (hiPSC)-derived motoneurons. The microfluidic channel was asymmetrically designed to generate the concentration gradients and a micropillar array was used to trap and culture the motoneuron spheroids containing motoneurons for 9 days. We optimized the concentration gradients in the microfluidic device using a computational fluid dynamics (CFD) model. We also observed that the motoneuron spheroid-derived neurite network was generated in response to the concentration gradients of riluzole in the microfluidic device. Therefore, this microfluidic gradient device could be useful for screening of various drugs for neurological disease applications.

Dynamin-related protein 1 controls the migration and neuronal differentiation of subventricular zone-derived neural progenitor cells.

Mitochondria are important in many essential cellular functions, including energy production, calcium homeostasis, and apoptosis. The organelles are scattered throughout the cytoplasm, but their distribution can be altered in response to local energy demands, such as cell division and neuronal maturation. Mitochondrial distribution is closely associated with mitochondrial fission, and blocking the fission-promoting protein dynamin-related protein 1 (Drp1) activity often results in mitochondrial elongation and clustering. In this study, we observed that mitochondria were preferentially localized at the leading process of migratory adult neural stem cells (aNSCs), whereas neuronal differentiating cells transiently exhibited perinuclear condensation of mitochondria. Inhibiting Drp1 activity altered the typical migratory cell morphology into round shapes while the polarized mitochondrial distribution was maintained. With these changes, aNSCs failed to migrate, and neuronal differentiation was prevented. Because Drp1 blocking also impaired the mitochondrial membrane potential, we tested whether supplementing with L-carnitine, a compound that restores mitochondrial membrane potential and ATP synthesis, could revert the defects induced by Drp1 inhibition. Interestingly, L-carnitine fully restored the aNSC defects, including cell shrinkage, migration, and impaired neuronal differentiation. These results suggest that Drp1 is required for functionally active mitochondria, and supplementing with ATP can restore the defects induced by Drp1 suppression.

Effects of extradural cortical stimulation on motor recovery in a rat model of subacute stroke.

PURPOSE: Previous studies demonstrated that administering extradural cortical stimulation (ECS) to rats during the acute phase of a photothrombotic infarct enhances motor recovery. However, the effect of ECS during the subacute phase was unknown. We aimed to evaluate the effects of ECS on motor recovery in a rat model of subacute photothrombotic stroke. METHODS: Photothrombotic ischemic injury to the left sensorimotor cortex (SMC) was induced in 41 male Sprague-Dawley rats using Rose-bengal dye (20 mg/kg) and cold light. The rats were randomly divided into two groups: ECS on infarcted SMC (ECS group) and no ECS on infarcted SMC (non-stimulated group). The ECS group received continuous ECS for 14 days starting from day 5 after the stroke onset. Behavioral training with the single-pellet reaching task (SPRT) was performed daily for all of the rats from the fifth day after stroke onset. After 19 days, brain sections were immunostained to allow the quantification of infarct volumes and the evaluation of the neuronal markers. RESULTS: The SPRT scores showed significantly faster and greater improvement in the ECS group than in the non-stimulated group. There were no significant differences in infarct size. However, in the ECS group, significantly more doublecortin-labeled cells were identified close to the penumbra region of the cerebral cortex. CONCLUSIONS: ECS in the subacute phase improved the behavior motor function in the stroke rat model, and induced a significant axonal sprouting in the peri-infarct area.

Altered expression of synaptotagmin 13 mRNA in adult mouse brain after contextual fear conditioning.

Contextual fear memory processing requires coordinated changes in neuronal activity and molecular networks within brain. A large number of fear memory-related genes, however, still remain to be identified. Synaptotagmin 13 (Syt13), an atypical member of synaptotagmin family, is highly expressed in brain, but its functional roles within brain have not yet been clarified. Here, we report that the expression of Syt13 mRNA in adult mouse brain was altered following contextual fear conditioning. C57BL/6 mice were exposed to a novel context and stimulated by strong electrical footshock according to a contextual fear conditioning protocol. After 24 h, the mice were re-exposed to the context without electrical footshock for the retrieval of contextual fear memory. To investigate the relationship between Syt13 and contextual fear memory, we carried out in situ hybridization and analyzed gene expression patterns for Syt13 at four groups representing temporal changes in brain activity during contextual fear memory formation. Contextual fear conditioning test induced significant changes in mRNA levels for Syt13 within various brain regions, including lateral amygdala, somatosensory cortex, piriform cortex, habenula, thalamus, and hypothalamus, during both acquisition and retrieval sessions. Our data suggest that Syt13 may be involved in the process of contextual fear memory.

Effect of retinoic acid on epithelial differentiation and mucin expression in primary human corneal limbal epithelial cells.

PURPOSE: Retinoic acid (RA) is essential for epithelial differentiation and maintenance of the mucous phenotype. This study investigated the effect of RA on corneal epithelial differentiation and mucin expression in a primary human corneal limbal epithelial cell (HCLEC) culture model. METHODS: HCLECs were grown in RA-supplemented media at various concentrations (0, 10(-9) to 10(-6) M). Stratified HCLECs were examined using immunohistochemical or immunofluorescent staining for p63, ABCG2, CK3, CK19, and Western blotting for ABCG2 and CK12 to assess differentiation. Ultrastructural morphology was investigated using scanning and transmission electron microscopy. They were incubated with rose bengal dye to examine barrier function. The effects of RA on the expression of MUC1, -4, and -16 were analyzed by immunohistochemistry, quantitative real-time PCR and Western blot analysis. RESULTS: HCLEC grown without RA showed hyperkeratosis, whereas those grown with 10(-8) to 10(-7) M RA induced non-keratinized stratified epithelium with a normal appearance. Under these conditions, p63, ABCG2, CK3, CK19, MUC1, -4, and -16 staining patterns were similar to in vivo limbal epithelium. A higher concentration (10(-6) M) of RA resulted in abnormal differentiation. HCLECs grown with RA were tightly apposed and maintained intact barrier function against dye penetration. In addition, MUC1, -4, and -16 expressions were highly associated with RA concentrations. CONCLUSIONS: This study showed that cultured HCLEC could mimic physiologic and functional phenotypes by controlling RA concentrations in medium. Also, our results suggested modulating effect of RA on differentiation and mucin expression in corneal epithelium.

Enhanced hypothalamic leptin signaling in mice lacking dopamine D2 receptors.

The dopamine D(2) receptor (D2R) plays a critical role in diverse neurophysiological functions. D2R knock-out mice (D2R(-/-)) show reduced food intake and body weight while displaying an increased basal energy expenditure level, compared with their wild type littermates. Thus, these mice show a lean phenotype. D2R(-/-) mice displayed increased leptin sensitivity, and leptin injection induced increased phosphorylation of the hypothalamic signal transducer and activator of transcription 3 (STAT3) in D2R(-/-) mice relative to wild type littermates. Using double immunofluorescence histochemistry, we have demonstrated that D2Rs are present in leptin-sensitive STAT3-positive cells in the arcuate nucleus of the hypothalamus and that leptin injection induces STAT3 phosphorylation in hypothalamic neurons expressing D2Rs. Stimulation of D2R by the D2R agonist quinpirole suppressed the leptin-induced STAT3 phosphorylation and nuclear trans-localization of phospho-STAT3 in the hypothalamus of wild type mice. However, this regulation was not detected in the D2R(-/-) mice. Treatment of D2R agonist and antagonist could modulate the leptin-induced food intake and body weight changes in wild type mice but not in D2R(-/-) mice. Together, our findings suggest that the interaction between the dopaminergic system and leptin signaling in hypothalamus is important in control of energy homeostasis.

Regulation of AHI1 expression in adult rat brain: Implication in hypothalamic feeding control.

Recent studies revealed that Abelson helper integration site 1 (AHI1) plays a role in brain development. However, little is known about the role of AHI1 in adult brain. To directly assess the role of AHI1 in the adult brain, we cloned full-length cDNA of rat AHI1 and observed prominent expression of AHI1 in the hypothalamus, which contributes mainly to the control of energy homeostasis. Furthermore, we demonstrated that food deprivation caused induction of AHI1 in the hypothalamus and subsequent re-feeding down-regulated AHI1 expression, suggesting the involvement of AHI1 in feeding control. Moreover, the expression of AHI1 was increased in serum-depleted Neuro2A cells and restored by subsequent insulin treatment. Furthermore, treatment in food-deprived rat with intraperitoneal glucose also reduced the increased AHI1 expression. These results demonstrate that AHI1 expression can be regulated through diet and suggest the novel role of AHI1 in feeding behavior.