The allergy-associated chemokine receptors CCR3 and CCR5 can be inactivated by the modified chemokine NNY-CCL11.

BACKGROUND: CC chemokine ligand 11 (CCL11) is the outstanding member of all described CC chemokine receptor 3 (CCR3) ligands and is shown to be selective for this receptor. However, it also activates CCR5 but only in the micromolar range. The in vivo activity of CCL11 is expected to be temporally restricted, as it is degraded by specific proteases such as the dipeptidyl-peptidase IV (DP4), also termed CD26. Based on the approach to inactivate chemokine receptors in allergic disease models as has been demonstrated for DP4-resistant n-nonanoyl (NNY)-CCL14 and for amino-oxypentane (AOP)-CCL5, it is tempting to study similar compounds derived from CCL11. METHODS: Synthesis of NNY-CCL11 was performed and it was characterized for biological functions in human and mouse eosinophils as well as in cell lines stably transfected either with human CCR3 or CCR5. Resistance to DP4 treatment was also investigated. RESULTS: The functional activities of NNY-CCL11 mediated via CCR3 show an almost identical pattern to CCL11 with respect to intracellular calcium mobilization and CCR3 internalization. N-terminal cleavage of CCL11 by preincubation with DP4 results in a reduced capacity to internalize CCR3, while preincubation of NNY-CCL11 shows no influence. In contrast to CCL11, NNY-CCL11 also activates CCR5+ cell lines and human monocytes in the nanomolar range, being about 100 times more potent than CCL11. CONCLUSIONS: n-Nonanoyl-CCL11 represents a compound with dual activity restricted to CCR3 and CCR5. Because of its receptor-inactivating capacity and stability against DP4 degradation, NNY-CCL11 is a suitable tool for the decoding of the pathophysiological mechanisms of allergic diseases.

Differential regulation of human platelet responses by cGMP inhibited and stimulated cAMP phosphodiesterases.

Platelets contain two cAMP phosphodiesterases (PDEs) which regulate intracellular cAMP levels, cGMP-inhibited cAMP PDE (PDE3A) and cGMP-stimulated PDE (PDE2A). Using the PDE3 inhibitor, milrinone and the PDE2 inhibitor, erythro-9-(2-hydroxyl-3-nonyl)adenine (EHNA), we have explored the contribution of each PDE to the regulation of platelet function. Inhibition of PDE2 resulted in higher levels of intracellular cAMP than inhibition of PDE3A suggesting this PDE may be the more important regulator of cAMP in human platelets. However, a concentration-dependent inhibition of agonist-induced aggregation was observed with milrinone while little effect was seen with EHNA. In addition, we observed a concentration-dependent inhibition in the increase of intracellular Ca2+ with PDE3 inhibition and significantly less with PDE2 inhibition. PDE3 inhibition also resulted in a concentration-dependent increase in cAMP-mediated phosphorylation of the vasodilator-stimulated phospho-protein (VASP) whereas there was no significant increase with PDE2 inhibition. In each of these experiments, synergism was noted with the combination of milrinone and EHNA. These results suggest that cAMP pools may be localized and the various PDEs regulate specific pools. These data also suggest that inhibitors of PDE3A may be more effective antiplatelet agents.

Trace and delay eyeblink conditioning: contrasting phenomena of declarative and nondeclarative memory.

We tested the proposal that trace and delay eyeblink conditioning aref fundamentally different kinds of learning. Strings of one, two, three, or four trials with the conditioned stimulus (CS) alone and strings of one, two, three, or four trials with paired presentations of both the CS and the unconditioned stimulus (US) occurred in such a way that the probability of a US was independent of string length. Before each trial, participants predicted the likelihood of the US on the next trial. During both delay (n = 20) and trace (n = 18) conditioning, participants exhibited high expectation of the US following strings of CS-alone trials and low expectation of the US following strings of CS-US trials--a phenomenon known as the gambler's fallacy. During delay conditioning, conditioned responses (CRs) were not influenced by expectancy but by the associative strength of the CS and US. Thus, CR probability was high following a string of CS-US trials and low following a string of CS-alone trials. The results for trace conditioning were opposite. CR probability was high when expectancy of the US was high and low when expectancy of the US was low: The results show that trace and delay eyeblink conditioning are fundamentally different phenomena. We consider how the findings can be understood in terms of the declarative and nondeclarative memory systems that support eyeblink classical conditioning.

Single-cue delay eyeblink conditioning is unrelated to awareness.

We examined the importance of awareness for eyeblink conditioning by directly comparing single-cue delay eyeblink conditioning and single-cue trace eyeblink conditioning. During single-cue delay conditioning, participants who became aware of the stimulus contingencies early in the conditioning session conditioned no better than those who became aware later in the session or did not become aware. Thus, the level of awareness was unrelated to the overall level of conditioning across the session. In contrast, awareness of the stimulus contingencies early in the session predicted the success of single-cue trace conditioning. These data, together with earlier findings, show that awareness is irrelevant to single-cue delay eyeblink conditioning but is critical for single-cue trace eyeblink conditioning. The findings from the present study are related to previous findings for differential (CS+ and CS-) eyeblink conditioning and awareness.

Perceptual learning, awareness, and the hippocampus.

Declarative memory depends on the hippocampus and related medial temporal lobe and diencephalic structures. Declarative memory has usually been found to be available to conscious recollection. A recent study (Chun and Phelps, Nat Neurosci 1999;2:844-847) found that damage to the medial temporal lobe (including the hippocampus) impaired performance on a perceptual learning task, yet the learning was accomplished in the absence of memory for the stimuli. This finding raised the possibility that some hippocampus-dependent tasks may be inaccessible to awareness and may be performed without evoking conscious memory processes. Using the same task, we show that when damage is confined largely to the hippocampal formation, perceptual learning is intact. Thus, the available data suggest that damage limited to the hippocampal formation does not impair nonconscious (nondeclarative) memory. Further, the data do not contradict the idea that hippocampus dependent memory is accessible to conscious recollection. Finally, perceptual learning was impaired in patients, with extensive damage to the medial temporal lobe and with additional variable damage to lateral temporal cortex.

Residues F16-G33 and A784-N823 within platelet thrombospondin-1 play a major role in binding human neutrophils: evaluation by two novel binding assays.

The thrombospondin-1 (TSP1) structural requirements within its heparin-binding domain (HBD)(30 kd) or within the other domains of the molecule (450 kd) that interact with neutrophils (PMNs) have not been delineated. Synthetic peptides based on the HBD, a TSP1 proteolytic fragment lacking the HBD, a large C-terminal domain of TSP1 (210 kd), a TSP1 recombinant fragment (rTSP1(784-932)), and a monoclonal antibody directed against the TSP1 type 3 repeats (mAb D4.6) were utilized to map such structural requirements on TSP1. Synthetic peptides containing a heparin-binding motif and encompassing residues F16-G33 or A74-S95 of TSP1 competed quantitatively with iodine 125-labeled TSP1 for binding to heparinagarose beads. However, only F16-G33 was a competitor of TSP1 binding to PMNs, suggesting that the sequence F16-G33 within the HBD plays a role in PMN binding. The interaction site within the 450-kd fragment was further narrowed. A TSP1 -derived proteolytic fragment (210 kd), a recombinant TSP1 fragment (rTSP1(784-932)), and a type 3 repeat anti-TSP1 monoclonal antibody (mAb D4.6) competed for the binding of 125I-labeled TSP1 to PMNs. The N-terminal of rTSP1(784-932) and C-terminal sequence analysis of TSP1-210 kd delineated the structural requirements for the second binding region for PMNs-namely, residues A784-N823.

The visual paired-comparison task as a measure of declarative memory.

Performance on the visual paired-comparison task depends on the integrity of the hippocampal formation in humans, monkeys, and, for an analogous task, in rats. The present study sought additional evidence in healthy volunteers concerning the nature of this task. We found that performance on the visual paired-comparison task was predictive of subsequent recognition memory performance whereas perceptual priming was unrelated to subsequent recognition memory performance. The results are consistent with the data from lesions and suggest that performance on the visual paired-comparison task measures a form of declarative memory.

Parallel acquisition of awareness and trace eyeblink classical conditioning.

Trace eyeblink conditioning (with a trace interval >/=500 msec) depends on the integrity of the hippocampus and requires that participants develop awareness of the stimulus contingencies (i.e., awareness that the conditioned stimulus [CS] predicts the unconditioned stimulus [US]). Previous investigations of the relationship between trace eyeblink conditioning and awareness of the stimulus contingencies have manipulated awareness or have assessed awareness at fixed intervals during and after the conditioning session. In this study, we tracked the development of knowledge about the stimulus contingencies trial by trial by asking participants to try to predict either the onset of the US or the onset of their eyeblinks during differential trace eyeblink conditioning. Asking participants to predict their eyeblinks inhibited both the acquisition of awareness and eyeblink conditioning. In contrast, asking participants to predict the onset of the US promoted awareness and facilitated conditioning. Acquisition of knowledge about the stimulus contingencies and acquisition of differential trace eyeblink conditioning developed approximately in parallel (i.e., concurrently).

Awareness predicts the magnitude of single-cue trace eyeblink conditioning.

Studies of differential eyeblink conditioning (CS+ and CS-) have demonstrated that successful conditioning requires awareness of the stimulus contingencies and that delay conditioning does not. Two experiments were carried out to determine whether awareness is also important for single-cue trace eyeblink conditioning. In experiment 1, participants who performed a secondary, attention-demanding task emitted significantly fewer conditioned eyeblink responses than participants who watched a silent movie during the conditioning session. In experiment 2, participants who became aware of the stimulus contingencies early in the conditioning session emitted significantly more conditioned responses during the remainder of the session than participants who became aware later in the session or who never became aware. These results indicate that awareness is important for single-cue trace eyeblink conditioning, just as it is for differential trace conditioning. The relationship between awareness and trace eyeblink conditioning is discussed in the light of these and other recent findings.

Impaired recognition memory on the Doors and People Test after damage limited to the hippocampal region.

There have been conflicting reports about the importance of the hippocampal region for recognition memory. Vargha-Khadem et al. (1997) described three patients who became amnesic early in life as a result of damage apparently limited to the hippocampal region. One of these patients (Jon) performed normally on the recognition portion of the Doors and People Test but was severely impaired in recall. To compare adult-onset amnesia directly with these early-onset cases, we tested six amnesic patients on the Doors and People Test. Three of the patients have damage thought to be limited to the hippocampal region. All six patients were markedly impaired on both the recall and recognition portions of the test. To account for the difference between our adult-onset cases and the early-onset case (Jon), we suggest that some compensation for Jon's injury occurred during development, either due to functional reorganization of cortex adjacent to the hippocampus or as the result of learned strategies.

Optimised separation of E- and Z- isomers of tamoxifen, and its principal metabolites using reversed-phase high performance liquid chromatography.

A reversed phase isocratic high-performance liquid chromatographic method is reported in which a formal structured procedure, the solvent selectivity triangle, was applied to predict the mobile phase composition giving baseline resolution of the clinically important triphenylethylene antioestrogenic agent (Z)-tamoxifen, its principal (Z)-metabolites, and also the clinically relevant (E)-geometric isomers of tamoxifen and 4-hydroxytamoxifen. The technique of solvent selectivity triangle was used to select the optimal organic modifier parameter for use with a Hichrom ODS 1 column, to achieve baseline separation of six triphenylethylene solutes. The detection system utilised post-column ultraviolet irradiation to convert solutes into their respective photocyclisation products, followed by fluorescence detection (lambda[ex] = 254 nm, lambda[em] = 360 nm), and the low detection limit for tamoxifen in serum of 0.1 microM. The optimal mobile phase composition was determined to be methanol-acetonitrile-water-trichloroacetic acid (50:31:18.9:0.1, v/v, pH 2.9). A single stage liquid-liquid extraction method for determination of triphenylethylene drugs in serum was developed. Reproducible recoveries for the (Z)-geometric isomers of tamoxifen (84 +/- 3%) and its principal metabolites including Metabolite Y (94 +/- 3%), N-desmethyltamoxifen (94 +/- 3%) and 4-hydroxytamoxifen (92 +/- 3%) were achieved, though more variable results were obtained for their corresponding (E)-geometric isomers (71 +/- 7% and 70 +/- 10%, respectively).

Patient awareness of genetic and environmental risk factors in non-insulin-dependent diabetes mellitus--relevance to first-degree relatives.

Primary preventative strategies may be useful in non-insulin-dependent (Type 2) diabetes mellitus (NIDDM), where weight reduction and physical exercise may help to counteract the increased risk to first-degree relatives of affected patients. To assess the extent of patient's awareness of these and related issues, 100 NIDDM patients were selected to complete a questionnaire. Forty-four per cent had a family history of NIDDM. Forty per cent and 35%, respectively, were aware that the patient's siblings and children were at an increased risk of developing NIDDM. Sixty-three per cent and 35%, respectively, were aware that obesity and physical inactivity increased the risk of developing NIDDM. After appropriate counselling about these risk factors, all 100 patients claimed that they would be advising their first-degree relatives. However, at 4 months review, 53% of patients had informed their first-degree relatives of the increased genetic risk to them. Of these patients, 83%, 74%, and 23% had provided relevant advice on diet, weight reduction, and physical exercise, respectively, as a means of reducing the risk of first-degree relatives developing NIDDM. We conclude that only a small proportion of patients are aware of the increased genetic susceptibility of their first-degree relatives to develop NIDDM and that weight reduction and regular physical exercise can reduce this risk. But, after appropriate education, a significant proportion of NIDDM patients did inform their close relatives of these issues and this may prove to be a useful strategy in the primary prevention of NIDDM.

Comparaçäo das propriedades mecânicas das hastes femorais bloqueadas AO-ASIF-FMRP: parte II - hastes implantadas em fêmures humanos "in vitro" / Comparison of the mechanical properties of AO-ASIF and FMRP blockaded femoral nails: part II - nails implanted in human femora \"in vitro\"

Nesta segunda parte, foi analisado o comportamento mecânico de hastes AO e FMRP femorais bloqueadas de l2mm de diâmetro e 400mm de comprimento, implantadas em dez pares de fêmures humanos frescos. Foi ressecado segmento diafisário extenso, resultando em um fragmento proximal de l2cm, um distal de l4cm e um gap entre os dois de l6cm. As hastes foram travadas inicialmente de forma usual.Foram feitos testes de inclinaçao da baste dentro dos fragmentos distal e proximal, nos sentidos ântero-posterior e medial-lateral e testes de carga axial excêntrica. Os espécimes foram analisados em máquina universal de testes. Os testes de inclinaçao mostraram superioridade estatisticamente significante dos espécimes FMRP da fixaçao no fragmento proximal e uma forte tendência à melhor fixaçao também no fragmento distal. Para tornar semelhante a distância entre os parafusos de travamento distais das bastes FMRP e AO, os parafusos dos espécimes FMRP foram mudados da posiçao convencional, o que enfraqueceu significantemente a fixaçao nas inclinaçoes, indicando a importância da inserçao em posiçao afastada dos dois parafusos distais de travamento no sistema FMRP. O segmento distal foi adicionalmente encurtado para 12 e 10cm, o que nao modificou de forma importante a fixaçao analisada nos testes de inclinaçao. O fato foi imputado ao já avantajado canal medular do fragmento distal antes da ressecçao. A fixaçao dependia, entao, só dos parafusos distais de bloqueio e nao do apoio cortical da diáfise. Os testes de carga axial excêntrica mostraram nítida superioridade do sistema FMRP em todos os espécimes, os quais sofreram deformaçao significantemente menor e resistiram à carga máxima que o sistema AO. Relevância clínica - Ernbora exista a possibilidade do uso de hastes mais calibrosas no sistema AO, para hastes de l2mm de diâmetro o comportamento mecânico é nitidamente superior no sistema FMRP. Os dados obtidos referendam o uso clínico do sistema FMRP, a julgar pelo lado da resistência mecânica. Estes resultados encorajam o uso clínico do sistema FMRP, a julgar pelo lado da resistência mecânica.

Production of a hemolytic factor by Candida albicans.

Candida albicans exhibits hemolytic activity when grown on glucose-enriched blood agar. This activity is present on intact organisms, and it is secreted into the culture medium. Hemoglobin released from lysed erythrocytes can restore the transferrin-inhibited growth of C. albicans. We conclude that C. albicans expresses a hemolytic factor which allows it to acquire iron from host erythrocytes.

Insulin-like growth factor I in follicular development and function in postpartum beef cows.

The objective of this study was to determine whether nutrition affects follicular growth and(or) steroid and insulin-like growth factor I (IGF-I) concentrations in follicular fluid. Beginning 6 d after calving, Hereford-cross cows (n = 28) were fed either 14 (ad libitum) or 7 (restricted) kg.animal1.d-1 of chopped alfalfa-brome hay. Half the cows in each treatment were ovariectomized on d 20 (OVX-20) and the remaining half on d 35 (OVX-35) postpartum. Cow weight and condition score were recorded weekly, and blood was collected thrice weekly for determination of insulin, IGF-I, glucose, and free fatty acid (FFA) concentrations. At ovariectomy, follicular fluid from each follicle greater than or equal to 4 mm in diameter was aspirated for determination of IGF-I, progesterone (P4), and estradiol-17 beta (E2) concentrations. Restricted cows lost more weight after calving than did ad libitum cows (P less than .0001), although all cows lost similar amounts of body condition (time postpartum, P = .008). Concentrations of FFA were elevated (P less than .0001) in restricted cows from wk 2 through 5 after calving but did not change with time in ad libitum cows. Plasma concentrations of glucose were lower in restricted than in ad libitum cows (59.6 +/- .4 vs 61.8 +/- .4 mg/dl; P = .05), but insulin and IGF-I were similar (P greater than .10) between dietary treatments.(ABSTRACT TRUNCATED AT 250 WORDS)

Relapsing polychondritis: bone marrow and circular fibrous nodules in the aorta.

A case is reported of extensive aortic calcification, bone formation, and haemopoietic tissue in a woman with relapsing polychondritis. An additional feature was 'clock face' nodules of collagen in the aorta.

Effects of passive immunization of growing guinea-pigs with an insulin-like growth factor-I monoclonal antibody.

The physiological importance of circulating as opposed to locally produced insulin-like growth factor-I (IGF-I) has not been determined. By using a passive immunoneutralization technique, our objectives were to evaluate the role of circulating IGF-I in the regulation of animal growth and pituitary GH content. A monoclonal antibody (MAb) to IGF-I, generated in our laboratory, has an affinity (Ka) of 0.13 litres/pmol for recombinant human IGF-I (rhIGF-I). Cross-reactivities of recombinant des-tripeptide IGF-I and recombinant bovine IGF-II were approximately 40 and 8% respectively. This MAb inhibited binding of purified hIGF-I to human placental membranes. In a radioimmunoassay based on displacement of 125I-labelled rhIGF-I from the MAb, displacement curves generated with dilutions of acid-gel chromatography extracts of guinea-pig serum and rhIGF-I standards were parallel. Twenty-four, 3-week-old male guinea-pigs were treated with the IGF-I MAb, a bovine herpes virus-I (BHV-I) MAb (control MAb) or vehicle (phosphate-buffered saline) (n = 8 per group). Treatments were administered i.p. every 3 days for 24 days at a dose of 20 mg/kg body weight. Blood was obtained on day 23 (48 h after treatment) and on day 25 (24 h after treatment). In a liquid-phase assay, serum from the IGF-I MAb-treated group bound 38 +/- 8% (mean +/- S.E.M.) (day 23) and 56 +/- 7% (day 25) of an 125I-labelled rhIGF-I trace at a final dilution of 1:10,000. Because of the development of an anti-mouse immune response in the guinea-pigs, these parameters would probably have been much greater during the first 2 weeks of the trial. Of the total IGF-I in serum, 50 +/- 5% and 61 +/- 4% could be immunoprecipitated with an excess of rabbit anti-mouse immunoglobulin in samples from days 23 and 25 respectively. Comparisons between the groups treated with IGF-I MAb and BHV-I MAb revealed no significant differences in whole animal growth rate, growth of individual tissues, or pituitary GH content. Mean serum concentrations of IGF-I were 69 and 99% greater in the IGF-I MAb-treated group than in the BHV-I MAb-treated group on days 23 and 25 respectively. These differences probably resulted from an extension of the half-life of IGF-I in serum of animals treated with the IGF-I MAb.(ABSTRACT TRUNCATED AT 400 WORDS)

Serum concentrations of IGF-I in postpartum beef cows.

Four experiments assessed changes in serum IGF-I under various physiologic conditions in postpartum cows. In Exp. 1, anestrous suckled cows (n = 25) were infused for 6 d with either saline or glucose at two different infusion rates. In Exp. 2, anestrous cows (n = 29) received either a saline (weaned and suckled controls) or 3 g/d phlorizin (weaned phlorizin) infusion for 3 d. Calves from the weaned groups were removed from 15 h before and throughout infusions. In Exp. 3, cycling suckled cows (n = 20) received prostaglandin F2 alpha (PGF2 alpha) when the 5-d saline or phlorizin infusion began. In Exp. 4, suckled cows (n = 20) had ad libitum access to feed or received 50% of control feed consumption from 30 to 40 d postpartum. Increasing glucose availability (Exp. 1) increased (P less than .05) serum IGF-I by 30 to 35%. IGF-I remained stable after weaning (Exp. 2) in phlorizin-infused cows (128.8 +/- 12.7 ng/ml), but increased (P less than .05) by 3 d after calf removal in weaned control cows (152.2 +/- 7.5 ng/ml). IGF-I also remained stable in phlorizin-infused cows following PGF2 alpha injection (Exp. 3), but increased in control cows by 2 d after PGF2 alpha (156.8 +/- 18.3 on d 2 vs. 133.7 +/- 9.8 ng/ml pre-injection; P less than .05) and remained elevated (P less than .05) during the periovulatory period. In cows receiving restricted feed intake (Exp. 4), IGF-I decreased by approximately 50% within 4 d of feed restriction (71.3 +/- 9.4 vs 137.4 +/- 16.6 ng/ml; P less than .05).(ABSTRACT TRUNCATED AT 250 WORDS)