Marked Reduction In Sensitivity Research Articles

Modulation of Pain-Associated Behaviors in Sickle Mice Using Low-Intensity Transcranial Focused Ultrasound

Pain is a major comorbidity of sickle cell disease (SCD), which is the most genetic diathesis affecting mostly African Americans in the United States. Opioids are used to reduce pain, however the increased risk of drug abuse, overdose, and addiction has led to an urgent need to develop non-pharmacologic approaches to treat pain safely and effectively. Low-intensity transcranial focused ultrasound (tFUS) is a non-invasive brain stimulation method featured with high precision and deep brain penetration, which holds great promise in managing pain. Here, we report how tFUS modulates pain-related behaviors in wild-type mice and humanized sickle mice by targeting specific brain regions involved in the processing and perception of pain. We used wild-type mice (C57BL/6J) and humanized transgenic BERK mice on a mixed genetic background (FVB/N, 129, DBA/2, C57BL/6, and Black Swiss) murine α and β globin knockouts (α−/−, β−/−) with sickle mice expressing >99% human sickle hemoglobin S (HbS), demonstrating severe SCD. Mice were generated in-house by cross-breeding homozygous (SS) males with hemizygous (AS) females. All studies in mice were approved by IACUC of Carnegie Mellon University and University of California at Irvine, and complied with NIH guidelines. The tFUS was applied to hindlimb of the primary somatosensory cortex (S1HL) at the left hemisphere of the brain, and the effect of tFUS stimulation was further investigated by comparing with control groups such as sham treatment (applying tFUS to a vicinity region of S1HL) and negative control (not applying tFUS while keeping the same experimental procedures). A modified hot-plate behavior test was used to assess unilateral effects of tFUS by comparing withdrawal responses of contralateral (right) and ipsilateral (left) hind paws. The nociceptive responses to noxious heat stimuli were blindly assessed and analyzed in terms of ∆hind paw withdrawal latency (∆hPWL) (s) (= contralateral hPWL- ipsilateral hPWL) before and after specific treatment. We observed a marked reduction in sensitivity to noxious heat stimuli in tFUS-treated male and female wild-type mice. Compared to the baseline value, tFUS stimulation with lower pulse repetition frequency (PRF) (i.e., 40 Hz) at left S1HL led to a significant increase in the averaged ∆hPWL (***p<0.001, N=8 male wild-type mice for 3 min after tFUS; *p<0.05, N=8 for 8 min after tFUS; **p<0.01, N=8 female wild-type mice for 3 min after tFUS; *p<0.05, N=8 for 8 min after tFUS; Friedman test), but not with the sham treatment (p>0.05, N=8 male and female wild-type mice for 3 and 8 min after sham treatment; Friedman test). Next, we assessed whether tFUS could modulate heat hyperalgesia in female sickle mice. Fig. 1 shows that tFUS stimulation with PRF of 40 Hz at left S1HL resulted in a significant change of ∆hPWL in the sickle mice compared to pre-stimulation baseline (**p<0.01, N=10 for 3 and 8 min after tFUS; Friedman test). This effect was specific to tFUS stimulation as a remarkable difference of averaged ∆hPWL between the sham treatment/negative control and pre-sham/negative control baseline was not observed (p>0.05, N=10 for 3 and 8 min after sham treatment and negative control; Friedman test), which suggests that tFUS stimulation with a lower PRF at S1HL can attenuate heat hyperalgesia in the sickle mice. In addition, when tFUS with higher PRF (i.e., 3 kHz) was applied at left S1HL, we found a negative averaged value of ∆hPWL based on the decreased contralateral (right) hind paw latency compared to latency of ipsilateral (left) hind paw, which is significantly different from the behavioral responses to heat stimuli with the PRF of 40 Hz (###p<0.001, N=10 for 3 min after tFUS with PRF of 40 and 3 kHz; ####p<0.0001, N=10 for 8 min after tFUS with PRF of 40 and 3 kHz; Kruskal-Wallis test). Since, tFUS stimulation with a higher PRF leads to exacerbation of heat hyperalgesia in the sickle mice, it is critical to optimize the level of PRF to achieve an analgesic effect. We show that tFUS stimulation can be effective in non-invasively attenuating heat pain sensitivity in wild-type mice and heat hyperalgesia in humanized sickle mice at a lower PRF, but exacerbating at a higher PRF by stimulating pre-identified pain-processing brain circuit. These observations have a translational potential for establishing an effective, non-addictive, non-pharmacological, non-invasive device-based neuromodulation technique for managing sickle cell pain. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Influence of Age on the Diagnosis of Myocardial Infarction.

Background:The 99th centile of cardiac troponin, derived from a healthy reference population, is recommended as the diagnostic threshold for myocardial infarction, but troponin concentrations are strongly influenced by age. Our aim was to assess the diagnostic performance of cardiac troponin in older patients presenting with suspected myocardial infarction.Methods:In a secondary analysis of a multicenter trial of consecutive patients with suspected myocardial infarction, we assessed the diagnostic accuracy of high-sensitivity cardiac troponin I at presentation for the diagnosis of type 1, type 2, or type 4b myocardial infarction across 3 age groups (<50, 50–74, and ≥75 years) using guideline-recommended sex-specific and age-adjusted 99th centile thresholds.Results:In 46 435 consecutive patients aged 18 to 108 years (mean, 61±17 years), 5216 (11%) had a diagnosis of myocardial infarction. In patients <50 (n=12 379), 50 to 74 (n=22 380), and ≥75 (n=11 676) years, the sensitivity of the guideline-recommended threshold was similar at 79.2% (95% CI, 75.5–82.9), 80.6% (95% CI, 79.2–82.1), and 81.6% (95% CI, 79.8–83.2), respectively. The specificity decreased with advancing age from 98.3% (95% CI, 98.1–98.5) to 95.5% (95% CI, 95.2–95.8), and 82.6% (95% CI, 81.9–83.4). The use of age-adjusted 99th centile thresholds improved the specificity (91.3% [90.8%–91.9%] versus 82.6% [95% CI, 81.9%–83.4%]) and positive predictive value (59.3% [57.0%–61.5%] versus 51.5% [49.9%–53.3%]) for myocardial infarction in patients ≥75 years but failed to prevent the decrease in either parameter with increasing age and resulted in a marked reduction in sensitivity compared with the use of the guideline-recommended threshold (55.9% [53.6%–57.9%] versus 81.6% [79.8%–83.3%].Conclusions:Age alters the diagnostic performance of cardiac troponin, with reduced specificity and positive predictive value in older patients when applying the guideline-recommended or age-adjusted 99th centiles. Individualized diagnostic approaches rather than the adjustment of binary thresholds are needed in an aging population.

Renal Artery Dimensions and Variation on Computed Tomography Angiogram: A Cross-sectional Study.

Introduction A knowledge of renal artery dimension is necessary for characterizing the physiological change as well as pathological process undergoing in the renal artery likely stenosis, ectasia, or aneurysm. This study aims to explore the anatomy of the renal artery with regards to its dimension, relation, and branching and identify the common anatomical variation. Materials and Methods The study was a retrospective hospital record-based study conducted during the period of one year from 2019 January to 2019 December. All abdominal CT angiograms were reviewed for renal artery anatomy and variation. Abnormal Abdominal CT angiograms were excluded from the study. Normal anatomy, dimensions, and variation of renal vasculature were assessed. ResultsA total of 110 patients included the inclusion criteria and was included in the study. The renal length and size of the renal artery were significantly larger on the left side. The renal artery diameter showed positive correlation with renal length (r= 0.432; p&lt;0.001). No variation of the renal size of renal artery diameter was however noted with sex. Supernumerary arteries were seen in 33(30%) patients. The optimum cut-off value of renal artery diameter for predicting accessory renal artery was 5.35 or less which yielded a sensitivity of 75% and specificity of 70%. (AUC-0.79; p&lt;0.001). When 4.15 mm was taken as cut-off, the specificity increased to 96% with a marked reduction in sensitivity to 3.6%. Conclusion Renal artery diameter is dependent on laterality, kidney size, and presence of accessory artery and independent of sex.

Effects of IVIg treatment on autoantibody testing in neurological patients: marked reduction in sensitivity but reliable specificity.

Therapy of autoimmune diseases of the central and peripheral nervous system with intravenous IgG immunoglobulin (IVIg) is well established. Since IVIg is produced from pooled human plasma, autoantibodies can be found in IVIg products and, accordingly, in patient sera after transfusion. The de novo evidence or disappearance of anti-neural autoantibodies after IVIg treatment has so far not been systematically examined. We screened 50 neurological patients before and after IVIg treatment for classical onconeural and the most common neurological surface autoantibodies as well as for ganglioside autoantibodies and 23 different antinuclear autoantibodies using immunoblot or cell-based indirect immunofluorescence assays. Furthermore, we screened 31 neurological patients with previously known seropositivity for disappearance of the corresponding antibody after treatment. After IVIg treatment, 90% of all sera were de novo positive for antinuclear antibodies, especially for Ro-52. In contrast, 94% of all sera did not show any de novo-positive anti-neural antibodies. In the remaining three cases, titers were very low. Importantly, 12.9% of all tested sera of patients with known antibody positivity turned false negative after IVIg treatment and titers were falsely low in 37% of the remaining sera. Here, we present for the first time results of a broad screening for clinically relevant autoantibodies before and after IVIg treatment in neurological patients. We identified a high specificity but reduced sensitivity for anti-neural antibody testing after IVIg transfusion. In contrast, antinuclear antibody testing is not reliable after IVIg treatment. These results are of high practical importance for diagnostic of neuroimmunological diseases.

Effects of p53 Status of Tumor Cells and Combined Treatment With Mild Hyperthermia, Wortmannin or Caffeine on Recovery From Radiation-Induced Damage

BackgroundThe aim of the study was to examine the dependency of p53 status and the usefulness of mild hyperthermia (MHT) as an inhibitor of recovery from radiation-induced damage, referring to the response of quiescent (Q) tumor cell population.MethodsHuman head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector (SAS/neo) were injected subcutaneously into left hind legs of nude mice. Tumor-bearing mice received 5-bromo-2’-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells. They received high dose-rate γ-ray irradiation (HDR) immediately followed by localized MHT (40 °C for 2 h), or caffeine or wortmannin administration, or low dose-rate γ-ray irradiation simultaneously with localized MHT or caffeine or wortmannin administration. Nine hours after the start of irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU.ResultsSAS/neo tumor cells, especially intratumor Q cell populations, showed a marked reduction in sensitivity due to the recovery from radiation-induced damage, compared with the total or Q tumor cells within SAS/mp53 tumors that showed little recovery capacity. The recovery from radiation-induced damage was thought to be a p53-dependent event. In both total and Q tumor cells within SAS/neo tumors, especially the latter, MHT efficiently suppressed the reduction in sensitivity caused by leaving an interval between HDR irradiation and the assay and decreasing the irradiation dose-rate, as well as the combination with wortmannin administration.ConclusionsFrom the viewpoint of solid tumor control as a whole, including intratumor Q-cell control, non-toxic MHT is useful for suppressing the recovery from radiation-induced damage, as well as wortmannin treatment combined with γ-ray irradiation.

Abstract A091: Therapeutic targeting of the p300/CBP bromodomain for the treatment of castration-resistant prostate cancer

Abstract Background: Sustained androgen receptor pathway activation is the hallmark of castration-resistant prostate cancer (CRPC). Therapeutic strategies for CRPC include non-ligand-binding domain targeted degradation of androgen receptor (AR) and AR variants (ARV). E1A binding protein (p300) and CREB binding protein (CBP) are two closely related histone acetyl transferase proteins that are critical transcriptional regulators of the androgen receptor. We have developed CCS1477, which is a potent, selective, and orally active small-molecule inhibitor of the bromodomain of p300/CBP, and investigated its role in regulating androgen receptor expression and function. Methods: Binding affinity of CCS1477 to p300, CBP and BRD4 was measured in a surface plasmon resonance (SPR) assay. Potency and functional activity (proliferation, cell cycle analysis, and biomarker knockdown) was demonstrated in a panel of prostate cells lines representing hormone-responsive (LNCaP), hormone-independent (DU145, PC3), and castration-resistant disease (22Rv1, VCaP, LNCaP-AR). A JQ1 resistant cell line was generated by culturing cells in increasing doses of JQ1 over a period of approximately 9 months and used to differentiate potency and functional activity of BET vs p300/CBP inhibition in vitro. In vivo efficacy, linked to inhibition of biomarkers, was determined in 22Rv1 and LNCaP xenograft models. Results: CCS1477 binds to p300 and CBP with high affinity (KD=1.3/1.7nM) and selectivity (KD=222nM; BRD4). It is a potent inhibitor of cell proliferation in castration-resistant cell lines (IC50= 96nM,22Rv1; 49nM,VCaP; 150nM,LNCaP-AR) with minimal effect in AR-ve lines (PC3, DU145). These inhibitory effects on cell proliferation were accompanied by a decrease in the number of cells in S phase and an increase in the number of cells in the G1/G0 phase of the cell cycle. We have developed a JQ1 resistant 22Rv1 cell line and observed a marked reduction in sensitivity (proliferation end point) to JQ1 as expected, which is accompanied by cross-resistance to a variety of other BET inhibitors, including iBET-151 and OTX-015. In contrast, and as evidence for a differentiated mechanism, sensitivity to CCS1477 is retained in this JQ1 resistant line. Inhibition of the bromodomain of p300/CBP downregulates AR-FL, AR-V7 and c-Myc protein in 22Rv1 cells by Western blot and this is accompanied by profound inhibition of c-Myc, KLK3 and TMPRSS2 genes measured by qPCR. The in vivo PK properties of CCS1477 are consistent with qd or qod oral dosing in mouse. CCS1477 dosed at 10mg/kg qd, 20mg/kg qd, or 30mg/kg qod caused complete tumor growth inhibition over 28 days in a 22Rv1 xenograft model, including extended duration in the absence of the drug for a further 24 days. This was accompanied by complete inhibition of plasma PSA and significant knockdown of tumor AR-FL, AR-V7, and c-Myc protein as well as c-Myc and TMPRSS2 mRNA expression. At 100mg/kg, a single dose of CCS1477 induces expression of cleaved PARP and when dosed orally every three days, causes tumor regression in the 22Rv1 model. Furthermore, CCS1477 results in almost complete tumor growth inhibition in a bicalutamide-resistant LNCaP xenograft model, when given alone or in combination with enzalutamide. Conclusions: Taken together, these data support the concept that therapeutic targeting of the p300/CBP bromodomain results in a novel and differentiated approach to targeting androgen receptor pathway activation in castration-resistant prostate cancer. Citation Format: Nigel Brooks, Neil Pegg, Jenny Worthington, Barbara Young, Amy Prosser. Therapeutic targeting of the p300/CBP bromodomain for the treatment of castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A091.

The Effect of p53 Status on Radio-Sensitivity of Quiescent Tumor Cell Population Irradiated With γ-Rays at Various Dose Rates.

BackgroundThe aim of the study was to clarify the effect of p53 status of tumor cells on radio-sensitivity of solid tumors following γ-ray irradiation at various dose rates, referring to the response of intratumor quiescent (Q) cells.MethodsHuman head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector (SAS/neo) were injected subcutaneously into hind legs of nude mice. Tumor bearing mice received 5-bromo-2’-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells. They received γ-rays at a high, middle or low dose rate. Immediately or 9 h after the high dose-rate irradiation (HDR, 2.5 Gy/min), or immediately after the middle (MDR, 0.039 Gy/min) or low (LDR, 0.00098 Gy/min) dose-rate irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU.ResultsFollowing γ-ray irradiation, SAS/neo tumor cells, especially intratumor Q cells, showed a marked reduction in sensitivity due to the recovery from radiation-induced damage, compared with the total or Q cells within SAS/mp53 tumors that showed little repair capacity. The recovery capacities following γ-ray irradiation were greater in Q than total cell population and increased in the following order of 9 h after HDR < MDR < LDR. Thus, the difference in radio-sensitivity between the total (P + Q) and Q cells after γ-ray irradiation increased in the same order.ConclusionTo secure controlling solid tumors as a whole, difference in sensitivity between total and Q tumor cells especially in solid tumors irrespective of p53 status has to be suppressed as irradiation dose rate decreases, for instance, through employing combined method for enhancing the response of Q tumor cells.

The Effect of p53 Status of Tumor Cells on Radiosensitivity of Irradiated Tumors With Carbon-Ion Beams Compared With γ-Rays or Reactor Neutron Beams.

BackgroundThe aim of the study was to clarify the effect of p53 status of tumor cells on radiosensitivity of solid tumors following accelerated carbon-ion beam irradiation compared with γ-rays or reactor neutron beams, referring to the response of intratumor quiescent (Q) cells.MethodsHuman head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector (SAS/neo) were injected subcutaneously into hind legs of nude mice. Tumor-bearing mice received 5-bromo-2’-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells. They received γ-rays or accelerated carbon-ion beams at a high or reduced dose-rate. Other tumor-bearing mice received reactor thermal or epithermal neutrons at a reduced dose-rate. Immediately or 9 hours after the high dose-rate irradiation (HDRI), or immediately after the reduced dose-rate irradiation (RDRI), the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU.ResultsThe difference in radiosensitivity between the total (P + Q) and Q cells after γ-ray irradiation was markedly reduced with reactor neutron beams or carbon-ion beams, especially with a higher linear energy transfer (LET) value. Following γ-ray irradiation, SAS/neo tumor cells, especially intratumor Q cells, showed a marked reduction in sensitivity due to the recovery from radiation-induced damage, compared with the total or Q cells within SAS/mp53 tumors that showed little repair capacity. In both total and Q cells within both SAS/neo and SAS/mp53 tumors, carbon-ion beam irradiation, especially with a higher LET, showed little recovery capacity through leaving an interval between HDRI and the assay or decreasing the dose-rate. The recovery from radiation-induced damage after γ-ray irradiation was a p53-dependent event, but little recovery was found after carbon-ion beam irradiation. With RDRI, the radiosensitivity to reactor thermal and epithermal neutron beams was slightly higher than that to carbon-ion beams.ConclusionFor tumor control, including intratumor Q-cell control, accelerated carbon-ion beams, especially with a higher LET, and reactor thermal and epithermal neutron beams were very useful for suppressing the recovery from radiation-induced damage irrespective of p53 status of tumor cells.

The Role of Cys-298 in Aldose Reductase Function

Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.

Abstract SY01-01: Adaptive protein and phosphoprotein networks which promote therapeutic sensitivity or acquired resistance

Abstract SY01-01: Adaptive protein and phosphoprotein networks which promote therapeutic sensitivity or acquired resistance

Aerial audiograms of Steller and California sea lions measured using auditory steady-state response methods.

Detection of aerial vocal signals by conspecifics is important in the reproductive behavior of the otariid pinnipeds. However, aerial hearing sensitivity measurements have only been obtained for a few otariid individuals that were trained to participate in behavioral experiments. In order to expand upon this small data set, auditory steady-state response (ASSR) methods were used to examine the aerial hearing sensitivity of Steller and California sea lions. Although ASSR thresholds were elevated relative to behavioral thresholds reported for otariids, the ASSR audiograms of the majority of individuals were similar to each other and to behavioral audiograms in terms of relative sensitivity. A marked reduction in sensitivity with increasing frequency regularly occurred between 16 and 32 kHz, indicating a consistent high-frequency cutoff. The reliability of the ASSR audiograms for both species suggests that behavioral aerial audiograms that exist for a few Steller and California sea lion individuals can be appropriately extrapolated to larger populations. The similarity of the ASSR audiograms among the Steller and California sea lions supports the notion that the otariid pinnipeds form a functional hearing group, with similar aerial hearing in terms of sensitivity and frequency range of hearing. [Work supported by ONR and NOAA Ocean Acoustics Program.]

Kinase switching in mesenchymal-like non-small cell lung cancer lines contributes to EGFR inhibitor resistance through pathway redundancy

NSCLC cells with a mesenchymal phenotype have shown a marked reduction in sensitivity to EGFR inhibitors, though the molecular rationale has remained obscure. Here we find that in mesenchymal-like tumor cells both tyrosine phosphorylation of EGFR, ErbB2, and ErbB3 signaling networks and expression of EGFR family ligands were decreased. While chronic activation of EGFR can promote an EMT-like transition, once having occurred EGFR family signaling was attenuated. We investigated the mechanisms by which mesenchymal-like cells bypass EGFR signaling and acquire alternative routes of proliferative and survival signaling. Mesenchymal-like NSCLC cells exhibit aberrant PDGFR and FGFR expression and autocrine signaling through these receptors can activate the MEK-ERK and PI3K pathways. Selective pharmacological inhibition of PDGFR or FGFR receptor tyrosine kinases reduced cell proliferation in mesenchymal-like but not epithelial NSCLC cell lines. A metastable, reversible EMT-like transition in the NSCLC line H358 was achieved by exogenous TGFbeta, which served as a model EMT system. The H358/TGFbeta cells showed many of the attributes of established mesenchymal-like NSCLC cells including a loss of cell-cell junctions, a loss of EGF-family ligand expression, a loss of ErbB3 expression, increased EGFR-independent Mek-Erk pathway activation and reduced sensitivity to EGFR inhibition. Notably an EMT-dependent acquisition of PDGFR, FGFR and TGFbeta receptors in H358/TGFbeta cells was also observed. In H358/TGFbeta cells both PDGFR and FGFR showed functional ligand stimulation of their intrinsic tyrosine kinase activities. The findings of kinase switching and acquired PDGFR and FGFR signaling suggest investigation of new inhibitor combinations to target NSCLC metastases.

Dependency of repair of radiation-induced damage on p53 status and efficient inhibition of the repair with wortmannin treatment in vivo, with reference to the effect on intratumor quiescent cell population

To examine the dependency of p53 status and the effect of wortmannin on the repair of radiation-induced damage, referring to the response of intratumor quiescent (Q) cells. Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector (SAS/neo) were injected subcutaneously into left hind legs of nude mice. Tumor-bearing mice received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all intratumor proliferating (P) cells. They received high dose-rate gamma-ray irradiation (HDRI) immediately followed by caffeine or wortmannin administration, or reduced dose-rate gamma-ray irradiation simultaneously with caffeine or wortmannin administration. Nine hours after the start of irradiation, the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (Q cells) was determined using immunofluorescence staining for BrdU. SAS/neo tumor cells, especially intratumor Q cell populations, showed a marked reduction in sensitivity due to the repair of radiation-induced damage, compared with the total or Q cell populations within SAS/mp53 tumors that showed little repair capacity. In both total and Q cell populations within SAS/neo tumors, wortmannin efficiently suppressed the reduction in sensitivity caused by leaving an interval between HDRI and the assay and decreasing the irradiation dose rate. The repair of radiation-induced damage was thought to be a p53-dependent event. From the viewpoint of tumor control, including intratumor Q-cell control, wortmannin treatment in combination with gamma-ray irradiation is thought to be useful for suppressing the repair of radiation-induced damage.

Transient Receptor Potential Vanilloid 1 Mediates Hyperalgesia and Is Up-Regulated in Rats With Chronic Pancreatitis

The neurobiologic basis of pancreatic hyperalgesia in chronic pancreatitis (CP) is understood poorly and there is a need to identify novel therapeutic targets. Our aim was to study the role of the transient receptor potential vanilloid 1 (TRPV1), a key integrator of noxious stimuli, in the pathogenesis of pancreatic pain in a rat model of CP. CP was induced in rats by intraductal injection of trinitrobenzene sulfonic acid. TRPV1 currents in pancreas-specific DRG neurons were measured using perforated patch-clamp techniques. Reverse-transcription polymerase chain reaction was used to measure mRNA expression of TRPV1 in these neurons after laser capture microdissection. Immunofluorescence and Western blot analysis, using TRPV1-specific antibodies, also were performed. Pancreatic hyperalgesia was assessed by rat's nocifensive behavior to electrical stimulation of the pancreas. CP was associated with a 4-fold increase in capsaicin-induced current density (P < .02), along with an increase in the proportion of pancreas-specific DRG neurons that responded to capsaicin (52.9% in controls vs 79.0% in CP; P < .05). CP also was associated with a significant increase in TRPV1 expression both at the messenger RNA and protein level in whole thoracic DRGs and pancreas-specific sensory neurons. Systemic administration of the TRPV1 antagonist SB-366791 markedly reduced both visceral pain behavior and referred somatic hyperalgesia in rats with CP, but not in control animals. TRPV1 up-regulation and sensitization is a specific molecular mechanism contributing to hyperalgesia in CP and represents a useful target for treating pancreatic hyperalgesia caused by inflammation.

The treatment of enteric fever

The treatment of enteric fever

Determination of viral load thresholds in cervical scrapings to rule out CIN 3 in HPV 16, 18, 31 and 33‐positive women with normal cytology

An increased high-risk human papillomavirus (hrHPV) viral load in cervical scrapings has been proposed as a determinant for high-grade cervical intraepithelial neoplasia (CIN) and cervical cancer (> or =CIN 2), but data so far for HPV types different from HPV 16 are limited and inconsistent. In addition, a viral load threshold to distinguish hrHPV positive women without > or =CIN 2 still has not been defined. Here, we used baseline cervical scrapings of women with normal cytology participating in a large population-based cervical screening trial (i.e. POBASCAM) who were GP5+/6+-PCR positive for 4 common hrHPV types, i.e. HPV 16, 18, 31 or 33, as a reference to arbitrarily define various viral load thresholds (i.e. 25th, 33rd, 50th, 67th and 75th percentiles of the lowest viral load values) for distinguishing women having single infections with these types without high-grade CIN. Viral load assessment was performed by real time type-specific PCR. The viral load threshold values were subsequently validated on abnormal cervical scrapes of 162 women with underlying, histologically confirmed CIN lesions containing 1 of these 4 hrHPV types. All 59 women with CIN 3 had viral load levels that were higher than those of 33% of the women with normal cytology containing the respective hrHPV type detectable by GP5+/6+-PCR (i.e. higher than the 33rd percentile of viral load). By using this 33rd percentile viral load cut-off, sensitivity for CIN 3 of 100% (95% CI 93.9-100) was obtained. Hence, application of this viral load threshold would increase the specificity of HPV testing for HPV 16, 18, 31 and 33-associated prevalent CIN 3 without the cost of a marked reduction in sensitivity. In practice, on the basis of viral load analysis, a less aggressive management can be foreseen for 33% of the women with normal cytology participating in a population-based screening program who are GP5+/6+-PCR positive for HPV 16, 18, 31 or 33.

Evolution of multi-drug resistant hepatitis B virus during sequential therapy

Multi-drug resistant hepatitis B virus (HBV) has been reported in hepatitis B patients who received sequential antiviral therapy. In vitro studies showed that HBV constructs with mutations resistant to lamivudine and adefovir have marked reduction in sensitivity to combination of lamivudine and adefovir, whereas constructs with mutations resistant to either drug remain sensitive to the other drug. We conducted this study to determine whether mutations conferring resistance to multiple antiviral agents co-locate on the same HBV genome in vivo and to describe the evolution of these mutations. Sera from six patients who had been found to have multi-drug resistant HBV mutations to lamivudine+adefovir, lamivudine+hepatitis B immunoglobulin (HBIG), or lamivudine+entecavir on direct sequencing were cloned after nested polymerase chain reaction (PCR). Analysis of 215 clones from 11 samples with multi-drug resistant mutations on direct sequencing showed that 183 (85%) clones had mutations to both therapies on the same genome; 31 clones had lamivudine-resistant mutants only. Clonal analysis of serial samples from three patients showed progressive evolution from all clones with lamivudine-resistant HBV mutations only to mixtures of clones that have multi-drug resistant mutations and clones that have lamivudine-resistant HBV mutations only, and ultimately all clones having multi-drug resistant HBV mutations. In conclusion, mutations conferring resistance to multiple antiviral agents co-locate on the same viral genome, suggesting that combination therapy directed against mutants resistant to each treatment may not be adequate in suppressing multi-drug resistant HBV. De novo combination therapy may prevent the emergence of multi-drug resistant mutants.

Functional heterogeneity of ryanodine receptor mutations associated with sudden cardiac death.

Point mutations in the cardiac ryanodine receptor (RyR2) mediate abnormal intracellular Ca(2+) release and are associated with stress-induced ventricular tachycardia (VT), leading to sudden cardiac death (SCD). Although the precise molecular basis of RyR2 dysfunction in SCD remains controversial, there is consensus that the mutations characterised to date all exhibit gain-of-function Ca(2+) release properties following cell stimulation. We investigated the functional impact of a distinct set of SCD-linked RyR2 mutations (L(433)P, N(2386)I, R(176)Q/T(2504)M) on intracellular Ca(2+) handling. We expressed full-length recombinant human wild-type (WT) and SCD-linked RyR2 mutations in human embryonic kidney (HEK) cells, and profiled the spatial and amplitude characteristics of caffeine-evoked Ca(2+) release through homo-tetrameric channels in living cells using rapid confocal laser scanning microscopy. Analysis of the precise mode of Ca(2+) release in HEK cells expressing RyR2 mutants demonstrated profound differences when compared with WT channels. The SCD-linked RyR2 mutations characterised in this study exhibited heterogeneous Ca(2+) release profiles, including the novel observation that one of the mutants, (L(433)P), exhibited a marked reduction in sensitivity to channel activation. However, all SCD-linked RyR2 mutations characterised in this study resulted in an increased duration of elevated cytoplasmic Ca(2+) levels following channel activation. Our live cell-based data demonstrates functional heterogeneity of Ca(2+) release through SCD-linked RyR2 mutants, suggesting that the mechanistic basis of RyR2 dysfunction in SCD may be more complex than previously anticipated. These findings may have profound consequences for the therapeutic modulation of RyR2 in stress-induced VT and SCD.

Insulin resistance in healthy prepubertal twins*1

Objectives To evaluate insulin sensitivity (SI) in prepubertal twins and to examine the relation to reduced birth weight, prematurity, and peroxisome proliferator-activated receptor-γ (PPARγ) polymorphism. Study design Fifty twins (birth weight SDS, −0.7 ± 0.2; gestation, 33.5 ± 0.5 weeks; and body mass index SDS, −0.04 ± 0.2) were studied at 8.2 ± 0.3 years. SI was measured by Bergman's minimal model from a 90 minutes frequently sampled intravenous glucose test. Twenty control children (height SDS, −1.7 ± 0.3; birth weight SDS, −0.3 ± 0.2; and gestation of 39.2 ± 0.7 weeks) were also evaluated at 7.0 ± 0.4 years. The PPARγ T-variant polymorphism was evaluated in 41 twins. Values are expressed as mean ± SEM, or 95% confidence intervals. Results SI was reduced in twins compared with control subjects, (12.7 [11-15] versus 23.0 [16.8-31.4] 10−4 min−1 μU/mL, respectively, P = .005). The reduction in SI was independent of prematurity and birth weight and zygosity (P<.0001). There was no difference in SI, even in twin pairs with >20% difference in birth weight (P = .9). The PPARγ heterozygote T-variant polymorphism was present in 7 of 41, with a further reduction in SI (P = .03) and a later gestation (P = .03). These twins also had increased fat mass (P = .02) but with similar fat free mass (P = .14). Conclusions Twin children, independent of prematurity or birth weight, had a marked reduction in SI. To use twins as a model to study the fetal origins of adult diseases for glucose homeostasis is not valid.

An N-terminal histidine regulates Zn(2+) inhibition on the murine GABA(A) receptor beta3 subunit.

1. Whole-cell currents were recorded from Xenopus laevis oocytes and human embryonic kidney cells expressing GABA(A) receptor beta3 subunit homomers to search for additional residues affecting Zn(2+) inhibition. These residues would complement the previously identified histidine (H267), present just within the external portal of the ion channel, which modulates Zn(2+) inhibition. 2. Zinc inhibited the pentobarbitone-gated current on beta3(H267A) homomers at pH 7.4, but this effect was abolished at pH 5.4. The Zn(2+)-sensitive spontaneous beta3 subunit-mediated conductance was also insensitive to block by Zn(2+) at pH 5.4. 3. Changing external pH enabled the titration of the Zn(2+) sensitive binding site or signal transduction domain. The pK(a) was estimated at 6.8 +/- 0.03 implying the involvement of histidine residues. 4. External histidine residues in the beta3 receptor subunit were substituted with alanine, in addition to the background mutation, H267A, to assess their sensitivity to Zn(2+) inhibition. The Zn(2+) IC(50) was unaffected by either the H119A or H191A mutations. 5. The remaining histidine, H107, the only other candidate likely to participate in Zn(2+) inhibition, was substituted with various residues. Most mutants were expressed at the cell surface but they disrupted functional expression of beta3 homomers. However, H107G was functional and demonstrated a marked reduction in sensitivity to Zn(2+). 6. GABA(A) receptor beta3 subunits form functional ion channels that can be inhibited by Zn(2+). Two histidine residues are largely responsible for this effect, H267 in the pore lining region and H107 residing in the extracellular N-terminal domain.