Kikyo-to for Acute Upper Respiratory Tract Infection-Associated Sore Throat Pain: A Multicenter Randomized Controlled Trial.

Objectives: Kikyo-to (Kt), a herbal medicine composed of glycyrrhiza root (Chinese licorice) and Platycodon root extracts (Chinese bellflower), is commonly used in Japan for relief of throat symptoms related to acute upper respiratory tract infection (URTI). Its effectiveness on URTI-associated sore throat pain over 30 min is examined here in comparison with a placebo. Design: Randomized double-blinded multicenter trial. Settings/Location: Two local Japanese medical centers with primary care. Subjects: Patients aged 20-65 years with URTI-related sore throat. Interventions: Patients were randomized to receive either 2500 mg of Kt, or 2500 mg of placebo (lactose). Randomization was stratified by age (< 45 vs. ≥ 45 years) and baseline sore throat score according to visual analogue scale (VAS) (< 50 vs. ≥ 50). Outcome measures: Primary outcome was change to sore throat score according to VAS 30 min after administration of Kt. Perceived impact of the sore throat on daily life at 30 mins after administration was another outcome. Outcomes are analyzed in the intention-to-treat population. Results: Among 70 participants, (Kt group: 36; placebo group: 34), each group contained 34 patients for analysis (n = 68, total). Difference between the groups in the mean change of sore throat score according to VAS at 30 min was without statistical significance (Kt 15.3, placebo 17.2; p = 0.66). Patients reporting that their sore throat had a moderate or greater impact on daily life were also similar in proportion between the groups (Kt 61.8% vs. placebo 55.9%; p = 0.80). Side effects were not reported. Conclusions: Kt did not relieve acute URTI-associated sore throats significantly more than a placebo. (UMIN trial ID: UMIN000035591).

Severe factor X deficiency in three unrelated Palestinian patients is caused by homozygosity for the mutation c302delG-correlation with thrombin generation and thromboelastometry.

Factor X (FX) is one of the vitamin K-dependent serine proteases, which forms the prothrombinase complex converting prothrombin into thrombin. To search for mutations in F10 gene giving rise to severe FX deficiency and to study the contribution of thrombin generation and thromboelastometry as a tool for evaluation of hemostasis. Mutations in the F10 gene were sought by direct sequencing of all the eight exons and intron/exon boundaries. Thrombin generation and thromboelastometry were performed. Three unrelated Palestinian patients had undetectable FX level (<1 U/dl). All patients were found to be homozygous for c302delG, a new frameshift mutation in the F10 gene causing a stop codon at amino acid 73. The mutant allele was not detected among 152 Palestinians analyzed. Thrombin generation was examined in one of the patients 4 days after fresh frozen plasma was applied, when his FX level was 2 U/dl. Minute thrombin generation was observed, as compared to normal thrombin generation in heterozygotes for the mutation and a healthy control. Thromboelastometry revealed prolonged lag phase when patient's platelet-poor plasma and platelet-rich plasma were tested, with a slightly decreased initial clot formation rate, as compared to carriers' and control sample. Genetic analysis disclosed a unique mutation causing a severe phenotype. Thrombin generation assay may serve as a quick tool for confirming severe deficiency until the specific mutation is identified. Thrombin generation can also serve for monitoring and optimizing treatment. The correlation of thromboelastometry assay and severe FX deficiency is less striking.

A case of atypical hemolytic uremic syndrome due to anti-factor H antibody in a patient presenting with a factor XII deficiency identified two novel mutations.

A 9-year-old boy with pallor and macrohematuria showed hemolytic anemia, thrombocytopenia and renal failure. There was no history of diarrhea and the stool culture was negative. A diagnosis of atypical hemolytic uremic syndrome (HUS) was confirmed; however, the cause of the prolonged activated partial thromboplastin time (APTT) was unknown. Plasma exchange and hemodialysis were performed because of progressive hemolytic anemia and renal dysfunction. Fresh frozen plasma was administered frequently to correct the prolonged APTT after hemolysis was controlled and C3 levels had recovered. Factor H (FH) and factor I (IF) levels were normal and we did not detect mutations of FH, IF and membrane cofactor protein. Further investigation revealed the presence of anti-FH antibody in the patient's plasma and a deficiency of coagulation factor XII. Analysis of the patient's coagulation system displayed <3% functional activity of factor XII, whereas levels of other coagulation factors were within the normal range. Two novel mutations (W222G and R447S) were identified upon analysis of the factor XII gene in this patient. Moreover, further investigation revealed that compound heterozygous mutations were present in two of the patient's three siblings, while the third sibling only had a mutation at W222G. The patient was treated for atypical HUS; however, no treatment was required for factor XII deficiency as he did not display a hemorrhagic tendency. We report here a rare case of atypical HUS due to anti-FH antibody presenting with a coagulation factor XII deficiency.

Factor XII Osaka: abnormal factor XII with partially defective prekallikrein cleavage activity.

A healthy Japanese male had reduced factor XII (FXII) activity (35%) in contrast to normal antigen levels (81%). The F12 of this proband had a 9775G to C mutation in exon 10 and an 11276G to A mutation in exon 13 that resulted in two amino acid substitutions of Ala324Pro (GCG→CCG) in the proline-rich connecting region and Gly531Glu (GGG→GAG) near the active Ser544 in the catalytic domain. His father had the nucleotide 46T/T and a heterozygous 9775G/C mutation. The FXII activity (32%) and antigen level (38%) of the father were about half that of normal individuals with 46T/T, suggesting a heterozygous cross reacting material (CRM)-negative deficiency. His mother had a 46C/T and heterozygous 11276G/A mutation, and 80% FXII activity was incompatible with the corresponding antigen level (125%), suggesting a heterozygous CRM-positive deficiency. The substitution of Ala324Pro probably caused the CRM-negative mutation and the Gly531Glu caused the CRM-positive mutation. We developed three methods based on chromogenic substrates to assay the distinct functions of FXII, namely its autoactivation on a negatively charged surface, activation by kallikrein cleavage and the prekallikrein cleavage activity of FXIIa. The ratios of autoactivated FXIIa/FXII antigen (0.80-1.10) and of kallikrein-induced FXIIa/FXII antigen (0.86-1.00) in plasma from the proband were within normal ranges, whereas those of FXIIa-induced kallikrein/FXII antigen were reduced to 0.41-0.45. In conclusion, the 9775G to C and 11276G to A mutations of F12 led to a CRM-negative and -positive FXII deficiency, and the F12 with 11276A produced a dysfunctional type of FXII with a partial defect (0.41-0.45) in prekallikrein cleavage activity.

Evaluation of diabetic neuropathy using the tone-entropy analysis, a noninvasive method to estimate the autonomic nervous function.

It is clinically important to diagnose diabetic neuropathy at the early stage. In this study, the toneentropy analysis of electrocardiogram was applied to diabetic patients to evaluate its usability in the screening of diabetic neuropathy. Consecutive 102 diabetic patients were invited to the study. Electrocardiogram was obtained and analyzed for the tone and the entropy using an original software developed previously. Nerve conduction velocity (NCV) was examined on the median, the posterior tibial and the sural nerves. Patients were divided into quartile subpopulations according to the NCVs in the analysis. Both the tone and the entropy significantly correlated with NCVs, while coefficient of variation in R-R intervals did not show a significant correlation. The correlation was most significant between the entropy and the NCV on the sural nerve. When a multivariate analysis (ordinary regression) was applied to examine independent effects of the factors influencing the NCV on the sural nerve, the entropy was the most potent independent factor (beta = 1.14 +/- 0.32, P = 0.0004) along with sex (beta = 0.43 +/- 0.19, P = 0.02) and BMI (beta = 0.11 +/- 0.05, P = 0.04). The tone-entropy analysis on electrocardiogram may be a promising non-invasive screening method for diabetic neuropathy.

[Structural homology between streptolysin O (SLO) produced by streptococcus pyogenes and SLO-like protein produced by non-pathogenic streptococci and cross-reactivity of antibody against SLO-like protein to SLO].

Nine clones of non-pathogenic streptococci were isolated from the pharynges of seven healthy subjects, and grown on sheep blood agar plates with a hemolysis or gamma hemolysis, then cultured in LB broth for 16 hrs. Purified streptolysin O (SLO) purchased from Sigma Chemical Co. (Sigma-SLO), SLO antigen as a latex agglutination reagent from A company (A-SLO) and supernatants from four culture media were electrophoresed on 12% SDS-polyacrylamide gel and transferred to PVDF membranes. Immunological analyses of antibodies against SLO in healthy sera and proteins in culture medium demonstrated that healthy sera contained an antibody recognizing Sigma-SLO, A-SLO and a protein of the same size as SLO (SLO-like protein) in culture medium. These findings suggest that healthy subjects develop an antibody directed against SLO-like protein produced by non-pathogenic streptococci, and that this antibody cross-reacts with Sigma-SLO and A-SLO. Using DNA from Streptococcus pyogenes and non-pathogenic streptococci, the SLO gene and SLO-like protein gene were analyzed by direct sequencing with oligonucleotide primers designed to cover no. 74 to approximately 1900 of the SLO gene. There were three different bases resulting in amino acid substitution between the SLO gene and SLO-like protein gene, namely 101Lys (AAA) of SLO to Asn (AAT), 175Met (ATG) to Arg (AGG) and 185Asp (GAT) to Asn (AAT). Remaining 560 residues of 563 amino acids constituting SLO-like protein were homologous to SLO. Non-pathogenic streptococci on the pharynges of healthy subjects produce an SLO-like protein composed of amino acids similar to those of SLO, which induces an antibody against this SLO-like protein in serum. It is likely that an antibody against SLO-like protein in healthy sera cross-reacts with SLO and causes a pseudo-positive reaction on ASO measurement by the latex agglutination method using SLO antigen.

Reduction in protein S activity during normal pregnancy.

We investigated the serial changes in blood protein S (PS) and related proteins in 11 normal pregnant women. The PS activity decreased significantly in the third trimester and reached minimum levels (23.3%) one hour after delivery. Although the PS activity was reduced markedly below the normal limits, all the women delivered safely. The mechanisms that cause the reduction in PS activity and the clinically dangerous conditions involving PS activity during pregnancy warrant further investigation.

Molecular characterization of 3 factor V mutations, R2174L, V1813M, and a 5-bp deletion, that cause factor V deficiency.

We identified 3 mutations in the factor V (FV) gene (F5) associated with FV deficiency in 3 unrelated Japanese patients. Patient 1 had severe bleeding symptoms (plasma FV activity, <1%; FV antigen, 9%) and was a compound heterozygote for a novel 5-bp deletion in exon 22 and the V1813M mutation. Patient 2 had moderate bleeding symptoms (plasma FV activity, <1%; FV antigen, 4%) and was homozygous for the V1813M mutation. Patient 3 had very mild symptoms (plasma FV activity, 1%; FV antigen, 5%) and was homozygous for the novel R2174L mutation. A study of recombinant protein expression revealed that the FV coagulant-specific activities in conditioned media for the FV-R2174L and FV-V1813M mutants were reduced to approximately 40% and 28% of wild-type FV, respectively. The amounts of FV-R2174L protein and messenger RNA in the platelets of patient 3 were similar to those of healthy subjects; however, the amount of FV-V1813M protein in patient 2 was decreased. Our data suggest that the severity of the bleeding tendency in patients with FV deficiency is correlated not only with plasma FV activity but also with the amount of FV protein in the platelets.

[Immunological analysis of pseudo-positive reaction in healthy adults at anti-streptolysin O measurement by latex agglutination method].

Streptolysin O (SLO) is a toxic immunogenic protein produced by Streptococcus pyogenes (S. pyogenes). The latex agglutination photometric immunoassay with latex coated by SLO (latex agglutination method) has been most widely used for determination of antibody to SLO (ASO). We measured ASO levels by latex agglutination method in serum specimens collected from 159 healthy individuals and eight patients with S. pyogenes, who had a positive S. pyogenes culture from throat swabs. A significant frequency(about 15%) had positive ASO levels (> 200 unit/ml) in healthy individuals aged < 20 (47 individuals) and 20-29 (80 individuals), respectively, however, none of 30 or over (32 individuals) were positive. The SLO specimen purchased from Sigma Chemical Co. showed at least three protein bands on SDS-PAGE; one was considered SLO protein because it had a molecular weight of 64 kD and of the remaining proteins, one had a molecular weight higher and the other had a molecular weight lower than 64 kD. Serum antibodies among the healthy adults to Sigma-SLO specimen consisted of IgG class in the majority, with little IgM class. Immunoblotting analysis revealed that serum antibodies of the patients recognized the 64-kD protein (SLO), and serum antibodies of the healthy adults recognized different proteins from SLO. It appeared that many of the healthy adults with a positive ASO level had antibodies different from ASO, resulting in pseudo-positive ASO values when ASO was measured by latex agglutination method using SLO specimen containing non-specific proteins. Improvement of commercial kits using this method is required.

Temporal expression of immunoreactivity for heat shock protein 25 (Hsp25) in the rat periodontal ligament following transection of the inferior alveolar nerve.

The present study examined the immunohistochemical localization of heat shock protein 25 (Hsp25) during the regeneration of nerve fibers and Schwann cells in the periodontal ligament of the rat lower incisor following transection of the inferior alveolar nerve. In the untreated control group, the periodontal ligament of rat incisor did not contain any Hsp25-immunoreaction. On postoperative day 3 (PO 3d), a small number of Schwann cells with slender cytoplasmic processes exhibited Hsp25-immunoreactivity. From PO 5d to PO 21d, Hsp25-positive nerve fibers and Schwann cells drastically increased in number in the alveolar half of the ligament. Although the axons of some regenerating Ruffini-like endings also showed Hsp25-immunoreactions, the migrated Schwann cells were devoid of Hsp25-immunoreaction. Thereafter, Hsp25-positive structures decreased in number gradually to disappear from the periodontal ligament by PO 56d. This temporal expression of Hsp25 in the periodontal ligament well-reflected the regeneration process of the nerve fibers. Hsp25 in the regenerating nerve fibers and denervated Schwann cells most likely serves in modulating actin dynamics and as a cellular inhibitor of apoptosis, respectively.

The antiplatelet aggregation effects of aspirin suppositories.

To confirm that aspirin suppositories are an effective treatment for acute ischemic stroke, we examined the suppressive effects of 200-mg aspirin suppositories on platelet aggregation. Aspirin suppositories suppressed platelet aggregation induced by ADP or collagen, and the suppression continued for 24 h. There was no significant difference in suppression of platelet aggregation between aspirin administered by suppository and orally given aspirin. These results suggest that aspirin suppositories are a useful treatment for acute ischemic stroke.