Spontaneous immunological activities in the target tissue of vitiligo-prone Smyth and vitiligo-susceptible Brown lines of chicken.

Introduction: Vitiligo is an acquired de-pigmentation disorder characterized by the post-natal loss of epidermal melanocytes (pigment-producing cells) resulting in the appearance of white patches in the skin. The Smyth chicken is the only model for vitiligo that shares all the characteristics of the human condition including: spontaneous post-natal loss of epidermal melanocytes, interactions between genetic, environmental and immunological factors, and associations with other autoimmune diseases. In addition, an avian model for vitiligo has the added benefit of an easily accessible target tissue (a growing feather) that allows for the repeated sampling of an individual and thus the continuous monitoring of local immune responses over time. Methods: Using a combination of flow cytometry and gene expression analyses, we sought to gain a comprehensive understanding of the initiating events leading to expression of vitiligo in growing feathers by monitoring the infiltration of leukocytes and concurrent immunological activities in the target tissue beginning prior to visual onset and continuing throughout disease development. Results: Here, we document a sequence of immunologically significant events, including characteristic rises in infiltrating B and αß T cells as well as evidence of active leukocyte recruitment and cell-mediated immune activities (CCL19, IFNG, GZMA) leading up to visual vitiligo onset. Examination of growing feathers from vitiligo-susceptible Brown line chickens revealed anti-inflammatory immune activities which may be responsible for preventing vitiligo (IL10, CTLA4, FOXP3). Furthermore, we detected positive correlations between infiltrating T cells and changes in their T cell receptor diversity supporting a T cell-specific immune response. Conclusion: Collectively, these results further support the notion of cell-mediated immune destruction of epidermal melanocytes in the pulp of growing feathers and open new avenues of study in the vitiligo-prone Smyth and vitiligo-susceptible Brown line chickens.

Establishment of Rat Models of Different Pathological Types of Hypersensitivity Pneumonitis Using Pigeon Droppings.

BACKGROUND: The pathogenesis and pulmonary histopathological characteristics of hypersensitivity pneumonitis (HP) are not yet fully understood. Therefore, we established animal models of HP of different stages, aiming to provide support for research on this disease. METHODS: We established rat models of pigeon breeder's lung of different pathological types by creating freeze-dried allergen powder from fresh pigeon feathers, dander, and other droppings. Freeze-dried allergen powder suspensions of pigeon droppings were used to establish 2 rat models of HP, one by aerosol inhalation and one by airway instillation, and the rats were sacrificed after different lengths of time to observe the pathological changes in their lung tissues. RESULTS: By the 40th week after allergen inhalation, granulomas were the main changes in the model, without fibrotic changes. When using airway instillation to establish the model, at the 20th week, group 1 (low dose + twice/week) and group 2 (medium dose + twice/week) showed granuloma changes, but no fibrosis; group 3 (high dose + once/week) and group 4 (high dose + twice/week) both showed obvious pulmonary fibrotic changes, but the death rate of rats in group 4 was greater. CONCLUSIONS: Both aerosol inhalation and airway instillation of freeze-dried pigeon allergen powder can successfully establish an HP model. The airway instillation method can cause pulmonary fibrotic changes in a short time, and the pulmonary pathological changes of animal models manifest with an obvious time-dose effect.

Local and systemic inflammatory responses to lipopolysaccharide in broilers: new insights using a two-window approach.

The inflammatory response involves a complex interplay of local tissue activities designed to recruit leukocytes and proteins from the blood to the infected tissue. For egg-type chickens, we established the growing feather (GF) as an accessible tissue test site to monitor tissue responses to injected test-material. For commercial broilers, whose health depends to a large extent on innate immune system functions, the GF test system offers an important novel window to directly assess their natural defenses. This study was conducted to adapt the GF test system for use in broilers, and use it to simultaneously examine local (GF) and systemic (blood) inflammatory responses initiated by GF pulp injection of lipopolysaccharide (LPS). Specifically, GF of 12 male and 12 female, 5-week-old broilers were injected with LPS (16 GF/chicken; 1 µg LPS/GF). Blood and GF were collected at 0 (before), 6, and 24 h after GF injection. GF pulp was used to determine leukocyte-infiltration and gene-expression profiles, reactive-oxygen-species generation, and superoxide dismutase (SOD) activity. Blood was used to determine blood cell profiles and SOD activity. A time effect (P ≤ 0.05) was observed for most aspects examined. In GF, LPS injection resulted in heterophil and monocyte infiltration reaching maximal levels at 6 and 24 h, respectively. Reactive-oxygen-species generation, SOD activity, and mRNA levels of IL-1ß, IL-8, IL-6, IL-10, and cathelicidin B1 were elevated, whereas those of TNF-α, LITAF, SOD1, and SOD2 decreased after LPS injection. In blood, levels of heterophils and monocytes were elevated at 6 h, lymphocytes and RBC decreased at 6 h, and thrombocytes and SOD activity increased at 24 h. Assessment of LPS-induced activities at the site of inflammation (GF) provided novel and more relevant insights into temporal, qualitative, and quantitative aspects of inflammatory responses than blood. Knowledge generated from this dual-window approach may find direct application in identification of individuals with robust, balanced innate defenses and provide a platform for studying the effects of exogenous treatments (e.g., nutrients, probiotics, immunomodulators, etc.) on inflammatory responses taking place in a complex tissue.

Feather duvet lung.

A 43-year-old non-smoker was referred with a 3-month history of malaise, fatigue and breathlessness. Blood avian precipitins were strongly positive. Lung function testing confirmed a restrictive pattern with impaired gas transfer. A 'ground glass' mosaic pattern was seen on CT imaging, suggestive of hypersensitivity pneumonitis. Although he had no pet birds, on closer questioning he had recently acquired a duvet and pillows containing feathers. His symptoms, chest radiograph and lung function tests improved after removal of all feather bedding, and he was also started on oral corticosteroid therapy. Our case reinforces the importance of taking a meticulous exposure history and asking about domestic bedding in patients with unexplained breathlessness. Prompt recognition and cessation of antigen exposure may prevent the development of irreversible lung fibrosis.

Frequency of goose and duck down causation of hypersensitivity pneumonitis within an 80-patient cohort.

BACKGROUND: A variety of antigens have been identified as causative of hypersensitivity pneumonitis (HP), which is characterized by inflammation to the lung parenchyma that is induced by exposure. Goose and duck down (GDD) bedding is often overlooked by physicians as a potential cause, yet the use of GDD has markedly increased in recent years, paralleling an increased frequency of reports of GDD-induced HP. OBJECTIVE: To determine the frequency of GDD as the causative antigen in patients with HP who use bedding that contains GDD. METHODS: Patients referred with a working diagnosis of HP underwent a detailed environmental history. Those who were using GDD were asked to remove it as an avoidance procedure. Signs, symptoms, spirometry, and inflammatory markers were followed up at weekly intervals for up to 1 month to determine the effect of remediation. RESULTS: Eighty patients with HP were seen during an 8-year period. Thirty-two patients (40%) were using GDD bedding. Of these 32 patients, 12 (37.5% of those exposed and 15% of the total HP population experienced remission (or nonprogression) of disease by simply avoiding GDD bedding. Eleven (92%) of these 12 patients were female. In patients with GDD-induced HP, lung biopsy patterns were varied. CONCLUSION: Approximately one-third of patients with HP, who slept with GDD, had persistent improvement or remission with simple avoidance. The higher incidence of GDD-induced HP in females may be hormonal and/or sociocultural related. Lung biopsy findings were across the spectrum of histopathologic patterns. Avoidance-challenge techniques were effective in confirming diagnoses and causation and mitigating the need for additional remediation.

Preservation potential of keratin in deep time.

Multiple fossil discoveries and taphonomic experiments have established the durability of keratin. The utility and specificity of antibodies to identify keratin peptides has also been established, both in extant feathers under varying treatment conditions, and in feathers from extinct organisms. Here, we show localization of feather-keratin antibodies to control and heat-treated feathers, testifying to the repeatability of initial data supporting the preservation potential of keratin. We then show new data at higher resolution that demonstrates the specific response of these antibodies to the feather matrix, we support the presence of protein in heat-treated feathers using ToF-SIMS, and we apply these methods to a fossil feather preserved in the unusual environment of sinter hot springs. We stress the importance of employing realistic conditions such as sediment burial when designing experiments intended as proxies for taphonomic processes occurring in the fossil record. Our data support the hypothesis that keratin, particularly the ß-keratin that comprises feathers, has potential to preserve in fossil remains.

Extensive protein hydrolyzation is indispensable to prevent IgE-mediated poultry allergen recognition in dogs and cats.

BACKGROUND: The central premise for the commercialization of diets with hydrolyzed ingredients is that the small-sized digested peptides would be unable to crosslink allergen-specific IgE at the surface of tissue mast cells and induce their degranulation. Evidence for the validity of this concept to diagnose food allergies in dogs and cats is limited, however. Our objectives were to study the recognition of standard and variably hydrolyzed poultry extracts by sera from dogs and cats with elevated chicken-specific serum IgE. RESULTS: Forty sera from dogs and 40 from cats with undetectable, low, medium or high serum levels of chicken-specific IgE were tested by ELISA on plates coated with the positive controls chicken, duck and turkey meat extracts and the negative controls beef meat (dogs) or wheat (cats). Plates were also coated with a non-hydrolyzed chicken meal, and mildly- or extensively-hydrolyzed poultry feather extracts. The frequencies of dogs with positive IgE against the various extracts were: chicken meat: 100%, duck and turkey meats: 97%, beef meat: 3%, non-hydrolyzed chicken meal: 73%, mildly-hydrolyzed poultry feathers: 37% and extensively-hydrolyzed poultry feathers: 0%. For cats, these respective percentages were (with wheat replacing beef as a negative control): 100, 84, 97, 7, 7, 0 and 0%. To detect any allergenic cross-reactivity between poultry meat-based and feather hydrolysate-derived extracts, an IgE ELISA inhibition was also done. Ten canine sera with the highest level of anti-poultry IgE in the previous experiment were incubated overnight with a previously optimized 50 µg amount of each of the extracts used above. We performed ELISA on plates coated with chicken, duck or turkey meats with or without inhibitors. The median inhibition percentages after incubation with the non-hydrolyzed chicken meal were ~22%, with the mildly-hydrolyzed poultry feathers: 14-22%, and those with the extensively-hydrolyzed poultry feathers: 5 to 10%; the last inhibition level was similar to that of the beef meat negative control. CONCLUSIONS: Altogether, these results suggest that an extensive-but not partial-hydrolyzation of the poultry feather extract is necessary to prevent the recognition of allergenic epitopes by poultry-specific IgE.

T lymphocytes dominate local leukocyte infiltration in response to intradermal injection of functionalized graphene-based nanomaterial.

Graphene-based nanomaterials (GBN) have many potential biomedical applications. However, information regarding their biological properties and interactions with cells and/or soluble factors within a complex tissue is limited. The objective of this study was to use the growing feather (GF) of chickens as a minimally invasive cutaneous test-site to assess and monitor leukocyte recruitment in response to intradermal GBN injection. Specifically, the dermis of 20 GFs per chicken was injected with 10 µl of phosphate-buffered saline (PBS)-vehicle or 10 µl of 300 µg ml-1 oxygen-functionalized (f) GBN (6 chickens/treatment). GFs were collected before- (0) and at 0.25, 1, 2, 3, 4, 5, and 7 days post-injection and used for leukocyte-population analysis of immunofluorescently stained pulp cell suspensions or histological examination. Based on flow-cytometric cell population analysis, lymphocytes and macrophages were the major leukocyte-populations infiltrating GFs in response to f-GBN presence. Compared with PBS-controls, levels of T cells (γδ-, αß-, CD4- and CD8-T cells) were greatly elevated in f-GBN-injected GFs within 6 h and remained elevated throughout the 7-day examination period. f-GBN's effects on local tissue leukocyte recruitment were not reflected in the blood, except for a higher percentage of lymphocytes on 7 days. These observations together with a visual examination of f-GBN-injected GF tissue-sections suggest a delayed-type hypersensitivity-like, inflammatory cell-mediated response to the non-biodegradable f-GBN. The GF 'in vivo test-tube'system together with blood sampling provided unique insight into the time-course, qualitative, and quantitative aspects of immune system activities initiated by the presence of f-GBN in a complex tissue of a living animal. Copyright © 2017 John Wiley & Sons, Ltd.

The growing feather as a dermal test site: Comparison of leukocyte profiles during the response to Mycobacterium butyricum in growing feathers, wattles, and wing webs.

Using the response to Mycobacterium butyricum as the test-immune response, the main goal of this study was to demonstrate the suitability of the growing feather (GF) as a dermal test site and window into in vivo cellular/tissue responses (US-Patent 8,216,551). Using M. butyricum immunized chickens, the specific objectives were to: 1) compare the leukocyte infiltration response to intra-dermally injected M. butyricum in GF, wattles, and wing webs; 2) use GF as the test site to monitor leukocyte response profiles to recall antigen in the same individuals; and 3) gain new knowledge regarding the local response to M. butyricum in chickens. For objective 1, chickens were euthanized for tissue collection at 4 to 6, 24, 48, and 72 h after intra-dermal antigen injection. Leukocyte infiltration profiles were determined using immunochemical and conventional histology. Data from this study established the similarities between the cellular response in GF, wattles, and wing webs and uncovered many advantages of working with GF. For objective 2, antigen was injected into multiple GF per individual. GF were collected before and at 0.25, 1, 2, 3, and 7 d post injection and processed for cell population analysis by flow cytometry. Advantages of the approach used in objective 2 included a technically easier, more comprehensive, and more objective leukocyte profile analysis; same-day data acquisition; and, most importantly, easy, minimally invasive sample collection from the same individual throughout the study. Both studies contributed new knowledge regarding the local cutaneous response to M. butyricum in M. butyricum immunized chickens and confirmed the cell-mediated nature of the immune response to M. butyricum (e.g., elevated levels [P < 0.05] of T cells [CD4+ and CD8+], macrophages and MHC class II+-cells on days one to 3 post injection in M. butyricum- compared to PBS-injected tissues). The use of GF as an "in vivo test tube" to monitor local innate and adaptive immune activities will find direct application in vaccine development, as well as in the assessment and optimization of immune system development and function in poultry.

[Improved IgG Antibody Diagnostics of Feather Duvet Lung by an Antibody Screening Test]. / Optimierte IgG-Antikörperdiagnostik der Bettfedern-Alveolitis mittels EAA-Suchtest.

The underdiagnosed feather duvet lung, an extrinsic allergic alveolitis (hypersensitivity pneumonitis) caused by duck and goose feathers, can be more frequently diagnosed, if duck and goose feather antibodies are included in the panel of the routinely applied IgG antibody screening test. This does not necessarily require extending the screening test to include duck and goose feather antigens. By analysing 100 sera with duck and goose antibodies we found that the commonly used pigeon and budgerigar antibodies can also screen for feather duvet antibodies. All examined sera lacking pigeon and budgerigar antibodies also lacked clear-cut duck and goose feather antibodies. The examined sera with strong pigeon or budgerigar antibodies always also contained feather duvet antibodies. However, sera with medium or low concentrated pigeon or budgerigar antibodies are not always associated with feather duvet antibodies. In the light of these observations, we find that 71% of the duck and goose antibody analyses would be dispensable without essential loss of quality, if the results of screening for pigeon and budgerigar antibodies were incorporated into the procedure of a step-by- step diagnostics.

Paediatric feather duvet hypersensitivity pneumonitis.

A previously well 12-year-old boy was admitted with a second insidious episode of dyspnoea, dry cough, anorexia, weight loss and chest pain. At admission, he had an oxygen requirement, significantly impaired lung function and reduced exercise tolerance. Initial forced expiratory volume in 1 s was 26%; a 3 min exercise test stopped at 1 min 50 when saturations dropped to 85%. CT scan showed ground-glass nodularity with lymphadenopathy. Bronchoalveolar lavage (BAL) for Pneumocystis carinii pneumonia and viruses were negative, and microbiology results for the BAL were reported in the absence of histology. This is because at the time the BAL samples were collected, a lung biopsy was performed. The biopsy was consistent with hypersensitivity pneumonitis. Echo was normal and CT pulmonary angiography negative. After taking a thorough history, exposure to feather duvets prior to each episode was elicited. IgG of avian precipitants was raised at 10.6 mgA/L (normal <10 mgA/L). Clinical improvement began with avoidance of exposure, while the boy was an inpatient. Antigen avoidance continued on discharge. He continues to improve since discharge. The condition was diagnosed as hypersensitivity pneumonitis secondary to exposure to antigens from feather duvets.

Enzymatic hydrolyzed feather peptide, a welcoming drug for multiple-antibiotic-resistant Staphylococcus aureus: structural analysis and characterization.

This study aimed to explore the bactericidal activity of a feather-degraded active peptide against multiple-antibiotic-resistant (MAR) Staphylococcus aureus. An antibacterial peptide (ABP) was isolated from the chicken feathers containing fermented media of Paenibacillus woosongensis TKB2, a keratinolytic soil isolate. It was purified by HPLC, and its mass was found to be 4666.87 Da using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectroscopy. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of this peptide were 22.5 and 90 µg/ml, respectively. SEM study revealed the distorted cell wall of the test strain along with pore formation. The possible reason for bactericidal activity of the peptide is due to generation of reactive oxygen species (ROS), resulting in membrane damage and leakage of intracellular protein. Complete sequence of the peptide was predicted and retrieved from the sequence database of chicken feather keratin after in silico trypsin digestion using ExPASy tools. Further, net charge, hydrophobicity (77.7 %) and molecular modelling of the peptide were evaluated for better understanding of its mode of action. The hydrophobic region (17 to 27) of the peptide may facilitate for initial attachment on the bacterial membrane. The ABP exhibited no adverse effects on RBC membrane and HT-29 human cell line. This cytosafe peptide can be exploited as an effective therapeutic agent to combat Staphylococcal infections.

Quantification of rHVT-F genome load in feather follicles by specific real-time qPCR as an indicator of NDV-specific humoral immunity induced by day-old vaccination in SPF chickens.

The purpose of this study was to look for a reliable molecular method for confirmation of uptake of recombinant turkey herpesvirus vaccine against Newcastle disease (rHVT-F) and for use as a valuable prediction tool of Newcastle disease virus (NDV)-specific immune response in chickens deprived of maternally derived antibody (MDA). A quantitative real-time polymerase chain reaction (real-time qPCR) specific to rHVT-F was developed. The method was applied to various tissue samples taken from specific pathogen free (SPF) chickens experimentally inoculated at day-old with one dose of rHVT-F vaccine over a 6-week period. Among the tested tissues, the rHVT-F vaccine was detected predominantly in the bursa of Fabricius (BF) and the lung for the first week, followed by a progressive decline from 9 days onwards. Then, an increase of genome load was observed in the feather follicles (FF) with a peak at 2 weeks, rising to a level almost 10(3)-fold greater than in the other tissues. Importantly, the rHVT-F genome load in FF appeared to be strongly correlated to the humoral immunity specific to NDV as evaluated by haemagglutination inhibition (HI) test and NDV-specific IgG, IgM and IgA ELISAs. This is the first report of quantification of rHVT-F vaccine in FF and its correlation with the induction of ND-specific immune response in chickens with no MDA. Our data indicate that the application of this real-time qPCR assay on FF samples taken from chickens in the field may be used to confirm rHVT-F vaccine administration and uptake with the important added benefit of offering a non-disruptive sampling procedure.

Sensitization to aeroallergens in atopic dermatitis patients: association with concomitant allergic diseases.

BACKGROUND: A few reports demonstrate the relationship between IgE sensitization to aeroallergens in atopic dermatitis (AD) patients and other allergic diseases and parameters. OBJECTIVES: The objective of this study was to evaluate, if there is a significant relationship between the sensitization to common aeroallergens in AD patients and the occurrence of asthma bronchiale, rhinitis and other atopic parameters. METHODS: Sensitization to dust, mites, animal dander and bird feather was examined (skin prick test, specific IgE) and the relationship with the occurrence of asthma bronchiale, rhinitis, duration of AD, family history and onset of AD was evaluated. RESULTS: Two hundred and eighty-eight patients were examined - 90 men and 198 women. According to our results, IgE sensitization to animal dander, dust and mites may increase the risk of developing asthma or rhinitis. Persistent lesions of AD occur more often in patients with sensitization to animal dander, mites and dust. Patients with the sensitization to bird feather have the onset of AD more often above 5 years of age and in these patients, there is no relationship with the positive data about atopy in the family history. CONCLUSION: There is a greater likelihood of developing other allergic diseases in atopic dermatitis patients who suffer from sensitisation to animal dander, mites, and dust. Thus, prompt management of atopic dermatitis and allergy to inhallant allergens that develop in early infancy may be a successful method for preventing of atopic march.

The proteomics of feather development in pied flycatchers (Ficedula hypoleuca) with different plumage coloration.

The genetic theory of morphological evolution postulates that form evolves largely by changing the expression proteins that are functionally conserved. It follows that understanding the function of proteins during different phases of development as well as the mechanisms by which the functions are modified is a prerequisite for understanding evolutionary change. Male pied flycatchers exhibit marked phenotypic variation in their breeding plumage. This variation has repeatedly been shown to have adaptive significance, but the molecular basis of this variation is not known. Here, we characterize the proteome of developing pied flycatcher feathers from differently pigmented males and also introduce a new method for examining the effect sizes of expression differences in protein interaction networks. Approximately 300 proteins were identified in the developing feathers of males. Gene products associated with cellular transport, cell metabolism and protein synthesis formed a large part of the developing feather proteome. Sixty-five proteins associated with the development of the epidermis and/or pigmentation were detected in the data. The examination of expression level differences of protein-protein interaction networks revealed an immunological signalling-related network to exhibit significantly higher expression in black compared to brown males. Additionally, indications of differences in energy balance and oxidative stress related characteristics were detected. Together, these results provide new insight into the molecular mechanisms and evolutionary significance of plumage colour variation.

[Domestic hypersensitivity pneumonitis]. / Pneumopathies d'hypersensibilité domestiques. Alvéolites allergiques extrinsèques domestiques.

INTRODUCTION: Domestic hypersensitivity pneumonitis (HP) cases are relatively widespread, with an overall annual incidence of approximately 1/100,000 reported in a British study covering several million patients. All-causes mortality is three times higher within HP-affected patients than amongst the general population. STATE OF THE ART: Cases of HP are usually diagnosed as 'farmer's lung' (FL) and 'bird fancier's lung' (BFL) diseases, however we suggest that other domestic causes, such as humidifier lung, hot tub pneumonitis, feather duvet and domestic exposure to moulds may be more frequent than widely suggested. Usually, the diagnosis is established on the basis of characteristic clinical, functional, radiological and broncho-alveolar lavage findings or recurrence of respiratory symptoms after returning home. PERSPECTIVES: In the absence of a common cause (FL or BFL), physicians must have a high index of clinical suspicion and should consider an environmental antigen source. Detailed questioning of HP patients on their living conditions and, where appropriate, a home inspection conducted by an environmental health expert are necessary for identifying causative antigens. CONCLUSION: The cornerstone of therapy is antigen avoidance.

Carotenoid-based ornaments of female and male American goldfinches (Spinus tristis) show sex-specific correlations with immune function and metabolic rate.

Conspicuous ornamentation has been linked to immunological and physiological condition in males of many species. In species where both sexes are ornamented, it is unclear whether the signal content of ornaments differs between males and females. We examined the immunological and physiological correlates of carotenoid-based bill and plumage ornamentation in American goldfinches Spinus tristis, a species in which bright orange bills are sexually monomorphic but yellow plumage is sexually dimorphic during the breeding season. Because bill color is dynamic over short periods while plumage color is static over longer time frames, we tested whether these signals have the potential to provide temporal information about immunity and condition. In both sexes, bill color (but not plumage color) was negatively related to leukocyte differential, a measure of recent stress, while plumage color (but not bill color) was positively related to resting metabolic rate. In females, bill color also positively correlated with immunoglobulin Y, a component of acquired immunity, while plumage color positively predicted natural antibody levels, a component of innate immunity. In males, neither bill color nor plumage color predicted immune function, suggesting that the mechanisms underlying these signals vary with sex. Our results demonstrate that dynamic signals such as bill coloration do not merely reflect the same information provided by static signals but that these two classes of signal provide information about different temporal aspects of phenotypic quality. Furthermore, our results indicate that a signal expressed in both sexes has the potential to provide different information depending on the sex of the bearer.

IFN-γ, IL-21, and IL-10 co-expression in evolving autoimmune vitiligo lesions of Smyth line chickens.

The Smyth line (SL) of chicken is an excellent animal model for human autoimmune vitiligo. In SL vitiligo (SLV), postnatal loss of melanocytes in feathers appears to be due to cell-mediated immunity. In this study, leukocyte infiltration and associated expression (RNA) of immune function-related cytokines in growing feathers were investigated throughout SLV development and progression. Both leukocyte infiltration and cytokine expression levels started to increase near visible SLV onset (early SLV), reached peak levels during active SLV, and decreased to near pre-vitiligo levels after complete loss of melanocytes. Specifically, significant increases were noticed in relative proportions of T cells, B cells, and major histocompatibility complex (MHC) II-expressing cells during active SLV. Levels of T-cell infiltration were higher than those of B cells, with more CD8+ than CD4+ cells throughout SLV. Elevated leukocyte infiltration in early and active SLV was accompanied by increased levels of cytokine expression, especially in IFN-γ, IL-10, and IL-21. Low expression of IL-4 and IL-17 did not suggest important roles of Th2 and Th17 cells in SLV pathogenesis. Taken together, SLV appears to be a Th1-polarized autoimmune disease, whereby IFN-γ expression is strongly associated with parallel increases in IL-10 and IL-21, particularly during early and active stages of SLV.