Dog Allergens Research Articles

Allergens on desktop surfaces in preschools and elementary schools of urban children with asthma.

Desktop dust has been studied as a source of food allergen, but not as a source of potential aeroallergen exposure. Thirty-six wiped samples from desktop surfaces were collected from preschools and schools. Samples were analyzed for detectable levels of common aeroallergens including Alternaria, cockroach, dog, dust mite, cat, mouse, and rat allergens by immunoassay. Mouse allergen was the most prevalent, detectable in 97.2% of samples. Cat allergen was detectable in 80.6% of samples, and dog allergen was detectable in 77.8% of samples. Other allergens were not as prevalent. Mouse was the only allergen that was highly correlated with settled floor dust collected from the same rooms (r=0.721, P<0.001). This is the first study to detect aeroallergens on desktop surfaces by using moist wipes. Allergens for mouse, cat, and dog were highly detectable in wipes with mouse desktop surface levels correlating with levels in vacuumed floor dust.

The homologous lipocalins Can f 1 and Lcn-1 induce different immune responses

Background Why and when the immune system provokes TH2 mediated allergic immune responses is still not well understood. Dendritic cells are one of the master regulators during the induction of allergic airway inflammation providing stimuli for TH2 cell differentiation. Most of the major as well as minor mammalian allergens causing respiratory sensitization belong to the lipocalin family. We investigated two homologous lipocalins, the endogenous non-allergenic human Lcn-1 and the major respiratory dog allergen Can f 1 to study their effects on human monocyte derived dendritic cells in order to initiate allergic immune responses.

Allergens and β-Glucans in Dutch Homes and Schools: Characterizing Airborne Levels

BackgroundIndoor air quality has an effect on respiratory health. Children are more vulnerable to a decreased indoor air quality as their lungs are still developing. We measured levels of allergens and β-(1,3)-glucans in 19 school buildings and determined whether measured levels could be reproduced. School levels were compared to those in 169 homes and the effect of building characteristics on both home and school exposure was explored.MethodsElectrostatic Dust fall Collectors were placed in school buildings for 8 weeks and in homes for 2 weeks to collect settled airborne dust. Cat, dog, and mouse allergen levels, domestic mite antigen levels and β-(1,3)-glucans were measured in the extracts from the collectors. Results were corrected for sampling duration. Using questionnaire data, relations between measured levels and building and classroom characteristics were explored.ResultsIn schools, exposure levels were highest in classrooms and were influenced by the socioeconomic status of the children, the season measurements were performed, moisture status of the building and pet ownership. Repeated measurements in different seasons and over the years showed significantly different levels. Home exposure was influenced by socioeconomic status, occupancy and pet ownership. Domestic mite antigen was found in higher levels in extracts from homes compared to schools while pet allergen levels were 13 times higher in schools compared to homes without pets. For mouse allergen overall levels of exposure were low but still two times higher in schools compared to homes. Levels of β-(1,3)-glucans were also approximately two times higher in schools than in homes.ConclusionExposure levels of several allergens and β-(1,3)-glucans in schools differ over time and are higher than in homes. For children, exposure levels measured at school could contribute to their total exposure as especially animal allergen levels can be much higher in schools compared to homes.

Endotoxin, Ergosterol, Fungal DNA and Allergens in Dust from Schools in Johor Bahru, Malaysia- Associations with Asthma and Respiratory Infections in Pupils

There are few studies on associations between respiratory health and allergens, fungal and bacterial compounds in schools in tropical countries. The aim was to study associations between respiratory symptoms in pupils and ethnicity, chemical microbial markers, allergens and fungal DNA in settled dust in schools in Malaysia. Totally 462 pupils (96%) from 8 randomly selected secondary schools in Johor Bahru, Malaysia, participated. Dust was vacuumed from 32 classrooms and analysed for levels of different types of endotoxin as 3-hydroxy fatty acids (3-OH), muramic acid, ergosterol, allergens and five fungal DNA sequences. Multiple logistic regression was applied. Totally 13.1% pupils reported doctor’s diagnosed asthma, 10.3% wheeze and 21.1% pollen or pet allergy. Indian and Chinese children had less atopy and asthma than Malay. Carbon dioxide levels were low (380–690 ppm). No cat (Fel d1), dog (Can f 1) or horse allergens (Ecu cx) were detected. The levels of Bloomia tropicalis (Blo t), house dust mite allergens (Der p 1, Der f 1, Der m 1) and cockroach allergens (Per a 1 and Bla g 1) were low. There were positive associations between levels of Aspergillus versicolor DNA and daytime breathlessness, between C14 3-OH and respiratory infections and between ergosterol and doctors diagnosed asthma. There were negative (protective) associations between levels of C10 3-OH and wheeze, between C16 3-OH and day time and night time breathlessness, between cockroach allergens and doctors diagnosed asthma. Moreover there were negative associations between amount of fine dust, total endotoxin (LPS) and respiratory infections. In conclusion, endotoxin at school seems to be mainly protective for respiratory illness but different types of endotoxin could have different effects. Fungal contamination measured as ergosterol and Aspergillus versicolor DNA can be risk factors for respiratory illness. The ethnical differences for atopy and asthma deserve further attention.

A Study On Allergy Sensitization Of Dense Urban Dwellers To Dog and Cat Allergens Using The Skin Prick Test

Pet allergen exposure has been a long debate amongst experts. Studies in different countries have produced rather conflicting results. This is a cross-sectional study that aims to demonstrate cutaneous allergy sensitization to cats and dogs of Filipinos living in a dense urban area where pets and strays are commonly seen. One hundred fifteen subjects (n=115) were tested for cutaneous skin prick test reactivity to dog and cat allergens. There were 73 female and 42 males with 31 pediatric subjects. Among the subjects (n=115), 54.8% (n=63) were pet owners while 45.2% (n=52) have no pets. Using chi-square, skin prick test reactivity to dog and cat allergens were compared to patient demographic data and patient pet ownership profile. Our results show that neither gender nor age correlates to skin prick test reactivity to both allergens. Comparing skin test reactivity to dog allergen, there was no significant difference between those who are dog owners versus those who do not own dogs (p-value 0.183). Cat owners have significant increase in cat allergen skin reactivity (p-value 0.031). In conclusion, this study shows that regardless if you owned a dog or a cat, the event of having a positive dog skin prick is the same. In contrast, if one owns a cat, there is a higher event of having cat allergen sensitization compared to not owning cats at all. Lastly, those without pets in general do not have a significantly lower incidence of skin test reactivity showing that animal allergens cannot be fully avoided.

The skin microbiome in healthy and allergic dogs.

BackgroundChanges in the microbial populations on the skin of animals have traditionally been evaluated using conventional microbiology techniques. The sequencing of bacterial 16S rRNA genes has revealed that the human skin is inhabited by a highly diverse and variable microbiome that had previously not been demonstrated by culture-based methods. The goals of this study were to describe the microbiome inhabiting different areas of the canine skin, and to compare the skin microbiome of healthy and allergic dogs.Methodology/Principal FindingsDNA extracted from superficial skin swabs from healthy (n = 12) and allergic dogs (n = 6) from different regions of haired skin and mucosal surfaces were used for 454-pyrosequencing of the 16S rRNA gene. Principal coordinates analysis revealed clustering for the different skin sites across all dogs, with some mucosal sites and the perianal regions clustering separately from the haired skin sites. The rarefaction analysis revealed high individual variability between samples collected from healthy dogs and between the different skin sites. Higher species richness and microbial diversity were observed in the samples from haired skin when compared to mucosal surfaces or mucocutaneous junctions. In all examined regions, the most abundant phylum and family identified in the different regions of skin and mucosal surfaces were Proteobacteria and Oxalobacteriaceae. The skin of allergic dogs had lower species richness when compared to the healthy dogs. The allergic dogs had lower proportions of the Betaproteobacteria Ralstonia spp. when compared to the healthy dogs.Conclusions/SignificanceThe study demonstrates that the skin of dogs is inhabited by much more rich and diverse microbial communities than previously thought using culture-based methods. Our sequence data reveal high individual variability between samples collected from different patients. Differences in species richness was also seen between healthy and allergic dogs, with allergic dogs having lower species richness when compared to healthy dogs.

Microarrayed dog, cat, and horse allergens show weak correlation between allergen-specific IgE and IgG responses⋆

Microarrayed dog, cat, and horse allergens show weak correlation between allergen-specific IgE and IgG responses⋆

Sensitization rates of causative allergens for dogs with atopic dermatitis: detection of canine allergen-specific IgE.

Allergen-specific IgE serology tests became commercially available in the 1980s. Since then these tests have been widely used to diagnose and treat allergic skin diseases. However, the relationship between a positive reaction and disease occurrence has been controversial. The purpose of this study was to evaluate allergens using a serologic allergy test in dogs with atopic dermatitis (AD). Dogs clinically diagnosed with AD (n=101) were tested using an allergen-specific IgE immunoassay. Among the total 92 environmental and food allergens, house dust and house dust mites were the most common. Several allergens including airborne pollens and molds produced positive reactions, and which was considered increasing allergens relating to the climate changes. The presence of antibodies against staphylococci and Malassezia in cases of canine AD was warranted in this study. Additionally, strong (chicken, turkey, brown rice, brewer's yeast, and soybean) and weakly (rabbit, vension, duck, and tuna) positive reactions to food allergens could be used for avoidance and limited-allergen trials.

A cross-sectional analysis of pet-specific immunoglobulin E sensitization and allergic symptomatology and household pet keeping in a birth cohort population

It is unknown whether family members with detectable specific immunoglobulin E (sIgE) and/or allergic symptoms to pets are more or less likely to reside in a household with pets. We cross-sectionally investigated potential relationships between family members' allergic sensitization and symptoms to dogs and cats and current household pet-keeping practices, using birth cohort data. Blood samples taken from children enrolled in a birth cohort and their biological mothers and fathers, when the children were aged 18 years, were assessed for sIgE to dog and cat allergens. Interviews assessed subjects' self-reported pet exposure symptoms, current household pet-keeping practices, and socioeconomic characteristics. Overall, household dog or cat keeping was not associated with sIgE to these animals and/or self-reported allergic symptoms in the presence of these animals, even after controlling for factors such as education and household income. In subgroup analyses, current household dog keeping among dog-symptomatic teens (n = 40) was significantly lower than among teens who were not dog symptomatic (n = 289), at 48.8 and 61.1%, respectively (p = 0.036). Current household cat keeping was significantly lower among cat-symptomatic mothers (n = 27) compared with mothers who were not cat symptomatic (n = 120), at 24.3 and 37.0%, respectively (p = 0.015). However, when considering those who were both sensitized and reported symptoms, only the mother and cat-keeping associations persisted (p = 0.049). When cat-sensitized mothers report allergic symptoms to cats, these pets may be less likely to be kept in homes. Elevated dog and cat allergen sIgE does not appear to be associated with the keeping of these pets.

Cat and dog sensitization in pet shop workers

Sensitivity and symptoms related to animal proteins have been investigated in various occupational groups. However, data from pet shops are limited. To investigate rates of sensitivity to cats and dogs among pet shop workers, to assess the relationship between sensitivity, allergen levels and symptoms and to investigate whether passive transport from pet shops to homes is possible. Pet shop workers underwent interviews with a questionnaire adapted from the European Community Respiratory Health Survey. Dust samples for allergen detection were collected from pet shops using a vacuum cleaner. Skin tests were performed with common allergens. Dust samples were also obtained from the houses of 7 workers and 12 control subjects. Fifty-one workers from 20 pet shops were included in the study. Thirteen (25%) workers reported work-related symptoms. Four workers had sensitivity to animal allergens. The mean cat/dog allergen levels from pet shops were 15.7 and 3.2 µg/g, respectively. There was no significant relationship between cat/dog allergen levels and work-related symptoms and sensitivity to pets. None of the dust samples collected from the homes of pet shop workers contained cat allergens. Dog allergen was detected in only one house (0.58 µg/g). Neither cat nor dog allergens were found in the homes of the 12 control subjects. Although a quarter of pet shop workers reported work-related symptoms, sensitivity to cat and dog was low. These findings suggest that work-related symptoms may be due to other factors than cat and dog sensitivity.

Cat, dog and house dust mite allergen levels on children’s soft toys

Objective: Children’s soft toys are known to harbour house dust mite (HDM) allergens, but little is known whether they harbour cat or dog allergens. The objective of the study was to measure cat (Fel d 1), dog (Can f 1) and HDM allergens on children’s soft toys. Methods: Dust was collected from 40 children’s soft toys and their mattresses. Data were collected on pet ownership. Dust samples were analysed for Fel d 1, Can f 1, Der p 1 and Der f 1 by enzyme-linked immunosorbent assay (ELISA) and results are expressed as median levels with inter-quartile ranges. Results: Thirty-five (87.5%) soft toys had detectable Fel d 1 levels (median: 0.73 µg/g; inter-quartile range: 0.26–2.56 µg/g) while 34 (85%) had detectable Can f 1 levels (1.20 µg/g; 0.53–2.68). Correspondingly, 32 (80%) mattresses had detectable Fel d 1 levels (0.18 µg/g, 0.07–1.01) while 34 (85%) had detectable Can f 1 levels (0.50 µg/g; 0.33–1.06). All mattresses and soft toys had detectable HDM allergen (Der p 1 + Der f 1) levels with soft toys containing about three times higher levels than mattresses. In homes with cats (n = 10) Fel d 1 levels were higher on soft toys than homes without cats (2.49 versus 0.48 µg/g; p = 0.0009). In homes with dogs (n = 25) Can f 1 levels were generally higher on soft toys (1.38 versus 0.63 µg/g; p = 0.10). Conclusions: This study has shown that soft toys can harbour cat and dog allergen as well as HDM allergens, some with very high levels. Cat and dog ownership resulted in higher Fel d 1 and Can f 1 levels on soft toys and mattresses. The levels of Fel d 1, Can f 1 and HDM allergens on soft toys could be of importance to sensitized asthmatic children.

Effects of butorphanol versus dexmedetomidine sedation on intradermal allergen and histamine responses in dogs with atopic dermatitis

Sedation is commonly used during intradermal testing (IDT). Morphine and its derivatives have long been avoided because of their histamine-releasing effects. Butorphanol, an opioid agonist/antagonist, will not adversely affect IDT in dogs. Ten client-owned dogs diagnosed with atopic dermatitis. Dogs were randomized to be sedated with butorphanol (0.4 mg/kg) or dexmedetomidine (5 μg/kg). Routine IDT along with intradermal injections of various dilutions of histamine were performed on the lateral thorax, followed 7 days later by the alternative sedative and IDT on the opposite side. The injection sites were subjectively scored and objectively measured by one investigator, blinded to the sedatives, and compared between groups. When the mean wheal diameters from the objective measurements of all antigens, including saline and histamine dilutions, were compared, butorphanol was associated with significantly smaller reactions than dexmedetomidine (P = 0.0001). There was a high level of agreement between sedatives when positive reactions subjectively scored as ≥3+ were compared (κ = 0.91). When mean wheal diameters of histamine at concentrations of 1:100,000, 1:400,000, 1:1,600,000 and 1:6,400,000 were compared, there were no significant differences between sedative types. Moreover, the percentage agreement when comparing subjective interpretation of all histamine dilutions between sedations was high (κ = 0.90). However, there was only 69% agreement beyond chance when objective and subjective interpretations of all antigens were compared between sedative groups. Although butorphanol resulted in significantly smaller wheal size in comparison to dexmedetomidine, it did not affect the overall subjective interpretation of the results of IDT.

Can f 1 Levels in Hair and Homes of Different Dog Breeds: Lack of Evidence to Describe Any Dog Breed as Hypoallergenic

DW Vregegoor, T Willemse, MD Chapman. J Allergy Clin Immunol. 2012;130(4):904–909 To compare levels of, and determine the relationships between, the major dog allergen (Can f 1) from the hair and coat of various breeds of dogs and in the homes in which the dogs live. A total of 356 dogs (Labradoodles, Labrador retrievers, poodles, Spanish waterdogs, airedales, and a heterogeneous control group) were recruited from breeders, breeder associations, and a veterinary hospital in the Netherlands. In addition, 168 homes with 1 dog had floor and airborne dust samples collected and a survey of their 502 owners was conducted. Dog …

Associations between selected allergens, phthalates, nicotine, polycyclic aromatic hydrocarbons, and bedroom ventilation and clinically confirmed asthma, rhinoconjunctivitis, and atopic dermatitis in preschool children

Previous studies, often using data from questionnaires, have reported associations between various characteristics of indoor environments and allergic disease. The aim of this study has been to investigate possible associations between objectively assessed indoor environmental factors and clinically confirmed asthma, rhinoconjunctivitis, and atopic dermatitis. The study is a cross-sectional case-control study of 500 children aged 3-5years from Odense, Denmark. The 200 cases had at least two parentally reported allergic diseases, while the 300 controls were randomly selected from 2835 participating families. A single physician conducted clinical examinations of all 500 children. Children from the initially random control group with clinically confirmed allergic disease were subsequently excluded from the control group and admitted in the case group, leaving 242 in the healthy control group. For most children, specific IgE's against various allergens were determined. In parallel, dust samples were collected and air change rates were measured in the children's bedrooms. The dust samples were analyzed for phthalate esters, polycyclic aromatic hydrocarbons (PAH), nicotine, and various allergens. Among children diagnosed with asthma, concentrations of nicotine were higher (P<0.05) and cat allergens were lower (P<0.05) compared with the healthy controls; air change rates were lower for those sensitized (specific IgE+) compared with those not sensitized (specific IgE-, P<0.05); and dust mite allergens were higher for specific IgE+ cases compared with healthy controls (P<0.05). When disease status was based solely on questionnaire responses (as opposed to physician diagnosis), significant associations were found between di(2-ethylhexyl) phthalate (DEHP) and dog allergens in dust and current wheeze.

Allergens as Immunomodulatory Proteins: The Cat Dander Protein Fel d 1 Enhances TLR Activation by Lipid Ligands

Allergic responses can be triggered by structurally diverse allergens. Most allergens are proteins, yet extensive research has not revealed how they initiate the allergic response and why the myriad of other inhaled proteins do not. Among these allergens, the cat secretoglobulin protein Fel d 1 is a major allergen and is responsible for severe allergic responses. In this study, we show that similar to the mite dust allergen Der p 2, Fel d 1 substantially enhances signaling through the innate receptors TLR4 and TLR2. In contrast to Der p 2, however, Fel d 1 does not act by mimicking the TLR4 coreceptor MD2 and is not able to bind stably to the TLR4/MD2 complex in vitro. Fel d 1 does, however, bind to the TLR4 agonist LPS, suggesting that a lipid transfer mechanism may be involved in the Fel d 1 enhancement of TLR signaling. We also show that the dog allergen Can f 6, a member of a distinct class of lipocalin allergens, has very similar properties to Fel d 1. We propose that Fel d 1 and Can f 6 belong to a group of allergen immunomodulatory proteins that enhance innate immune signaling and promote airway hypersensitivity reactions in diseases such as asthma.

A novel TLR-8 agonist attenuates nasal symptoms/congestion in both dog and human allergen challenge studies

Methods (Dog) Ragweed (RW)-sensitized dogs (n=5) were treated with VTX-378 (100, 500 or 1000 μg/dog), given ~ 24 hours prior to RW challenge. Two VTX-378 pretreatments of 250 or 1000 μg/dog, spaced either 3, 4 or 7 days apart were also evaluated. Changes in nasal cavity volumes (congestion) were determined by acoustic rhinometry. (Human) Subsequently, a randomized, double blind, placebo-controlled study was conducted in-season, and enrolled 80 adults with confirmed atopy to grass pollen. Two dosing regimens were compared to placebo: ascending dose (GrpA; 25, 50, 75, 100 μg) and fixed dose (GrpB; 62.5 μg x 4 doses). Subjects were dosed on Days 1, 8, 15, and 22 with VTX-1463. On day 24, subjects underwent grass allergen exposure. The primary endpoints were the average change over 6 hours of allergen exposure in Total Nasal Symptom Score (TNSS; sum of scores for nasal congestion, itching, sneezing and rhinorrhea) and Active Anterior Rhinometry (ARR).

Do allergic dogs exhibit features of a severe steroid-resistant allergic airway phenotype?

Background Most asthmatic patients respond favorably to corticosteroid therapy, however, a subpopulation of patients (5-10%) display persistent immune activation and airway inflammation, despite high doses of oral/inhaled corticosteroids. The development of more predictive animal models that incorporate the severe steroid-resistant phenotype are needed to better define translational biomarkers and therapeutic targets for this patient population.

In vitro effects of CpG oligodeoxynucleotides delivered by gelatin nanoparticles on canine peripheral blood mononuclear cells of atopic and healthy dogs – a pilot study

Cytosine-phosphate-guanine (CpG) oligodeoxynucleotides offer a novel promising immunotherapeutic approach for atopic dermatitis (AD) both in humans and animals. Gelatin nanoparticles (GNP) enhance and prolong CpG-associated immunomodulatory effects and minimize adverse effects both in vitro and in vivo. Information about the effects of this combination in dogs is lacking. The aim of this study was to evaluate immunological effects of CpG coupled to GNP on canine peripheral blood mononuclear cells (PBMCs) in vitro. Eight dogs with AD, diagnosed by standard criteria and with a concurrent immediate hypersensitivity to house dust mites were included. Control samples were taken from eight healthy, age-matched control dogs without history or evidence of cutaneous or systemic illness. Peripheral blood mononuclear cells of healthy and allergic dogs were incubated with CpG-GNP and the uptake of CpG-GNP was demonstrated using confocal laser scanning microscopy. Cell culture supernatant concentrations of interferon gamma (IFN-γ), interleukin (IL)-4, IL-6 and IL-10 were measured by Canine Cytokine Milliplex. No significant changes in IFN-γ and IL-4 were found when comparing PBMCs incubated with CpG and CpG-GNP with the negative controls in atopic and healthy dogs. Interleukin-6 was not detected in any of the groups. However, a statistically significant increase in IL-10 concentration was found after 24h stimulation with CpG-GNP compared with CpG alone both in atopic and healthy dogs. As IL-10 is considered an immunosuppressive cytokine playing a key role in peripheral tolerance; the reported CpG-GNP formulation could be a new approach in allergy treatment.

Microbial content of household dust associated with exhaled NO in asthmatic children

Exhaled nitric oxide (eNO) is increasingly used as a non-invasive measure of airway inflammation. Despite this, little information exists regarding the potential effects of indoor microbial components on eNO. We determined the influence of microbial contaminants in house dust and other indoor environmental characteristics on eNO levels in seven-year-olds with and without a physician-diagnosis of asthma. The study included 158 children recruited from a birth cohort study, and 32 were physician-diagnosed as asthmatic. The relationship between eNO levels and exposures to home dust streptomycetes, endotoxin, and molds was investigated. Streptomycetes and endotoxin were analyzed both as loads and concentrations in separate models. Dog, cat, and dust mite allergens also were evaluated. In the multivariate exposure models, high streptomycetes loads and concentrations were significantly associated with a decrease in eNO levels in asthmatic (p<0.001) but not in healthy children. The presence of dog allergen, however, was associated with increased levels of eNO (p=0.001). Dust endotoxin was not significant. The relationship between eNO and indoor exposure to common outdoor molds was u-shaped. In non-asthmatic children, none of the exposure variables was significantly associated with eNO levels. To our knowledge, this is the first study demonstrating a significant association between microbial components in the indoor environment and eNO levels in asthmatic children. This study demonstrates the importance of simultaneously assessing multiple home exposures of asthmatic children to better understand opposing effects. Common components of the indoor Streptomyces community may beneficially influence airway inflammation.

Family and home characteristics correlate with mold in homes

Previously, we demonstrated that infants residing in homes with higher Environmental Relative Moldiness Index were at greater risk for developing asthma by age seven. The purpose of this analysis was to identify the family and home characteristics associated with higher moldiness index values in infants’ homes at age one. Univariate linear regression of each characteristic determined that family factors associated with moldiness index were race and income. Home characteristics associated with the moldiness index values were: air conditioning, carpet, age of the home, season of home assessment, and house dust mite allergen. Parental history of asthma, use of dehumidifier, visible mold, dog and cat allergen levels were not associated with moldiness index. Results of multiple linear regression showed that older homes had 2.9 units higher moldiness index (95% confidence interval [CI]=0.4, 5.4), whereas homes with central air conditioning had 2.5 units lower moldiness index (95% CI=−4.7, −0.4). In addition, higher dust mite allergen levels and carpeting were positively and negatively associated with higher moldiness index, respectively. Because older homes and lack of air conditioning were also correlated with race and lower income, whereas carpeting was associated with newer homes, the multivariate analyses suggests that lower overall socioeconomic position is associated with higher moldiness index values. This may lead to increased asthma risk in homes inhabited by susceptible, vulnerable population subgroups. Further, age of the home was a surrogate of income, race and carpeting in our population; thus the use of these factors should carefully be evaluated in future studies.