Publications by authors named "Kei Kozawa"

13 Publications

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The CD44/COL17A1 pathway promotes the formation of multilayered, transformed epithelia.

Curr Biol 2021 May 28. Epub 2021 May 28.

Department of Breast and Endocrine Surgery (Surgery II), Nagoya University Graduate School of Medicine, Nagoya, Japan.

At the early stage of cancer development, oncogenic mutations often cause multilayered epithelial structures. However, the underlying molecular mechanism still remains enigmatic. By performing a series of screenings targeting plasma membrane proteins, we have found that collagen XVII (COL17A1) and CD44 accumulate in RasV12-, Src-, or ErbB2-transformed epithelial cells. In addition, the expression of COL17A1 and CD44 is also regulated by cell density and upon apical cell extrusion. We further demonstrate that the expression of COL17A1 and CD44 is profoundly upregulated at the upper layers of multilayered, transformed epithelia in vitro and in vivo. The accumulated COL17A1 and CD44 suppress mitochondrial membrane potential and reactive oxygen species (ROS) production. The diminished intracellular ROS level then promotes resistance against ferroptosis-mediated cell death upon cell extrusion, thereby positively regulating the formation of multilayered structures. To further understand the functional role of COL17A1, we performed comprehensive metabolome analysis and compared intracellular metabolites between RasV12 and COL17A1-knockout RasV12 cells. The data imply that COL17A1 regulates the metabolic pathway from the GABA shunt to mitochondrial complex I through succinate, thereby suppressing the ROS production. Moreover, we demonstrate that CD44 regulates membrane accumulation of COL17A1 in multilayered structures. These results suggest that CD44 and COL17A1 are crucial regulators for the clonal expansion of transformed cells within multilayered epithelia, thus being potential targets for early diagnosis and preventive treatment for precancerous lesions.
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http://dx.doi.org/10.1016/j.cub.2021.04.078DOI Listing
May 2021

Evaluation of varicella vaccine effectiveness during outbreaks in schools or nurseries by cross-sectional study.

Vaccine 2021 05 22;39(21):2901-2905. Epub 2021 Apr 22.

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192, Japan.

Objective: The aim of this study was to elucidate vaccine effectiveness (VE) during varicella outbreaks in schools and nurseries in Japan.

Methods: An outbreak was defined as emergence of three or more cases of varicella within 21 days at the same institute. Clinical information such as varicella vaccination status, and history of varicella was collected. If a child had varicella during the outbreak, information about absences, fever, and disease severity was collected.

Results: From September 2018 to January 2020, four outbreaks were reported around our institute from three elementary schools and one nursery. A total of 676 children were analyzed in this study. Seventy-six children (11.2%) were unvaccinated, 309 (45.7%) had received one dose of vaccine, and 291 (43.0%) had received two doses. Most children in Pre-K2 (1-2 years old) to Pre-K6 (5-6 years old), who were the targets of the national immunization schedule, received two doses. Meanwhile, most children older than third grade received single dose. Seventy-five children (11.1%) had varicella. Varicella prevalence from Pre-K5 to the third grade was greater than 10%. The adjusted VEs of single- and two-dose of varicella vaccine were 57.8% and 89.0%. The number of days absent was significantly longer in unvaccinated children than single-dose recipients (P = 0.0145). Unvaccinated children had significantly more severe skin eruptions than single-dose recipients (P = 0.0046) and two-dose recipients (P = 0.0258).

Conclusions: Although VEs of single-dose varicella vaccination during outbreaks was not high, the VE of two-dose vaccination was similar to that in a previously reported case-control study.
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http://dx.doi.org/10.1016/j.vaccine.2021.04.009DOI Listing
May 2021

Inherited chromosomally integrated human herpesvirus 6 and autoimmune connective tissue diseases.

J Clin Virol 2020 11 5;132:104656. Epub 2020 Oct 5.

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

Background: Entire genome of human herpesvirus 6 (HHV-6) that integrates into human chromosomes is called chromosomally integrated HHV-6 (ciHHV-6). Several viral infections have been suggested to be involved in autoimmune connective tissue diseases (CTDs). Reactivated HHV-6 from the integrated viral genome can induce immune responses against the virus. Thus, it is plausible that ciHHV-6 is associated with autoimmune CTDs.

Objectives: We sought to determine whether the prevalence of ciHHV-6 was significantly higher in patients with autoimmune CTDs than in a healthy population.

Study Design: A total of 846 peripheral blood samples collected from autoimmune CTD patients were analyzed. Since there was a large number of samples, they were pooled into 24 samples per group. Copy numbers of HHV-6 DNA were measured by real-time PCR. The threshold level for distinguishing between ciHHV-6 and active viral infection and the reliability of pooled DNA analysis were examined as initial validation experiments.

Results: The threshold level was 1.6 × 10^6 copy/mL in whole blood. The reliability of pooled DNA analysis to identify one ciHHV-6 sample among 23 HHV-6 DNA-negative samples was high. No HHV-6 DNA was detected in any of the pooled DNA samples collected from the patients. The probability of the present study including the 846 autoimmune CTD patient's samples was statistically not different with a healthy Japanese population which was 0.2 % or 0.6 %.

Conclusions: There was no significant difference in the prevalence of ciHHV-6 between a healthy population and patients with autoimmune CTDs.
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http://dx.doi.org/10.1016/j.jcv.2020.104656DOI Listing
November 2020

Trend in varicella patients 4 years after implementation of universal two-dose varicella vaccination in Japan.

Vaccine 2020 10 30;38(46):7331-7336. Epub 2020 Sep 30.

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

Objective: To elucidate the trend and clinical spectrum of virologically diagnosed varicella patients after implementation of universal vaccination as a national immunization program in Japan.

Patients And Methods: Study subjects were patients suspected of varicella, less than 15 years of age, who visited 14 pediatric clinics in the Nagoya VZV Study Group from September 2015 to August 2019. Practitioners collected patient samples and information such as backgrounds, clinical symptoms, and previous immunization status. All patients were confirmed as having varicella based on molecular diagnostic assays.

Results: Varicella zoster virus (VZV) DNA was detected in swab samples from 506 (83.1%) of the 609 suspected patients. The 455 varicella patients for whom vaccination status was available were divided into two groups: 180 universal vaccination targets and 275 non-targets. Numbers of monthly varicella patients decreased gradually during the observation period. In the 2016/17 season, the seasonal epidemic of varicella became undetectable in the universal vaccination target group, and starting in the 2017/18 season, it was obscured even in the non-target group. The median age of patients was significantly lower in the universal vaccination target group (3 years) than the non-target group (7 years) (P < 0.001). Vaccination status differed significantly between the two groups (P < 0.001). Most varicella patients were in the non-target group, especially those who had been vaccinated once (60.4%). Frequency of fever (P < 0.001) and number of skin rashes at the time of the first hospital visit (P = 0.001) were significantly higher in the non-target group.

Conclusions: Although the number of childhood varicella patients declined after implementation of national immunization with two doses of varicella vaccination, sporadic outbreaks still occurred, mainly in the non-universal vaccination target group. Insufficient vaccination of members of this group is likely to be a major reason for small local outbreaks.
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http://dx.doi.org/10.1016/j.vaccine.2020.09.038DOI Listing
October 2020

Inherited Chromosomally Integrated Human Herpesvirus 6 Is a Risk Factor for Spontaneous Abortion.

J Infect Dis 2021 May;223(10):1717-1723

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Japan.

Background: Human herpesvirus 6 (HHV-6) can be genetically transmitted from parent to child as inherited chromosomally integrated HHV-6 (iciHHV-6). HHV-6 reactivation occurs in pregnant women with iciHHV-6. We found no sex differences in the frequency of index cases with iciHHV-6 but inheritance from the father was more common. We evaluated the association between iciHHV-6 status and spontaneous abortion.

Methods: iciHHV-6 was confirmed by high viral DNA copy numbers in whole blood and somatic cells. The origin of integrated viral genome, paternal or maternal, was examined using the same method. The pregnancy history of 23 mothers in families with iciHHV-6 and 285 mothers in families without iciHHV-6 was abstracted.

Results: Of 23 iciHHV-6 index cases, 8 mothers and 15 fathers had iciHHV-6. Spontaneous abortion rates in mothers with and mothers without/fathers with iciHHV-6 and mothers in families without iciHHV-6 were 27.6%, 10.3%, and 14.8%, respectively (P = .012). Mothers with iciHHV-6 (odds ratio [OR], 6.41; 95% confidence interval [CI], 1.10-37.4) and maternal age at the most recent pregnancy ≥40 years (OR, 3.91; 95% CI, 1.30-11.8) were associated with 2 or more spontaneous abortions.

Conclusions: Mothers with iciHHV-6 is a risk factor for spontaneous abortion.
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http://dx.doi.org/10.1093/infdis/jiaa606DOI Listing
May 2021

Immune response against SARS-CoV-2 in pediatric patients including young infants.

J Med Virol 2021 03 29;93(3):1776-1779. Epub 2020 Sep 29.

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Japan.

Pediatric cases of the coronavirus disease 2019 (COVID-19) are generally mild or asymptomatic, and are usually detected by virological examination following close contact with COVID-19 patients, often the children's parents. The detailed clinical features and virological data of pediatric patients with COVID-19, particularly young infants, remain unclear. Here, the clinical and virological characteristics of four children with COVID-19 including two young infants were investigated. One- and 4-month-old boys with COVID-19 were both asymptomatic, and seroconversion was demonstrated. These findings suggest that even young infants can mount an immune response against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), despite having weaker immune defenses than adolescents and adults. Three-year-old boy, who was SARS-CoV-2-negative, was admitted to the same room as his SARS-CoV-2-positive father due to the lack of caregivers. Although he was asymptomatic, he had seroconverted to SARS-CoV-2. Eleven-year-old boy, who was sibling of the 3-year-old boy, was also SARS-CoV-2-negative. He was isolated in his own room and did not seroconvert. If young children are SARS-CoV-2 negative, they should be isolated from their SARS-CoV-2-positive parents. This may be difficult in practice, if parents with COVID-19 are the only available caregivers. In such situations, the most appropriate measures should be taken for each patient.
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http://dx.doi.org/10.1002/jmv.26493DOI Listing
March 2021

ZAK Inhibitor PLX4720 Promotes Extrusion of Transformed Cells via Cell Competition.

iScience 2020 Jul 30;23(7):101327. Epub 2020 Jun 30.

Division of Molecular Oncology, Institute for Genetic Medicine, Hokkaido University Graduate School of Chemical Sciences and Engineering, Sapporo 060-0815, Japan; Department of Molecular Oncology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan. Electronic address:

Previous studies have revealed that, at the initial step of carcinogenesis, transformed cells are often eliminated from epithelia via cell competition with the surrounding normal cells. In this study, we performed cell competition-based high-throughput screening for chemical compounds using cultured epithelial cells and confocal microscopy. PLX4720 was identified as a hit compound that promoted apical extrusion of RasV12-transformed cells surrounded by normal epithelial cells. Knockdown/knockout of ZAK, a target of PLX4720, substantially enhanced the apical elimination of RasV12 cells in vitro and in vivo. ZAK negatively modulated the accumulation or activation of multiple cell competition regulators. Moreover, PLX4720 treatment promoted apical elimination of RasV12-transformed cells in vivo and suppressed the formation of potentially precancerous tumors. This is the first report demonstrating that a cell competition-promoting chemical drug facilitates apical elimination of transformed cells in vivo, providing a new dimension in cancer preventive medicine.
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http://dx.doi.org/10.1016/j.isci.2020.101327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371749PMC
July 2020

Coinfection With Human Herpesvirus (HHV)-6B in Immunocompetent, Healthy Individuals With Chromosomally Integrated HHV-6A.

J Pediatric Infect Dis Soc 2021 Mar;10(2):175-178

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Japan.

Immunocompetent sisters with chromosomally integrated human herpesvirus 6A (HHV-6A) transiently excreted HHV-6B genome in their saliva. They did not have past histories of exanthema subitum but had antibodies against HHV-6A and HHV-6B. This suggests that endogenous HHV-6A may modify the clinical features of HHV-6B coinfection.
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http://dx.doi.org/10.1093/jpids/piaa009DOI Listing
March 2021

Frequency of subclinical herpes zoster in pediatric hematology-oncology patients receiving chemotherapy: A retrospective cohort analysis.

J Med Virol 2020 08 18;92(8):1260-1265. Epub 2019 Dec 18.

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

Varicella-zoster virus (VZV) reactivation from the enteric nervous system can cause ileus (Ogilvie's syndrome) in adult patients. Since no pediatric cases have been described, we sought to retrospectively analyze VZV reactivation in pediatric hematology-oncology patients to determine whether VZV infection including subclinical VZV reactivation can induce gastrointestinal complications such as Ogilvie's syndrome. Thirty-five patients who received chemotherapy at our institution between September 2013 and June 2018 were included. Serum samples were collected weekly during hospitalization and every 3 months during outpatient maintenance chemotherapy. A real-time polymerase chain reaction assay was used to measure VZV DNA load in serum. The clinical features of patients with VZV infection were retrospectively analyzed. Of 1165 serum samples, 7 (0.6%) were positive for VZV DNA. VZV DNA was detected in 3 of 35 patients. In patient A, VZV DNA was detected during two episodes. The first episode involved varicella-like eruptions caused by the Oka VZV vaccine strain. The second episode involved herpes zoster (HZ) caused by the same strain. Patients B and C had a clinical course that was typical for HZ caused by wild-type VZV. No gastrointestinal symptoms were observed at the time of VZV infection in these three patients. VZV DNA was not detected in any other samples. No pediatric cases with Ogilvie's syndrome caused by VZV reactivation were demonstrated in this cohort. Additionally, no subclinical VZV reactivation was found in this cohort. Further study is needed to elucidate the precise incidence of pediatric Ogilvie's syndrome caused by VZV reactivation.
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http://dx.doi.org/10.1002/jmv.25650DOI Listing
August 2020

Clinical Characteristics of Primary HHV-6B Infection in Children Visiting the Emergency Room.

Pediatr Infect Dis J 2019 10;38(10):e248-e253

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Japan.

Objective: This cohort study, based on the design of a prior study in the United States, was conducted to elucidate the clinical features of primary human herpesvirus-6B (HHV-6B) infection.

Methods: Between June 2014 and May 2016, febrile children younger than 5 years who visited the emergency room (ER) and underwent blood examination were enrolled in this study.

Results: Fifty-nine (12%) of the 491 patients were diagnosed with primary HHV-6B infection. The rates of both simple and complex febrile seizure were significantly higher in patients with primary HHV-6B infection than in those without (P < 0.001 and P = 0.008, respectively). The median age at primary HHV-6B infection was 15 months. Forty-seven (79.7%) of the 59 patients with primary HHV-6B infection were younger than 2-year-old. Clinical features were compared between HHV-6B-infected patients older and younger than 2 years. The frequency of apparent infection (exanthema subitum) was significantly higher in the younger patients (P = 0.01). The median leukocyte (P = 0.01) and lymphocyte (P < 0.001) counts in the patients older than 2 years were significantly lower than those in the younger patients.

Conclusions: Primary HHV-6B infection accounted for 12% of ER visits. Secondary febrile seizures, in particular the complex type, were considered to be a major contributor to the disease burden of primary HHV-6B infection. The timing of primary HHV-6B infection occurred at older ages than in past reports, and the frequency of inapparent infection was higher in older patients.
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http://dx.doi.org/10.1097/INF.0000000000002379DOI Listing
October 2019

Chromosomally integrated human herpesvirus 6 in the Japanese population.

J Med Virol 2018 10 10;90(10):1636-1642. Epub 2018 Jul 10.

Department of Pediatrics, Fujita Health University School of Medicine, Toyoake, Aichi, Japan.

The objectives of the work are to elucidate the incidence and virological findings of chromosomally integrated human herpesvirus 6 (ciHHV-6) in Japanese population and to analyze an association between ciHHV-6 and the clinical manifestation of exanthema subitum (ES). Real-time polymerase chain reaction was performed to determine HHV-6 DNA loads in 2347 cord blood samples from healthy neonates (cohort A), febrile children less than 5 years old (cohort B), and hematopoietic cell transplant recipients (cohort C). CiHHV-6 was confirmed by detection of high copy numbers of viral DNA in somatic cells. The integration site was determined by fluorescent in situ hybridization analysis. In the ciHHV-6 subjects of cohorts A and B, HHV-6 antibody titers were measured, the history of ES was obtained, and the incidence of ES was compared with non-ciHHV-6 children without primary HHV-6B infection in the cohort B. CiHHV-6 was detected in 14 (0.60%) of the 2347 samples: A (6/1006, 0.60%), B (6/790, 0.76%), and C (2/551, 0.36%). The integration sites were on chromosome 22q in seven cases, Yp in two cases, and 17q and Xp in one case. No past history of ES was observed in 11 of the 12 subjects. Nine children with ciHHV-6 underwent serological analysis and were found to be positive for HHV-6 IgG antibodies. Incidence of ES was statistically higher in the control subjects than the ciHHV-6 subjects (P = 0.0039). In Japan, the frequency of ciHHV-6 was 0.60%. A high incidence of ciHHV-6A, specifically in chromosome 22, is a characteristic finding among the Japanese. CiHHV-6 may interfere with the clinical symptoms of primary HHV-6B infection.
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http://dx.doi.org/10.1002/jmv.25244DOI Listing
October 2018

Time-Series Analysis Comparing the Prevalence of Antibodies against Nine Viral Species Found in Umbilical Cord Blood in Japan.

Jpn J Infect Dis 2016 Jul 13;69(4):314-8. Epub 2015 Nov 13.

Department of Pediatrics, Konan Kosei Hospital.

In this study, we investigated the prevalence of antibodies against 9 viral species found in umbilical cord blood from 561 neonates in 2013. Serum IgG antibodies against the following viruses were measured: herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV-6), measles virus (MV), rubella virus (RV), mumps virus (MuV), and human parvovirus B19 (HPV B19). A survey questionnaire regarding past medical history and maternal immunization status for the vaccine-preventable diseases of varicella, measles, rubella, and mumps was simultaneously administered. The results were compared with previous data collected in 2001-2002 from 378 umbilical cord blood samples. Viral seroprevalence data were: HSV, 54%; VZV, 96%; EBV, 96%; CMV, 67%; HHV-6, 100%; MV, 95%; RV, 94%; MuV, 64%; and HPV B19, 55%. The seroprevalence of CMV, MV, and MuV were significantly lower in 2013 than in 2001-2002 (CMV, 76%; MV, 98%; MuV, 93%). Compared with the 2001-2002 data, the mean IgG antibody values of the 4 vaccine-preventable diseases were significantly lower, and vaccination coverage for those diseases among mothers was significantly higher. Thus, attention should be paid to antibody levels in women of childbearing age in the future.
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http://dx.doi.org/10.7883/yoken.JJID.2015.366DOI Listing
July 2016

Kinase-interacting substrate screening is a novel method to identify kinase substrates.

J Cell Biol 2015 Jun;209(6):895-912

Department of Cell Pharmacology, Graduate School of Medicine, Nagoya University, Showa-ku, Nagoya, Aichi 466-8550, Japan

Protein kinases play pivotal roles in numerous cellular functions; however, the specific substrates of each protein kinase have not been fully elucidated. We have developed a novel method called kinase-interacting substrate screening (KISS). Using this method, 356 phosphorylation sites of 140 proteins were identified as candidate substrates for Rho-associated kinase (Rho-kinase/ROCK2), including known substrates. The KISS method was also applied to additional kinases, including PKA, MAPK1, CDK5, CaMK1, PAK7, PKN, LYN, and FYN, and a lot of candidate substrates and their phosphorylation sites were determined, most of which have not been reported previously. Among the candidate substrates for Rho-kinase, several functional clusters were identified, including the polarity-associated proteins, such as Scrib. We found that Scrib plays a crucial role in the regulation of subcellular contractility by assembling into a ternary complex with Rho-kinase and Shroom2 in a phosphorylation-dependent manner. We propose that the KISS method is a comprehensive and useful substrate screen for various kinases.
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http://dx.doi.org/10.1083/jcb.201412008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4477863PMC
June 2015