Lauranell H. Burch

Loss-of-Function Mutation in Tryptophan Hydroxylase-2 Identified in Unipolar Major Depression

Dysregulation of central serotonin neurotransmission has been widely suspected as an important contributor to major depression. Here, we identify a (G1463A) single nucleotide polymorphism (SNP) in the rate-limiting enzyme of neuronal serotonin synthesis, human tryptophan hydroxylase-2 (hTPH2). The functional SNP in hTPH2 replaces the highly conserved Arg441 with His, which results in approximately 80% loss of function in serotonin production when hTPH2 is expressed in PC12 cells. Strikingly, SNP analysis in a cohort of 87 patients with unipolar major depression revealed that nine patients carried the mutant (1463A) allele, while among 219 controls, three subjects carried this mutation. In addition, this functional SNP was not found in a cohort of 60 bipolar disorder patients. Identification of a loss-of-function mutation in hTPH2 suggests that defect in brain serotonin synthesis may represent an important risk factor for unipolar major depression.

IL-35 production by inducible costimulator (ICOS)–positive regulatory T cells reverses established IL-17–dependent allergic airways disease

BACKGROUND Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined. OBJECTIVE We sought to identify the regulatory cells and molecules that suppress IL-17-dependent allergic airways disease. METHODS Mice were sensitized by means of airway instillations of ovalbumin together with low levels of LPS. Leukocyte recruitment to the lung and AHR were assessed after daily challenges with aerosolized ovalbumin. Flow cytometry, quantitative PCR, and gene-targeted mice were used to identify naturally arising subsets of regulatory T (Treg) cells and their cytokines required for the suppression of established allergic airway disease. RESULTS Allergic sensitization through the airway primed both effector and regulatory responses. Effector responses were initially dominant and led to airway inflammation and IL-17-dependent AHR. However, after multiple daily allergen challenges, IL-17 production and AHR decreased, even though pulmonary levels of T(H)17 cells remained high. This loss of AHR was reversible and required the expansion of a Treg cell subset expressing both forkhead box protein 3 and inducible costimulator. These Treg cells also expressed the regulatory cytokines IL-10, TGF-β, and IL-35. Whereas IL-10 and TGF-β were dispensable for suppression of AHR, IL-35 was required. CONCLUSION IL-35 production by inducible costimulator-positive Treg cells can suppress IL-17 production and thereby reverse established, IL-17-dependent AHR in mice. Targeting this pathway might therefore be of therapeutic value for treating allergic asthma in human subjects.

Plasminogen Alleles Influence Susceptibility to Invasive Aspergillosis

Invasive aspergillosis (IA) is a common and life-threatening infection in immunocompromised individuals. A number of environmental and epidemiologic risk factors for developing IA have been identified. However, genetic factors that affect risk for developing IA have not been clearly identified. We report that host genetic differences influence outcome following establishment of pulmonary aspergillosis in an exogenously immune suppressed mouse model. Computational haplotype-based genetic analysis indicated that genetic variation within the biologically plausible positional candidate gene plasminogen (Plg; Gene ID 18855) correlated with murine outcome. There was a single nonsynonymous coding change (Gly110Ser) where the minor allele was found in all of the susceptible strains, but not in the resistant strains. A nonsynonymous single nucleotide polymorphism (Asp472Asn) was also identified in the human homolog (PLG; Gene ID 5340). An association study within a cohort of 236 allogeneic hematopoietic stem cell transplant (HSCT) recipients revealed that alleles at this SNP significantly affected the risk of developing IA after HSCT. Furthermore, we demonstrated that plasminogen directly binds to Aspergillus fumigatus. We propose that genetic variation within the plasminogen pathway influences the pathogenesis of this invasive fungal infection.

Mechanisms of allergy and clinical immunologyIL-35 production by inducible costimulator (ICOS)–positive regulatory T cells reverses established IL-17–dependent allergic airways disease

BACKGROUND Recent evidence suggests that IL-17 contributes to airway hyperresponsiveness (AHR); however, the mechanisms that suppress the production of this cytokine remain poorly defined. OBJECTIVE We sought to identify the regulatory cells and molecules that suppress IL-17-dependent allergic airways disease. METHODS Mice were sensitized by means of airway instillations of ovalbumin together with low levels of LPS. Leukocyte recruitment to the lung and AHR were assessed after daily challenges with aerosolized ovalbumin. Flow cytometry, quantitative PCR, and gene-targeted mice were used to identify naturally arising subsets of regulatory T (Treg) cells and their cytokines required for the suppression of established allergic airway disease. RESULTS Allergic sensitization through the airway primed both effector and regulatory responses. Effector responses were initially dominant and led to airway inflammation and IL-17-dependent AHR. However, after multiple daily allergen challenges, IL-17 production and AHR decreased, even though pulmonary levels of T(H)17 cells remained high. This loss of AHR was reversible and required the expansion of a Treg cell subset expressing both forkhead box protein 3 and inducible costimulator. These Treg cells also expressed the regulatory cytokines IL-10, TGF-β, and IL-35. Whereas IL-10 and TGF-β were dispensable for suppression of AHR, IL-35 was required. CONCLUSION IL-35 production by inducible costimulator-positive Treg cells can suppress IL-17 production and thereby reverse established, IL-17-dependent AHR in mice. Targeting this pathway might therefore be of therapeutic value for treating allergic asthma in human subjects.

Donor polymorphisms in Toll-like receptor-4 influence the development of rejection after renal transplantation

Abstract:  Background:  Although innate immunity is crucial to host defense against pathogens, the extent to which innate immune mechanisms participate in the rejection of allogenic tissues in humans is unknown. We hypothesize that activation of innate immunity through Toll‐like receptors (TLRs) critically regulates the development of renal allograft rejection. We have recently demonstrated decreased acute rejection in lung transplant recipients heterozygous for either of two functional polymorphisms in TLR4 associated with endotoxin hyporesponsiveness. In the present investigation, we sought to evaluate the role of innate immune activation through TLR4, in either donor or recipient, upon the development of renal allograft rejection.

Genetic regulation of rejection and survival following human lung transplantation by the innate immune receptor CD14.

We have developed the hypothesis that genetic polymorphisms which alter the expression or function of innate immune receptors contribute to the marked interindividual differences in the onset and severity of lung transplant rejection. In this analysis, we considered the effects of a common promotor polymorphism of the lipopolysaccharide receptor CD14 associated with increased transcriptional activity upon the development of posttransplant rejection and graft survival. Genotyping was performed in 226 lung transplant recipients well characterized with regards to clinical outcomes. An earlier onset of acute rejection, bronchiolitis obliterans syndrome (BOS) and worse posttransplant graft survival due to greater BOS related deaths was evident in patients with the CD14 –159 TT genotype (TT). The adverse effect upon graft survival of the TT genotype remained significant in a multivariate Cox model (Hazard Ratio 1.65, 95% CI, 1.03–2.64, p‐value = 0.04) after adjusting for other important covariates. Furthermore, TT patients have significantly greater sCD14, TNF‐α and IFN‐γ in the peripheral blood implying a heightened state of innate immune activation drives the development of increased post‐transplant rejection. Inhibition of innate immune activation through CD14 represents a novel and potentially important therapeutic target to prevent post‐transplant rejection and improve outcomes after human lung transplantation.

Genetic Variation in Soluble Epoxide Hydrolase (EPHX2) Is Associated With Forearm Vasodilator Responses in Humans

Cytochrome P450-derived epoxyeicosatrienoic acids are potent vasodilators in preclinical models and are hydrolyzed by soluble epoxide hydrolase (EPHX2). Associations between the EPHX2 Lys55Arg and Arg287Gln polymorphisms and cardiovascular disease risk have been reported; however, their impact on vascular function in humans has not been investigated. In 265 volunteers (198 white, 67 black American), forearm blood flow was measured by strain-gauge venous occlusion plethysmography at baseline and in response to bradykinin, methacholine, and sodium nitroprusside. Forearm vascular resistance was calculated as mean arterial pressure/forearm blood flow. In white Americans, Lys55Arg genotype was associated with vasodilator response to bradykinin, such that forearm blood flow was significantly lower (P=0.043) and forearm vascular resistance was significantly higher (P=0.013) in Arg55 variant allele carriers compared to wild-type individuals. Significant associations were also observed with methacholine and sodium nitroprusside. In contrast, no relationship was observed in black Americans. In black Americans, Arg287Gln genotype was associated with vasodilator response to bradykinin. Although the difference in forearm blood flow did not reach statistical significance (P=0.058), forearm vascular resistance was significantly lower (P=0.037) in Gln287 variant allele carriers compared to wild-type individuals. Significant associations were also observed with methacholine and sodium nitroprusside. In white Americans, Gln287 variant allele carriers did not exhibit significantly higher forearm blood flow (P=0.128) or lower forearm vascular resistance (P=0.080). Genetic variation in EPHX2 is associated with forearm vasodilator responses in a bradykinin receptor- and endothelium-independent manner, suggesting an important role for soluble epoxide hydrolase in the regulation of vascular function in humans.

Association of polymorphisms of toll-like receptor 4 with a reduced prevalence of hay fever and atopy

BACKGROUND The response to innate immune stimuli seems to be critical to conditioning adaptive immunity. Early exposure to endotoxin initiates immune responses that have been shown to alter the risk of asthma and allergic diseases. The toll-like receptor 4 (TLR4) gene encodes the principal innate immunity receptor in humans for bacterial endotoxin. Polymorphisms in the TLR4 gene may regulate the effects of endotoxin exposure and could play a role in the development of asthma and atopy-related phenotypes. OBJECTIVE To investigate the association between TLR4 polymorphisms and allergic phenotypes in nonsmokers. METHODS The data from 915 nonsmoking students were available for the study. The TLR4 299 and 399 polymorphisms were genotyped using mouthwash samples. The TLR4 299 and 399 polymorphisms were grouped together to define the TLR4 polymorphic group. Skin prick tests were conducted in a subgroup of healthy participants. A brief questionnaire was administered to determine demographic characteristics and chronic health conditions. RESULTS The prevalence of hay fever was 0% in the TLR4 polymorphic group and 7.5% in the wild-type group (P = .01). After controlling for age group and sex using logistic regression, the odds of having hay fever were reduced by 88% (P = .009) in the TLR4 polymorphic group compared with the wild-type group. In a subgroup analysis, the association between TLR4 polymorphisms and atopy was only observed among females. CONCLUSIONS To our knowledge, this study is the first to report an association between TLR4 polymorphisms and atopy-related phenotypes in a nonsmoking population. Further investigation of the role of TLR4 polymorphisms in asthma and atopy-related phenotypes is warranted.

Damage-induced localized hypermutability

Genome instability continuously presents perils of cancer, genetic disease and death of a cell or an organism. At the same time, it provides for genome plasticity that is essential for development and evolution. We address here the genome instability confined to a small fraction of DNA adjacent to free DNA ends at uncapped telomeres and double-strand breaks. We found that budding yeast cells can tolerate nearly 20 kilobase regions of subtelomeric single-strand DNA that contain multiple UV-damaged nucleotides. During restoration to the double-strand state, multiple mutations are generated by error-prone translesion synthesis. Genome-wide sequencing demonstrated that multiple regions of damage-induced localized hypermutability can be tolerated, which leads to the simultaneous appearance of multiple mutation clusters in the genomes of UV- irradiated cells. High multiplicity and density of mutations suggest that this novel form of genome instability may play significant roles in generating new alleles for evolutionary selection as well as in the incidence of cancer and genetic disease.

E-NTPDases in human airways: Regulation and relevance for chronic lung diseases

Chronic obstructive lung diseases are characterized by the inability to prevent bacterial infection and a gradual loss of lung function caused by recurrent inflammatory responses. In the past decade, numerous studies have demonstrated the importance of nucleotide-mediated bacterial clearance. Their interaction with P2 receptors on airway epithelia provides a rapid ‘on-and-off’ signal stimulating mucus secretion, cilia beating activity and surface hydration. On the other hand, abnormally high ATP levels resulting from damaged epithelia and bacterial lysis may cause lung edema and exacerbate inflammatory responses. Airway ATP concentrations are regulated by ecto nucleoside triphosphate diphosphohydrolases (E-NTPDases) which are expressed on the mucosal surface and catalyze the sequential dephosphorylation of nucleoside triphosphates to nucleoside monophosphates (ATP → ADP → AMP). The common bacterial product, Pseudomonas aeruginosa lipopolysaccharide (LPS), induces an acute reduction in azide-sensitive E-NTPDase activities, followed by a sustained increase in activity as well as NTPDase 1 and NTPDase 3 expression. Accordingly, chronic lung diseases, including cystic fibrosis (CF) and primary ciliary dyskinesia, are characterized by higher rates of nucleotide elimination, azide-sensitive E-NTPDase activities and expression. This review integrates the biphasic regulation of airway E-NTPDases with the function of purine signaling in lung diseases. During acute insults, a transient reduction in E-NTPDase activities may be beneficial to stimulate ATP-mediated bacterial clearance. In chronic lung diseases, elevating E-NTPDase activities may represent an attempt to prevent P2 receptor desensitization and nucleotide-mediated lung damage.