Coexpression of Fas/FasL and Bax on brain and infiltrating T cells in the central nervous system is closely associated with apoptotic cell death during autoimmune encephalomyelitis.

Recent studies have suggested that autoimmune inflammation elicited in the central nervous system (CNS) is subsided by apoptotic cell death of inflammatory cells. To elucidate the molecular mechanism of apoptosis of infiltrating T and other cells occurring in the CNS during autoimmune encephalomyelitis, we determined the type of apoptotic cells and the localization of apoptosis-related molecules (Fas, FasL, Bax, Bcl-2 and active caspase 3) by immunohistochemistry. Double labeling with the TUNEL method and cell-type markers showed that infiltrating T cells and microglia/macrophages underwent apoptosis, while astrocytes and neurons did not. Staining for apoptosis-related molecules revealed that infiltrating T cells and microglia/macrophages, but not astrocytes and neurons, expressed both Fas-FasL and Bax. The distribution and cell type of active caspase 3-positive cells were essentially the same as those of TUNEL-positive cells. These findings suggest that coexpression of Fas/FasL and Bax is closely associated with apoptotic cell death of infiltrating T cells and microglia in the CNS. Furthermore, astrocytes which express Fas and FasL, but not Bax, may play an important role in regulating inflammation in the CNS by inducing apoptotic cell death of infiltrating T cells and microglia, both of which have an inflammation-promoting nature.

Competitive PCR quantification of pro- and anti-inflammatory cytokine mRNA in the central nervous system during autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the central nervous system that can be induced by immunization with myelin basic protein (MBP)/complete Freund's adjuvant and serves as a model for multiple sclerosis. Recent studies have suggested that cytokines play a crucial role in the clinical course of EAE. To clarify the roles of cytokines in EAE, we examined levels of interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta1 (TGF-beta1) and interleukin-10 (IL-10) mRNA in isolates from infiltrating inflammatory cells in EAE lesions induced in Lewis rats. The non-radioactive and sensitive competitive PCR method was employed to quantify the relative amounts of cytokine mRNA. Levels of both IFN-gamma and TNF-alpha mRNA were increased at the early stage of EAE and rapidly decreased at the peak stage. On the other hand, TGF-beta1 mRNA was demonstrated throughout the course of EAE as well as under normal conditions and its amount paralleled the severity of EAE. IL-10 mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR) under normal conditions, but was below the level of detection of competitive PCR. IL-10 mRNA expression peaked at the early stage of EAE and declined gradually thereafter. Taken together, these results suggest that IFN-gamma and TNF-alpha might play a crucial role in the development of EAE. Furthermore, it appears that the peak expression of IL-10 mRNA at the early stage and the following marked TGF-beta1 expression at the peak stage might represent an important endogenous mechanism to limit the extent of inflammation and to prevent relapse in the course of acute monophasic EAE.

Interaction between apoptotic cells and reactive brain cells in the central nervous system of rats with autoimmune encephalomyelitis.

To elucidate the role of brain cells in the immune regulation in the central nervous system (CNS), acute and chronic relapsing experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats and the location of apoptotic inflammatory cells and their interaction with astrocytes and microglia was investigated at various stages of the disease. Apoptotic cells detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) were few in number at day 10-12 post-immunization (PI), increased and peaked at day 13 PI. Then, these cells decreased gradually by day 21 PI. The most characteristic finding was that apoptotic cells were mainly distributed in the CNS parenchyma with only a few cells present in perivascular cuffs. Double staining by the TUNEL method and immunocytochemistry for astrocytes and microglia revealed that astrocytes were more closely associated with apoptotic cells than microglia. Apoptotic cell death may be one mechanism by which T cells are eliminated from the CNS. Furthermore, the present study suggests that astrocytes, rather than microglia, induce programmed cell death of infiltrating inflammatory cells.

Immunoscintigraphy of human tumors transplanted in nude mice with radiolabeled anti-ras p21 monoclonal antibodies.

Anti-ras p21 monoclonal antibody (RASK-3) was used for immunoscintigraphy of human cancer cell lines in nude mice. Iodine-125-labeled RASK-3 was injected into nude mice with either human colon cancers (FCC-1 or BM-314) or lung cancer (KNS-62). Clear images were obtained in all three cancers 7 days after the injection of antibody. No localization of 125I-labeled control monoclonal antibody was observed. The ratio of tissue/blood radioactivity and % ID/g in the tumor were significantly higher than other organs by Day 8. The specific localization index examined by 131I-RASK-3 and 125I-control monoclonal antibody was also higher in the tumor than in other tissues. In the in vitro study, binding of RASK-3 to tumor cells increased significantly by treatment of cells with either lysolecithin or periodate-lysine-paraformaldehyde, which confirmed the intracellular localization of ras p21. The mechanism by which anti-ras p21 antibodies accumulate in tumor sites could be the necrotic changes in tumor cells or changes in membrane permeability of non-necrotic cells. These results provide a strong rationale for the utilization of ras p21 as a target antigen in the imaging of a variety of human cancers.

Phasic loss of intercostal muscle activity occurring with rapid eye movements during REM sleep.

We tried to estimate the phasic motor inhibition occurring with rapid eye movements (REMs) during REM sleep in children by means of polysomnography. Phasic inhibition of intercostal muscle activity with REMs has been proved by averaging the integrated surface electromyograms in three children. The average latency from the onset of REMs to this inhibition was 38.0 ms, their average duration being 237.0 ms. We discussed the possibility that the REM-related phasic inhibition obtained here was involved in the brainstem-spinal cord inhibitory system functioning during REM sleep.

Cerebellar neurodegeneration in human hereditary DNA repair disorders.

Recent findings have focused attention on the role of apoptosis in neurodegenerative diseases, however, the apoptotic process in child-onset brain disorders has been little investigated. Xeroderma pigmentosum (XP) and Cockayne syndrome (CS) are hereditary disorders characterized by impaired DNA repair and neurodegeneration. We investigated apoptotic cell death in the cerebellum of five cases of XP group A (XPA), four cases of CS, and twelve controls, using TdT-mediated DIG-dUTP nick-end labeling (TUNEL) and immunohistochemical staining for bcl-2, bcl-x, p53, bax, BDNF and Trk B. The TUNEL-positive cells were found in the granule cells of the cerebellar cortex of two patients with XPA and two patients with CS, whereas such cells were not detected in the cerebellar cortex in controls. Upregulation of bcl-2 or BDNF was not observed, and bcl-x expression was not altered. Some patients showed nuclear expression of p53 in the granule cells and/or molecular layer, bax-positive glial cells in the cerebellar white matter, and a few Trk B-positive cells in the granular layer. These findings suggest that apoptotic cell death can be involved in the cerebellar degeneration in patients with hereditary defects in DNA repair mechanisms.

Selective uptake by cancer cells of liposomes coated with polysaccharides bearing 1-aminolactose.

We investigated the selective uptake of liposomes chemically modified by polysaccharides-cholesterol derivatives with 1-aminolactose (lactose) in two human hepatoma cell lines (HUH7 and Alexander), a human colon cancer cell line (FCC) and a human lung cancer cell line (KNS). The uptakes of the labeled liposomes alone (conventional liposomes), those with cholesterol pullulan (CHP) and with lactose (lactose CHP) were compared in four cancer cells and normal rat hepatocytes after 3 hours of incubation. The radioactivities of the lactose CHP were 4.4, 4, 3.4 and 4.4 times greater than those of CHP in HuH7, Alexander, FCC and KNS cells, respectively, after 3 hours of incubation. All the above differences were statistically significant (p < 0.01). No statistically significant differences were seen in the case of hepatocytes. Thus, cancer cells have a common affinity with lactose CHP liposomes, however, these mechanisms appear to have no connection with the galactose-specific asialoglycoprotein receptors of hepatocytes.

Impairment of respiratory rhythmogenesis and sequelae of bacterial meningitis.

A 9-year-old boy with respiratory disturbance associated with medullary lesions after pneumococcal meningitis is reported. Although he lives a normal daily life, he cannot cough or sneeze. A polysomnographic study revealed a low respiration rate and an irregular respiratory rhythm not only during REM sleep but also during slow wave sleep, and marked desaturation during sleep. Respiratory function tests including CO2 response revealed normal values. Magnetic resonance imaging demonstrated bilateral small lesions in the medulla. This patient is unusual because respiratory rhythm is impaired, without decreased ventilatory capacity or CO2 response, supporting the possibility that rhythmogenetic respiratory neurons are located in a limited area of the human medulla.

Variations in radioimmunoscintigraphic detection of tumor showed by five monoclonal antibodies to carcinoembryonic antigen.

Radioimmunoscintigraphy using mouse monoclonal antibodies to various parts of a carcinoembryonic antigen (CEA) molecule was performed. Four radiolabeled antibodies (F4-82, 28A, F3-30, F33-104) were injected into tumor transplanted nude mice to compare the accumulation of these antibodies in tumors. The four antibodies were accumulated selectively in CEA- producing tumors. The tumor visualization correlated with the tumor/blood radioactivity ratio, whereas the tumor/blood radioactivity ratio did not correlate with the in vitro percent binding to tumor cells or the in vivo percent injected dose in CEA-producing tumors. Among the four antibodies, F33-104 showed the highest tumor/blood radioactivity ratio and the best image quality in any CEA-producing tumor. These results suggest that the antibody which has a high tumor/blood ratio rather than high total tumor uptake may be useful for radioimmunoscintigraphy.

The mechanism of placental alkaline phosphatase induction in vitro.

The mechanism of placental alkaline phosphatase (PLAP) induction by prednisolone in a uterine cervical epidermoid cancer cell line SKG-IIIa was investigated in vitro by enzyme-cytochemistry, enzyme immunoassay, Northern and Southern blot analysis, and in situ hybridization. Enzyme-cytochemical alkaline phosphatase (ALP) staining and immunoassay revealed increased levels of PLAP (heat-stable ALP) in prednisolone-treated cells. Northern blot analysis and in situ hybridization showed increased amounts of PLAP mRNA. Southern blot analysis indicated that PLAP was not a product of an amplified or rearranged gene. These findings suggest that the induction of PLAP mRNA in SKG-IIIa cells by prednisolone in turn increased the levels of PLAP.

Immunohistochemical analysis of brainstem lesions in infantile spasms.

Whether the cerebral or subcortical lesions are involved in the pathogenesis in infantile spasms (IS) remains to be determined. To investigate the functional lesions of the subcortical structures in IS, the brainstem expression of neurotransmitters, neuropeptides and calcium-binding proteins in IS autopsy cases of lissencephaly and of perinatal hypoxic ischemic encephalopathy (HIE/IS) was investigated. The IS patients consisted of four subjects each of lissencephaly and HIE. They suffered from both West and Lennox-Gastaut syndromes. The healthy and disease controls were composed of four subjects without neuromuscular disorders and six cases of HIE (HIE/C), neither of whom had the epileptic syndrome. In these subjects the expressions of tryptophan hydroxylase (TrH), tyrosine hydroxylase (TH), parvalbumin (PV), methionine-enkephalin (ME) and substance P (SP) were immunohistochemically determined in serial sections of the midbrain, pons and medulla oblongata. The immunoreactivity of neurons and neuronal processes for TH was altered in the mesencephalic periaqueductal gray matter, locus ceruleus, and dorsal vagal nucleus in the patients. The HIE/IS cases showed reduced TrH-immunoreactivity in the medullary raphe nuclei. The brainstem auditory tract was poorly discernible on anti-PV immunostaining in the IS patients. The immunoreactivity for ME in the spinal trigeminal nucleus was severely affected in the IS patients, while that for SP was comparatively well preserved. It is suggested that the presence of common brainstem lesions in IS is irrespective of etiologies. It is intriguing that some of the changes seemed to be interrelated with the neurophysiological abnormalities being reported in IS patients.