A novel modified thaw-siphon method for extracting cryoprecipitate: The Bokutoh-siphon method.

BACKGROUND: Cryoprecipitate (CRYO) is neither produced nor supplied by the Japanese Red Cross Society. A novel CRYO extraction method established in-house by modifying a thaw-siphon technique was demonstrated in this study. STUDY DESIGN AND METHODS: A pack of fresh frozen plasma was thawed and equally divided into two bags for CRYO extraction by different methods. CRYO was extracted from the blood plasma using a standard centrifugation method and our modified thaw-siphon method (Bokutoh-siphon method; B method). The two different CRYOs extracted were analyzed to compare the differences in the amount of fibrinogen recovered, clotting factors extracted, and clotting activity. RESULTS: The amount of fibrinogen in the CRYO extracted using the B-siphon method was similar to that obtained using the standard method (recovery of fibrinogen: B-siphon method: 71.2% vs. standard method: 61.0%). The amount of clotting XIII factor extracted using the B-siphon method was significantly lower than those extracted using the standard method. On the other hand, clotting II, V factors, and C1q esterase inhibitor not concentrated in CRYO content from the B-siphon method were significantly higher than that from the standard method. CONCLUSION: A new in-house CRYO preparation method was established by modifying a previously used thaw-siphon method. A coagulation factor-rich CRYO was extracted from plasma frozen at -40°C along with the first fraction of thawed plasma, without using a large-capacity refrigerated centrifuge for blood bags.

Characteristics of internalization of NMDA-type GluRs with antibodies to GluN1 and GluN2B.

To characterize internalization of NMDA-type glutamate receptors (GluRs) by antibodies to NMDA-type GluRs, we produced rabbit antibodies to N-terminals of human GluN1 and GluN2B, and examined internalization of NMDA-type GluRs in HEK293T cells using confocal microscopy. Internalization of NMDA-type GluRs occurred from at least 10 min after incubation with antibodies to GluN1 and or GluN2B and was temperature-dependent. These findings confirm that antibodies to N-terminals of GluN1 and GluN2B present in the cerebrospinal fluid of patients with NMDAR encephalitis can mediate prompt internalization of NMDA-type GluR complexes.

Effects of in-house cryoprecipitate on transfusion usage and mortality in patients with multiple trauma with severe traumatic brain injury: a retrospective cohort study.

BACKGROUND: Hypofibrinogenaemia is a common complication of multiple trauma with severe traumatic brain injury (Abbreviated Injury Scale score of the head ≥4; body ≥3). In Japan, neither fibrinogen concentrate nor cryoprecipitate is permitted to treat acquired hypofibrinogenaemia with the purpose of rapidly restoring a haemostatic level of fibrinogen. The aim of this study was to investigate transfusion usage and mortality in patients with multiple trauma and severe traumatic brain injury who were given a cryoprecipitate prepared in-house, comparing those administered the product early or later. MATERIAL AND METHODS: We prepared and produced cryoprecipitate from fresh-frozen plasma beginning in March 2013. We performed a retrospective cohort study of patients admitted to our single tertiary medical centre with severe multiple trauma with traumatic brain injury from March 2013 to June 2018, sorting them into those given the cryoprecipitate infusion within 90 minutes of admission (Early group) and those given it more than 90 minutes after admission (Late group). Clinical outcomes were compared between the two groups using chi-square or Fisher's exact tests and the Wilcoxon test as appropriate. RESULTS: There were 26 and 16 patients in the Early and Late groups, respectively. The 24-hour mortality tended to be lower in the Early group than in the Late group (8 vs 13%, respectively). The patients were more severely anaemic and thrombocytopenic after haemostatic therapy in the Late group than in the Early group. Transfusion usage in the Early group was lower than that in the Late group (red blood cells: 7±1 units vs 17±3 units, p<0.05; fresh-frozen plasma: 9±1 units vs 16±3 units, p<0.05; platelet concentrate: 3±1 units vs 15±4 units, p<0.05, respectively). DISCUSSION: Early administration of an in-house cryoprecipitate may reduce transfusion usage in patients with multiple trauma with severe traumatic brain injury.

Initial Results of Empirical Cryoprecipitate Transfusion in the Treatment of Isolated Severe Traumatic Brain Injury: Use of In-house-produced Cryoprecipitate.

Acute coagulopathy is common after traumatic brain injury (TBI), particularly in severe cases of acute subdural hemorrhage (ASDH). Although acute coagulopathy is associated with poor outcomes, the optimal treatment strategy remains unknown. Here, we report the initial results of an empirical cryoprecipitate transfusion strategy that we developed as an early intervention for acute coagulopathy after TBI. We performed chart reviews of adult patients (aged ≥18 years) who received early cryoprecipitate transfusion after admission to our institution with a diagnosis of severe TBI (Glasgow Coma Scale ≤8) and ASDH from March 2013 to December 2016. We compared the outcomes of these patients with those who were treated before the implementation of the cryoprecipitate transfusion strategy (January 2011-February 2013). During the study period, 33 patients received early cryoprecipitate transfusion and no acute transfusion-related adverse event was reported. The rate of coagulopathy development within 24 h after admission was lower in these patients (23%) than in the controls (49%), but the difference was not significant (P = 0.062). The in-hospital mortality rate was 36% in patients receiving early cryoprecipitate transfusion and 52% in controls. After adjusting for confounding factors, the in-hospital mortality rate was significantly lower in the intervention period [adjusted odds ratio: 0.25, 95% confidence interval (CI): 0.08-0.78, P = 0.017]. In summary, we analyzed initial results of a cryoprecipitate transfusion strategy in patients with severe isolated TBI and ASDH. No acute transfusion-related adverse event was observed, and early transfusion of the in-house-produced cryoprecipitate may have reduced rates of coagulopathy development and in-hospital mortality.

Pre-transfusion testing for Ebola virus disease patients in serious communicable infectious diseases hospitals in Tokyo: A cross-sectional study.

BACKGROUND: Ebola virus disease (EVD) was endemic to Africa in 2014-2016. Supportive therapies have been shown to improve the outcome of EVD, and additional supportive therapy including blood transfusion therapy and external circulation could be needed in the event of a future global outbreak. However, pre-transfusion testing policies and guidelines have not yet been established in Japan. METHODS: We conducted a cross-sectional study of blood transfusion therapy for EVD patients at three designated hospitals for serious communicable diseases in Tokyo. In each hospital, we surveyed blood transfusion therapy policy, blood transfusion protocol, presence of a specialist in the department of transfusion medicine, facility capacity for pre-transfusion compatibility testing, and types of personal protective equipment available. RESULTS: One hospital had a cross-matched compatible blood transfusion policy, one had a cross-matched compatible blood transfusion policy only when the patient's ABO and RhD type is previously known, and the third had not created a policy. Two hospitals had a department of transfusion medicine. These two hospitals had a special testing unit for serious communicable diseases, while the other had a portable unit for testing. There were no major differences noted in available personal protective equipment. CONCLUSION: Policies and protocols differ among hospitals. The choice of blood transfusion policy and pre-transfusion testing is largely dependent on equipment and human resources. Further discussion is required to develop national guidelines for blood transfusion therapy in patients with serious communicable diseases, including countermeasures against complications and ethical issues related to the safety of patients and healthcare workers.

Use of potassium adsorption filter for the removal of ammonia and potassium from red blood cell solution for neonates.

BACKGROUND: Ammonia in the plasma usually does not pass through the blood-brain barrier (BBB). However, it can affect the brain as a neurotoxin in neonates with anemia of prematurity. Excess intake of ammonia should therefore be restricted in conditions involving BBB breakdown, such as in premature neonates. A potassium adsorption filter (PAF) can remove not only potassium, but also ammonia from red blood cell (RBC) solution. PAF for neonates (PAF-n) has been recently introduced using small satellite packs. We evaluated the effects of PAF-n on the removal of ammonia and potassium from RBC solution in small satellite packs. STUDY DESIGN AND METHODS: RBC solutions were obtained from the Japanese Red Cross Society. Two units of RBC solution (280 mL) were divided into four satellite packs (70 mL/pack). The RBC solution was passed through PAF-n (Kawasumi Laboratories Inc.) that was primed with saline (100 mL) before use. The concentrations of ammonia and potassium were measured in the solution before and after filtration (four samples of 10 mL each of filtered RBC solution) by Biomedical Laboratories. RESULTS: Approximately 47 to 82 and 84% to 93% of ammonia and potassium were removed from the RBC solution, respectively, without dilution with saline. CONCLUSION: PAF-n can remove ammonia and potassium from RBC solution in small satellite packs. PAF-n could therefore improve the clinical prognosis of neonates with poorly developed BBB by limiting the delivery of excess ammonia found in the RBC solution.

Effects of potassium adsorption filters on the removal of ammonia from blood products.

BACKGROUND: Although ammonia in plasma does not usually pass through the blood-brain barrier (BBB), in cases of traumatic brain injury it may do so, acting as a neurotoxin on the brain. Excess intake of ammonia should be restricted in conditions involving BBB breakdown, such as traumatic brain injury. Washing is a method to remove ammonia from blood products, but fresh-frozen plasma and albumin products cannot be washed. A potassium adsorption filter (PAF) can remove not only potassium, but also ammonia from red blood cell solutions. We, therefore, examined the effects of a PAF on the removal of ammonia from a range of blood products. MATERIALS AND METHODS: Ammonia concentrations were measured in expired red blood cell solutions, fresh-frozen plasma, and platelet concentrates and purchased albumin products before and after filtration through a PAF. The PAF was primed with saline, which was removed before the filter was used. RESULTS: The percentages of ammonia removal from the red blood cell solutions, fresh-frozen plasma, plasma concentrates, 20% albumin and 5% albumin were approximately 76-87%, 21-31%, 53%, 77-92% and 49-63%, respectively. DISCUSSION: A PAF appears capable of removing ammonia from a range of blood products, although the reason for the lesser effect on the ammonia concentration in fresh-frozen plasma compared to other blood products remains unknown. We hypothesise that, by lowering ammonia levels in blood products, the PAF could improve the clinical prognosis of neonates with an underdeveloped BBB or patients with BBB breakdown following traumatic brain injury.

Semi-quantitative analyses of antibodies to N-methyl-d-aspartate type glutamate receptor subunits (GluN2B & GluN1) in the clinical course of Rasmussen syndrome.

OBJECTIVE: In Rasmussen syndrome (RS), in addition to the predominant involvement of cytotoxic T cells, heterogeneous autoantibodies against neural molecules are also found, but their function has not been elucidated. We examined antibodies to N-methyl-d-aspartate (NMDA) type glutamate receptor (GluR) subunits (GluN2B & GluN1) semi-quantitatively in cerebrospinal fluid (CSF) samples from RS patients, and evaluated their changes over time and their roles in immunopathogenesis. METHODS: Autoantibodies against N-terminal and C-terminal of GluN2B and GluN1 were examined in 40 CSF samples collected from 18 RS patients 5 to 180 months after the onset of RS. Epileptic patients without infectious etiology or progressive clinical course served as disease controls (n=23). Synthesized peptides encoding the extracellular and intracellular domains of human GluN2B and GluN1 subunits were used as antigens in ELISA. We defined the cut-off for these antibodies as mean +2 standard deviations (optimal density) of the disease controls. MRI were evaluated according to the MRI staging proposed by Bien et al. (2002b, Neurology 58, 250). RESULTS: CSF levels of antibodies against N-terminal and C-terminal of GluN2B were higher in RS patients than in disease controls (p<0.01). Likewise, CSF levels of antibodies against N-terminal and C-terminal of GluN1 were also higher in RS patients than in disease controls (p<0.01). All four antibodies tested were below cut-off levels in almost all CSF samples collected within one year from epilepsy onset. The proportions of CSF samples with these antibodies above cut-off levels were highest from 12 to 23 months after epilepsy onset, and declined after 24 months. CSF levels of these antibodies were higher when seizure occurred daily than when seizure occurred less frequently (p<0.01), and were higher at MRI stage 3 than at MRI stages 0, 2 and 4 (p<0.05), except for anti-GluN1-CT antibody at stage 2. CONCLUSIONS: Broad epitope recognition spectrum and delayed production of autoantibodies to NMDA type GluR in CSF of RS patients suggest that the autoantibodies are produced against NMDA type GluR antigens derived from cytotoxic T cell-mediated neuronal damages. These antibodies may impact the pathophysiology of RS in the most active stage, and could be a marker for active inflammation in the clinical course of RS. Further studies including passive transfer of the antibodies to mice may reveal the pivotal roles of the antibodies in RS.

Chronic subdural hematoma in elderly patient with EDTA-dependent pseudothrombocytopenia recently treated with aspirin and warfarin: case report.

A 78-year-old man who had a history of myocardial and cerebral infarction and who was treated with aspirin and warfarin, presented with left chronic subdural hematoma. Cerebral computed tomography showed severe brain compression of hematoma with midline shift, indicating the need for emergent surgery. The hematology and clotting tests upon admission revealed severe thrombocytopenia (platelet count, 1.3 × 10(4)/µL) with normal clotting activity. Because platelet aggregation was evident in the smear, we re-examined the patient for hematology using tubes that contained heparin, showing also low platelet count (2.3 × 10(4)/µL). The day on admission, we performed irrigation and drainage of the chronic subdural hematoma through single burr-hole craniostomy. During surgery, we used 10 units of platelet concentrates (PCs) for the reason that the patient was taking aspirin and coagulopathy derived from low platelet count could not be excluded. After surgery, we re-evaluated the hematology of the blood stored in tubes that contained ethylenediaminetetraacetic acid (EDTA) with or without kanamycin (KM). Treatment with KM dissociated EDTA-induced platelet aggregation and revealed platelet counts with highest accuracy (no KM treatment, 1.3 × 10(4)/µL; KM treatment, 15.2 × 10(4)/µL). This phenomenon is called EDTA-Dependent Pseudothrombocytopenia (PTCP) defined as falsely low platelet counts reported by automated hematology analyzers due to platelet aggretgation. Awareness of the phenomenon will enable neurosurgeons to manage patients with PTCP appropriately and clinical laboratory especially in emergency hospital is recommended to prepare for the hematological tubes being added KM in routine analysis, resulting in preventing mistaken diagnosis.

Influence of CYP2C19 polymorphism and concomitant antiepileptic drugs on serum clobazam and N-desmethyl clobazam concentrations in patients with epilepsy.

OBJECTIVE: The aims of this study were to identify the factors influencing the metabolism of clobazam (CLB) and its active metabolite [N-desmethyl clobazam (NCLB)] and to evaluate the NCLB concentration as an indicator for CYP2C19 polymorphism in epileptic patients. METHODS: A total of 302 serum samples from 238 Japanese patients were evaluated. The ratios of the serum CLB and NCLB concentrations to the CLB dose (CD ratios) were calculated and compared with CYP2C19 phenotypes. RESULTS: The mean CD ratio of NCLB in extensive metabolizers (EM: *1/*1), intermediate metabolizers (IM: *1/*2 or *1/*3), and poor metabolizers (PM: *2/*2, *3/*3, or *2/*3) was 3.1, 4.9, and 21.6 (µg/mL)/(mg/kg), respectively. In the EM and IM groups, the concomitant use of hepatic enzyme inducers (phenytoin and carbamazepine) reduced the CD ratio of CLB and increased that of NCLB. In the PM group, these inducers also decreased the CD ratio for CLB but did not elevate the CD ratio for NCLB. Using multiple regression analysis, body weight showed a positive correlation with an increased CD ratio for NCLB. The concomitant use of zonisamide and stiripentol also elevated the CD ratio for NCLB in the EM and IM groups, but that of the PM group was almost unchanged. When the cut-off value of the CD ratio for NCLB was set as 10.0 (µg/mL)/(mg/kg) for predicting the CYP2C19 PM status, the sensitivity and specificity were 94.4% and 95.7%, respectively. CONCLUSIONS: The interaction between NCLB and other antiepileptic drugs showed marked differences among CYP2C19 phenotypes. Measurement of the serum NCLB concentration is clinically useful for identifying the PM phenotype.

Hypocholesterolemia in patients with polycythemia vera.

Polycythemia vera (PV) is characterized by low serum total cholesterol despite its association with vascular events such as myocardial and cerebral infarction. Serum cholesterol level has not been used as a diagnostic criterion for PV since the 2008 revision of the WHO classification. Therefore, we revisited the relationship between serum lipid profile, including total cholesterol level, and erythrocytosis. The medical records of 34 erythrocytosis patients (hemoglobin : men, > 18.5 g/dL ; women, > 16.5 g/dL) collected between August 2005 and December 2011 were reviewed for age, gender, and lipid profiles. The diagnoses of PV and non-PV erythrocytosis were confirmed and the in vitro efflux of cholesterol into plasma in whole blood examined. The serum levels of total cholesterol, low-density-lipoprotein cholesterol (LDL-Ch), and apolipoproteins A1 and B were lower in PV than in non-PV patients. The in vitro release of cholesterol into the plasma was greater in PV patients than in non-PV and non-polycythemic subjects. Serum total cholesterol, LDL-Ch, and apolipoproteins A1 and B levels are lower in patients with PV than in those with non-PV erythrocytosis. The hypocholesterolemia associated with PV may be attributable to the sequestration of circulating cholesterol into the increased number of erythrocytes.

[Development of rapid genotyping methods for single nucleotide polymorphisms of cytochrome P450 2C9 (CYP2C9) and cytochrome P450 2C19 (CYP2C19) and their clinical application in pediatric patients with epilepsy].

Genetic polymorphism of cytochrome P450 2C9 (CYP2C9) and cytochrome P450 2C19 (CYP2C19) is widely known to contribute to interindividual differences in the pharmacokinetics of some antiepileptic drugs. We developed a rapid detection assay of polymorphisms of CYP2C9 and CYP2C19, using the Light Cycler(®) polymerase chain reaction (PCR) system. Using this assay, we examined polymorphisms in 20 Japanese pediatric patients prescribed phenytoin for the treatment of epilepsy, and classified their polymorphisms into four groups: group I, CYP2C9*1/*1 and CYP2C19*1/*1; group II, CYP2C9*1/*1 and CYP2C19*1/*2 or *1/*3; group III, CYP2C9*1/*1 and CYP2C19*2/*2; and group IV, CYP2C9*1/*3 and CYP2C19*1/*2 or *1/*3. The mean maximal elimination rates (V(max)) in groups I, II, III and IV were 13.1, 11.2, 10.2 and 8.0 mg/day/kg, respectively, with statistically significant differences among groups (p=0.012, Kruskal-Wallis analysis). The intrinsic metabolic activity (V(max)/K(m)) of groups I, II, III and IV were 2.9, 2.2, 1.5 and 1.1 l/day/kg, respectively (p=0.009), again with significant differences among groups. These findings indicate that polymorphism of CYP2C9 and CYP2C19 plays an important role in phenytoin metabolism in children. With a total processing time for this assay of less than 3 hours, prediction of the optimal phenytoin dosage based on the CYP2C9 and CYP2C19 genotypes will be possible before commencement of therapy, resulting in the prevention of phenytoin overdoses in pediatric patients with epilepsy.

Relationship between clotting activity and phosphatidylserine expression on erythrocyte membranes in polycythemia vera patients with the JAK2 V617F mutation.

INTRODUCTION: Polycythemia vera (PV) accompanies the clinical course of thrombosis. Phosphatidylserine (PS) expression on the plasma membrane has been known to be one of place where the coagulation system activates. We studied the relationship between clotting factor activity and PS expression on the erythrocyte membrane in patients with erythrocytosis. METHODS: The coagulation test and PS expression in 23 patients with erythrocytosis were measured. PS expression was determined indirectly by measuring annexin V binding to erythrocytes using fluorescence activated cell sorter analysis (FACS). RESULTS: The activity of clotting factors (II, V, VII, VIII, von Willebrand factor, IX, X) was significantly lower in PV than in the mutation-negative erythrocytosis. There was a significant correlation between reduced activity of clotting factors such as V, X, and IX and increased PS expression of the erythrocyte membrane. CONCLUSION: Increased expression of PS on the erythrocyte membrane may reduce the activities of clotting factors in PV patients with JAK2 V617F mutation.

Clinical and epidemiological features of Clostridium perfringens bacteremia: a review of 18 cases over 8 year-period in a tertiary care center in metropolitan Tokyo area in Japan.

OBJECTIVE: Clostridial sepsis has a very poor prognosis, owing to the life-threatening combination of shock and acute massive hemolysis. No papers have described the clinical features of clostridial sepsis cases in Japan. Therefore, we retrospectively examined the clinical features of patients with systemic inflammatory response syndrome (SIRS) from whose blood cultures Clostridium perfringens was isolated. SUBJECTS AND MATERIALS: Blood samples were obtained from SIRS patients and cultured between January 1, 2001 and June 30, 2009. The samples were retrospectively reviewed, and 18 samples were positive for C. perfringens. The medical records of these 18 patients were reviewed for age, gender, underlying disease, past illnesses, results of physical and laboratory testing, and radiographic data. RESULTS: All patients were diagnosed with SIRS. Fifteen patients (83.3%) were >65 years old -mean age, 75±2 years (range, 59-88 years). There were more men (13) than women (5). The blood cultures were obtained from patients in various wards: tertiary care center (8), emergency room (5), surgical ward (4), and medical ward (1). Hepatobiliary tract diseases such as gallbladder stones and hepatic carcinoma were the most frequent underlying diseases (8). Five patients died, resulting in an overall mortality rate at 30 days of 27%. In the non-survival group, patients presented with septic shock (4) and gas-forming infection (2), and with significantly lower fibrinogen levels than those in the survival group. Septic shock at initial presentation was significantly associated with 30-day mortality for C. perfringens infection. DISCUSSION AND CONCLUSION: There were no specific characteristics among clinical features of C. perfringens infection accompanied with SIRS. This may indicate that, in emergency rooms, diagnosing and initiating appropriate treatment for C. perfringens infection may be considerably difficult. It is important to be especially vigilant in identifying patients with C. perfringens infection underlying SIRS, and accompanied by shock.

[Retrospective study on CT findings on the sternal bone after bone marrow aspiration procedure in hematological patients].

We retrospectively studied the CT findings of sternal bone in 129 patients with hematological diseases who underwent bone marrow aspiration in Tokyo Metropolitan Bokutoh Hospital between August 2005 and July 2007. Sternal findings demonstrated sternal foramen (3 cases), incomplete fracture (1 case) and irregularity and decreased bone density of the visceral cortical bone (first intercostal space: 66 cases, third intercostal space: 3 cases). Irregularity and decreased bone density of the visceral cortical bone were significantly related to findings of decreased bone density in the third lumbar spine, indicating osteoporosis. These findings suggest that sternal bones demonstrated various bone diseases such as sternal foramen, fracture and osteoporosis, and that bone marrow aspiration from sternal bones might not be as safe as previously thought.

[Acute limbic encephalitis and NMDA type-glutamate receptor].

We compared clinical characteristics and autoantibodies against GluRepsilon2 between 95 patients with nonparaneoplastic non-herpetic acute limbic encephalitis (NPNHALE) and 19 patients with non-herpetic acute encephalitis accompanying ovarian teratoma (NHAE-OT). Onset age (mean +/- SD) was 27.7 +/- 18.6 years old in NPNHALE, 27.5 +/- 6.5 in NHALE-OT. Preceding factors were found in 63.8% of patients with NPNHALE and 89.5% of patients with NHALE-OT (Fisher's exact test, p = 0.025), and major preceding factors were upper respiratory infections or fever in both groups. Symptoms at the onset were disorder of behavior and talk > seizures > impairment of consciousness in NPNHALE, and disorder of behavior and talk > seizures > disorientation in NHALE-OT. Symptoms at the acute stage were similar between NPNHALE and NHAE-OT, but duration of hospital stay was longer in NHAE-OT (209.0 days) than NPNHALE (87.5 days) (Mann Whitney test, p<0.0001). At the onset, cell counts in CSF were 51.6 +/- 66.4/mm3 and protein levels were 35.4 +/- 14.7 mg/dl, and IgG levels were 6.6 +/- 4.2 mg/dl in NHAE-OT, and these data were not significantly different between NPNHALE and NHAE-OT. In acute stage, autoantibodies against whole molecule of GluRepsilon2 in CSF were detected in 51.8% (29/56) of adult NPNHALE, and 40% (6/15) of NHAE-OT patients by immunoblot. These autoantibodies in both groups included epitopes to n-terminal of GluRepsilon2. Antibodies against NMDAR complex (Dalmau's method) in CSF were detected in 90.9% (10/11) of NHAE-OT patients.

[Autoantibodies against glutamate receptors in patients with encephalitis].

We examined autoantibodies against GluRepsilon2 in patients with acute encephalitis, who were categorized into localized encephalitis and widespread encephalitis. Patients with localized encephalitis are defined as patients showing psychic symptoms (illusions, anxiety and distraction etc.), solitary seizures and/or very mild impairment of consciousness in the initial stage. Patients with widespread encephalitis are defined as patients showing a profound loss of consciousness and or convulsive status in the initial stage. In 24 patients with localized encephalitis, immunoglobulin (Ig) M autoantibodies against GluRepsilon2 tended to appear in CSF in the acute stage (0-20 days after onset of neurological symptoms) or recovery stage (21-60 days after onset of neurological symptoms) of encephalitis. In 22 patients with widespread encephalitis, IgM autoantibodies against GluRepsilon2 in CSF tended to appear in the recovery stage (21-60 days after onset of neurological symptoms) or chronic stage (>60 days after onset of neurological symptoms) of encephalitis. All patients with localized encephalitis had autoantibodies to the extracellular N epitope. However, no patients with widespread encephalitis had autoantibodies to the extracellular N epitope in acute stages. These data may suggest that GluR autoimmunity contributes to the onset of localized encephalitis.

A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo.

BACKGROUND: The GATA-1haematopoietic enhancer (G1HE), located between 3.9 and 2.6 kb 5' to the haematopoietic first exon, is essential for GATA-1 gene transcription in erythroid cells. However, G1HE is not sufficient to confer tissue specificity on the GATA-1 gene in vivo, indicating that additional regulatory sequences are necessary. RESULTS: We demonstrate here that two other upstream promoter elements containing a double GATA motif or two CACCC boxes are also indispensable for reporter gene expression in erythroid cells in the transgenic mouse. The combination of these three cis-acting regions was sufficient for reporter expression in primitive erythroid cells, as demonstrated by linking the elements together into a 659 bp artificial (GdC) minigene. The minigene activated the transcription of a reporter gene from either the endogenous or an exogenous thymidine kinase promoter, retaining cell type-specificity. The addition of a 320 bp fragment in the first intron to the GdC minigene sustained reporter expression in the definitive stage. Moreover, a line of transgenic mouse that expressed GATA-1 cDNA under the control of the complete 979 bp minigene rescued GATA-1 germ line mutant mice from embryonic lethality. CONCLUSIONS: A combination of four distinct sequence motifs co-operatively serve as a fundamental functional unit for GATA-1 erythroid transcription in vivo.