Insulin resistance and intestinal integrity in children with and without HIV infection in Uganda.

OBJECTIVES: The risk of cardiometabolic complications in children with perinatally acquired HIV infection (PHIVs) and in perinatally HIV-exposed but uninfected children (HEUs) and its relationship to systemic inflammation and markers of gut integrity are not well established. In this current study, we assed insulin resitance in PHIV compared to HEUs and HIV unexposed uninfected children and explored potential association with intestinal damage biomarkers. METHODS: This was a cross-sectional study in PHIVs, HEUs and HIV-unexposed, uninfected children (HUUs) aged 2-10 years enrolled in Uganda. PHIVs were on stable antiretroviral therapy (ART) with HIV viral load  < 400 HIV-1 RNA copies/mL. Insulin resistance was estimated using the homeostasis model assessment of insulin resistance (HOMA-IR). We measured markers of systemic inflammation, monocyte activation and gut integrity. Kruskal-Wallis tests were used to compare markers by HIV status; Pearson correlation and multiple linear regressions were used to assess associations of the HOMA-IR index with biomarkers of intestinal damage and translocation. RESULTS: Overall, 172 participants were enrolled in the study (57 PHIVs, 59 HEUs and 56 HUUs). The median age was 7.8 [interquartile range (IQR) 6.39, 8.84] years, 55% were female and the median body mass index (BMI) was 15 (IQR 14.3, 15.8) kg/m2 . Among PHIVs, the median CD4% was 37%, and 93% had viral load ≤ 20 copies/mL. PHIVs had higher waist:hip ratio, high-density lipoprotein (HDL) cholesterol, triglycerides and HOMA-IR index than the other groups (P ≤ 0.02). Factors correlated with insulin resistance included higher BMI and HDL cholesterol and lower soluble tumour necrosis factor receptor I (sTNFRI) (P ≤ 0.02). There was no correlation between any of the other inflammatory or gut biomarkers and HOMA-IR index (P ≥ 0.05). After adjusting for age and sTNFRI, BMI remained independently associated with the HOMA-IR index (ß = 0.16; P < 0.01). CONCLUSIONS: Despite viral suppression, Ugandan PHIVs have disturbances in glucose metabolism. Higher BMI, and not immune activation or alteration of gut integrity, was associated with insulin resistance in this population.

Tildrakizumab (MK-3222), an anti-interleukin-23p19 monoclonal antibody, improves psoriasis in a phase IIb randomized placebo-controlled trial.

BACKGROUND: Tildrakizumab is a high-affinity, humanized, IgG1/κ, anti-interleukin (IL)-23p19 monoclonal antibody that does not bind human IL-12 or p40 is being developed for the treatment of chronic plaque psoriasis. OBJECTIVES: To evaluate the safety and efficacy of subcutaneous tildrakizumab in patients with moderate-to-severe chronic plaque psoriasis. METHODS: A three-part, randomized, double-blind, phase IIb trial was conducted in 355 adults with chronic plaque psoriasis. Participants were randomized to receive subcutaneous tildrakizumab (5, 25, 100, 200 mg) or placebo at weeks 0 and 4 (part I) and every 12 weeks thereafter until week 52 (part II). Study drug was discontinued at week 52 and participants were followed through week 72 (part III). Primary efficacy end point was Psoriasis Area and Severity Index (PASI) 75 response at week 16. Adverse events (AEs) and vital signs were monitored throughout the study. RESULTS: At week 16, PASI 75 responses were 33·3% (n = 14), 64·4% (n = 58), 66·3% (n = 59), 74·4% (n = 64) and 4·4% (n = 2) in the 5-, 25-, 100- and 200-mg tildrakizumab and placebo groups, respectively (P ≤ 0·001 for each tildrakizumab dose vs. placebo). PASI 75 response was generally maintained through week 52; only eight of 222 participants who achieved PASI 75 response at week 52 and continued to part III relapsed following discontinuation up to week 72. Possible drug-related serious AEs included bacterial arthritis and lymphoedema (part I), and melanoma, stroke, epiglottitis and knee infection (part II). CONCLUSIONS: Tildrakizumab had treatment effects that were superior to placebo, maintained for 52 weeks of treatment, and persisted for 20 weeks after cessation. Tildrakizumab was generally safe and well tolerated. These results suggest that IL-23p19 is a key target for suppressing psoriasis.

Cigarette smoke mediates epigenetic repression of miR-217 during esophageal adenocarcinogenesis.

Although microRNAs (miRs) have been implicated in the pathogenesis of various human malignancies, limited information is available regarding mechanisms by which these noncoding RNAs contribute to initiation and progression of tobacco-induced esophageal cancers. In this study, array and quantitative reverse transcriptase-PCR techniques were used to examine miR expression in immortalized esophageal epithelia (IEE) and esophageal adenocarcinoma (EAC) cells cultured in normal media with or without cigarette smoke condensate (CSC). Under relevant exposure conditions, CSC significantly decreased miR-217 expression in these cells. Endogenous levels of miR-217 expression in cultured EAC cells (EACC)/primary EACs were significantly lower than those observed in IEE/ paired normal esophageal tissues. RNA crosslink immunoprecipitation, quantitative reverse transcriptase-PCR (qRT-PCR) and immunoblot experiments demonstrated direct interaction of miR-217 with kallikrein 7 (KLK7), encoding a putative oncogene not previously implicated in EAC. Repression of miR-217 correlated with increased levels of KLK7 in primary EACs, particularly those from smokers. Chromatin and methylated DNA immunoprecipitation experiments demonstrated that CSC-mediated repression of miR-217 coincided with DNMT3b-dependent hypermethylation and decreased occupancy of nuclear factor 1 within the miR-217 genomic locus. Deoxyazacytidine induced miR-217 expression and downregulated KLK7 in EACC; deoxyazacytidine also attenuated CSC-mediated miR-217 repression and upregulation of KLK7 in IEE and EACC. Overexpression of miR-217 significantly decreased, whereas overexpression of KLK7 increased proliferation, invasion and tumorigenicity of EACC. Collectively, these data demonstrate that epigenetic repression of miR-217 contributes to the pathogenesis of EAC via upregulation of KLK7 and suggest that restoration of miR-217 expression may be a novel treatment strategy for these malignancies.

Interleukin-23 is sufficient to induce rapid de novo gut tumorigenesis, independent of carcinogens, through activation of innate lymphoid cells.

Chronic inflammation has been associated with increased risk for developing gastrointestinal cancer. Interleukin-23 (IL-23) receptor signaling has been correlated with inflammatory bowel disease pathogenesis, as well as promotion of tumor growth. However, little is known about the relative potential for IL-23-directed causality in gut tumorigenesis. We report that IL-23 transgene expression was sufficient to induce rapid (3-4 weeks) de novo development of intestinal adenomas with 100% incidence. Initiation of tumorigenesis was independent of exogenous carcinogens, Helicobacter colonization, or pre-existing tumor-suppressor gene mutations. Tumorigenesis was mediated by Thy1(+)IL-23R(+) innate lymphoid cells (ILC3), in part, through IL-17 responses as tumor development was inhibited in RAG(-/-) × IL-17(-/-) double knockout mice. Remarkably, IL-23 initiation of tumorigenesis by resident ILCs consistently occurred before recruitment of conspicuous inflammatory infiltrates. Our results reveal an explicit role for IL-23-mediated initiation of gut tumorigenesis and implicate a key role for IL-23R(+) ILC3 in the absence of overt cellular infiltrate recruitment.

Lifestyle factors correlate with the risk of late pelvic symptoms after prostatic radiotherapy.

AIMS: Although technological improvements are reducing the risks of late side-effects of radiotherapy for prostate cancer, the influence of lifestyle has been less well examined. The aim of this study was to correlate the effects of exercise, body mass index and smoking on the incidence and severity of late pelvic symptoms after radical radiotherapy for prostate cancer. MATERIALS AND METHODS: In total, 440 men completed a questionnaire consisting of the Vaizey rectal symptoms score, the National Cancer Institute common symptoms scores for rectal bleeding, erectile function and urinary incontinence, the General Practice Physical Activity Questionnaire and questions regarding smoking, height and weight. The effect of each lifestyle factor on pelvic symptoms was investigated using a non-parametric multivariate ANOVA (Kruskal-Wallis) test and the chi-squared test. RESULTS: At the time of the survey, 63.3% of men were overweight or obese (body mass index > 25 kg/m(2)) and 71% were inactive or moderately inactive. Active men had significantly lower rectal symptoms scores (P < 0.001) and better erectile (P < 0.001) and urinary function (P < 0.01). Men smoking more than five a day had higher rectal symptoms scores (P < 0.001), as did overweight men (P < 0.05). CONCLUSION: The data show lower late pelvic symptoms after radiotherapy among non-smokers and physically active individuals with a body mass index <25. Although these results would ideally require confirmation in a prospective study, we now include advice on lifestyle in our pre-radiotherapy information pack. The high percentage of obesity and inactivity among this cohort of prostate cancer survivors revealed in this study has prompted the development of an exercise/weight reduction programme in our unit.

Subcortical volumetric reductions in adult Niemann-Pick disease type C: a cross-sectional study.

BACKGROUND AND PURPOSE: Voxel-based analysis has suggested that deep gray matter rather than cortical regions is initially affected in adult Niemann-Pick type C. We sought to examine a range of deep gray matter structures in adults with NPC and relate these to clinical variables. MATERIALS AND METHODS: Ten adult patients with NPC (18-49 years of age) were compared with 27 age- and sex-matched controls, and subcortical structures were automatically segmented from normalized T1-weighted MR images. Absolute volumes (in cubic millimeters) were generated for a range of deep gray matter structures and were compared between groups and correlated with illness variables. RESULTS: Most structures were smaller in patients with NPC compared with controls. The thalamus, hippocampus, and striatum showed the greatest and most significant reductions, and left hippocampal volume correlated with symptom score and cognition. Vertex analysis of the thalamus, hippocampus, and caudate implicated regions involved in memory, executive function, and motor control. CONCLUSIONS: Thalamic and hippocampal reductions may underpin the memory and executive deficits seen in adult NPC. Volume losses in other subcortical regions may also be involved in the characteristic range of motor, psychiatric, and cognitive deficits seen in the disease.

Pontine-to-midbrain ratio indexes ocular-motor function and illness stage in adult Niemann-Pick disease type C.

BACKGROUND AND PURPOSE: Niemann-Pick disease type C (NPC) is a progressive neurovisceral disorder associated with dystonia, ataxia and a characteristic gaze palsy. Neuropathological studies have demonstrated brainstem atrophy associated with neuronal inclusions and loss, and neurofibrillary tangles, although it is not known whether this pathology can be detected in vivo or how these changes relate to illness variables, particularly ocular-motor changes. Our aim was to utilize a method for brainstem atrophy, validated in progressive supranuclear palsy (PSP), in a group of adult patients with NPC, and explore its relationship to illness variables and ocular-motor functioning. METHODS: We calculated the midbrain and pontine area, and pontine-to-midbrain ratio (PMR) from midsagittal images of 10 adult patients with NPC and 27 age- and gender-matched controls. Measures were correlated with illness variables, and measures of horizontal saccadic functioning. RESULTS: Pontine-to-midbrain ratio was 14% higher in the NPC group, but this difference was not significant. However, PMR showed a significant positive correlation with duration of illness and a measure of illness severity. Furthermore, PMR was significantly negatively correlated with saccadic peak velocity and gain, and self-paced saccadic performance. CONCLUSIONS: Pontine-to-midbrain ratio was increased in adult patients with NPC compared to controls, although not to the same degree as previously described in PSP, which also presents with significant gaze palsy. These changes were driven predominantly by progressive midbrain atrophy. The strong correlation with illness and ocular-motor variables suggests that it may be a useful marker for illness progression in NPC.

Size and shape of the corpus callosum in adult Niemann-Pick type C reflects state and trait illness variables.

BACKGROUND AND PURPOSE: Variable alterations to the structure of the corpus callosum have been described in adults with NPC, a neurometabolic disorder known to result in both white and gray matter pathology. This study sought to examine the structure of the callosum in a group of adult patients with NPC and compared callosal structure with a group of matched controls, and to relate callosal structure with state and trait illness variables. MATERIALS AND METHODS: Nine adult patients with NPC were matched to control subjects (n = 26) on age and sex. The corpus callosum was segmented from the midsagittal section of T1-weighted images on all subjects, and total area, length, bending angle, and mean thickness were calculated. In addition, 39 regional thickness measures were derived by using a previously published method. All measures were compared between groups, and analyzed alongside symptom measures, biochemical parameters, and ocular-motor measures. RESULTS: The callosal area and mean thickness were significantly reduced in the patient group, and regional thickness differences were greatest in the genu, posterior body, isthmus, and anterior splenium. Global callosal measures correlated significantly with duration of illness and symptom score, and at trend level with degree of filipin staining. Measures of reflexive saccadic peak velocity and gain, and self-paced saccades, correlated strongly with total callosal area. CONCLUSIONS: Callosal structure and size reflect both state and trait markers in adult NPC, and they may be useful biomarkers to index both white and gray matter changes that reflect illness severity and progression.

Frequent homozygous deletion of the LKB1/STK11 gene in non-small cell lung cancer.

LKB1/STK11 is a tumor suppressor and a negative regulator of mammalian target of rapamycin signaling. It is inactivated in 30% of lung cancer cell lines but only 5-15% of primary lung adenocarcinomas. There is evidence that homozygous deletion (HD) of chromosome 19p at the LKB locus contributes to the inactivation of the gene in primary human lung cancers. Here, we used several complementary genetic approaches to assess the LKB1 locus in primary non-small cell lung cancers (NSCLCs). We first analyzed 124 NSCLC cases for allelic imbalance using eight microsatellite markers on chromosome 19p, which revealed an overall rate of 65% (80 of 124) loss of heterozygosity (LOH). We next used chromogenic in situ hybridization (CISH) to directly examine the chromosomal status of the LKB1 locus. In all, 65 of 124 LOH tested samples were available for CISH and 58 of those (89%) showed either loss of one copy of chromosome 19p (LOH, 40 of 65 cases, 62%) or both copies (HD 18 of 65 cases, 28%). The occurrence of HD was significantly more frequent in Caucasian (35%) than in African-American patients (6%) (P=0.04). A total of 62 of 124 samples with LOH at one or both markers immediately flanking the LKB1 gene were further analyzed by directly sequencing the complete coding region, which identified 7 of 62 (11%) tumors with somatic mutations in the gene. Jointly, our data identified total inactivation of the LKB1 gene by either HD or LOH with somatic mutation in 39% of tested samples, whereas loss of chromosome 19p region by HD or LOH at the LKB1 region occured in 90% of NSCLC.

Gender dimorphism in siblings with schizophrenia-like psychosis due to Niemann-Pick disease type C.

We describe the differential presentation of schizophrenia-like psychosis in two siblings with the 'variant' biochemical presentation of adult Niemann-Pick disease type C. The male sibling presented with psychosis at age 16 years and cognitive and motor disturbance at age 25 years, whereas his elder sister, sharing the same mutation but showing less severe biochemical, neuroimaging and ocular motor parameters, presented with a similar schizophrenia-like illness with associated cognitive and motor disturbance at age 31 years. Their illness onset, course and response to treatment mirrors the sex dimorphism seen in schizophrenia, and is suggestive of an interaction between the neurobiology of their metabolic disorder and sex differences in neurodevelopment.

Saccades in adult Niemann-Pick disease type C reflect frontal, brainstem, and biochemical deficits.

BACKGROUND: The autosomal recessive disorder Niemannn-Pick type C (NPC) presents in adulthood with psychosis or cognitive deficits associated with supranuclear gaze palsies. While saccadic innervation to the extraocular muscles is generated in the brainstem, the frontal lobes play an integral role in the initiation of volitional saccades and the suppression of unwanted reflexive saccades. No study has examined the frontally driven volitional control of saccadic eye movements in NPC. OBJECTIVE: To examine self-paced and antisaccades as well as reflexive saccades in adult patients with NPC, a disorder known to affect brainstem and frontal cortical function. METHODS: Three biochemically confirmed adult patients with NPC were compared with 10 matched controls on horizontal saccadic and antisaccadic measures using an infrared limbus eye tracker. Patients' cholesterol esterification and filipin staining, Mini-Mental State performance, and NPC symptom level were rated. RESULTS: Reflexive saccade latency ranged from shorter to longer than normal, reflexive saccade gain was reduced, asymptotic peak velocity was reduced, fewer self-paced saccades were generated, and increased errors on antisaccades were made by patients compared to controls. Patients with more severe biochemical, cognitive, and symptom deficits performed most poorly on brainstem and frontal ocular motor measures. Paradoxically, less severe illness was associated with an abnormally reduced saccadic latency. CONCLUSIONS: Ocular motor measures provide an index of disease severity in Niemannn-Pick type C (NPC) and may be a useful adjunct for monitoring the illness progress and medication response. Reduced saccadic latency may result from inadequate fixation input from abnormally functioning frontal eye fields in NPC.

Neurons in dopamine-rich areas of the rat medial midbrain predominantly encode the outcome-related rather than behavioural switching properties of conditioned stimuli.

Midbrain dopamine neurons are phasically activated by a variety of sensory stimuli. It has been hypothesized that these activations contribute to reward prediction or behavioural switching. To test the latter hypothesis we recorded from 131 single neurons in the ventral tegmental area and retrorubral field of thirsty rats responding during a modified go/no-go task. One-quarter (n = 33) of these neurons responded to conditioned stimuli in the task, which varied according to the outcome with which they were associated (saccharin or quinine solution) and according to whether they triggered a switch in the ongoing sequence of the animal's behaviour ('behavioural switching'). Almost half the neurons (45%) responded differentially to saccharin- vs. quinine-conditioned stimuli; the activity of a minority (15%) correlated with an aspect of behavioural switching (mostly exhibiting changes from baseline activity in the absence of a behavioural switch) and one-third (33%) encoded various outcome-switch combinations. The strongest response was excitation to the saccharin-conditioned stimulus. Additionally, a proportion (38%) of neurons responded during outcome delivery, typically exhibiting inhibition during saccharin consumption. The neurons sampled did not fall into distinct clusters on the basis of their electrophysiological characteristics. However, most neurons that responded to the outcome-related properties of conditioned stimuli had long action potentials (> 1.2 ms), a reported characteristic of dopamine neurons. Moreover, responses to saccharin-conditioned stimuli were functionally akin to dopamine responses found in the macaque and rat nucleus accumbens responses observed within the same task. In conclusion, our data are more consistent with the reward-prediction than the behavioural switching hypothesis.

Development of a behavioral task measuring reward "wanting" and "liking" in rats.

It has been suggested that reward "wanting" and "liking" are mediated by separable brain systems. To facilitate neuropharmacological and neurophysiological research on this issue we developed a behavioral task with putative measures of reward "wanting" and "liking" available on a trial-by-trial basis. We were able to test whether our measures were sensitive to changes in thirsty rats' "wanting" and "liking" of liquid reward by manipulating its delay, taste and volume. We found that three of our putative "wanting" measures (anticipatory errors, reaction time and reward collection latency) were affected by upcoming reward delay and/or taste and our putative "liking" measure (post-reward licking) was sensitive to variations in reward taste and volume. To cross-validate our measures with previous pharmacological work we tested rats following acute, systemic administration of drug compounds that globally enhance serotonin and noradrenaline (imipramine), dopamine (GBR 12909) and opioid (morphine) function. Imipramine augmented the effects of delay and taste on reward "wanting", GBR 12909 attenuated the effects of delay on reward "wanting" and the effects of taste on reward "liking", and morphine reduced the effect of delay on a measure of reward "wanting". Since morphine failed to affect reward "liking" but has been previously found to enhance reward "liking" in taste reactivity tests, our measure requires further pharmacological validation. However, this task shows potential to assess the specific neural mechanisms that contribute to the impact of reward parameters on "wanting" and "liking".

Rat nucleus accumbens neurons predominantly respond to the outcome-related properties of conditioned stimuli rather than their behavioral-switching properties.

It has been proposed that nucleus accumbens neurons respond to outcome (reward and punishment) and outcome-predictive information. Alternatively, it has been suggested that these neurons respond to salient stimuli, regardless of their outcome-predictive properties, to facilitate a switch in ongoing behavior. We recorded the activity of 82 single-nucleus accumbens neurons in thirsty rats responding within a modified go/no-go task. The task design allowed us to analyze whether neurons responded to conditioned stimuli that predicted rewarding (saccharin) or aversive (quinine) outcomes, and whether the neural responses correlated with behavioral switching. Approximately one third (28/82) of nucleus accumbens neurons exhibited 35 responses to conditioned stimuli. Over 2/3 of these responses encoded the nature of the upcoming rewarding (19/35) or aversive (5/35) outcome. No response was selective solely for the switching of the rat's behavior, although the activity of approximately one third of responses (11/35) predicted the upcoming outcome and was correlated with the presence or absence of a subsequent behavioral switch. Our data suggest a primary functional role for the nucleus accumbens in encoding outcome-predicting information and a more limited role in behavioral switching.

Circulating clonal CLA(+) and CD4(+) T cells in Sezary syndrome express the skin-homing chemokine receptors CCR4 and CCR10 as well as the lymph node-homing chemokine receptor CCR7.

BACKGROUND: Adhesion molecules and chemokine receptors are involved in tissue-specific homing of T cells to the skin and play an important role in the pathophysiology of cutaneous lymphoma. It has recently been reported that the chemokine CCL27 expressed by keratinocytes attracts lymphocytes bearing the chemokine receptor CCR10. OBJECTIVES: To investigate the expression of CCR4, CCR7 and CCR10 on skin-homing CLA(+) and CD4(+) T cells in the peripheral blood of patients with Sezary syndrome (SS), a rare leukaemic variant of cutaneous T-cell lymphoma. METHODS: Lymphocytes from five patients with SS, six patients with mycosis fungoides and four healthy volunteers were isolated and analysed using flow cytometry. Additionally, the T-cell receptor (TCR)-Vbeta CDR3 regions were cloned and sequenced in two patients. RESULTS: We found that CCR4 is expressed on almost all CLA(+) and CD4(+) memory T cells. Using monoclonal antibodies specific for single TCR-Vbeta chains we identified malignant T cells in four patients with SS. Importantly, we found that most but not all malignant Sezary cells expressed the skin-homing chemokine receptor CCR10. Additionally, we found that a significant proportion of these cells also expressed the lymph node-homing chemokine receptor CCR7. CONCLUSIONS: Our results support the concept that chemokine receptors play an important role in the pathophysiology of SS and suggest that the malignant clone may represent an expansion of skin-homing cutaneous 'central' memory T cells in the peripheral blood of these patients.

Second-order stimuli do not always increase overall response rates in second-order schedules of reinforcement in the rat.

RATIONALE: Second-order schedules of reinforcement have been used extensively to model reward-seeking and drug-seeking behaviour. Second-order stimuli within second-order schedules have been shown to enhance response rates during operant responding for natural reinforcers and drug reinforcers. This has led some to view second-order schedules of drug reinforcement as a model maintained of drug-seeking in addicts by drug-associated stimuli. However, the functional role of the second-order stimulus within second-order schedules is complex. OBJECTIVE: We investigated the role of second-order stimuli within a second-order schedule of reinforcement [FI 4 min (FR10: S)] maintained by sweetened water reinforcement. METHODS: Eight rats were trained to press a bar on a second-order schedule of reinforcement and tested in the presence and absence of the second-order stimulus. RESULTS: In contrast to most previous work, overall bar-pressing rates were significantly increased when the second-order stimulus was omitted (second-order stimulus omission: 0.17 Hz (+/-0.04, 95% CI); second-order stimulus present: 0.13 Hz (+/-0.04, 95% CI)). However, second-order stimuli also changed the pattern of responding whereby rats would make a bout of bar presses prior to the presentation of the second-order stimulus and then pause briefly after the second-order stimulus. In the absence of second-order stimuli, responding was uniformly high. Control measures, such as the ability of the second-order stimulus to evoke checking for the primary reinforcers, indicated that the second-order stimulus was associated with the primary reinforcer. CONCLUSIONS: These results demonstrated that although second-order stimuli maintained responding and caused the rat to check for primary reinforcement, overall response rates were increased when the second-order stimuli were omitted. This has implications for interpreting the results of studies where overall response rates within second-order schedules have been the only measure used to assess the effects of potential anti-addiction drugs. Future studies could be improved by performing a second-order stimulus omission test analysing both the overall response rates and the temporal organization of responding with respect to the second-order stimulus.

Functional significance of XPD polymorphic variants: attenuated apoptosis in human lymphoblastoid cells with the XPD 312 Asp/Asp genotype.

Recent molecular epidemiological studies have identified polymorphisms in the XPD gene that are associated with increased risk of brain gliomas and head, neck, lung, and skin cancers. However, the functional significance of these polymorphic variants in altering cell processes such as cell cycle checkpoints, DNA repair, and apoptosis is uncertain. We have cloned the XPD variants Lys751Gln, Asp312Asn, and Lys751Gln-Asp312Asn into a pcDNA-3.1-expression vector. Using these constructs, we did not find any detectable difference in either in vitro binding with wild-type p53 or in DNA repair proficiency as measured by host cell reactivation assay. We then genotyped 34 different lymphoblastoid cell lines from six Centre d'Etude du Polymorphisme Humaine (CEPH)/Utah pedigree families and a CEPH/French pedigree family for polymorphisms at codons 751 and 312 and assessed their apoptotic response after either UV or ionized radiation exposure. The lymphoblastoid cell lines with homozygous or heterozygous Asp at codon 312 have similar apoptotic rates, whereas cell lines with homozygous Asn at codon 312 showed a 2.5-fold increased response to UV (P = 0.005; Student's t test). This is the first report known to us of a functional polymorphism in a gene involved in DNA damage-induced apoptosis. However, the presence of Lys or Gln at codon 751 did not influence the apoptotic response to UV. The diminished apoptotic response of cells containing the 312 Asp allele could both allow the survival and selective clonal expansion of carcinogen-damaged cells and be a mechanistic explanation for the increased risk of cancer at diverse tissue sites.

Smoking increases carcinogenic polycyclic aromatic hydrocarbons in human lung tissue.

Tobacco smoke is a major source of human exposure to polycyclic aromatic hydrocarbons (PAHs). The concentration of PAHs in lung tissue would reflect an individual's dose, and its variation could perhaps reflect cancer risk. Eleven PAHs were measured in 70 lung tissue samples from cancer-free autopsy donors by gas chromatography-mass spectrometry. There were 37 smokers and 33 nonsmokers as estimated by serum cotinine concentration. The sum of PAH concentrations was higher in smokers (P = 0.01), and there was a dose-response relationship for greater smoking (P < 0.01). Smoking increased the concentration of five PAHs including benzo(a)pyrene, which increased approximately 2-fold. The risk for increasing carcinogenic PAHs (odds ratio, 8.20; 95% confidence interval, 2.39-28.09) was 3-fold compared with noncarcinogenic PAHs (odds ratio, 2.61; 95% confidence interval, 0.75-9.12). A higher concentration of PAHs was detected in the lung tissue of males, although the estimated smoking was similar in males and females. Race was not associated with PAH concentrations overall, but PAH concentrations appeared to be higher in African-American males than in any other group. Age was weakly correlated with an increase in fluoranthene and pyrene. The measurement of PAHs in human lung tissue can be used to estimate the actual dose to the target organ.

Discovery of a novel antitumor benzolactone enamide class that selectively inhibits mammalian vacuolar-type (H+)-atpases.

A series of naturally occurring compounds reported recently by multiple laboratories defines a new small-molecule class sharing a unique benzolactone enamide core structure and diverse biological actions, including inhibition of growth of tumor cells and oncogene-transformed cell lines. Here we show that representative members of this class, including salicylihalamide A, lobatamides A-F, and oximidines I and II inhibit mammalian vacuolar-type (H+)-ATPases (V-ATPases) with unprecedented selectivity. Data derived from the NCI 60-cell antitumor screen critically predicted the V-ATPase molecular target, while specific biochemical assays provided confirmation and further illumination. The compounds potently blocked representative V-ATPases from human kidney, liver, and osteoclastic giant-cell tumor of bone but were essentially inactive against V-ATPases of Neurospora crassa and Saccharomyces cerevisiae and other membrane ATPases. Essential regulation of pH in cytoplasmic, intraorganellar, and local extracellular spaces is provided by V-ATPases, which are ubiquitously distributed among eukaryotic cells and tissues. The most potent and selective V-ATPase inhibitors heretofore known were the bafilomycins and concanamycins, which do not discriminate between mammalian and nonmammalian V-ATPases. Numerous physiological processes are mediated by V-ATPases, and aberrant V-ATPase functions are implicated in many different human diseases. Previous efforts to develop therapeutic pharmacological modulators of V-ATPases have been frustrated by a lack of synthetically tractable and biologically selective leads. Therefore, availability of the unique benzolactone enamide inhibitor class may enable further elucidation of functional and architectural features of mammalian versus nonmammalian V-ATPase isoforms and provide new opportunities for targeting V-ATPase-mediated processes implicated in diverse pathophysiological phenomena, including cancer.

The differential diagnosis of epilepsy, pseudoseizures, dissociative identity disorder, and dissociative disorder not otherwise specified.

The authors review the co-occurrences of dissociative symptoms and disorders with epilepsy and pseudo-seizures and examine newer diagnostic instruments that assist in accurate diagnosis of persons with concomitant seizure behaviors and dissociative symptoms. They also review seizure behaviors and electroencephalographic findings in persons with dissociative identity disorder (DID) and dissociative disorder not otherwise specified (DDNOS) and dissociative symptoms in persons with epilepsy and with pseudoseizures. Dissociative symptoms in 15 patients with epilepsy and 15 with pseudo-seizures were examined using the Dissociative Experiences Scale (DES) and the Structured Clinical Interview for DSM-IV Dissociative Disorders (SCID-D). On the SCID-D, pseudo-seizure patients had significantly higher dissociative symptom scores than epileptic patients, but DES scores did not reliably distinguish epileptic and pseudo-seizure patients. Misdiagnosis of persons with seizures and dissociative symptoms can be avoided by careful adherence to DSM dissociative disorder criteria, the use of video-EEG monitoring, and systematic assessment of dissociative symptoms with the SCID-D.