Organization of the trigeminal and facial motor nuclei in the hagfish, Eptatretus burgeri: a retrograde HRP study

We studied the trigeminal and facial motor nuclei of the hagfish by the retrograde HRP method. We distinguished 4 components in a single column of the motor nuclei of the trigeminal nerve and the facial nerve, viz., the pars magnocellularis of the trigeminal motor nucleus (mVm), the anterior part of the pars parvocellularis of the trigeminal motor nucleus (mVp1), the posterior part of the pars parvocellularis of the trigeminal motor nucleus (mVp2) and the facial motor nucleus (mVII). Although in Nissl preparations only the mVm could be distinguished from the rest of the nucleus, the boundaries of the other 3 components were clearly demarcated in HRP preparations. Intramuscular injections into two representative antagonistic jaw muscles revealed that there was no apparent topological organization of the neurons pertaining to the opening and closing muscles in the mVm and mVp1, but both antagonistic muscles were innervated bilaterally. Although the hagfish does possess a cartilaginous jaw, the organization pattern of the motor nuclei of the jaw muscles seems to be the most primitive of all living vertebrates.     

Different origin of leiomyoblastoma by immunohistochemical study.

Leiomyoblastoma has been regarded as a neoplasm of smooth muscle origin. With recent progress in immunohistostaining techniques, many clinicopathological discrepancies have been pointed out about the origin of leiomyoblastoma. It has been claimed that gastrointestinal non-epithelial tumors should be regarded as stromal tumors in order to study their origin. In the present study, we performed various forms of immunohistostaining in seven cases of leiomyoblastoma to determine their origin. One case expressed desmine and muscle specific actin and was considered to be derived from smooth muscle. Four neoplasms expressed X-100 protein (two cases were also NSE positive) and were thought to be derived from the nerve. Two cases were of unknown derivation. These results suggest that the cells of leiomyoblastoma may arise from a primitive to totipotential cell of neural lineages that may anomalously express smooth muscle filaments.     

Whole genome association study of rheumatoid arthritis using 27 039 microsatellites

A major goal of current human genome-wide studies is to identify the genetic basis of complex disorders. However, the availability of an unbiased, reliable, cost efficient and comprehensive methodology to analyze the entire genome for complex disease association is still largely lacking or problematic. Therefore, we have developed a practical and efficient strategy for whole genome association studies of complex diseases by charting the human genome at 100 kb intervals using a collection of 27,039 microsatellites and the DNA pooling method in three successive genomic screens of independent case-control populations. The final step in our methodology consists of fine mapping of the candidate susceptible DNA regions by single nucleotide polymorphisms (SNPs) analysis. This approach was validated upon application to rheumatoid arthritis, a destructive joint disease affecting up to 1% of the population. A total of 47 candidate regions were identified. The top seven loci, withstanding the most stringent statistical tests, were dissected down to individual genes and/or SNPs on four chromosomes, including the previously known 6p21.3-encoded Major Histocompatibility Complex gene, HLA-DRB1. Hence, microsatellite-based genome-wide association analysis complemented by end stage SNP typing provides a new tool for genetic dissection of multifactorial pathologies including common diseases.     

Immunological circumvention of multiple organ metastases of multidrug resistant human small cell lung cancer cells by mouse-human chimeric anti-ganglioside GM2 antibody KM966

The formation of metastases in multiple organs and acquired multi-drug resistance (MDR) are the major obstacles for treatment of human small-cell lung cancer (SCLC). To explore the possibility of immunological overcoming of multiple-organ metastases produced by refractory SCLC, we established the MDR variant (SBC-3/DOX), expressing P-glycoprotein, of parental SBC-3 cells by culturing with gradually increasing concentration of adriamycin. Both SBC-3 and SBC-3/DOX cells expressed a high amount of ganglioside GM2, an ideal target of SCLC cells. A mouse-human chimeric anti-GM2 monoclonal antibody (KM966) induced antibody-dependent cellular cytotoxicity (ADCC) mediated by human mononuclear cells (lymphocytes and monocytes) and complement-dependent cytotoxicity (CDC) mediated by human AB serum against SBC-3/DOX cells to a similar extent compared with parental SBC-3 cells. Pretreatment of human effector cells with various cytokines induced further enhancement of the KM966-dependent ADCC against SBC-3/DOX cells. Intravenous injection of SBC-3 or SBC-3/DOX cells into natural killer (NK) cell-depleted severe combined immunodeficient (SCID) mice developed metastases in multiple organs (liver, kidneys and lymph nodes). Interestingly, SBC-3/DOX cells produced metastases more rapidly than SBC-3 cells, suggesting more aggressive phenotype of SBC-3/DOX cells than their parental cells in vivo. Systemic treatment with KM966, given on days 2 and 7, drastically inhibited the formation of multiple-organ metastases produced by both SBC-3 and SBC-3/DOX cells, indicating that KM966 can eradicate metastasis by SCLC cells irrespective of MDR phenotype. These findings suggest that the mouse-human chimeric KM966 targets the GM2 antigen, and might be useful for the immunological circumvention of multiple-organ metastases of refractory SCLC. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effect of thromboxane A2 synthetase inhibitor on immediate-type hypersensitivity reactions

The effect of the thromboxane (TX) A2 synthetase inhibitor, OKY-046, on human leukocyte histamine release and bronchial hypersensitivity in asthmatic subjects was evaluated. It was found that OKY-046 inhibited IgE- and Ca2+ ionophore A23187-mediated leukocyte histamine release in a dose-dependent fashion (IC50: 1.0 and 3.0 X 10(-3) M, respectively) and that OKY-046 could diminish bronchial hypersensitivity, determined by leukotriene D4 inhalation, following a 2-week oral medication. These data suggest that the TXA2 synthetase inhibitor can produce favorable effects upon the course of immediate-type hypersensitivity reactions.     

Direct in vivo protein transduction into a specific restricted brain area in rats

Attempts at protein transduction into specific restricted brain areas have remained unsuccessful. We attempted targeted, direct in vivo protein transduction by microinjecting beta-galactosidase (beta-gal) with hemagglutinating virus of Japan envelope (HVJ-E) vector into the rat nucleus tractus solitarius (NTS). The medulla oblongata including the NTS was removed 6h post-injection and cryostat sections were histochemically stained to detect beta-gal enzymatic activity. beta-gal-positive cells were present in these sections as was beta-gal activity determined by colorimetric analysis. beta-gal-positive cells were not present in the rats microinjected only beta-gal protein without HVJ-E vector. Our findings suggest that direct in vivo protein transduction into specific restricted brain areas is possible. The type of targeted delivery system we present may have wide applications in the administration of therapeutic proteins to the central nervous system.     

Density heterogeneity of eosinophil leucocytes: induction of hypodense eosinophils by platelet-activating factor.

We have examined the induction of hypodense eosinophils by platelet-activating factor (PAF), a mediator which may be involved in eosinophil activation in allergic diseases. Guinea-pig eosinophils were incubated with buffer or PAF and applied to continuous Percoll density gradients. Cellular density ranged from 1.0142 to 1.1369 g/ml. Peak eosinophil density in control was 1.0887 +/- 0.0008 g/ml (mean +/- SEM), and 91.1 +/- 1.4% of eosinophils were distributed between 1.0810 and 1.1000 g/ml. Preincubation of eosinophils with PAF(10(-7) M) resulted in a time-dependent and non-cytolytic increase of the number of hypodense eosinophils, with peak densities after incubation for 1 hr and 2 hr of 1.0834 +/- 0.0014 (n = 4, P less than 0.05) and 1.0755 +/- 0.0007 g/ml (n = 6, P less than 0.01), respectively. After incubation for 2 hr, 82.0 +/- 4.9% (n = 6) eosinophils showed a density lower than 1.080 g/ml. Lyso-PAF, the inactive precursor and metabolite of PAF, at a concentration of 10(-7) M had no effect on cell density. The specific PAF receptor antagonist WEB 2086 (10(-6) M) inhibited the PAF-induced density shift by 87.0 +/- 5.3%. Our results demonstrate that a single mediator is able to induce the formation of hypodense eosinophils. We conclude that the appearance of hypodense eosinophils in allergic diseases such as asthma may occur, at least in part, in response to inflammatory mediators which activate these cells.     

Determination of medullasin levels for the diagnosis of multiple sclerosis

Medullasin levels in granulocytes of patients with neurological diseases and healthy volunteers were determined by the enzyme immunoassay using mouse monoclonal antibodies against human medullasin and o-phenylenediamine-H2O2 as the detection system of the enzyme activity. One hundred twenty-one out of 159 patients with multiple sclerosis (76.1%) showed positive results (above means of normals + 2SD) in this test, while only 16.9% (24/142) of patients with non-inflammatory neurological diseases had positive results. This enzyme immunoassay method for medullasin is considered to be an useful paraclinical test for the diagnosis of multiple sclerosis.     

Involvement of platelet activating factor (PAF) in ovalbumin antigen-induced late asthmatic response and increase of airway hyperresponsiveness in a guinea pig experimental model of asthma

Platelet activating factor, a potent chemical mediator, has been implicated in the pathogenesis of asthma in terms of inflammatory cell recruitment and activation. We have recently demonstrated that repeated antigen (ovalbumin; OA) exposure by inhalation to guinea pigs results in a development of late asthmatic response (LAR) in more than 50% of the animals and significant increase in airway hyperresponsiveness (AH). We have studied the effect of WEB 2086, a specific PAF receptor-antagonist, on this model. Respiratoly resistance (Res) of guinea pigs was measured by a oscillation technique and AH was evaluated by the provocative concentration of aerosols of histamine causing 200% increase of Rrs over the baseline Rrs (PC200 Hist). Four out of 5 actively sensitized and diphenhydramine-pretreated animals developed LAR 3 to 9 hr after allergen (20 mg/ml OA, 10 min inhalation)-induced immediate bronchoconstriction (LAR). Treatment with WEB 2086 (3 mg/kg intravenously) 30 min before and 3 hr after the exposure suppressed LAR clearly without affecting the IAR. Significant increase in AH from 2.80 +/- 0.03 to 2.51 +/- 0.01 and 2.60 +/- 0.08 (p less than 0.05, n = 8) of PC200 Hist (mg/ml, log) was observed 24 hr and 5 day after the OA exposure, respectively. The WEB 2086 treatment also prevented the increase of AH after the OA exposure (PC200 Hist; 2.82 +/- 0.09 before the challenge 2.80 +/- 0.07 and 2.75 +/- 0.09 24 hr and 5 days after, respectively. n = 8). Administration of WEB 2086 did not affect baseline Rrs and PC200 Hist in normal guinea pigs without any antigen challenge. We conclude that WEB 2086 is capable of preventing the development of LAR and increase in AH, and thus PAF may play an important causal role in LAR and increased AH observed in asthma.