Low dose antibiotic ingestion potentiates systemic and microbiome changes induced by silver nanoparticles.

Changes in the mammalian gut microbiome are linked to the impairment of immunological function and numerous other pathologies. Antimicrobial silver nanoparticles (AgNPs) are incorporated into numerous consumer products (e.g., clothing, cosmetics, food packaging), which may directly impact the gut microbiome through ingestion. The human health impact of chronic AgNP ingestion is still uncertain, but evidence from exposure to other antimicrobials provides a strong rationale to assess AgNP effects on organ function, immunity, metabolism, and gut-associated microbiota. To investigate this, mice were gavaged daily for 5 weeks with saline, AgNPs, antibiotics (ciprofloxacin and metronidazole), or AgNPs combined with antibiotics. Animals were weighed daily, assessed for glucose tolerance, organ function, tissue and blood cytokine and leukocyte levels. At the end of the study, we used 16S rDNA amplicon and whole-metagenome shotgun sequencing to assess changes in the gut microbiome. In mice exposed to both AgNPs and antibiotics, silver was found in the stomach, and small and large intestines, but negligible amounts were present in other organs examined. Mice exposed to AgNPs alone showed minimal tissue silver levels. Antibiotics, but not AgNPs, altered glucose metabolism. Mice given AgNPs and antibiotics together demonstrated slower weight gain, reduced peripheral lymphocytes, and elevated splenic, but not circulatory markers of inflammation. 16S rDNA profiling of cecum and feces and metagenomic sequencing of fecal DNA demonstrated that combined AgNP-antibiotic treatment also significantly altered the structure and function of the gut microbiota, including depletion of the indicator species Akkermansia muciniphila. This study provides evidence for possible biological effects from repeated ingestion of AgNP-containing consumer products when antibiotics are also being used and raises concern that an impaired gut microbiome (e.g., through antibiotic use) can potentiate the harm from chemical exposures such as AgNPs.

Maternal Separation Induces Orofacial Mechanical Allodynia in Adulthood.

It is well known that exposure to maternal separation (MS) in early life causes plastic changes in the nervous system in adulthood, occasionally resulting in ubiquitous chronic pain. However, the pathogenic mechanisms of pain hypersensitivity remain unclear. Here, the authors examined the involvement of corticosterone in orofacial mechanical hypersensitivity induced by MS. To establish a rat model of MS, pups were placed in isolated cages 180 min/d and kept in a temperature-controlled environment at 22 ± 2 °C for 14 d. Mechanical allodynia in the whisker pad skin in adulthood was induced by MS and was significantly suppressed by successive postnatal subcutaneous administration of the glucocorticoid receptor antagonist mifepristone. Corticosterone levels were increased in the serum of MS rats, and successive postnatal administration of subcutaneous corticosterone to naive rats induced mechanical allodynia in the whisker pad skin. The number of P2X3 receptor-immunoreactive (P2X3R-IR) trigeminal ganglion (TG) neurons innervating the whisker pad skin was significantly increased in MS rats and decreased following subcutaneous administration of mifepristone. The number of P2X3R-IR TG neurons innervating the whisker pad skin was also significantly increased following successive postnatal administration of subcutaneous corticosterone in naive rats. Moreover, the mechanical allodynia was suppressed 30 min after administration of the P2X3R antagonist A317491 to the whisker pad skin in MS rats. These findings suggest that the increase in P2X3R-IR TG neurons innervating the whisker pad skin via enhanced neonatal corticosterone signaling by MS plays an important role in orofacial mechanical allodynia in adulthood.

Alendronate does not prevent long bone fragility in an inactive rat model.

The lack of estrogen and inactivity are both important in the pathogenesis of osteoporosis in elderly women, and there have been no appropriate rodent studies to examine the effects of common bisphosphonates on these two components separately. We compared the efficacy of alendronate (ALN) on the long bones of aged female rats, which were sedentary, estrogen deficient, or both. The rats were either forced to remain in a sitting position or allowed to walk in standard cages with or without ALN administration. The 8-week experimental period began 5 weeks after ovariectomy or sham surgery. Parameters of the hindlimb bones were determined by a three-point bending test, peripheral quantitative computed tomography, microfocus computed tomography, confocal laser Raman microspectroscopy, and dynamic histomorphometry. Regardless of ovariectomy, ALN was ineffective against the deterioration of breaking stress caused by sitting even though the trabecular bone mineral density was significantly higher in the sitting-ALN groups. Toughness was significantly deficient in the ovariectomy sitting-ALN group. This was in agreement with the bone geometry with a greater marrow space. Sitting also increased the mineral-to-matrix ratio and the carbonate-to-phosphate ratio, both indicative of aged bone. A greater loss of proteinaceous amide intensity compared with mineral intensity resulted in an increased mineral-to-matrix ratio in the presence of ALN. Sitting resulted in deficits in the quality and the geometry of cortical bone, resulting in fragility. The use of bisphosphonates, such as ALN, may provide a therapy best suited for osteoporotic individuals whose daily activity is not limited.

Disability, HRQoL and resource use among chronic and episodic migraineurs: results from the International Burden of Migraine Study (IBMS).

BACKGROUND: Migraine imposes significant burden on patients, their families and health care systems. In this study, we compared episodic to chronic migraine sufferers to determine if migraine status predicted headache-related disability, health-related quality of life (HRQoL) and health care resource utilization. METHODS: A Web-based survey was administered to panelists from nine countries. Participants were classified as having chronic migraine (CM), episodic migraine (EM) or neither using a validated questionnaire. Data collected and then analyzed included sociodemographics, clinical characteristics, Migraine Disability Assessment, Migraine-Specific Quality of Life v2.1, Patient Health Questionnaire and health care resource utilization. FINDINGS: Of the respondents, 5.7% had CM and 94.3% had EM, with CM patients reporting significantly more severe disability, lower HRQoL, higher levels of anxiety and depression and greater health care resource utilization compared to those with EM. INTERPRETATION: These results provide evidence that will enhance our understanding of the factors driving health care costs and will contribute to development of cost-effective health care strategies.

Threonine 74 of MOB1 is a putative key phosphorylation site by MST2 to form the scaffold to activate nuclear Dbf2-related kinase 1.

Mammalian nuclear Dbf2-related (NDR) kinases (LATS1 and 2, NDR1 and 2) play a role in cell proliferation, apoptosis and morphological changes. These kinases are regulated by mammalian sterile 20-like kinases (MSTs) and Mps one binder (MOB) 1. Okadaic acid (OA), which activates MST2, facilitates the complex formation of MOB1, MST2 and NDR1 in HEK293FT cells. The in vitro biochemical study demonstrates the phosphorylation of MOB1 by MST2. The phosphorylated MOB1 alone is capable to partially activate NDR1 in vitro, but MST2 is also required for the full activation. The knockdown of MOB1 or MST2 abolishes the OA-induced NDR1 activation in HEK293FT cells. Among MOB1 mutants, in which each serine or threonine residue is replaced with alanine, MOB1 T74A and T181A mutants fail to activate NDR1. Thr74, but not Thr181, is phosphorylated by MST2 in vitro, although MOB1 is also phosphorylated by MST2 at other site(s). The interaction of MOB1 T74A with NDR1 is barely enhanced by OA treatment. These findings indicate that the phosphorylation of MOB1 at Thr74 by MST2 is essential to make a complex of MOB1, MST2 and NDR1, and to fully activate NDR1.

Rapid and accurate measurement of anti-A/B IgG antibody in ABO-unmatched living donor liver transplantation by surface plasmon resonance.

High anti-blood group A or B (anti-A/B) immunoglobulin G (IgG) haemagglutination titres are associated with poor graft survival in ABO-unmatched liver transplantation. We have previously reported that the surface plasmon resonance (SPR) method can be used to measure anti-A/B IgG levels in the plasma very quickly and quantitatively. The aim of this study was to brush up this SPR method. The anti-A/B IgG antibodies (Abs) were purified from the plasma of healthy volunteers by affinity chromatography and used to establish standard curves for the SPR and flow cytometry (FCM) methods. The haemagglutination test tube (TT), FCM and SPR methods were then used to measure the changes over time in the anti-A/B IgG titres of 25 ABO-unmatched living donor liver transplantation (LDLT) recipients. The standard curve permitted the SPR values for the anti-A/B IgG titres to be expressed in microg mL(-1) units. The SPR measurements of the anti-A/B IgG levels in the LDLT recipients correlated very well with the FCM values, whereas the TT values correlated poorly with either method. Furthermore, the SPR method accurately detected the effects of plasma exchange. In conclusion, the SPR method is an accurate, time- and labour-saving method for measuring anti-A/B IgG titres that can be easily standardized.

Ligand-of-Numb protein X is an endocytic scaffold for junctional adhesion molecule 4.

Junctional adhesion molecule 4 (JAM4) is a cell adhesion molecule that interacts with a tight junction protein, membrane-associated guanylate kinase inverted 1 (MAGI-1). Our previous studies suggest that JAM4 is implicated in the regulation of paracellular permeability and the signalings of hepatocyte growth factor. In this study, we performed yeast two-hybrid screening to search for an unidentified JAM4-binding protein and obtained one isoform of Ligand-of-Numb protein X1 (LNX1), LNXp70, that is an interactor of Numb. Ligand-of-Numb protein X1 is expressed in kidney glomeruli and intestinal epithelial cells, where JAM4 is also detected. Immunoprecipitation from kidney lysates supports the in vivo interaction of proteins. Biochemical studies reveal that JAM4 directly binds the second PDZ domain of LNX1 through its carboxyl terminus. Junctional adhesion molecule 4, LNX1 and Numb form a tripartite complex in vitro and are partially colocalized in heterologous cells. Ligand-of-Numb protein X1 facilitates endocytosis of JAM4 and is involved in transforming growth factor beta -induced redistribution of JAM4 in mammary epithelial cells. Experiments using dominant-negative constructs and RNA interference insure that Numb is necessary for the LNX1-mediated endocytosis of JAM4. All these findings indicate that LNX1 provides an endocytic scaffold for JAM4 that is implicated in the reorganization of cell junctions.

Hereditary paroxysmal ataxia with mental retardation: a clinicopathological study in relation to episodic ataxia type 2.

A case of hereditary acetazolamide-responsive paroxysmal ataxia with mild mental retardation in an autopsied Japanese man is described. His ataxic attacks had occurred for approximately 65 years since the age of 6. One of his daughters had severe mental retardation and epilepsy, and the other had paroxysmal ataxic attacks and mild mental retardation. Analysis of the subject's CACNA1A gene and that in his daughter revealed neither mutations nor CAG expansion. Neuropathologically, cortical degeneration consisting of the marked loss of Purkinje and granule cells was found exclusively in the cerebellar vermis. This was consistent with findings at autopsy for cases reported as spinocerebellar ataxia 6. In addition, there were minor anomalies, such as hypoplastic cerebellum and brainstem, heterotopic Purkinje cells, and cortical microdysgenesis of the temporal lobe. It is considered that the cerebellar cortical degeneration and the minor malformations found in the brain are closely related to one another, rather than having occurred independently.

Long-term outcome in 306 males with alcoholism.

The subjects of this study were 306 male alcoholics who lived in Osaka, Japan, and who were initially diagnosed with alcoholism at a psychiatric institution between 1972 and 1983. Follow-up studies were done on three occasions: 1 March 1985 (Time 1), 1 November 1988 (Time 2) and 1 March 1992 (Time 3). We followed up 232 (75.8%) of the 306 male alcoholics. By the end of the study period 110 (35.9%) of the subjects were deceased. Regarding cross-sectional sobriety status, from Time 1 to Time 3 the complete abstinence rate changed from 16.0 to 18.6%, excessive drinking rate was from 13.1 to 9.8%, and controlled drinking rate was from 6.9 to 9.8%. The longitudinal sobriety status of 122 living patients during the 5 years before the close of this study were: rate of stable abstinence, 28.7%; unstable abstinence, 21.3%; controlled drinking, 12.3%; and relapse 37.7%. Such variables as being without public assistance at the time of the initial diagnosis of alcoholism and attending a self-help group soon after the initial treatment were associated with stable abstinence. Age (20-39 years) and receiving outpatient treatment at the time of the initial treatment also emerged as predictors of survival. However, those variables, except attending a self-help group soon after the initial treatment, might merely indicate severity of alcoholism. For improving treatment results, it may be most important to provide a treatment environment within the residential area so that alcoholics may receive treatment at an early stage of alcoholism and attend a self-help group.

Effects of amino acids on the amidation of polyaromatic carboxylic acids by Bacillus cereus.

The soil bacterium Bacillus cereus Tim-r01 efficiently transformed polyaromatic carboxylic acids (PACA) such as 4-biphenylcarboxylic acid (4-BPCA), 4-biphenylacetic acid, and 4-phenoxybenzoic acid into their corresponding amides. The amidation activity was expressed at 37 degrees C (pH 7-8) in the presence of grown cells in nutrients under an aerobic atmosphere. Other strains of B. cereus, IFO 3001 and IAM 1229, also gave the amide from 4-BPCA. In phosphate-buffered saline (PBS), the addition of normal amino acids was essential, while sulfur-containing amino acids such as methionine and cysteine drastically inhibited the amidation. Tracer experiments using N-15-isoleucine and N-15-alanine showed that the nitrogen atom of the amide came from an amino group of amino acids but not from ammonia or alkylamines.

Experimental allergic myositis in SJL/J mouse. Reappraisal of immune reaction based on changes after single immunization.

SJL/J mice have been subjected to immunization with wide varieties of antigens to produce models of autoimmune disorders including experimental myositis. They also have a defect in dysferlin gene and spontaneously develop muscle fiber degeneration, a condition akin to limb-girdle type muscular dystrophy and Miyoshi myopathy. To know whether muscle inflammation of SJL mice after immunization with muscle fractions really represents immune-mediated myositis or no more than an epiphenomenon of muscle degeneration due to dysferlin defect, we studied immunological parameters after immunization with rabbit myosin B fraction. Initial infiltration of macrophages and CD4+ lymphocytes on day 11 was followed by increase in number of CD8+ cells. Such increase was not observed in the nontreated and adjuvant controls. Some infiltrating cells were interferon gamma (IFN-gamma) positive. Furthermore, increased expression of the signal transducers and activator of transcription 1 (STAT-1) and interferon regulatory factor 1 (IRF-1) mRNA was shown in the first 2 weeks. These results indicate Th1 system activity in the muscle, rather than simple dysferlin deficiency, particularly 1-3 weeks after immunization. Thus it is concluded that an immune-mediated myositis is taking place at this stage. This model can be helpful in understanding pathomechanisms involved in the early stage of human myositides. It has also important implications concerning immune reactions associated with transplantation or gene therapy for muscular dystrophies.

Cause-specific mortality risk among male alcoholics residing in the Osaka metropolitan area.

We evaluated the mortality risk among 306 male alcoholics living in Osaka, Japan, at the time of initial diagnosis between 1972 and 1983, with regard to the cause of death, length of time from diagnosis, and participation in an alcohol abstinence self-help group. By the closing date on 1 March 1992, 110 of the 306 alcoholics had died, yielding an observed-to-expected (O/E) ratio of 4.5 [95% confidence interval (CI) = 3.7-5.4]. The alcoholics had significantly elevated mortality risks from all malignant neoplasms (O/E = 2.1, 95%CI = 1.2-3.3), esophageal cancer (O/E = 8.4, 95%CI = 1.7-24.5), diseases of the circulatory system (O/E = 4.4, 95%CI = 3.0-6.2), liver cirrhosis (O/E = 15.9, 95%CI = 10.2-23.6), diseases of the genitourinary system (O/E = 6.3, 95%CI = 1.3-18.5), and external death (O/E = 10.3, 95%CI = 6.3-15.8). The mortality risk from all causes still remained significantly high beyond the tenth year following initial diagnosis (O/E = 2.6, 95%CI = 1.0-6.2). The mortality risks from liver cirrhosis and external death (such as suicide) were highest within the first year following diagnosis, and were still high beyond the tenth year. A significantly high mortality risk from diseases of the circulatory system was observed between the first and ninth years, and the mortality risk from all malignant neoplasms was significantly elevated beyond 10 years following diagnosis. Alcoholics who did not join a self-help group soon after the initial institutional treatment had different cause-specific and time-specific mortality risks from those who did join a self-help group. These findings show the importance of long-term clinical follow-up of male alcoholics, taking into consideration the cause-specific mortality.

Matrix metalloproteinase-2 in dentin matrix mineralization.

In the serum-free culture medium of bovine odontoblasts we detected active gelatinolytic metalloproteinases, matrix metalloproteinase (MMP)-2 and MMP-9 (gelatinases A and B). The activity of MMP-2, in particular, appeared suddenly around day 21 in the culture, coinciding with the development of odontoblastic cell processes and the loss of alkaline phosphatase. Reverse transcriptase-polymerase chain reaction analysis of these odontoblasts demonstrated that messages of MMP-2 but not MMP-9 increased significantly between day 15 and day 21. The in vitro observation indicates that medium conditioned by these odontoblasts and containing significant amounts of MMP-2 degrades not only the collagenous substrates but also purified dentin phosphophoryn as well. We have also observed that dephosphorylated dentin phosphoprotein becomes a better substrate for casein kinase II after limited proteolysis with MMP-2. These results support our working hypothesis that MMP-2-mediated proteolytic processing is an important step in accelerating the process of dentin matrix maturation, which includes phosphorylation and subsequent mineralization. As has been suggested previously, extracellular phosphorylation of matrix proteins is an important step in biomineralization both in bone and in dentin (Mikuni-Takagaki et al., J Bone Miner Res 1995;10:231-42; Zhu et al., Biochem J 1997; 323:637-43). Our present histochemical analysis in MMP-2 knockout mice confirms the concept with the delayed formation of mineralized tissues, dentin, and bone.

Cytotoxic T cell activity against the peptide, AYRARALEL, from Yo protein of patients with the HLA A24 or B27 supertype and paraneoplastic cerebellar degeneration.

OBJECTIVE: To determine a peptide that reacts with cytotoxic T cells (CTL) of patients with paraneoplastic cerebellar degeneration and anti-Yo antibodies with either HLA A24 or B27 supertype. METHOD: We studied CTL activity of four patients, three were HLA A24-positive and one did not have HLA A24 but had B27 supertype. After an incubation of mononuclear cells with or without peptide and IL-2, CD8-rich fraction was prepared by treatment with Magnetic Cell Sorting system (MACS) twice. CTL activity was calculated by 51Cr release from transfectant, C1RA*2402 as target cells. The peptide-binding assay was examined by flow cytometry. RESULTS: Two of three HLA A 24-positive patients demonstrated CTL activity against the Yo peptide, AYRARALEL. CTL activity was found to be 19.5% and 11.7% at the effector/target (E/T) ratio of 23:1 and 11:1, respectively. A patient who did not have HLA A24 but had A2 and B27 supertype possessed a CTL activity of 19.4% with 15:1 as E/T ratio. The peptide could bind to HLA A*2402 molecules but not to A*0201. CONCLUSIONS: We showed CTL activity in two of three Japanese patients with HLA A24 by using HLA A*2402 transfectant cells as the target. In addition, we identified the first Japanese patient who had B27 supertype, and suggested that the same peptide, AYRARALEL, could be recognized by CTL in this patient.

[Clinical features of autosomal dominant spinocerebellar ataxias genetically unclassified into any known triplet-repeat diseases].

To clarify the clinical features of genetically unclassified autosomal dominant spinocerebellar ataxias (AD-SCAs) treated in our hospital, we retrospectively analyzed the clinical findings of patients who could not be molecularly classified into SCA types 1 through 3, 6 through 8, 12 and dentato-rubro-pallido-luysian atrophy (DRPLA). The clinical findings of 19 patients from 15 different families which form 14 percent of all patients with different types of AD-SCAs (136 patients, 105 families) were collected. Based on these, 17 patients from 13 families showed late-onset (mean age at onset: 46 years old) and slowly progressive truncal and limb ataxias of cerebellar origin. Some of them showed gaze-evoked nystagmus, increased deep reflexes without Babinski sign, tremor, mildly decreased vibration sense and mild rectourinary disturbances. All but one were still ambulatory after 15 years of their mean disease duration. An earlier disease onset in successive generations was observed in 8 families. Brain MRIs revealed cerebellar atrophy predominantly in the anterior vermis in all the patients and mild brainstem atrophy in only 3 patients. Although the clinical phenotype manifested by most of the genetically unclassified AD-SCAs is similar to that of SCA6, whether the phenotype is caused by expansion of triplet repeats or some mutations in a single gene or different mutations in various genes is still unclarified.