Skin recruitment of monomyeloid precursors involves human herpesvirus-6 reactivation in drug allergy.

BACKGROUND: In drug-induced hypersensitivity syndrome (DIHS), latent human herpesvirus (HHV)-6 is frequently reactivated in association with flaring of symptoms such as fever and hepatitis. We recently demonstrated an emergence of monomyeloid precursors expressing HHV-6 antigen in the circulation during this clinical course. METHODS: To clarify the mechanism of HHV-6 reactivation, we immunologically investigated peripheral blood mononuclear cells (PBMCs), skin-infiltrating cells, and lymphocytes expanded from skin lesions of patients with DIHS. RESULTS: The circulating monomyeloid precursors in the patients with DIHS were mostly CD11b(+) CD13(+) CD14(-) CD16(high) and showed substantial expression of skin-associated molecules, such as CCR4. CD13(+) CD14(-) cells were also found in the DIHS skin lesions, suggesting skin recruitment of this cell population. We detected high levels of high-mobility group box (HMGB)-1 in blood and skin lesions in the active phase of patients with DIHS and showed that recombinant HMGB-1 had functional chemoattractant activity for monocytes/monomyeloid precursors in vitro. HHV-6 infection of the skin-resident CD4(+) T cells was confirmed by the presence of its genome and antigen. This infection was likely to be mediated by monomyeloid precursors recruited to the skin, because normal CD4(+) T cells gained HHV-6 antigen after in vitro coculture with highly virus-loaded monomyeloid precursors from the patients. CONCLUSIONS: Our results suggest that monomyeloid precursors harboring HHV-6 are navigated by HMGB-1 released from damaged skin and probably cause HHV-6 transmission to skin-infiltrating CD4(+) T cells, which is an indispensable event for HHV-6 replication. These findings implicate the skin as a cryptic and primary site for initiating HHV-6 reactivation.

Factors modifying the frequency of spontaneous activity in gastric muscle.

The cellular mechanisms that determine the frequency of spontaneous activity were investigated in gastric smooth muscles isolated from the guinea-pig. Intact antral muscle generated slow waves periodically; the interval between slow waves was decreased exponentially by depolarization of the membrane to reach a steady interval value of about 7 s. Isolated circular muscle bundles produced slow potentials spontaneously or were evoked by depolarizing current stimuli. Evoked slow potentials appeared in an all-or-none fashion, with a refractory period of approximately 2-3 s. Low concentrations of chemicals that modify intracellular signalling revealed that the refractory period was causally related to the activity of protein kinase C (PKC). Activation of PKC increased and inhibition of PKC activity decreased the frequency of slow potentials. Chemicals that inhibit mitochondrial functions reduced the frequency of slow waves. Inhibition of internal Ca(2+)-store activity decreased the amplitude, but not the frequency of slow potentials, suggesting that the amplitude is causally related to Ca(2+) release from the internal store. The results suggest that changes in [Ca(2+)](i) caused by the activity of mitochondria may play a key role in determining the frequency of spontaneous activity in gastric pacemaker cells.

Asymptomatic portal vein obstruction after hepatobiliary resection: early detection by Doppler ultrasonography.

We report two different types of portal vein obstruction after liver resection: portal vein thrombosis due to steal phenomenon via a splenorenal shunt, and kinking of the skeletonized left portal vein after right hepatic lobectomy with caudate lobectomy. The two cases of portal vein obstruction were asymptomatic without any suggestive laboratory findings. Only routine Doppler ultrasonography detected portal vein obstruction which was successfully treated by emergency operation.

Doppler sonography of hepatic arterial blood flow velocity after percutaneous transhepatic portal vein embolization.

OBJECTIVE: This study was conducted to elucidate the changes in hepatic arterial blood flow after portal vein embolization. SUBJECTS AND METHODS: We prospectively measured the flow velocity and resistive index of the common, right, and left hepatic arteries, using Doppler sonography, in 21 patients who underwent embolization of the right portal vein. The measurements were performed before and 1, 3, 5, 7, and 14 days after embolization. We assessed the changes in liver volume with a volumetric study using CT. RESULTS: After embolization, flow velocity in the common hepatic artery increased significantly (p < 0.0001). Flow velocity in the right hepatic artery also increased significantly (p < 0.0001), with a significant decrease in resistive index (p < 0.0001). The flow velocity and resistive index of the left hepatic artery were unchanged. The increase in flow velocity in the right hepatic artery significantly correlated with that in the common hepatic artery (r = 0.514, p < 0.05). The calculated volume of the embolized right hepatic lobe significantly (p < 0.0001) decreased, from 685 +/- 32 cm(3) before embolization to 568 +/- 28 cm(3) after embolization. The atrophy rate of the right hepatic lobe significantly correlated with the increase in flow velocity in the right hepatic artery (r = 0.700, p < 0.0005). CONCLUSION: Portal vein embolization induces an increase in hepatic arterial blood flow velocity in the embolized hepatic segments, resulting from an increase in common hepatic arterial flow, but not from a steal phenomenon due to decreased hepatic arterial blood flow in the nonembolized hepatic segments. This observation may be explained by the simple mechanical effect of interposing a slower flowing stream (portal flow) in the path of a faster flowing stream (arterial flow).

Inhibitory actions of indomethacin on electrical and mechanical responses produced by nerve stimulation in circular smooth muscle of the guinea-pig gastric fundus.

The effects of indomethacin on electrical and mechanical responses produced by transmural nerve stimulation (TNS) were investigated in isolated circular smooth muscle of the guinea-pig gastric fundus. TNS evoked a cholinergic excitatory junction potential (e.j.p.). The e.j.p.s were inhibited by 1-10 microM indomethacin, in a concentration-dependent manner, with no marked alteration of the resting membrane potential. Exogenously applied acetylcholine caused a depolarization of the membrane that was not altered by indomethacin. TNS evoked a cholinergic twitch contraction at low frequencies (0.1 Hz). A train of TNS's at high frequency (1 Hz) produced a transient contraction with a subsequent sustained relaxation. Indomethacin reduced the resting tension and inhibited these TNS-induced contractions. Application of Nomega-nitro-L-arginine (NOLA), an inhibitor of nitric oxide (NO) synthesis, increased the amplitude of twitch contractions, and altered transient contractions to tetanic contractions during TNS at a frequency of 1 Hz, also with an increased amplitude. In the presence of NOLA, indomethacin (5 microM) again reduced the resting tension and inhibited TNS-induced contractions. This inhibition was greater for twitch contractions than for tetanic contractions. Nifedipine reduced the TNS-induced contractions, while addition of indomethacin further reduced the amplitude of contractions. Contractions produced by low concentrations of acetylcholine (0.1 microM) were inhibited by indomethacin, while those produced by 1 microM were not. These results indicate that the inhibitory actions of indomethacin on TNS-induced contractions do not involve enhanced production of NO or selective inhibition of voltage-gated Ca-channels. Prejunctional autoregulatory mechanisms may also not be altered by indomethacin. As indomethacin inhibits the enzyme cyclooxygenase, it is speculated that endogenously produced prostaglandins exert excitatory actions on gastric smooth muscle, and act mainly postjunctionally to facilitate spontaneous and neurogenic electrical and mechanical activity.

The quantitative analysis of three action modes of volatile anesthetics on purple membrane.

We quantitatively assessed the spectroscopic changes of purple membrane in relation to the concentrations of a volatile anesthetic. As reported previously, volatile anesthetics show three modes of action on purple membrane. By using an anesthetic for which the concentration in solution could be determined spectroscopically and by applying modified analytical methods regarding the M-intermediate lifetime, we were able to clarify the quantitative relation between anesthetic concentration and each mode of action, a relation which in the past has only been described qualitatively. We also determined through the measurement of transient pH changes with pyranine that the proton pump efficiency per photochemical cycle in an action mode induced with low concentrations of anesthetic does not change from that of the native state. Moreover, we dynamically obtained the individual M-bacteriorhodopsin difference spectrum of each state at room temperature using our flash photolysis system equipped with a wavelength-tunable dye laser. These results demonstrated again that we should clearly distinguish different action modes of anesthetics according to their concentrations.

Structural and functional differences of two forms of GTP-binding protein, Gq, in the cephalopod retina.

The major GTP-binding protein (G-protein) in the rhabdomeric photoreceptor membranes of the squid (Watasenia scintillans) has been identified as a Gq-class G-protein. Anti-Gq alpha antibodies recognized a protein not only in the photoreceptor membranes but also in soluble fractions of the retina. The 42 kD protein in the soluble fractions (soluble Gq alpha) had the same molecular mass and the same reactivities to anti-Gq antibodies as those of membrane-bound Gq alpha. The G beta subunit was scarcely detected in the soluble fractions, being found mostly in the membrane fraction, indicating soluble Gq alpha exists in monomeric form. Soluble Gq alpha had no effect on the GTPase activity of the photoreceptor membranes, suggesting that it does not interact with photoactivated rhodopsin or G beta gamma. Soluble Gq alpha would be an inactive form of Gq alpha. In the retina of Octopus fangsiao, soluble Gq alpha was scarcely detected after dark adaptation, but increased during subsequent light exposure and decreased on returning to dark adaptation. These results with Octopus suggest that functional membrane-bound Gq alpha is converted to soluble Gq alpha on exposure to light. Transformation of membrane-bound Gq alpha into the soluble form by hydroxylamine suggests that the difference between membrane-bound and soluble Gq alpha is associated with the attachment of fatty acid(s).

Regulation of squid visual phospholipase C by activated G-protein alpha.

Phospholipase C (PLC) is the key enzyme in the phototransduction cascade of invertebrate rhabdomeric photoreceptors. In addition to 130 kDa PLC, a 95 kDa protein recognized by antibody against the catalytic site of PLC was found in the squid retina. The PLC-like 95 kDa protein (95 kDa PLC) was produced from 130 kDa PLC by an intrinsic protease in the presence of calcium. The 130 kDa PLC was stimulated by the active form of Gq-class G-protein alpha (Gq alpha), but the 95 kDa PLC was not, although their PLC activity was similar. A 35 kDa fragment, the counterpart of 95 kDa PLC, was not recognized by antibodies against catalytic site or N-terminal site of the 130 kDa PLC, indicating that the cleavage site is on the C-terminal side beyond the catalytic site. In the presence of a large excess of the 35 kDa fragment, 95 kDa PLC was stimulated by Gq alpha to a similar extent as intact 130 kDa PLC. These results indicate that the C-terminal polypeptide of PLC is necessary for regulation of its enzyme activity by Gq alpha. The uncoupling of PLC from Gq alpha, caused by limited proteolysis, is therefore a candidate regulatory mechanism of the phototransduction cascade in rhabdomeric photoreceptors.

Effects of dopamine on veins in humans: comparison with noradrenaline and influence of age.

OBJECTIVE: To compare the venoconstricting effect of dopamine with that of noradrenaline and to investigate the influence of age on the responsiveness to dopamine in human subjects. METHODS: In eight young and eight elderly male subjects, increasing doses of dopamine or noradrenaline were infused into a dorsal hand vein and its diameter was measured using a linear variable differential transformer. RESULTS: There was no significant difference between the maximum venoconstriction (Emax) for dopamine and that for noradrenaline. The infusion rate to induce 50% of Emax (ED50) for dopamine in the young and elderly subjects was 363 ng x min(-1) and 352 ng min(-1), and the ED50 for noradrenaline was 40.7 ng min(-1) and 43.8 ng x min(-1), respectively. Neither in the Emax nor in the ED50 for these drugs were there significant differences between the young and elderly subjects. CONCLUSION: The venoconstricting effect of dopamine is 5-20 times less than that of noradrenaline, and aging does not influence the responsiveness to dopamine and noradrenaline in human subjects.

Binding of volatile anesthetics to purple membranes studied by X-ray diffraction.

Volatile anesthetics, diiodomethane and trifluoroethyl iodide, acted on the purple membrane of Halobacterium halobium in two different modes depending on the concentration. At low concentration, the absorption maximum of bacteriorhodopsin shifted from 561 to 558 nm (BR558) and the M-intermediate of the photocycle decayed faster than the native one. Higher concentration induced a species absorbing maximally at 480 nm (BR480) and the long-lived M-intermediates. The X-ray study suggested that anesthetics bound specifically to the protein-lipid interfacial region within a trimer near the surface of membrane in BR558 and entered into the hydrophobic domain of the membrane in BR480.

Role of glucocorticoid in the upregulation of type I interleukin-1 receptor mRNA expression in hepatocytes of endotoxin-administrated mice.

The interleukin-1 (IL-1) signal is transduced through type I IL-1 receptor (IL-1RI). We have recently reported that lipopolysaccharide (LPS) upregulated IL-1RI mRNA expression in mouse liver in vivo and that IL-1 and IL-6 directly upregulated IL-1RI mRNA expression in primary cultured mouse hepatocytes. Glucocorticoid (GC) has been reported to increase IL-1 binding to the cell surface and the expression level of IL-1R mRNA in a variety of cell types. As serum GC level is elevated in an inflammatory response, we evaluated the role of GC in LPS-induced upregulation of IL-1RI mRNA in the mouse liver. When LPS was administered to adrenalectomized (ADX) mice, IL-1RI mRNA was upregulated at a level comparable to those of untreated or sham-operated mice. A high dose of dexamethasone (Dex), however, caused upregulation of the mRNA. When primary cultured mouse hepatocytes were treated with Dex, only a weak upregulation of IL-1RI mRNA was observed. However, Dex in combination with IL-1 or IL-6 markedly enhanced the IL-1RI mRNA expression. A marked upregulation of the mRNA was also induced by treatment with a combination of IL-1 and IL-6 in the absence of Dex, reflecting the observation in ADX mice. These results suggest that the upregulation of IL-1RI mRNA in response to LPS is induced by the interaction of IL-1 and IL-6 and that GC augments their effect.

A novel yeast gene, RHK1, is involved in the synthesis of the cell wall receptor for the HM-1 killer toxin that inhibits beta-1,3-glucan synthesis.

The HM-1 killer toxin from Hansenula mrakii is known to inhibit cell wall beta-1,3-glucan synthase of Saccharomyces cerevisiae and other sensitive strains of yeast. A number of mutants of Saccharomyces cerevisiae that show resistance to this toxin were isolated in order to clarify the killing mechanism of the toxin. These mutants, designated rhk (resistant to Hansenula killer), were classified into three complementation groups. A novel gene RHK1, which complements the killer-resistant phenotype of the largest complementation group rhk1, was isolated. DNA sequence analysis revealed an open reading frame that encodes a hydrophobic protein composed of 458 amino acids. Gene disruption followed by tetrad analysis showed that RHK1 is not essential and loss of RHK1 function endowed S. cerevisiae cells with complete killer resistance. A biochemical analysis suggested that RHK1 does not participate directly in the synthesis of beta-1,3-glucan but is involved in the synthesis of the receptor for the HM-1 killer toxin.

Molecular cloning, purification and characterization of two endo-1,4-beta-glucanases from Aspergillus oryzae KBN616.

Two endo-1,4-beta-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa, a pH optimum of 5.0 and temperature optimum of 55 degrees C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and temperature optimum of 45 degrees C.

Clinical experience of percutaneous cardiopulmonary support.

Recently, percutaneous cardiopulmonary support (PCPS) combined with femoro-femoral bypass without reservoir has become valued because of its quick and easy application. We developed a fully preconnected compact integrated cardiopulmonary bypass (CPB) unit (priming volume of 250 ml) with a blind pore membrane oxygenator (Kuraray Menox) for PCPS. From 1990 to 1995, PCPS was performed in 49 patients of whom 26 were weaned from support. In most cases, we applied this CICU in patients with no active bleeding (22 patients); in patients with active bleeding (n = 13), we used Medtronic's heparin-bonded close chest support pack (CCSP). Of these, PCPS was performed uneventfully for 2 h (median) in 8 elective cases; all of these patients were weaned or were switched to a left ventricular assist system (LVAS). In 8 urgent cases, such as those with low cardiac output syndrome, PCPS was performed for 4 days (median), 1 was weaned, and 2 CICU were cases switched to other procedures. In 32 cases of shock, 5 CICU patients were weaned, and 3 of them survived. Eight patients including 5 CICU patients and 1 CCSP patient were switched to operation or LVAS, and 2 CICU patients remain alive. From these data, PCPS has been shown to support the patient's circulation in the acute phase and earn time to switch to operation or LVAS; the quick and easy set-up of the CICU can improve the clinical results. The use of the Medtronic device broadened the indication for PCPS. The CCSP enlarged the indication of PCPS but could not improve the results. To improve the results, a heparin-bonded surface is desired.

[Clinical study of re-do surgery after Bentall-type operation].

Among patients who underwent reconstruction of the aortic root by Bentall-type procedure using a composite graft, we investigated patients who underwent reoperation for complications related to composite graft. We employed composite graft reconstruction of the aortic root in 155 patients for 16 years prior to December 1994. Annulo-aortic ectasia was observed in 112 patients, aortic dissection in 34, and aortitis without aneurysm formation in 9. The original Bentall procedure was performed in 36, the Cabrol method in 8, the interposition method in 26, and the Carrel patch method in 85 patients. Thirteen (8.4%) of these patients required reoperation for complications related to the composite graft. Three of 4 patients with graft infection early after surgery underwent reconstruction with composite graft, but died in the hospital. The remaining patient survived after combined treatment that included the graft washing with Iodine (Isozin) solution and omentopexy. Four patients developed pseudoaneurysm formation due to sutural insufficiency and 7 has prosthetic valve failure as late complications. Three of the 4 patients with pseudoaneurysm underwent reconstruction with a second composite graft by the interposition method, while one patient with aortitis required another reoperation. In the remaining one patient, the leak was directly close along with second aortic valve replacement. All prosthetic valves used in 7 patients with prosthetic valve failure were Ionescu-Shiley biological valves. Primary tissue failure was observed in 6 and prosthetic valve endocarditis in one patient. Second cardiac valve replacement using a mechanical valve was possible. All the patients who underwent late reoperation showed favorable results. Infection of the composite graft showed the poor prognosis, and prevention of infection is important. Sutural insufficiency at the anastomosed site can be prevented by appropriate surgical procedures such as reinforcing suture, but further countermeasures for sutural insufficiency were considered necessary for aortitis in conditions, such as Behcet's diseases.

[Valvular surgery in the patients more than 70 years old].

One hundred twenty-eight valvular surgeries in patients over 70-year-old were reviewed (AVR:58, MVR:38, AVR and MVR: 11, mitral valvuloplasty (MVP): 11, AVR + MVP:11, mitral valvuloplasty (MVP): 11, AVR + MVP: 8, others: 2). Concomitant CABG was performed in 7, Maze in 6, TVR in 5 and Bentall in 3 cases. Early deaths occurred in 17 patients (13%). The early mortality was 5% in AVR, 21% MVR, 18% in AVR and MVR, 0% in MVP and 38% in AVR and MVP. Late death occurred in 16 patients. Forty-three percent of the late deaths were cardiac death. The actuarial survival at 10 years was around 50% in all groups. In the aortic position, a mechanical valve was implanted in 47 cases and a bioprosthetic valve was implanted in 33 cases. In the mitral position, the mechanical valve was implanted in 37 cases and the bioprosthetic valve was implanted in 12 cases. The event free rate after AVR at 10 years was 37% in patients with the mechanical valve (3 cerebral hemorrhage, 2 PVE, 1 thromboembolism and 1 sudden death) and 46% in patients with the bioprosthetic valve (2 PVE and 1 primary tissue failure). The event free rate after MVR at 10 years was 84% in patients with mechanical valves (1 perivalvular leak, 1 PVE and 1 sudden death) and 75% in patients with bioprosthetic valves (1 PVE and 1 sudden death). Between mechanical valve group and bioprosthesis group, no statistically significance was found in the event free curve after AVR nor MVR. There was no valve related event after MVP. Considering the better durability of bioprostheses in the aortic position than in the mitral position, the presence of atrial fibrillation and necessity of warfarin anticoagulation, we conclude that a choice of a bioprosthetic valve could be acceptable in the aortic position, but may not be recommended in the mitral position.