John H. H. Williams

Homocysteine and cognitive decline in healthy elderly.

Serum homocysteine is increased, and correlates inversely with cognitive scores, in Alzheimer’s disease (AD), vascular dementia and ‘age-associated memory impairment’. Elevated levels might signal accelerated cognitive decline, although this remains to be established. We therefore repeated Mini-Mental State Examinations, together with additional ADAS-Cog assessments, in 32 healthy elderly individuals to determine whether prior homocysteine levels predicted cognitive changes over a 5-year period. Homocysteine predicted follow-up cognitive scores and rate of decline in cognitive performance independently of age, sex, education, renal function, vitamin B status, smoking and hypertension (p < 0.001). Homocysteine predicted word recall (p = 0.01), orientation (p = 0.02) and constructional praxis scores (p < 0.0001). One subject, with the second highest initial homocysteine, had developed probable AD at follow-up. Fasting total serum homocysteine appears to be an independent predictor of cognitive decline in healthy elderly and exerts a maximal effect on spatial copying skills.

A novel role for vitamin B12: Cobalamins are intracellular antioxidants in vitro

Oxidative stress is a feature of many chronic inflammatory diseases. Such diseases are associated with up-regulation of a vitamin B(12) (cobalamin) blood transport protein and its membrane receptor, suggesting a link between cobalamin and the cellular response to inflammation. The ability of cobalamin to regulate inflammatory cytokines suggests that it may have antioxidative properties. Here we show that cobalamins, including the novel thiolatocobalamins N-acetyl-l-cysteinylcobalamin and glutathionylcobalamin, are remarkably effective antioxidants in vitro. We also show that thiolatocobalamins have superior efficacy compared with other cobalamin forms, other cobalamins in combination with N-acetyl-l-cysteine (NAC) or glutathione (GSH), and NAC or GSH alone. Pretreatment of Sk-Hep-1 cells with thiolatocobalamins afforded robust protection (>90% cell survival) against exposure to 30 microM concentrations of the pro-oxidants homocysteine and hydrogen peroxide. The compounds inhibited intracellular peroxide production, maintained intracellular glutathione levels, and prevented apoptotic and necrotic cell death. Moreover, thiolatocobalamins are remarkably nontoxic in vitro at supraphysiological concentrations (>2 mM). Our results demonstrate that thiolatocobalamins act as powerful but benign antioxidants at pharmacological concentrations. Because inflammatory oxidative stress is a component of many conditions, including atherosclerosis, dementia, and trauma, their utility in treating such disorders merits further investigation.

Thermoregulation in rats: opposing effects of thyrotropin releasing hormone and its metabolite histidyl-proline diketopiperazine.

Abstract Intraventricular administration of histidyl-proline diketopiperazine to rats produces a dose-dependent hypothermia at 4° or 24°, but not at 31°. At 4°, administration of thyrotropin releasing hormone elicits a dose-dependent hypothermia up to 0.1 μmole/kg which is not evoked at higher doses. At 24°, thyrotropin releasing hormone administration results in no change in core temperature, whereas it induces hyperthermia at 31°. At 4°, thyrotropin releasing hormone antagonizes and thyrotropin releasing hormone antiserum potentiates the hypothermic effect of histidyl-proline diketopiperazine, suggesting opposing actions of thyrotropin releasing hormone and histidyl-proline diketopiperazine on thermoregulation.

Evaluation of heat shock protein 70 as a biomarker of environmental stress in Fucus serratus and Lemna minor

Heat shock proteins (Hsps) are known to be induced in response to short-term stress. The present study aimed to evaluate the potential of Hsp70 as a biomarker of stress produced by increased temperature, osmotic pressure, and exposure to cadmium and sodium chloride in marine macroalgae and fresh water plant species. An indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) was developed with a working range of 0.025–10 μg ml−1 using a monoclonal antibody raised against purified Hsp70 of Phaseolus aureus (mung bean). Fucus serratus (toothed wrack), Chondrus crispus (Stackhouse or Carrageen moss), Ulva lactuca (sea lettuce) and Lemna minor (common duckweed) sample extracts were stressed for up to 24 h and then tested in the IC-ELISA. The presence of Hsp70 and cross-reactivity of the monoclonal antibody was confirmed by Western blot. The heat shock response was confirmed in each species using a 2-h 42°C treatment. Following heat shock, Hsp70 concentrations increased to a peak at 2 h (F. serratus) or 4 h (L. minor), after which concentrations decreased. Osmotic and cadmium stresses also resulted in elevated Hsp70 concentrations in samples of F. serratus and L. minor when compared with unstressed controls. In both, osmotic and metal stress, the production of Hsp70 increased to a maximum and subsequently decreased as the stressor levels increased. Results suggest that Hsp70 IC-ELISA could potentially be applied to the detection of stress in these aquatic species, although it would probably be most effective when used in conjunction with other measurements to provide a stressor-specific biomarker profile or fingerprint.

Vitamin B12 and Redox Homeostasis: Cob(II)alamin Reacts with Superoxide at Rates Approaching Superoxide Dismutase (SOD)

We report a kinetic study of the reaction between superoxide and an important intracellular form of vitamin B(12), cob(II)alamin. Superoxide is implicated in the pathophysiology of many inflammatory diseases, whereas vitamin B(12) derivatives are often beneficial in their treatment. We found that cob(II)alamin reacts with superoxide at rates approaching those of superoxide dismutase itself, suggesting a probable mechanism by which vitamin B(12) protects against chronic inflammation and modulates redox homeostasis.

Heat shock proteins form part of a danger signal cascade in response to lipopolysaccharide and GroEL.

An increasing number of cell types, including peripheral blood mononuclear cells (PBMCs), have been demonstrated to release heat shock proteins (Hsps). In this paper we investigate further the hypothesis that Hsps are danger signals. PBMCs and Jurkat cells released Hsp70 (0·22 and 0·7 ng/106 cells, respectively) into medium over 24 h at 37°C. Release of Hsp70 was stimulated 10‐fold by GroEL (P < 0·001) and more than threefold by lipopolysaccharide (LPS) (P < 0·001). Although Hsp60 could be detected in the medium of cells cultured at 37°C for 24 h, the low rates of release were due probably to cell damage. Significant release of Hsp60 was observed when Jurkat cells were exposed to GroEL (2·88 ng/106 cells) or LPS (1·40 ng/106 cells). The data are consistent with the hypothesis that Hsp70 and Hsp60 are part of a danger signalling cascade in response to bacterial infection.

Osteoprotegerin is produced when prostaglandin synthesis is inhibited causing osteoclasts to detach from the surface of mouse parietal bone and attach to the endocranial membrane.

Osteoclast differentiation and activation is controlled, at least in part, by the counterbalancing influences of osteoprotegerin ligand (OPGL) and osteoprotegerin (OPG). Nonsteroidal anti-inflammatory drugs have been shown to inhibit bone loss in vivo and bone resorption in vitro, and this is associated with a loss of osteoclasts from the bone surface. We test the hypothesis that OPG mediates the inhibition of osteoclast activity that occurs with indomethacin in the mouse calvaria. Recombinant human OPG, like indomethacin, was found to cause osteoclasts to detach from the bone surface and attach to the adjacent endocranial membrane (periosteum). Recombinant human OPG also inhibited the stimulatory effect of prostaglandin E2 (PGE2), parathyroid hormone (PTH), and 1,25-dihydroxyvitamin D3 (1,25D3) on osteoclast adhesion to bone after an incubation with indomethacin. A function-blocking antibody to OPG and soluble human OPGL both inhibited the effect of indomethacin, leaving active osteoclasts on the bone. OPG activity was detected in the culture medium from indomethacin-treated bones and PTH, PGE2, 1,25D3, and dexamethasone all inhibited the production of OPG activity. We conclude that, in the absence of specific stimulators of bone resorption, OPG is produced by the mouse calvaria in vitro, which inhibits bone resorption by causing osteoclasts to detach from the bone surface.

Sensing danger—Hsp72 and HMGB1 as candidate signals

Molecules that behave as danger signals are produced when the body is perceived to be under attack, and they alert the immune system to the problem. The immune system can then mount an appropriate response. Two molecules that have received attention as potential danger signals are heat shock protein 72 (Hsp72) and high mobility group box 1 (HMGB1), which are intracellular proteins but are released when cells are under stress, in particular, when necrosis occurs. This review considers the similarities between these two molecules and then contrasts their mechanism of action and problems that can arise when they are overpresented in the extracellular environment. It is proposed that Hsp72 and HMGB1 are members of a suite of danger molecules that provide a fingerprint of the threat, or stressor, to tissue or organism integrity.

Antagonism of ethanol-induced decrease in rat brain cGMP concentration by histidyl-proline diketopiperazine, a thyrotropin releasing hormone metabolite.

Abstract Intraperitoneal administration of thyrotropin releasing hormone (50 μmol/kg) produced an approximately 2-fold increase in rat brain cGMP concentration within 15 min. Histidyl-proline diketopiperazine, a metabolite of thyrotropin releasing hormone, produced a similar effect, but the response was faster and shorter-lasting. Intraperitoneal administration of ethanol (1.5 g/kg) decreased brain cGMP concentration approximately 50% within 10–15 min; thyrotropin releasing hormone or histidyl-proline diketopiperazine, injected 5 min after ethanol, antagonized the ethanol-induced decrease in cGMP. Antagonism of the ethanol-induced decrease in the cGMP level required 10 μmol/kg of thyrotropin releasing hormone but was observed with 5 μmol/kg of histidyl-proline diketopiperazine. These data suggest that the metabolic conversion of thyrotropin releasing hormone to histidylproline diketopiperazine might explain the previous observation that thyrotropin releasing hormone elevated the level of brain cGMP and antagonized the ethanolinduced decrease in brain cGMP concentration.

Monocytes/macrophages express chemokine receptor CCR9 in rheumatoid arthritis and CCL25 stimulates their differentiation

IntroductionMonocytes/macrophages accumulate in the rheumatoid (RA) synovium where they play a central role in inflammation and joint destruction. Identification of molecules involved in their accumulation and differentiation is important to inform therapeutic strategies. This study investigated the expression and function of chemokine receptor CCR9 in the peripheral blood (PB) and synovium of RA, non-RA patients and healthy volunteers.MethodsCCR9 expression on PB monocytes/macrophages was analysed by flow cytometry and in synovium by immunofluorescence. Chemokine receptor CCR9 mRNA expression was examined in RA and non-RA synovium, monocytes/macrophages from PB and synovial fluid (SF) of RA patients and PB of healthy donors using the reverse transcription polymerase chain reaction (RT-PCR). Monocyte differentiation and chemotaxis to chemokine ligand 25 (CCL25)/TECK were used to study CCR9 function.ResultsCCR9 was expressed by PB monocytes/macrophages in RA and healthy donors, and increased in RA. In RA and non-RA synovia, CCR9 co-localised with cluster of differentiation 14+ (CD14+) and cluster of differentiation 68+ (CD68+) macrophages, and was more abundant in RA synovium. CCR9 mRNA was detected in the synovia of all RA patients and in some non-RA controls, and monocytes/macrophages from PB and SF of RA and healthy controls. CCL25 was detected in RA and non-RA synovia where it co-localised with CD14+ and CD68+ cells. Tumour necrosis factor alpha (TNFα) increased CCR9 expression on human acute monocytic leukemia cell line THP-1 monocytic cells. CCL25 induced a stronger monocyte differentiation in RA compared to healthy donors. CCL25 induced significant chemotaxis of PB monocytes but not consistently among individuals.ConclusionsCCR9 expression by monocytes is increased in RA. CCL25 may be involved in the differentiation of monocytes to macrophages particularly in RA.