i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4.

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an "i-body," the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor.

Peripheral quantitative computed tomography measures are associated with adult fracture risk: the Hertfordshire Cohort Study.

Peripheral quantitative computed tomography (pQCT) captures novel aspects of bone geometry that may contribute to fracture risk and offers the ability to measure both volumetric bone mineral density (vBMD) and a separation of trabecular and cortical compartments of bone, but longitudinal data relating measures obtained from this technique to incident fractures are lacking. Here we report an analysis from the Hertfordshire Cohort Study, where we were able to study associations between measures obtained from pQCT and DXA in 182 men and 202 women aged 60-75 years at baseline with incident fractures over 6 years later. Among women, radial cortical thickness (HR 1.72, 95% CI 1.16, 2.54, p=0.007) and cortical area (HR 1.91, 95% CI 1.27, 2.85, p=0.002) at the 66% slice were both associated with incident fractures; these results remained significant after adjustment for confounders (age, BMI, social class, cigarette smoking and alcohol consumption, physical activity, dietary calcium, HRT and years since menopause). Further adjustment for aBMD made a little difference to the results. At the tibia, cortical area (HR 1.58, 95% CI 1.10, 2.28, p=0.01), thickness (HR 1.49, 95% CI 1.08, 2.07, p=0.02) and density (HR 1.64, 95% CI 1.18, 2.26, p=0.003) at the 38% site were all associated with incident fractures with the cortical area and density relationships remaining robust to adjustment for the confounders listed above. Further adjustment for aBMD at this site did lead to attenuation of relationships. Among men, tibial stress-strain index (SSI) was predictive of incident fractures (HR 2.30, 95% CI 1.28, 4.13, p=0.005). Adjustment for confounding variables and aBMD did not render this association non-significant. In conclusion, we have demonstrated relationships between measures of bone size, density and strength obtained by pQCT and incident fracture. These relationships were attenuated but in some cases remained significant after adjustment for BMD measures obtained by DXA, suggesting that some additional information may be conferred by this assessment.

Engineered bacterially expressed polypeptides: assembly into polymer particles with tailored degradation profiles.

In nature, the sequence of amino acids in a protein is determined by the genetic code. Biosynthesis of polypeptides by bacteria can be used to exploit this natural process to afford exact control over properties such as molecular weight, chemical functionality, and structure. It is demonstrated how control over the positioning of functional groups can be used to tune the degradation of assembled polypeptide particles (see scheme).

Identification of critical positive charges in XIP, the Na/Ca exchange inhibitory peptide.

The peptides XIP (RRLLFYKYVYKRYRAGKQRG) and C28R2 (LRRGQILWFRGLNRIQTQIRVVKAFRSS) correspond to the autoinhibitory domains of the Na-Ca exchanger and the plasma membrane Ca pump, respectively. An increase of ionic strength reduced the inhibition of exchange activity by XIP and C28R2, consistent with an important role for electrostatic interactions. Sulfosuccinimidyl acetate (SNA)-modified XIP did not inhibit Na-Ca exchange. Because SNA modifies lysines, we conclude that at least one of the positive charges at the XIP lysine positions (7, 11, or 17) is important for inhibition. 2CK-XIP (RRLLFYRYVYRCYCAGRQKG) has cysteines at 12 and 14 and only one lysine (at 19).2CK-XIP inhibited Na-Ca exchange; thus positive charges at 12 and 14 are not essential. SNA-modified 2CK-XIP did not inhibit; thus a positive charge at 19 is important. Iodoacetic acid-modified 2CK-XIP inhibits the Na-Ca exchanger but not the PM Ca pump. These results show that the structural determinants for inhibition of the Na-Ca exchanger and the PM Ca pump are different, that positive charges at 7, 11, or 17 (or some combination) are more important than positive charges at 12 and 14 for inhibition by XIP of the Na-Ca exchanger.

Influence of increased adrenergic activity and magnesium depletion on cardiac rhythm in alcohol withdrawal.

OBJECTIVE: To investigate the prevalence of arrhythmias in alcoholic men during detoxification and its relation to neuroendocrine activation and electrolyte disturbances. DESIGN: Consecutive case-control study. SETTING: Primary and secondary care, detoxification ward. PATIENTS AND CONTROLS: 19 otherwise healthy alcoholic men (DSM-III-R) with withdrawal symptoms necessitating detoxification in hospital. 19 age matched, healthy non-alcoholic men as controls for Holter recordings. INTERVENTIONS: Treatment with chlomethiazole; additional treatment with carbamazepine in patients with previous seizures. MAIN OUTCOME MEASURES: Computer based analyses of mean heart rate and arrhythmias from 24 hour Holter recordings, 24 hour urinary excretion of adrenaline and noradrenaline, magnesium retention measured by means of intravenous loading test, and serum concentrations of electrolytes. RESULTS: The 24 hour mean heart rate was higher in the alcoholic men (97.4 beats/minute, 95% confidence interval (CI) 91.2 to 103.6) than in the controls (69.6 beats/minute, 95% CI 65.4 to 73.8, P < 0.001). However, there was no difference in diurnal heart rate variation. The prevalence of premature supraventricular depolarisations was lower in the alcoholic men (P < 0.05). Neither atrial fibrillation nor malignant ventricular arrhythmias occurred. The sinus tachycardia in the alcoholic men correlated with the concomitant urinary excretion of catecholamines (P < 0.05). The mean serum magnesium concentration was 0.78 mmol/l (95% CI 0.73 to 0.83) in the alcoholic men and 0.83 mmol/l (95% CI 0.81 to 0.85) in a reference population of 55 men aged 40. Magnesium depletion (defined as magnesium retention > 30%) was detected in 10 alcoholic men (53%). Three alcoholic men had serum potassium concentrations < or = 3.3 mmol/l on admission. CONCLUSION: Increased adrenergic activity, magnesium depletion, and hypokalaemia are often seen after heavy drinking, but in alcoholic men without clinical heart disease these changes were not accompanied by arrhythmias other than sinus tachycardia during detoxification in hospital.

Diagnosis of pyogenic liver abscess via liver scanning with indium-111 labelled granulocytes.

An 82-year-old man presented with a history indicating cancer or a developing pyogenic abscess in the liver. The latter diagnosis was established by a liver scan with indium-111 labelled granulocytes, whereas ultrasonography, computed tomography and percutaneous liver puncture were inconclusive.