Temporal Changes in Brain Perfusion in a Patient with Myoclonus and Ataxia Syndrome Associated with COVID-19.

Myoclonus and ataxia, with or without opsoclonus, have recently been recognized as a central nervous system syndrome associated with coronavirus disease-2019 (COVID-19). A 52-year-old Japanese man developed myoclonus and ataxia 16 days after the onset of COVID-19. Brain single-photon emission computed tomography (SPECT) revealed hyperperfusion in the cerebellum and hypoperfusion in the cerebral cortices with frontal predominance during the acute stage, which improved over two months. This study indicates that brain perfusion SPECT can be effective in detecting functional alterations in COVID-19-related myoclonus and ataxia.

A structured summary of a study protocol for a multi-center, randomized controlled trial (RCT) of COVID-19 prevention with Kampo medicines (Integrative Management in Japan for Epidemic Disease by prophylactic study: IMJEDI P1 study).

OBJECTIVE: We aimed to test our hypothesis that traditional Japanese (Kampo) medicine, hochuekkito (Hochu-ekki-to: HET) has a preventive effect for the symptoms on COVID-19. TRIAL DESIGN: The study is designed as a multi-center, interventional, parallel-group, randomized (1:1 ratio), investigator sponsored, two-arm study. PARTICIPANTS: Six thousand participants will be recruited from healthy hospital workers in 7 Japanese University Hospitals. INCLUSION CRITERIA: 1. Age from 20 to 75 years old at the time of registration 2. Asymptomatic and body temperature below 37°C at the time of registration 3. Capable of eating orally Exclusion criteria: 1. Previous upper respiratory inflammation due to viral infection (including suspected COVID-19) 2. Taking immunosuppressants 3. Allergic to the Kampo medicines used in this study 4. History of hypokalaemia, severe hypertension, severe liver dysfunction, and interstitial pneumonia 5. Regularly taking other Kampo medicines 6. Pregnant or possibly pregnant 7. Participating in other research 8. Judged to be unsuitable for this study by the doctor in charge INTERVENTION AND COMPARATOR: Kampo group: participants receive HET in 9 tablets 2 times per day for 8 weeks. CONTROL GROUP: participants receive placebo in the same dosage as the Intervention group - 9 tablets 2 times per day for 8 weeks. Placebo tablets are identical in appearance and package to HET. Taste of placebo is different from that of HET. The Ohsugi Pharmaceutical Co. Ltd, Osaka, Japan manufactured the placebo and HET. MAIN OUTCOMES: Primary outcome: Number of patients with a SARS-CoV-2 RNA by ploymerase chain reaction (PCR) positive result with at least one symptom (fever, cough, sputum, malaise, shortness of breath) during the 12-week study period (including the 4-week observation period after oral administration). SECONDARY OUTCOMES: 1. Period from infection to onset 2. Period from the appearance of symptoms to the disappearance of PCR positive 3. Number of days until the appearance or improvement of symptoms 4. Severe stage: presence of hospitalization 5. Shock stage: ICU management required for mechanical ventilation, shock vitals or failure of organ(s) other than lungs Safety endpoints include numbness in the hands and/or feet, edema, skin rash or other allergic symptoms, and gastric discomfort. RANDOMISATION: Patients are randomized (1:1 ratio) to each group using minimization implemented with the Electric data capture system (DATATRAK Enterprise Cloud), with balancing of the arms with age range (under 50 years of age or not) and having a history of risk factors for COVID-19 (cardiovascular disease, hypertension, diabetes, respiratory diseases). BLINDING (MASKING): Only participants will be randomized. NUMBERS TO BE RANDOMISED (SAMPLE SIZE): The main research hypothesis of this study is that Kampo medicines significantly prevent the onset of COVID-19. It is assumed that the infection rate before the administration of the drug under consideration will be 0% and that the incidence of COVID-19 thereafter will be 2- 3%, of which 70%-80% will show symptoms of COVID-19. Assuming that the pharmaceutical effect of the drug will be effective in 50% of patients and that the incidence rates in the placebo and drug groups will be 1.4%-2.4% and 0.7%-1.2%, respectively, the placebo is calculated at 2%, and the study drug at 1%. Since the frequency of verification is low and the number of cases will be large, we set a total of 10 analyses (9 interim analyses and a final analysis). Since the number of cases at the time of the final analysis will be 4,986 under the conditions of α = 0.05 and a power of 80% by the Peto method. We set at 600 cases in each interim analysis with an estimated dropout rate of 16.9%. Finally, the total number of cases is set to 6,000 with 3,000 in the placebo group and 3,000 in the HET group. TRIAL STATUS: Protocol version 1.3 of October 23rd , 2020. Recruitment start (expected): December 1st, 2020. Recruitment finish (expected): December 31st, 2022. TRIAL REGISTRATION: This trial is registered in the Japan Registry of Clinical Trials (jRCT) ( jRCTs031200150 ) on 14 October 2020. FULL PROTOCOL: The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest of expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol.

Comparison between neoadjuvant and adjuvant gemcitabine plus cisplatin chemotherapy for muscle-invasive bladder cancer.

AIM: Radical cystectomy plus platinum-based perioperative chemotherapy is a standard treatment for patients with clinically localized muscle-invasive bladder cancer. The standard perioperative chemotherapy is methotrexate, vinblastine, doxorubicin and cisplatin (MVAC). However, no prospective randomized trial has been published that compares neoadjuvant and adjuvant chemotherapy for bladder cancer. Moreover, the efficacy of perioperative chemotherapy with gemcitabine plus cisplatin (GC) has not been clarified. In this study we have compared the clinical outcomes between neoadjuvant and adjuvant chemotherapy in patients receiving GC. METHODS: We retrospectively reviewed the records of patients who were scheduled to be treated with a radical cystectomy plus perioperative chemotherapy with GC from 2005 to 2010 at our institution. The primary outcome measure was recurrence-free survival (RFS). RESULTS: A total of 42 patients received perioperative chemotherapy with GC (25 neoadjuvant, 17 adjuvant). The median number of cycles of GC administered to the two groups was not significantly different. The median duration of follow up was 28.6 months. During the follow-up period, recurrence was observed in nine and three patients in the neoadjuvant and adjuvant groups, respectively. The RFS rate at median follow up was 67 and 76% in the neoadjuvant and adjuvant groups, respectively. No significant difference in RFS at median follow up was observed between the two groups (P = 0.124). CONCLUSION: Our results showed no statistically significant difference in RFS between neoadjuvant and adjuvant GC chemotherapy for muscle-invasive bladder cancer. We expect to validate these findings in a prospective randomized trial.

The isolation and identification of apolipoprotein C-I in hormone-refractory prostate cancer using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry.

Androgens play a central role in prostate cancer pathogenesis, and hence most of the patients respond to androgen deprivation therapies. However, patients tend to relapse with aggressive prostate cancer, which has been termed as hormone refractory. To identify the proteins that mediate progression to the hormone-refractory state, we used protein-chip technology for mass profiling of patients' sera. This study included 16 patients with metastatic hormone-refractory prostate cancer who were initially treated with androgen deprivation therapy. Serum samples were collected from each patient at five time points: point A, pre-treatment; point B, at the nadir of the prostate-specific antigen (PSA) level; point C, PSA failure; point D, the early hormone-refractory phase; and point E, the late hormone-refractory phase. Using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we performed protein mass profiling of the patients' sera and identified a 6 640-Da peak that increased with disease progression. Target proteins were partially purified, and by amino acid sequencing the peak was identified as a fragment of apolipoprotein C-I (ApoC-I). Serum ApoC-I protein levels increased with disease progression. On immunohistochemical analysis, the ApoC-I protein was found localized to the cytoplasm of the hormone-refractory cancer cells. In this study, we showed an increase in serum ApoC-I protein levels in prostate cancer patients during their progression to the hormone-refractory state, which suggests that ApoC-I protein is related to progression of prostate cancer. However, as the exact role of ApoC-I in prostate cancer pathogenesis is unclear, further research is required.

Protein profiling in pancreatic juice for detection of intraductal papillary mucinous neoplasm of the pancreas.

BACKGROUND/AIMS: Malignant intraductal papillary mucinous neoplasm of the pancreas (IPMN) has a very poor prognosis, and there is no useful biomarker for an early diagnosis at present. A biomarker is expected to allow an early diagnosis of IPMNs and consequently lead to an improvement of the patients' prognosis. Recent advances in proteomic analysis are remarkable; therefore we explored novel biomarkers for IPMN using Surface-Enhanced Laser Desorption and Ionization (SELDI) Mass Spectrometry. METHODOLOGY: We collected pancreatic juice samples from 33 patients with IPMNs, 54 patients with pancreatic ductal carcinoma, and 31 with chronic pancreatitis. We analyzed the pancreatic juice samples using a SELDI ProteinChip system (Ciphergen Biosystems, Fremont, CA). RESULTS: We identified a 6240-Da peak whose expression in pancreatic juice from patients with IPMNs was significantly higher compared with that in other pancreatic diseases (P<0.01). This 6240-Da protein was partially purified and was identified as pancreatic secretory trypsin inhibitor (PSTI) by amino acid sequencing. The pancreatic juice PSTI levels, as measured by radioimmunoassay, were significantly higher in the IPMN group than in the other groups (P<0.001). When the diagnostic cutoff value of PSTI in pancreatic juice was set at 25000 ng/mL, the positive predictive value, negative predictive value, sensitivity, and specificity were respectively 89%, 83%, 48%, and 98%. CONCLUSIONS: PSTI levels of pancreatic juice in patients with IPMN were significantly higher than those in patients with other pancreatic diseases. The PSTI level in pancreatic juice may be useful for the diagnosis of IPMN.

Role of the genetic polymorphisms in the 5'-flanking region for transcriptional regulation of the human CYP2E1 gene.

BACKGROUND: The human cytochrome P-4502E1 (CYP2E1) has been known to be that are associated with alcohol-related diseases and various cancers. Difference in susceptibility in enzyme induction of the CYP2E1 by environmental factors such as ethanol may be associated with the risk of these diseases. In the 5'-flanking region of the human CYP2E1 gene, there were tandem repeat polymorphism (CYP2E1*1C*,1D) and RsaI polymorphism (CYP2E1*5A*,5B), which have been reported to be related to these diseases. Thus, we focused relationship between these polymorphisms and transcriptional regulation. The aim of this study was to determine the role of these polymorphisms for the transcriptional regulation of the human CYP2E1 gene in general transcription and during enzyme induction. METHODS: To determine the role of these polymorphisms, various constructs of the 5'-flanking region of the human CYP2E1 gene were prepared and transfected into HeLa cells and HepG2 cells treated or not with pyrazine, a well-known CYP2E1 inducer. RESULTS: The transcriptional activity of CYP2E1*1D was higher than that of CYP2E1*1C in HeLa cells. The -1,306 base pairs (bp) construct (-1,306 to +9) increased transcriptional activity compared with the 2.5 kb full-length construct. The suppressive effect of transcriptional activity by deletion of the region including CYP2E1*1C was greater than that by CYP2E1*1D in HeLa cells. The -580 bp construct (-580 to +9) decreased transcriptional activity compared with the -1,306 bp construct. The -1,306 bp construct also showed a similar tendency during pyrazine treatment. CONCLUSIONS: The region including the tandem repeat polymorphism was suggested to regulate transcription suppressively. Besides, it was speculated that the transcriptional activity of CYP2E1*1D was higher than that of CYP2E1*1C because transcriptional suppression of CYP2E1*1D was weaker than that of CYP2E1*1C. Also, it was confirmed that the region including RsaI polymorphism was associated with the promotion of transcription.

Detection of biomarkers for alcoholism by two-dimensional differential gel electrophoresis.

BACKGROUND: Up to now, gamma-glutamyltransferase (gamma-GTP) and carbohydrate-deficient transferrin (CDT) have been used as markers for alcoholism most widely, but they are not satisfactory regarding sensitivity/specificity. Therefore, for novel markers need to be searched. METHODS: To detect new biomarkers for alcoholism, albumin and immunoglobulinG were first removed from serum. Then, protein profiles of 12 serum samples before and after 3 months of abstinence treatment were examined using agarose 2-dimensional differential gel electrophoresis (agarose2-D DIGE). Two-dimensional differential gel electrophoresis images were analyzed using Shimadzu 2-D Evolution Software. RESULTS: Eight spots whose expression were significantly altered after abstinence were detected. Of these, 2 proteins increased and 6 proteins decreased after treatment. CONCLUSIONS: Altered expressions of several serum proteins after abstinence therapy were detected. They are promising markers for clinical application of alcoholism.

Expression of the fetal-oncogenic fibroblast growth factor-8/17/18 subfamily in human hematopoietic tumors.

The fibroblast growth factors (FGFs) are involved in hematopoiesis and tumorigenesis. However, little is known about the contribution of the FGFs identified within the past 10 years to leukemogenesis. To elucidate whether these FGFs (FGF-8, -9, -10, -11, -12, -13, -14, -16, -17, -18, -19, -20, and -21) are expressed in leukemic cells, we performed RT-PCR analyses using 28 cell lines. The members of a fetal-oncogenic subfamily, FGF-8/-17/-18, were often expressed (53.5%, 25.0%, and 32.1%) with the co-expression of their receptors. Realtime quantitative-PCR analysis showed that FGF-8/-17 were aberrantly expressed in patients with acute leukemia. Moreover, cell proliferation assays revealed the proliferation activity of FGF-17 on leukemic cells expressing its receptors. These results demonstrated that certain recently identified FGFs play an important role in the growth of leukemic cells, possibly with an autocrine mode of action, and that these FGFs will become novel biomarkers for hematopoietic tumors.

Centromere protein H is up-regulated in primary human colorectal cancer and its overexpression induces aneuploidy.

Chromosomal instability (CIN) has been recognized as a hallmark of human cancer and is caused by continuous chromosome missegregation during mitosis. Proper chromosome segregation requires a physical connection between spindle microtubules and centromeric DNA and this attachment occurs at proteinaceous structures called kinetochore. Several centromere proteins such as CENP-A and CENP-H are the fundamental components of the human active kinetochore, and inappropriate expression of the centromere proteins could be a major cause of CIN. We have previously shown that CENP-A was overexpressed in primary human colorectal cancer. In this study, we show that CENP-H was also up-regulated in all of 15 primary human colorectal cancer tissues as well as in CIN tumor cell lines. Surprisingly, transient transfection of CENP-H expression plasmid into the diploid cell line HCT116 remarkably induced aneupoidy. Moreover, CENP-H stable transfectant of mouse embryonic fibroblast/3T3 cell lines showed aberrant interphase micronuclei, characteristic of chromosome missegregation. In these CENP-H overexpressed cells, CENP-H completely disappeared from the centromere of mitotic chromosomes, which might be the cause of the chromosome segregation defect. These results suggest that the aberrant expression and localization of a kinetochore protein CENP-H plays an important role in the aneuploidy frequently observed in colorectal cancers.

Comparative analyses of four different methods of genotyping ALDH2.

BACKGROUND: A number of methods of genotyping single nucleotide polymorphisms (SNPs) are currently available, ranging from the traditional restriction fragment length polymorphism (RFLP) and single-stranded conformational polymorphism (SSCP) to more sophisticated technologies. We determined the utility of three novel methods by genotyping aldehyde dehydrogenase-2 (ALDH2). METHODS: DNA was isolated from blood samples of 241 control subjects and 74 patients with esophageal cancer. The utility of three novel genotyping methods-melting curve analysis using a LightCycler, SNaPshot analysis using an ABI PRISM 310 genetic analyzer, and denaturing high-performance liquid chromatography using a WAVE DNA fragment analysis system-were compared with that of conventional fluorescent-based polymerase chain reaction (PCR)-SSCP using an ALF express DNA sequencer. RESULTS: The frequency of the mutant ALDH2*2 allele was significantly higher in patients with esophageal cancer (27.7%) than in control subjects (16.2%; p < 0.01; habitual alcohol drinkers). The melting curve analysis was accurate, more rapid, and easier to use than the SNaPshot analysis or denaturing high-performance liquid chromatography analysis. Fluorescent-based PCR-SSCP proved useful for analyzing a large number of samples. CONCLUSION: Melting curve analysis using the LightCycler is suitable for the genotyping of small numbers of samples in a routine clinical setting; fluorescent-based PCR-SSCP analysis using the ALF express DNA sequencer can be used for large-scale genotyping in epidemiologic studies.

Identification of novel and downregulated biomarkers for alcoholism by surface enhanced laser desorption/ionization-mass spectrometry.

Since personal and verbal reporting of alcohol use is not necessarily accurate, objective markers to assess alcohol consumption are required. The currently available markers, however, are limited in sensitivity and specificity for screening of excessive alcohol drinkers. Therefore, searches for novel markers are warranted. Recently, surface enhanced laser desorption/ionization-time of flight-mass spectrometry (SELDI-TOF-MS) has been successfully used to detect disease-associated proteins in complex biological specimens. We used the ProteinChip SELDI technology to generate comparative protein profiles of the consecutive serum samples obtained during abstinence from a total of 16 chronic alcoholic patients hospitalized for a rehabilitation program. We recognized two peaks (5.9 and 7.8 kDa), both of which had been downregulated on admission, the expression level of which significantly increased after a one-week abstinence. These changes were also seen in nonresponders of gamma-glutamyltransferase. These two proteins were partially purified and subjected to amino acid sequencing. The 5.9 kDa protein was identified as a fragment of fibrinogen alphaE chain and the 7.8 kDa was a fragment of apoprotein A-II. These novel protein fragments may be promising biomarkers for excessive alcohol drinking.

[Postgraduate medical training in laboratory medicine and clinical genetics in Chiba University Hospital].

Compulsory postgraduate clinical training in laboratory medicine will start on a national basis in April 2004. It remains to be discussed how departments of laboratory medicine and/or divisions of clinical laboratory should contribute to the training program. I here summarize what we are planning to do in Chiba University Hospital together with my personal views on this topic. Two points are of note in our program. First, one-week training in the division of clinical laboratory is essential as a part of the 6-month training of internal medicine. The items in the program include Gram staining, white blood cell differential counting, urinary sediment study, and representative point-of-care testing. A program to follow the entire flow of laboratory tests(from withdrawal of blood to reporting of the test results) is also included to highlight a variety of pre-analytical errors. Furthermore, trainees are encouraged to learn how to work and co-operate with co-medical personnel. Second, our program aims at training postgraduate medical students to be clinical geneticists competent at genetic counseling. Genetic counseling is the process by which patients or relatives at risk of a disorder that may be hereditary are advised of the consequences of the disorder, the probability of developing or transmitting it and of the ways to test them. Thus, our final goal is to foster genetically oriented experts in laboratory medicine and clinical geneticists with a wide knowledge of laboratory medicine.

Transcriptional activity of the tandem repeat polymorphism in the 5'-flanking region of the human CYP2E1 gene.

BACKGROUND: There are remarkable interindividual differences in the expression of cytochrome P-4502E1(CYP2E1), which in turn may alter susceptibility to alcohol-related diseases and various cancers. We recently characterized the tandem repeat polymorphism in the 5'-flanking region of the human CYP2E1 gene and found that subjects with the homozygous mutant-type (A4/A4) may be at higher risk of developing esophageal cancer. In this study, we determined how this tandem repeat polymorphism alters transcriptional activities of the human CYP2E1 gene by transfection studies. METHODS: The 5'-flanking region (-2,562 base pair to +9 base pair) of the CYP2E1 gene from three individuals of different genotypes (A2/A2, A2/A4, A4/A4) was amplified by polymerase chain reaction. The polymerase chain reaction products placed in front of a luciferase reporter gene were transfected into human hepatoblastoma cells, human esophageal cancer cells, and human uterus cancer cells. Transcriptional activities were determined by the dual-luciferase assay. When indicated, ethanol (50 mM) was included in the culture medium. CYP2E1 messenger RNA levels in peripheral lymphocytes were measured by the real-time reverse transcription-polymerase chain reaction using the LightCycler system. RESULTS: The construct including the tandem repeat region exhibited luciferase activities in both A2 and A4 type. It was of note that the activity produced by the A4 allele was significantly greater than that by A2 allele in HeLa cells (p < 0.001). CYP2E1 messenger RNA levels in peripheral blood lymphocytes were comparable between the two genotypes. CONCLUSION: Transcriptional activity of the mutant allele of the tandem repeat polymorphism in the 5'-flanking region of the CYP2E1 gene is greater than that of the wild type.

Identification of the CAB2/hCOS16 gene required for the repair of DNA double-strand breaks on a core amplified region of the 17q12 locus in breast and gastric cancers.

We previously reported that CAB1 and c-ERBB-2 genes were found to be located in a core amplified region of the 17q12 locus, which is frequently amplified in various cancers. During identification of this core region, CAB2, a human homologue of the yeast COS16 required for the repair of DNA double-strand breaks was cloned. Autofluorescence analysis of cells transfected with its GFP fusion protein demonstrated that CAB2 translocates into vesicles, suggesting that overexpression of CAB2 may decrease intercellular Mn2+ by accumulating it in the vesicles, in the same way as yeast COS16. This is the first report identifying all of the genes on the core amplified region of the 17q12 locus in breast and gastric cancers.