Beta amyloid is focally deposited within the outer basement membrane in the amyloid angiopathy of Alzheimer''s disease. An immunoelectron microscopic study.

The fine structure of cerebral amyloid angiopathy, especially in small and presumably early deposits, was examined by immunolabeling of the beta/A4 protein in semithin and ultrathin sections from brains with Alzheimer's disease. The following findings emerged: 1) in large leptomeningeal arteries, small, focal amyloid deposits appear to consist of clusters of delicate (approximately 8 nm diameter) amyloid fibrils, not previously described, in the outermost part of the basement membrane (BM) at the media-adventitia junction; 2) in small leptomeningeal arteries and perforating cortical arterioles, small foci of delicate amyloid fibrils were observed within the BM. They appeared mostly in the outer portion of the BM, around intact smooth muscle cells, rather than in the subendothelial region. In larger and presumably more advanced deposits, coarse amyloid fibrils (approximately 10 nm) occupied the abluminal BM, and adjacent smooth muscle cells showed degeneration; and 3) in capillaries, small amounts of delicate (approximately 8 nm) amyloid fibrils, not previously described, were seen within the BM in the smallest discernible deposits. The BM at these sites was abnormally folded and layered. In larger deposits, amyloid fibrils appeared to extravasate from the outer BM of the capillary into the neuropil and were surrounded by astrocytic foot processes and/or microglia. Our results suggest that vascular amyloid fibrils may first be formed within the abluminal vascular BM, that is, outside of cells. The BM may trap degradative intermediates of the amyloid precursor protein that contain the beta/A4 region, and local proteases may then cleave them further to yield amyloidogenic fragments.     

Induction of brain tumors by a newly isolated JC virus (Tokyo-1 strain).

A newly isolated virus from a patient with progressive multifocal leukoencephalopathy (PML) (Tokyo-1 strain) was found serologically identical to JC virus (Mad-1 strain) and showed high neurooncogenicity in hamsters. Twenty-one animals inoculated intracerebrally with the virus developed brain tumors during a period that averaged 5 months. The tumors were cerebellar medulloblastoma (n = 20); plexus tumor (n = 2) occurred in 1 animal as a single tumor and in another in combination with a medulloblastoma. Thalamic gliomatosis was also present in 6 animals with medulloblastoma. Five mock-infected animals did not develop tumors. Medulloblastoma cells were shown to contain papovavirus T-antigen. In 20 animals examined the medulloblastoma showed a close resemblance to the human medulloblastoma in its histologic, immunocytochemical, and ultrastructural features. Examination of the incipient tumors indicated that the hamster medulloblastoma originated in cells in the neonatal external granular layer. Following infection the cells apparently migrated into the internal granular layer, carrying integrated virus genes and expressing phenotypical transformation. These findings confirm previous reports on the oncogenicity of virus isolates from PML (ZuRhein and Varakis, 1979), but are novel in that with this new isolate tumors could be induced with comparatively low levels of virus inocula.     

Axonal projection of descending pathways responsible for eliciting forelimb stepping into the cat cervical spinal cord

Summary The descending pathways responsible for eliciting forelimb stepping are located in the lateral funiculus (Yamaguchi 1986). In order to determine into which spinal segments the descending pathways project and to know the projections and functions of the other descending system, the ventral funicular pathways, we placed various lesions in the cervical spinal cord of decerebrate cats with the lower thoracic cord transected and studied their effects on forelimb stepping evoked by stimulation of the midbrain locomotor region. (1) The lateral funiculus was transected on one side. The operation removes descending input to all the segments caudal to the lesion. Experiments with serial transections from the caudal to rostral segment revealed that stepping activity of the limb on the lesioned side is reduced when the lesion is placed at the level between the C6 and C7 segment and then between C5 and C6. A slight reduction of activity was also observed after a lesion placed between C7 and C8. (2) Consistently, bilateral transection of the lateral funiculus at the level between C5 and C6 abolished stepping movements of both forelimbs. (3) The cervical cord was split in the parasagittal plane through the dorsal root entry. The operation removes the descending input to the segment in which the lesion is placed. The parasagittal lesions from the C1 to C6 did not abolish stepping activity, although a lesion placed between C5 and C6 could slightly affect stepping. The results, (1)–(3) suggest that the lateral funicular pathways project into the spinal segments mainly at the C6–C7 level with some rostrocaudal extension into C5 and C8. (4) Complete transections of the medial part of the spinal cord cut extensor bursts short and raised stepping frequency. Nevertheless, if the lesion at C1–C5 spared the ventromedial part of the ventral funiculus, it did not result in such high-frequency stepping or in weakened extensor activity. In the case of segments caudal to C6, medial transections which spared the corresponding region could result in such stepping. It is suggested that the pathways descending through the ventromedial part of the ventral funiculus in the rostral segments provide extensor activity during stepping. They may change their course in the more dorsal part of the ventral funiculus below the C6 and presumably project into the grey matter of more caudal segments.     

Constitutive Expression of a Bacterial Pattern Recognition Receptor, CD14, in Human Salivary Glands and Secretion as a Soluble Form in Saliva

Saliva contains a number of proteins and glycoproteins that protect oral tissues, but little is known about the role of human saliva in innate immunity. Here we showed that human major salivary gland cells constitutively expressed a bacterial pattern recognition receptor, CD14, by immunohistochemistry. Human salivary gland cells in culture express CD14 mRNA and a 55-kDa CD14 protein in, but not on the cells, and secrete a soluble form with the same molecular mass. Human whole saliva contains a 55-kDa CD14, and the concentration of parotid saliva was 10-fold higher than whole saliva, which is comparable to that of serum CD14. Levels of CD14 in unstimulated whole and parotid saliva were unchanged before and after a meal and between unstimulated and stimulated saliva, indicating that saliva CD14 is constitutively secreted into the oral cavity. In contrast, lipopolysaccharide (LPS)-binding protein was below the detectable level. The saliva CD14 is functionally active in that it mediated the activation of CD14-lacking intestinal epithelial cells by LPS in a Toll-like receptor 4-dependent manner. These results suggested that saliva CD14 is important for the maintenance of oral health and possibly intestinal homeostasis.     

Clinical study on the serum carcinoembryonic antigen, immunosuppressive acidic protein and C-reactive protein levels in patients with adult T cell leukemia

Serum carcinoembryonic antigen (CEA), immunosuppressive acidic protein (IAP) and C-reactive protein (CRP) levels were measured in patients with adult T cell leukemia (ATL) in order to clarify their significance in this disease. Mean (+/- SD) serum CEA levels in 11 patients with acute ATL (3.1 +/- 1.3 ng/ml) and 7 patients with smoldering ATL (3.1 +/- 0.5 ng/ml) were significantly higher than in sera of 222 healthy controls (2.4 +/- 1.3 ng/ml). However, the levels in 7 patients with chronic ATL and healthy controls showed no differences. On the other hand, mean (+/- SD) serum IAP levels in patients with acute ATL (928 +/- 395 micrograms/ml), chronic ATL (487 +/- 125 micrograms/ml) and smoldering ATL (429 +/- 90 micrograms/ml) were significantly higher than in sera of healthy controls (359 +/- 103 micrograms/ml). However, the levels in patients with smoldering ATL and healthy controls showed no differences. Serum IAP levels in crisis in chronic and smoldering ATL were similar to those in patients with acute ATL. 85% of ATL patients with IAP levels above 500 micrograms/ml had CRP levels above 1+. Serum CEA, IAP and CRP levels were serially measured in a number of patients. Serum IAP and CRP levels reflected each patient's clinical course more than serum CEA levels. Overall the simultaneous measurements of serum CEA, IAP and CRP levels revealed a potential usefulness for determination of ATL subtype, and serum IAP and CRP levels may provide a way to evaluate the effectiveness of chemotherapy.     

Enhancement of lipopolysaccharide-stimulated cyclooxygenase-2 mRNA expression and prostaglandin E2 production in gingival fibroblasts from individuals with Down syndrome

It is well known that Down syndrome (DS) is a premature ageing syndrome. Periodontal disease in individuals with DS develops rapidly and extensively in a relatively younger age bracket compared with that in healthy controls. The mechanisms involved in the periodontal inflammatory processes in DS patients are not fully understood. In the present study, the non-inflamed gingival fibroblasts isolated from seven patients with DS (DGF) and seven healthy controls (NDGF) were stimulated with lipopolysaccharide (LPS) derived from Actinobacillus actinomycetemcomitans (A. a.). We measured the level of prostaglandin E2 (PGE2) production by DGF and NDGF by radioimmunoassay, and also measured the mRNA expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) by using the real-time PCR method. We found the higher levels of LPS-stimulated COX-2 mRNA expression and PGE2 production in DGF when compared with those in NDGF. This study may indicate that overexpression of LPS-stimulated COX-2 induced a greater ability of DGF to produce PGE2, and that these phenomena may be responsible for the severer periodontal disease in DS patients.     

Tumor necrosis factor-alpha in the placenta is not elevated in pre-eclamptic patients despite its elevation in peripheral blood

PROBLEM: Tumor necrosis factor-alpha (TNF-alpha) is present in human placental and uterine cells at the early and late stages of gestation and promotes the regulation of trophoblast growth and invasion. We evaluated whether TNF-alpha levels in the placenta and blood of pre-eclamptic women differed from those with normal pregnancies. METHOD OF STUDY: The subjects were 39 pregnant women carrying single fetuses (21 normal-pregnant and 18 pre-eclamptic patients). Their average gestational age at entry was 38-39 weeks. Peripheral blood was collected before the onset of labor and separated serum was stored at -20 degrees C. A tissue segment of the placenta was cut and frozen in liquid nitrogen immediately after delivery at -80 degrees C. The frozen placental tissue was added to phosphate-buffered saline. The tissue was fully homogenized and centrifuged. Separated supernatant was stored at -80 degrees C. TNF-alpha levels in separated serum and TNF-alpha and total protein (TP) levels in separated supernatant were measured. The presence of TNF-alpha in the placenta was evaluated by immunohistochemistry in five pre-eclamptic and five normal-pregnant patients. RESULTS: Serum TNF-alpha levels were higher in pre-eclampsia than in normal pregnancies. However, TNF-alpha/TP levels in the placenta did not differ significantly between the two groups. As for TNF-alpha immunostaining of trophoblastic cells in the placenta, it was weak in three and moderate in two of the normal pregnancies, while it was absent in two, weak in one, and moderate in two in the pre-eclampsia group. CONCLUSIONS: We demonstrated no significant increase in TNF-alpha/TP levels in the placenta in pre-eclampsia despite a significant increase in serum TNF-alpha levels. There was no strong immunostaining for TNF-alpha detected by immunohistochemistry in the pre-eclampsia group. These findings suggest that TNF-alpha in the placenta is not a key cytokine to interfere with normal trophoblast invasion into the myometrium in pre-eclampsia, and that sources other than the placenta may contribute to the elevated levels of TNF-alpha found in the circulation of pre-eclamptic patients.     

Increased production of human immunodeficiency virus (HIV) in HIV-induced syncytia formation: An efficient infection process

Syncytia or multinucleated giant-cell formation is one of the major cytopathic effects induced by human immunodeficiency virus (HIV) infection. Cell fusion results from the strong interaction of CD4 molecules on the surface of the uninfected T cells and gp120, an external envelope glycoprotein of HIV on the infected T cells. We studied the production of HIV in fusion cells between MOLT-4 and virus-infected MOLT-4/HIV cells and found that HIV production was enhanced up to three- to fivefold, which showed a good correlation with the appearance and extent of syncytia formation. Blocking the fusion by monoclonal antibody against a binding epitope of CD4 molecule to gp120 decreased the HIV production significantly. Enhancement of HIV production was observed by more than five-fold in comparison with chronically infected cells, which were fusion free 20 hr postcocultivation. Electron microscopic observation also showed the presence of abundant HIV particles inside the fused cells and on the outer surface. AZT blocked the HIV augmentation of fused cells in coculture completely. Southern blot analysis revealed that both integrated and unintegrated HIV DNA were highly accumulated in fusion cells, as compared with fusion-free MOLT-4/HIV cells. Among unintegrated DNA, circular and linear DNA were accumulated to a similar degree. Northern blot hybridization showed that rapid enhancement of all three species of HIV-specific RNA containing genomic (9.2 kb) and subgenomic (4.3 and 1.9 kb) RNAs were found 20 hr postinfection in fusion cells. These data suggest that syncytia formation is an extremely active infection process of HIV, by which multiple rounds of reinfection might take place.     

Hemopoietic growth factors regulate the survival of human basophils in vitro.

Human basophils were purified from normal peripheral blood, using density gradient followed by negative panning selection. We tested the effects of hemopoietic growth factors on the survival of these basophils in vitro. In the absence of exogenous factors, basophils (purity greater than 90%) decreased in number rapidly. At day 7 only 11% of the cells remained alive in cultures; less than 1% of cells survived at day 14. Interleukin (IL)-3 maintained numbers of viable cells; cell viability was 67% at day 7 and 45% at day 14. Granulocyte-macrophage (GM)-colony-stimulating factor (CSF) exhibited slight effect on the survival; 33% of cells remained at day 7. Other growth factors including granulocyte (G)-CSF, macrophage (M)-CSF, and IL-4 had no significant effect on the survival of basophils at all. Morphological and functional characterization of cells maintained by IL-3 revealed that they belonged to the basophil lineage. These observations indicate that normal basophils possess functional receptors for IL-3 and GM-CSF and that both factors modulate immediate- and delayed-type hypersensitivity reactions by prolonging the life span of basophils.     

Association of natural tooth loss with genetic variation at the human matrix Gla protein locus in elderly women

Natural tooth loss represents a major medical issue within the elderly population, since it impairs masticatory function critical for oral intake of essential nutrition. Contribution of genetic factors has been implicated in the determination of natural tooth loss; degree of reduction in number of natural teeth remaining intact (NTI) varies among individuals; thus, heterogeneity in NTI might reflect genetic variation within the population. One candidate gene, the matrix Gla protein gene (MGP), has been implicated in the pathogenesis of bone loss through a repression of bone/tooth formation. We have investigated a possible association between the CA repeat polymorphism at the human MGP gene locus and the NTI in 458 elderly Japanese women. In 916 chromosomes tested, ten alleles of the polymorphic nucleotide repeat were observed (designated A1–A10), among which five alleles were regarded as major alleles to be tested for the association. Twenty-seven women who possessed an A6 allele (164 bp) had significantly higher NTI than the remaining participants (n=431), who did not carry an allele of that size (mean: 10.0 teeth vs 5.6 teeth; P=0.007, Mann-Whitney test). An eight-year longitudinal follow-up study of NTI suggested that the genetic variations at the MGP locus did not affect the rate of tooth loss in the elderly period. These results suggest that genetic variation at the MGP gene locus is associated with some determinants for tooth loss in elderly women.