Outbreaks of Campylobacteriosis Caused by Drinking Raw Milk in Japan: Evidence of Relationship Between Milk and Patients by Using Whole Genome Sequencing.

Raw milk may contain some infectious bacteria and usually requires pasteurization before drinking. In this study, we report rare outbreaks of campylobacteriosis associated with raw milk in Japan, and the application of whole genome sequencing (WGS) to studies on foodborne diseases. In August 2018, there were three outbreaks of campylobacteriosis, presumably caused by the consumption of unpasteurized raw milk, derived from the same farm; thus, these three outbreaks seemed to be associated with a single contaminant at the farm. Therefore, we analyzed Campylobacter jejuni isolates obtained at the three locations using several genetic methods. The sequence type of each isolate, revealed by multilocus sequence typing, was ST-61, and the profile determined using pulsed-field gel electrophoresis was the same; however, neither method could distinguish these from previously obtained strains. Subsequently, we performed WGS and single nucleotide variant (SNV) analysis that provided evidence of clonality, indicating that C. jejuni contamination was attributed to the farm. As in this study, evidence suggests that SNV analysis provides molecular biological support in cases with sufficient epidemiological information. Hence, similar analytical methods may be used in other sporadic cases to elucidate the relevance of the cases.

Loss of Cdc42 in Exocrine Acini Decreases Saliva Secretion but Increases Tear Secretion-A Potential Model of Exocrine Gland Senescence.

Cdc42 is a small GTPase essential for the cell cycle, morphogenesis, and cell adhesion, and it is involved in the polarity of epithelial cells. However, the functional roles of Cdc42 in exocrine glands, such as the maintenance of acini and water secretion, are not yet well understood. In this study, we generated acinar-cell-specific Cdc42 conditional knockout (Cdc42cKO) mice to assess their maintenance of acinar cells and physiological functions in the salivary glands (SGs) and lacrimal glands (LGs). Our data revealed that the loss of Cdc42 altered the luminal structures to bulging structures and induced acinar cell apoptosis in both the parotid glands (PGs) and LGs of Cdc42cKO mice. Interestingly, saliva secretion in response to pilocarpine stimulation was decreased in the Cdc42cKO group, whereas tear secretion was increased. Consistent with the water secretion results, protein expression of the water channel AQP5 in acinar cells was also decreased in the PGs but conversely increased in the LGs. Moreover, the changes that increased AQP5 expression in LGs occurred in the acinar cells rather than the duct cells. The present study demonstrates that Cdc42 is involved in the structural and survival maintenance of acinar cells in SGs and LGs. On the other hand, depletion of Cdc42 caused the opposite physiological phenomena between PGs and LGs.

Retinal damage alters gene expression profile in lacrimal glands of mice.

Early detection of such retinal diseases as glaucoma and age-related macular degeneration (AMD) is important to prevent blindness. There have been reports of changes in some components in the tears of glaucoma and AMD patients, suggesting tears' potential usefulness in screening for retinal diseases. We hypothesized that retinal damage might alter gene expression in the lacrimal gland, leading to those changes in tear components. We caused retinal damage in mice by intravitreal injection of N-methyl-d-aspartate (NMDA) or excessive light exposure. Hematoxylin and eosin staining showed no histological changes in the lacrimal glands of animals whose retinas had been damaged. However, RNA sequencing of lacrimal glands on the 3rd day after NMDA injection or light exposure revealed changes in the expression of 491 genes (268 up-regulated; 223 down-regulated) in the NMDA group and 531 genes (311 up-regulated; 220 down-regulated) in the light group. Further gene-set enrichment analysis indicated that both types of retinal damage activated the immune system in the lacrimal glands. This is the first demonstration that retinal damage can alter gene expression in the lacrimal glands, and it might lead to a novel non-invasive screening method for early detection of retinal diseases.

Arginase 1 is involved in lacrimal hyposecretion in male NOD mice, a model of Sjögren's syndrome, regardless of dacryoadenitis status.

KEY POINTS: Few reports have explored the possibility of involvement of non-inflammatory factors in lacrimal hyposecretion in Sjögren's syndrome (SS). RNA-sequencing analysis revealed that only four genes, including arginase 1, were downregulated in the lacrimal gland of SS model male mice (NOD mice) after onset of lacrimal hyposecretion and dacryoadenitis. Even in non-dacryoadenitis-type NOD mice, tear secretion and arginase 1 expression remained low. An arginase 1 inhibitor reduced tear secretion and partially reduced saliva secretion in BALB/c mice. The results indicate that a non-inflammatory factor, arginase 1, is involved in lacrimal hyposecretion in male NOD mice, regardless of dacryoadenitis status. ABSTRACT: Lacrimal fluid (tears) is important for preservation of the ocular surface, and thus lacrimal hyposecretion in Sjögren's syndrome (SS) leads to reduced quality of life. However, the cause(s) of lacrimal hyposecretion remains unknown, even though many studies have been conducted from the perspective of inflammation. Here, we hypothesized that a non-inflammatory factor induces lacrimal hyposecretion in SS pathology, and to elucidate such a factor, we conducted transcriptome analysis of the lacrimal glands in male non-obese diabetic (NOD) mice as an SS model. The NOD mice showed inflammatory cell infiltration and decreased pilocarpine-induced tear secretion at and after 6 weeks of age compared to age-matched BALB/c mice. RNA-sequencing analysis revealed that only four genes, including arginase 1, were downregulated, whereas many genes relating to inflammation were upregulated, in the lacrimal glands of male NOD mice after onset of lacrimal hyposecretion and dacryoadenitis (lacrimal gland inflammation). Changes in the level of arginase 1 expression were confirmed by real-time RT-PCR and western blot analysis. Furthermore, non-dacryoadenitis-type NOD mice were used to investigate the relationships among arginase 1 expression, lacrimal hyposecretion and dacryoadenitis. Interestingly, these NOD mice retained the phenotype of dacryoadenitis with regard to tear secretion and arginase 1 expression level. An arginase 1 inhibitor reduced tear secretion and partially reduced saliva secretion in BALB/c mice. In conclusion, a non-inflammatory factor, arginase 1, is involved in lacrimal hyposecretion in male NOD mice, regardless of dacryoadenitis status. These results shed light on the pathophysiological role of arginase 1 in SS (dry eye).

JPH203, a newly developed anti-cancer drug, shows a preincubation inhibitory effect on L-type amino acid transporter 1 function.

JPH203 is a novel anti-cancer drug targeting L-type amino acid transporter 1 (LAT1), which plays a primary role in the uptake of essential amino acids in tumor cells. Although a co-incubation inhibitory effect of JPH203 has been shown in a conventional uptake assay, its preincubation inhibitory effects have remained undetermined. Therefore, we aimed to characterize the preincubation inhibitory effects of JPH203 on LAT1 function using leucine uptake assays in LAT1-positive human colon cancer HT-29 cells. Preincubation of the cells with JPH203 (0.3 µM for 120 min) decreased the activity level to 30% of that in dimethylsulfoxide-treated cells. Similarly, in time-dependency analysis, preincubation of HT-29 cells with 10 µM JPH203 for 30, 60, and 120 min decreased the leucine uptake activity (42%, 32%, and 28% of that in control cells, respectively). Furthermore, the IC50 value of the combination of preincubation and co-incubation effects was lower than that of co-incubation inhibition alone (34.2 ± 3.6 nM vs. 99.2 ± 11.0 nM). In conclusion, we revealed that JPH203 has the capability to inhibit LAT1 function through preincubation effects. Moreover, preincubation synergistically enhances the co-incubation inhibitory effects. These findings provide a novel insight into the anti-cancer effects of JPH203 in cancer therapy.

Rapid attainment of target trough concentrations of tacrolimus for early improvement of clinical symptoms in patients with ulcerative colitis.

WHAT IS KNOWN AND OBJECTIVE: The target trough concentration of tacrolimus for ulcerative colitis is recommended to be 10-15 ng/mL in the initial two weeks and 5-10 ng/mL in the later phase. However, the effectiveness of rapid attainment of these target trough concentrations of tacrolimus in patients with ulcerative colitis is still unclear. In the present study, we evaluated the clinical efficacy and safety of rapid attainment of target trough concentrations of tacrolimus in patients with ulcerative colitis. METHODS: A prospective cohort was conducted at Gifu University Hospital in Gifu, Japan. Hospitalized patients who received tacrolimus for the treatment of ulcerative colitis between April 2009 and March 2017 were enrolled. Since June 2011, the initial loading dose of tacrolimus increased from 0.05 to 0.1-0.2 mg/kg/d, and the maintenance dose to achieve the target trough concentration was determined to be 12.5 ng/mL by proportional calculation with measured blood concentration. The period required to attain target trough concentration and the clinical efficacy before and after dosage modification was compared. RESULTS: The initial dose after dosage modification was significantly increased compared to that before dosage modification (0.10 [0.04-0.22], median [range] mg/kg/d vs 0.05 [0.03-0.05] mg/kg/d, P < 0.001). The period required to attain a target trough concentration over 10 ng/mL was significantly shortened by dosage modification (6 [4-14] days before dosage modification vs 4.5 [2-8] days after modification, P = 0.048). Further, stool frequency score was significantly improved after dosage modification, without affecting the incidence of adverse events. WHAT IS NEW AND CONCLUSION: Our findings suggest that rapid attainment of the target trough concentration of tacrolimus improves clinical symptoms in patients with ulcerative colitis.

Evaluation of the Microbiological Efficacy of a Single 2-Gram Dose of Extended-Release Azithromycin by Population Pharmacokinetics and Simulation in Japanese Patients with Gonococcal Urethritis.

The objective of this study was to analyze the relationship between the pharmacokinetic (PK)/pharmacodynamic (PD) parameters of a single 2-g dose of extended-release formulation of azithromycin (AZM-SR) and its microbiological efficacy against gonococcal urethritis. Fifty male patients with gonococcal urethritis were enrolled in this study. In 36 patients, the plasma AZM concentrations were measured using liquid chromatography-tandem mass spectrometry, the AZM MIC values for the Neisseria gonorrhoeae isolates were determined, and the microbiological outcomes were assessed. AZM-SR monotherapy eradicated N. gonorrhoeae in 30 (83%) of the 36 patients. AZM MICs ranged from 0.03 to 2 mg/liter. The mean value of the area under the concentration-time curve (AUC), estimated by population PK analysis using a two-compartment model, was 20.8 mg · h/liter. Logistic regression analysis showed that the PK/PD target value required to predict an N. gonorrhoeae eradication rate of ≥95% was a calculated AUC/MIC of ≥59.5. The AUC/MIC value was significantly higher in patients who achieved microbiological cure than in patients who achieved microbiological failure. Monte Carlo simulation using this MIC distribution revealed that the probability that AZM-SR monotherapy would produce an AUC/MIC exceeding the AUC/MIC target of 59.5 was 47%. Furthermore, the MIC distribution for strains isolated in this study was mostly consistent with that for strains currently circulating in Japan. In conclusion, in Japan, AZM-SR monotherapy may not be effective against gonococcal urethritis. Therefore, use of a single 2-g dose of AZM-SR either with or without other antibiotics could be an option to treat gonococcal urethritis if patients are allergic to ceftriaxone and spectinomycin or are diagnosed to be infected with an AZM-sensitive strain.

Involvement of cannabinoid receptor type 2 in light-induced degeneration of cells from mouse retinal cell line in vitro and mouse photoreceptors in vivo.

Earlier studies showed that the expressions of the agonists of the cannabinoid receptors are reduced in the vitreous humor of patients with age-related macular degeneration (AMD), and the cannabinoid type 2 receptor is present in the retinas of rats and monkeys. The purpose of this study was to determine whether the cannabinoid type 2 receptor is involved in the light-induced death of cultured 661W cells, an immortalized murine retinal cell line, and in the light-induced retinal degeneration in mice. Time-dependent changes in the expression and location of retinal cannabinoid type 2 receptor were determined by Western blot and immunostaining. The cannabinoid type 2 receptor was down-regulated in murine retinae and cone cells. In the in vitro studies, HU-308, a cannabinoid type 2 receptor agonist, had a protective effect on the light-induced death of 661W cells, and this effect was attenuated by SR144528, a cannabinoid type 2 receptor antagonist. Because the cannabinoid type 2 receptor is a G-protein coupled receptor and is coupled with Gi/o protein, we investigated the effects of the cAMP-dependent protein kinase (PKA). HU-308 and H89, a PKA inhibitor, deactivated PKA in retinal cone cells, and H89 also suppressed light-induced cell death. For the in vivo studies, a cannabinoid type 2 receptor agonist, HU-308, or an antagonist, SR144528, was injected intravitreally into mouse eyes before the light exposure. Electroretinography was used to determine the physiological status of the retinas. Injection of HU-308 improved the a- and b-waves of the ERGs and also the thickness of the outer nuclear layer of the murine retina after light exposure. These findings indicate that the cannabinoid type 2 receptor is involved in the light-induced retinal damage through PKA signaling. Thus, activation of cannabinoid type 2 receptor may be a therapeutic approach for light-associated retinal diseases.

Time-Dependent Decline in Serum Phenytoin Concentration With Heightened Convulsive Seizure Risk by Prolonged Administration of Fosphenytoin in Japanese: A Retrospective Study.

BACKGROUND: Because clinical data to confirm the safety and effectiveness of fosphenytoin, a prodrug of phenytoin, are insufficient, the length of administration of fosphenytoin is restricted. Nevertheless, some cases require fosphenytoin administration for more than a few days. The aim of this study was to retrospectively investigate the serum concentration of phenytoin in adult Japanese patients who received intravenous fosphenytoin therapy for more than 3 days. METHODS: Patients injected with intravenous fosphenytoin for more than 3 days at Gifu University Hospital between January 2012 and September 2014 were enrolled. Individual pharmacokinetic parameters were predicted by Bayesian estimation using NONMEM software, and the maintenance dose of fosphenytoin required to maintain the therapeutic trough concentration (10-20 mcg/mL) was calculated from the parameters. RESULTS: Among a total of 8 patients, the serum trough concentration of phenytoin decreased with each day after repeated injection of fosphenytoin. The incidence rate of significant convulsive seizures was increased time dependently (0% on day 1, 12.5% on day 2, 25% on day 3, and 66.7% on day 4 and after). Phenytoin clearance showed a time-dependent increase. The maintenance dose of fosphenytoin required to maintain the therapeutic trough concentration was simulated to be 779.8 ± 316.8 mg/d, a dose that was markedly higher than the actual maintenance dose (414.1 ± 55.7 mg/d). CONCLUSIONS: Prolonged use of fosphenytoin, for such patients as those with autoimmune-mediated encephalopathy accompanied with reflux disease and/or ileus, time dependently decreased the serum concentration of phenytoin and increased the risk of convulsion. Therefore, the maintenance dose should be increased to maintain the therapeutic serum concentration.

Characterization of an L-Carnitine Transport System in Murine Photoreceptor Cell Line.

While it is well known that L-carnitine [3-hydroxy-4-(trimethylazaniumyl)-butanoate] is an essential molecule for ß-oxidation, it provides anti-oxidative effects as well. Since these effects have been observed in photoreceptor cells, the carnitine's intracellular concentration is considered to play a protective role against oxidative damage to those cells. However, even though its high hydrophilicity makes it likely that carnitine import is accomplished via a dedicated host transport system, the specific uptake process into those cells is currently unknown. Therefore, in this study, we sought to identify and characterize photoreceptor cell carnitine uptake transporter(s) utilizing 661W cells as a photoreceptor cell model. The results of our uptake assays showed that carnitine was transported into 661W cells in a saturable manner (Km=5.5 mM), and that the activity was susceptible to extracellular pH and Na+. While these data suggest the involvement of a transporter in 661W cell carnitine uptake, the observed transport profile did not correspond to any of the currently known carnitine transporters such as organic cation/carnitine transporter 1 (Octn1), Octn2, Octn3, B0,+ and Ct2. In fact, in our experiments, the mRNA expressions for such carnitine transporters in 661W cells were consistently very low and the carnitine transporter substrates did not inhibit the uptake activities. Taken as a whole, our results indicate that carnitine is transported into 661W cells in a carrier-mediated manner. However, since its transport modes cannot be fully explained by known carnitine transporters, it is highly likely that photoreceptor cells utilize a unique molecularly-based carnitine uptake system.