Thyroid hormones and androgens differentially regulate gene expression in testes and ovaries of sexually mature Silurana tropicalis.

A series of ex vivo exposures using testicular and ovarian tissues of sexually mature Western clawed frogs (Silurana tropicalis) were designed to examine molecular mechanisms of thyroid hormone (TH) and androgen crosstalk sans hypophyseal feedback as well as investigate potential sex-specific differences. Tissues were exposed ex vivo to either triiodothyronine (T3), iopanoic acid (IOP), one co-treatment of IOP + 5α-dihydrotestosterone (5α-DHT), 5α-DHT, 5ß-dihydrotestosterone (5ß-DHT), or testosterone (T). Direct exposure to different androgens led to androgen specific increases in thyroid receptor and deiodinase transcripts in testes (trß and dio1) but a decrease in expression in ovaries (trß and dio3), suggesting that male and female frogs can be differently affected by androgenic compounds. Moreover, exposure to select androgens differentially increased estrogen-related transcription (estrogen receptor alpha (erα) and aromatase (cyp19)) and production (estradiol) in ovaries and testes indicating the activation of alternate metabolic pathways yielding estrogenic metabolites. Sex-steroid-related transcription (i.e., steroid 5α-reductase type 2 (srd5α2) and erα) and production (i.e., 5α-DHT) were also differentially regulated by THs. The presence and frequency of transcription factor binding sites in the putative promoter regions of TH- and sex steroid-related genes were also examined in S. tropicalis, rodent, and fish models using in silico analysis. In summary, this study provides an improved mechanistic understanding of TH- and androgen-mediated actions and reveals differential transcriptional effects as a function of sex in frogs.

Characterization of Ras k 1 a novel major allergen in Indian mackerel and identification of parvalbumin as the major fish allergen in 33 Asia-Pacific fish species.

BACKGROUND: Fish is a well-recognized cause of food allergy and anaphylaxis. The evolutionary and taxonomic diversity of the various consumed fish species pose a challenge in the identification and characterization of the major fish allergens critical for reliable diagnostics. Globally, fish is a rising cause of food allergy complicated by a large under-investigated variety of species as well as increasing global tourism and trade. This is the first comprehensive study on allergen profiles of heat-processed fish from Vietnam. OBJECTIVE: The aim of this study was to identify the major heat-stable allergens from frequently exported Asia-Pacific freshwater and marine fish and to characterize the major allergen parvalbumin (PV) from one of the most consumed and exported fish species from Asia, the Indian mackerel (Rastrelliger kanagurta). METHODS: Heated protein extracts from 33 fish species were separated by gel electrophoresis. PV isoforms were identified by immunoblotting utilizing 3 different PV-specific monoclonal and polyclonal antibodies and further characterized by mass spectrometry. IgE reactivity was investigated using sera from 21 patients with confirmed fish allergy. RESULTS: Heat-stable IgE-reactive PVs, with up to 5 isoforms per species, were identified in all 33 analysed fish species. In the Indian mackerel, 7 PV isoforms were identified by 2D-gel electrophoresis combined with mass spectrometric analyses. The amino acid sequence deduced from cDNA of the most expressed isoform showed a high identity (>90%) to PVs from 2 other mackerel species. CONCLUSIONS AND CLINICAL RELEVANCE: Different PVs were identified as the major heat-stable allergens in all 33 analysed freshwater and marine fish species from Vietnam, many of which are exported world-wide and 21 species that have never been investigated before. The Indian mackerel PV represents a novel fish allergen, now officially registered as Ras k 1. Improved diagnostics for fish allergy against Asia-Pacific species should be developed with focus on PV.

Minimal impact of extensive heating of hen's egg and cow's milk in a food matrix on threshold dose-distribution curves.

We analyzed reaction threshold data from 352 children undergoing open food challenges to hen's egg or cow's milk, either fresh or extensively heated into a muffin. There was no significant shift in dose-distribution curves due to the baking process, implying that existing threshold data for these allergens can be applied to allergen risk management, even when these allergens are heat-processed into baked foods.

The practice and perception of precautionary allergen labelling by the Australasian food manufacturing industry.

BACKGROUND: The precautionary allergen labelling (PAL) and Voluntary Incidental Trace Allergen Labelling (VITAL® ) tools were designed by industry to assist consumers with selecting safe foods for consumption. However, a sizeable proportion of food products bear no label, and it is unclear whether these products are free from allergens and therefore safe to consume or have simply not undergone a risk assessment and therefore remain unlabelled for that reason. OBJECTIVE: To assess the prevalence of unlabelled products that have undergone a risk assessment process and to examine the factors influencing industry's uptake of the VITAL® process. METHODS: A web-based questionnaire was distributed to Australasian food and grocery manufacturers. RESULTS: One hundred and thirty-seven Australasian manufacturers were contacted, and 59 questionnaires were returned (response rate: 43%). The respondents represented 454 different manufacturing sites. Manufacturers reported that 23% (95% CI 19-28) of products (n=102/434) that had been through the VITAL® risk assessment process had no PAL statement on the label. 34% (95% CI 30-38), (n=204/600) of products that had undergone another (non-VITAL® ) risk assessment process had no PAL statement. In examining the factors that influenced industry's uptake of the VITAL® process, 25 manufacturers reported on factors that influenced the uptake of the VITAL® process, 76% (CI 95% 55-91) reported that VITAL® was an effective tool because it was based on science; 52% (CI 95% 31-72) reported that it was too time-consuming and 36% (CI 95% 18-57) identified a concern with it not being endorsed by the government. CONCLUSION AND CLINICAL RELEVANCE: Currently, we estimate that at least 30% of products may have been through a risk assessment process and yet bear no PAL statement on the label. Permissive labelling could be incorporated onto these products if they have been assessed to be safe for consumption.

Allergenicity of bony and cartilaginous fish - molecular and immunological properties.

Allergy to bony fish is common and probably increasing world-wide. The major heat-stable pan-fish allergen, parvalbumin (PV), has been identified and characterized for numerous fish species. In contrast, there are very few reports of allergic reactions to cartilaginous fish despite widespread consumption. The molecular basis for this seemingly low clinical cross-reactivity between these two fish groups has not been elucidated. PV consists of two distinct protein lineages, α and ß. The α-lineage of this protein is predominant in muscle tissue of cartilaginous fish (Chondrichthyes), while ß-PV is abundant in muscle tissue of bony fish (Osteichthyes). The low incidence of allergic reactions to ingested rays and sharks is likely due to the lack of molecular similarity, resulting in reduced immunological cross-reactivity between the two PV lineages. Structurally and physiologically, both protein lineages are very similar; however, the amino acid homology is very low with 47-54%. Furthermore, PV from ancient fish species such as the coelacanth demonstrates 62% sequence homology to leopard shark α-PV and 70% to carp ß-PV. This indicates the extent of conservation of the PV isoforms lineages across millennia. This review highlights prevalence data on fish allergy and sensitization to fish, and details the molecular diversity of the two protein lineages of the major fish allergen PV among different fish groups, emphasizing the immunological and clinical differences in allergenicity.

Increases in anaphylaxis fatalities in Australia from 1997 to 2013.

BACKGROUND: Recent epidemiological studies indicate increases in Australian, UK and US hospital anaphylaxis admission rates. OBJECTIVES: The aim of this study was to determine whether Australian anaphylaxis fatalities are increasing in parallel and to examine the characteristics of fatalities recorded in the National Coronial Information System (NCIS). METHODS: Time trends in Australian anaphylaxis fatalities were examined using data derived from the Australian Bureau of Statistics (ABS) 1997-2013 and the NCIS 2000-2013, the latter providing additional information to verify cause and identify risk factors. RESULTS: The ABS recorded 324 anaphylaxis fatalities by cause: unspecified (n = 205); medication (n = 52); insect stings/tick bites (n = 41); food (n = 23); and blood products (n = 3). From 1997 to 2013, all-cause fatal anaphylaxis rates increased by 6.2%/year (95% CI: 3.8-8.6%, P < 0.0001) or from 0.054% to 0.099/10(5) population. Fatal food anaphylaxis increased by 9.7%/year (95% CI: 0.25-20%, P = 0.04) and unspecified anaphylaxis deaths by 7.8% (95% CI: 4.6-11.0, P < 0.0001). There was an insignificant change in medication-related fatalities (5.6% increase/year; 95% CI: 0.3% decrease to 11.8% increase, P = 0.06), and sting/bite fatalities remained unchanged. Hospital anaphylaxis admission rates for all-cause, food, unspecified and medication anaphylaxis increased at rates of 8%, 10%, 4.4% and 6.8%/year, respectively. A total of 147 verified NCIS deaths were examined in detail: medication- and sting/bite-related fatalities occurred predominantly in older individuals with multiple comorbidities. Upright posture after anaphylaxis was associated with risk of sudden death (all causes). Seafood (not nuts) was the most common trigger for food-related anaphylaxis deaths. CONCLUSIONS: Australian anaphylaxis fatality rates (most causes) have increased over the last 16 years, contrasting with UK- and US-based studies that describe overall lower and static overall anaphylaxis fatality rates (0.047-0.069/10(5) population).

Mechanisms of allergic disease - environmental and genetic determinants for the development of allergy.

Allergic disease can be viewed as an early manifestation of immune dysregulation. Environmental exposures including maternal inflammation, diet, nutrient balance, microbial colonization and toxin exposures can directly and indirectly influence immune programming in both pregnancy and the postnatal period. The intrauterine microclimate is critical for maternal and fetal immunological tolerance to sustain viable pregnancy, but appears susceptible to environmental conditions. Targeting aspects of the modern environment that promote aberrant patterns of immune response is logical for interventions aimed at primary prevention of allergic disease. Defining the mechanisms that underpin both natural and therapeutic acquisition of immunological tolerance in childhood will provide insights into the drivers of persistent immune dysregulation. In this review, we summarize evidence that allergy is a consequence of intrauterine and early life immune dysregulation, with specific focus on contributing environmental risk factors occurring preconception, in utero and in the early postnatal period. We explore the immunological mechanisms which underpin tolerance and persistence of allergic disease during childhood. It is likely that future investigations within these two domains will ultimately provide a road map for the primary prevention of allergic disease.

Baked egg food challenges - clinical utility of skin test to baked egg and ovomucoid in children with egg allergy.

BACKGROUND: Many children with IgE-mediated egg allergy can tolerate products containing extensively heated (baked) egg. Aside from food challenge, there are no tests which reliably predict tolerance to baked egg in egg-allergic individuals. OBJECTIVES: To determine if skin prick test (SPT) to baked egg (muffin) and ovomucoid can predict the outcome of baked egg challenges in egg allergic patients. METHODS: In this prospective study, children with a recent history of immediate allergic reactions to egg [and corroborative positive SPT or serum-specific IgE (ssIgE) to egg] or those with SPT/ssIgE > 95% PPV for egg allergy were invited to undergo an open standardized baked egg (muffin) challenge. SPT to egg white, ovomucoid, and fresh muffin were performed immediately prior to challenge. RESULTS: One hundred and forty-three egg allergic children underwent baked egg challenge and of these, 90 (63%) tolerated 1 g of egg protein in a baked muffin. Of the 53 positive challenges, eight (15%) had respiratory and/or cardiovascular symptoms. The median SPT diameters in positive challenges compared with negative challenges were baked muffin - 6.0 mm/4.0 mm and ovomucoid 7.5 mm/5.0 mm respectively. Receiver operating characteristic (ROC) curves were generated for SPT to baked egg and ovomucoid. The area under the curve was 0.68 for baked egg, and 0.67 for ovomucoid. A muffin SPT of < 2 mm had a negative predictive value of 88% and an ovomucoid SPT ≥ 11 mm had a positive predictive value of 100%. CONCLUSIONS AND CLINICAL RELEVANCE: A SPT of < 2 mm to muffin had a high negative predictive value to baked egg challenge. Ovomucoid SPT ≥ 11 mm was very likely to predict a reaction to baked egg. In these children, deferring the challenge would be appropriate.

Utility of intramuscular antibiotics for secondary prevention of early onset, asymptomatic 'suspected' neonatal sepsis.

OBJECTIVE: To evaluate the safety, efficacy and cost-benefit of intramuscular (IM) antibiotics administration in the secondary prevention of suspected neonatal sepsis in asymptomatic term neonates. STUDY DESIGN: Retrospective review of inborn asymptomatic full-term neonates with birth weights ≥2000 g who required sepsis evaluation and treatment with IM antibiotics were undertaken from July 2001 to July 2008. The IM antibiotic protocol was categorized as inadequate if the neonate became symptomatic, had positive blood or cerebrospinal fluid (CSF) cultures or was readmitted for sepsis within 2 weeks of nursery discharge. Data were analyzed to identify relationships between key indicators for the presence of neonatal bacterial infection, 'inadequate IM antibiotics protocol' and the rehospitalization rates. RESULT: There were 29 698 infants admitted to the newborn nursery (NBN) during the study period. A total of 5045 infants (17%) were evaluated for suspected neonatal sepsis; 421 neonates (8.3%) were treated with IM antibiotics for 48 to 72 h. Fourteen infants (3.3%) met criteria for 'inadequate IM antibiotics protocol'. Seven infants developed symptoms within the first 32 h of life, and seven infants had positive blood or CSF cultures, one of whom required rehospitalization. None of the infants were hemodynamically unstable or developed complications, including adverse events associated with IM drug administration. CONCLUSION: Use of IM antibiotics is a safe alternative to intravenous antibiotics in the secondary prevention of asymptomatic term newborns with presumed sepsis. There is a substantial cost savings in caring for asymptomatic neonates with presumed sepsis in the NBN compared with neonatal intensive care unit costs.

Analytical and biological considerations in the measurement of cell-associated CCR5 and CXCR4 mRNA and protein.

The accurate measurement of T cell-associated CC chemokine receptor type 5 (CCR5) and CXC chemokine receptor type 4 (CXCR4) expression, including expression of CCR5 and CXCR4 mRNA as an immune measure of immunologic response to highly active antiretroviral therapy (HAART) and newer agents, including entry inhibitors, is essential. Previous studies have reported alterations in lymphocyte cell membrane CCR5 expression that were related to blood collection and cell separation media. Clinical trials often require the transport of specimens to central laboratories for evaluation, resulting in significant time delays between specimen procurement and analysis. This study shows that CCR5 expression on naïve and memory T cells is influenced by blood collection media and specimen age. Peripheral blood collected in Streck Vacutainer tubes containing a cell stabilizer and fixative was found to improve detection of CCR5 expression compared to specimens collected in K2 EDTA anticoagulant. The selection of flow cytometry gating strategies for the identification of naïve and memory T-helper cells can also significantly influence the sensitivity of detection of CCR5 expression. Procedural methods are described that allow for the optimal measurement of naïve and memory T-helper cell CCR5 and CXCR4 expression as well as the quantitation of CCR5 and CXCR4 mRNA.

Cryopreservation decreases receptor PD-1 and ligand PD-L1 coinhibitory expression on peripheral blood mononuclear cell-derived T cells and monocytes.

The B7-CD28 immunoglobulin superfamily of costimulatory and coinhibitory ligands and their cell receptors play a critical role in modulating immune responses. Imbalances in these immune regulatory signals occur in pathological conditions characterized by chronic antigenic stimulation. Clinical studies often rely on the use of cryopreserved peripheral blood mononuclear cells (PBMC) to evaluate cellular immune responses. The impact of cryopreservation on these coinhibitory ligands and their cell receptors is unknown. In our studies, cryopreservation significantly reduced the expression of both PD-1 and PD-L1 on PBMC-derived CD3+/CD8+ T cells and CD45+/CD14+ monocytes obtained from adult control subjects. Blockade of PD-1, PD-L1, and PD-L2 using both freshly isolated and cryopreserved PBMC led to higher levels of phytohemagglutinin (PHA) and Candida-induced gamma interferon (IFN-gamma), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-alpha) with no effect on IL-10 production. Coinhibitory signaling blockade of freshly isolated, PHA-stimulated PBMC from normal adult controls and human immunodeficiency virus (HIV)-infected subjects led to increased production of IL-4 and IL-5. Candida-stimulated PBMC preferentially induced IFN-gamma and TNF-alpha production, with reduced production of IL-2 and IL-10. This is in contrast to high levels of IFN-gamma, IL-2, and TNF-alpha production with PHA-stimulated cells. The effects of coinhibitory blockade on PHA and Candida-induced lymphoproliferation were varied, with freshly isolated PBMC from adult control subjects and HIV-infected patients yielding higher levels of lymphoproliferation in response to PD-1/PD-L1 blockade. Immune function studies employing cryopreserved cells may lead to increased T-cell effector cytolytic and regulatory immune responses.

Food allergy: is strict avoidance the only answer?

It is an immunological paradigm that avoidance of food allergen may reduce the risk or prevent immunological reactions and conversely that a greater exposure increases the magnitude of the immune response. Consequently, food allergen avoidance has been recommended to reduce the risk of sensitization in infants and to prevent clinical reactions in children with positive skin prick tests (SPT). In the latter setting, it is hoped that avoidance may either promote or at least not retard the development of tolerance. Animal studies, however, have demonstrated that tolerance to food allergens may be induced by either large (high zone tolerance) or small (low zone tolerance) doses, whereas doses in between may actually stimulate immune responses. In this review, we discuss whether strict allergen avoidance is always the most appropriate strategy for preventing or managing IgE-mediated food allergy.

Splanchnic tissue oxygenation, but not brain tissue oxygenation, increases after feeds in stable preterm neonates tolerating full bolus orogastric feeding.

OBJECTIVE: The objective of this prospective, observational study was to test the hypothesis that tissue oxygenation in the splanchnic bed compared with tissue oxygenation in the cerebral circulation changes after feeding in preterm neonates who are tolerating full bolus orogastric feeds. STUDY DESIGN: Clinically stable premature neonates with postmenstrual age between 32 and 35(6/7) weeks who were tolerating full bolus feedings were studied before feeding and 1 h after feeding using near-infrared spectroscopy. The ratio of oxygenated to reduced hemoglobin (tissue oxygenation index, TOI) in the splanchnic circulation bed was divided by the TOI in the cerebral circulation, thereby yielding the cerebro-splanchnic oxygenation ratio (CSOR). We compared TOI and CSOR before and after feeding. As the changes in TOI and CSOR had non-Gaussian distribution, nonparametric statistics were used. RESULT: Among 32 infants, CSOR increased significantly after feeding (median difference 0.08; range -0.48, +0.58; P=0.011), whereas pulse oximetry did not change significantly (P=0.600). The change in CSOR with feeding was associated with a significant increase in splanchnic TOI (preprandial median 43.8, range 25.2-68.4 vs postprandial 47.5, range 25.8-70.8; P=0.013), without any significant change in brain TOI (preprandial median 64.9, range 44.5-75.4 vs postprandial 58.9, range 42.2-72.3; P=0.153). CONCLUSION: This study indicates that CSOR and splanchnic TOI, but not brain TOI, increase significantly after feeding in stable preterm infants who are tolerating full orogastric feeds.

Frequency analysis of air and skin temperature in neonates in servo-controlled incubators.

OBJECTIVE: To test a new system designed for digital recording of skin and air temperature, thereby allowing analysis of cyclic changes in temperature in neonates in servo-controlled incubators. METHODS: Analysis of cyclic changes of serial skin and air temperature in asymptomatic infants in servo-controlled incubators adjusting heat output to the patient's temperature. RESULTS: In nine asymptomatic neonates ranging from 25 to 40 weeks of gestational age, analysis showed peaks of coherence (squared correlation) between skin and air temperature measurements at periods ranging between 1.5 and 3 h. CONCLUSION: We have established a new system to study cyclic changes in skin and air temperature in neonates in servo-controlled incubators. The analysis of this pilot study suggests that the most important changes in skin and air temperature in asymptomatic neonates occur at a periodicity of 1.5 to 3 h, which is similar to that previously described for neonatal temperature. Additional data are required to determine whether this new system may be useful in neonatal care.

Osteomyelitis and pyogenic sacroiliitis: A difficult diagnosis.

Pyogenic sacroiliitis is an uncommon infection often diagnosed late because of poor localization of symptoms and inadequate physical examination. We describe a 12-year-old girl whose osteomyelitis and pyogenic sacroiliitis was initially misdiagnosed and discuss examination, investigation and management of the condition. This case highlights the importance of thorough physical examination and the consideration of septic arthritis in an unusual joint.

Furosemide for symptomatic patent ductus arteriosus in indomethacin-treated infants.

BACKGROUND: Inhibition of prostaglandin synthesis mediates closure of the ductus arteriosus and renal side effects after indomethacin administration. Because furosemide increases prostaglandin production, it could potentially help prevent indomethacin-related toxicity but also decrease ductal response to indomethacin. OBJECTIVES: The primary objectives of this review were to assess (1) whether furosemide affects the incidence of failure of ductal closure after indomethacin and that of indomethacin-related toxicity and (2) the effect of furosemide on mid-term and long-term outcome. The secondary objective was to determine whether the effect of furosemide on renal function and water balance depends on prior extracellular volume (assessed by blood urea nitrogen [BUN]/creatinine ratio). SEARCH STRATEGY: We searched electronic databases (Medline, Embase and Cochrane) and selected abstract books, without language restriction. SELECTION CRITERIA: We selected studies with (1) random allocation to either indomethacin alone or indomethacin and furosemide and (2) analysis of either short-term risk-benefit ratio of furosemide, mid- or long-term outcome, or the relationship between extracellular volume at study entry and changes in renal function. DATA COLLECTION AND ANALYSIS: We assessed studies for possible bias and for quality of assessment of ductal patency. We assessed categorical variables using relative risk and absolute risk reduction. We assessed the effects of furosemide on renal function and fluid balance by comparing changes from baseline in the treatment group with those in controls. Subsets were determined a priori based on BUN/creatinine ratio at study entry. MAIN RESULTS: All 3 studies fulfilling the entry criteria had limitations, including possible or definite bias. There was substantial heterogeneity among studies. Furosemide administration did not significantly increase the risk of failure of ductal closure; however, sample size was insufficient to rule out even a 31% increase. In the subset with initial BUN/creatinine ratio > 20 mg/mg, 2 of 18 patients receiving furosemide could not complete a 3-dose course of indomethacin because of toxicity. Minimal or no information was available about any of the other main outcome variables. Furosemide increased urine output regardless of the initial BUN/creatinine ratio, leading to a 5% weight loss during a 3-dose course, an undesired effect in patients with initial BUN/creatinine ratio > 20 mg/mg. Furosemide increased creatinine clearance only in patients with initial BUN/creatinine ratio <20 mg/mg. REVIEWER'S CONCLUSIONS: There is not enough evidence to support the administration of furosemide to premature infants treated with indomethacin for symptomatic patent ductus arteriosus. Furosemide appears to be contraindicated in the presence of dehydration in those infants.