Ontogeny of neurotensin-containing neuron system of the rat: immunohistochemical analysis--II. Lower brain stem

The ontogeny of the neurotensin neuron system in the lower brain stem of the rat was investigated by means of indirect immunofluorescent method. Neurotensin-like immunoreactivity-containing cells first appear in the primordium of the n. tractus solitarii, n. tractus spinalis nervi trigemini, reticular formation just medial to the latter nucleus, n. reticularis parvocellularis, n. laterodorsalis tegmenti, and midbrain reticular formation of the fetus at gestational day 17. At gestional day 18, neurotensin-immunoreactive cells newly appear in the n. raphe dorsalis. Between gestational day 19 and postnatal day 7, the animals show a remarkable increase in number of immunoreactive cells and fibers in various lower brain stem areas except for n. tractus spinalis nervi trigemini and n. tractus solitarii. Moreover, during this stage, neurotensin-immunoreactive cells located in the n. prepositus hypoglossi and n. vestibularis lateralis appear for the first time at birth and postnatal day 5, respectively. Since postnatal day 7, although the majority of immunoreactive cells located in the lower brain stem decrease in number as the rats grow, immunoreactive cells in the n. tractus spinalis nervi trigemini, on the contrary, increase in number from after birth until postnatal day 10, and maintain more or less their immunoreactivity even in the adult rat. In addition, neurotensin-immunoreactive cells in the nucleus of the solitary tract increase in number during the fetal period, reach the maximum content at postnatal day 7-10, and maintain their immunoreactivity even in the adult rats. Thus, the present study demonstrated that neurotensin-like immunoreactive structures appear at a very early ontogenetical stage, suggesting that neurotensin plays an important role in the development of the lower brain stem of the rat. In addition, the present study further showed that neurotensin-immuno-reactivity shows various fluctuations during the ontogeny, suggesting multiple functions of neurotensin in the central nervous system.     

Fas-mediated cytotoxicity by gammadelta T cells during acute rejection in xenotransplantation of spheroidal aggregate-cultured hepatocytes

Xenogeneic transplantation has recently become a subject of interest for the transplantation community due to the current organ shortage, which could be partially or even totally solved by the development of this strategy. However, xenogenetic rejection remains a formidable barrier preventing such use in a clinical setting. The spheroidal aggregate-cultured hepatocytes of WKA rats were injected into the spleen of C3H mice, and quantitative assessment of transplanted xenogeneic hepatocytes using 99mTc-GSA demonstrated that hepatocytes decreased dramatically 2 days after transplantation (day 2) and few viable hepatocytes in spheroids were detected on day 3. The NK activity significantly increased on day 1, and gammadelta receptor/FasL-expressing T cells appeared on day 2. These results suggested that xenogeneic cytotoxicity consisted of gammadelta T cells through the Fas/Fas ligand system, as well as non-T-cell-mediated cellular response, in the MHC-unrestricted pathway in this intrasplenically transplanted xenogeneic hepatocyte model.     

Cell-mediated cytotoxicity in acute rat cardiac allograft rejection: An immunological and ultrastructural study

Summary To clarify the immune mechanism of cytotoxicity in acute cardiac allograft rejection, we observed interactions between cardiocytes and mononuclear cells using immunohistochemistry and light and electron microscopy. All allografted WKA rat hearts transplanted to F344 recipients stopped beating by the 7th day after the transplantation. The population of helper/inducer T cells (Th) and IL2R⁺ cells was large for the first 3 days, whereas that of cytotoxic/suppressor T cells (Tc-s) and macrophages increased from the 4th day. TheTh/Tc-s ratios were more than 2.0 until the 3rd day, then decreased to less than 1.0. In circulating T lymphocytes; theTh/Tc-s ratios were under 1.0 on the 1st, 6th and 7th days. Electron microscopically IL2R⁺ cells, Tc-s and macrophages were often seen in close contact with the plasma membrane of the cardiocytes. The majority of IL2R⁺ cells are NK cells, Tc-s andTh. Of these, the population of Tc-s was small until the 3rd day. Thus, NK cells play a pivotal role in the early stage of the rejection, and Tc-s and macrophages then aggravate cell-mediated cardiocyte injury.     

Pharmacological characterization of mouse ear PCA

Effects of some antiallergic agents on homologous passive cutaneous anaphylaxis (PCA) in mouse ear were investigated by means of assessing the amount of extravasated dye. Antihistamines (chlorpheniramine and diphenhydramine) and antiserotonins (methysergide and cyproheptadine) suppressed mouse ear PCA significantly. In contrast, an antagonist of slow reacting substance of anaphylaxis (SRS-A) (FPL 55712) and an inhibitor of SRS-A synthesis (AA-861) did not suppress the reaction. beta-Adrenergic stimulants (isoproterenol and salbutamol) and theophylline, which elevate cyclic AMP (cAMP) levels, also suppressed mouse ear PCA significantly. The antiallergic agents, N(3',4'-dimethoxycinnamoyl)anthranilic acid(N-5') and ketotifen suppressed mouse ear PCA significantly, but disodium cromoglycate (DSCG) failed to suppress the reaction.     

New complex t(2;11;17)(p21;q23;q11), a variant form of t(2;11), associated with del(5)(q23q32) in myelodysplastic syndrome-derived acute myeloblastic leukemia

The t(2;11)(p21;q23) is a rare recurrent aberration observed in myelodysplastic syndrome (MDS) and acute myeloblastic leukemia (AML). It has been suggested that t(2;11) is specifically associated with a deletion of the long arm of chromosome 5 (5q). A 63-year-old man was initially diagnosed as AML with del(5)(q23q32) as a sole abnormality. At relapse, t(2;11;17)(p21;q23;q11) in association with del(5q) appeared in 14 of 20 cells by G-banding. Spectral karyotyping confirmed three derivative chromosomes, der(11)t(2;11), der(17)t(11;17), and der(2)t(2;17). Fluorescence in situ hybridization analysis with a probe for MLL demonstrated that the breakpoint at 11q23 was telomeric to the MLL gene. Nine of 10 reported cases with t(2;11) and del(5q) had MDS including 5q- syndrome and four of them evolved to AML, as observed in the present case. Our results indicated that t(2;11;17) was a secondary genetic change, which appeared during disease progression after del(5q) was observed. Furthermore, considering another reported case, the MLL gene seems to be not involved in the pathogenesis of MDS/AML with t(2;11) and del(5q).     

Glial Fibrillary Acidic Protein: Dynamic Property and Regulation by Phosphorylation

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein of astroglia, and belongs to the type III subclass of IF proteins. IF proteins are composed of an amino-terminal HEAD domain, a central ROD domain and a carboxyterminal TAIL domain. GFAP, with a molecular mass of ˜50 KDa, has the smallest HEAD domain among type III IF proteins. Despite its insolubility, GFAP is in dynamic equilibrium between assembled filaments and unassembled subunits, as demonstrated using fluo-rescently labeled GFAP molecules. Like other IF proteins, assembly of GFAP is regulated by phosphory-lation-dephosphorylation of the HEAD domain by altering its charge. This regulation of GFAP assembly contributes to extensive remodeling of glial frameworks in mitosis. Another type III IF protein, vimentin, colocalizes with GFAP in immature, reactive or radial glia, thereby indicating that vimentin has an important role in the build up of the glial architecture.     

Genetic analysis of control of proliferation in fibroblastic cells in culture. I. Isolation and characterization of mutants temperature-sensitive for proliferation or survival of untransformed diploid rat cell line 3Y1

Mutants temperature sensitive for proliferation or survival were isolated from an untransformed diploid clone of fibroblastic rat cells (3Y1), according to an isolation protocol that selected for mutants defective at 38.5C (selection temperature) in undergoing the transition from quiescent to proliferating state while maintaining viability at 38.5 C. Of the 108 temperature-sensitive clones isolated, 32 were examined for survival in sparse cultures at 39.8 C (nonpermissive temperature) and classified into four classes. Results of temperature shift-up experiments suggest that functions defective in 11 of the 32 mutants are necessary not only for changing from the quiescent to proliferating state but also for maintenance of the proliferating state. Of the 32 mutants, 17 were assigned to eight complementation groups. Results of the physiological characterization of the representative mutants of each of the eight complementation groups are presented.On leave of absence from the Division of Child Neurology, Institute of Neurological Sciences, Tottori University, School of Medicine.     

Genetic classification of combined hepatocellular-cholangiocarcinoma

Combined hepatocellular-cholangiocarcinoma (combined HCC/ CC) is a rare form of liver neoplasms showing both hepatocellular (HCC) and bile duct differentiation (CC). In an attempt to clarify the clonality and genetic/phenotypic relationships in the evolution of these neoplasms, we microdissected multiple HCC and CC foci and studied allelic status of chromosome arms 1p, 1q, 3p, 4q, 5q, 6q, 8p, 9p, 10q, 11q, 13q, 16q, 17p, 17q, 18q, and 22q. Overall, the highest frequency of loss of heterozygosity (LOH) was seen on 4q and 17p, followed by 8p and 16q. Of the 11 cases studied, 3 cases did not show any of the identical allelic losses between HCC and CC foci, indicating the biclonal nature. The remaining 8 cases showed multiple allelic losses shared between both components, strongly suggestive of a single clonal derivation. Moreover, 4 of the 8 cases showed additional or divergent allelic losses at more than 1 chromosomal locus only in HCC and/or CC foci. Thus, this heterogeneity was shown to affect the phenotypic diversity of the tumor. Summarizing the genetic patterns, combined HCC/CC could be classified into the following 3 possibilities: (1) collision tumor in which 2 independent neoplastic clones develop at close proximity; (2) single clonal tumor with homogeneous genetic background in both components--histological diversity is thus a manifestation of divergent differentiation potential of a single clone; (3) single clonal process in which genetic heterogeneity in the process of clonal evolution within the tumor parallels histologic diversity; therefore, the tumor in this category is mainly composed of mosaics of closely related subclones.     

Inhibitory effect of the immunosuppressant FK506 on apoptotic cell death induced by HIV-1 gp120

Human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120 may play a central role in inducing immunoregulatory disorders after HIV infection. The apoptotic death of normal human peripheral blood mononuclear cells was induced by priming with gp120 followed by stimulation with an anti-T cell receptor (TCR) antibody. Tumor necrosis factor- produced by gp120-binding macrophages may be important to induce this cell death. Treatment of gp120-primed cells with an immunosuppressant (FK506) before TCR signaling inhibited apoptotic cell death, and this blocking effect of FK506 was concentration dependent. FK506 did not have any influence on cell growth and viability over the range of concentrations tested. These findings suggest that FK506 is a potentially useful drug in delaying the onset of AIDS after HIV infection.