The calcium-independent protein kinase C participates in an early process of CD3/CD28-mediated induction of thymocyte apoptosis.

Thymocyte negative selection eliminates self-reactive clones and involves both a T-cell receptor (TCR)/CD3-mediated signal and a costimulatory signal, which can be delivered via CD28. Anti-CD3/anti-CD28-triggered apoptosis in isolated CD4+CD8+ thymocytes in vitro provides a basic model for negative selection. Effects of isoform-selective and non-isoform-selective inhibitors of protein kinase C (PKC) on this apoptotic process suggest that activation of Ca2+-independent PKC isoforms during the first 2-3 hr of culture is essential for inducing apoptosis, and that Ca2+-dependent PKC isoforms may be influential, but not essential, for apoptosis. To assess the CD3/CD28-mediated activation of PKC in the apoptotic process, we prepared CD4+CD8+ thymocytes (without contamination with cells that had received negative or positive selection signals in vivo) by establishing TCR-transgenic mice with RAG-2-deficient and non-selecting major histocompatibility complex (MHC) backgrounds, in addition to a CD4+CD8+ thymocyte-enriched population from normal mice. Translocation of Ca2+-independent PKC from the cytosolic fraction to the particulate fraction of CD4+CD8+ thymocytes was induced by CD3/CD28-mediated stimulation, but not by CD3- or CD28-mediated stimulation alone, and peaked 2 hr after the start of culture. The kinase activity of the translocated Ca2+-independent PKC was dependent on cofactors in vitro, indicating that novel (n)PKC, but not atypical (a)PKC or a proteolytic PKC fragment, was responsible for the activity. Immunoblotting analysis indicated that the nPKC-theta isoform was the major contributor among nPKC isoforms, and that the classical (c)PKC-alpha isoform was the major contributor among cPKC isoforms. These results suggest that activation of nPKC (especially the theta isoform) in CD4+CD8+ thymocytes is involved in a pathway for negative selection.

Role of secretory IgA, secretory component, and eosinophils in mucosal inflammation.

Eosinophils and their products are important in the pathophysiology of allergic inflammation in mucosal tissues. Secretory component (SC) bound to IgA mediates transepithelial transport of IgA. As another biological activity of SC, we have reported that secretory IgA (sIgA) and SC preferentially activate human eosinophils. When eosinophils were stimulated with immobilized sIgA, degranulation and superoxide production were greater than when stimulated with serum IgA. In contrast, neutrophils responded similarly to sIgA and serum IgA. Superoxide production by eosinophils stimulated with cytokines was enhanced synergistically by immobilized SC, while SC showed no effect on neutrophil activation. Eosinophil superoxide production stimulated with sIgA was abolished by anti-CD18 mAb, suggesting that beta2 integrins might be crucial for this reaction. There are several reports that SC and sIgA may play important roles in regulating eosinophil functions in vivo in diseases associated with mucosal eosinophilia and in various allergic diseases. It is speculated that eosinophils in the mucosa are activated by SC or sIgA, and that subsequent degranulation and superoxide production are induced.

Stimulation of the beta(2) integrin, alpha(M)beta(2), triggers tyrosine phosphorylation and cellular degranulation on human eosinophils.

Activation of human eosinophils by specific extracellular stimuli triggers the cellular degranulation response. Because cellular adhesion is critical for this eosinophil degranulation, we have tested the hypothesis that ligation of the beta(2) integrin, alpha(M)beta(2) (Mac-1, CD11b/CD18), leads to intracellular signaling events that contribute to the eosinophil activation response. Recently, we found that engagement of beta(2) integrin using two different approaches, such as cell adhesion induced by IL-5 or direct ligation of alpha(M)beta(2), triggered tyrosine phosphorylation of Cbl, the product of the c-cbl proto-oncogene, paxillin, a cytoskeletal protein, an unidentified 115-kD protein, and subsequent cellular degranulation. The results of this study indicate that engagement of alpha(M)beta(2) on eosinophils triggers an intracellular signaling cascade leading to cellular degranulation. Tyrosine phosphorylation of Cbl, paxillin, and a 115-kD protein may play important roles in adhesion-dependent cellular functions of eosinophils.

Altered cholesterol metabolism in human apolipoprotein E4 knock-in mice.

Thevarepsilon4 allele of apolipoprotein E (apoE) is associated with an increased risk of developing Alzheimer's disease (AD). To accurately determine the isoform-specific effects of human apoE on brain functions under physiological and pathological situations, we created mice expressing human apoE4 isoform in place of mouse apoE by utilizing the gene-targeting technique on the embryonic stem cells (knock-in). The homozygousvarepsilon4 (4/4) mice correctly expressed human apoE4 in the serum and the brain. The human apoE in the brain was found primarily in the astrocytes as was the mouse apoE in the wild-type (+/+) mice. In the 4/4 mice, the serum cholesterol level was 2.5-fold that of the +/+ littermate controls on a regular diet. This marked elevation was accounted for by an accumulation of very low and low density lipo-proteins. In the brains of the 4/4 mice, however, the amounts of total cholesterol and phospholipids were significantly decreased compared with the +/+ littermates. These findings indicate that cholesterol and lipid metabolism is markedly altered in the 4/4 mice. Our human apoE4 knock-in mice will be useful in clarifying the role of apoE in the etiologies of AD and cardiovascular diseases in relation to cholesterol and lipid metabolism.

[Serum amyloid A levels in patients with hemophagocytic syndrome].

Concentrations of serum amyloid A protein (SAA) were measured in patients with hemophagocytic syndrome (HPS). There was a significant correlation between SAA and ferritin (p = 0.0003), while there was no significant correlation between C-reactive protein (CRP) and ferritin. These results indicate that SAA could be a useful clinical marker for activity of HPS.

Mouse Pitx2 deficiency leads to anomalies of the ventral body wall, heart, extra- and periocular mesoderm and right pulmonary isomerism.

Pitx2, a bicoid-related homeobox gene, is involved in Rieger's syndrome and the left-right (L-R) asymmetrical pattern formation in body plan. In order to define the genomic structure and roles of Pitx2, we analyzed the genomic structure and generated Pitx2-deficient mice with the lacZ gene in the homeobox-containing exon of Pitx2. We were able to show that among three isoforms of Pitx2, Pitx2c shows asymmetrical expression whereas Pitx2a, Pitx2b and Pitx2c show symmetrical expression. In Pitx2(-)(/)(-) embryos there was an increase in mesodermal cells in the distal end of the left lateral body wall and an amnion continuous with the lateral body wall thickened in its mesodermal layer. These changes resulted in a failure of ventral body wall closure. In lung and heart in which Pitx2 is expressed asymmetrically, right pulmonary isomerism, atrioventricular canals with prominent swelling, and juxtaposition of the atrium were detected. The hearts failed to develop tricuspid and mitral valves and a common atrioventricular valve forms. Further, dysgenesis of the Pitx2(-)(/)(-) extraocular muscle and thickening of the mesothelial layer of cornea were observed in the ocular system where Pitx2 is expressed symmetrically, and these resulted in enophthalmos. The present study shows that Pitx2 expressed in various sites participates in morphogenesis through three types of actions: the involvement of asymmetric Pitx2 expression in the entire morphogenetic process of L-R asymmetric organs; the involvement of asymmetric Pitx2 expression in the regional morphogenesis of asymmetric organs; and finally the involvement of symmetric Pitx2 expression in the regional morphogenesis of symmetric organs.

Intracellular signaling pathways in human eosinophil activation: role of a beta2 integrin, alphaMbeta2.

Cellular adhesion is crucial roles for eosinophil effector functions such as degranulation. Here, we focused on the role of a beta2 integrin, alphaMbeta2, in intracellular signaling pathways of human eosinophil activation. We found that the ligation of alphaMbeta2 triggers the activation of an intracellular signaling cascade, including protein tyrosine phosphorylation and phosphoinositide turnover, and subsequent cellular degranulation in human eosinophils. These signaling events may play important roles in adhesion-dependent cellular functions of eosinophils.

Sulochrin inhibits eosinophil activation and chemotaxis.

OBJECTIVE: Because eosinophils likely play important roles in the pathophysiology of allergic diseases, specific inhibitors of eosinophils may be desirable to treat such diseases. To evaluate the capacity of a novel compound, sulochrin, as an inhibitor of eosinophilic inflammation, we examined the effects of this compound on various effector functions of eosinophils. MATERIALS AND METHODS: We examined the effects of sulochrin on degranulation of human eosinophils stimulated with platelet-activating factor (PAF) or Sepharose 4B beads coated with secretory IgA (sIgA) or IgG. The effects of sulochrin on other effector functions of human eosinophils, including superoxide anion (O2-) production, leukotriene (LT) C4 release, and interleukin (IL)-8 production induced by sIgA-beads were also studied. Finally, using PAF and LTB4 as chemoattractants, we evaluated the potency of sulochrin to inhibit eosinophil migration in vitro and in vivo. RESULTS: Sulochrin inhibited EDN release by eosinophils stimulated with sIgA-beads. IgG-beads and PAF in a concentration-dependent manner; IC50 values were 0.75 microM, 0.30 microM and 0.03 microM. Eosinophil O2- production, LTC4 release, and IL-8 production were also inhibited by sulochrin. Furthermore, PAF-induced chemotaxis of human eosinophils and LTB4-induced chemotaxis of guinea pig eosinophils were abolished by 1 microM of sulochrin. Finally, sulochrin potently inhibited LTB4-induced infiltration of eosinophils into the skin of guinea-pig in vivo. CONCLUSIONS: These results suggest that sulochrin is a potent inhibitor of various effector functions of eosinophils. Sulochrin and its derivatives may be useful in the development of therapeutic approaches for patients with allergic diseases.

Interaction with secretory component stimulates effector functions of human eosinophils but not of neutrophils.

Eosinophils and their products are important in the pathophysiology of allergic inflammation in mucosal tissues. Secretory component bound to IgA mediates transepithelial transport of IgA and confers increased stability on the resultant secretory IgA; however, the effect of secretory component on the biologic activity of IgA is unknown. Here, we report that secretory IgA and secretory component preferentially activate human eosinophils. When eosinophils were stimulated with immobilized secretory IgA, degranulation and superoxide production were two- to threefold greater than when stimulated with serum IgA. In contrast, neutrophils responded similarly to secretory IgA and serum IgA. Flow cytometric analysis showed that eosinophils bound to purified secretory component. The binding of 125I-labeled secretory component was inhibited by unlabeled secretory component or secretory IgA but not by serum IgA. Superoxide production by eosinophils stimulated with cytokines or IgG was enhanced synergistically by immobilized secretory component; secretory component showed no effect on neutrophil activation. Finally, anti-CD18 mAb abolished eosinophil superoxide production stimulated with secretory IgA or secretory component but not with serum IgA, suggesting a crucial role for beta2 integrins in eosinophil interactions with secretory IgA or secretory component. Thus, secretory component plays important roles in activating eosinophil functions but not neutrophil functions. This preferential interaction between secretory component and eosinophils may provide a novel mechanism to regulate mucosal tissue inflammation.

Malignant hyperthermia during epidural anesthesia.

We anesthesized a patient susceptible to malignant hyperthermia (MH) three different times by epidural anesthesia with different types of local anesthetics. His skinned fiber test showed a marked acceleration of calcium (Ca2+)-induced Ca2+ release (CICR). When ester type local anesthetic was used for ankle contracture repair, MH signs appeared following the release of the tourniquet. CICR test is reliable for diagnosing different types of MH.

In vitro differentiation and commitment of CD4+ CD8+ thymocytes to the CD4 lineage, without TCR engagement.

Thymocyte positive selection is based on protection of immature CD4/CD8 double-positive (DP) thymocytes from apoptosis and their differentiation into CD4 or CD8 single-positive (SP) cells. Intracellular signals essential for positive selection appear to be induced through the TCR and some of the accessory molecules including LFA-1, CD4 and CD8 upon interaction with thymic stromal cells. The signals, however, still remain to be identified. Since physiological levels of glucocorticoids potentially induce or enhance thymocyte apoptosis even in vivo, the signals are likely to inhibit the apoptotic effect of glucocorticoids. We have previously shown that proper cross-linking of TCR-CD3 with LFA-1, CD4 or CD8 inhibited glucocorticoid-induced thymocyte apoptosis in vitro, and that a proper combination of the calcium ionophore, ionomycin and the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), mimicked the inhibitory effect. Here we determined whether this combination of ionomycin and PMA induces differentiation of isolated DP thymocytes from normal and TCR transgenic mice. We found that pretreatment of DP thymocytes with ionomycin and PMA followed by 1 day culture of the cells without the reagents resulted in the differentiation of the cells into CD4 SP and CD4+ CD8lo T cells that have mostly committed to the CD4 lineage. The changes in expression of other differentiation markers were also in good accordance with those associated with positive selection, except the final maturation. The results indicate that moderate and transient increases in intracellular Ca2+ level and PKC activity induce differentiation and commitment of DP thymocytes to the CD4 lineage, and suggested that the biochemical pathway leading to positive selection is based on a similar mechanism.

[MRI findings in patients with vertigo and dizziness possibly arising from vertebrobasilar insufficiency].

In order to evaluate diagnostic usefulness of MRI in vertebrobasilar insufficiency (VBI), we performed magnetic resonance imaging (MRI) and MR angiography (MRA) in 90 patients presenting vertigo and dizziness as an initial and cardinal complaint. High signals observed by T2-weighted imaging in the basal ganglia (44.4%) or pontine base (48.9%) were more frequently seen in the possible VBI group than in the controls (p < 0.001). The electronystagmographical abnormalities were commonly observed in the patients with a high signal in the pontine base, reflecting diffuse ischemic lesion in the territory of the vertebrobasilar system. Vertebral artery asymmetry (45.6%) or basilar artery twisting (41.1%) as shown by MRA was also significantly more frequent in the patients than in the controls (p < 0.05). In conclusion, MRI and MRA were considered to be useful in making a clinical diagnosis of VBI in such patients.

Two cases of protruding atherosclerotic plaque with mobile projections in the aortic arch.

We recently studied two patients with cerebral embolism, in whom transesophageal echocardiography revealed protruding atherosclerotic plaques with freely mobile projections in the aortic arch. Ultrasonic imaging showed that the carotid artery was normal, and transthoracic and transesophageal echocardiography did not reveal a cardiac embolic source in either case. In one patient, we observed that an atherosclerotic plaque became ulcerated and developed mobile projections over the course of a year. More consideration should be given to the thoracic aorta as a source of embolism in patients with unexplained stroke.

[Clinical significance of pontine high signals identified on magnetic resonance imaging].

Spin-echo magnetic resonance imaging (MRI) was evaluated to 530 cases in order to investigate the clinical, significance of pontine high signals. The subjects comprised 109 cases of pontine infarction with high signal on T2-weighted image and low signal on T1-weighted image (PI group), 145 of pontine high signal with high signal on T2-weighted image but normal signal on T1-weighted image (PH group) and 276 of age-matched control without abnormality either on T1 or T2-weighted images (AC group). Subjective complaints such as vertigo-dizziness were more frequent in the PH group than in the PI group. In both PI and PH groups, periventricular hyperintensity as well as subcortical high signals in the supratentorium were more severe than in the AC group. These degrees were higher in the PI group than in the PH group. In conclusion, PH as well as PI may result from diffuse arteriosclerosis and PH is considered to be an early finding of pontine ischemia.

Influence of environmental mite antigen on anti-mite antibody production in mice.

To investigate the effects of environmental mite antigen on anti-mite antibody production in mice, a mite-free breeding system was established. The amount of Dermatophagoides farinae (Df) antigen was measured using the ELISA inhibition method. Df antigen was detected in the wood-chip bedding used in our conventional breeding system for 1 week (2.55 +/- 0.79 micrograms/g sample) and 2 weeks (8.96 +/- 3.27 micrograms/g sample). Df antigen was also detected in the standard diet (0.4 micrograms/g sample) in the conventional system, whereas no antigen was detected in the case of the mite-free breeding system. Neither anti-Df IgE nor IgG antibody was detected before immunization, either in mice raised in the mite-free breeding system or in mice raised in the conventional system. Primary and secondary antibody production after immunizations with 1 or 10 micrograms Df antigen was studied in the mouse bred in the conventional and in the mite-free breeding system. Primary anti-Df IgE antibody response of the conventional group was greater than that in the mite-free group after immunization with either dose of 1 and 10 micrograms Df antigen. Primary anti-Df IgG antibody response was greater in the conventional than the mite-free group after immunization with 1 microgram Df antigen. Secondary IgE and IgG responses of the conventional group were greater than the mite-free group after immunizations with 10 micrograms Df antigen. These results suggest that anti-Df antibody production is enhanced by chronic exposure to Df antigen in the environment.

[Magnetic resonance angiography in 12 patients with Wallenberg's syndrome].

Magnetic resonance angiography (MRA) was performed in 12 patients with Wallenberg's syndrome (WS) in chronic stage to investigate the lesion of the vertebral artery (VA). The laterality of infarction in the medulla oblongata was confirmed with MRI: the right in four patients and the left in eight. In this study, time-of-flight MRA was evaluated. Coronal and OM sections in MRA were examined, because the former is useful to observe asymmetry and curve of the cervical VA, and the latter is useful to observe asymmetry and stenosis of the intracranial VA. In nine out of 12 patients, the ipsilateral VA was rather hypoplastic compared to the contralateral VA, and the flow of the hypoplastic VA stopped before and after entering the intracranial region. The contralateral VA of the hypoplastic cases showed curve in the neck in five patients and stenosis in the intracranial portion in one patient. A comparative study was conducted between vertebral angiogram (VAG) and MRA in four patients. Both VAG and MRA were useful in detecting hypoplasia and stoppage of the flow. It was considered that VA hypoplasia is frequently responsible for WS, and that MRA is useful in screening the vascular lesion as a non-invasive procedure to reveal changes of the bilateral VA.

Partial anomalous pulmonary venous return showing anomalous venous return to the azygos vein.

Partial anomalous pulmonary venous return (PAPVR) is a congenital heart disease with a reported incidence of autopsied case. The location of the anomalous pulmonary venous return is usually the right atrium, superior vena cava (SVC), and sometimes the brachiocephalic vein, inferior vena cava (IVC) or coronary venous sinus. Recently we experienced a rare case of PAPVR showing anomalous right total pulmonary venous return to the azygos vein. Furthermore, downward translocation of the right upper lobe bronchus was evident. This rare case is reported along with a review of the related literature.

[The correlation between vertebral artery asymmetry and pontine infarction--an MR angiography study].

The purpose of this study is to evaluate the correlation between variation of the vertebral artery (VA) and the incidence of pontine infarction. A total of 206 patients were examined using magnetic resonance imaging (MRI) and 3-dimension time-of-flight MR angiography (MRA) of the brain. Of these, 54 patients had pontine infarctions (23 symptomatic and 31 asymptomatic), and the majority of them were located in the pontine base. The sites of dominant lesion in the pons were right in 18 cases, left in 8 cases, and bilateral in 28 cases. The number of patients with VA asymmetry (the ratio of internal diameters 1:2 or more) were 89 (43.2%). Of these, 67 patients had small diametric VA of right side, and 22 of left side. Among the 117 patients with normal VA pattern, 19 (16.2%) had infarction, while among the 89 patients with VA asymmetry, 35 (39.3%) had infarction. The patients with small diametric VA of right side significantly had infarctions in the same side of the pons. The results of this study suggest that VA asymmetry is considered to be one of the risk factors of pontine infarction and that MRA can be useful in the examination of the cerebral artery as a valuable and non-invasive screening method.