Investigating the release of inflammatory cytokines in a human model of incontinence-associated dermatitis.

Incontinence-associated dermatitis (IAD) is a painful complication in elderly patients, leading to reduced quality of life. Despite recent attention, its underlying inflammatory mechanisms remain poorly understood. This study was designed to quantify the release of inflammatory cytokines in a human model of IAD. The left volar forearm of ten healthy volunteers was exposed to synthetic urine and synthetic faeces for 2 h, simulating the effects of urinary and faecal incontinence, respectively, and the subsequent cytokine response compared to that of an untreated control site. Inflammatory cytokines were collected using both the Sebutape® absorption method and dermal microdialysis and quantified using immunoassays. Results from the former demonstrated an upregulation in IL-1α, IL-1RA and TNF-α. Synthetic urine caused a higher median increase in IL-1α from baseline compared to synthetic faeces, whereas synthetic faeces were associated with significantly higher median TNF-α levels compared to synthetic urine (p = 0.01). An increase in IL-1α/IL-1RA ratio was also observed with significant differences evident following exposure to synthetic urine (p = 0.047). Additionally, microdialysis revealed a time-dependent increase in IL-1ß and IL-8 following exposure of up to 120 min to synthetic urine and synthetic faeces, respectively. This study demonstrated the suitability of both sampling approaches to recover quantifiable cytokine levels in biofluids for the assessment of skin status following exposure to synthetic fluids associated with incontinence. Findings suggest some differences in the inflammatory mechanisms of IAD, depending on moisture source, and the potential of the cytokines, IL-1α and TNF-α, as responsive markers of early skin damage caused by incontinence.

The potential role of human HIV-1 TAT-Interactive Protein 2 levels in the pathogenesis of contact dermatitis

Background/aim: Human HIV-1 TAT interactive protein 2 (HTATIP2/TIP30) is a gene that is extensively expressed in human tissues as well as in tumor tissues. This study aimed to explore the potential role of HTATIP2/TIP30 in contact dermatitis (CD), which is one of the most common inflammatory cutaneous conditions. Materials and methods: This cross-sectional study involved adult patients with acute contact dermatitis who were admitted to the outpatient dermatology clinic of a tertiary hospital and healthy adult volunteers without any cutaneous or systemic diseases. The blood concentration of HTATIP2/TIP30 was measured using ELISA kits. Results: The research sample consisted of 31 patients with CD (18 males, 13 females) and 20 healthy control subjects (14 males, 6 females). The mean ages of the patients with CD and healthy volunteers were 37 and 30 years, respectively (p > 0.05). The mean value of serum HTATIP2/TIP30 levels in patients with CD was 1.65 ng ml­1, which is 0.60 ng ml­1 in the control group (p = 0.02) Conclusion: In this study, serum levels of HTATIP2/TIP30 were statistically significantly higher in patients with CD when compared to healthy controls. This outcome may indicate possible role of HTATIP2/TIP30 in the pathogenesis of CD.

Platelets are important for the development of immune tolerance: Possible involvement of TGF-ß in the mechanism.

Platelets have diverse roles in immune processes in addition to their key functions in haemostasis and thrombosis. Some studies imply that platelets may be possibly related to the immune tolerance induction. However, the role of platelets in the development of immune tolerance is not fully understood. The purpose of this study was to investigate the role of platelets in the development of regulatory mechanisms responsible for cutaneous inflammation using a mouse model of low zone tolerance (LZT). Mice were treated with 2,4,6-trinitro-1-chlorobenzene (TNCB) 8 times every other day for tolerance induction with administration of anti-platelet antibody or control antibody during the tolerance induction phase every 3 days. After the treatment for the tolerance induction, mice were sensitized and then challenged with TNCB. The contact hypersensitivity (CHS) was significantly decreased at 24 hours after challenge in the mice with LZT than in those without LZT. Platelet depletion via administration of anti-platelet antibody reversed the inhibition of CHS and reduced the frequency of Foxp3+ Tregs in the inflamed skin and draining lymph nodes in mice with LZT. In addition, repeated low-dose skin exposure resulted in elevated plasma levels of transforming growth factor (TGF)-ß1. Interestingly, platelet depletion reduced plasma TGF-ß1 levels of mice with LZT. Furthermore, the CHS response was reduced by administration of recombinant TGF-ß1 during platelet depletion in mice with LZT. Administration of anti-TGF-ß antibody reversed the inhibition of the CHS responses. These results suggest that platelets are involved in the induction of immune tolerance via the release of TGF-ß1.

The Role of Interleukin-19 in Contact Hypersensitivity.

Interleukin (IL)-19 is a member of the IL-10 family of interleukins and is an immuno-modulatory cytokine produced by the main macrophages. The gastrointestinal tissues of IL-19 knockout mice show exacerbated experimental colitis mediated by the innate immune system and T cells. There is an increasing focus on the interaction and relationship of IL-19 with the function of T cells. Contact hypersensitivity (CHS) is T cell-mediated cutaneous inflammation. Therefore, we asked whether IL-19 causes CHS. We investigated the immunological role of IL-19 in CHS induced by 1-fluoro-2,4-dinitrofluorobenzene as a hapten. IL-19 was highly expressed in skin exposed to the hapten, and ear swelling was increased in IL-19 knockout mice. The exacerbation of the CHS response in IL-19 knockout mice correlated with increased levels of IL-17 and IL-6, but no alterations were noted in the production of interferon (IFN)γ and IL-4 in the T cells of the lymph nodes. In addition to the effect on T cell response, IL-19 knockout mice increased production of inflammatory cytokines. These results show that IL-19 suppressed hapten-dependent skin inflammation in the elicitation phase of CHS.

Influence of the season on vitamin D levels and regulatory T cells in patients with polymorphic light eruption.

The exact mechanisms of photohardening in polymorphic light eruption (PLE) are still unknown, but medical photohardening was shown to increase regulatory T cell (Treg) numbers in the blood of PLE patients, similar to natural hardening. Furthermore, oral vitamin D supplementation increased peripheral Tregs in healthy individuals. We herein report on a post hoc analysis of 26 screened PLE patients of a clinical trial (ClinicalTrials.gov No. NCT01595893), in which the influence of the progressing season was investigated on baseline CD4+CD25+FoxP3+CD127- Treg numbers by flow cytometry and Treg suppressive function by co-culture assays with T effector cells as a secondary endpoint, together with 25-hydroxy vitamin D (25(OH)D) serum levels at the study's screening visit, taking place in the period from January to June. The mean 25(OH)D serum level of all patients was 33.2 ng ml(-1). Ten of those patients (38.5%) were identified with low 25(OH)D levels (<30 ng ml(-1)). Significantly higher baseline 25(OH)D serum levels (plus 34.4%; P = 0.0182) as well as higher relative Treg percentages in CD4+ population (plus 62.8%; P = 0.0157) and in total lymphocyte population (plus 59.6%; P = 0.0372) and higher absolute Treg numbers (plus 100.2%; P = 0.0042) were observed in the late spring/early summer period (April to June) compared to the winter period (January to February). No significant relationship was observed when Treg numbers and function were correlated with 25(OH)D levels. These data indicate that in PLE patients Treg numbers and their suppressive function are independent of vitamin D serum levels and suggest that UV light and/or other seasonal factors may affect these cells via the non-vitamin D related pathway(s).

Stat5 gene dosage in T cells modulates CD8+ T-cell homeostasis and attenuates contact hypersensitivity response in mice.

BACKGROUND: Contact hypersensitivity assay (CHS) faithfully models human allergies. The Stat5 transcription factors are essential for both lymphocyte development and acute immune responses. Although consequences of Stat5 ablation and transgenic overexpression for the lymphocyte development and functions have been extensively studied, the role of Stat5 gene dosage in contact allergies has not been addressed. OBJECTIVE: We investigated the effect of Stat5 gene dosage modulation in contact allergies using CHS in mice. METHODS: Transgenic animals heterozygous for the germline Stat5 null allele were subjected to CHS. To dissect cell type sensitive to Stat5 gene dosage, animals with Stat5 haplo-insufficiency in T cells, where one Stat5 allele was removed by Lck-Cre-mediated deletion (Stat5(ΔT/+)), were tested by CHS. Frequency of T cells, B cells, and monocytes were analyzed in Stat5(ΔT/+) and wild-type animals by flow cytometry. Proliferation of Stat5(ΔT/+) CD8(+) T cells was studied in vitro by stimulation with IL-4 and IL-2 cytokines, and changes in the expression of Stat5 target genes were assayed by quantitative real-time PCR assay. RESULT: Haplo-insufficiency of Stat5 in T cells leads to the reduction in CD8(+) T cells in all lymphoid organs and attenuates CHS response. Stat5(ΔT/+) CD8(+) T cells failed to fully activate Stat5-dependent expression of cell cycle/survival target genes, such as Bcl2 and Pim1, and to proliferate efficiently in response to IL-2 and IL-4 cytokine. CONCLUSION: Our data identify Stat5 as a dose-dependent regulator of CD8(+) T-cell functions in contact allergies and suggest that modulation of Stat5 dosage could be used to target contact allergies in humans.

Decrease of serum adenine nucleotide hydrolysis in an irritant contact dermatitis mice model: potential P2X7R involvement.

Extracellular adenosine 5'-triphosphate (ATP) has significant effects on a variety of pathological conditions and it is the main physiological agonist of P2X7 purinergic receptor (P2X7R). It is known that ATP acting via purinergic receptors plays a relevant role on skin inflammation, and P2X7R is required to neutrophil recruitment in a mice model of irritant contact dermatitis (ICD).The present study investigated the effects of chemical irritant croton oil (CrO) upon ATP, ADP, and AMP hydrolysis in mice blood serum, and the potential involvement of P2X7R. The topical application CrO induced a decrease on soluble ATP/ADPase activities (~50 %), and the treatment with the selective P2X7R antagonist, A438079, reversed these effects to control level. Furthermore, we showed that CrO decreased cellular viability (52.6 % ± 3.9) in relation to the control and caused necrosis in keratinocytes (PI positive cells). The necrosis induced by CrO was prevented by the pre-treatment with the selective P2X7R antagonist A438079. The results presented herein suggest that CrO exerts an inhibitory effect on the activity of ATPDase in mouse serum, reinforcing the idea that ICD has a pathogenic mechanism dependent of CD39. Furthermore, it is tempting to suggest that P2X7R may act as a controller of the extracellular levels of ATP.

An Animal Model of Trichloroethylene-Induced Skin Sensitization in BALB/c Mice.

Trichloroethylene (TCE) is a major occupational hazard and environmental contaminant that can cause multisystem disorders in the form of occupational medicamentosa-like dermatitis. Development of dermatitis involves several proinflammatory cytokines, but their role in TCE-mediated dermatitis has not been examined in a well-defined experimental model. In addition, few animal models of TCE sensitization are available, and the current guinea pig model has apparent limitations. This study aimed to establish a model of TCE-induced skin sensitization in BALB/c mice and to examine the role of several key inflammatory cytokines on TCE sensitization. The sensitization rate of dorsal painted group was 38.3%. Skin edema and erythema occurred in TCE-sensitized groups, as seen in 2,4-dinitrochlorobenzene (DNCB) positive control. Trichloroethylene sensitization-positive (dermatitis [+]) group exhibited increased thickness of epidermis, inflammatory cell infiltration, swelling, and necrosis in dermis and around hair follicle, but ear painted group did not show these histological changes. The concentrations of serum proinflammatory cytokines including tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-2 were significantly increased in 24, 48, and 72 hours dermatitis [+] groups treated with TCE and peaked at 72 hours. Deposition of TNF-α, IFN-γ, and IL-2 into the skin tissue was also revealed by immunohistochemistry. We have established a new animal model of skin sensitization induced by repeated TCE stimulations, and we provide the first evidence that key proinflammatory cytokines including TNF-α, IFN-γ, and IL-2 play an important role in the process of TCE sensitization.

IL-13: a marker of chromium contact allergy.

BACKGROUND: Allergic contact dermatitis is a frequent, often disabling disease caused by countless substances. Patch testing remains the gold standard test to identify the causative agent; however, it is subjective, time-consuming and not completely safe. Alternative methods were tried, but significant success has only been achieved with nickel. OBJECTIVE: Develop an alternative or complementary allergic contact dermatitis diagnostic test. METHODS: We compared the lymphocyte proliferative rate and cytokine production (IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17 and RANTES) between 18 chromium allergic patients and 19 controls. RESULTS: The lymphocyte proliferation test and some of the cytokines tested (IFN-γ, IL-2, IL-5, IL-12 and IL-13) were able to discriminate allergic patients. However, striking results were only achieved using IL-13, leading to an accuracy of about 90%. CONCLUSIONS: If further studies confirm the data found, IL-13 could be used as an alternative or complementary test to detect chromium contact allergy whereas lymphocyte proliferation test, IFN-γ, IL-2, IL-5 and IL-12 detections may serve as additional diagnostic tests.

Varying allergen composition and content affects the in vivo allergenic activity of commercial Dermatophagoides pteronyssinus extracts.

BACKGROUND: Diagnosis and immunotherapy of house-dust mite (HDM) allergy is still based on natural allergen extracts. The aim of this study was to analyze commercially available Dermatophagoides pteronyssinus extracts from different manufacturers regarding allergen composition and content and whether variations may affect their allergenic activity. METHODS: Antibodies specific for several D. pteronyssinus allergens (Der p 1, 2, 5, 7, 10 and 21) were used to analyze extracts from 10 different manufacturers by immunoblotting. Sandwich ELISAs were used to quantify Der p 1 and Der p 2 in the extracts. Mite-allergic patients (n = 45) were skin-tested with the extracts and tested for immunoglobulin E (IgE) reactivity to a panel of 10 mite allergens (Der p 1, 2, 4, 5, 7, 8, 10, 14, 20 and 21) by dot blot. RESULTS: Only Der p 1 and Der p 2 were detected in all extracts but their concentrations and ratios showed high variability (Der p 1: 6.0-40.8 µg ml(-1); Der p 2: 1.7-45.0 µg ml(-1)). At least 1 out of 4 allergens (i.e. Der p 5, 7, 10 and 21) was not detected in 8 of the studied extracts. Mite-allergic subjects showed different IgE reactivity profiles to the individual mite allergens, the extracts showed different allergenic activity in skin-prick tests and false-negative results. CONCLUSIONS: Commercially available D. pteronyssinus extracts lack important allergens, show great variability regarding allergen composition and content and some gave false-negative diagnostic test results in certain patients.

Identification of C3 beta chain as the human serum eosinophil cytotoxicity inhibitor.

An eosinophil cytotoxicity inhibitor (ECI) was purified from serum of a human subject with severe allergic dermatitis. Molecular weight of the isolated polypeptide (75,000) and its NH2-terminal amino acid sequence identified it as the beta chain of the C3 complement component (apparently free, but perhaps attached to very small fragments of the alpha chain). Free beta chain, prepared from normal plasma by reduction of C3, inhibited both eosinophil cytotoxicity and neutrophil adherence functions, with half-maximal activity at approximately 250 ng/ml. Apparently free C3 beta chain was detected in certain human biological fluids associated with inflammation; the presence of C3 beta chain correlated with ECI activity. This evidence demonstrates a potential role for free C3 beta chain as a suppressor of eosinophil and neutrophil functions in inflammation.

Anti-inflammatory effects of Glehnia littoralis extract in acute and chronic cutaneous inflammation.

Glehnia littoralis (Umbelliferae) is a traditional medicine used in Korea, China, and Japan to treat the immune-related diseases. However, its anti-inflammatory activities and mechanisms remain to be defined. We investigated the effects of 70% ethanolic extract from G. littoralis (GLE) on skin inflammation in mice. Production of proinflammatory cytokines (IL-1ß and TNF-α), activation of myeloperoxidase (MPO), and histological indicators were examined in acute and chronic skin inflammation using 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced mouse ear edema. We also performed acetic acid-induced vascular permeability tests. GLE treatment at 200 mg/kg inhibited topical edema in the mouse ear, leading to substantial reductions in skin thickness and tissue weight, inflammatory cytokine production, neutrophil-mediated MPO activity, and several histopathological indicators. Furthermore, GLE effectively reduced inflammatory damage induced by chronic TPA exposure and significantly inhibited the vascular permeability induced by acetic acid in mice. These results suggest that G. littoralis is an effective anti-inflammatory agent in murine phorbol ester-induced dermatitis and may have therapeutic potential in a variety of immune-related cutaneous diseases.

Amphiregulin is not essential for induction of contact hypersensitivity.

BACKGROUND: Amphiregulin (AR) is expressed in Th2 cells, rather than Th1 cells, and plays an important role in Th2 cell/cytokine-mediated host defense against nematodes. We also found earlier that AR mRNA expression was strongly upregulated in inflamed tissue during Th2 cell/cytokine-mediated fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS), suggesting a contribution of AR to the induction of those responses. METHODS: To elucidate the role of AR in the induction of FITC- or dinitrofluorobenzene (DNFB)-induced CHS, AR-deficient mice were sensitized and/or challenged with FITC or DNFB epicutaneously. The levels of FITC-mediated skin dendritic cell (DC) migration and FITC-specific lymph node cell proliferation and cytokine production were assessed by flow cytometry, [3H]-thymidine incorporation and ELISA, respectively, after FITC sensitization. The degree of ear swelling, the activities of myeloperoxidase (MPO) and eosinophil peroxidase (EPO) in inflammatory sites and the levels of FITC-specific immunoglobulin (Ig) in sera were determined by histological analysis, colorimetric assay and ELISA, respectively, after FITC challenge. RESULTS: DC migration and FITC-specific lymph node cell proliferation and cytokine production were normal in the AR-deficient mice. Ear swelling, tissue MPO and EPO activities and FITC-specific serum Ig levels were also similar in AR-deficient and -sufficient mice. CONCLUSIONS: Amphiregulin is not essential for the induction of FITC- or DNFB-induced CHS responses in mice.

Enhanced contact hypersensitivity by delayed T-helper 2 response in BALB/c mice.

T-helper (Th) 1/Th2 balance determines the direction of contact hypersensitivity (CHS). To clarify the immunopathogenesis of contact dermatitis, 2,4-dinitrofluorobenzene (DNFB)-induced CHS reaction was compared between the BALB/c and C57BL/6 mice. The two strains were sensitized with DNFB systemically and challenged with DNFB locally. The CHS reaction in BALB/c mice was intense compared with that in C57BL/6 mice at 24 and 48 hours post-DNFB challenge. The dermal lesions were characterized by infiltration of lymphocytes, eosinophils, neutrophils, and macrophages including CD4+ and CD8+ T cells, and interleukin (IL)-4-producing(+) and interferon (IFN)-gamma+ cells in BALB/c mice. In C57BL/6 mice, the composition of inflammatory cells was same as those in BALB/c mice except for eosinophils, CD4+ T cells, and IL-4+ cells. There was no increase in the number of mast cells in the two strains. Local and systemic productions of IL-4 and IFN-gamma in BALB/c mice were higher than those in C57BL/6 mice. Although blood IgE values increased in BALB/c mice, but not in C57BL/6 mice, at 48 hours postchallenge, its value was low. The delayed Th2-like response together with Th1-like response in BALB/c mice may induce strong CHS reaction compared with C57BL/6 mice, which may dominantly develop Th1-like reaction. Moreover, mast cell and IgE do not appear to be involved in delayed CHS.

Allergens in atopic dermatitis.

Allergens play an essential role in atopic dermatitis, either intrinsic or extrinsic. They provoke cutaneous inflammation via IgE-dependent and cell-mediated immune reactions. Food allergens have a well-known contribution to disease activity of atopic dermatitis, especially in infants and young children. However, the importance of inhaled allergens is still under investigation. For clinical implication, identification of individualized allergens is an ideal strategy for better control of atopic dermatitis and avoidance of atopic march. The aim of this article is to discuss the common allergens in atopic dermatitis (AD), the specificity and sensitivity of laboratory tests for allergens, and the clinical effect of various preventions.

[Eosinophilia, ECP and Ecp/Eo ratio in allergic and non-allergic diseases]. / Eozynofilia, ECP, wskaznik ECP/Eo w chorobach alergicznych i niealergicznych.

Eosinophilia in peripheral blood occurs in allergic as well as non-allergic diseases. In the research there were 26 patients with eosinophil level above 5% encountered in leukocyte smear of peripheral blood encountered. Patients were split into 3 groups: patients with allergic atopic disease (ANN), patients with non-atopic (contact dermatitis) disease and patients with upper airways infection and/or parasitic disease. In the researched groups there were no statistically significant differences of ECP level in serum and of ECP/Eo ratio detected. There were also correlations between ECP and ECP/Eo ratio for every researched group analyzed. The highest correlation was observed in patients with contact dermatitis and in non-atopic patients in general. Initial results show uselessness of applied research methods in the routine differential diagnosis of eosinophilia.

Anti-inflammatory effect of water-soluble complex of 1'-acetoxychavicol acetate with highly branched ß-1,3-glucan on contact dermatitis.

The anti-inflammatory effect on contact dermatitis of the water solubilized 1'-Acetoxychavicol Acetate (ACA) by complexation with ß-1,3-glucan isolated form Aureobasidium pullulans black yeast is reported. It is well-known that ACA possesses a function to inhibit the activation of NF-κB by which genes encoding proinflammatory cytokines, chemokines, and growth factors are regulated. However, because ACA is quite insoluble in water, its usefulness has been extremely limited. On the other hand, a triple-helical polysaccharide ß-1,3-glucan can include hydrophobic compounds into intrastrand hydrophobic cavity and solubilize poorly water-soluble compounds. In this study, solubilization of ACA by complexation with highly branched ß-1,3-glucan was achieved. The effect of anti-inflammatory response of water-soluble ACA complex with ß-1,3-glucan was confirmed in vitro and in vivo.

Role of UVB-induced serum factor(s) in suppression of contact hypersensitivity in mice.

Ultraviolet, 280-320 nm (UVB), irradiation of the shaved dorsal skin of mice results in suppression of the development of contact hypersensitivity (CHS) to antigens applied subsequently to a distant nonirradiated skin site. Serum from BALB/cAnNCr mice exposed to a single dose of UVB radiation (8.6 X 10(4) J/m2) was evaluated for its ability to induce suppression of CHS to 2-chloro-1,3,5-trinitrobenzene (TNCB), a contact allergen, after transfer to normal recipients. Serum from UVB-irradiated donors was capable of inducing immunosuppression only when collected and transferred within a restricted time period, i.e., approximately 2-6 h post irradiation, and at least 400 microliters of serum per recipient was required. Serum from UVB-irradiated donors was sufficient to induce splenic suppressor cells in recipient mice.

Inflammatogenic properties of bacterial DNA following cutaneous exposure.

Bacterial DNA and oligodeoxynucleotides containing cytosine-phosphate-guanosine sequences and thereby mimicking prokaryotic DNA, have recently been shown to exert potent immunostimulatory properties. As skin normally harbors bacteria, and as the bacterial content and the levels of bacterial degradation products increase during skin infection, we analyzed the potential inflammatogenic role of bacterial DNA and oligodeoxynucleotides in a mouse model of cutaneous inflammation. Bacterial DNA from Staphylococcus aureus was injected intradermally into mice and its inflammatogenic properties were compared with synthetic phosphodiester and phosphorothioate cytosine-phosphate-guanosine- or GpC-containing oligodeoxynucleotides. A peak inflammatory infiltrate in the skin was seen already 2 d after injection with either bacterial DNA or the phosphodiester cytosine-phosphate-guanosine-oligodeoxynucleotides. In contrast, nuclease-resistant phosphorothioate cytosine-phosphate-guanosine-induced dermatitis peaked 7 d after intradermal injection. The inflammatory infiltrates consisted mainly of macrophages, and depletion of this cell population resulted in a significant (p=0.0001) decrease in the severity of inflammation, which suggests that macrophages play a central part in inflammatory responses in the skin following exposure to cytosine-phosphate-guanosine-containing oligodeoxynucleotides. A significant decrease in local inflammatory infiltrate was also seen in mice with deficiencies in neutrophil or lymphocyte populations, which indicates that these cell populations may also be involved in mediating inflammatory signals after the injection of immunostimulatory DNA sequences. In summary, our results suggest that bacterial DNA is an important virulence determinant and inflammatory stimulus during skin infections.

Detection of hapten erythrocyte conjugates in vitro and in vivo.

The studies presented demonstrate that immunofluorescent techniques are capable of detecting picryl chloride erythrocyte protein conjugates formed in vitro and in vivo, following infusion of picryl chloride into normal, sensitized and tolerant guinea pigs. These experiments and the finding that picryl erythrocyte stromata prepared in vitro, and in vivo from infused recipients, have the capacity fo sensitize fresh animals, support the view that infused picryl chloride conjugates with the red cell membrane in vivo.