Tadayoshi Kosugi

The Relationship Between Microvessel Density, the Expression of Vascular Endothelial Growth Factor (VEGF), and the Extension of Nasopharyngeal Carcinoma

Objective The present study was aimed at clarifying whether the microvessel density (MVD) and the e‐pression of vascular endothelial growth factor (VEGF) were related to the degree of local invasion and metastasis in nasopharyngeal carcinoma (NPC).

Expression of p16, nm23-H1, E-cadherin, and CD44 Gene Products and Their Significance in Nasopharyngeal Carcinoma

Objective The present study was aimed to determine whether p16/MTS1, nm23‐H1, E‐cadherin, and CD44 proteins were expressed in nasopharyngeal carcinoma (NPC) and whether those expressions were pathologically significant in the progress of NPC.

Hemostatic disturbances observed in patients with snakebite in south China.

To investigate the hematological disorders after snakebite, we measured the maximum platelet aggregation rate (MAR), antithrombin III (AT-III) activity, alpha(2)-plasmin inhibitor (alpha(2)-PI) activity, concentration of fibrinogen (Fg) and fibrin degradation products (FDP) in 25 samples from 17 patients with snakebite in south China. The results obtained in the patients before application of antivenom and patients with Ophiophagus hannah (Oh.) bite were as follows: (1) the mean MAR values were significantly decreased in the case of the snakebites from Vipera russellii (Vr.) and Trimeresurus mucrosquamatus (Tm.); (2) the mean activities of AT-III were decreased in all patients in the present study; 3) the mean activities of alpha(2)-PI were significantly decreased in patients bitten by Deinagkistrodon acutus (Da.), Agkistrodon halys (Ah.), Vr., Trimeresurus stejnegeri (Ts.), Tm. and Naja naja atra (Nn.); (4) the mean concentrations of Fg were markedly decreased in patients bitten by Da., Ah., Vr., Ts. and Tm.; and (5) the mean levels of FDP were significantly increased in cases of Da., Vr. and Ts. bite, but not in Ah., Tm., Nn. and Oh. bite. The results of the present study indicate that disorders of platelet aggregation and the coagulation-fibrinolysis system are liable to occur in patients with snakebite from Da., Ah., Vr., Ts., Tm. and Nn. Furthermore, it appeared that disseminated intravascular coagulation (DIC) was evoked in some patients. Specific antivenom was found to be useful for improving the hemostatic disturbances after snakebite from Ah. and Nn.

Congenital cricoid stenosis.

A case of congenital cricoid stenosis in a 9‐year‐old girl was reported. The stenosis was confirmed by direct laryngoscopy and laryngotracheograms performed through the tracheostoma. The excess cartilaginous tissue was removed under a midline incision of the cricoid cartilage (cricoidplasty). Seventeen cases of cricoid stenosis reported since 1960 were studied and the clinical entity was discussed. On the basis of the embryology of the larynx, the mechanism of origin of this malformation was also discussed.

Production of a monoclonal dinitrophenyl-specific rat IgE and establishment of an IgE capture ELISA for estimating the concentration of rat IgE antibodies to dinitrophenyl-Ascaris suum.

A hybridoma producing monoclonal rat IgE antibodies of antidinitrophenyl (anti-DNP) specificity was generated by fusion of Sp2/0-Ag 14 (SP2) mouse myeloma cells and spleen cells from a DNP-Ascaris-sensitized Brown-Norway rat. Subsequently, the supernatant of the hybridoma (FE-3) was applied to an affinity column of DNP-bovine serum albumin-Sepharose 4B. The adsorbed protein fraction was pooled, concentrated, and further purified using Sephadex G-200. The molecular weight of the isolated protein was approximately 200,000 by SDS-PAGE, and the protein reacted with peroxidase (POD) mouse antirat myeloma IgE on Western blotting. Rabbit antibodies against DNP-specific rat IgE were also prepared by immunizing Japanese white rabbits with monoclonal DNP-specific rat IgE. These antibodies against DNP-specific rat IgE were applied to an affinity column of normal rat serum-Sepharose 4B and monoclonal DNP-specific rat IgG2b-Sepharose 4B to remove any other reactive substances apart from IgE contained in the serum proteins of the rat sensitized with DNP-Ascaris. On ELISA, it was found that the specificity of POD rabbit antibodies against DNP-specific rat IgE for monoclonal DNP-specific rat IgE was the same as that for rat myeloma IgE (IR 162). In addition, determinations of the monoclonal DNP-specific rat IgE revealed that the sensitivity of ELISA using POD-rabbit antibodies against DNP-specific rat IgE [POD-RA(DNP)RE] was higher than that using POD goat antibodies against rat myeloma IgE. Furthermore, an IgE capture ELISA employing the above-mentioned RA(DNP)RE was established for estimating the rat IgE antibodies to DNP-Ascaris suum. A good correlation was found between the antigen-specific IgE antibodies in the serum of Wistar rats estimated by this IgE capture ELISA and those estimated by passive cutaneous anaphylaxis.

Hematological studies on DIC-like findings observed in patients with snakebite in south China

To clarify the characteristics of the hematological disturbances evoked by snakebite, we measured the antithrombin III (AT-III) activity, alpha2-plasmin inhibitor (alpha2-PI) activity, fibrinogen concentration (Fg) and level of fibrin degradation products (FDP) in 21 patients envenomed by several snakes in south China between August 1998 and October 1999. The hematological changes observed were as follows: the mean activities of AT-III were decreased in patients bitten by Ophiophagus hannah (Oh.), Bungarus fasciatus (Bf.), Hydrophis cyanocinctus (Hc.), Rhabdophis subminiatus (Rs.), and Trimeresurus stejnegeri (Ts.), while those of alpha2-PI were decreased in all patients in the present study; Fg was not detectable in the case of Rs. bite, and the Fg concentration after Ts., Oh., Hc. and Bf. bites also decreased markedly thereby increasing the mean levels of FDP in all patients. It thus appeared that DIC-like syndrome was caused in patients envenomed by snakebite. In the present study, we found that patients who were bitten by Rs., which is still being classified as a non-venomous snake, exhibited complete defibrinogenation and severe hemorrhage without any evidence of severe multiple organ damage. We also found that patients with Ts. bite showed marked hemostatic disturbance without severe multiple organ damage. It is considered that such a discrepancy between the hematological findings and clinical symptoms could be a characteristic phenomenon of the DIC-like syndrome induced by snakebite, especially by Rs. and Ts. bites.

Urokinase-type plasminogen activator and plasminogen activator inhibitor antigen in tissue extracts of paranasal sinus mucous membranes affected by chronic sinusitis and antrochoanal polyps

Abstract We examined the effect of pH on the extraction of urokinase-type plasminogen activator (u-PA) and plasminogen activator inhibitor-1 (PAI-1) from paranasal sinus mucous membrane associated with chronic sinusitis and antrochoanal polyps. The specific activity of u-PA extracted with buffer at pH 7.4 was stronger than that extracted with buffer at pH 4.2. The antigen level of u-PA extracted with the acidic buffer was significantly higher than that extracted with the neutral buffer. In contrast, the difference in antigen levels of PAI-1 extracted with the acidic buffer and neutral buffer was not significant. Based on these results, we inferred that the u-PA-PAI-1 complex was extracted by the acidic buffer and the activity of u-PA was therefore decreased.

Pharmacokinetics of habutobin in rabbits

Following the administration of habutobin, the fibrinogen level in the circulating blood of the rabbits decreased. These results showed that the activity of habutobin was retained in vivo. The plasma level of habutobin was determined by a ELISA-double sandwich method. The pharmacokinetics of habutobin from Trimeresurus flavoviridis venom was studied in rabbits following i.v. administration of 50 micrograms kg-1 of habutobin. The time course of the plasma concentration of habutobin fitted a two-compartment open model. The half-life of the distribution phase was 4.43 +/- 1.28 min and that of the elimination phase was 50.42 +/- 7.89 min. The area under the plasma concentration-time curve (AUC) was 38.69 +/- 6.68 micrograms min ml-1. The total body clearance was 3.82 +/- 1.08 ml min-1. When the steady state was reached, the concentration ratio of habutobin in the tissue (Ct) to that in the plasma (Cc), Ct:Cc was 0.47:1. These findings suggest that relatively little habutobin tended to remain in the tissue.

Production of a monoclonal antibody against the thrombin-like enzyme, habutobin, from Trimeresurus flavoviridis venom

We succeeded in producing a monoclonal antibody to the thrombin-like enzyme, habutobin, which was purified from crude venom of the snake Trimeresurus flavoviridis. The monoclonal antibody obtained belonged to IgG1, and its light chain consisted of a kappa-chain. The monoclonal antibody reacted specifically with habutobin and crude venom from T. flavoviridis but did not react with human thrombin or bovine thrombin on Western blotting. The concentration of habutobin and crude venom of T. flavoviridis, in vitro, could be measured by means of ELISA using the monoclonal antibody. Furthermore, the ELISA-double sandwich method employing this monoclonal antibody may represent a reliable method for determining the habutobin levels in the circulating blood.

Argatroban, specific thrombin inhibitor, induced phenotype change of cultured rabbit vascular smooth muscle cells

To investigate whether argatroban ((2R,4R)-4-methyl-1-[N(2)-((RS)-3-methyl-1,2,3,4-tetrahydro-8-quinolinesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid hydrate, a selective thrombin inhibitor, exerts a direct action on phenotype conversion of vascular smooth muscle cells, cultured rabbit aortic vascular smooth muscle cells were employed. Myosin heavy chain isoforms (SM1, SM2, and SMemb) mRNA expressions were evaluated by in situ hybridization and reverse transcription-polymerase chain reaction (RT-PCR). After the cells were incubated in serum-free medium containing argatroban (10 and 50 microg/ml) and platelet-derived growth factor (PDGF)-BB (10 and 50 ng/ml) for 3 h, total RNA was extracted. In situ hybridization demonstrated that myosin heavy-chain isoform mRNAs were homogenously expressed in argatroban- and PDGF-BB-treated cells. RT-PCR revealed that SM1/SM2 mRNA expressions were not changed with argatroban, while SMemb mRNA expression was increased to 1.6-fold with a statistical significance (P<0.05). Treatment with argatroban (10 and 50 microg/ml) at 24 h did not change SM1/SM2 mRNA expressions. Although SMemb mRNA expression was slightly increased, there was no statistical significance. Other phenotype markers including plasminogen activator inhibitor-1 (PAI-1) and beta-actin mRNAs were also significantly increased by argatroban. In conclusion, argatroban can directly induce phenotype conversion of vascular smooth muscle cells with the resultant up-regulation of SMemb, PAI-1, and beta-actin mRNAs.