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Routine histopathologic examination of hemorrhoidectomy specimens is controversial having been described as not useful and expensive with few of these common cases demonstrating incidental lesions. However, unexpected premalignant and malignant lesions have been detected on excised hemorrhoids. The high-risk human papillomavirus (HR-HPV) types associated with these incidentally identified high-grade lesions are presently unknown. We aimed to identify cases of incidental high-grade anal intraepithelial neoplasia (HG-AIN) and anal squamous cell carcinoma incidentally discovered on hemorrhoidectomy specimens, genotype HR-HPVs from these lesions, and assess p53 and p16 expression by immunohistochemistry to identify risk factors for their development. With institutional approval, cases with associated demographics from 1995 to 2015 were reviewed to identify and confirm incidental HG-AIN or squamous cell carcinoma in hemorrhoidectomy specimens. Genotyping for HR-HPV types and immunohistochemical staining for p53 and p16 was performed. Statistical analysis comparing HPV genotypes, p53 and p16 staining, and potential risk factors by the Fisher exact test was performed. In the largest series of incidental high-grade lesions on hemorrhoidectomy, HPV 16 was the most common HR-HPV detected though multiple-type infections were common including some HPV 16/18-negative cases. By genotyping, HPV 39 was significantly associated with IV-drug abuse history (P=0.0015) and HIV-positive status (P=0.037), whereas HPV 58 detection correlated with chemotherapy-induced immunosuppression (P=0.029). There was frequent overlap between p53 staining and HPV positivity, particularly when HPV 31 was detected. We also identified several mimickers of HG-AIN that may present diagnostic challenges in these specimens. Our data support continued routine examination of hemorrhoidectomy specimens and suggest that adjunctive studies such as immunohistochemistry for challenging cases may be useful.

Objective: A vast majority of cervicovaginal intraepithelial lesions are caused by high-risk human papillomaviruses (HPVs). The Pap test has been the sole method used for the screening of cervicovaginal squamous intraepithelial lesions (SIL). Recently, the FDA approved an HPV-DNA assay as a method of primary screening. We report on a series of FDA-approved HPV-DNA test-negative SIL with HPV genotyping, using an alternative method on the corresponding surgical biopsy specimens. Study Design: A retrospective review identified cytology-positive HPV-negative cases over a 15-month period at a tertiary care gynecologic oncology institution. Corresponding biopsies were reviewed and genotyped for high-risk HPVs. Results: Of the 18,200 total cases, 17 patients meeting the study criteria were selected with 27 surgical specimens corresponding to their cytologic diagnoses. Four patients with high-grade lesions were identified, 3 of whom (75%) were positive for HPV. One of these 4 patients (25%) showed high-grade SIL on biopsies from 4 separate sites in the cervix and vagina. Multiviral HPV infections were frequent. Conclusions: We discuss the relevance of cotesting for screening cervical SILs and emphasize that false-negative results are possible with the FDA-approved HPV screening assay, also in patients with high-grade SIL. These cases may be detectable by cytologic examination and this suggests that the Pap test remains an important diagnostic tool.

Traumatic brain injury (TBI) is a global problem and causes long-term disability in millions of individuals. This is a major problem for both military- and civilian-related populations. The prevalence of sleep disorders in individuals with TBI is very high, yet mostly unrecognized. Approximately 46% of all chronic TBI patients have sleep disorders, which require nocturnal polysomnography and the Multiple Sleep Latency Test for diagnosis. These disorders include sleep apnoea (23% of all TBI patients), post-traumatic hypersomnia (11%), narcolepsy (6%) and periodic limb movements (7%). Over half of all TBI patients will have insomnia complaints, most often with less severe injury and after personal assault, and half of these may be related to a circadian rhythm disorder. Hypothalamic injury with decreased levels of wake-promoting neurotransmitters such as hypocretin (orexin) and histamine may be involved in the pathophysiology of excessive sleepiness associated with TBI. These sleep disorders result in additional neurocognitive deficits and functional impairment, which might be attributed to the original brain injury itself and thus be left without specific treatment. Most standard treatment regimens of sleep disorders appear to be effective in these patients, including continuous positive airway pressure for sleep apnoea, pramipexole for periodic limb movements and cognitive behavioural therapy for insomnia. The role of wake-promoting agents and CNS stimulants for TBI-associated narcolepsy, post-traumatic hypersomnia and excessive daytime sleepiness requires further study with larger numbers of patients to determine effectiveness and benefit in this population. Future research with multiple collaborating centres should attempt to delineate the pathophysiology of TBI-associated sleep disorders, including CNS-derived hypersomnia and circadian rhythm disturbances, and determine definitive, effective treatment for associated sleep disorders.

There are ~12,000 new cases per year in the United States of spinal cord injury (SCI) with life expectancies from 11 to 14 years (ventilator dependent) to 44 years (non-ventilator dependent). Those with SCI (C2-C8) are at great risk for developing hypoventilation, especially during sleep, and this risk increases along with the risk of sleep disordered breathing as they age. Most will have significantly reduced vital capacity and ventilatory reserve because of interruption of neural pathways to the diaphragm, chest, and abdomen, resulting in a restrictive ventilatory impairment with intact diffusing capacity. Diagnosis entails measurement of pCO (2) with capnography both awake and during sleep, optimally along with polysomnography to evaluate for all forms of sleep disordered breathing. Treatment options include diaphragmatic pacing, full positive pressure ventilation through tracheostomy, and noninvasive positive pressure ventilation. Some may require mechanical ventilation only during sleep.

Objective: We present biochemical characterization of the previously described 14 kDa, 37 kDa, and 52 kDa immunophilins and a newly identified 5–8 kDa immunophilin. Design and methods: Proteins were tested for the following enzymatic activities—rotamase, G3PDH, protein kinase C, cAMP dependent protein kinase—and for the ability to inhibit calcineurin phosphatase when complexed with tacrolimus (FK506). Results: The 5–8 kDa protein, like the other minor immunophilins, lacks rotamase activity. Since the 37 kDa possesses G3PDH activity, the 5–8 kDa protein, 14 kDa protein, and 52 kDa protein were all tested and found to lack G3PDH activity. Additional work shows that none of the minor immunophilins possess protein kinase C or cyclic AMP-dependent protein kinase activity and that the 37 kDa and 5–8 kDa and probably the 52 kDa proteins are capable of inhibiting calcineurin phosphatase when bound to tacrolimus.

Objectives: To determine the pediatric reference ranges for iron, cortisol, CK, CKMB, and troponin I. Methods: Iron and CK were measured on the Vitros analyzer (Johnson and Johnson) while CKMB, troponin I, and cortisol were measured on the Immuno I (Bayer Corp.). Pediatric reference ranges were determined on hospitalized patients using the Hoffmann approach. Results: Pediatric reference ranges were obtained for iron ( am and pm ) and cortisol ( am and pm ). Ranges were also obtained for CKMB, troponin I, and total CK. Conclusion: This work represents an expansion in our knowledge base on pediatric reference ranges. For iron, the 97.5th percentiles were significantly higher in the pm than in the am . The diurnal fluctuation in 97.5th percentiles for cortisol was only 10–20%. Pediatric reference ranges for CKMB were not previously available and are important especially in the first year of life. The elevated Troponin I is found in the first year of life also represents new data.

Objectives: There is a controversy regarding the role of cyclosporine (CsA) metabolites in both immunosuppression and toxicity, and measurement of the parent drug is commonly recommended. High performance liquid chromatography (HPLC) is the method commonly used for specific measurement of the parent drug, but is very time consuming. Antibody techniques are available but vary in specificity. Mixed lymphocyte culture assay (MLC) is a functional bioassay for the measurement of CsA which measures both parent drug and active metabolites. Because it is time consuming and labor intensive, it is not practical to use the MLC to monitor patient’s CsA levels. The objective of this study is to evaluate the degree of cross-reactivity or interference among two different CsA immunoassays [(Immunoassay: CYCLO-Trac-RIA, Monoclonal-TDX; and two radioreceptor assays (RRA) (52 kDa immunophilin and cyclophilin)] with seven cyclosporine metabolites (AM19, AM1c9, AM4n9, AM1, AM9, AM1c, AM4n). The results are compared with a previously published MLC assay for the same metabolites. Methods: 500 ng/mL of each of the CsA metabolites was assayed in spiked blood samples with both RRA using 52 kDa immunophilin and commercial cyclophilin and two commonly used commercial immunoassay procedures. The results were compared to those obtained with the previously published MLC assay. Results and Conclusion: The CYCLO-Trac-radioimmunoassay showed minimal cross-reactivity with all of the seven CsA metabolites tested and is more specific to parent CsA than the current Abbott monoclonal procedure for the measurement of CsA. However the cross-reactivity of the seven metabolites using the Abbott monoclonal assay matched closely with their pharmacological potency as measured in the MLC assay. The RRAs showed greater cross-reactivity for most of the CsA metabolites tested than that found in the immunoassay procedures.

We describe a quantitative radioreceptor assay (RRA) for quantifying FK-506 in whole blood. FK-506 extracted from whole blood with a cyclohexyl-sorbent column competes with [3H]dihydro-FK-506 for binding to a partially purified preparation of FK-506 binding protein (FK-BP). Free and protein-bound FK-506 are separated on LH 20 Sephadex chromatographic columns. We compared the results of this method with those of an enzyme immunoassay that uses a monoclonal antibody: r = 0.97, Sy/x = 0.039. Between-day precisions (CV) at FK-506 concentrations of 8 and 17 micrograms/L were 9.2% and 8.2%, respectively. Within-run precisions were 5.9%, 8.1%, and 9.4%, respectively, at 4, 8, and 15 micrograms/L. Analytical recovery, evaluated at 5, 10, 15, 20, and 25 micrograms/L for FK-506 added to whole blood, ranged from 98% to 103%. The assay can reliably quantify FK-506 blood concentrations between 1.0 and 25 micrograms/L.

THE DIAGNOSIS OF SLEEP APNEA TYPICALLY INVOLVES AN OVERNIGHT POLYSOMNOGRAM WITH CONTINUOUS MONITORING OF SEVERAL PHYSIOLOGIC parameters and surrogate measures of airflow (nasal pressure [Pn], oronasal thermistor, expired CO2 waveform [PECO2]) using contact sensors.1–5 A subject can come in contact with at least 20 such sensors during a study. These sensors and the wires can influence not only the usual sleep architecture, but also the posture of the patient during the study night.6–8 Contact sensors like nasal prongs and oronasal thermistors are routinely used as surrogate measures of airflow because they are less obtrusive than the reference-standard airflow sensor, namely, the pneumotachometer.1,9,10 However, the presence of the surrogate airflow sensors and the associated wires in the proximity of the subject's oronasal area can still cause discomfort to the patient during sleep. Contact sensors also have the potential to misalign during the study, requiring repositioning at the bedside by a technician, to obtain good-quality data. We have demonstrated that measurement of breathing rates is feasible at a distance using thermal infrared imaging (TIRI).11 The method is based on second-order statistics of the presence or absence of the hot expiratory plume in the vicinity of the nostrils. We have also studied a more advanced method that is based on wavelet analysis of the thermal imaging signal on the nostrils themselves.12,13 When analyzing the thermal imaging signal, we extracted the full breathing waveform (rate and amplitude). Consistent segmentation of the nostril area and facial tissue-tracking algorithms14 enabled sustained monitoring of breathing, resulting in functionality equivalent to that of a thermistor, delivered in a contact-free manner. In 5-minute recordings of 20 awake healthy individuals, agreement between breathing waveforms from the virtual (imaging) and contact thermistors was found to be greater than 90%. The hardware and software system that was developed to implement the latest method is called automatic thermal monitoring system (ATHEMOS). The centerpiece of the system is a midwave infrared camera (FLIR® SC-6000), supported by a black body for calibration and a pan-tilt mechanism for positioning. The system is controlled and the acquired imaging signals are processed by real-time custom software developed in our labs. ATHEMOS and the imaging method that we reported on in a previous publication12 constitute the TIRI portion of the present study. The objective in this current study was to determine the efficacy of TIRI to detect apnea and hypopnea by using conventional surrogate airflow sensors as comparison standards during polysomnography. Thus, our hypothesis was that there would be a very high degree of chance-corrected agreement (κ ≥ 0.6) between TIRI and each of the conventional flow sensors, such as thermistor, Pn, and PECO2 in the detection of apnea and hypopnea. Some of the data from this manuscript has been presented at scientific meetings and published in the form of abstracts.15,16

Mast cell degranulation is a highly regulated, calcium-dependent process, which is important for the acute release of inflammatory mediators during the course of many pathological conditions. We previously found that Synaptotagmin-2, a calcium sensor in neuronal exocytosis, was expressed in a mast cell line. We postulated that this protein may be involved in the control of mast cell-regulated exocytosis, and we generated Synaptotagmin-2 knock-out mice to test our hypothesis. Mast cells from this mutant animal conferred an abnormally decreased passive cutaneous anaphylaxis reaction on mast cell-deficient mice that correlated with a specific defect in mast cell-regulated exocytosis, leaving constitutive exocytosis and nonexocytic mast cell effector responses intact. This defect was not secondary to abnormalities in the development, maturation, migration, morphology, synthesis, and storage of inflammatory mediators, or intracellular calcium transients of the mast cells. Unlike neurons, the lack of Synaptotagmin-2 in mast cells was not associated with increased spontaneous exocytosis.

The present paper proposes a novel methodology to monitor sleep apnea through thermal imaging. First, the nostril region is segmented and it is tracked over time via a network of cooperating probabilistic trackers. Then, the mean thermal signal of the nostril region, carrying the breathing information, is analyzed through wavelet decomposition. The experimental set included 22 subjects (12 men and 10 women). The sleep-disordered incidents were detected by both thermal and standard polysomnographic methodologies. The high accuracy confirms the validity of the proposed approach, and brings non-obtrusive clinical monitoring of sleep disorders within reach.

Potentially toxic levels of oxyradicals (superoxide and hydroxyl radicals) are formed in vivo during many metabolic reactions, including oxidation of catecholamines (reviewed in Halliwell and Gutteridge 1984; 1989). In cell membranes, hydroxyl radical:~ can initiate lipid peroxidation resulting in altered membrane structure and function. Under physiological conditions, cells are protected against oxyradical-mediated damage primarily by the cooperative and sequential actions of the enzymes superoxide dismutase (SODL glutathione peroxidase (GSHPx), and catalase (CAT), which are critical components of the antioxidant defence system (AODS). There has been limited investigation of oxyradical metabolism in schizophrenia. Most investigators (Michelson et M 1977; Golse et a! 1978; Abdalla et al 1986), bat no;. all (Stnet et al 1983), have reported increased red blood cell (RBC) SOD activity m schizo

Objectives: To determine the effect of gestational age and birth weight (BW) on troponin I (TnI) and creatine kinase MB fraction (CKMB) levels during the first year of life. Methods: Troponin I and CKMB levels were determined in infants less than 1 year of age using the Immuno I (Bayer Corp.). Results: Troponin I fractions were greatest in the preterm infant; the levels decreased significantly with increasing gestational age and BW, (p = 0.008 and p = 0.005, respectively). The CKMB levels did not exhibit a significant difference between the preterm and term infant groups when assessed for the effects of gestational age or BW (p = 0.12 vs. p = 0.35). Neither TnI nor CKMB levels were significantly different between preterm survivors and nonsurvivors (p = 0.31; p = 0.34, respectively). TnI levels were elevated in critically ill patients without documented myocardial infarction, and without a comparable rise in CKMB. Conclusion: The higher TnI levels during the first 3 months of life may indicate programmed cell death, or apoptosis. This may be especially true in the preterm infant in which the greatest values were documented.

Objectives: We have previously identified three minor immunophilins of molecular weights 37 kDa, 14 kDa, and 5–8 kDa capable of binding tacrolimus and sirolimus. Design and methods: When tested against pure preparations of five sirolimus metabolites, the 14 kDa protein had almost no cross-reactivity, the 37 kDa protein cross-reacted from a high of 23.2% to

Objectives: We have previously identified a minor immunophilin of 52 kDa molecular weight capable of binding tacrolimus and sirolimus. Because immunophilins are capable of binding both parent drug and metabolites and HPLC assays are typically used to assess parent drug in clinical situations, we used this immunophilin in a radioreceptor assay (RRA) to determine if any metabolites not included in the HPLC measurement would bind to the immunophilin and be associated with thrombocytopenia in patients receiving sirolimus. Design and methods: We tested 51 steady-state trough whole blood samples from non-thrombocytopenic patients and 51 steady-state trough samples from thrombocytopenic patients and compared them to HPLC measurements of parent drug in the same samples. We also tested whole blood samples spiked with authentic sirolimus metabolites using RRA to ascertain the effect these metabolites have on the technique. Results: We found minimal cross-reactivity in this assay for sirolimus metabolites (binding ranged from

Objective: Sirolimus (RAPA) is a new immunosuppressive drug currently in Phase III clinical trials in combination with cyclosporine A (CsA). The toxicity profiles for CsA and RAPA are only partially overlapping, with RAPA toxicity consisting primarily of hyperlipidemia and myelodepression but without the nephrotoxicity, neurotoxicity, and hepatotoxicity, which are seen with CsA. Patients in the clinical trial are being monitored using HPLC or LC/MS/MS assays; there is no immunoassay for RAPA reported to date. We have previously reported a radioreceptor assay (RRA) for RAPA, which has an excellent correlation with the HPLC assay (r = 0.997). The RRA has several advantages including excellent precision, sensitivity, rapid turnaround time, and a one-step extraction procedure. We report the evaluation of blood samples from patients who were exhibiting RAPA toxicity and comparison of the RRA results with the HPLC results. Methods: EDTA whole blood specimens (n = 42) were obtained from six renal transplant recipients taking RAPA and CsA and exhibiting decreased platelet counts. Thirty-two samples from patients without decreased platelet counts were also received. The samples were analyzed with the RRA and the results were compared to those obtained with the HPLC assay. Results: By HPLC, the results ranged from 3.2–72.6 μg/L RAPA with 43% of the results ≥30 μg/L. With the RRA, the range was 7.7–83.0 μg/L RAPA equivalents, with 60% of the results ≥30 μg/L. The RRA results are distinctly higher than the HPLC results all along the range. The correlation between the two assays was 0.861, with a slope of 0.966 and a Y-intercept of 11.1. Conclusion: Since the RRA is consistently higher than HPLC concentration in patients with decreased platelet counts, but correlates well in patients with no signs of toxicity, the RRA may be useful for monitoring patients for toxicity, by giving a better indication of increasing degree of immunosuppression than the HPLC assay.

To evaluate the Point-of-Care (POC) i-STAT system for measuring blood gases (pH, pCO2, pO2) and whole blood electrolytes (sodium, Na+, potassium, K+ and ionized calcium, iCa2+) in the neonatal and pediatric intensive care units.The i-STAT system was evaluated for imprecision, necessity of running quality control and accuracy. Comparison of patients' samples analyzed by the i-STAT system and the Ciba Coming 288 blood gas analyzer were performed. The reliability of the i-STAT system when performed by non-laboratory personnel was assessed.The system was evaluated for imprecision and linearity using three concentrations of aqueous standards. Except for pO2, the %CVs were3.0 for all the analytes (pH, pCO2, Na+, K+ and iCa2+) at all the three concentrations. Using whole blood studies the precision data gave %CVs that were3.5 for all the analytes. Linearity studies showed good linearity over the five different concentrations tested. Comparison of the i-STAT and the Ciba Coming 288 blood gas analyzer was assessed by split sample measurement. Patients' results from the i-STAT correlated well with the Ciba Coming 288 blood gas analyzer (r = 0.99 for pH, pCO2 and pO2 and 0.95 to 0.99 for Na+ and K+) with the exception of iCa2+ (r = 0.73). There was no significant difference in the results when operated by PICU/NICU nurses or laboratory personnel. A further study was made to assess whether routine quality control (QC) samples are necessary when using the i-STAT system. The regression analysis (slope and correlation coefficient) of the results from instruments run with and without QC samples gave results close to 1, indicating that there is no need to run additional quality control (QC).The POC testing analyzer i-STAT is a reliable alternative to the traditional blood gas analyzers and provides marginal improvement in turnaround time when compared with the service received from the PICU/NICU laboratory. Costs need to be carefully controlled.

Objective: The isolation and partial characterization of a 37 kDa minor immunophilin from the Jurkat cell line which binds to cyclosporine (CsA), Tacrolimus (FK506) and Sirolimus (RAPA). Design and Methods: Using standard protein purification steps including isoelectric focusing and cation exchange chromatography, we have isolated and purified to homogeneity a minor immunophilin from the Jurkat cell line which has a molecular weight of 37 kDa. Binding properties for immunosuppressive drugs CsA, FK506 and RAPA were assessed by Scatchard and displacement analysis. The amino acid analysis and the protein sequences were also studied. Results: The immunophilin was purified to homogeneity and the molecular weight corresponds to 37 kDa. Saturation experiments using 3Hdihydro FK506 gave a Kd of 4.5 nM and the Bmax of 117 nmol/mg protein. Displacement studies using 3Hdihydro FK506 and RAPA gave a Kd of 0.8 nM. For CsA binding, the protein showed somewhat less avid binding. The amino acid composition was in close agreement with the amino acid composition of uracil DNA glycosylase which corresponds to part of the monomer of glyceraldehyde 3 phosphate dehydrogenase (G3PD). Protein digestion gave at least 3 peptides. The primary sequence of the first of these matched 7 of 8 residues of human liver nuclear uracil DNA glycosylase. The 37 kDa immunophilin was found to have G3PD activity not inhibited by FK506. Conclusions: The amino acid analysis, protein sequences, binding properties and G3PD activity indicate that this 37 kDa immunophilin is different from any other known Immunophilins.

Objectives: To develop a radioreceptor assay (RRA) for sirolimus (rapamycin, RAPA). Methods: A direct methanol extraction was used to prepare 45 patient samples for the RRA. Results were compared to the results obtained previously using high-performance liquid chromatography (HPLC). Between-run precision, recovery, and drug interference studies were also performed. Results: The RRA is sensitive to 1.0 μg/L RAPA equivalents in whole blood. Comparison with HPLC yielded a correlation coefficient for 45 patient samples of 0.977. Between-run precision at 2.5, 7.5, 12.5, and 20 μg/L showed coefficients of variation (CVs) of 12.9, 9.2, 8.5, and 5.9%, respectively. Recoveries from the extraction procedure were 93% at 7.5 μg/L and 103% at 12.5 μg/L. Drug interference studies showed no interference in the RRA by cyclosporine (CsA), dexamethasone, prednisone, or methotrexate. Conclusion: We have demonstrated that the RRA for RAPA correlates well with HPLC, and has excellent precision and recovery. The procedure is far less time-consuming and complex than HPLC and has potential for automation.

Objectives: To compare the binding characteristics of four rapamycin (RAPA) metabolites to the 14 and 52 kDa minor immunophilins with their pharmacologic activity, as measured by the mixed-lymphocyte culture (MLC) assay. Methods: Four RAPA metabolites were isolated by HPLC from the urine of renal transplant patients. Each metabolite was evaluated at 40 μg/L for its pharmacologic activity using the MLC assay. The results of the MLC assay were compared to those obtained using the radioreceptor assay (RRA), which measured the binding characteristics of equal concentrations of the metabolites to the 14 and 52 kDa minor immunophilins. Results: Each of the four RAPA metabolites showed low immunosuppressive activity by MLC. RM2 showed the highest activity, with 9% of parent RAPA activity. RM1, 3, and 4 showed 2%, 8%, and 4% activity, respectively. Only RM1 was found to bind significantly to either minor immunophilin, with 21% of parent binding to the 14 kDa protein and 25% of parent binding to the 52 kDa protein. RM2, 3, and 4 bound to both proteins with ⩽2% of parent binding. Conclusion: We have demonstrated that the RRA for these four RAPA metabolites shows little cross-reactivity. There is no commercially available immunoassay for RAPA at present. The RRA, therefore, provides an excellent way to rapidly assess efficacy/toxicity of RAPA in patients receiving the drug.

Objectives : Evaluation of the precision, accuracy, and user-friendliness of the Technician Immuno I. Calculation of pediatric reference ranges for ferritin and endocrine tests run on Immuno I. Methods : Precision and accuracy were measured using controls and method comparison studies. Pediatric reference ranges were calculated by comparing the Immuno I results for 100 patients with those of the Abbott IMx and TDx. The regression equation obtained was then used to convert the [Mx and TDx reference ranges to reference ranges for the Immuno I. Results : The Immuno I provided both accurate and precise measurement of drugs and endocrine hormones. Pediatric reference ranges were obtained for ferritin and all endocrine tests. Conclusion : The Immuno I is user-friendly and provides reliable measurement of both the drugs tested and endocrine tests on a micro-sample. Reagent and curve stability are excellent.

Specific binding proteins (immunophilins, 12–17 kDa) have been described in the cytosol for the immunosuppressant drugs cyclosporine, FK-506, and rapamycin. We describe the identification of a low abundant minor immunophilin (14 kDa) from calf thymus. Saturation experiments using dihydro[ 3 ]-FK-506 gave a K d of 2 n M and the B max nmol/mg protein. At saturation, 71.3 nmol of FK-506 is bound per mg of the 14 kDa protein (71 nmol) giving a drug/protein ratio of 1.0. Competition experiments using 3 H-dihydro FK-506 and rapamycin showed dis placement of rapamycin, with K d in the range of 40 n M . The 14 kDa immunophilin does not have peptidyl-prolyl cis-trans isomerase activity with any of the four substrates investigated. Initial amino acid analysis and protein sequencing data indicate that the immunophilin is different from both the 12 kDa FK-506 binding protein and any other known protein.

Time-dependent changes in levels of the antioxidant enzymes, superoxide dismutasae (SOD), glutathione peroxidase (GSHPOD), and catalase (CAT) after cortical focal ischemia in rat indicate that: (1) primary and peri-ischemic tissues differ in both rate and the magnitude of oxyradical-induced ischemic injury, and (2) ischemic tissue remains vulnerable to oxyradical damage as long as 72 h after ischemia since the antioxidant enzyme levels remain at or below basal levels. After 72h, the increased levels of these enzymes are sufficient to protect tissue against oxyradical damage. GM1 ganglioside (10 mg/kg, im) further increased the already elevated levels of the enzymes after ischemia, thereby indicating the GM1 treatment increases the capacity of ischemic tissue to protect against oxyradical injury.

Background Both Peak Oxygen Uptake (peak VO2), from cardiopulmonary exercise testing (CPET) and the distance walked during a Six-Minute Walk Test (6 MWD) are used for following the natural history of various diseases, timing of procedures such as transplantation and for assessing the response to therapeutic interventions. However, their relationship has not been clearly defined. Methods We determined the ability of 6 MWD to predict peak VO2 using data points from 1,083 patients with diverse cardiopulmonary disorders. The patient data came from a study we performed and 10 separate studies where we were able to electronically convert published scattergrams to bivariate points. Using Linear Mixed Model analysis (LMM), we determined what effect factors such as disease entity and different inter-site testing protocols contributed to the magnitude of the standard error of estimate (SEE). Results The LMM analysis found that only 0.16 ml/kg/min or about 4% of the SEE was due to all of the inter-site testing differences. The major source of error is the inherent variability related to the two tests. Therefore, we were able to create a generalized equation that can be used to predict peak VO2 among patients with different diseases, who have undergone various exercise protocols, with minimal loss of accuracy. Although 6 MWD and peak VO2 are significantly correlated, the SEE is unacceptably large for clinical usefulness in an individual patient. For the data as a whole it is 3.82 ml/kg/min or 26.7% of mean peak VO2. Conversely, the SEE for predicting the mean peak VO2 from mean 6 MWD for the 11 study groups is only 1.1 ml/kg/min. Conclusions A generalized equation can be used to predict peak VO2 from 6 MWD. Unfortunately, like other prediction equations, it is of limited usefulness for individual patients. However, the generalized equation can be used to accurately estimate mean peak VO2 from mean 6 MWD, among groups of patients with diverse diseases without the need for cardiopulmonary exercise testing. The equation is

BACKGROUND:Adenosine is generated in response to cellular stress and damage and is elevated in the lungs of patients with chronic lung disease. Adenosine signaling through its cell surface receptors serves as an amplifier of chronic lung disorders, suggesting adenosine-based therapeutics may be beneficial in the treatment of lung diseases such as chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF). Previous studies in mouse models of chronic lung disease demonstrate that the key components of adenosine metabolism and signaling are altered. Changes include an up-regulation of CD73, the major enzyme of adenosine production and down-regulation of adenosine deaminase (ADA), the major enzyme for adenosine metabolism. In addition, adenosine receptors are elevated. METHODOLOGY/PRINCIPAL FINDINGS:The focus of this study was to utilize tissues from patients with COPD or IPF to examine whether changes in purinergic metabolism and signaling occur in human disease. Results demonstrate that the levels of CD73 and A(2B)R are elevated in surgical lung biopsies from severe COPD and IPF patients. Immunolocalization assays revealed abundant expression of CD73 and the A(2B)R in alternatively activated macrophages in both COPD and IPF samples. In addition, mediators that are regulated by the A(2B)R, such as IL-6, IL-8 and osteopontin were elevated in these samples and activation of the A(2B)R on cells isolated from the airways of COPD and IPF patients was shown to directly induce the production of these mediators. CONCLUSIONS/SIGNIFICANCE:These findings suggest that components of adenosine metabolism and signaling are altered in a manner that promotes adenosine production and signaling in the lungs of patients with COPD and IPF, and provide proof of concept information that these disorders may benefit from adenosine-based therapeutics. Furthermore, this study provides the first evidence that A(2B)R signaling can promote the production of inflammatory and fibrotic mediators in patients with these disorders.