Discovery of Affinity-Based Probes for Btk Occupancy Assays

Bruton's tyrosine kinase (Btk) is an attractive target for the treatment of a wide array of B-cell malignancies and autoimmune diseases. Small-molecule covalent irreversible Btk inhibitors targeting Cys481 have been developed for the treatment of such diseases. In clinical trials, probe molecules are required in occupancy studies to measure the level of engagement of the protein by these covalent irreversible inhibitors. The result of this pharmacodynamic (PD) activity provides guidance for appropriate dosage selection to optimize inhibition of the drug target and correlation of target inhibition with disease treatment efficacy. This information is crucial for successful evaluation of drug candidates in clinical trials. Based on the pyridine carboxamide scaffold of a novel solvent-accessible pocket (SAP) series of covalent irreversible Btk inhibitors, we successfully developed a potent and selective affinity-based biotinylated probe 12 (2-[(4-{4-[5-(1-{5-[(3aS,4S,6aR)-2-oxo-hexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanamido}-3,6,9,12-tetraoxapentadecan-15-amido)pentanoyl]piperazine-1-carbonyl}phenyl)amino]-6-[1-(prop-2-enoyl)piperidin-4-yl]pyridine-3-carboxamide). Compound 12 has been used in Btk occupancy assays for preclinical studies to determine the therapeutic efficacy of Btk inhibition in two mouse lupus models driven by TLR7 activation and type I interferon.     

Epidemiological study of hypertension and its determinants in an urban population of North India

OBJECTIVES: To determine age-specific prevalence of hypertension and blood pressure (BP) levels in relation to diet and lifestyle factors in North Indians. DESIGN AND SETTING: Cross-sectional survey in 20 randomly selected streets in Moradabad, North India. SUBJECTS AND METHODS: A total of 1806 subjects from North India (904 males and 902 females) age range 25-64 years. The survey methods were as follows: dietary diaries for 7 days food intake record; BP measurements; physician administered questionnaire and anthropometric measurements. Diagnosis of hypertension was based on new World Health Organization/International Society of Hypertension (WHO/ISH) criteria. Risk factors were assessed based on WHO guidelines. RESULTS: The prevalence of hypertension according to WHO/ISH criteria was 23.7% and by old WHO criteria 13.3%. In the WHO/ISH hypertensive group, isolated diastolic hypertension was present in 47.3% males and 40.6% females. Males have a slightly higher prevalence than females in the young age group, however, the prevalence rates are comparable in the older age groups. In both sexes, the prevalence rates and BP level increased with older age. Multivariate analysis revealed that age, higher body mass index, central obesity and higher socioeconomic status were independently and strongly associated with hypertension in both sexes. Higher dietary fat and salt intake and lower physical activity were weakly but significantly associated with hypertension. CONCLUSION: Association of higher socioeconmic status, higher body mass index and central obesity in North Indian adults with higher fat intake, lower physical activity and higher prevalence and level of hypertension indicate that these populations may benefit by decreasing the dietary fat intake and increasing physical activity, with an aim to decrease central obesity for decreasing hypertension in North Indians.     

Protein analysis of human von Ebner saliva and a method for its collection from the foliate papillae

The lingual serous glands of von Ebner are located close to the foliate and circumvallate papillae. Saliva secreted by these glands provides the immediate environment of the taste buds, and it has been hypothesized that it modulates taste perception. The purpose of this study was to develop a technique for collection of unstimulated and stimulated saliva from human von Ebner glands. Saliva was collected under resting conditions and after application of various gustatory stimuli (sweet, sour, salt, and bitter) by insertion of periostrips into the folds of the foliate papillae of healthy human volunteers. Stimulated saliva was also collected in glass microcapillaries or micropipettes. The flow-rates of unstimulated von Ebner saliva were 2.3 +/- 0.6 (S.E.) microL/min and 4.5 +/- 1.2 (S.E.) microL/min with 1% citric acid stimulation. The protein content was 2.5 +/- 0.5 (S.E.) mg/mL. The SDS gel electrophoretic profile of von Ebner saliva revealed two protein bands of Mr 18,000 that were identified on Western blots as von Ebner gland (VEG) proteins. Although lingual lipase activity was detected at very low levels by enzyme assay, this protein was not detected on Western blots. This collection technique should prove useful for analysis of specific functions associated with secretion from von Ebner glands.     

P300 latency: abnormal in sleep apnea with somnolence and idiopathic hypersomnia, but normal in narcolepsy

To evaluate cognitive abnormalities in excessive daytime sleepiness (EDS) using cognitive evoked potentials (P300), and to evaluate if P300 measures differentiate among disorders of EDS, a series of EDS subjects were administered a polysomnogram, auditory and visual P300 testing using 31 scalp electrodes, and a multiple sleep latency test. P300 variables were compared with those of normal subjects. Forty normal subjects ages 16 to 65 years, and 69 EDS patients ages 16 to 65 years were used. Of these, 39 had profound obstructive sleep apnea (OSA, Respiratory Disturbance Index or RDI > 80/h sleep) with severe somnolence (Mean Sleep Latency < 5 min). Twenty-two had idiopathic hypersomnia (IH). Eight had narcolepsy. The normals and the three EDS groups did not differ in age. IH and profound OSA patients had longer visual P300 latency than normals or narcolepsy patients (p < 0.05). (p < 0.05). IH and profound OSA patients had longer auditory P300 latency than normals. They had smaller auditory P300 amplitude than narcolepsy patients. There were visual P300 latency topographic differences between normals and profound OSA patients. In conclusion, IH and profound OSA patients show cognitive evoked potential evidence of cognitive dysfunction. Narcolepsy patients do not show such evidence. Visual P300 latency differentiates among disorders of EDS.     

Quantitative evaluation of pancreatic enhancement during dual-phase helical CT

PURPOSE: To determine the improvement in pancreatic enhancement at helical computed tomography (CT) performed with an early delay after administration of contrast material compared with that performed with a standard delay. MATERIALS AND METHODS: Dual-phase helical CT of the abdomen was performed in 120 patients with a 150-mL bolus of contrast material infused at 5 mL/sec. Early and standard delayed scanning was performed beginning at 20 seconds and 49-71 seconds, respectively. Regions of interest were measured in the head, body, and tail of the pancreas in 92 patients. The difference in enhancement between early and standard delayed scanning was calculated. RESULTS: Mean pancreatic enhancement was 82 HU +/- 3 (standard error) with an early delay, whereas enhancement on standard delay scans was 62 HU +/- 2 (P < .001). An improvement in enhancement greater than 10 HU was attained in 66 of 92 cases (72%). CONCLUSION: Pancreatic enhancement at helical CT with an early delay after contrast material administration is often significantly greater than the enhancement seen with a standard delay when a monophasic, rapidly infused bolus of contrast material is used.     

Vesicoureteral reflux and ureteropelvic junction obstruction: association, treatment options and outcome

PURPOSE: We investigated the association, treatment options and outcomes of patients with ureteropelvic junction obstruction and concomitant vesicoureteral reflux. MATERIALS AND METHODS: We analyzed 6,790 consecutive pediatric urology records at our university. Treatment options included observation, and primary pyeloplasty, ureteroneocystostomy and nephroureterectomy. Hydronephrosis, reflux and obstruction were judged as resolved, improved, unchanged or worse. RESULTS: A total of 1,140 patients had vesicoureteral reflux, 224 had ureteropelvic junction obstruction and 41 had both conditions (39 ipsilateral and 6 contralateral kidneys). There was no increased risk of obstruction in patients with reflux when all grades of reflux were grouped (odds ratio 1.26, confidence interval 0.91 to 1.71). In contrast, subgroup analysis of patients with high grade reflux demonstrated a 5-fold increased risk of obstruction (odds ratio 5.0, confidence interval 2.4 to 10.8). One patient was lost to followup. Observation of 6 kidneys led to resolution of reflux in 3 (50%), resolution of obstruction in 3 (50%) and resolution or improvement of hydronephrosis in 4 (67%). Primary pyeloplasty was done on 29 kidneys with 10 (35%) requiring subsequent ureteroneocystostomy. At latest followup hydronephrosis resolved or improved in 24 patients (83%), vesicoureteral reflux resolved or improved in 19 (66%) and ureteropelvic junction obstruction resolved in all. Primary ureteroneocystostomy was performed on 5 kidneys, all of which required subsequent pyeloplasty. Hydronephrosis resolved in 3 patients (60%), and reflux and obstruction resolved in all. Two patients treated with primary nephroureterectomy, and 1 who underwent concomitant pyeloplasty and ureteroneocystostomy have had no subsequent urological problems. One patient awaits primary pyeloplasty. CONCLUSIONS: High grade vesicoureteral reflux is associated with ureteropelvic junction obstruction. No association with low or intermediate grade reflux was demonstrated. While some patients may be monitored expectantly, in our series pyeloplasty or nephrectomy was required in 81% and ureteroneocystostomy was required in 36%. In no case did primary ureteroneocystostomy protect against the subsequent need for pyeloplasty.     

Incisional hernia after laparoscopic nephrectomy with intact specimen removal: caveat emptor

The choline-containing phosphoglycolipid, MfGL-II, is the major polar lipid of Mycoplasma fermentans PG18. Anti-MfGL-II antisera raised in rabbits using the purified MfGL-II as an immunogen were employed in immunogold electron microscopic and immunofluorescence studies showing that MfGL-II is uniformly distributed and exposed on the cell surface of M. fermentans cells. The specificity of the antibodies was determined by immunostaining of lipid extracts separated by thin layer chromatography. The antibodies recognize lipids specific to M. fermentans but did not cross-react with lipid extracts of M. penetrans, M. capricolum, M. gallisepticum or Acholeplasma laidlawii. As phosphocholine almost completely abolished antibody interaction with MfGL-II in an ELISA assay it is suggested that the anti-MfGL-II repertoire is composed primarily of anti-phosphocholine antibodies. The anti-MfGL-II antisera inhibit the attachment of M. fermentans to Molt-3 lymphocytes suggesting that MfGL-II plays a major role in M. fermentans-host cell interaction.