Proteoglycans in human long-term bone marrow cultures: biochemical and ultrastructural analyses

Proteoglycans within the extracellular matrix of human bone marrow have been implicated in the process of hematopoiesis, but little is known about the structure and composition of these macromolecules in this tissue. Hematopoietically active human long-term bone marrow cultures were incubated with medium containing 35S-sulfate and 3H-glucosamine as labeling precursors. Proteoglycans present in the medium and cell layer were extracted with 4 mol/L guanidine HCI and purified by diethylaminoethyl (DEAE)-Sephacel ion exchange and molecular sieve chromatography. Both culture compartments contain a large chondroitin sulfate proteoglycan (MI, CI) that eluted in the void volume of a Sepharose CL-4B column and contained glycosaminoglycan chains of molecular weight (mol wt) approximately 38,000. A second population of sulfate-labeled material was identified as a broad heterogenous peak (MII, CII) that was included on Sepharose CL-4B at Kav = 0.31. This material when chromatographed on Sepharose CL-6B could be further separated into a void peak (MIIa, CIIa) and an included peak eluting at Kav = 0.39 (MIIb, CIIb). The void peaks (MIIa, CIIa) were susceptible to chondroitinase ABC digestion (99%) but slightly less susceptible to chondroitinase AC digestion (90%). Papain digestion of these peaks revealed them to be proteoglycans with glycosaminoglycan chains of mol wt approximately 38,000. The included peaks on Sepharose CL-6B (MIIb, CIIb) from both medium and cell layer compartments resisted digestion with papain, indicating the presence of glycosaminoglycan chains of mol wt approximately 38,000 either free or attached to a small peptide. Although this material was susceptible to chondroitinase ABC (98%), it was considerably less susceptible to chondrotinase AC (approximately 60%), indicating that it contained dermatan sulfate. A small amount of heparan sulfate proteoglycan was also identified but constituted only approximately 10% of the total sulfated proteoglycan extracted from these cultures. Additionally, approximately 40% of the incorporated 3H- activity radioactivity was present as hyaluronic acid. Electron microscopy revealed a layer of adherent cells covered by a mat containing ruthenium red-positive granules that were connected by thin filaments. The extracellular matrix layer above the adherent cells contained a mixture of hematopoietic cells. Chondroitinase ABC treatment of the cultures completely removed the ruthenium red-positive granules overlying the cells and resulted in a loss of approximately 70% of the 35S-sulfate-labeled material from the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blood cell dynamics in P-selectin-deficient mice

P-selectin is expressed on the surfaces of activated platelets and endothelium where it mediates binding to leukocytes. P-selectin- deficient mice were shown to exhibit peripheral neutrophilia (Mayadas et al: Cell 74:541, 1993). We now show that this is not caused by changes in bone marrow precursors nor by a lack of neutrophil margination. Both P-selectin-positive and -negative animals displayed similar increases in peripheral blood neutrophil numbers after injection of epinephrine. However, clearance of 51Chromium-labeled neutrophils is delayed in mice deficient for P-selectin, indicating that the neutrophilia is at least in part the result of delayed removal. We detected no obvious alterations in lymphocyte differentiation, distribution, or adhesion to high endothelial venules in peripheral lymph nodes. Through intravital microscopy, we examined the impact of P-selectin deficiency on leukocyte/endothelial interaction beyond the initial stages of inflammation. Four hours after the administration of an inflammatory irritant, leukocyte rolling was observed even in the absence of P-selectin. There were significantly fewer rolling cells relative to wild-type mice, and their velocity was reduced. Moreover, in the peritonitis model, the number of peritoneal macrophages in wild-type mice increased threefold at 48 hours, whereas the macrophages in the mutant mice remained near baseline levels. Thus, whereas P-selectin is known to be involved in early stages of an inflammatory response, our results indicate that it is additionally responsible for leukocyte rolling and macrophage recruitment in more prolonged tissue injury.     

Individual responses to novelty are associated with differences in behavioral and neurochemical profiles

Experimental animals can be differentiated on the basis of their horizontal or vertical activity to high responders (HR) and low responders (LR) upon exposure to a novel environment. These individual differences have been associated with behavioral and neurobiological differences in a number of experimental procedures used for studying sensitivity to psychostimulants, anxiety, depression, and cognitive function. In the present study, we differentiated the rats to HR and LR based on their vertical activity upon exposure to a novel environment. Additionally, we ascertained whether HR and LR rats differ in a battery of tests such as passive avoidance (PA), object recognition (OR), and the water-maze (WM) that provide indices for cognitive function and the forced swim test (FST), an animal model of affective responsivity and antidepressant-like activity. Potential differences in neurochemical indices between the two phenotypes were also examined. HR rats displayed impaired non-spatial object recognition memory, but enhanced spatial performance, as compared to LR rats. FST induced "depressive-like" symptoms in both phenotypes that were differently manifested in HR versus LR rats. Neurochemical findings revealed distinct differences in serotonergic and dopaminergic activity in the striatum and the prefrontal cortex of HR as compared to LR rats. The above results show that HR and LR rats exhibit important differences in a battery of tests related to cognitive performance or affective responsivity, which may be associated with differences in certain neurobiological parameters.     

Helicobacter pylori infection in hospital workers over a 5-year period: correlation with demographic and clinical parameters

Background: We aimed to determine whether any of various groups of medical and nonmedical staff in a large acute care hospital were at increased risk of acquiring Helicobacter pylori infection over a 5-year period, and we also aimed to identify risk factors or symptoms related to H. pylori positivity and seroconversion. Methods: A total number of 437 subjects, aged 36.8 ± 7.7 years (range, 23–60 years)—employees of our hospital—were tested by immunoassay for serum IgG antibodies against H. pylori. Subjects were assigned to four main groups: (I) nursing staff (n = 249; aged 34.7 ± 7 years); (II) administrative and technical staff (n = 127; aged 39.2 ± 8.1 years); (III) medical staff (n = 31; aged 42.4 ± 4.9 years); and (IV) paramedical staff (blood donor department) (n = 30; aged 37.6 ± 8.5 years). Differences in age and educational level between these four groups were statistically highly significant (P < 0.0001). Each subject completed a questionnaire containing several clinical and demographic parameters. The same cohort of individuals was tested 5 years later. Results: The overall seroprevalence of H. pylori infection was 45.5%, and in each group (I, II, III, and IV) being 48.6%, 44.1%, 41.9%, and 30% respectively. Logistic regression analysis revealed that the risk of infection by H. pylori was significantly higher in group I compared with group II (odds ratio [OR], 1.91; 95% confidence interval [CI], 1.04–3.52; P = 0.037). The H. pylori positivity increased with age: 40.6% for those aged 23–40 years and 57.5% for those aged 41–60 years (P = 0.001). The level of education was inversely associated with H. pylori infection (P = 0.001). During the 5-year observation, 59 of 238 (24.8%) subjects initially negative for H. pylori infection became positive, thus giving an annual seroconversion rate of 4.95%. Logistic regression analysis revealed that the seroconversion rate was significantly higher in group I compared with group II (28.1% vs 21.1%; OR, 2.34; 95% CI, 1.08–5.07; P = 0.03). The rate of seroconversion was higher in subjects aged 35–55 years compared with subjects aged 23–34 years (32% vs 17.5%; P = 0.009). Subjects who were positive for H. pylori infection in both examinations had a higher percentage of heartburn (P = 0.029), regurgitation (P = 0.023), and nausea (P = 0.037) compared with those who were negative in both examinations. Differences between those who were continuously negative for H. pylori infection and those who seroconverted during the observation period were not significant. Conclusions: In this longitudinal study of workers in a large acute care hospital in Greece it was found that nursing staff had a significantly higher risk of infection compared with administrative and technical staff. Age was significantly positively related both to H. pylori infection and to seroconversion. The level of education was strongly related to the prevalence, but not to the incidence of H. pylori infection. The presence of infection over the time was associated with a higher percentage of heartburn, regurgitation, and nausea compared with subjects who were continuously negative for H. pylori infection. Received: November 1, 2001 / Accepted: March 8, 2002     

A detailed behavioral analysis of the acute motor effects of caffeine in the rat: involvement of adenosine A<Subscript>1</Subscript> and A<Subscript>2A</Subscript> receptors

Rationale There is no consensus on the contribution of adenosine A₁ and A_2A receptor blockade to motor-activating effects of caffeine.Objective Our aim was to use a detailed and continuous observational method to compare the motor effects induced by caffeine with those induced by selective A₁ and A_2A receptor antagonists.Methods The behavioral repertoire induced by systemic administration of caffeine (3, 10, and 30 mg/kg), A₁ receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 1.2, 4.8 and 7.2 mg/kg), and A_2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium salt (MSX-3; 1, 3, and 10 mg/kg) was analyzed. The effects of pretreatment with the selective A₁ receptor agonist N ⁶-cyclopentyladenosine (CPA; 0.1 mg/g) and the selective A_2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxyamidoadenosine (CGS 21680; 0.2 mg/kg) on the pattern of motor activation induced by caffeine, CPT, or MSX-3 were also examined.Results The pattern of behavioral activation induced by caffeine was better mimicked by CPT than by MSX-3. Coadministration of CPT and MSX-3 gave different results depending on the dose and the type of behavioral response. CPA was more effective at decreasing the activating effects of caffeine and CPT than those of CGS 21680. On the other hand, CGS 21680 was more effective at decreasing the activating effects of MSX-3 than those of caffeine or CPT. Factor analysis revealed a complex three-dimensional behavioral profile for caffeine that was similar to the profile for CPT and was different from the profile for MSX-3.Conclusions The results indicate a predominant role for A₁ receptors in the motor-activating effects of acutely administered caffeine.