Anti-inflammatory activity of c(ILDV-NH(CH2)5CO), a novel, selective, cyclic peptide inhibitor of VLA-4-mediated cell adhesion.

1. Small, N- to C-terminal cyclized peptides containing the leucyl-aspartyl-valine (LDV) motif from fibronectin connecting segment-1 (CS-1) have been investigated for their effects on the adhesion of human T-lymphoblastic leukaemia cells (MOLT-4) to human plasma fibronectin in vitro mediated by the integrin Very Late Antigen (VLA)-4 (alpha4beta1, CD49d/CD29). 2. Cyclo(-isoleucyl-leucyl-aspartyl-valyl-aminohexanoyl-) (c(ILDV-NH(CH2)5CO)) was approximately 5 fold more potent (IC50 3.6+/-0.44 microM) than the 25-amino acid linear CS-1 peptide. Cyclic peptides containing two more or one less methylene groups had similar potency to c(ILDV-NH(CH2)5CO) while a compound containing three less methylene groups, c(ILDV-NH(CH2)2CO), was inactive at 100 microM. 3. c(ILDV-NH(CH2)5CO) had little effect on cell adhesion mediated by two other integrins, VLA-5 (alpha5,beta1, CD49e/CD29) (K562 cell adhesion to fibronectin) or Leukocyte Function Associated molecule-1 (LFA-1, alphabeta2, CD11a/CD18) (U937 cell adhesion to Chinese hamster ovary cells transfected with intercellular adhesion molecule-1) at concentrations up to 300 microM. 4. c(ILDV-NH(CH2)5CO) inhibited ovalbumin delayed-type hypersensitivity or oxazolone contact hypersensitivity in Balb/c mice when dosed continuously from subcutaneous osmotic mini-pumps (0.1-10 mg kg(-1) day(-1)). Maximum inhibition (approximately 40%) was similar to that caused by the monoclonal antibody PS/2 (7.5 mg kg(-1) i.v.) directed against the alpha4 integrin subunit. 5. c(ILDV-NH(CH2)5CO) also inhibited oxazolone contact hypersensitivity when dosed intravenously 20 h after oxazolone challenge (1-10 mg kg(-1)). Ear swelling was reduced at 3 h and 4 h but not at 1 h and 2 h post-dose (10 mg kg(-1)). 6. Small molecule VLA-4 inhibitors derived from c(ILDV-NH(CH2)5CO) may be useful as anti-inflammatory agents.     

The influences of age and caffeine on psychomotor and cognitive function

Rationale: The response to caffeine is affected by a number of factors, including age. Older subjects may be more sensitive to the objective effects than younger but report fewer subjective effects. Objective: This study assessed the influence of age on the effects of caffeine on a variety of psychomotor, cognitive and subjective tests. Methods: Forty-eight healthy subjects, male and female, were recruited, 24 in the age range 20–25 and 24 in the range 50–65 years. All subjects were regular moderate caffeine drinkers and were not withdrawn from caffeine before entry to the study. A double-blind parallel group design was used with two groups of 12 subjects in each age range. One group in each age range received placebo and the other 250 mg caffeine B.P. A range of tests was used to assess psychomotor, cognitive and subjective functioning before and 1 h post-treatment. Results: Before treatment, young subjects generally performed better than older on psychomotor and cognitive tests. On the subjective tests, however, older subjects rated themselves as more alert and less tired than the younger ones. After placebo, performance and alertness improved in the younger group but declined in the older. After caffeine there were improvements in psychomotor performance and cognitive functioning in both groups, particularly in offsetting declining performance over time in the older subjects. It also produced subjective improvements in alertness. One factor to emerge was that on most assessments older subjects were better earlier in the day whereas in younger subjects performance did not show the same magnitude of decline throughout the day. Conclusions: Caffeine induced small but significant improvements in vigilance and psychomotor performance. Received: 27 October 1998/Final version: 15 February 1999     

Chimaeric G alpha proteins: their potential use in drug discovery

Approaches that allow ligand occupancy of a wide range of G protein-coupled receptors to be converted into robust assays amenable to relatively high-throughput analysis are ideal for screening for novel ligands at this class of receptor. Many attempts have been made to design universal ligand-screening systems such that any GPCR can be screened using a common assay end-point. Manipulation of the G protein within the assay system offers the possibility of achieving this. To better understand the domains involved in the interactions between G protein-coupled receptors, G proteins and effector polypeptides and the fine details of these contacts, a wide range of chimaeric G protein alpha subunits have been produced. Graeme Milligan and Stephen Rees discuss the information generated by such studies and the ways in which such chimaeric G proteins can be integrated into assay systems for drug discovery.     

Immune response of the draining and distal lymph nodes during the progressive grwoth of a chemically-induced transplantable rat hepatoma.

The immunological and histological changes occurring in the lymph node draining the site of a progressively growing intramuscular tumour (D192A) implant were monitored during a 4-week time course. Cell-mediated cytotoxicity against hepatoma-D192A and 15-day rat embryo cell targets, was detected with cells derived from the draining "lumbar" lymph node 4 days after tumour implantation and persisted up to the 2nd week of tumour growth, decreasing rapidly during the 3rd week. The observed lymph-node anergy demonstrated in cytotoxicity tests correlated with the histological findings, in that an initial marked paracortical (T-dependent) response also declined towards the end of the 3rd week of tumour growth. The B-dependent cortex showed active lymphocyte follicles in the 2nd week of the time course, and plasma-cell production continued until the experiment was terminated. These changes were shown to occur with the progressive increase in lymph-node mass. Serum antibody specific for the developing tumour was detected during the latter stages of tumour growth. The immunological and histological changes displayed were out of phase with those shown by the draining lymph node.     

Value of a radionuclide limb blood flow technique in the assessment of percutaneous balloon and dynamic angioplasty

In a prospective study, a radionuclide technique was used to evaluate the limb blood flow (LBF) changes in 30 patients undergoing dynamic (n=15) or balloon (n=15) angioplasty for arterial occlusions or stenoses, respectively. The results were compared with Doppler Ankle Brachial Index (DABI) and treadmill exercise tests. Whilst LBF values (ml of blood flow per 100 ml of limb volume per min) were significantly lower in limbs with arterial occlusion than stenosis (4.5±0.46 and 6.4±0.74, respectively; P<0.05), DABI provided no discrimination. Immediately after balloon angioplasty, there was a fall in DABI, from 0.60±0.05 to 0.47±0.04 (P<0.05), which rose 24 h later to 0.73±0.02 (P<0.01). Following dynamic angioplasty, DABI improved from 0.60±0.05 to 0.66±0.02 (P<0.05). At 3 weeks, the LBF improved from 4.6±0.66 to 11.1±0.53 (P<0.001) following dynamic angioplasty and from 6.2±0.68 to 8.53±0.81 (P<0.001) following balloon angioplasty. Normal LBF (> 10 ml/100 ml per min) was achieved in 80% of patients who underwent successful dynamic angioplasty but in only 36% of the balloon group (P<0.05, ²-test). Reproducibility of repeated LBF measurements in control limbs was superior to that of DABI. This was indicated by a lower coefficient of variation, 13.8% compared with 25.2%, and a higher correlation coefficient, r=0.79 compared with 0.27. Treadmill exercise tests were invalid or impossible in 30% of all occasions. There was a good correlation between the improvement in maximum walking distance on the treadmill and that in LBF (r=0.84, P<0.05). In conclusion, measurement of LBF using radionuclide technique is a potentially valuable method for the assessment of patients undergoing percutaneous angioplasty. LBF results are reproducible and correlate well with the improvement in walking ability, which cannot always be assessed objectively. Offprint requests to: A.A. Gehani     

Chronic prenatal exposure to carbon monoxide results in a reduction in tyrosine hydroxylase-immunoreactivity and an increase in choline acetyltransferase-immunoreactivity in the fetal medulla: implications for Sudden Infant Death Syndrome

Maternal cigarette smoking during pregnancy is associated with a significantly increased risk of Sudden Infant Death Syndrome (SIDS). This study investigated the effects of prenatal exposure to carbon monoxide (CO), a major component of cigarette smoke, on the neuroglial and neurochemical development of the medulla in the fetal guinea pig. Pregnant guinea pigs were exposed to 200 p.p.m CO for 10 h per day from day 23-25 of gestation (term = 68 days) until day 61-63, at which time fetuses were removed and brains collected for analysis. Using immunohistochemistry and quantitative image analysis, examination of the medulla of CO-exposed fetuses revealed a significant decrease in tyrosine hydroxylase-immunoreactivity (TH-IR) in the nucleus tractus solitarius, dorsal motor nucleus of the vagus (DMV), area postrema, intermediate reticular nucleus, and the ventrolateral medulla (VLM), and a significant increase in choline acetyltransferase-immunoreactivity (ChAT-IR) in the DMV and hypoglossal nucleus compared with controls. There was no difference between groups in immunoreactivity for the m2 muscarinic acetylcholine receptor, substance P- or met-enkephalin in any of the medullary nuclei examined, nor was there evidence of reactive astrogliosis. The results show that prenatal exposure to CO affects cholinergic and catecholaminergic pathways in the medulla of the guinea pig fetus, particularly in cardiorespiratory centers, regions thought to be compromised in SIDS.     

Pseudoaneurysm of the medial inferior genicular artery following anterior cruciate ligament reconstruction

Pseudoaneurysm is a rare complication of surgery or trauma around the knee. A 30-year-old man presented 10 days following anterior cruciate ligament repair with a 2 cm pulsatile swelling on the medial side of the knee. Angiography demonstrated a pseudoaneurysm of the medial inferior genicular artery. Surgical exploration and ligation of the feeding vessel to the aneurysm was performed and the patient made a full recovery. Vascular injury must be suspected in patients presenting with a haemarthrosis or pulsatile swelling following surgery on the knee.