George W. Bernard

Ineffective phagocytosis of amyloid-β by macrophages of Alzheimer's disease patients

The defective clearance of amyloid-beta (Abeta) in the brain of Alzheimers disease (AD) patients is unexplained. The immunohistochemical studies of the frontal lobe and hippocampus show perivascular and intraplaque infiltration by blood-borne macrophages containing intracellular Abeta but only inefficient clearance of beta deposits. Neurons and neuronal nuclei, respectively, express interleukin-1beta and the chemokine RANTES, which could induce the inflammatory cell infiltration. To clarify the pathophysiology ofbeta clearance, we examined Abeta phagocytosis by monocytes and macrophages isolated from the blood of age-matched patients and controls. Control monocytes display excellent differentiation into macrophages and intracellular phagocytosis of Abeta followed by beta degradation or export. AD monocytes show poor differentiation and only surface uptake of Abeta and suffer apoptosis. HLA DR and cyclooxygenase-2 are abnormally expressed on neutrophils and monocytes of AD patients. AD patients have higher levels of intracellular cytokines compared to controls. Thus Abeta clearance is not restricted to brain microglia and involves systemic innate immune responses. In AD, however, macrophage phagocytosis is defective, which may elicit compensatory response by the adaptive immune system.

Innate immunity and transcription of MGAT-III and Toll-like receptors in Alzheimer's disease patients are improved by bisdemethoxycurcumin.

We have tested a hypothesis that the natural product curcuminoids, which has epidemiologic and experimental rationale for use in AD, may improve the innate immune system and increase amyloid-β (Aβ) clearance from the brain of patients with sporadic Alzheimers disease (AD). Macrophages of a majority of AD patients do not transport Aβ into endosomes and lysosomes, and AD monocytes do not efficiently clear Aβ from the sections of AD brain, although they phagocytize bacteria. In contrast, macrophages of normal subjects transport Aβ to endosomes and lysosomes, and monocytes of these subjects clear Aβ in AD brain sections. Upon Aβ stimulation, mononuclear cells of normal subjects up-regulate the transcription of β-1,4-mannosyl-glycoprotein 4-β-N-acetylglucosaminyltransferase (MGAT3) (P < 0.001) and other genes, including Toll like receptors (TLRs), whereas mononuclear cells of AD patients generally down-regulate these genes. Defective phagocytosis of Aβ may be related to down-regulation of MGAT3, as suggested by inhibition of phagocytosis by using MGAT3 siRNA and correlation analysis. Transcription of TLR3, bditTLR4, TLR5, bditTLR7, TLR8, TLR9, and TLR10 upon Aβ stimulation is severely depressed in mononuclear cells of AD patients in comparison to those of control subjects. In mononuclear cells of some AD patients, the curcuminoid compound bisdemethoxycurcumin may enhance defective phagocytosis of Aβ, the transcription of MGAT3 and TLRs, and the translation of TLR2–4. Thus, bisdemethoxycurcumin may correct immune defects of AD patients and provide a previously uncharacterized approach to AD immunotherapy.

Ultrastructural observations of initial calcification in dentine and enamel

An electron microscopic study of developing mandibular molars of Swiss-Webster mice indicates that the pattern of initial hydroxyapatite formation in dentine is identical with woven bone. Initial calcification loci are the cellular extensions or “buds” of odontoblasts into the predentine. Many of these cellular elements have intact plasma membranes when crystallization has just begun. These membranes disappear when calcification is more extensive. Hydroxyapatite growing radially from these initial calcification loci becomes spheroidal. These spheroids coalesce to form seams of the first formed dentine, mantle dentine. Later circumpulpal calcification develops from the previously formed territorial zone of calcified mantle dentine. When mantle dentine forms a continuous layer, the basement lamina underlying the ameloblasts disappears. Pre-enamel protein is secreted which almost immediately calcifies. The first enamel crystals are coextensive with hydroxyapatite of dentine at the dentino-enamel junction. The dentinal hydroxyapatitic territorial domain appears to be the source of secondary crystallization growth centers for enamel crystals.

Curcuminoids enhance amyloid-β uptake by macrophages of Alzheimer's disease patients

Treatment of Alzheimers disease (AD) is difficult due to ignorance of its pathogenesis. AD patients have defects in phagocytosis of amyloid-beta (1-42) (Abeta) in vitro by the innate immune cells, monocyte/macrophages and in clearance of Abeta plaques [5]. The natural product curcuminoids enhanced brain clearance of Abeta in animal models. We, therefore, treated macrophages of six AD patients and 3 controls by curcuminoids in vitro and measured Abeta uptake using fluorescence and confocal microscopy. At baseline, the intensity of Abeta uptake by AD macrophages was significantly lower in comparison to control macrophages and involved surface binding but no intracellular uptake. After treatment of macrophages with curcuminoids, Abeta uptake by macrophages of three of the six AD patients was significantly (P<0.001 to 0.081) increased. Confocal microscopy of AD macrophages responsive to curcuminoids showed surface binding in untreated macrophages but co-localization with phalloidin in an intracellular compartment after treatment. Immunomodulation of the innate immune system by curcuminoids might be a safe approach to immune clearance of amyloidosis in AD brain.

The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide.

Silverman and Kruger (Somatosens. Res. 5(2):157-175; 1987) reported that sensory nerve fibers of the dental pulp secrete calcitonin gene-related peptide (CGRP). These are localized exactly where secondary or tertiary dentin calcification occurs. Recently we found that CGRP has an osteogenic stimulating effect by increasing the number and size of bone colonies in vitro. The purpose of this study is to test whether there is a relationship between the effects of different doses of CGRP and bone colony numbers and/or size. Rat CGRP in different dosages (0.4, 4 and 40 micrograms/ml in BGJb medium) was added daily to 3 million light density (LD) bone marrow white cells which were harvested from adult Sprague-Dawley rats with the Ficoll-Paque density gradient separation method, then seeded onto a previously prepared feeder layer of fibroblasts in Petri dishes. Seven days after adding CGRP, in the controls without CGRP there were 2 bone colonies; with 0.4 microgram of CGRP there were 4 colonies; with 4 micrograms of CGRP there were 6 colonies; with 40 micrograms there were 9 colonies, indicating there was a significant increase in number of bone colonies with an increase in dose of CGRP between individual groups, respectively (p less than 0.0005 and p less than 0.0001). In another experiment, intravenous injection of 10 micrograms of rCGRP/kg body weight was performed two hours before surgery. LD bone marrow white cells were collected and seeded onto a feeder layer in Petri dishes exactly as described above.(ABSTRACT TRUNCATED AT 250 WORDS)

Phagocytosis of Amyloid-β and Inflammation: Two Faces of Innate Immunity in Alzheimer's Disease

Abstract Innate immunity provides the first line of defense by recognizing pathogen-associated microbial patterns and inducing key co-stimulatory molecules and cytokines, which activate the mechanisms of the adaptive immune response. Innate immune cells perform phagocytosis, which can clear pathogens and tissue waste products but may also contribute to tissue injury due to harmful side effects of inflammation. Genetic studies of APOE4, cytokine polymorphisms and overexpression of inflammatory genes suggest that chronic inflammation may have adverse effects in patients with sporadic Alzheimers disease. However, a vaccine against amyloid-beta induced beneficial clearance of amyloid-beta deposits, possibly through Fc receptor-mediated stimulation of microglial and macrophage uptake. A reconciliation of these two pathogenetic mechanisms is crucial to future progress in immune diagnosis and effective therapy. This may be possible by extending our recent observations suggesting that phagocytosis of amyloid-beta by macrophages is excellent in normal subjects but is deficient in most AD patients. Thus increased proinflammatory cytokine levels and activated microglia and macrophages in patients may be compensatory for defective clearance of amyloid-beta. Consequently, therapeutic interventions that increase phagocytosis of amyloid-beta might decrease brain inflammation as well as reduce inflammation-induced neurodegeneration. Finally, peripheral blood leukocytes are a superior model system for investigation of innate immune dysfunction in Alzheimers disease.

Neurogenic substance P stimulates osteogenesis in vitro.

Previous studies have shown that there is colocalization of substance P (SP) and calcitonin gene-related peptide (CGRP) immunoreactive nerve fibers in bone, periosteum and bone marrow. Because SP may also possibly play a role in bone formation, we decided to test whether it has an osteogenic stimulating effect on developing bone in vitro. To this end, 0.4, 4 and 40 micrograms/ml of SP in BGJb medium was added daily to 3 million light density (LD) bone marrow white cells which were separated by Ficoll-Paque density gradient separation then seeded onto a previously prepared fibroblast feeder layer in Petri dishes. Seven days after adding SP, in the control without SP there were 2 bone colonies; with 0.4 micrograms of SP there were 3 colonies; with 4 micrograms there were 5 colonies; with 40 micrograms there were 7 colonies. In addition, there was an increase in the size of bone colonies in the SP-added group. The results indicated that SP had a dose-related osteogenic stimulating effect. The increase in the number and size of bone colonies by SP was probably caused by stimulating stem cell mitosis, osteoprogenitor cell differentiation or osteoblastic activity.

The effect of hyaluronan on mouse intramembranous osteogenesis in vitro

Abstract Hyaluronan (HA) is an almost ubiquitous component of extracellular matrices. Early in embryogenesis mesenchymal cells migrate, proliferate and differentiate, in part, because of the influence of HA. Because many of the features of embryogenesis are revisited during wound repair, including bone fracture repair, this study was initiated to evaluate whether HA has an effect on calcification and bone formation in an in vitro system of osteogenesis. Enzyme-digested calvarial mesenchymal cells from 13-day-old mouse embryos were cultured in BGJb medium with rooster comb hyaluronan in seven different molecular weights (30, 40, 90, 160, 550, 660, and 1300 kDa). The dosages for each molecular weight were 0.5, 1.0 and 2.0 mg/ml. HA was added once to the medium at the plating of cells. After 10 days in culture, with low molecular weight hyaluronan (30 and 40 kDa) bone colonies were identifiable on a base of confluent fibroblasts. The number of colonies was larger than controls, particularly in the 1.0 and 2.0 mg/ml dosages of both 30 and 40 kDa of HA. Hyaluronan of high molecular weight, no matter what the dose, showed no significant bone colony formation, with apparent cell growth inhibition. Higher molecular weights were thereafter not included in this study. No statistically significant difference in the size of colonies was found when compared to controls in the 30 and 40 kDa bone colonies no matter what the dose.

The Ultrastructural Interface of Bone Crystals and Organic Matrix in Woven and Lamellar Endochondral Bone

Endochondral bone consists of three calcified regions: calcified cartilage, woven bone, and lamellar bone. Woven or immature bone begins when osteoblastic extrusions or buds calcify; from initial calcification loci, bone crystals grow radially to form spheroidal bone nodules. Nodules eventually coalesce into bone seams. Lamellar bone develops as a sequel to the calcification of cartilage or woven bone. Hydroxyapatite grows into lamellar bone and then is oriented parallel to collagen. Except for the stage of cartilage calcification, intramembranous and endochondral osteogenesis are similar.

Lamellar membrane encircled viruses in the erythrocytes of Rana pipiens.

In the erythrocytes of adult leopard frogs, viral particles were identified by the electron microscope. These viruses were identified in the inclusion seen previously by conventional blood staining methods in the light microscope. The viruses, whose nucleoid probably contained DNA, were usually polygonal, primarily hexagonal, rarely circular and consisted of two electron lucent zones enclosing an electron dense zone between them. In relation to the cytoplasm, the viruses exhibited an unusual lamellar phenomenon composed of stacked membranes.