Cleavage of Host Cytokeratin-6 by Lysine-Specific Gingipain Induces Gingival Inflammation in Periodontitis Patients

Background/PurposeLysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion.MethodsK6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay.ResultsWe identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359–378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt.ConclusionKgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.     

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs.     

Insular Gray Matter Volume and Objective Quality of Life in Schizophrenia

Improving quality of life has been recognized as an important outcome for schizophrenia treatment, although the fundamental determinants are not well understood. In this study, we investigated the association between brain structural abnormalities and objective quality of life in schizophrenia patients. Thirty-three schizophrenia patients and 42 age-, sex-, and education-matched healthy participants underwent magnetic resonance imaging. The Quality of Life Scale was used to measure objective quality of life in schizophrenia patients. Voxel-based morphometry was performed to identify regional brain alterations that correlate with Quality of Life Scale score in the patient group. Schizophrenia patients showed gray matter reductions in the frontal, temporal, limbic, and subcortical regions. We then performed voxel-based multiple regression analysis in these regions to identify any correlations between regional gray matter volume and Quality of Life Scale scores. We found that among four subcategories of the scale, the Instrumental Role category score correlated with gray matter volume in the right anterior insula in schizophrenia patients. In addition, this correlation was shown to be mediated by negative symptoms. Our findings suggest that the neural basis of objective quality of life might differ topographically from that of subjective QOL in schizophrenia.     

A Naturally Occurring Canine Model of Autosomal Recessive Congenital Stationary Night Blindness

Congenital stationary night blindness (CSNB) is a non-progressive, clinically and genetically heterogeneous disease of impaired night vision. We report a naturally-occurring, stationary, autosomal recessive phenotype in beagle dogs with normal daylight vision but absent night vision. Affected dogs had normal retinas on clinical examination, but showed no detectable rod responses. They had “negative-type” mixed rod and cone responses in full-field ERGs. Their photopic long-flash ERGs had normal OFF-responses associated with severely reduced ON-responses. The phenotype is similar to the Schubert-Bornschein form of complete CSNB in humans. Homozygosity mapping ruled out most known CSNB candidates as well as CACNA2D4 and GNB3. Three remaining genes were excluded based on sequencing the open reading frame and intron-exon boundaries (RHO, NYX), causal to a different form of CSNB (RHO) or X-chromosome (NYX, CACNA1F) location. Among the genes expressed in the photoreceptors and their synaptic terminals, and mGluR6 cascade and modulators, reduced expression of GNAT1, CACNA2D4 and NYX was observed by qRT-PCR in both carrier (n = 2) and affected (n = 2) retinas whereas CACNA1F was down-regulated only in the affecteds. Retinal morphology revealed normal cellular layers and structure, and electron microscopy showed normal rod spherules and synaptic ribbons. No difference from normal was observed by immunohistochemistry (IHC) for antibodies labeling rods, cones and their presynaptic terminals. None of the retinas showed any sign of stress. Selected proteins of mGluR6 cascade and its modulators were examined by IHC and showed that PKCα weakly labeled the rod bipolar somata in the affected, but intensely labeled axonal terminals that appeared thickened and irregular. Dendritic terminals of ON-bipolar cells showed increased Goα labeling. Both PKCα and Goα labeled the more prominent bipolar dendrites that extended into the OPL in affected but not normal retinas. Interestingly, RGS11 showed no labeling in the affected retina. Our results indicate involvement of a yet unknown gene in this canine model of complete CSNB.     

Multi-Acupuncture Point Injections and Their Anatomical Study in Relation to Neck and Shoulder Pain Syndrome (So-Called Katakori) in Japan

Katakori is a symptom name that is unique to Japan, and refers to myofascial pain syndrome-like clinical signs in the shoulder girdle. Various methods of pain relief for katakori have been reported, but in the present study, we examined the clinical effects of multi-acupuncture point injections (MAPI) in the acupuncture points with which we empirically achieved an effect, as well as the anatomical sites affected by liquid medicine. The subjects were idiopathic katakori patients (n = 9), and three cadavers for anatomical investigation. BL-10, GB-21, LI-16, SI-14, and BL-38 as the WHO notation were selected as the acupuncture point. Injections of 1 mL of 1% w/v mepivacaine were introduced at the same time into each of these points in the patients. Assessment items were the Pain Relief Score and the therapeutic effect period. Dissections were centered at the puncture sites of cadavers. India ink was similarly injected into each point, and each site that was darkly-stained with India ink was evaluated. Katakori pain in the present study was significantly reduced by MAPI. Regardless of the presence or absence of trigger points, pain was significantly reduced in these cases. Dark staining with India ink at each of the points in the anatomical analysis was as follows: BL-10: over the rectus capitis posterior minor muscle and rectus capitis posterior major muscle fascia; GB-21: over the supraspinatus muscle fascia; LI-16: over the supraspinatus muscle fascia; SI-14: over the rhomboid muscle fascia; and BL-38: over the rhomboid muscle fascia. The anatomical study suggested that the drug effect was exerted on the muscles above and below the muscle fascia, as well as the peripheral nerves because the points of action in acupuncture were darkly-stained in the spaces between the muscle and the muscle fascia.     

Hepatocyte Nuclear Factor 4 Alpha Is a Key Factor Related to Depression and Physiological Homeostasis in the Mouse Brain

Major depressive disorder (MDD) is a common psychiatric disorder that involves marked disabilities in global functioning, anorexia, and severe medical comorbidities. MDD is associated with not only psychological and sociocultural problems, but also pervasive physical dysfunctions such as metabolic, neurobiological and immunological abnormalities. Nevertheless, the mechanisms underlying the interactions between these factors have yet to be determined in detail. The aim of the present study was to identify the molecular mechanisms responsible for the interactions between MDD and dysregulation of physiological homeostasis, including immunological function as well as lipid metabolism, coagulation, and hormonal activity in the brain. We generated depression-like behavior in mice using chronic mild stress (CMS) as a model of depression. We compared the gene expression profiles in the prefrontal cortex (PFC) of CMS and control mice using microarrays. We subsequently categorized genes using two web-based bioinformatics applications: Ingenuity Pathway Analysis and The Database for Annotation, Visualization, and Integrated Discovery. We then confirmed significant group-differences by analyzing mRNA and protein expression levels not only in the PFC, but also in the thalamus and hippocampus. These web tools revealed that hepatocyte nuclear factor 4 alpha (Hnf4a) may exert direct effects on various genes specifically associated with amine synthesis, such as genes involved in serotonin metabolism and related immunological functions. Moreover, these genes may influence lipid metabolism, coagulation, and hormonal activity. We also confirmed the significant effects of Hnf4a on both mRNA and protein expression levels in the brain. These results suggest that Hnf4a may have a critical influence on physiological homeostasis under depressive states, and may be associated with the mechanisms responsible for the interactions between MDD and the dysregulation of physiological homeostasis in humans.     

Investigating the Influence of Visual Function and Systemic Risk Factors on Falls and Injurious Falls in Glaucoma Using the Structural Equation Modeling

Purpose

To investigate the relationship between visual function and the risks of falling and injurious falls in subjects with primary open angle glaucoma (POAG)

Methods

Questionnaires were conducted in 365 POAG patients to assess history of falls and falls with injury and general patient health. Structural equation modeling (SEM) was used to investigate the relationship between visual function, as measured by a patient’s binocular integrated visual field and visual acuity (VA), general health and the risks of falling and injurious falls.

Results

Among the 365 subjects, 55 subjects experienced falls in the past year. A significant difference was observed in worse-eye VA between the faller and non-faller groups (p = 0.03). SEM of fallers obtained a Root Mean Square Error of Approximation (RMSEA) of 0.035 and a Comparative Fit Index (CFI) of 0.99. The 95% confidence intervals (CI) of regression coefficients from this model suggested better VA and worse VA were significant risk factors for falling. Among the 55 fallers, 22 subjects experienced an associated injury. There was a significant difference in gender between the non-injurious and injurious faller groups (p = 0.002). SEM of injurious fallers obtained a RMSEA of 0.074 and a CFI of 0.97. In this SEM model, the 95% CI of regression coefficients suggested gender and average total deviation values in the lower peripheral visual field were significant risk factors for an injurious fall.

Conclusions

This study suggests that worse-eye and better-eye VAs are associated with falls. Furthermore, patients with inferior visual field loss and females were found to be at greater risk of injurious falls.     

Different combinations of Notch ligands and receptors regulate V2 interneuron progenitor proliferation and V2a/V2b cell fate determination

The broad diversity of neurons is vital to neuronal functions. During vertebrate development, the spinal cord is a site of sensory and motor tasks coordinated by interneurons and the ongoing neurogenesis. In the spinal cord, V2-interneuron (V2-IN) progenitors (p2) develop into excitatory V2a-INs and inhibitory V2b-INs. The balance of these two types of interneurons requires precise control in the number and timing of their production. Here, using zebrafish embryos with altered Notch signaling, we show that different combinations of Notch ligands and receptors regulate two functions: the maintenance of p2 progenitor cells and the V2a/V2b cell fate decision in V2-IN development. Two ligands, DeltaA and DeltaD, and three receptors, Notch1a, Notch1b, and Notch3 redundantly contribute to p2 progenitor maintenance. On the other hand, DeltaA, DeltaC, and Notch1a mainly contribute to the V2a/V2b cell fate determination. A ubiquitin ligase Mib, which activates Notch ligands, acts in both functions through its activation of DeltaA, DeltaC, and DeltaD. Moreover, p2 progenitor maintenance and V2a/V2b fate determination are not distinct temporal processes, but occur within the same time frame during development. In conclusion, V2-IN cell progenitor proliferation and V2a/V2b cell fate determination involve signaling through different sets of Notch ligand–receptor combinations that occur concurrently during development in zebrafish.