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The tadalafil-like compounds have appeared recently as adulterants in drinks and healthcare dietary supplements sourced from medicinal and edible food, which may cause illness and even death. In this work, the rationality of haptens was explored by computational chemistry and molecular simulation theories such as frontier molecular orbital (FMO)-based softness (S), three-dimensional (3D) structure, surface electrostatic potential (ESP), and lipophilic potential (LP). An antiserum from hapten H5 with the highest softness and maintaining the appropriate three-dimensional (3D) structure showed the optimal immunoassay performance, indicating an increasing softness was a critical factor for effective hapten. Based on the antibody induced by hapten H5, an indirect competitive enzyme-linked immunosorbent assay (icELISA) method for detecting multiple tadalafil-like adulterants was established. The icELISA showed a limit of detection (LOD), 50% inhibition concentration (IC

The regulatory community is transitioning to the use of nonanimal methods for dermal sensitization assessments; however, some in vitro assays have limitations in their domain of applicability depending on the properties of chemicals being tested. This study explored the utility of epidermal sensitization assay (EpiSensA) to evaluate the sensitization potential of complex and/or "difficult to test" chemicals. Assay performance was evaluated by testing a set of 20 test chemicals including 10 methacrylate esters, 5 silicone-based compounds, 3 crop protection formulations, and 2 surfactant mixtures; each had prior in vivo data plus some in silico and in vitro data. Using the weight of evidence (WoE) assessments by REACH Lead Registrants, 14 of these chemicals were sensitizers and, six were nonsensitizers based on in vivo studies (local lymph node assay [LLNA] and/or guinea pig studies). The EpiSensA correctly predicted 16/20 materials with three test materials as false positive and one silane as false negative. This silane, classified as weak sensitizer via LLNA, also gave a "false negative" result in the KeratinoSens™ assay. Overall, consistent with prior evaluations, the EpiSensA demonstrated an accuracy level of 80% relative to available in vivo WoE assessments. In addition, potency classification based on the concentration showing positive marker gene expression of EpiSensA was performed. The EpiSensA correctly predicted the potency for all seven sensitizing methacrylates classified as weak potency via LLNA (EC3 ≥ 10%). In summary, EpiSensA could identify dermal sensitization potential of these test substances and mixtures, and continues to show promise as an in vitro alternative method for dermal sensitization.

Aggregation of IgE bound to high affinity IgE receptor (FceRI) by multivalent antigen induces mast cell activation. Reportedly, disaggregation of aggregated FceRI immediately terminated degranulation, and formation of co-ligated FceRI and low affinity IgG receptor FcγRIIB blocked degranulation by inhibitory signal via SH2-containing inositol 5'-phosphatase 1 (SHIP1) phosphorylation. However, their molecular mechanisms to inhibit mast cell activation have been unclear in detail. Herein, we found that addition of excess monomeric hapten (TNP-alanine) to multivalent antigen (TNP-OVA)-activated rat basophilic leukemia cells and mouse bone marrow-derived mast cells induced immediate and transient Syk dephosphorylation, which was previously phosphorylated by TNP-OVA addition. Syk dephosphorylation correlated to rapidly decreased intracellular Ca2+ concentrations ([Ca2+ ]i ), terminated degranulation, and suppressed cytokine production through inhibition of Akt and ERK phosphorylation. Addition of hapten-specific IgG monoclonal antibody (anti-TNP IgG1) to activated mast cells induced translocation of SHIP1 to the plasma membrane and its phosphorylation, indicating that co-ligation of FceRI and FcγRIIB after FceRI aggregation can lead to SHIP1 activation. SHIP1 phosphorylation led to gradually decreased [Ca2+ ]i , weak inhibition of degranulation, and strong inhibition of cytokine production. Our findings clearly show the inhibitory mechanism of cell function in activated mast cells by operating Fc receptor crosslinking.

Early warning systems for monitoring toxic events may benefit from the availability of monoclonal antibodies enabling the sensitive and specific detection of anatoxin-a, a cyanotoxin involved in numerous cases of animal poisoning resulting from toxic algal blooms in freshwaters. Through the synthesis of three functionalized derivatives of anatoxin-a, we have succeeded in generating the first-ever reported immunoreagents (bioconjugates and antibodies) suitable for the development of immunoanalytical approaches aimed at rapid and onsite detection of this harmful cyanotoxin.

Several lines of evidence have clarified that the key transmission pathways of itching sensation travel from the periphery to the central nervous system (CNS). Despite the functional significance of gastrin‐releasing peptide (GRP) and its cognate receptor in the itch processing mechanism in the spinal dorsal horn (SDH), the roles of GRP‐expressing (GRP+) neurons in different regions remain unclear. This study aimed to determine whether GRP+ neurons in the CNS directly modulated itch processing. To specifically activate spinal and supraspinal GRP neurons by the designer receptors exclusively activated by designer drugs (DREADDs) system, CAG‐LSL‐Gq‐DREADD mice were crossed with GRP‐Cre mice, resulting in the development of GRP‐hM3Dq mice. Immunohistochemistry showed that hM3Dq was highly expressed in the SDH and brainstem closely related to sensory processing. The intraperitoneal, intrathecal, or intracerebroventricular administration of clozapine‐N‐oxide, an agonist of hM3Dq, strongly elicited dermatome‐dependent itch‐related scratching behavior, but did not change pain sensitivity. Importantly, GRP‐Gq‐DREADD‐mediated scratching behavior in GRP‐hM3Dq mice was not affected by the ablation of transient receptor potential vanilloid 1+ sensory C‐fibers, and it was also observed to a similar degree under chronic itch conditions. Furthermore, there were no significant sex differences in the scratching behavior elicited by GRP‐Gq‐DREADD, suggesting that itch‐dominant roles of central GRP+ neurons might be common in both sexes, at least under normal physiological conditions. These novel findings not only contribute to understanding the functional roles of central GRP+ neurons further, but also propose the development of future effective therapeutics for intractable itching., Chemogenetic activation of spinal GRP+ neurons by CNO administration in GRP‐hM3Dq mice elicited robust dermatome‐dependent scratching behavior in male and female mice, suggesting that itch‐processing roles of GRP+ neurons might be common in both sexes under physiological condition.

In human medicine, narrow-band ultraviolet B (NB-UVB) phototherapy has been used to treat various T-cell-mediated skin diseases. However, the effect of NB-UVB on inflamed canine skin remains uncertain.To investigate the effect of NB-UVB phototherapy on the skin of dogs with hapten-induced contact dermatitis.Seven healthy beagles without skin problems.Dogs were irradiated with varying doses of NB-UVB to determine the minimal erythema dose (MED). After determining the MEDs of six dogs (excluding one of the seven whose skin did not show a visible reaction), we investigated the effect of NB-UVB on their inflamed skin by topically applying 2,4-dinitrochlorobenzene (DNCB), which causes type 1 helper T cell (Th1)- and cytotoxic T-cell (Tc)1-induced skin inflammation. We then irradiated the skin with NB-UVB. We analysed the treated skin samples via histopathological and immunohistochemical methods, and TdT-mediated dUTP nick-end labelling (TUNEL) to demonstrate apoptotic cells. We also analysed the cytokine gene transcription via real-time quantitative reverse transcription PCR.The NB-UVB MEDs caused mild inflammatory changes yet no severe epidermal exfoliations in the irradiated skin. In DNCB-treated skin irradiated by the NB-UVB MEDs, TUNEL-positive dermal apoptotic cells were increased significantly compared with those of DNCB-treated, nonirradiated skin. INF-γ and TNF-α transcription levels in DNCB-treated, irradiated skin were significantly lower than those in the DNCB-treated, nonirradiated skin.Phototherapy using NB-UVB MEDs attenuated cutaneous Th1 and Tc1 cytokine responses with minimal skin damage in a canine model of hapten-induced contact dermatitis.En médecine humaine, la photothérapie ultraviolette B (NB-UVB) à bande étroite a été utilisée pour traiter diverses maladies de la peau à médiation par les lymphocytes T. Cependant, l'effet de NB-UVB sur la peau canine enflammée reste incertain.Étudier l'effet de la photothérapie NB-UVB sur la peau de chiens atteints de dermatite de contact induite par l'haptène.Sept beagles en bonne santé sans problèmes de peau. MÉTHODES ET MATÉRIEL: Les chiens ont été irradiés avec des doses variables de NB-UVB pour déterminer la dose minimale d'érythème (DEM). Après avoir déterminé les MED de six chiens (à l'exclusion de l'un des sept dont la peau n'a pas montré de réaction visible), nous avons étudié l'effet du NB-UVB sur leur peau enflammée en appliquant localement du 2,4-dinitrochlorobenzène (DNCB), qui provoque un inflammation de type 1 de la peau induite par les lymphocytes T auxiliaires (Th1) et les lymphocytes T cytotoxiques (Tc)1. Nous avons ensuite irradié la peau avec du NB-UVB. Nous avons analysé les échantillons de peau traités via des méthodes histopathologiques et immunohistochimiques, et le marquage dUTP nick-end (TUNEL) médié par TdT pour démontrer les cellules apoptotiques. Nous avons également analysé la transcription du gène des cytokines par PCR de transcription inverse quantitative en temps réel. RÉSULTATS: Les MED NB-UVB ont provoqué de légers changements inflammatoires, mais aucune exfoliation épidermique grave de la peau irradiée. Dans la peau traitée au DNCB irradiée par les MED NB-UVB, les cellules apoptotiques dermiques positives pour TUNEL ont augmenté de manière significative par rapport à celles de la peau non irradiée traitée au DNCB. Les niveaux de transcription d'INF-γ et de TNF-α dans la peau irradiée traitée au DNCB étaient significativement inférieurs à ceux de la peau non irradiée traitée au DNCB.La photothérapie utilisant des MED NB-UVB a atténué les réponses des cytokines cutanées Th1 et Tc1 avec des lésions cutanées minimales dans un modèle canin de dermatite de contact induite par l'haptène.INTRODUCCIÓN: en medicina humana, la fototerapia ultravioleta B de banda estrecha (NB-UVB) se ha utilizado para tratar diversas enfermedades de la piel mediadas por células T. Sin embargo, el efecto de NB-UVB sobre la piel canina inflamada sigue siendo incierto. OBJETIVOS: investigar el efecto de la fototerapia NB-UVB en la piel de perros con dermatitis de contacto inducida por hapteno. ANIMALES: Siete perros Beagle sanos sin problemas en la piel. MÉTODOS Y MATERIALES: los perros se irradiaron con dosis variables de NB-UVB para determinar la dosis mínima de eritema (MED). Después de determinar las MEDs de seis perros (excluyendo uno de los siete cuya piel no mostró una reacción visible), investigamos el efecto de NB-UVB en su piel inflamada aplicando tópicamente 2,4-dinitroclorobenceno (DNCB), que causa inflamación de la piel por tipo 1 linfocito T colaborador (Th1) y linfocito T citotóxico (Tc) 1. Luego irradiamos la piel con NB-UVB. Analizamos las muestras de piel tratadas a través de métodos histopatológicos e inmunohistoquímicos, y marcaje de extremos rotos (Nick-end labelling) dUTP mediado por TdT (TUNEL) para demostrar células apoptóticas. También analizamos la transcripción de genes de citoquinas mediante PCR cuantitativa de transcripción inversa en tiempo real. RESULTADOS: las NB-UVB MEDs causaron cambios inflamatorios leves pero no exfoliaciones epidérmicas graves en la piel irradiada. En la piel tratada con DNCB irradiada por las MEDs de NB-UVB, las células apoptóticas dérmicas positivas a TUNEL aumentaron significativamente en comparación con las de la piel no irradiada tratada con DNCB. Los niveles de transcripción de INF-γ y TNF-α en la piel irradiada tratada con DNCB fueron significativamente más bajos que los de la piel no irradiada tratada con DNCB. CONCLUSIÓN Y RELEVANCIA CLÍNICA: la fototerapia con NB-UVB MEDs atenuó las respuestas de las citoquinas cutáneas Th1 y Tc1 con un daño cutáneo mínimo en un modelo canino de dermatitis de contacto inducida por hapteno.In der Humanmedizin wird die Schmalband Ultraviolet B (NB-UVB) Phototherapie verwendet, um verschiedene T-Zell-mediierte Hauterkrankungen zu behandeln. Die Auswirkung von NB-UVB auf entzündete Hundehaut bleibt jedoch noch unklar.Eine Untersuchung der Auswirkung von NB-UVB Phototherapie auf die Haut von Hunden mit Hapten-induzierter Kontaktdermatitis.Sieben gesunde Beagles ohne Hautprobleme.Die Hunde wurden mit unterschiedlichen Dosen an NB-UVB bestrahlt, um die minimale Erythem Dosis (MED) zu bestimmen. Nach Bestimmung der MEDs bei sechs Hunden (einer der sieben Hunde wurde ausgenommen, da seine Haut keine sichtbare Reaktion zeigte), untersuchten wir die Auswirkung von NB-UVB auf ihre entzündete Haut durch topische Verabreichung von 2,3-Dinitrochlorobenzen (DNCB), welches eine Typ 1 Helfer T Zell (Th1)- und zytotoxische T-Zell (Tc)1-induzierte Hautentzündung verursacht. Danach wurde die Haut mit NB-UVB bestrahlt. Wir analysierten die behandelten Hautproben mittels histopathologischer und immunhistochemischer Methoden und TdT-mediierter dUTP Nick-Ende Färbung (TUNEL), um apoptotische Zellen zu demonstrieren. Wir analysierten auch die Zytokin Gentranskription mittels Real-Time Quantitativer Reverser Transkriptions PCR.Die NB-UVB MEDs verursachten in der bestrahlten Haut milde entzündliche Veränderungen, jedoch keine hochgradigen epidermalen Exfoliationen. Bei der DNCB-behandelten Haut, die mit NB-UVB MEDS bestrahlt worden war, nahm die Anzahl der TUNEL-positiven dermalen apoptotischen Zellen im Vergleich zu den mit DNCB-behandelter, nicht bestrahlter Haut signifikant zu. INF-γ und TNF-α Transkriptionswerte bei DNCB-behandelter, bestrahlter Haut waren signifikant niedriger als jene in der DNCB-behandelten, nicht bestrahlten Haut.Phototherapie mittels NB-UVB MEDS verminderte die kutane Th1 und Tc1 Zytokin Antwort bei minimalen Hautveränderungen in einem Hundemodell von Hapten-induzierter Kontaktdermatitis.背景: ヒトでは、ナローバンド紫外線B(NB-UVB) 光線療法は、様々なT細胞介在性皮膚疾患の治療に用いられている。しかし、炎症を起こした犬の皮膚に対するNB-UVBの効果はまだ不明である。 目的: 本研究の目的は、ハプテン誘発性接触皮膚炎のイヌの皮膚に対するNB-UVB光線療法の効果を調査することであった。 被検動物: 皮膚に問題のない健常ビーグル7頭。 材料と方法: 犬に様々な線量のNB-UVBを照射し、最小紅斑量 (MED) を決定した。6頭の犬 (皮膚が目に見える反応を示さなかった7頭のうち1頭を除く) のMEDを決定した後、1型ヘルパーT細胞 (Th1) および細胞障害性T細胞 (Tc)1誘発性皮膚炎症を引き起こす2,4-ジニトロクロロベンゼン (DNCB) を局所的に塗布することで、炎症を起こした皮膚に対するNB-UVBの効果を調査した。次に、この皮膚にNB-UVBを照射した。照射した皮膚サンプルは、病理組織学的および免疫組織化学的手法により解析し、TdT-mediated dUTP nick-end labelling (TUNEL)によりアポトーシス細胞を検出した。また、リアルタイム定量逆転写PCRでサイトカイン遺伝子の転写を解析した。 結果: NB-UVB MEDは、照射された皮膚に軽度の炎症性変化を引き起こしたが、重度の表皮剥離はなかった。NB-UVB MEDを照射したDNCB処理皮膚では, TUNEL陽性の真皮アポトーシス細胞がDNCB処理した非照射皮膚に比べて有意に増加した。DNCB処理した照射皮膚のINF-γおよびTNF-α転写レベルは、DNCB処理した非照射皮膚の転写レベルに比べて有意に低かった。 結論と臨床的妥当性: NB-UVB MEDを用いた光線療法は、ハプテン誘発性接触皮膚炎イヌモデルにおいて、皮膚の損傷を最小限に抑えながら、皮膚のTh1およびTc1サイトカイン反応を弱めた。.背景: 在人类医学中, 窄谱中波紫外线(NB-UVB)光疗已被用于治疗各种T细胞介导的皮肤病。然而, NB-UVB对犬发炎皮肤的影响仍不确定。 目的: 半抗原诱导出犬接触性皮炎,研究NB-UVB光疗对皮肤的影响。 动物: 7只健康比格犬, 无皮肤问题。 方法和材料: 用不同剂量的NB-UVB照射犬, 测定最小红斑剂量(MED)。在确定了6只犬的MED后 (不包括1只皮肤无可见反应的犬) , 我们研究NB-UVB对发炎皮肤的影响,通过局部涂抹2,4-二硝基氯苯(DNCB),引起1型辅助性T细胞(Th1)和细胞毒性T细胞(Tc)1诱导的皮肤炎症。然后我们用NB-UVB照射皮肤。我们通过组织病理学和免疫组织化学方法分析了治疗的皮肤样本, 并通过TdT介导的dUTP缺口末端标记(TUNEL)证明凋亡细胞。我们还通过实时定量逆转录PCR分析了细胞因子基因转录。 结果: NB-UVB MED在照射皮肤中引起轻度炎症变化, 但无重度表皮剥脱。DNCB处理、经NB-UVB MEDs照射皮肤, TUNEL阳性真皮凋亡细胞较DNCB处理、未照射皮肤显著增加。DNCB处理、照射皮肤的INF-γ和TNF-α转录水平显著低于DNCB处理、未照射皮肤。 结论和临床相关性: 在半抗原诱导的接触性皮炎犬模型中, 使用NB-UVB MED的光疗减弱了皮肤Th1和Tc1细胞因子反应, 减轻了皮肤损伤。.Na medicina humana, a fototerapia ultravioleta B de banda curta (NB-UVB) tem sido usada para tratar várias doenças cutâneas mediadas por células T. No entanto, o efeito de NB-UVB na pele canina inflamada permanece incerto.Investigar o efeito da fototerapia NB-UVB na pele de cães com dermatite de contato induzida por hapteno.Sete beagles saudáveis sem diagnóstico de dermatopatia. MÉTODOS E MATERIAIS: Os cães foram irradiados com doses variadas de NB-UVB para determinar a dose eritema mínima (MED). Depois de determinar as MEDs de seis cães (excluindo um dos sete cuja pele não mostrou uma reação visível), investigamos o efeito de NB-UVB em sua pele inflamada aplicando topicamente 2,4-dinitroclorobenzeno (DNCB), que causa inflamação cutânea mediada por células T-helper 1 (Th1) e por células T citotóxicas (Tc) 1. Em seguida, irradiamos a pele com NB-UVB. Analisamos as amostras de pele tratadas por meio de métodos histopatológicos e imuno-histoquímicos e marcação com dUTP nick-end (TUNEL) mediada por TdT para demonstrar células apoptóticas. Também analisamos a transcrição do gene da citocina via PCR de transcrição reversa quantitativa em tempo real.As MEDs de NB-UVB causaram leves alterações inflamatórias, mas não geraram esfoliação epidérmica grave na pele irradiada. Na pele tratada com DNCB irradiada pelas MEDs de NB-UVB, as células apoptóticas dérmicas positivas para TUNEL aumentaram significativamente em comparação com aquelas da pele não irradiada tratada com DNCB. Os níveis de transcrição de INF-γ e TNF-α em pele irradiada tratada com DNCB foram significativamente mais baixos do que na pele não irradiada tratada com DNCB. CONCLUSÃO E RELEVÂNCIA CLÍNICA: A fototerapia com MEDs de NB-UVB atenuou as respostas cutâneas de citocinas Th1 e Tc1 com injúria cutânea mínima em um modelo canino de dermatite de contato induzida por hapteno.

In human dendritic cells (DCs), we previously demonstrated in vitro that syndecan-1 (SDC1) is downregulated during maturation correlating with enhanced motility. We investigated the effects of SDC1 on DC migration in vivo during TNCB(2,4,6-trinitro-1-chlorobenzene)-induced cutaneous hypersensitivity reaction (CHS) in mice. We show that DC in SDC1-deficient mice migrated faster and at a higher rate to lymph nodes draining the hapten-painted skin. Adoptive transfer of SDC1-deficient hapten- and fluorochrome-labelled DC into wild-type (WT) mice led to increased and faster migration of DC to paracortical lymph nodes, and to a stronger CHS compared to WT DC. In SDC1-/- mice, CCR7 remains longer on the DC surface within the first 15-minutes maturation (after LPS-induced maturation). In addition, a time-dependent upregulation of CCL2, CCL3, VCAM1 and talin was found during maturation in SDC1-/- DC. However, no difference in T-cell-stimulating capacity of SDC1-deficient DC was found compared to WT DC. Mechanistically, SDC1-deficient DC showed enhanced migration towards CCL21 and CCL19. This may result from functional overexpression of CCR7 in SDC1-/- DC. Increased and accelerated migration of otherwise functionally intact SDC1-deficient DC leads to an exacerbated CHS. Based on our results, we conclude that SDC1 on DC negatively regulates DC migration.

Single-domain antibodies (sdAbs), the variable domains of camelid heavy chain-only antibodies, are generally thought to poorly recognize nonproteinaceous small molecules and carbohydrates in comparison with conventional antibodies. However, the structures of anti-methotrexate, anti-triclocarban and anti-cortisol sdAbs revealed unexpected contributions of the non-hypervariable “CDR4” loop, formed between β-strands D and E of framework region 3, in binding. Here, we investigated the potential role of CDR4 in sdAb binding to a hapten, 15-acetyl-deoxynivalenol (15-AcDON), and to carbohydrates. We constructed and panned a phage-displayed library in which CDR4 of the 15-AcDON-specific sdAb, NAT-267, was extended and randomized. From this library, we identified one sdAb, MA-232, bearing a 14-residue insertion in CDR4 and showing improved binding to 15-AcDON by ELISA and surface plasmon resonance. On the basis of these results, we constructed a second set of phage-displayed libraries in which the CDR4 and other regions of three hapten- or carbohydrate-binding sdAbs were diversified. With the goal of identifying sdAbs with novel glycan-binding specificities, we panned the library against four tumor-associated carbohydrate antigens but were unable to enrich binding phages. Thus, we conclude that while CDR4 may play a role in binding of some rare hapten-specific sdAbs, diversifying this region through molecular engineering is probably not a general solution to sdAb carbohydrate recognition in the absence of a paired VL domain.

Summary Hapten‐binding antibodies have for more than 50 years played a pivotal role in immunology, paving the way to antibody generation (as haptens are very important and robust immunogens), to antibody characterization (as the first structures generated more than 40 years ago were those of hapten binders), and enabled and expanded antibody engineering technologies. The latter field of engineered antibodies evolved over many years and many steps resulting in recombinant humanized or human‐derived antibody derivatives in multiple formats. Today, hapten‐binding antibodies are applied not only as reagents and tools (where they still play an important part) but evolved also to engineered targeting and pretargeting vehicles for disease diagnosis and therapy. Here we describe recent applications of hapten‐binding antibodies and of engineered mono‐ and bispecific hapten‐binding antibody derivatives. We have designed and applied these molecules for the modulation of the pharmacokinetic properties of small compounds or peptides. They are also integrated as additional binding entities into bispecific antibody formats. Here they serve as non‐covalent or covalent coupling modules to haptenylated compounds, to enable targeted payload delivery to disease tissues or cells.

Protein arginine phosphorylation is a post-translational modification (PTM) that is important for bacterial growth and virulence. Despite its biological relevance, the intrinsic acid lability of phosphoarginine (pArg) have impaired studies of this novel PTM. Herein, we report for the first time the development of phosphonate-amidines and sulfonate-amidines as isosteres of pArg and then use these mimics as haptens to develop the first high affinity sequence independent anti-pArg specific antibody. Employing this anti-pArg antibody, we further showed that arginine phosphorylation is induced in Bacillus subtilis during oxidative stress. Overall, we expect this antibody to see widespread use in analyzing the biological significance of arginine phosphorylation. Additionally, the chemistry reported here will facilitate the generation of pArg mimetics as highly potent inhibitors of the enzymes that catalyze arginine phosphorylation/dephosphorylation.

Diphencyprone (DPCP) is a hapten that induces delayed-type hypersensitivity (DTH) reactions. MicroRNAs (miRNAs) are short non-coding RNAs that negatively regulate gene expression and have been implicated in various inflammatory skin diseases, but their role in DTH reactions is not well understood. We generated global miRNA expression profiles (using next-generation sequencing) of DPCP reactions in skin of seven healthy volunteers at 3, 14 and 120 days after challenge. Compared to placebo-treated sites, DPCP-challenged skin at 3 days (peak inflammation) had 127 miRNAs significantly deregulated. At 14 days (during resolution of inflammation), 43 miRNAs were deregulated and, at 120 days (when inflammation had completely resolved), six miRNAs were upregulated. While some miRNAs have been observed in psoriasis or atopic dermatitis, most of the deregulated miRNAs have not yet been studied in the context of skin biology or immunology. Across the three time points studied, many but not all miRNAs were uniquely expressed. As various miRNAs may influence T cell activation, this may indicate that the miRNAs exclusively expressed at different time points function to promote or resolve skin inflammation, and therefore, may inform on the paradoxical ability of DPCP to treat both autoimmune conditions (alopecia areata) and conditions of ineffective immunity (melanoma).

Drug hypersensitivity reactions (DHRs) may be caused by immunologic and non-immunologic mechanisms. According to the World Allergy Organization, drug allergy (DA) encompasses the subgroup of immunologic DHRs which are mediated either by specific antibodies or specific T lymphocytes. Due to the immunologic memory, DA reactions bear an increased risk for dramatically enhanced reactions on re-exposure. Some current concepts of DA were described decades ago. Drug allergies to soluble macromolecular protein drugs such as biopharmaceuticals are predominantly T cell-dependent drug-specific antibody responses leading to IgE-or IgG-mediated allergy. However, most drugs are too small to be directly recognized by specific B and T cells. Immune reactions to low-molecular drugs have been explained by the hapten model: a hapten drug can bind covalently to soluble autologous proteins (e.g. serum albumin). Resulting compounds may then be recognized by matching B cell receptors (BCRs) and induce a specific T cell-dependent IgE-or IgG-antibody production. Drug haptens may bind to extra- or intracellular proteins, which are processed and presented by various professional antigen-presenting cells (APCs). Depending on the APC, they may induce not only specific antibody production, but also non-immediate T cell-mediated DA. More recently, a supplementary effector mechanism for non-immediate DA to low-molecular drugs has been described, namely the pharmacological interaction of native low-molecular drugs with immune receptors (p-i-concept). Low-molecular drugs may directly and reversibly attach to immune receptors. These non-covalent interactions may modify the affinity between autologous major histocompatibility complex (MHC), presented peptides and specifically primed T cell receptors (TCRs) and thereby stimulate T cells. A special type of p-i-reaction has been recently described between the antiviral drug abacavir and the F pocket of HLA-B*57:01. This interaction causes an alteration of the MHC-presented self-peptide repertoire and may consecutively lead to a kind of auto-reactivity. Such types of reactions can explain the strong MHC-HLA associations which have been found for some T cell-mediated DHRs.

Virus nanoparticles (VNPs) have been applied as carrier proteins for effective vaccine development. In this paper, we report the usage of tobacco mosaic virus (TMV) as a carrier for the display of the small molecule estriol (E3), a weakly immunogenic hapten. A highly efficient copper (I)-catalyzed azide-alkyne cycloaddition reaction (CuAAC) was performed for the conjugation of E3 onto TMV capsid at tyrosine (Tyr) 139, by which the antigen density could be controlled. The immune properties of these constructs were evaluated in mice. We found that a strong and long-term antibody response was elicited by conjugating a high density of small molecular haptens on TMV through an oligo(ethylene glycol) (OEG) linker, likely due to the effective activation of B-cells. This study suggests that TMV can serve as a promising platform to induce strong humoral immune responses and that the optimized conjugation strategy was critical to produce high quality antibodies.

Humanized hapten-binding IgGs were designed with an accessible cysteine close to their binding pockets, for specific covalent payload attachment. Individual analyses of known structures of digoxigenin (Dig)- and fluorescein (Fluo) binding antibodies and a new structure of a biotin (Biot)-binder, revealed a “universal” coupling position (52+2) in proximity to binding pockets but without contributing to hapten interactions. Payloads that carry a free thiol are positioned on the antibody and covalently linked to it via disulfides. Covalent coupling is achieved and driven toward complete (95–100%) payload occupancy by spontaneous redox shuffling between antibody and payload. Attachment at the universal position works with different haptens, antibodies, and payloads. Examples are the haptens Fluo, Dig, and Biot combined with various fluorescent or peptidic payloads. Disulfide-bonded covalent antibody-payload complexes do not dissociate in vitro and in vivo. Coupling requires the designed cysteine and matching payload thiol because payload or antibody without the Cys/thiol are not linked (

The immune response against hapten is T-cell-dependent, and so requires the uptake, processing and presentation of peptides on MHC class II molecules by antigen-presenting cells to the specific T cell. Some haptens, following conjugation to the available free amines on the surface of the carrier protein, can reduce its immunogenicity. The purpose of this study was to explore the mechanism by which this occurs. Four proteins were tested as carriers and six molecules were used as haptens. The immune response to the carrier proteins was reduced > 100-fold by some of the haptens (termed carrier immunogenicity reducing haptens – CIRH), whereas other haptens did not influence the protein immunogenicity (carrier immunogenicity non-reducing haptens – nCIRH). Conjugation of the protein to a CIRH affected protein degradation by lysosomal cathepsins, leading to the generation of peptides that differ in length and sequence from those derived from the same native protein or that protein modified with nCIRH. Injection of CIRH-conjugated protein into mice induced an increase in the population of regulatory T cells. The results of this study provide a putative mechanism of action for the reduction of immune response to haptenated proteins.

Stevens-Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN) are life-threatening cutaneous adverse drug reactions that induce widespread epidermal necrosis. Recent advances in pharmacogenomic studies have provided evidence of genetic predispositions to SJS/TEN. Several concepts have been proposed to explain the pathogenesis of severe cutaneous adverse drug reactions. In the hapten concept, small molecules called haptens elicit an immune response only when attached to proteins. The "p-i" concept postulates that the causative drugs can stimulate cells by binding directly and reversibly to immune receptors. In addition, there is the idea that drugs alter the antigen by binding to the human leukocyte antigen pocket. With regard to keratinocyte death, several cell death mediators, such as FasL, granulysin and annexin A1, have been proposed as playing a role in SJS/TEN pathogenesis. A subset of T lymphocytes, including regulatory T cells, also may play a role in SJS/TEN.

Atopic dermatitis and allergic contact dermatitis are both common skin diseases having an immune pathogenesis. There has been considerable interest about their inter-relationships with regard to altered susceptibility. Recent investigations have shed new light on this important question, and in this article, we explore whether there is evidence that atopic dermatitis affects the risk of contact sensitization and allergic contact dermatitis. The use of topical products to treat xerotic and inflamed skin in atopic dermatitis often results in a higher prevalence of sensitization to, for example, fragrances and other ingredients in emollients. Moreover, the prevalence of metal allergy seems to be increased, probably due to compromised chelation of the metals in the stratum corneum of patients with atopic dermatitis. However, conversely, the T-helper cell 2 bias that characterizes immune responses in atopic dermatitis appears to lower the risk of contact sensitization compared to healthy controls. Based on these observations, we conclude that multiple factors affect the association between atopic dermatitis and contact sensitization, and that these need to be appreciated in the clinical management of atopic dermatitis patients.

Increasing evidence suggests that antibodies can have stimulatory effects on T-cell immunity. However, the contribution of circulating antigen-specific antibodies on MHC class I cross-priming in vivo has not been conclusively established. Here, we defined the role of circulating antibodies in cross-presentation of antigen to CD8(+) T cells. Mice with hapten-specific circulating antibodies, but naϊve for the T-cell antigen, were infused with haptenated antigen and CD8(+) T-cell induction was measured. Mice with circulating hapten-specific antibodies showed significantly enhanced cross-presentation of the injected antigen compared with mice that lacked these antibodies. The enhanced cross-presentation in mice with circulating antigen-specific antibodies was associated with improved antigen capture by APCs. Importantly, CD11c(+) APCs were responsible for the enhanced and sustained cross-presentation, although CD11c(-) APCs had initially captured a significant amount of the injected antigen. Thus, in vivo formation of antigen-antibody immune complexes improves MHC class I cross-presentation, and CD8(+) T-cell activation, demonstrating that humoral immunity can aid the initiation of systemic cellular immunity. These findings have important implications for the understanding of the action of therapeutic antibodies against tumor-associated antigens intensively used in the clinic nowadays.

ONZIN is abundantly expressed in immune cells of both the myeloid and lymphoid lineage. Expression by lymphoid cells has been reported to further increase after cutaneous exposure of mice to antigens and haptens capable of inducing contact hypersensitivity (CHS), suggesting that ONZIN has a critical role in this response. Here, we report that indeed ONZIN-deficient mice develop attenuated CHS to a number of different haptens. Dampened CHS responses correlated with a significant reduction in pro-inflammatory IL-6 at the challenge site in ONZIN-deficient animals, compared with wild-type controls. Together the study of these animals indicates that loss of ONZIN impacts the effector phase of the CHS response through the regulation of pro-inflammatory factors.

Dendritic cells (DCs) have the ability to present antigen and play a critical role in the induction of the acquired immune response. Skin DCs uptake antigen and subsequently migrate to regional draining lymph nodes (LNs), where they activate naive T cells. Here we show that the water/glycerol channel protein aquaporin 7 (AQP7) is expressed on epidermal and dermal DCs and involved in the initiation of primary immune responses. AQP7-deficient DCs showed a decreased cellular uptake of low-molecular-mass compounds (fluorescein isothiocyanate and Lucifer yellow) and high-molecular-mass substances (ovalbumin and dextran), suggesting that AQP7 is involved in antigen uptake. AQP7-deficient DCs also exhibited reduced chemokine-dependent cell migration in comparison to wild-type DCs. Consistent with these in vitro results, AQP7-deficient mice demonstrated a reduced accumulation of antigen-retaining DCs in the LNs after antigen application to the skin, which could be attributed to decreased antigen uptake and migration. Coincidentally, AQP7-deficient mice had impaired antigen-induced sensitization in a contact hypersensitivity model. These observations suggested that AQP7 in skin DCs is primarily involved in antigen uptake and in the subsequent migration of DCs and is responsible for antigen presentation and the promotion of downstream immune responses.

Inflammatory bowel disease (IBD) results from dysfunctional interactions between the intestinal immune system and microbiota, influenced by host genetic susceptibility. Because a key feature of the pathology is intestinal epithelial damage, potential disease factors have been traditionally analyzed within the background of chemical colitis models in mice. The zebrafish has greatly complemented the mouse for modeling aspects of disease processes, with an advantage for high content drug screens. Larval zebrafish exposed to the haptenizing agent trinitrobenzene sulfonic acid (TNBS) displayed impaired intestinal homeostasis and inflammation reminiscent of human IBD. There was a marked induction of pro-inflammatory cytokines, the degradative enzyme mmp9 and leukocytosis. Enterocolitis was dependent on microbiota and Toll-like receptor signaling, that can be ameliorated by antibiotic and anti-inflammatory drug treatments. This system will be useful to rapidly interrogate in vivo the biological significance of the IBD candidate genes so far identified and to carry out pharmacological modifier screens.

We present the MALDI-TOF/TOF-MS analyses of various hapten – bovine serum albumin (BSA) neoglycoconjugates obtained by squaric acid chemistry coupling of the spacer-equipped, terminal monosaccharide of the O-specific polysaccharide of Vibrio cholerae O1, serotype Ogawa, to BSA. These analyses allowed not only to calculate the molecular masses of the hapten – BSA neoglycoconjugates with different hapten – BSA ratios (4.3, 6.6 and 13.2) but, more importantly, also to localize the covalent linkages (conjugation sites) between the hapten and the carrier protein. Determination of the site of glycation was based on comparison of the MALDI-TOF/TOF-MS analysis of the peptides resulting from the digestion of BSA with similar data resulting from the digestion of BSA glycoconjugates, followed by sequencing by MALDI-TOF/TOF-MS/MS of the glycated peptides. The product-ion scans of the protonated molecules were carried out with a MALDI-TOF/TOF-MS/MS tandem mass spectrometer equipped with a high-collision energy cell. The high-energy collision-induced dissociation (CID) spectra afforded product ions formed by fragmentation of the carbohydrate hapten and amino acid sequences conjugated with fragments of the carbohydrate hapten. We were able to identify three conjugation sites on lysine residues (Lys235, Lys437 and Lys455). It was shown that these lysine residues are very reactive and bind lysine specific reagents. We presume that these Lys residues belong to those that are considered to be sterically more accessible on the surface of the tridimensional structure. The identification of the y-series product ions was very useful for the sequencing of various peptides. The series of a- and b-product ions confirmed the sequence of the conjugated peptides. Copyright c � 2010 John Wiley & Sons, Ltd.

Rigorous consideration of the consequences of antibody bivalence in the published competitive kinetic procedure for quantifying the solution characteristics of an antigen-antibody interaction in solution has rendered redundant the practice of substituting the Fab fragment for the antibody to ensure validity of the analysis of results in terms of theory developed for a univalent analyte. Although the quantitative expressions differ for univalent and bivalent analytes, the additional contribution arising from bivalence is likely to be well within the limits of experimental uncertainty in the measured binding constant.

We described the production and characterization of antibodies against three important groups of neuro-active haptens, e.g., neurotransmitters and biogenic amines. First, from the tryptophane metabolic pathway: tryptamine, serotonin, 5-hydroxy-indole acetic acid, and melatonin. Secondly, the tyrosine metabolic pathway: tyramine, dopamine, dihydroxyphenyl acetic acid, and norepinephrine. Thirdly, antibodies against excitatory and inhibitory neurotransmitters: glycine, glutamate, glutamine, and GABA. Immunogenic conjugates were prepared after linking haptens to carrier proteins. Most antibodies displayed high specificity against corresponding neuro-active haptens conjugated in vitro and in situ in biological specimens, but not to closely related conjugated metabolites, precursors, pharmaceuticals, agonists, antagonists, or free neuro-active haptens. Conjugated norepinephrine was highly tolerant in different animal species and produced incidentally a short specific antibody response.

Background: Linalool is a widely used fragrance terpene. Pure linalool is not allergenic or a very weak allergen, but autoxidizes on air exposure and the oxidation products can cause contact allergy. Oxidized (ox.) linalool has previously been patch tested at a concentration of 2.0% in petrolatum (pet.) in 1511 patients, and 1.3% positive patch test reactions were observed. Objective: To investigate the optimal patch test concentration for detection of contact allergy to ox. linalool. Methods: Four concentrations of ox. linalool (2.0%, 4.0%, 6.0%, 11.0% pet.) were tested in 3418 consecutive dermatitis patients. Results: Ox. linalool 2.0%, 4.0%, 6.0%, and 11.0% pet. detected positive patch test reactions in 0.83%, 3.2%, 5.3%, and 7.2% of the tested patients, respectively. The doubtful reactions increased with rising concentrations but relatively less, giving 5.1%, 6.4%, and 7.3% doubtful reactions, respectively, for ox. linalool 4.0%, 6.0%, and 11.0% pet. Few irritative reactions were seen. Conclusions: Raising the patch test concentration for ox. linalool gave a better detection of contact allergy, as many as 5-7% positive patch test reactions were detected. We suggest a patch test concentration of ox. linalool 6.0% pet. for future patch testing, giving a dose per unit area of 2.4 mg/cm(2) when 20 mg test substance is tested in small Finn Chambers (R).

Leucocyte-associated immunoglobulin-like receptor-1 (LAIR-1) is a membrane receptor of the immunoglobulin (Ig) superfamily that is expressed on most types of haematopoietic cells, and delivers inhibitory signals through interacting with collagens. In order to elucidate the immunological functions of LAIR-1 in vivo, we established transgenic mice expressing a chimeric protein composed of the extracellular domain of LAIR-1 fused with an Ig tag (LAIR-1-Ig), which acts as a decoy by competing with endogenous LAIR-1. The transgenic mice showed an increased susceptibility for development of contact hypersensitivity (CHS), an experimental model of allergic contact dermatitis, in association with enhanced hapten-specific T-cell responses. When T cells from the hapten-sensitized donor mice were transferred into non-sensitized recipients, treatment of either donor mice or recipient mice with LAIR-1-Ig protein accelerated CHS, suggesting a potentially negative role of LAIR-1 in both the sensitization and the elicitation of hapten-reactive T cells. In vitro assays revealed that LAIR-1 decreased the production of interleukin-6 and interleukin-12 in dendritic cells, and inhibited the proliferation and cytokine production of naïve and memory T cells along with G(0)/G(1) cell cycle arrest. Collectively, our findings suggest that LAIR-1 plays a crucial inhibitory role in CHS by regulating antigen-presenting cell and T-cell functions.

The thrombopenic effects of immune complexes were studied in 32 rabbits, immunized actively with aspirin. The test animals were exposed to hapten-protein conjugates with a hapten density ranging from 0.6 to 38, and to pure hapten. The resulting thrombopenia and leucopenia correlated closely with the presence of immune complexes in the serum as detected by the platelet aggregation sedimentation pattern test (P1.A.). This platelet-aggregation activity sedimented mainly in the 19S fraction. The effects induced by polyvalent antigens where dose-dependent and could be modified by a prior injection of aspirin. Expectedly, monovalent antigens were only marginally effective.

The extraction of lipopolysaccharides (LPS) from smooth strains of Salmonella typhimurium for structural studies using methylation analysis is preferably done by hot phenol-water method on a cell-wall preparation obtained by mechanical disintegration of γ-irradiated bacteria. The LPS obtained by this method was less contaminated than LPS extracted by hot phenol-water from acetone dried bacteria or bacteria pretreated with 1 per cent formaldehyde. LPS for structural analysis from rough mutants is, due to its lipophilic character, preferably extracted with a mixture of phenol, chloroform and petroleum ether. The presence of the precursor of the O side-chain, LI-hapten, in the different LPS- and supernatant fractions was assessed by gel precipitation tests. The LPS forms a line near the antigen well whereas the low molecular weight hapten forms a line near the antiserum well. The hapten was not formed in excess in the smooth strain S. typhimurium LT2. In the rough mutant studied, S. typhimurium SL 733, O-antigenic material was found in both the LPS-preparation (probably as a covalently linked O side-chain) and the supernatant (as a hapten).

An O-haptenic material (LI-fraction) was isolated from cytoplasmic and cell-wall preparations of the rough mutant Salmonella typhimurium 395 MR10. The yield from the cell-wall preparation, virtually free from cytoplasmic contamination, was approximately ten times higher than that from the cytoplasmic preparation. The LI-fraction obtained from cell-walls was less contaminated with extraneous polysaccharide material. After purification of the LI-fraction with ribonuclease treatment and Sephadex G-100 gel chromatography structural studies using methylation analysis could be performed. The hapten showed the same structural features as the O side-chains of the parent strain S. typhimurium 395 MS. The average number of repeating units in the hapten was 23 as compared to 11 in the O side-chain of the parent strain. Molecular weight determination with Sepharose 6 B gel chromatography revealed that the LI-fraction was polydisperse with a weight average of 26,300 and a number average of 21,800.

Some properties of the antibodies involved in the immune complex-induced platelet aggregation (Pl.A.) reaction were analyzed in the present study. Rabbits were immunized with a hapten-protein conjugate (NIP-chicken globulin) and the primary response (3 week) IgG was fractionated. The Pl.A. reactivities of these fractions were found to correlate with the titres of haptenated phage inactivation (HPI) when the values were adjusted to represent equal antibody concentrations (measured by hapten binding capacity). The minimum amount of primary response anti-NIP IgG detectable with the Pl.A. method was estimated to be between 1.7 and 14.6 μg/ml. Complement fixing antibodies were detected with approximately similar sensitivity. The titres of lightly haptenated phage inactivation did not correlate to the Pl.A. reactivities, suggesting that the role of very high-affinity antibodies is not critical to the Pl.A. reaction. The affinity requirements of the Pl.A. method was concluded to be close to the standard HPI method (heavy coupling), but considerably lower than that of the HPI method with lightly coupled phage.

Acute intravascular haemolysis and renal failure developed while a patient was taking triamterene. A direct antiglobulin test with a polyvalent reagent was positive. Serum caused agglutination of normal red cells in the presence of triamterene and caused an increase of partial haemolysis of both trypsin-treated erythrocytes and red cells from a patient with paroxysmal nocturnal haemoglobinuria (PNH) in the presence of complement. From the results of antibody-neutralization test and treatment with 2-mercaptoethanol, the presence of IgM antibody with Λ light chain could be demonstrated. The triamterene seemed to bind strongly to the red cells in vitra but in vivo there was no detectable adsorption to red cells. Haptenic inhibition was not demonstrated. From these results, it was assumed that this antibody caused acute intravascular haemolysis by immune complex mechanism. The antibody was found to cross-react with methotrexate which has a structure similar to that of triamterene.

In contrast to its favourable effects on Langerhans cell (LC) differentiation, transforming growth factor (TGF)-beta1 has been reported to prevent dendritic cells from maturing in response to tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, or lipopolysaccharide (LPS). We first characterized the effects of TGF-beta1 on dendritic cell function by testing the response of TGF-beta1-treated monocyte-derived dendritic cells (MoDCs) to maturation stimuli that LCs receive in the epidermis, namely, haptens, ATP and ultraviolet (UV). TGF-beta1 treatment, which augmented E-cadherin and down-regulated dendritic cell-specific ICAM3-grabbing non-integrin on MoDCs, significantly suppressed their CD86 expression and hapten-induced expression of IL-1beta and TNF-alpha mRNA and protein. As TGF-beta1-treated MoDCs lacked Langerin expression, we demonstrated the suppressive effects of TGF-beta1 on haematopoietic progenitor cell-derived dendritic cells expressing both CD1a and Langerin. These suppressive effects of TGF-beta1 increased with the duration of treatment. Furthermore, TGF-beta1-treated MoDCs became resistant to apoptosis/necrosis induced by high hapten, ATP or UV doses. This was mainly attributable to dampened activation of p38 mitogen-activated protein kinase (MAPK) in TGF-beta1-treated MoDCs. Notably, although ATP or hapten alone could only induce CD86 expression weakly and could not augment the allogeneic T-cell stimulatory function of TGF-beta1-treated MoDCs, ATP and hapten synergized to stimulate these phenotypic and functional changes. Similarly, 2,4-dinitro, 1-chlorobenzene (DNCB) augmented the maturation of TGF-beta1-treated MoDCs upon co-culture with a keratinocyte cell line, in which ATP released by the hapten-stimulated keratinocytes synergized with the hapten to induce their maturation. These data may suggest that TGF-beta1 protects LCs from being overactivated by harmless environmental stimulation, while maintaining their ability to become activated in response to danger signals released by keratinocytes.

The synthesis and characterization of monofluoroacetyl (MFAc) functionalized haptens are described. These were covalently bound to polypeptide carriers (bovine serum albumin and poly-D-lysine) by primary amine/succinimide ester or primary amine/acid chloride coupling. Epitopic densities of the resulting antigens were determined by both 19F n.m.r. and picryl sulfonic acid assays. 19F n.m.r. experiments defining the stability of MFAc ester and amide linkages as a function of media (including in vitro), time, and temperature are presented. These results indicate that MFAc functionalized antigens are well suited for further immunologic studies.

The alpha-amino group of bovine Growth Hormone was selectively modified with TNBS with no detectable changes in growth promoting activity. TNP was used as hapten for the production of antibodies against the end terminal region of bGH. The region near the alpha-amino group is not involved in the binding of bGH to rat liver cells, and it seems to be away from the part of the molecule that interacts with the cell binding sites.

Proteins are renowned for their specificity of function. There is, however, accumulating evidence that many proteins, from enzymes to antibodies, are functionally promiscuous. Promiscuity is of considerable physiological importance. In the immune system, cross-reactive or multispecific antibodies are implicated in autoimmune and allergy conditions. In most cases, however, the mechanism behind promiscuity and the relationship between specific and promiscuous activities are unknown. Are the two contradictory? Or can a protein exhibit several unrelated activities each of which is highly specific? To address these questions, we studied a multispecific IgE antibody (SPE7) elicited against a 2,4-dinitrophenyl hapten (DNP). SPE7 is able to distinguish between closely related derivatives such as NP (nitrophenol) and DNP, yet it can also bind a number of unrelated ligands. We find that, like DNP, the cross-reactants are themselves bound specifically-close derivatives of these cross-reactants show very low or no binding to SPE7. It has been suggested that cross-reactivity is simply due to "hydrophobic stickiness", nonspecific interactions between hydrophobic ligands and binding sites. However, partitioning experiments reveal that affinity for SPE7 is unrelated to ligand hydrophobicity. These data, combined with crystal structures of SPE7 in complex with four different ligands, demonstrate that each cross-reactant is bound specifically, forming different hydrogen bonds dependant upon its particular chemistry and the availability of complementary antibody residues. SPE7 is highly homologous to the germline antinitrophenol (NP) antibody B1-8. By comparing the sequences and binding patterns of SPE7 and B1-8, we address the relationship between affinity maturation, specificity, and cross-reactivity.

Allergen-specific immunoglobulin E (IgE) mediates immediate-type hypersensitivity reactions and plays a central role in allergic diseases. Although antigen-driven B-cell maturation and isotype switching occur within germinal centres (GCs), the role of GCs in IgE production is poorly understood. In view of this, we investigated the development of IgE-expressing cells within GCs in response to an extensively characterized antigen, 2-phenyloxazolone (phOx). The phOx-specific IgE-expressing cells localized within GCs 7 days after immunization, and peaked in number on day 11. Surprisingly, very few IgE-positive cells were found in the T-cell areas of the lymph node. Flow cytometric studies confirmed that IgE was expressed by B cells and was not the result of trapping by follicular dendritic cells. The specificity of the antibody response was confirmed by microdissection and reverse transcription-polymerase chain reaction using phOx-specific IgE primers. IgE-positive cells were primarily found within GCs while, in contrast, many IgG1-positive cells could also be detected outside GCs in the T-cell areas. Taken together, these data highlight the importance of GCs in the production of antigen-specific IgE antibody.

While it was realized as early as 30 to 40 years ago that antibody—antigen reactions occur very rapidly (Hooker and Boyd, 1935; Mayer and Heidelberger, 1942), quantitative kinetic studies of both antibody—antigen and antibodyhapten reactions have come into prominence only during the past decade. Only within recent years have specialized techniques become available which satisfy criteria for accurate and meaningful kinetic measurements.

The glucocorticoid-induced tumour necrosis factor receptor family related gene (GITR) has been reported to be expressed on the activated T and CD4(+)CD25(+) regulatory T cells (Treg). Signalling triggered by GITR not only neutralizes the suppressive effect of Treg cells, but also augments activation, proliferation and cytokine production of effector T cells. To test the role of GITR in 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis - a murine model of mucosal inflammation - TNBS-injected Balb/c mice were treated with agonistic anti-GITR monoclonal antibody (mAb). Anti-GITR treatment increased the death rate compared to rat IgG-treated mice. Typically, death occurred within 4 days after the TNBS injection when the mice were treated with anti-GITR. The mice that survived anti-GITR treatment suffered from severe inflammation in their entire intestines. CD4(+) T-depletion protected the mice from colitis; even an anti-GITR effect was not apparent. In contrast, CD8(+) T depletion showed less protective than did CD4(+) T depletion. Stimulation of GITR enhanced the production of proinflammatory cytokines including interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-12. It also enhanced the humoral response such as serum levels of IgG(2b) and IgA, which was completely dependent on CD4(+) T cells. Taken together, this study demonstrated that GITR signalling on CD4(+) T cells is involved in the development and progress of colitis by enhancing both T helper type 1 (Th1) and Th2 type responses.

p-Phenylenediamine (PPD) is a commonly used hair-dye and a potent skin allergen. The mechanism of sensitization is unknown, as PPD is protein unreactive. We studied Bandrowski's base (BB), a PPD trimer, as well as 1,4-benzoquinone (BQ), a PPD hapten. PPD patch-test positive patients were patch-tested to BB and BQ. All tests were negative to 0.01% BQ and 0.01% BB. Five of 14 (35.7%) tested had true positive reactions to 0.1% BQ. One percent BQ was found to be irritant. Seven of 43 tested (16%) were positive to either 0.1% or 1% BB. The positive reactions to BB were weak, even when PPD reactions were strong. Mice lymph node assay gave EC3 values of 0.14% for PPD compared with 0.03% for BB. Therefore, BB is approximately 10 times more potent than PPD, taking into account the molarity. We suggest that while PPD may act as a prohapten, there is probably a spectrum of antigenic determinants in vivo. BB may be bound or metabolized by keratinocytes before it reacts with Langerhans cells.

It has been known for 15 years that the chicken epidermis contains ATPase+ and major histocompatibility complex class II-positive (MHCII+) dendritic cells. These cells were designated as Langerhans cells but neither their detailed phenotype nor their function was further investigated. In the present paper we demonstrate a complete overlapping of ATPase, CD45 and vimentin staining in all dendritic cells of the chicken epidermis. The CD45+ ATPase+ vimentin+ dendritic cells could be divided into three subpopulations: an MHCII+ CD3- KUL01+ and 68.1+ (monocyte-macrophage subpopulation markers) subpopulation, an MHCII- CD3- KUL01- and 68.1- subpopulation and an MHCII- CD3+ KUL01- and 68.1- subpopulation. The first population could be designated as chicken Langerhans cells. The last population represents CD4- CD8- T-cell receptor-alphabeta- and -gammadelta- natural killer cells with cytoplasmic CD3 positivity. The epidermal dendritic cells have a low proliferation rate as assessed by bromodeoxyuridine incorporation. Both in vivo and in vitro experiments showed that dendritic cells could be mobilized from the epidermis. Hapten treatment of epidermis resulted in the decrease of the frequency of epidermal dendritic cells and hapten-loaded dendritic cells appeared in the dermis or in in vitro culture of isolated epidermis. Hapten-positive cells were also found in the so-called dermal lymphoid nodules. We suggest that these dermal nodules are responsible for some regional immunological functions similar to the mammalian lymph nodes.

The systemic immune response of joining-chain (J-chain)-deficient mice (J(-/-) mice) on a C57BL/6 background against the hapten 4-hydroxy-3-nitrophenyl (NP) was analysed. While primary IgG responses to the hapten were similar to those observed in WT control animals, secondary immune responses were compromised both at the level of serum IgG and the number of responding B cells. The repertoire switch from lambda to kappa in secondary immune responses was diminished in J(-/-) mice. The number of somatic mutations introduced in the V(H) 186.2 gene during the primary immune response was reduced, while the frequency of affinity-increasing mutations in position 33 was similar. By adoptive transfer experiments it could be shown that the compromised secondary immune response was transferred with T cells from J(-/-) mice. Thus, J-chain-deficient mice have a selective defect in T helper cell function during B cell immune responses, resulting in a deficiency in the formation of B cell memory.