Utility of DNA, RNA, Protein, and Functional Approaches to Solve Cryptic Immunodeficiencies.

PURPOSE: We report a female infant identified by newborn screening for severe combined immunodeficiencies (NBS SCID) with T cell lymphopenia (TCL). The patient had persistently elevated alpha-fetoprotein (AFP) with IgA deficiency, and elevated IgM. Gene sequencing for a SCID panel was uninformative. We sought to determine the cause of the immunodeficiency in this infant. METHODS: We performed whole-exome sequencing (WES) on the patient and parents to identify a genetic diagnosis. Based on the WES result, we developed a novel flow cytometric panel for rapid assessment of DNA repair defects using blood samples. We also performed whole transcriptome sequencing (WTS) on fibroblast RNA from the patient and father for abnormal transcript analysis. RESULTS: WES revealed a pathogenic paternally inherited indel in ATM. We used the flow panel to assess several proteins in the DNA repair pathway in lymphocyte subsets. The patient had absent phosphorylation of ATM, resulting in absent or aberrant phosphorylation of downstream proteins, including γH2AX. However, ataxia-telangiectasia (AT) is an autosomal recessive condition, and the abnormal functional data did not correspond with a single ATM variant. WTS revealed in-frame reciprocal fusion transcripts involving ATM and SLC35F2 indicating a chromosome 11 inversion within 11q22.3, of maternal origin. Inversion breakpoints were identified within ATM intron 16 and SLC35F2 intron 7. CONCLUSIONS: We identified a novel ATM-breaking chromosome 11 inversion in trans with a pathogenic indel (compound heterozygote) resulting in non-functional ATM protein, consistent with a diagnosis of AT. Utilization of several molecular and functional assays allowed successful resolution of this case.

Lysinibacillus massiliensis Panniculitis Masquerading as Erythema Nodosum: A Case Report.

Lysinibacillus massiliensis, formerly Bacillus massiliensis, is an environmental Gram-positive bacillus that is generally non-pathogenic. Rare case reports in immunosuppressed patients have described sepsis with this organism. In this study, we report a case of L massiliensis as a cause of infectious panniculitis mimicking erythema nodosum after infusion of autologous adipose-derived stem cells in an immunosuppressed patient with refractory Crohn's disease. This case highlights the importance of care providers to consider exposures and host factors when interpreting culture results with otherwise benign organisms.

Vitamin D and food allergies in children: A systematic review and meta-analysis.

BACKGROUND: Vitamin D insufficiency has been associated with immune dysfunction and linked to the epidemic of atopic diseases in the Western hemisphere, yet there are studies with conflicting results, and the risk has not been quantified uniformly across studies. OBJECTIVE: To perform a systematic review and meta-analysis to evaluate and quantify if vitamin D deficiency is associated with the presence and persistence of food allergy. METHODS: A systematic review was undertaken to assess for the association between food allergy and vitamin D status in children. RESULTS: A total of 368 citations relevant to this systematic review were identified. In the whole review, 5105 children were included. We did not find a significant association between 25 hydroxy vitamin D (25(OH)D) status and risk of food allergy in children (odds ratio [OR] 1.35 [95% confidence interval {CI}, 0.79-2.29]; p = 0.27, I2 = 58.3%). We conducted subgroup analyses based on different cutoffs of the 25(OH)D status (20 versus 30 ng/mL). Only one study used 30 ng/mL and found that children with <30 ng/mL were more likely to report food allergy than children with a 25(OH)D status of ≥30 ng/mL (OR 2.04 [95% CI, 1.02-4.04]; p = 0.04). Four studies compared children with a 25(OH)D status of <20 ng/mL to children with a 25(OH)D status of ≥20 ng/mL and found no significant differences (OR 1.18 [95% CI, 0.62-2.27]; p = 0.62, I2 = 62.7%). CONCLUSION: Based on the studies analyzed, this systematic review did not identify a significant association between vitamin D status and food allergy. Interpretation of the included studies was limited by a lack of a standard definition for vitamin D deficiency and insufficient knowledge regarding the optimal vitamin D status needed to impact immune function. Longitudinal studies are warranted to assess if vitamin D might contribute to the development of food allergy.

Sublingual Immunotherapy Dosing Regimens: What Is Ideal?

Sublingual immunotherapy (SLIT) is a treatment for allergic respiratory diseases that has demonstrated efficacy and safety. Several formulations of SLIT are now available worldwide for treatment of allergic rhinitis (AR). Grass tablets containing 15 to 25 µg of group 5 major allergen reduced combined AR symptoms and medication use by 23% to 41% in 3 treatment years and 2 follow-up years. Ragweed pollen tablets (12 µg of Ambrosia artemisiifolia 1) and liquid extracts (50 µg of Ambrosia artemisiifolia 1) reduced combined AR symptoms and medication use by 26% and 43%, respectively. House dust mite tablets containing 300 index of reactivity (16 µg of Dermatophagoides pteronyssinus 1 and 68 µg of Dermatophagoides farinae 1) reduced AR symptoms by 17.9% and 17.0% in 1 treatment year and 1 follow-up year, respectively. A different house dust mite tablet (12 standardized quality house dust mite) was able to reduce the risk of asthma exacerbation compared with placebo (hazard ratio, 0.69; 95% CI, 0.50-0.96). Most adverse events were local and mild to moderate in severity. For SLIT products reviewed herein, effective doses range from 1.12 to 84 µg of major allergen(s). However, allergen content is not uniformly standardized, can be expressed in arbitrary or proprietary units (depending on the manufacturer), and assays for determination of allergen content are highly variable. Thus, results from one study of a given product cannot be extrapolated to other products. Despite these limitations, this Clinical Management Review aims to provide practitioners with relevant information on the dosing of selected SLIT formulations in the treatment of allergic respiratory disease.

Hematologic Malignancies Identified in Patients with Hypereosinophilia and Hypereosinophilic Syndromes.

BACKGROUND: Certain hematologic malignancies are associated with hypereosinophilia or tissue eosinophilia. It is unclear if patients with hypereosinophilia are more likely to develop one of these malignancies. OBJECTIVE: This study sought to quantify the specific hematologic malignancies that developed in patients with preexisting hypereosinophilia. METHODS: Adult patients with eosinophilia associated with the development of hematologic malignancy were identified by a retrospective review of the Mayo Clinic patient database between 2000 and 2013. RESULTS: Of 2642 patients identified with eosinophilia, hypereosinophilia, or hypereosinophilic syndrome, 25 (aged 28.8 to 86.1 years; 13 male; 12 female) had a diagnosis of either lymphoma or leukemia. The majority of these patients had non-Hodgkin lymphoma (17 of 25). T-cell-derived lymphomas were more common (12 of 17) than B-cell-derived lymphomas (4 of 17). In patients with leukemia (8 of 25), chronic lymphocytic leukemia (4 of 8) was most common, followed by chronic eosinophilic leukemia (3 of 8). Approximately 5.1% of patients with hypereosinophilia developed a hematologic malignancy. On average, the malignancy developed 30.0 ± 42.7 months after the onset of hypereosinophilia. CONCLUSIONS: The development of hematologic malignancies in this referral population with eosinophilia was rare (0.2%), but more common in those with hypereosinophilia (5.1%). Non-Hodgkin's lymphomas, particularly T-cell-derived malignancies, were most commonly diagnosed. Patients with preexisting hypereosinophilia were diagnosed with hematologic conditions that were rarer within the general population.

Symptom-Based Clustering in Chronic Rhinosinusitis Relates to History of Aspirin Sensitivity and Postsurgical Outcomes.

BACKGROUND: Symptoms burden in chronic rhinosinusitis (CRS) may be assessed by interviews or by means of validated tools such as the 22-item SinoNasal Outcome Test (SNOT-22). However, when only the total SNOT-22 scores are used, the pattern of symptom distribution and heterogeneity in patient symptoms is lost. OBJECTIVES: To use a standardized symptom assessment tool (SNOT-22) on preoperative symptoms to understand symptom heterogeneity in CRS and to aid in characterization of distinguishing clinical features between subgroups. METHODS: This was a retrospective review of 97 surgical patients with CRS. Symptom-based clusters were derived on the basis of presurgical SNOT-22 scores using unsupervised analysis and network graphs. Comparison between clusters was performed for clinical and demographic parameters, postsurgical symptom scores, and presence or absence of a history of aspirin sensitivity. RESULTS: Unsupervised analysis reveals coclustering of specific symptoms in the SNOT-22 tool. Using symptom-based clustering, patients with CRS were stratified into severe overall (mean total score, 90.8), severe sinonasal (score, 62), moderate sinonasal (score, 40), moderate nonsinonasal (score, 37) and mild sinonasal (score, 16) clusters. The last 2 clusters were associated with lack of history of aspirin sensitivity. The first cluster had a rapid relapse in symptoms postoperatively, and the last cluster demonstrated minimal symptomatic improvement after surgery. CONCLUSION: Symptom-based clusters in CRS reveal a distinct grouping of symptom burden that may relate to aspirin sensitivity and treatment outcomes.

Ventricular septal defect and bivalvular endocarditis.

A 63-year-old man presented with generalized fatigue, chills, malaise, dyspnea, intermittent fevers, and 50-pound weight loss of 4 months' duration. Blood cultures were positive for pan-sensitive Streptococcus anginosus. Transesophageal echocardiography showed an 11 mm × 3 mm mobile mass attached to the mitral valve, a 16 mm × 16 mm mobile mass attached to the pulmonary valve, and a small membranous ventricular septal defect. The patient received 12 weeks of intravenous (IV) antibiotics with eventual resolution of the masses. Multi-valve endocarditis involving both the left and right chambers is rarely reported without prior history of IV drug use or infective endocarditis. Our case emphasizes the importance of careful assessment for ventricular septal defects or extra-cardiac shunts in individuals who present with simultaneous right and left-sided endocarditis.

Morphine-induced trafficking of a mu-opioid receptor interacting protein in rat locus coeruleus neurons.

Opiate addiction is a devastating health problem, with approximately 2million people currently addicted to heroin or non-medical prescription opiates in the United States alone. In neurons, adaptations in cell signaling cascades develop following opioid actions at the mu opioid receptor (MOR). A novel putative target for intervention involves interacting proteins that may regulate trafficking of MOR. Morphine has been shown to induce a re-distribution of a MOR-interacting protein Wntless (WLS, a transport molecule necessary for secretion of neurotrophic Wnt proteins), from cytoplasmic to membrane compartments in rat striatal neurons. Given its opiate-sensitivity and its well-characterized molecular and cellular adaptations to morphine exposure, we investigated the anatomical distribution of WLS and MOR in the rat locus coeruleus (LC)-norepinephrine (NE) system. Dual immunofluorescence microscopy was used to test the hypothesis that WLS is localized to noradrenergic neurons of the LC and that WLS and MOR co-exist in common LC somatodendritic processes, providing an anatomical substrate for their putative interactions. We also hypothesized that morphine would influence WLS distribution in the LC. Rats received saline, morphine or the opiate agonist [d-Ala2, N-Me-Phe4, Gly-ol5]-enkephalin (DAMGO), and tissue sections through the LC were processed for immunogold-silver detection of WLS and MOR. Statistical analysis showed a significant re-distribution of WLS to the plasma membrane following morphine treatment in addition to an increase in the proximity of gold-silver labels for MOR and WLS. Following DAMGO treatment, MOR and WLS were predominantly localized within the cytoplasmic compartment when compared to morphine and control. In a separate cohort of rats, brains were obtained from saline-treated or heroin self-administering male rats for pulldown co-immunoprecipitation studies. Results showed an increased association of WLS and MOR following heroin exposure. As the LC-NE system is important for cognition as well as decisions underlying substance abuse, adaptations in WLS trafficking and expression may play a role in modulating MOR function in the LC and contribute to the negative sequelae of opiate exposure on executive function.

MOR is not enough: identification of novel mu-opioid receptor interacting proteins using traditional and modified membrane yeast two-hybrid screens.

The mu-opioid receptor (MOR) is the G-protein coupled receptor primarily responsible for mediating the analgesic and rewarding properties of opioid agonist drugs such as morphine, fentanyl, and heroin. We have utilized a combination of traditional and modified membrane yeast two-hybrid screening methods to identify a cohort of novel MOR interacting proteins (MORIPs). The interaction between the MOR and a subset of MORIPs was validated in pulldown, co-immunoprecipitation, and co-localization studies using HEK293 cells stably expressing the MOR as well as rodent brain. Additionally, a subset of MORIPs was found capable of interaction with the delta and kappa opioid receptors, suggesting that they may represent general opioid receptor interacting proteins (ORIPS). Expression of several MORIPs was altered in specific mouse brain regions after chronic treatment with morphine, suggesting that these proteins may play a role in response to opioid agonist drugs. Based on the known function of these newly identified MORIPs, the interactions forming the MOR signalplex are hypothesized to be important for MOR signaling and intracellular trafficking. Understanding the molecular complexity of MOR/MORIP interactions provides a conceptual framework for defining the cellular mechanisms of MOR signaling in brain and may be critical for determining the physiological basis of opioid tolerance and addiction.

Expression of GPR177 (Wntless/Evi/Sprinter), a highly conserved Wnt-transport protein, in rat tissues, zebrafish embryos, and cultured human cells.

GPR177 is an evolutionarily conserved transmembrane protein necessary for Wnt protein secretion. Little is currently known, however, regarding expression of GPR177, especially in vertebrate species. We have developed an antiserum against GPR177, and used it to examine expression of GPR177 in human tissue culture cells, adult mouse, and rat tissues, as well as developing zebrafish embryos. In rodents, GPR177 is expressed in virtually all tissue types and brain regions examined. In zebrafish, GPR177 polypeptides are expressed throughout embryogenesis, and are detectable as early as 1 hr post-fertilization. In situ hybridization analysis reveals that gpr177 mRNA expression is prominent in embryonic zebrafish brain and ear. Structural studies suggest that GPR177 is modified by N-linked sugars, and that the protein contains an even number of transmembrane segments. The relatively ubiquitous expression of GPR177 suggests that this protein may serve to regulate Wnt secretion in a variety of embryonic and adult tissue types.

Interaction of the mu-opioid receptor with GPR177 (Wntless) inhibits Wnt secretion: potential implications for opioid dependence.

BACKGROUND: Opioid agonist drugs produce analgesia. However, long-term exposure to opioid agonists may lead to opioid dependence. The analgesic and addictive properties of opioid agonist drugs are mediated primarily via the mu-opioid receptor (MOR). Opioid agonists appear to alter neuronal morphology in key brain regions implicated in the development of opioid dependence. However, the precise role of the MOR in the development of these neuronal alterations remains elusive. We hypothesize that identifying and characterizing novel MOR interacting proteins (MORIPs) may help to elucidate the underlying mechanisms involved in the development of opioid dependence. RESULTS: GPR177, the mammalian ortholog of Drosophila Wntless/Evi/Sprinter, was identified as a MORIP in a modified split ubiquitin yeast two-hybrid screen. GPR177 is an evolutionarily conserved protein that plays a critical role in mediating Wnt protein secretion from Wnt producing cells. The MOR/GPR177 interaction was validated in pulldown, coimmunoprecipitation, and colocalization studies using mammalian tissue culture cells. The interaction was also observed in rodent brain, where MOR and GPR177 were coexpressed in close spatial proximity within striatal neurons. At the cellular level, morphine treatment caused a shift in the distribution of GPR177 from cytosol to the cell surface, leading to enhanced MOR/GPR177 complex formation at the cell periphery and the inhibition of Wnt protein secretion. CONCLUSIONS: It is known that chronic morphine treatment decreases dendritic arborization and hippocampal neurogenesis, and Wnt proteins are essential for these processes. We therefore propose that the morphine-mediated MOR/GPR177 interaction may result in decreased Wnt secretion in the CNS, resulting in atrophy of dendritic arbors and decreased neurogenesis. Our results demonstrate a previously unrecognized role for GPR177 in regulating cellular response to opioid drugs.

The presence of Lys27 instead of Asn27 in human phospholamban promotes sarcoplasmic reticulum Ca2+-ATPase superinhibition and cardiac remodeling.

BACKGROUND: Phospholamban (PLN) is an inhibitor of the Ca2+ affinity of sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2). The amino acid sequence of PLN is highly conserved, and although all species contain asparagine (Asn), human PLN is unique in containing lysine (Lys) at amino acid 27. METHODS AND RESULTS: Human PLN was introduced in the null background. Expression of human PLN, at similar levels to mouse wild-type PLN, resulted in significant decreases in the affinity of SERCA2 for Ca2+, attributed to unique spatial conformation of this PLN form and increases in its monomeric active unit compared with mouse PLN. The increased inhibition by human PLN was associated with attenuated cardiac contractility in the intact-animal, organ, and cardiomyocyte levels and with depressed calcium kinetics. These inhibitory effects could not be fully reversed even on maximal isoproterenol stimulation. There were no alterations in the expression levels of SERCA2, calsequestrin, ryanodine receptor, and FKBP12, although the sodium/calcium exchanger and the L-type Ca2+ channel expression levels were upregulated. The depressed function resulted in increased heart/body weight ratios and phosphorylation levels of Akt, p38, and Erk1/2. CONCLUSIONS: Human PLN may play a more inhibitory role than that of other species in Ca2+ cycling. Expression of human PLN in the mouse is compensated by alterations in Ca2+-handling proteins and cardiac remodeling in an effort to normalize cardiac contractility. Thus, the unique amino acid sequence of human PLN may be critical in maintaining a high cardiac reserve, which is of paramount importance in the regulation of human cardiac function.