In vitro pharmacological effects of peficitinib on lymphocyte activation: a potential treatment for systemic sclerosis with JAK inhibitors.

OBJECTIVES: Peficitinib, a novel Janus kinase (JAK) inhibitor, demonstrated promising results in treating RA in phase 3 clinical trials. This in vitro study was undertaken to characterize the pharmacological properties of peficitinib and investigate the involvement of JAK and signal transducer and activator of transcription (STAT) pathways in the pathological processes of SSc, which is also an autoimmune disease. METHODS: Phosphorylation levels of STAT molecules were assessed in peripheral blood mononuclear cells collected from patients with RA or SSc and healthy subjects, and in skin specimens obtained from 19 patients with SSc. In vitro inhibition of STAT phosphorylation and cytokine/chemokine production by peficitinib, tofacitinib and baricitinib were also characterized. RESULTS: Higher spontaneous STAT1 or STAT3 phosphorylation was observed in peripheral T-cells and monocytes from patients with RA and SSc compared with healthy subjects. In skin sections from patients with SSc, phosphorylated STAT3-positive cells were found in almost all cases, irrespective of disease subtype or patient characteristics. Conversely, phosphorylated STAT1-positive cells were observed only in samples from untreated patients with diffuse disease of short duration. Peficitinib inhibited STAT phosphorylation induced by various cytokines, with comparable efficacy to tofacitinib and baricitinib. Peficitinib also suppressed cytokine and chemokine production by peripheral blood mononuclear cells and skin fibroblasts. CONCLUSION: Our results suggest that JAK/STAT pathways are constitutively activated in SSc and RA, and that the JAK inhibitor may represent a novel therapeutic option for SSc.

Long-term Hyperglycemia Naturally Induces Dental Caries but Not Periodontal Disease in Type 1 and Type 2 Diabetic Rodents.

Periodontal disease (PD) in patients with diabetes is described as the sixth complication of diabetes. We have previously shown that diabetes increases dental caries, and carious inflammation might have a strong effect on the adjacent periodontal tissue in diabetic rodent models. However, the possibility that hyperglycemia may induce PD in diabetic animals could not be completely eliminated. The goal of this study was to confirm the presence of PD in diabetic animal models by preventing carious inflammation with fluoride administration. F344 rats injected with alloxan (type 1 diabetic model) and db/db mice (type 2 diabetic model) were given either tap water alone or tap water containing fluoride. A cariostatic effect of fluoride was evident in the diabetic animals. Meanwhile, fluoride treatment drastically attenuated periodontal inflammation in addition to preventing dental caries. Furthermore, with fluoride treatment, periodontitis was notably nonexistent in the periodontal tissue surrounding the normal molars, whereas the caries-forming process was clearly observed in the teeth that were enveloped with persistent periodontitis, suggesting that enhanced periodontal inflammation might have been derived from the dental caries in the diabetic rodents rather than from the PD. In conclusion, long-term hyperglycemia naturally induces dental caries but not PD in type 1 and type 2 diabetic rodents.

Investigation of Mechanisms for MK-801-Induced Neurotoxicity Utilizing Metabolomic Approach.

Single treatment of rats with the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801 induces neuronal cell degeneration and death in the retrosplenial/posterior cingulate cortex (RS/PC) region, along with local cerebral glucose utilization. However, the relationship between this neuronal cell degeneration and death and glucose utilization remains unclear. To investigate the mechanism of MK-801-induced neurotoxicity and its relation to glucose utilization, changes in endogenous metabolites in the RS/PC region of MK-801 treated rats were assessed using metabolomics. Inverse correlation between citrulline and arginine levels suggested increased nitric oxide (NO) production. In addition, decreased levels of purine metabolites suggested enhanced xanthine oxidase activity accompanied with reactive oxygen species (ROS) production. Histopathological analysis confirmed that the production of ROS in the RS/PC region was increased by MK-801 and that the nonspecific NO synthase inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) prevented MK-801-induced neuronal cell death. These results suggest that NO increases oxidative stress-related cell death. Increased levels of metabolites of glucose metabolism suggested enhanced energy production via glycolysis. To confirm the relationship between NO and glucose utilization, positron emission tomography (PET) imaging with [(18)F] fluoro-2-deoxy-d-glucose ([(18)F] FDG) was conducted. [(18)F] FDG-PET imaging accompanied by co-treatment of L-NAME with MK-801 demonstrated that L-NAME ameliorated MK-801-induced glucose utilization.In conclusion, MK-801 induces NO and ROS production in the RS/PC region, which might subsequently induce oxidative stress and in turn neuronal cell death. In addition, MK-801-induced NO production increased glucose utilization and affected glucose metabolism, the imbalance of which might generate additional oxidative stress related to neuronal cell death.

Assessment of Alloxan-Induced Diabetic Rats as a Periodontal Disease Model Using a Selective Cyclooxygenase (COX)-2 Inhibitor.

Several recent studies have reported that alloxan-treated rats with long-term hyperglycemia can develop naturally occurring periodontal disease (PD). Our previous studies detected dental caries in the same model. Therefore, these two lesions of different etiologies are expected to occur concurrently. In this study, we evaluated the use of diabetic rats as a PD model by employing a selective COX-2 inhibitor reported to be effective against PD. Six-week-old female F344 rats were divided into 3 groups: intact rats (control), alloxan-induced diabetic rats fed a standard diet (AL) and alloxan-induced diabetic rats fed a diet containing 0.01% etodolac (AL+Et). The animals were euthanized at 26 weeks of age, and their oral tissues were examined histopathologically. Gingivitis, marginal periodontitis and alveolar bone resorption were markedly enhanced along with dental caries in the AL group compared with the control group. However, the COX-2 inhibitor had no effect on periodontal inflammation in the AL+Et group. In addition, in the AL group, periodontitis was notably nonexistent around the normal molars, and gingivitis was scarcely worse than that in the control group. In the diabetic rats, the progression of periodontal inflammation was closely correlated with the severity of adjacent dental caries, and marginal periodontitis was frequently continuous with apical periodontitis. In conclusion, an alloxan-induced diabetic rat is not a model of PD but of dental caries. It is probable that in this model, hyperglycemia may enable crown caries to progress to apical periodontitis, while the associated inflammation may rostrally expand to surrounding periodontal tissue.

The effect of food hardness on the development of dental caries in alloxan-induced diabetic rats.

We have previously shown that dental caries may be produced in diabetic rodent models fed with noncariogenic standard diets; however, many studies usually add large amounts of sugar to the diet to induce dental caries. Moreover, the physical properties of cariogenic diets have been reported as an important factor in the formation of caries. The aim of this study was to clarify the effect of the hardness of non-cariogenic diets on the development of dental caries in diabetic rodents. Seven-week-old female F344 rats were divided into 4 groups: intact rats fed with a standard pelletized or powdered diet and alloxan-induced diabetic rats fed with a standard pelletized or powdered diet. All of the rats were sacrificed at 52 weeks of age for morphological examinations on their dental tissue. Dental caries had developed and extended to all the molars in the diabetic rats that were fed with both the pelletized and powdered diets. Moreover, the lesion was significantly enhanced in the powdered diet group compared to that in the pelletized diet group. In conclusion, food hardness is an important factor influencing the development of dental caries in diabetic rats.

Glycemic control with insulin prevents progression of dental caries and caries-related periodontitis in diabetic WBN/KobSlc rats.

We have previously reported that dental caries progress in spontaneously and chemically induced diabetic rodent models. The aim of this study was to clarify the relationship between hyperglycemia and dental caries by evaluating the preventive effect of glycemic control with insulin on the progression of the lesions in diabetic rats. Male WBN/KobSlc rats aged 15 weeks were divided into groups of spontaneously diabetic rats (intact group), spontaneously diabetic rats with insulin treatment (INS group), alloxan-induced prolonged diabetic rats (AL group), and alloxan-induced prolonged diabetic rats with insulin treatment (AL + INS group). The animals were killed at 90 weeks of age, and their oral tissue was examined. Dental caries and periodontitis were frequently detected in the intact group, and the lesions were enhanced in the AL group (in which there was an increased duration of diabetes). Meanwhile, glycemic control with insulin reduced the incidence and severity of dental caries and periodontitis in the INS group, and the effects became more pronounced in the AL + INS group. In conclusion, glycemic control by insulin prevented the progression of dental caries and caries-related periodontitis in the diabetic rats.

Alloxan-induced hyperglycemia causes rapid-onset and progressive dental caries and periodontitis in F344 rats.

We have previously shown that diabetes increases dental caries, and periodontitis might be a secondary change resulting from dental caries in spontaneous diabetic rodent models. However, the lesions in these models were slow to manifest, and the intensity and frequency were mild and varied among individuals. The goal of this study was to confirm the reproducibility of caries development in chemically induced diabetic rats and investigate whether alloxan, which induces immediate and severe hyperglycemia in experimental animals, increases the lesions. Female F344 rats were examined 13 and 26 weeks after dosing of alloxan. Alloxan injection induced severe hyperglycemia in two-thirds of the rats. Progressive molar caries and periodontitis were already induced in all diabetic rats 13 weeks after dosing of alloxan, although the lesions were not observed in nondiabetic rats. Histopathologically, dental caries initially developed in the crown, then spread into the dental root, entered the periodontal connective tissue via the apical foramen, and progressed to periodontitis. In conclusion, alloxan-induced severe hyperglycemia is capable of causing rapid-onset and progressive dental caries and periodontitis in rats.

Diabetes enhances dental caries and apical periodontitis in caries-susceptible WBN/KobSlc rats.

Many epidemiologic studies have suggested that diabetes may be an important risk factor for periodontal disease. To determine whether diabetes induces or enhances periodontal disease or dental caries, dental tissue from diabetic male and nondiabetic female WBN/KobSlc rats and male and female age-matched nondiabetic F344 rats was analyzed morphologically and morphometrically for these 2 types of lesions. Soft X-ray examination revealed that the incidence and severity of both molar caries and alveolar bone resorption were much higher in male WBN/KobSlc rats with chronic diabetes than in nondiabetic female rats of the same strain. Histopathologic examination showed that dental caries progressed from acute to subacute inflammation due to bacterial infections and necrosis in the pulp when the caries penetrated the dentin. In the most advanced stage of dental caries, inflammatory changes caused root abscess and subsequent apical periodontitis, with the formation of granulation tissue around the dental root. Inflammatory changes resulted in resorption of alveolar bone and correlated well with the severity of molar caries. Our results suggest that diabetic conditions enhance dental caries in WBN/KobSlc rats and that periodontal lesions may result from the apical periodontitis that is secondary to dental caries.

Morphological study on dental caries induced in WBN/KobSlc rats (Rattus norvegicus) fed a standard laboratory diet.

In our previous studies, WBN/KobSlc was characterized as a rat strain in which only males began to develop pancreatitis, and then presented with diabetic symptoms. In the course of studying their pancreatic inflammation, we detected molar caries in prediabetic males feeding on a standard diet (CRF-1) widely used for experimental animals. The purpose of this study is to confirm whether the WBN/KobSlc strain is caries-susceptible to the diet reported to be non-cariogenic, and to examine the effect of a prediabetic condition on their dental caries. For a morphological study, 25 male WBN/KobSlc rats aged 3.2-7.8 months and 24 females of the same strain aged 3.3-6.6 months were used, along with 10 males and 10 females of 8.2-month-old F344 rats. Marked dental caries were detected in the mandibular molars of male and female WBN/KobSlc rats regardless of pancreatitis, although no similar changes were observed in any teeth of the F344 strain fed the same diet. Soft X-ray examination revealed that the caries began in the crown and progressed horizontally and vertically, and that a severe radiolucent lesion extensively expanded to the entire crown, corresponding to a macroscopically deleted molar. The caries had gradually developed mainly in the second mandibular molar from more than 3.5 months of age, while none were seen in any rats before that time. The WBN/KobSlc rats were caries-susceptible even to the standard laboratory diet, and pancreatitis was not directly associated with the onset of dental caries in this strain.

Polypoid eosinophilic cystitis with pseudosarcomatous proliferative tissue in a dog.

A dog presented with hematuria, and two small polypoid masses were detected in the urinary bladder. Histopathologically, the masses were located in the mucosal or submucosal layer. That tissue consisted of a random proliferation of spindle-shaped, round and pleomorphic cells with single or multiple large atypical nuclei and abundant cytoplasm, and eosinophil infiltration. These large cells were confirmed by immunohistochemical staining as fibroblasts, myofibroblasts and macrophages. Mitotic figure was rarely seen. These masses were diagnosed as eosinophilic polypoid cystitis with pseudosarcomatous proliferative tissue, since they consisted of a wide variety of cells and showed low growth activity.

Semi-quantitative immunohistochemical analysis of male rat-specific alpha2u-globulin accumulation for chemical toxicity evaluation.

We purified male rat urinary alpha(2u)-globulin, prepared the antibody in rabbits, and improved an immunohistochemical detection method using this antibody for male rat-specific alpha(2u)-globulin accumulation appearing as hyaline droplets in the kidneys. Our prepared antibody reacted specifically with alpha(2u)-globulin in both immunohistochemical and Western blotting analyses, furthermore, and the graded immuno-reactivities on the slide were well associated with computational image analyzing results. Using this method, we retrospectively analyzed the renal sections from the toxicity studies of 12 nephrotoxic chemicals, which had already been conducted under the Japanese Existing Chemicals Survey Program. We demonstrated that the hyaline droplets induced by treatment with 10 chemicals (1,4-dibromobenzene, dicyclopentadiene, 3,4-dimethylaniline, 1,4-dicyanobenzene, tetrahydrothiophene-1,1-dioxide, 1,3-dicyanobenzene, acenaphthene, 3,4-dichloro-1-butene, 3a,4,7,7a-tetrahydro-1H-indene and 3,5,5-trimethylhexan-1-ol) were directly associated with alpha(2u)-globulin accumulation. This immunohistochemical method is convenient for applying, even retrospectively, paraffin sections from general toxicity studies and could be useful for qualifying male rat-specific hyaline droplets consisting of alpha(2u)-globulin and renal risk in humans.