A polycistronic transgene design for combinatorial genetic perturbations from a single transcript in Drosophila.

Experimental models that capture the genetic complexity of human disease and allow mechanistic explorations of the underlying cell, tissue, and organ interactions are crucial to furthering our understanding of disease biology. Such models require combinatorial manipulations of multiple genes, often in more than one tissue at once. The ability to perform complex genetic manipulations in vivo is a key strength of Drosophila, where many tools for sophisticated and orthogonal genetic perturbations exist. However, combining the large number of transgenes required to establish more representative disease models and conducting mechanistic studies in these already complex genetic backgrounds is challenging. Here we present a design that pushes the limits of Drosophila genetics by allowing targeted combinatorial ectopic expression and knockdown of multiple genes from a single inducible transgene. The polycistronic transcript encoded by this transgene includes a synthetic short hairpin cluster cloned within an intron placed at the 5' end of the transcript, followed by two protein-coding sequences separated by the T2A sequence that mediates ribosome skipping. This technology is particularly useful for modeling genetically complex diseases like cancer, which typically involve concurrent activation of multiple oncogenes and loss of multiple tumor suppressors. Furthermore, consolidating multiple genetic perturbations into a single transgene further streamlines the ability to perform combinatorial genetic manipulations and makes it readily adaptable to a broad palette of transgenic systems. This flexible design for combinatorial genetic perturbations will also be a valuable tool for functionally exploring multigenic gene signatures identified from omics studies of human disease and creating humanized Drosophila models to characterize disease-associated variants in human genes. It can also be adapted for studying biological processes underlying normal tissue homeostasis and development that require simultaneous manipulation of many genes.

Germline mutations of breast cancer susceptibility genes through expanded genetic analysis in unselected Colombian patients.

BACKGROUND: In Colombia and worldwide, breast cancer (BC) is the most frequently diagnosed neoplasia and the leading cause of death from cancer among women. Studies predominantly involve hereditary and familial cases, demonstrating a gap in the literature regarding the identification of germline mutations in unselected patients from Latin-America. Identification of pathogenic/likely pathogenic (P/LP) variants is important for shaping national genetic analysis policies, genetic counseling, and early detection strategies. The present study included 400 women with unselected breast cancer (BC), in whom we analyzed ten genes, using Whole Exome Sequencing (WES), know to confer risk for BC, with the aim of determining the genomic profile of previously unreported P/LP variants in the affected population. Additionally, Multiplex Ligation-dependent Probe Amplification (MLPA) was performed to identify Large Genomic Rearrangements (LGRs) in the BRCA1/2 genes. To ascertain the functional impact of a recurrent intronic variant (ATM c.5496 + 2_5496 + 5delTAAG), a minigene assay was conducted. RESULTS: We ascertained the frequency of P/LP germline variants in BRCA2 (2.5%), ATM (1.25%), BRCA1 (0.75%), PALB2 (0.50%), CHEK2 (0.50%), BARD1 (0.25%), and RAD51D (0.25%) genes in the population of study. P/LP variants account for 6% of the total population analyzed. No LGRs were detected in our study. We identified 1.75% of recurrent variants in BRCA2 and ATM genes. One of them corresponds to the ATM c.5496 + 2_5496 + 5delTAAG. Functional validation of this variant demonstrated a splicing alteration probably modifying the Pincer domain and subsequent protein structure. CONCLUSION: This study described for the first time the genomic profile of ten risk genes in Colombian women with unselected BC. Our findings underscore the significance of population-based research, advocating the consideration of molecular testing in all women with cancer.

Early Life and Childhood Environmental Exposures, More Than Genetic Predisposition, Influence Age of Diagnosis in a Diverse Cohort of 2952 Patients With IBD.

BACKGROUND AND AIMS: Inflammatory bowel disease (IBD) develops from a combination of genetic and environmental factors. The aim of this study was to determine the contribution of established environmental risk factors and genetic risk on age of IBD diagnosis in a diverse cohort. METHODS: IBD patients in clinic completed detailed questionnaires. Blood was drawn for genetic analysis. Environmental risk factors and age of diagnosis were analyzed by ethnicity (Hispanic/Latinx or non-Hispanic White [NHW] individuals) and IBD subtype (ulcerative colitis or Crohn's disease [CD]). Weighted genetic risk scores and environmental risk scores were developed. We examined the relationship between environmental risk scores, genetic risk scores, and age of diagnosis. RESULTS: A total of 2952 patients were included: 58.9% had CD. A total of 46.83% were of Hispanic background. Early life exposures like cesarean delivery and being born in a developed country were associated with a younger age of IBD diagnosis. Childhood exposures such as frequent plastic water bottle use and having more than 1 bathroom at home were associated with a younger age of IBD. Hispanic and NHW individuals shared similar susceptibilities to environmental exposures. Environmental factors explained 21% of the variance in age of CD diagnosis and 39% in ulcerative colitis. In models incorporating genetic risk score and environmental risk score, the environment was the only significant factor associated with younger age of IBD diagnosis in all groups. CONCLUSIONS: Early life and childhood exposures impact IBD diagnosis and influence Hispanic and NHW individuals similarly. A cumulative environmental risk score contributes more to age of IBD diagnosis than genetic risk.

Role of Mitochondria in Inflammatory Bowel Diseases: A Systematic Review.

Mitochondria are key cellular organelles whose main function is maintaining cell bioenergetics by producing ATP through oxidative phosphorylation. However, mitochondria are involved in a much higher number of cellular processes. Mitochondria are the home of key metabolic pathways like the tricarboxylic acid cycle and ß-oxidation of fatty acids, as well as biosynthetic pathways of key products like nucleotides and amino acids, the control of the redox balance of the cell and detoxifying the cell from H2S and NH3. This plethora of critical functions within the cell is the reason mitochondrial function is involved in several complex disorders (apart from pure mitochondrial disorders), among them inflammatory bowel diseases (IBD). IBD are a group of chronic, inflammatory disorders of the gut, mainly composed of ulcerative colitis and Crohn's disease. In this review, we present the current knowledge regarding the impact of mitochondrial dysfunction in the context of IBD. The role of mitochondria in both intestinal mucosa and immune cell populations are discussed, as well as the role of mitochondrial function in mechanisms like mucosal repair, the microbiota- and brain-gut axes and the development of colitis-associated colorectal cancer.

Synergistic Deoxynucleoside and Gene Therapies for Thymidine Kinase 2 Deficiency.

OBJECTIVE: Autosomal recessive human thymidine kinase 2 (TK2) mutations cause TK2 deficiency, which typically manifests as a progressive and fatal mitochondrial myopathy in infants and children. Treatment with pyrimidine deoxynucleosides deoxycytidine and thymidine ameliorates mitochondrial defects and extends the lifespan of Tk2 knock-in mouse (Tk2KI ) and compassionate use deoxynucleoside therapy in TK2 deficient patients have shown promising indications of efficacy. To augment therapy for Tk2 deficiency, we assessed gene therapy alone and in combination with deoxynucleoside therapy in Tk2KI mice. METHODS: We generated pAAVsc CB6 PI vectors containing human TK2 cDNA (TK2). Adeno-associated virus (AAV)-TK2 was administered to Tk2KI , which were serially assessed for weight, motor functions, and survival as well as biochemical functions in tissues. AAV-TK2 treated mice were further treated with deoxynucleosides. RESULTS: AAV9 delivery of human TK2 cDNA to Tk2KI mice efficiently rescued Tk2 activity in all the tissues tested except the kidneys, delayed disease onset, and increased lifespan. Sequential treatment of Tk2KI mice with AAV9 first followed by AAV2 at different ages allowed us to reduce the viral dose while further prolonging the lifespan. Furthermore, addition of deoxycytidine and deoxythymidine supplementation to AAV9 + AAV2 treated Tk2KI mice dramatically improved mtDNA copy numbers in the liver and kidneys, animal growth, and lifespan. INTERPRETATION: Our data indicate that AAV-TK2 gene therapy as well as combination deoxynucleoside and gene therapies is more effective in Tk2KI mice than pharmacological alone. Thus, combination of gene therapy with substrate enhancement is a promising therapeutic approach for TK2 deficiency and potentially other metabolic disorders. ANN NEUROL 2021;90:640-652.

Translation and validation of the “personal evaluation of transitions in treatment (PETIT)” scale for people with schizophrenia.

To adapt the ‘Personal Evaluation of Transitions in Treatment (PETIT)’ scale into Spanish and analyse its psychometric properties on schizophrenic population.

Cardiomyopathy and altered integrin-actin signaling in Fhl1 mutant female mice.

X-linked scapuloperoneal myopathy (X-SM), one of Four-and-a-half LIM 1 (FHL1) related diseases, is an adult-onset slowly progressive myopathy, often associated with cardiomyopathy. We previously generated a knock-in mouse model that has the same mutation (c.365 G > C, p.W122S) as human X-SM patients. The mutant male mouse developed late-onset slowly progressive myopathy without cardiomyopathy. In this study, we observed that heterozygous (Het) and homozygous (Homo) female mice did not show alterations of skeletal muscle function or histology. In contrast, 20-month-old mutant female mice showed signs of cardiomyopathy on echocardiograms with increased systolic diameter [wild-type (WT): 2.74 ± 0.22 mm, mean ± standard deviation (SD); Het: 3.13 ± 0.11 mm, P < 0.01; Homo: 3.08 ± 0.37 mm, P < 0.05) and lower fractional shortening (WT: 31.1 ± 4.4%, mean ± SD; Het: 22.7 ± 2.5%, P < 0.01; Homo: 22.4 ± 6.9%, P < 0.01]. Histological analysis of cardiac muscle revealed frequent extraordinarily large rectangular nuclei in mutant female mice that were also observed in human cardiac muscle from X-SM patients. Western blot demonstrated decreased Fhl1 protein levels in cardiac muscle, but not in skeletal muscle, of Homo mutant female mice. Proteomic analysis of cardiac muscle from 20-month-old Homo mutant female mice indicated abnormalities of the integrin signaling pathway (ISP) in association with cardiac dysfunction. The ISP dysregulation was further supported by altered levels of a subunit of the ISP downstream effectors Arpc1a in Fhl1 mutant mice and ARPC1A in X-SM patient muscles. This study reveals the first mouse model of FHL1-related cardiomyopathy and implicates ISP dysregulation in the pathogenesis of FHL1 myopathy.

Low-Fat, High-Fiber Diet Reduces Markers of Inflammation and Dysbiosis and Improves Quality of Life in Patients With Ulcerative Colitis.

BACKGROUND & AIMS: A high-fat diet has been associated with an increased risk of ulcerative colitis (UC). We studied the effects of a low-fat, high-fiber diet (LFD) vs an improved standard American diet (iSAD, included higher quantities of fruits, vegetables, and fiber than a typical SAD). We collected data on quality of life, markers of inflammation, and fecal markers of intestinal dysbiosis in patients with UC. METHODS: We analyzed data from a parallel-group, cross-over study of 17 patients with UC in remission or with mild disease (with a flare within the past 18 mo), from February 25, 2015, through September 11, 2018. Participants were assigned randomly to 2 groups and received a LFD (10% of calories from fat) or an iSAD (35%-40% of calories from fat) for the first 4-week period, followed by a 2-week washout period, and then switched to the other diet for 4 weeks. All diets were catered and delivered to patients' homes, and each participant served as her or his own control. Serum and stool samples were collected at baseline and week 4 of each diet and analyzed for markers of inflammation. We performed 16s ribosomal RNA sequencing and untargeted and targeted metabolomic analyses on stool samples. The primary outcome was quality of life, which was measured by the short inflammatory bowel disease (IBD) questionnaire at baseline and week 4 of the diets. Secondary outcomes included changes in the Short-Form 36 health survey, partial Mayo score, markers of inflammation, microbiome and metabolome analysis, and adherence to the diet. RESULTS: Participants' baseline diets were unhealthier than either study diet. All patients remained in remission throughout the study period. Compared with baseline, the iSAD and LFD each increased quality of life, based on the short IBD questionnaire and Short-Form 36 health survey scores (baseline short IBD questionnaire score, 4.98; iSAD, 5.55; LFD, 5.77; baseline vs iSAD, P = .02; baseline vs LFD, P = .001). Serum amyloid A decreased significantly from 7.99 mg/L at baseline to 4.50 mg/L after LFD (P = .02), but did not decrease significantly compared with iSAD (7.20 mg/L; iSAD vs LFD, P = .07). The serum level of C-reactive protein decreased numerically from 3.23 mg/L at baseline to 2.51 mg/L after LFD (P = .07). The relative abundance of Actinobacteria in fecal samples decreased from 13.69% at baseline to 7.82% after LFD (P = .017), whereas the relative abundance of Bacteroidetes increased from 14.6% at baseline to 24.02% on LFD (P = .015). The relative abundance of Faecalibacterium prausnitzii was higher after 4 weeks on the LFD (7.20%) compared with iSAD (5.37%; P = .04). Fecal levels of acetate (an anti-inflammatory metabolite) increased from a relative abundance of 40.37 at baseline to 42.52 on the iSAD and 53.98 on the LFD (baseline vs LFD, P = .05; iSAD vs LFD, P = .09). The fecal level of tryptophan decreased from a relative abundance of 1.33 at baseline to 1.08 on the iSAD (P = .43), but increased to a relative abundance of 2.27 on the LFD (baseline vs LFD, P = .04; iSAD vs LFD, P = .08); fecal levels of lauric acid decreased after LFD (baseline, 203.4; iSAD, 381.4; LFD, 29.91; baseline vs LFD, P = .04; iSAD vs LFD, P = .02). CONCLUSIONS: In a cross-over study of patients with UC in remission, we found that a catered LFD or iSAD were each well tolerated and increased quality of life. However, the LFD decreased markers of inflammation and reduced intestinal dysbiosis in fecal samples. Dietary interventions therefore might benefit patients with UC in remission. ClinicalTrials.gov no: NCT04147598.

Leukocyte and Dried Blood Spot Arylsulfatase A Assay by Tandem Mass Spectrometry.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays were developed to measure arylsulfatase A (ARSA) activity in leukocytes and dried blood spots (DBS) using deuterated natural sulfatide substrate. These new assays were highly specific and sensitive. Patients with metachromatic leukodystrophy (MLD) and multiple sulfatase deficiency (MSD) displayed a clear deficit in the enzymatic activity and could be completely distinguished from normal controls. The leukocyte assay reported here will be important for diagnosing MLD and MSD patients and for monitoring the efficacy of therapeutic treatments. ARSA activity was measured in DBS for the first time without an antibody. This new ARSA DBS assay can serve as a second-tier test following the sulfatide measurement in DBS for newborn screening of MLD. This leads to an elimination of most of the false positives identified by the sulfatide assay.

Genes Involved in Galactooligosaccharide Metabolism in Lactobacillus reuteri and Their Ecological Role in the Gastrointestinal Tract.

Strains of Lactobacillus reuteri are commonly used as probiotics due to their demonstrated therapeutic properties. Many strains of L. reuteri also utilize the prebiotic galactooligosaccharide (GOS), providing a basis for formulating synergistic synbiotics that could enhance growth or persistence of this organism in vivo In this study, in-frame deletion mutants were constructed to characterize the molecular basis of GOS utilization in L. reuteri ATCC PTA-6475. Results suggested that GOS transport relies on a permease encoded by lacS, while a second unidentified protein may function as a galactoside transporter. Two ß-galactosidases, encoded by lacA and lacLM, sequentially degrade GOS oligosaccharides and GOS disaccharides, respectively. Inactivation of lacL and lacM resulted in impaired growth in the presence of GOS and lactose. In vitro competition experiments between the wild-type and ΔlacS ΔlacM strains revealed that the GOS-utilizing genes conferred a selective advantage in media with GOS but not glucose. GOS also provided an advantage to the wild-type strain in experiments in gnotobiotic mice but only on a purified, no sucrose diet. Differences in cell numbers between GOS-fed mice and mice that did not receive GOS were small, suggesting that carbohydrates other than GOS were sufficient to support growth. On a complex diet, the ΔlacS ΔlacM strain was outcompeted by the wild-type strain in gnotobiotic mice, suggesting that lacL and lacM are involved in the utilization of alternative dietary carbohydrates. Indeed, the growth of the mutants was impaired in raffinose and stachyose, which are common in plants, demonstrating that α-galactosides may constitute alternate substrates of the GOS pathway.IMPORTANCE This study shows that lac genes in Lactobacillus reuteri encode hydrolases and transporters that are necessary for the metabolism of GOS, as well as α-galactoside substrates. Coculture experiments with the wild-type strain and a gos mutant clearly demonstrated that GOS utilization confers a growth advantage in medium containing GOS as the sole carbohydrate source. However, the wild-type strain also outcompeted the mutant in germfree mice, suggesting that GOS genes in L. reuteri also provide a basis for utilization of other carbohydrates, including α-galactosides, ordinarily present in the diets of humans and other animals. Collectively, our work provides information on the metabolism of L. reuteri in its natural niche in the gut and may provide a basis for the development of synbiotic strategies.

Inflammatory Cytokine Profile in Crohn's Disease Nonresponders to Optimal Antitumor Necrosis Factor Therapy.

BACKGROUND: A significant number of patients receiving therapy with antitumor necrosis factor (TNF) agents for Crohn's disease experience primary or secondary nonresponse. The aim of this study was to assess whether patients with nonresponse to anti-TNF agents have increased expression of alternative cytokine pathways. METHODS: We designed a prospective, cross-sectional study that included patients with Crohn's disease receiving anti-TNF undergoing colonoscopy with adequate serum trough drug levels (≥8 µg/mL) and without anti-drug antibodies. Inflammatory cytokines and cell adhesions markers measured included intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1, interleukin (IL)-8, IL-1ß, and IL-6. The primary outcome was the presence of active endoscopic inflammation defined as the presence of at least 1 ulceration ≥5 mm. RESULTS: In total, 47 patients were included. Patients with active inflammation had significantly higher levels of ICAM-1 and IL-1ß when compared with those without intestinal inflammation (45.9 vs. 35.8 ng/mL, P<0.0001 and 3.2 vs. 1.5 pg/mL, P=0.002, respectively). There were no significant differences in the other study variables. Using receiving operating curves, ICAM and IL-1ß had a good correlation (receiver operating characteristic ≥0.8) with inflammation in this cohort of patients with "anti-TNF resistance." The results were similar in the group of patients with previous anti-TNF exposure. CONCLUSION: Our study suggests that patients who have active inflammation with seemingly adequate serum anti-TNF levels have increased levels of specific inflammatory pathways that may serve as biomarkers of nonresponse as well as potential targets of therapy in anti-TNF nonresponders.

General anesthesia safety in progressive leukodystrophies: A retrospective study of patients with Krabbe disease and metachromatic leukodystrophy.

BACKGROUND: Krabbe disease and metachromatic leukodystrophy are leukodystrophies characterized by neurologic degeneration and early death. Patients often require general anesthesia for diagnostic and therapeutic interventions. METHODS: A retrospective review of medical records was conducted for patients with Krabbe disease and metachromatic leukodystrophy receiving general anesthesia at a large children's hospital between 2012 and 2017. Patient complications and American Society of Anesthesiologists Physical Status were recorded for all procedures. The Neurodevelopment in Rare Disorders classification system was created to categorize the severity of the patient's disease progression based on clinical markers. Descriptive and inferential statistics were used to compare: (a) complication rate of affected patients vs the general hospital population; (b) the accuracy of the novel Neurodevelopment in Rare Disorders classification system vs American Society of Anesthesiologists Physical Status regarding the assessment of complication risk; (c) complication rate in patients with hematopoietic stem cell transplantation vs those without transplantation; (d) complication rate in immunosuppressed patients vs nonimmunosuppressed patients; and (e) complication rate of the three most commonly performed procedures. RESULTS: A total of 96 patients underwent 287 procedures. Of these, 11 cases had complications, yielding a rate of 3.8%. This is significantly higher than the overall complication rate at our institution of 0.246%. Statistical analysis showed better correlation between the Neurodevelopment in Rare Disorders classification system and complication rate than American Society of Anesthesiologists Physical Status and complication rate. The system also showed better accuracy in differentiating low-risk and high-risk patients. No statistically significant difference in complication rate was found for patients with transplantation vs those without transplantation or for immunosuppressed vs nonimmunosuppressed patients. Of the three most common procedures, central catheter placement/removal exhibited the highest complication rate. CONCLUSIONS: Although the complication rate for patients with Krabbe disease and metachromatic leukodystrophy is higher than the general population, most complications were mild and self-limiting. These results suggest that, in experienced hands, general anesthesia is well tolerated in most children. Findings show that the Neurodevelopment in Rare Disorders classification system is a better indicator for assessing complication risk in patients with Krabbe and metachromatic leukodystrophy than American Society of Anesthesiologists Physical Status.

Association of Antibodies to VAR2CSA and Merozoite Antigens with Pregnancy Outcomes in Women Living in Yaoundé, Cameroon.

Plasmodium falciparum infections are serious in pregnant women, because VAR2CSA allows parasitized erythrocytes to sequester in the placenta, causing placental malaria (PM). In areas of endemicity, women have substantial malarial immunity prior to pregnancy, including antibodies to merozoite antigens, but produce antibodies to VAR2CSA only during pregnancy. The current study sought to determine the importance of antibodies to VAR2CSA and merozoite antigens in pregnant women in Yaoundé, Cameroon, where malaria transmission was relatively low. A total of 1,377 archival plasma samples collected at delivery were selected (at a 1:3 ratio of PM-positive [PM+] to PM-negative [PM-] women) and screened for antibodies to full-length VAR2CSA and 7 merozoite antigens. Results showed that many PM+ women and most PM- women lacked antibodies to VAR2CSA at delivery. Among PM+ women, antibodies to VAR2CSA were associated with a reduced risk of having high placental parasitemia (odds ratio [OR], 0.432; confidence interval [CI], 0.272, 0.687; P = 0.0004) and low-birth-weight (LBW) babies (OR = 0.444; CI, 0.247, 0.799; P = 0.0068), even during first pregnancies. Among antibodies to the 7 merozoite antigens, i.e., AMA1, EBA-175, MSP142, MSP2, MSP3, MSP11, and Pf41, only antibodies to MSP3, EBA-175, and Pf41 were associated with reduced risk for high placental parasitemias (P = 0.0389, 0.0291, and 0.0211, respectively) and antibodies to EBA-175 were associated with reduced risk of premature deliveries (P = 0.0211). However, after adjusting for multiple comparisons significance declined. Thus, in PM+ women, antibodies to VAR2CSA were associated with lower placental parasitemias and reduced prevalence of LBW babies in this low-transmission setting.

Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency.

OBJECTIVE: Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2-/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. METHODS: To test these hypotheses, we assessed two therapies in Tk2-/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. RESULTS: We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2-/- animals compared to dCMP+dTMP. INTERPRETATION: Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652.

Theoretical perspectives on central chemosensitivity: CO2/H+-sensitive neurons in the locus coeruleus.

Central chemoreceptors are highly sensitive neurons that respond to changes in pH and CO2 levels. An increase in CO2/H+ typically reflects a rise in the firing rate of these neurons, which stimulates an increase in ventilation. Here, we present an ionic current model that reproduces the basic electrophysiological activity of individual CO2/H+-sensitive neurons from the locus coeruleus (LC). We used this model to explore chemoreceptor discharge patterns in response to electrical and chemical stimuli. The modeled neurons showed both stimulus-evoked activity and spontaneous activity under physiological parameters. Neuronal responses to electrical and chemical stimulation showed specific firing patterns of spike frequency adaptation, postinhibitory rebound, and post-stimulation recovery. Conversely, the response to chemical stimulation alone (based on physiological CO2/H+ changes), in the absence of external depolarizing stimulation, showed no signs of postinhibitory rebound or post-stimulation recovery, and no depolarizing sag. A sensitivity analysis for the firing-rate response to the different stimuli revealed that the contribution of an applied stimulus current exceeded that of the chemical signals. The firing-rate response increased indefinitely with injected depolarizing current, but reached saturation with chemical stimuli. Our computational model reproduced the regular pacemaker-like spiking pattern, action potential shape, and most of the membrane properties that characterize CO2/H+-sensitive neurons from the locus coeruleus. This validates the model and highlights its potential as a tool for studying the cellular mechanisms underlying the altered central chemosensitivity present in a variety of disorders such as sudden infant death syndrome, depression, and anxiety. In addition, the model results suggest that small external electrical signals play a greater role in determining the chemosensitive response to changes in CO2/H+ than previously thought. This highlights the importance of considering electrical synaptic transmission in studies of intrinsic chemosensitivity.

Hispanics Coming to the US Adopt US Cultural Behaviors and Eat Less Healthy: Implications for Development of Inflammatory Bowel Disease.

INTRODUCTION: The incidence of inflammatory bowel disease (IBD) among US Hispanics is rising. Adoption of an American diet and/or US acculturation may help explain this rise. AIMS: To measure changes in diet occurring with immigration to the USA in IBD patients and controls, and to compare US acculturation between Hispanics with versus without IBD. Last, we examine the current diet of Hispanics with IBD compared to the diet of Hispanic controls. METHODS: This was a cross-sectional study of Hispanic immigrants with and without IBD. Participants were recruited from a university-based GI clinic. All participants completed an abbreviated version of the Stephenson Multi-Group Acculturation Scale and a 24-h diet recall (the ASA-24). Diet quality was calculated using the Healthy Eating Index (HEI-2010). RESULTS: We included 58 participants: 29 controls and 29 IBD patients. Most participants were Cuban or Colombian. Most participants, particularly those with IBD, reported changing their diet after immigration (72% of IBD and 57% of controls). IBD participants and controls scored similarly on US and Hispanic acculturation measures. IBD patients and controls scored equally poorly on the HEI-2010, although they differed on specific measures of poor intake. IBD patients reported a higher intake of refined grains and lower consumption of fruits, whereas controls reported higher intake of empty calories (derived from fat and alcohol). CONCLUSION: The majority of Hispanics change their diet upon immigration to the USA and eat poorly irrespective of the presence of IBD. Future studies should examine gene-diet interactions to better understand underlying causes of IBD in Hispanics.

Psychopathological profile and prevalence of dual pathology on patients with alcoholic dependence undergoing outpatient treatment.

AIMS: Assess the prevalence of dual pathology in patients with alcohol dependence and describe the psychopathological profile of mental disorders, impulsiveness, ADHD presence and craving. METHOD: It is a cross-sectional study about dual pathology, carried out on 102 patients undergoing outpatient treatment. The presence of dual pathology is established by means of the MINI-5 interview and the MCMI-III test; DSM-IV being used as the alcohol abuse criteria. Impulsiveness, ADHD presence, craving and quality of life were measured through SIS, ASRSv1, MACS and SF-36. RESULTS: The prevalence of dual pathology ranges from 45.1% to 80.4% according to MCMI-III and MINI-5, respectively. The most frequent pathologies are current major depressive episodes, followed by current generalized anxiety disorders, suicide risk and current dysthymia disorders; 73.2% of dual patients present a moderate and intense global score according to MACS, 56.1% got a meaningful score in impulsiveness according to SIS and 41.5% has highly consistent symptoms with ADHD. As regards quality of life, 53.7% of the sample had bad mental health. In the case of dual patients consuming other substances, 30% had a history of bipolar disorders and 10% had a high suicide risk. CONCLUSIONS: The prevalence of psychiatric comorbidity in patients with alcohol dependence undergoing outpatient treatment varies depending on the detection method, MINI being the one identifying a greater number of cases. More than half of dual patients present impulsive behavior, a bad mental health state and high craving levels. Special attention should be paid to dual patients consuming other substances.

The association of tissue anti-TNF drug levels with serological and endoscopic disease activity in inflammatory bowel disease: the ATLAS study.

OBJECTIVE: The aim of this study was to assess the correlation between serum and intestinal anti-tumour necrosis factor (TNF) levels, and their relationship to endoscopic disease activity and levels of TNF. DESIGN: Cross-sectional study of 30 patients receiving treatment with infliximab or adalimumab for Crohn's disease or UC. For each patient, a sample of serum was matched to tissue biopsies. Endoscopic and histological disease activity was recorded for each tissue sample. RESULTS: There was a significant positive correlation between anti-TNF in serum and tissue (r=0.3920, p=0.002), especially in uninflamed tissue (r=0.50, p<0.001), but not with those samples that had inflammation (r=0.19, p=0.54). Anti-TNF concentration in tissue correlated with degree of endoscopic inflammation, except for tissue with severe inflammation in which anti-TNF levels were again lower (mean normalised anti-TNF in tissue: uninflamed=0.93, mild=2.17, moderate=13.71, severe=2.2 inflammation (p=0.0042)). The ratio of anti-TNF-to-TNF in tissue was highest in uninflamed areas and lowest in severely inflamed areas. Patients with active mucosal disease had a higher rate of serum to tissue drug level mismatch when compared to those in remission (73.3% vs 33.3%, respectively; p=0.03). CONCLUSIONS: Our data suggest that local tissue inflammation characterised by high levels of TNF serves as a sink for anti-TNF. We further postulate that some patients with high serum anti-TNF levels have active disease because tissue levels of anti-TNF are insufficient to neutralise local TNF production.

A case-control study of innate immunity pathway gene polymorphisms in Puerto Ricans reveals association of toll-like receptor 2 +596 variant with asthma.

BACKGROUND: For many years, African Dust Storms (ADE) has been thought to be associated with high prevalence of asthma in Puerto Rico (PR). Endotoxins (ENX) have been associated with ADE particulate matter (PM) and are known to promote pro-inflammatory responses in lung cells of susceptible individuals through the Toll-like receptor (TLR2/4) signaling pathways. Genetic variants are plausible contributors to such susceptibility. Therefore, we have evaluated a series of nine single nucleotide polymorphisms (SNPs) in TLR genes, which have been correlated positive and negatively to asthma prevalence and/or risk, in the Puerto Rican asthmatic population. METHODS: The following SNPs were evaluated in 62 asthmatics and 61 controls through Taqman® Real Time PCR Assay: TLR4 (+896A/G, +1196C/T, -6687A/G); TLR2 (+596C/T, -16934 T/A, +399A/G, +1349C/T) and CD14 (-159C/T, +1188C/G). Genotypes were assessed for asthma association employing an odds ratio (OR) analysis. RESULTS: Minor allele frequencies (n = 123) were determined for those variants as 0.07, 0.06, 0.35, 0.35, 0.37, 0.29, 0.04, 0.35 and 0.11, respectively. Two (+596C/T, +399A/G) TLR2 SNPs showed to be more represented in the asthmatic group by 89 % and 65 %, respectively. TLR4 SNP +896A/G analysis revealed only 1 G/G genotype (2 %) on the asthmatic group. The CD14 SNPs were similarly represented in the Puerto Rican population. Only the TLR2 +596 SNP was found to be significantly associated to asthma (OR = 3.24 for CT, 2.71 for TT) and particularly to females. CONCLUSIONS: The identification of TLR SNPs will reveal potential candidates for gene-environment interactions in Puerto Ricans. As far as we know this is the first study to evaluate this type of TLR gene polymorphisms in Puerto Rican asthmatics, contributing to the current knowledge in the Hispanic population.

Concentrations of 6-thioguanine nucleotide correlate with trough levels of infliximab in patients with inflammatory bowel disease on combination therapy.

BACKGROUND & AIMS: In patients with inflammatory bowel diseases, the combination of infliximab and thiopurines (such as 6-thioguanine) is more effective treatment than monotherapy. We assessed the correlation between serum levels of 6-thioguanine (6-TGN) and infliximab levels or antibodies to infliximab (ATI). METHODS: We performed a cross-sectional study of 72 patients receiving maintenance therapy with infliximab and a thiopurine for inflammatory bowel disease at the Crohn's and Colitis Center of the University of Miami, FL. We collected clinical, endoscopic, and biochemical data, and levels of thiopurine metabolites. The primary outcomes were trough level of infliximab and the presence of ATI. RESULTS: Levels of 6-TGN correlated with those of infliximab (ρ, 0.53; P < .0001). The cut-off point of 6-TGN that best predicted a higher level of infliximab was 125 pmol/8 × 10(8) red blood cells (RBCs) (area under receiver operating characteristic, 0.86; P < .001). Patients in the lowest quartile of 6-TGN had infliximab levels that were similar to patients on no thiopurines (4.3 vs. 4.8 mcg/mL, respectively; P = .8). An infliximab level of 8.3 mcg/mL or greater was associated with mucosal healing. Only 8 patients (11%) had detectable ATI. Patients with 6-TGN levels less than 125 pmol/8 × 10(8) RBCs were significantly more likely to have ATI (odds ratio, 1.3; 95% confidence interval, 2.3-72.5; P < .01). CONCLUSIONS: Although 6-TGN levels of greater than 230 pmol/8 × 10(8) RBCs have been associated with improved outcomes in patients on monotherapy, a level of 6-thioguanine of 125 pmol/8 × 10(8) RBCs or greater may be adequate to achieve therapeutic levels of infliximab. In the long term, this may minimize the toxicity for patients on combination therapy.