Decreased Alkaline Phosphatase Research Articles

Hypophosphatasia: A Unique Disorder of Bone Mineralization.

Hypophosphatasia (HPP) is a rare genetic disease characterized by a decrease in the activity of tissue non-specific alkaline phosphatase (TNSALP). TNSALP is encoded by the ALPL gene, which is abundantly expressed in the skeleton, liver, kidney, and developing teeth. HPP exhibits high clinical variability largely due to the high allelic heterogeneity of the ALPL gene. HPP is characterized by multisystemic complications, although the most common clinical manifestations are those that occur in the skeleton, muscles, and teeth. These complications are mainly due to the accumulation of inorganic pyrophosphate (PPi) and pyridoxal-5′-phosphate (PLP). It has been observed that the prevalence of mild forms of the disease is more than 40 times the prevalence of severe forms. Patients with HPP present at least one mutation in the ALPL gene. However, it is known that there are other causes that lead to decreased alkaline phosphatase (ALP) levels without mutations in the ALPL gene. Although the phenotype can be correlated with the genotype in HPP, the prediction of the phenotype from the genotype cannot be made with complete certainty. The availability of a specific enzyme replacement therapy for HPP undoubtedly represents an advance in therapeutic strategy, especially in severe forms of the disease in pediatric patients.

Endothelial to Mesenchymal Transformation-derived Activated Fibroblast Behavior in a 3D Culture Environment

Objective: In calcific aortic valve disease (CAVD), glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) and hyaluronic acid (HA) are present in the normally collagen-rich aortic valve fibrosa near calcified nodules. Previous work using a 3D in vitro model has shown that GAGs can also promote endothelial to mesenchymal transformation (EndMT). The role of mesenchymal transformation in the progression of CAVD remains unclear. The goal of this study is to investigate the role of GAGs and EndMT on valve cell behavior in a 3D, in vitro model of healthy and diseased valves. Methods: Three dimensional in-vitro cultures, representing healthy (collagen 1 hydrogels) or diseased (collagen 1 hydrogels + GAGs) valve tissue, were seeded with Porcine Aortic Valve Endothelial Cells (PAVEC) and Porcine Aortic Valve Interstitial Cells (PAVIC). Cell cultures were grown for 14 days in regular medium and then evaluated for calcified nodule formation, alkaline phosphatase activity, proliferation and protein expression relating to EndMT activity Results: Introduction of GAGs into the in vitro model resulted in decreased alkaline phosphatase activity. Hydrogels containing CS conditions induced the greatest calcification, as measured with Alzarin Red quantification, while gels containing HA conditions did not have calcification that was significantly different than controls. Proliferation results suggest high levels of cell division activity, for both PAVEC and PAVIC in CS conditions. Protein expression analysis indicated increased EndMT in hydrogels containing GAGs. Conclusions: 3D, in vitro models of the aortic valve simulated progression toward late stage CAVD. Further investigation of the downstream role of EndMT within the aortic valve microenvironment is critical to subsequent improvement in treatment options.

Diagnosis of Hypophosphatasia in Adults Presenting With Metatarsal Stress Fracture: Proof-of-Concept for a Case-Finding Strategy.

ABSTRACTHypophosphatasia (HPP) is caused by loss‐of‐function mutations in ALPL resulting in decreased alkaline phosphatase (ALP) activity. Metatarsal stress fracture (MSF) is a common clinical feature of hypophosphatasia in adults. In this study, the primary objectives were to determine whether new cases of ALPL variants could be identified in patients with MSF and who also had serum ALP concentration below the reference range and to phenotype their clinical course. Electronic health records were queried for adult patients with MSF using International Classification of Disease codes (ICD‐9, ICD‐10CM) and ALP measurements. Patients with ALP levels below the normal limit were invited to receive mutational analysis of ALPL and to complete the following surveys: the Short Form 36 version 2 (SF36v2), the Brief Pain Inventory‐Short Form (BPI), and the Health Assessment Questionnaire Disability Index (HAQ‐DI). Cases with and controls without ALPL pathogenic variants were compared by survey scores and clinical variables relevant to fracture. In 1611 patients with MSF presenting to a podiatry clinic (10/1/2011–10/1/2017), 937 had ALP measurement, of whom 13 (1.4%) had ALP levels below the lower normal limit. In eight patients consenting to participate, two had heterozygous pathogenic ALPL variants. ALPL variants were found in 2 of 1611 patients (0.12%) with MSF, 2 patients of 937 (0.21%) in those with MSF and any ALP measurement, and 2 of 13 patients (15%) in MSF and decreased ALP level. Cases versus controls rated lower scores on eight of eight SF36v2 scales (range, 0–100); higher scores for worst pain (8.0 vs. 0.8) and average pain (6.0 vs. 0.7) on the BPI (range, 0–10); and higher standard disability score (1.4 vs. 0) on the HAQ‐DI (range, 0–3). These data provide proof‐of‐concept for HPP case identification in patients presenting to a podiatry clinic with MSF, suggesting a search for historically low ALP levels may be a useful step for consideration of HPP diagnosis, and supports a prospective study to determine an optimal case‐finding strategy. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

In vitro acaricidal activity of green synthesized nickel oxide nanoparticles against the camel tick, Hyalomma dromedarii (Ixodidae), and its toxicity on Swiss albino mice

The green synthesized nanoparticles have been determined as a novel pesticide against arthropod pests. This study was designed to evaluate the in vitro acaricidal activity of green synthesized nickel oxide nanoparticles (NiO NPs) using aqueous extract of Melia azedarach ripened fruits against different developmental stages of the camel tick Hyalomma dromedarii in addition to their toxic effect on laboratory animals. The synthesized NiO NPs were characterized by UV-visible (UV-Vis) spectroscopy, Fourier transforms infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and energy dispersive X-ray spectroscopy (EDS). The UV-Vis spectra of the NiO NPs showed an absorption peak at 307nm. FTIR analysis showed the possible functional groups used for capping and stabilization of NiO NPs with strong bands at 3416.2 and 1626.6cm-1. The SEM images of the NiO NPs exhibited a size ranging from 21 to 35nm. The immersion test was used for the in vitro application of the synthesized NiO NPs on the various tick stages (egg, nymph, larva, and adult). Mortality percentages and LC50 values of each tick stage were calculated. The oviposition and hatchability of the engorged females were monitored for the survived tick after treatment. The LC50 values for NiO NPs on embryonated eggs, larvae, and engorged nymphs were 5.00, 7.15, and 1.90mg/mL, respectively. The egg productive index (EPI), egg number, and hatchability (%) were lower in females treated with the NiO NPs than in control ticks. The toxicity of the NiO NPs on laboratory animals was also investigated using Swiss albino mice by oral dose of 500mg/kg/day administration for five consecutive days. The hematological, biochemical, and histopathological changes were evaluated. The hematological analysis showed significant increase in the level of white blood cells (WBC) and hemoglobin (Hb). Biochemical analysis showed non-significant decrease in alkaline phosphatase (ALP) and alanine amino transferase (ALT). We concluded that NiO NPs have a significant acaricidal activity as demonstrated on eggs, larvae, engorged nymphs, and fully fed females of H. dromedarii. From a toxicological point of view further in vivo investigations are needed to determine the mechanism of toxic effect of NiO NPs.

Oral Acute Toxicity, Influence on the Gastrointestinal Microbiota and in Vivo Anti-Salmonellosis Effect of Zizyphus Lotus (L.) and Ruta Chalepensis (L.) Essential Oils

Introduction: The aim of this study was to evaluate the chemical composition of Z. lotus (ZL) and R. chalepensis (RC) Essential Oils (EOs), the oral acute toxicity, influence on the gastrointestinal (GI) microbiota, and the in vivo anti-salmonellosis effect. Materials and Methods: The EOs were isolated using the steam distillation process, and bioactive components were identified by GC-MS analysis. Oral acute toxicity, influence on the GI flora composition, and the anti-salmonellosis effect were elucidated using in vivo methods on experimental animals. Results: The GC-MS allowed us to identify 33 and 58 components in Z. lotus and R. chalepensis, respectively. Di-isooctyl phthalate (89.857%) was found to be the major compound identified in ZL. The main compounds in RC were 2-undecanone (26.528 %) followed by 2-nonanone (13.404 %). The LD50 of EOs was found to be greater than 5000 mg/kg. Also, no negative influence to intestinal microbiota was detected. An important decrease in S. enterica ssp arizonae cells achieving a bactericidal effect was recorded in rats treated with the EOs of both plants at a dose of 400 mg/kg. In parallel, an important significant (p <0.05) increase in lymphocyte number was observed for all tested animals. A decrease in alkaline phosphatase (ALP), alanine aminotransferase(ALT), and aspartate aminotransferase (AST) levels were observed. Furthermore, a reduced blood sedimentation rate (ESR) was recorded in treated animals. Conclusions: The Z. lotus and R. chalepensis act effectively as anti-salmonellosis agents, which support the use of these plants to cure gastrointestinal infections.

Chlorogenic acid co-administration abates tamoxifen-mediated reproductive toxicities in male rats: An experimental approach.

Reports over the years have demonstrated toxic side effect-including reproductive toxicity- of tamoxifen (TAM), a drug of choice in the management of primary breast cancer. Chlorogenic acid (CGA), a dietary polyphenol, reportedly elicits beneficial pharmacological effects. However, the impact of CGA on TAM-associated reproductive toxicity is absent in the literature. We, therefore, experimented on CGA's effect and TAM-mediated reproductive toxicity in rats. Cohorts of rats were treated with TAM (50mg/kg) or co-treated with CGA (25 or 50mg/kg) for 14 consecutive days. The result showed that treatment of CGA significantly increases testosterone, LH, and FSH levels compared to the TAM group. However, prolactin level was markedly decreased after pretreatment of CGA in TAM-treated rats. CGA abated TAM-induced decreases acid phosphatase, alkaline phosphatase, and antioxidant enzymes in the testis. CGA alleviated TAM-facilitated surges of reactive oxygen and nitrogen species, myeloperoxidase, nitric oxide, interleukin-1β, and tumor necrosis factor-alpha in rats epididymis and testes. Additionally, CGA increased anti-inflammatory cytokine -interleukin-10-, suppressed caspase-3 activity, and reduced pathological lesions in the examined organs of rats co-treated with CGA and TAM. CGA phytoprotective effect improved reproductive function occasioned by TAM-mediated toxicities in rats, by abating oxido-inflammatory damages and downregulating apoptotic responses. PRACTICAL APPLICATIONS: CGA protects against the damaging oxido-inflammatory responses incumbent on TAM metabolism. As an antioxidant abundant in plant-derived foods, CGA reportedly protects against inflammatory damage, hypertension, and neurodegenerative diseases. We present evidence that CGA ameliorates TAM-induced reproductive dysfunction by suppressing oxidative and inflammation stress downregulate apoptosis and improve reproductive function biomarker in rats.

Effect of parathyroidectomy on bone tissue biomarkers and body composition in patients with chronic kidney disease and secondary hyperparathyroidism.

Loss of renal function may induce secondary hyperparathyroidism (s-HPT), which triggers several complications leading to an extreme decline in quality of life and increased mortality in affected patients. We evaluated whether parathyroidectomy (PTx), as surgical treatment for s-HPT, modifies body composition, and hormones involved in the protein-energy metabolism of affected patients. Overall, 30 s-HPT patients were evaluated at two times, before PTx (pre PTx) and 6 months after PTx (post PTx). Patients were evaluated by biochemistry analysis, anthropometry, electrical bioimpedance (BIA), food intake diary, handgrip strength, and modified global subjective nutritional assessment (SGA). After PTx, patients showed decreased serum levels of total and ionic calcium, as well as decreased alkaline phosphatase and PTH, and increased 25 (OH) vitamin D. These results demonstrate that PTx was efficient to correct part of the mineral disorder. We also observed an increase in caloric intake, body weight, body mass index (BMI), phase angle, handgrip strength, SGA score, and a decreasing in the percentage of weight loss. The osteocalcin concentration of both carboxylated (cOC) and undercarboxylated form was diminished post PTx. The cOC correlated with bone metabolism markers and SGA score. PTx modified body composition improving nutritional status and preventing the progression of weight loss with increased of energy intake, BMI, handgrip strength, phase angle of BIA, and SGA score. The present study also suggests an association of cOC with bone markers and SGA score. Further studies are needed to better clarify these associations with larger sample size.

Metabolic status of rabbits under physiological adaptation to cage husbandry technology

The article considers changes in biochemical blood parameters depending on the degree of adaptation process manifesting on the plantar surface of hind limbs of different sex rabbits and physiological state under cage husbandry technology. Groups of stud bucks (1st series), pregnant and lactating does (2nd and 3rd series of experiments) were formed according to the stages of structural changes depending on the stress of physiological regulatory processes of adaptation (the systematics proposed by R.M. Baevsky). The authors studied the following factors in serum: total protein, protein fractions (albumin, α-, β-, γ-globulin), creatinine, total bilirubin, urea, uric acid, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, glucose triacylglycerol and cholesterol. At the stressed stage of regulatory systems the group of bucks showed a reduction of total bilirubin on the background of decrease of ALT, AST and alkaline phosphatase, which can testify to activation of liver function at this stage, and increase in bilirubin, ALT, AST with a simultaneous decrease in alkaline phosphatase, noted at the overstress stage of physiological adaptation regulatory processes, can testify to decrease in the functional activity of hepatocytes.Pregnant rabbits have the most pronounced compensatory reactions in the third stage of the adaptation process: the increase in absolute and relative γ-globulin values indicates the activation of immune factors. An increase in creatinine and urea levels during the gestation period, against a background of developing adaptive changes on the plantar surface of the feet, indicates a decrease in the filtration capacity of the renal tubules.Lactating animals experience an increased physiological load on the liver, which is expressed by an increase in triacylglycerols throughout the adaptation.

Intravascular Lymphoma in Canine - Clinical, Pathological and Immunohistochemical Aspects

Background: Intravascular lymphoma (IL) is a rare disease characterized by presence of neoplastic lymphocytes in the lumen of blood vessels. Any tissue can be affected; however, the most frequently compromised areas of the body are the skin and the central nervous system (CNS). The clinical signs and macroscopic alterations caused by IL are nonspecific and are usually secondary to a continuous proliferative disorder, which leads to occlusion of the blood vessels with consequent thrombosis, hemorrhage and infarction. The objective of this work is to report a case of IL in a dog.Case: An 8-year-old male Rottweiler dog was referred to the Veterinary Hospital of the Federal University of Paraná with a history of cerebellar ataxia that had not improved after treatment. At the neurological examination, cerebellar ataxia and deficit of nasal reflex were detected. A complete blood count showed presence of slight anemia (4.8 million red blood cells/mm3; normal ranges= 5.5-8.5 million/mm3) and thrombocytopenia (176.000/mm3; normal ranges= 250.000-500.000/mm3). Biochemical tests revealed a small increase in alanine aminotransferase (42 IU/L; normal ranges= 14-38 IU/L) and a slight decrease in alkaline phosphatase (49 IU/L; normal ranges= 90-170 IU/L). The cerebrospinal fluid (CSF) exhibited an increased protein concentration (147.3 mg/dL; normal ranges= 18-44 mg/dL) and pleocytosis (8 cells/µL; normal ranges= up to 5 cells/µL). No alterations were observed at radiographic and ultrasonographic exams. A clinical treatment was prescribed; however, in view of the worsening of the clinical signs, euthanasia was performed, and the body was submitted to a Laboratory of Pathology. At necropsy, moderate multifocal ulcers were observed in the oral cavity and ventral side of the tongue; moderately infarcted areas were detected in the spleen. Additional alterations such as fatty liver degeneration, glomerulonephritis, and pulmonary edema were also observed. Consequently, fragments from various tissues were collected, fixed in 10% formaldehyde, and processed for paraffin embedding and microtomy. Sections with a thickness of 5 µm were cut, and stained with hematoxylin and eosin. Microscopically, the cerebral cortex exhibited a marked number of round neoplastic cells with well-defined edges and scant cytoplasm filling the vascular lumen. Similar cells were observed in the lumen of the blood vessels in the cerebellum, spinal cord, liver, lungs, kidneys, and mucocutaneous junction. The cerebellum, mucocutaneous junction, and kidneys were submitted to immunohistochemical evaluation. The results were consistent with T cell lymphoma in the telencephalon, in the mucocutaneous junction, and in the cerebellum; however, the neoplastic cells observed in the kidneys were not positively stained by the antibodies used.Discussion: Since, in this neoplasia, there is no formation of solid tumors as in other lymphomas, the diagnosis is more difficult both clinically and macroscopically, and hence a combination of immunohistochemistry and microscopy are indispensable. Immunohistochemistry for detection of the markers CD3, CD45RA, CD20, PAX5 and CD79a was essential to determine the cell type especially in the mucocutaneous junction, telencephalon, and cerebellum. Absence of staining for any of these markers on the neoplastic cells in the kidneys may be associated with a high degree of cellular undifferentiation, which worsens the prognosis. This case report highlights the importance of the combination of histopathological and immunohistochemical examinations with laboratory tests and the clinical picture of the animal for the diagnosis of this neoplasia, which can be difficult to identify.

Organ weight, serum biochemistry and gut microbial counts of Thryonomys swinderianus fed diet supplemented with Vernonia amygdalina leaf meal

The study investigated the effect of Vernonia amygdalina (VA) leaf supplementation on organ weight and gut microbial count of Thryonomys swinderianus. Thirty-six weaner grasscutters of mixed sexes were allotted randomly to six treatments (A, B, C, D, E, and F) in a completely randomized design for a nutritional trial of 8 weeks. At the end of the experiment, caecum, kidney, liver, lungs, intestine and the heart were excised to determine their weights and the intestinal/caecal contents and scrapings were obtained to determine the fungi and bacteria population. Data collected were analyzed using one-way Analysis of Variance (ANOVA). The VA supplementation had significant effect (p&lt;0.05) on relative organs with a range of 0.47 – 0.60%, 0.40 – 0.62%, 1.20 – 2.83%, 0.52 - 0.58%, 5.53 – 12.91%, 714.00 – 960.00g, 494.00 – 781.00g and 479.00 – 747.00g for lungs, heart, liver, kidney, intestine, live weight, eviscerated and carcass weight of the grasscutter respectively, with a smaller relative weight of the kidney from 3g/kg VA supplementation upwards. The serum biochemical parameters were also significantly affected by VA supplementation. There was a decrease in alkaline phosphatase, alanine aminotransferase and aspartate aminotransferase but were within the normal range required for rodents. Total protein and Albumin were significantly improved by the supplement while total bilirubin was variable with a significant decrease prior to 4gVA supplementation. Microbial populations in the intestine and ceacum were reduced to varying extent by the supplement. It could be concluded that VA supplementation reduced intestinal and caecal microbes and increased the live and carcass weights but decreased relative weights of selected organs in the grasscutter.

Clinical and prognostic value of metabolic disorders in non-alcoholic fatty liver disease

A comprehensive assessment of metabolic parameters in patients with non-alcoholic fatty liver disease and based on them the development of prognostic criteria for the development of liver fibrosis. 288 patients with non-alcoholic fatty liver disease at the stage of steatosis were examined. The patients underwent ultrasound examination of the hepatobiliary system using the SONIX OP apparatus (Canada), the FibroMax test, and liver elastography using the AIXPLORER apparatus (France). The parameters of anthropometry, lipid spectrum, protein and pigment metabolism, cytolysis, cholestasis, bilirubin, insulin in the blood serum were evaluated. The methods of statistical forecasting are used. The clinical picture in patients with non-alcoholic fatty liver disease at the stage of steatosis was nonspecific and was due to the presence of concomitant diseases of the digestive system and the cardiovascular system. A kettle index of up to 30 was observed in 26.5% of patients, from 30 to 34.9 in 37.5%, from 35 to 39.9 in 29.7% of patients, more than 40 in 6.3% of patients. The results obtained during the biochemical analysis of blood serum indicate the presence of atherogenic dyslipidemia, an increase in the level of total protein, bilirubin, transaminases, gamma-glutamyl transpeptidase and a decrease in alkaline phosphatase in relation to the control group. Against the background of elevated glucose levels, an increase in basal insulin levels was observed (p=0.001). HOMA-IR insulin resistance was increased in patients (p=0.01). When analyzing the results of the study, the main prognostic criteria (body mass index and atherogenicity coefficient) in the diagnosis of non-alcoholic fatty liver disease in the stage of steatosis with high specificity (97.8%) and sensitivity (96.2%) were determined. In determining the risk of progression of non-alcoholic fatty liver disease into liver fibrosis, the model of prognostic criteria included insulin and triglycerides with a specificity of 93.8% and a sensitivity of 85.7%. Among many variable factors, we have identified the main prognostic criteria that are highly likely to diagnose non-alcoholic fatty liver disease at an early stage and determine the risk of progression to liver fibrosis.

Effectiveness and safety of obeticholic acid in a Southern European multicentre cohort of patients with primary biliary cholangitis and suboptimal response to ursodeoxycholic acid.

Obeticholic acid (OCA) was recently approved as the only on-label alternative for patients with primary biliary cholangitis (PBC) with intolerance or suboptimal response to ursodeoxycholic acid (UDCA). However, few data are available outside clinical trials. To assess the effectiveness and safety of OCA in a real-world cohort of patients with non-effective UDCA therapy. Open-label, prospective, real-world, multicentre study, enrolling consecutive patients who did not meet Paris II criteria, from 18 institutions in Spain and Portugal. Effectiveness was assessed by the changes in GLOBE and UK-PBC scores from baseline. POISE and Paris II criteria were evaluated after 12months of OCA . Liver fibrosis was evaluated by FIB-4 and AST to platelet ratio index (APRI). One hundred and twenty patients were eligible, median time since PBC diagnosis 9.3 (4.0-13.8) years, 21.7% had cirrhosis, and 26.7% received had previous or concomitant treatment with fibrates. Seventy-eight patients completed at least 1year of OCA. The Globe-PBC score decreased to 0.17 (95% CI 0.05 to 0.28; P=0.005) and the UK-PBC score decreased to 0.81 (95% CI -0.19 to 1.80; P=0.11). There was a significant decrease in alkaline phosphatase of 81.3U/L (95% CI 42.5 to 120; P<0.001), ALT 22.1U/L (95% CI 10.4 to 33.8; P<0.001) and bilirubin 0.12mg/dL (95% CI 0 to 0.24; P=0.044). FIB-4 and APRI remained stable. According to the POISE criteria, 29.5% (23 out of 78) achieved response. The adverse events rate was 35%; 11.67% discontinued (8.3% due to pruritus). This study supports data from phase III trials with significant improvement of PBC-Globe continuous prognostic marker score among OCA-treated patients with good tolerability.

Role of Zip1 in the regulation of NPY expression by MLT to promote fracture healing in rats.

Our previous study documented that melatonin (MLT) induced the osteogenic differentiation of mesenchymal stem cells (MSCs) and promoted the healing of femoral fractures in rats via the neuropeptide Y (NPY)/neuropeptide Y1 receptor (NPY1R) signaling pathway. MLT treatment upregulated the expression of the zinc uptake transporter zinc transporter 1 (Zip1) in nerve cells. Prior research demonstrated that oral zinc upregulated NPY expression. MSCs were isolated from rat bone marrow and identified using flow cytometry in our study. The results showed that MLT treatment upregulated NPY and NPY1R levels in MSCs with osteogenic differentiation, which was accompanied by upregulated Zip1 expression. However, the MLT-induced osteogenic differentiation of MSCs was reversed after interference of Zip1 expression. It was confirmed by the decreased alkaline phosphatase (ALP) level; downregulated activities of type I collagen α1 chain (COL1A1), osteocalcin (OCN), runt-related transcription factor 2 (Runx2) and ALP; and reduced mineralized nodule formation. MLT promoted fracture healing in rats with femoral fracture, which was accompanied by increased expression of NPY and NPY1R and significantly increased expression of Zip1. In contrast, the silencing of Zip1 expression reversed MLT-mediated fracture healing. In summary, Zip1 participated in the regulation of the NPY/NPY1R signaling pathway via MLT to promote the osteogenic differentiation of MSCs and fracture healing.

Mandibular-related bone metabolism in orchiectomized rats treated with sex hormones

ObjectiveThis study aimed to compare the impact of testosterone and estrogen replacement therapy on mandibular bone density and bone-related markers. DesignForty male adult rats were randomly allocated to 4 experimental groups (n = 10/group): Sham (Control); Orchiectomy; Orchiectomy plus testosterone replacement; and orchiectomy plus estradiol replacement. Twenty-four days after orchiectomy, the hemi-mandibles were collected and processed for analysis of microhardness in cortical and trabecular bone, radiographic bone density and histomorphometric evaluation. Serum was collected for the analysis of calcium, phosphorus, alkaline phosphatase and magnesium. ResultsThe orchiectomy group had the lowest mandibular bone density (p < 0.01) and also their serum levels of alkaline phosphatase were higher than all other experimental groups (p < 0.001). Estradiol replacement significantly reduced microhardness when compared to orchiectomy in cortical bone (p < 0.05). Both testosterone and estrogen replacement reverted orchiectomy impact on this parameter (p < 0.01); and decreased alkaline phosphatase to levels comparable to the Sham-Control group. The effect of estrogen was more pronounced than testosterone, and a statistically significant difference was observed between Sham-Control and testosterone replacement (p < 0.05) but not between Sham-Control and estradiol replacement groups. ConclusionOur findings demonstrated that both estradiol and testosterone replacement therapies play a role in mandibular bone metabolism, but suggest different pathways.

Effects of cadmium on osteoblast cell line: Exportin 1 accumulation, p-JNK activation, DNA damage and cell apoptosis

Cadmium is an environmental metal pollutant that has been a focus of research in recent years, which is reported to cause bone disease; however, its skeletal toxicity and the mechanism involved are not yet fully known. Therefore, this study used MC3T3-E1 subclone 14 cells to determine the mechanism of cadmium toxicity on bone. Cadmium chloride (Cd) significantly reduced cell viability in a concentration-dependent manner. Exposure to Cd inhibited osteoblast-related proteins (Runx2, Col-1, STC2) and decreased alkaline phosphatase (ALP) activity. Cd caused Exportin-1 accumulation and induced DNA damage. Cd significantly down-regulated caspase 9 and induced cleaved-PARP, cleaved-caspase 3 protein level. Treatment with JNK inhibitor, SP600125, suppressed cadmium-induced elevation in the ratio of phosphorylation of JNK to JNK. Inhibition of caspase with pan-caspase inhibitor, Z-VAD-FMK, prevented MC3T3-E1 subclone 14 cells from cadmium-induced reduction of Runx2, STC2, caspase 9, and accumulation of cleaved PARP and cleaved caspase 3. Cd-induced cell survival enhanced by SP600125 but rescued by Z-VAD-FMK or KPT-335. These results suggest that cadmium cytotoxicity on bone involved exportin 1 accumulation, phosphorylation of JNK, induction of DNA damage and pro-apoptosis, which was induced by activation of caspase-dependent pathways.

WWP2 Promotes Odontoblastic Differentiation by Monoubiquitinating KLF5

WW domain-containing E3 Ub-protein ligase 2 (WWP2) belongs to the homologous to E6AP C-terminus (HECT) E3 ligase family. It has been explored to regulate osteogenic differentiation, chondrogenesis, and palatogenesis. Odontoblasts are terminally differentiated mesenchymal cells, which contribute to dentin formation in tooth development. However, it remained unknown whether WWP2 participated in odontoblast differentiation. In this study, WWP2 was found to be expressed in mouse dental papilla cells (mDPCs), odontoblasts, and odontoblastic-induced mDPCs by immunohistochemistry and Western blotting. Besides, WWP2 expression was decreased in the cytoplasm but increased in the nuclei of differentiation-induced mDPCs. When Wwp2 was knocked down, the elevated expression of odontoblast marker genes (Dmp1 and Dspp) in mDPCs induced by differentiation medium was suppressed. Meanwhile, a decrease of alkaline phosphatase (ALP) activity was observed by ALP staining, and reduced formation of mineralized matrix nodules was demonstrated by Alizarin Red S staining. Overexpression of WWP2 presented opposite results to knockdown experiments, suggesting that WWP2 promoted odontoblastic differentiation of mDPCs. Further investigation found that WWP2 was coexpressed and interacted with KLF5 in the nuclei, leading to ubiquitination of KLF5. The PPPSY (PY2) motif of KLF5 was essential for its physical binding with WWP2. Also, cysteine 838 (Cys838) of WWP2 was the active site for ubiquitination of KLF5, which did not lead to proteolysis of KLF5. Then, KLF5 was confirmed to be monoubiquitinated and transactivated by WWP2, which promoted the expression of KLF5 downstream genes Dmp1 and Dspp. Deletion of the PY2 motif of KLF5 or mutation of Cys838 of WWP2 reduced the upregulation of Dmp1 and Dspp. Besides, lysine (K) residues K31, K52, K83, and K265 of KLF5 were verified to be crucial to WWP2-mediated KLF5 transactivation. Taken together, WWP2 promoted odontoblastic differentiation by monoubiquitinating KLF5.

Alkaline Phosphatase And Prostate-Specific Antigen Dynamics And Survival Outcomes In Patients With Metastatic Prostate Cancer Receiving Radium-223

Radium-223 (Ra-223) is a targeted alpha-emitter for metastatic castrate-resistant prostate cancer (mCRPC) patients that demonstrated overall survival (OS) benefit compared to placebo in the ALSYMPCA trial. A secondary analysis of the ALSYMPCA trial demonstrated that decreased alkaline phosphatase (ALP) values at 12 weeks after treatment initiation were associated with improved OS. We further investigate prostate-specific antigen (PSA) and ALP dynamics in a large real-world cohort of patients with mCRPC receiving Radium-223. The Flatiron Health database is a nationwide longitudinal de-identified database derived from electronic health record (EHR) data from 280+ cancer clinics (∼800 sites of care) representing 2.2 million patients in the United States. We included patients with mCRPC treated with Ra-223 between 2013 and 2017 with complete ALP and PSA data. We specified a priori the following factors in a Cox proportional hazards regression for OS: age, performance status, year of treatment, baseline PSA and ALP, occurrence of a PSA or ALP decrease at any point during treatment or within 12 weeks of last administration, timing of response (before cycle 3 versus after cycle 3). Date of first Radium-223 administration was considered the index date for time-dependent analyses. We analyzed data from 586 patients with mCRPC receiving Ra-223 at a median follow-up of 9.8 months, with 252 patients (29.7%) having received prior docetaxel or cabazitaxel. In total, 447 patients (76.3%) achieved any ALP decrease and 250 patients (42.3%) achieved an ALP decrease ≥30%. Of those who achieved an ALP decrease, the response occurred before cycle 3 in 301 patients (67.3%). The median percent change from baseline to ALP nadir was 27.9% (interquartile range [IQR] 10.3% to 46.8%). With respect to PSA, 192 patients (32.7%) achieved any PSA decrease and 124 patients (21.1%) achieved a PSA decrease ≥30%. The median percent change from baseline to PSA nadir was 43.9% (IQR 19.9% to 80.4%). In our survival analysis, we identified age, performance status, ALP value at baseline, PSA value at baseline, presence of an ALP decrease (hazard ratio [HR] 0.61; 95% CI 0.42-0.90; p = 0.012), and presence of a PSA decrease (HR 0.47; 95% CI 0.33-0.65; p = 0.001) as significant predictors of OS. Notably, timing of ALP decrease before or after cycle 3 (HR 0.76; 95% CI 0.57-1.02; p = .066) was not significantly associated with survival. We analyzed PSA and ALP dynamics in the largest reported series of patients receiving Ra-223 based on real-world data. We identified baseline ALP and PSA values, presence of an ALP decrease, and presence of a PSA decrease as predictors of OS. Approximately one-third of patients will have a delayed ALP response after cycle 3; however, timing of ALP response was not significantly associated with OS.

Signal transducer and activator of transcription 3 positively regulates osteoblastic differentiation in MC3T3-E1 cells.

Signal transducer and activator of transcription 3 (STAT3) plays a pivotal role in osteoblastic differentiation. However, the exact role of STAT3 in osteogenic differentiation of the pre-osteoblastic cell line MC3T3-E1 is still controversial. In this study, we demonstrated that eradication of STAT3 signaling by the inhibitors cryptotanshinone (CPT, a STAT3-specific inhibitor) or STAT3 siRNA both suppressed osteogenic differentiation of MC3T3-E1 cells, with a decrease in alkaline phosphatase (ALP) activity, protein expressions of the osteogenic differentiation markers Collagen I (ColI), ALP, and osteocalcin (OCN), and reduced matrix mineralization capacity at the terminal stage of osteogenic differentiation. However, the inhibition of STAT3 by CPT did not affect MC3T3-E1 cell proliferation. To further clarify the effect of STAT3 on osteogenic differentiation of MC3T3-E1 cells, we forced STAT3 expression and found that this ameliorated osteogenic differentiation. Thus, our results confirmed that STAT3 is a likely positive regulator of osteogenic differentiation in MC3T3-E1 cells. These findings may provide a basis for the development of more efficient and controllable protocols for osteoblastic differentiation and facilitate their use in regenerative medicine. In addition, our results provide novel insights into the effect of the STAT3 antagonist CPT on modulation of osteogenesis.

Transcutaneous Neuromodulation improved inflammation and sympathovagal ratio in patients with primary biliary ssscholangitis and inadequate response to Ursodeoxycholic acid: a pilot study

BackgroundAt present, ursodeoxycholic acid (UDCA) is internationally recognized as a therapeutic drug in clinic. However, about 40% Primary Biliary Cholangitis (PBC) patients are poor responders to UDCA. It has been demonstrated that Transcutaneous Neuromodulation (TN) can be involved in gut motility, metabolism of bile acids, immune inflammation, and autonomic nerve. Therefore, this study aimed to explore the effect of TN combined with UDCA on PBC and related mechanisms.MethodsAccording to inclusion and exclusion criteria, 10 healthy volunteers and 15 PBC patients were recruited to control group and TN group, respectively. PBC patients were alternately but blindly assigned to group A (TN combined with UDCA) and group B (sham-TN combined with UDCA), and a crossover design was used. The TN treatment was performed via the posterior tibial nerve and acupoint ST36 (Zusanli) 1 h twice/day for 2 weeks. T test and nonparametric test were used to analyze the data.Results1. TN combined with UDCA improved the liver function of PBC patients shown by a significant decrease of alkaline phosphatase and gamma-glutamyltransferase (γ-GT) (P < 0.05). 2. The treatment also decreased serum IL-6 levels (P < 0.05), but not the level of Tumor Necrosis Factor-α, IL-1β or IL-10. 3. TN combined with UDCA regulated autonomic function, enhanced vagal activity, and decreased the sympathovagal ratio assessed by the spectral analysis of heart rate variability (P < 0.05). 4. There was no change in 13 bile acids in serum or stool after TN or sham-TN.ConclusionsTN cssombined with UDCA can significantly improve the liver function of PBC patients. It is possibly via the cholinergic anti-inflammatory pathway. TN might be a new non-drug therapy for PBC. Further studies are required.Trial registrationThe study protocol was registered in Chinese Clinical Trial Registry (number ChiCTR1800014633) on 25 January 2018.

Toxicity study of Chlorella vulgaris water extract on female Sprague Dawley rats by using the Organization for Economic Cooperation and Development (OECD) Guideline 420

Chlorella vulgaris, a coccoid green eukaryotic microalga, is a popular food supplement and has been marketed as a nutritional supplement in Asia. Despite numerous reports on the antioxidant property of this species, its toxic effect has not been extensively studied. The aim of this study was to determine the acute oral toxic effect of C. vulgaris on female Sprague Dawley (SD) rats. An acute oral toxicity assessment of C. vulgaris was performed based on the Organization for Economic Cooperation and Development (OECD) Guideline 420. These guidelines are divided into a sighting study and a main study. In the sighting study, C. vulgaris extract was administered orally in sequential doses of 5, 50, 300 and 2000 mg kg−1 body weight (BW) with one rat used for each dosage. Clinical observation and mortality were observed for each rat to monitor signs of toxicity for 14 days. BW and relative organ weight (ROW) were recorded. Blood was collected for liver function test (LFT). Gross pathological evaluations were performed after euthanasia of rats. Haematoxylin and eosin (H&E) staining of the liver and kidney was also performed. The main study was performed based on the highest dose in the sighting study that did not exhibit any signs of toxicity and mortality. For 14 days, clinical observations, BW, and ROW were recorded. Blood from rats in the main study was also measured for LFT, and gross pathological evaluations and H&E staining of the liver and kidney were also performed. The highest dose of 2000 mg kg−1 BW C. vulgaris was used in the main study. No toxicity and mortality were observed in both the sighting study and the main study. Increases in BW were observed in both studies. Gross pathological evaluations showed no signs of organ abnormalities on each organ in the sighting and main studies. The ROW also showed no differences. The values of biochemical parameters in the liver function test showed no differences in the sighting study. However, in the main study, significant decreases in alkaline phosphatase (ALP) and aspartate aminotransferase (AST) were observed on day 14 compared with day 0 (p < 0.05). Chlorella vulgaris showed no toxicity at the dose of 2000 mg kg−1 BW. In conclusion, C. vulgaris can be categorized as unclassified according to the Globally Harmonised Classification System (GHS) for chemical substances and mixtures.