Monoclonal enolase-1 blocking antibody ameliorates pulmonary inflammation and fibrosis.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic fatal disease with limited therapeutic options. The infiltration of monocytes and fibroblasts into the injured lungs is implicated in IPF. Enolase-1 (ENO1) is a cytosolic glycolytic enzyme which could translocate onto the cell surface and act as a plasminogen receptor to facilitate cell migration via plasmin activation. Our proprietary ENO1 antibody, HL217, was screened for its specific binding to ENO1 and significant inhibition of cell migration and plasmin activation (patent: US9382331B2). METHODS: In this study, effects of HL217 were evaluated in vivo and in vitro for treating lung fibrosis. RESULTS: Elevated ENO1 expression was found in fibrotic lungs in human and in bleomycin-treated mice. In the mouse model, HL217 reduced bleomycin-induced lung fibrosis, inflammation, body weight loss, lung weight gain, TGF-ß upregulation in bronchial alveolar lavage fluid (BALF), and collagen deposition in lung. Moreover, HL217 reduced the migration of peripheral blood mononuclear cells (PBMC) and the recruitment of myeloid cells into the lungs. In vitro, HL217 significantly reduced cell-associated plasmin activation and cytokines secretion from primary human PBMC and endothelial cells. In primary human lung fibroblasts, HL217 also reduced cell migration and collagen secretion. CONCLUSIONS: These findings suggest multi-faceted roles of cell surface ENO1 and a potential therapeutic approach for pulmonary fibrosis.

Rapid culture of human keratinocytes in an autologous, feeder-free system with a novel growth medium.

BACKGROUND AIMS: The ability to culture human keratinocytes is beneficial in the treatment of skin injury and disease, as well as for testing chemicals in vitro as a substitute for animal testing. RESULTS: We have identified a novel culture medium for the rapid growth of keratinocytes from human skin. "Kelch's medium" supports keratinocyte growth that is as rapid as in the classical Rheinwald and Green method, but without the need for cholera toxin or xenogeneic feeder cells. It enables keratinocytes to out-compete co-cultured autologous fibroblasts so that separation of the epidermis from the dermis is no longer required before keratinocyte culture. Enzymatic digests of whole human skin can therefore be used to generate parallel cultures of autologous keratinocytes, fibroblasts and melanocytes simply by using different cell culture media. CONCLUSIONS: This new keratinocyte medium and the simplified manufacturing procedures it enables are likely to be beneficial in skin engineering, especially for clinical applications.

Elicitation of potent neutralizing antibodies in obese mice by ISA 51-adjuvanted SARS-CoV-2 spike RBD-Fc vaccine.

Vaccination is considered to be the most effective countermeasure to prevent and combat the global health threats of COVID-19. People with obesity are at a greater risk of hospitalization, life-threatening illness, and adverse outcomes after having COVID-19. Therefore, a safe and effective COVID-19 vaccine for obese individuals is urgently needed. In the study, the vaccine composed of the ISA 51 adjuvant and the SARS-CoV-2 spike (S) receptor-binding domain (RBD) in conjugation with the human IgG1 Fc fragment (named as ISA 51-adjuvanted RBD-Fc vaccine) was developed and inoculated in the regular chow diet (RCD) lean mice and the high-fat diet (HFD)-induced obese mice. The S protein-specific IgG titers were largely induced in an increasing manner along with three doses of ISA 51-adjuvanted RBD-Fc vaccine without causing any harmful side effect. In the HFD mice, the S protein-specific IgG titers can be quickly observed 2 weeks post the first inoculation. The antisera elicited by the ISA 51-adjuvanted RBD-Fc vaccine in the RCD and HFD mice exhibited potent SARS-CoV-2 neutralizing activities in the plaque reduction neutralization test (PRNT) assays and showed similar specificity for recognizing the key residues in the RBD which were involved in interacting with angiotensin-converting enzyme 2 (ACE2) receptor. The immune efficacy of the ISA 51-adjuvanted RBD-Fc vaccine in the HFD mice can be sustainably maintained with the PRNT50 values of 1.80-1.91×10-3 for at least 8 weeks post the third inoculation. Collectively, the RBD-Fc-based immunogen and the ISA 51-adjuvanted formulation can be developed as an effective COVID-19 vaccine for obese individuals. KEY POINTS: • The ISA 51-adjuvanted RBD-Fc vaccine can induce potent SARS-CoV-2 neutralizing antibodies in the obese mouse • The antibodies elicited by the ISA 51-adjuvanted RBD-Fc vaccine can bind to the key RBD residues involved in interacting with ACE2 • The immune efficacy of the ISA 51-adjuvanted RBD-Fc vaccine can be sustainably maintained for at least 8 weeks post the third inoculation.

Aspergillus oryzae Fermentation Extract Alleviates Inflammation in Mycoplasma pneumoniae Pneumonia.

The filamentous fungus Aspergillus oryzae, also known as koji mold, has been used for centuries in the production of fermented foods in East Asia. A. oryzae fermentation can produce enzymes and metabolites with various bioactivities. In this study, we investigated whether A. oryzae fermentation extract (AOFE) has any effect on Mycoplasma pneumoniae (Mp) pneumonia. We performed solid-state fermentation of A. oryzae and obtained the ethanol extract. AOFE was analyzed by HPLC, and the major component was identified to be kojic acid. In vitro, AOFE suppressed Mp growth and invasion into A549 lung epithelial cells as determined by the gentamicin protection assay. AOFE treatment also suppressed Mp-stimulated production of tumor necrosis factor (TNF)-α and interleukin (IL)-6 at mRNA and protein levels in murine MH-S alveolar macrophages. In a mouse model of Mp pneumonia, Mp infection induced a marked pulmonary infiltration of neutrophils, which was significantly reduced in mice pre-treated orally with AOFE. AOFE administration also suppressed the production of proinflammatory cytokines and chemokines in the lungs. Collectively, our results show that AOFE has the potential to be developed into a preventive/therapeutic agent for Mp pneumonia.

Detection of Anticipatory Dynamics between a Pair of Zebrafish.

Anticipatory dynamics (AD) is unusual in that responses from an information receiver can appear ahead of triggers from the source, and direction of information flow (DIF) is needed to establish causality. Although it is believed that anticipatory dynamics is important for animals' survival, natural examples are rare. Time series (trajectories) from a pair of interacting zebrafish are used to look for the existence of AD in natural systems. In order to obtain the DIF between the two trajectories, we have made use of a special experimental design to designate information source. However, we have also used common statistical tools such as Granger causality and transfer entropy to detect DIF. In our experiments, we found that a majority of the fish pairs do not show any anticipatory behaviors and only a few pairs displayed possible AD. Interestingly, for fish in this latter group, they do not display AD all the time. Our findings suggest that the formation of schooling of fish might not need the help of AD, and new tools are needed in the detection of causality in AD system.

Photoinduced arylation of chloroarenes in flow: synthesis of unsymmetrical biaryls.

A photoflow method is presented for a radical-based coupling of unactivated arenes and aryl chlorides. The process proceeded smoothly at ambient temperature under metal-free conditions. Of note is that the reaction conditions are fine-tuned for chloroarenes with different electronic properties. While the reactivity profile of aryl chlorides is generally known to be inferior to those of the corresponding iodides and bromides, we demonstrate that the title protocol is efficient in converting readily available and inexpensive chloroarenes into unsymmetrical biaryl products.

Migratory cues controlling B-lymphocyte trafficking in human lymph nodes.

B-cell migration within lymph nodes (LNs) is crucial to adaptive immune responses. Chemotactic gradients are proposed to drive migration of B cells into follicles, followed by their relocation to specific zones of the follicle during activation, and ultimately egress. However, the molecular drivers of these processes and the cells generating chemotactic signals that affect B cells in human LNs are not well understood. We used immunofluorescence microscopy, flow cytometry and functional assays to study molecular mechanisms of B-cell migration within human LNs, and found subtle but important differences to previous murine models. In human LNs we find CXCL13 is prominently expressed at the follicular edge, often associated with fibroblastic reticular cells located in these areas, whereas follicular dendritic cells show minimal contribution to CXCL13 expression. Human B cells rapidly downregulate CXCR5 on encountering CXCL13, but recover CXCR5 expression in the CXCL13-low environment. These data suggest that the CXCL13 gradient in human LNs is likely to be different from that proposed in mice. We also identify CD68+ CD11c+ PU.1+ tingible body macrophages within both primary and secondary follicles as likely drivers of the sphingosine-1-phosphate (S1P) gradient that mediates B-cell egress from LNs, through their expression of the S1P-degrading enzyme, S1P lyase. Based on our findings, we present a model of B-cell migration within human LNs, which has both similarities and interesting differences to that proposed for mice.

Interleukin-1 receptor (IL-1R) mediates epilepsy-induced sleep disruption.

BACKGROUND: Sleep disruptions are common in epilepsy patients. Our previous study demonstrates that homeostatic factors and circadian rhythm may mediate epilepsy-induced sleep disturbances when epilepsy occurs at different zeitgeber hours. The proinflammatory cytokine, interleukin-1 (IL-1), is a somnogenic cytokine and may also be involved in epileptogenesis; however, few studies emphasize the effect of IL-1 in epilepsy-induced sleep disruption. We herein hypothesized that IL-1 receptor type 1 (IL-1R1) mediates the pathogenesis of epilepsy and epilepsy-induced sleep disturbances. We determined the role of IL-1R1 by using IL-1R1 knockout (IL-1R1 -/- KO) mice. RESULTS: Our results elucidated the decrease of non-rapid eye movement (NREM) sleep during the light period in IL-1R -/- mice and confirmed the somnogenic role of IL-1R1. Rapid electrical amygdala kindling was performed to induce epilepsy at the particular zeitgeber time (ZT) point, ZT13. Our results demonstrated that seizure thresholds induced by kindling stimuli, such as the after-discharge threshold and successful kindling rates, were not altered in IL-1R -/- mice when compared to those obtained from the wildtype mice (IL-1R +/+ mice). This result suggests that IL-1R1 is not involved in kindling-induced epileptogenesis. During sleep, ZT13 kindling stimulation significantly enhanced NREM sleep during the subsequent 6 h (ZT13-18) in wildtype mice, and sleep returned to the baseline the following day. However, the kindling-induced sleep alteration was absent in the IL-1R -/- KO mice. CONCLUSIONS: These results indicate that the IL-1 signal mediates epilepsy-induced sleep disturbance, but dose not participate in kindling-induced epileptogenesis.

Distinct binding of amyloid imaging ligands to unique amyloid-ß deposited in the presubiculum of Alzheimer's disease.

Non-invasive determination of amyloid-ß peptide (Aß) deposition with radioligands serves for the early diagnosis and clarification of pathogenetic mechanisms of Alzheimer's disease (AD). The polymorphic binding site on multimeric Aß for current radioligands, however, is little understood. In this study, we investigated the binding of several radioligands including (11)C-Pittsburgh Compound B ((11)C-PiB), (3)H-AZD2184, and two recently developed compounds, (125)I-DRM106 and (125)I-DRK092, with unique presubicular Aß deposits lacking interaction with the commonly used amyloid dyes FSB. (11)C-PiB, (3)H-AZD2184, and (125)I-DRK092 showed overt binding to presubicular Aß deposits, while (125)I-DRM106 barely bound to these aggregates despite its strong binding in the hippocampal CA1 sector. Unlike neuritic plaques in the CA1, Aß lesions in the presubiculum were not accompanied by inflammatory gliosis enriched with 18-kDa translocator protein (TSPO). Thus, there are at least two different components in Aß aggregates providing distinct binding sites for the current amyloid radioligands, and one of these binding components is distinctly present in the presubicular Aß deposits. Amyloid radioligands lacking affinity for this component, such as (125)I-DRM106, may selectively capture Aß deposits tightly associated with TSPO neuroinflammation and neurodegeneration as exemplified by CA1 neuritic plaques. Hence, comparative autoradiographic assessments of radioligand binding in CA1 and presubiculum could serve for the development of an amyloid PET imaging agent visualizing neurotoxicity-related Aß pathologies. Non-invasive determination of amyloid-ß peptide (Aß) serves for the early diagnosis and clarification of pathogenetic mechanisms of Alzheimer's disease (AD). We found that there are at least two different amyloid components in hippocampal CA1 and presubiculum providing distinct binding sites for the current amyloid radioligands. Comparative analysis for radioligand binding in these two regions could serve for developing novel imaging agents selectively visualizing neurotoxicity-related Aß pathologies.

Sphingosine-1-phosphate lyase is expressed by CD68+ cells on the parenchymal side of marginal reticular cells in human lymph nodes.

Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141(high) podoplanin(+), CD90(+), ICAM1(+), and VCAM1(+) but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68(+) APCs. CD68(+) APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.

A novel TLR2-triggered signalling crosstalk synergistically intensifies TNF-mediated IL-6 induction.

Toll-like receptors (TLR) recognize pathogens and trigger the production of vigorous pro-inflammatory cytokines [such as tumour necrosis factor (TNF)] that induce systemic damages associated with sepsis and chronic inflammation. Cooperation between signals of TLR and TNF receptor has been demonstrated through the participation of TNF receptor 1 (TNFR) adaptors in endotoxin tolerance. Here, we identify a TLR2-mediated synergy, through a MyD88-independent crosstalk, which enhances subsequent TNF-mediated nuclear factor-kappa B activation and interleukin-6 induction. Membrane-associated adaptor MAL conduces the link between TNF receptor-associated factor 6 (TRAF6) and TNFR-associated death domain, leading to a distinctive K63-ubiquitinylated TRAF6 recruitment into TNFR complex. In summary, our results reveal a novel route of TLR signal that synergistically amplifies TNF-mediated responses, indicating an innovative target for inflammation manipulation.

Identification of germinal centres in the lymph node of a patient with hyperimmunoglobulin M syndrome associated with congenital rubella.

BACKGROUND: The hyper immunoglobulin M syndrome (HIM) associated with congenital rubella infection (rHIM) is an extremely rare disorder, where patients have elevated serum IgM in association with reduced IgG and IgA. We have previously shown that in contrast to X-linked HIM (XHIM), a patient with well-characterised rHIM is able to express functional CD40 ligand, undergo immunoglobulin isotype switching and to generate memory B cells. Here we describe the ultrastructural features of an excised lymph node from this patient. METHODS: An inguinal lymph node was surgically removed and examined histologically as well as by immunohistochemistry. It was then stained with multiple fluorescent dyes to visualize the cellular interactions within the node. Flow cytometry was undertaken on a cellular suspension from the node. FINDINGS: Our patient has normal lymph node architecture by light microscopy. Immunohistochemistry studies showed the presence of scattered germinal centres. Polychromatic immunofluorescence staining showed disruption of the architecture with mostly abnormal germinal centres. A small number of relatively intact germinal centres were identified. Both IgM and IgG bearing cells were identified in germinal centres. INTERPRETATION: In contrast to XHIM where germinal centres are absent, the presence of small numbers of relatively normal germinal centres explain our previous identification of isotype switched memory B cells in rHIM.

Synthesis and biological evaluation of novel radioiodinated imidazopyridine derivatives for amyloid-ß imaging in Alzheimer's disease.

Non-invasive detection for amyloid-ß peptide (Aß) deposition has important significance for the early diagnosis and medical intervention for Alzheimer's disease (AD). In this study, we developed a series of imidazopyridine derivatives as potential imaging agents for single-photon emission computed tomography (SPECT). Two of them, compounds DRK092 and DRM106, showed higher affinity for synthetic human Aß 1-40 fibrils than did the well-known amyloid-imaging agent IMPY. A metabolite analysis revealed brain-permeable radioactive metabolites of (125)I-labeled DRK092 and IMPY; no radioactive metabolites from (125)I-labeled DRM106 ([(125)I]DRM106) were detected. In addition, in vitro autoradiography clearly demonstrated specific binding of [(125)I]DRM106 in the hippocampal region of AD enriched with Aß plaques. Thus, our results strongly suggested that compound DRM106 can be used as an imaging agent for SPECT to detect Aß deposition in AD brain.

Identification of a key pathway required for the sterile inflammatory response triggered by dying cells.

Dying cells stimulate inflammation, and this response is thought to contribute to the pathogenesis of many diseases. Very little has been known, however, about how cell death triggers inflammation. We found here that the acute neutrophilic inflammatory response to cell injury requires the signaling protein myeloid differentiation primary response gene 88 (Myd88). Analysis of the contribution of Myd88-dependent receptors to this response revealed only a minor reduction in mice doubly deficient in Toll-like receptor 2 (Tlr2) and Tlr4 and normal responses in mice lacking Tlr1, Tlr3, Tlr6, Tlr7, Tlr9, Tlr11 or the interleukin-18 receptor (IL-18R). However, mice lacking IL-1R showed a markedly reduced neutrophilic inflammatory response to dead cells and tissue injury in vivo as well as greatly decreased collateral damage from inflammation. This inflammatory response required IL-1alpha, and IL-1R function was required on non-bone-marrow-derived cells. Notably, the acute monocyte response to cell death, which is thought to be important for tissue repair, was much less dependent on the IL-1R-Myd88 pathway. Also, this pathway was not required for the neutrophil response to a microbial stimulus. These findings suggest that inhibiting the IL-1R-Myd88 pathway in vivo could block the damage from acute inflammation that occurs in response to sterile cell death, and do so in a way that might not compromise tissue repair or host defense against pathogens.

Activation of antitumor immune responses by Ganoderma formosanum polysaccharides in tumor-bearing mice.

Fungi of the genus Ganoderma are basidiomycetes that have been used as traditional medicine in Asia and have been shown to exhibit various pharmacological activities. We recently found that PS-F2, a polysaccharide fraction purified from the submerged culture broth of Ganoderma formosanum, stimulates the maturation of dendritic cells and primes a T helper 1 (Th1)-polarized adaptive immune response in vivo. In this study, we investigated whether the immune adjuvant function of PS-F2 can stimulate antitumor immune responses in tumor-bearing mice. Continuous intraperitoneal or oral administration of PS-F2 effectively suppressed the growth of colon 26 (C26) adenocarcinoma, B16 melanoma, and sarcoma 180 (S180) tumor cells in mice without adverse effects on the animals' health. PS-F2 did not cause direct cytotoxicity on tumor cells, and it lost the antitumor effect in mice with severe combined immunodeficiency (SCID). CD4(+) T cells, CD8(+) T cells, and serum from PS-F2-treated tumor-bearing mice all exhibited antitumor activities when adoptively transferred to naïve animals, indicating that PS-F2 treatment stimulates tumor-specific cellular and humoral immune responses. These data demonstrate that continuous administration of G. formosanum polysaccharide PS-F2 can activate host immune responses against ongoing tumor growth, suggesting that PS-F2 can potentially be developed into a preventive/therapeutic agent for cancer immunotherapy.

Proliferating macrophages in human tumours show characteristics of monocytes responding to myelopoietic growth factors.

Macrophages play essential roles in maintaining tissue homeostasis and immune defence. However, their extensive infiltration into tumours has been linked to adverse outcomes in multiple human cancers. Within the tumour microenvironment (TME), tumour-associated macrophages (TAMs) promote tumour growth and metastasis, making them prime targets for cancer immunotherapy. Recent single-cell analysis suggest that proliferating TAMs accumulate in human cancers, yet their origins and differentiation pathways remain uncertain. Here, we show that a subpopulation of CD163+ TAMs proliferates in situ within the TME of melanoma, lung cancer, and breast cancer. Consistent with their potential role in suppressing anti-tumour activities of T cells, CD163+ TAMs express a range of potent immunosuppressive molecules, including PD-L1, PD-L2, IL-10, and TGF-ß. Other phenotypic markers strongly suggested that these cells originate from CD14+ CCR2+ monocytes, a cell population believed to have minimal capacity for proliferation. However, we demonstrate in vitro that certain myelopoietic cytokines commonly available within the TME induce robust proliferation of human monocytes, especially the combination of interleukin 3 (IL-3) and Macrophage Colony-Stimulating Factor 1 (M-CSF). Monocytic cells cultured with these cytokines efficiently modulate T cell proliferation, and their molecular phenotype recapitulates that of CD163+ TAMs. IL-3-driven proliferation of monocytic cells can be completely blocked by IL-4, associated with the induction of CDKN1A, alongside the upregulation of transcription factors linked to dendritic cell function, such as BATF3 and IRF4. Taken together, our work suggests several novel therapeutic routes to reducing immunosuppressive TAMs in human tumours, from blocking chemokine-mediated recruitment of monocytes to blocking their proliferation.

Seven-colour multiplex immunochemistry/immunofluorescence and whole slide imaging of frozen sections.

Multiplex Immunochemistry/Immunofluorescence (mIHC/IF) aims to visualise multiple biomarkers in a single tissue section and is especially powerful when used on slide scanners coupled with digital analysis tools. mIHC/IF is commonly employed in immuno-oncology to characterise features of the tumour microenvironment (TME) and correlate them with clinical parameters to guide prognostication and therapy. However, mIHC/IF can be applied to a wide range of organisms in any physiological or disease context. Recent innovation has extended the number of markers that can be detected using slide scanners well beyond the 3-4 markers typically reported in traditional fluorescence microscopy. However, these methods often require sequential antibody staining and stripping, and are not compatible with frozen tissue sections. Using fluorophore-conjugated antibodies, we have established a simple mIHC/IF imaging workflow that enables simultaneous staining and detection of seven markers in a single section of frozen tissue. Coupled with automated whole slide imaging and digital quantification, our data efficiently revealed the tumour-immune complexity in metastatic melanoma. Computational image analysis quantified the immune and stromal cell populations present in the TME as well as their spatial interactions. This imaging workflow can also be performed with an indirect labelling panel consisting of primary and secondary antibodies. Our new methods, combined with digital quantification, will provide a valuable tool for high-quality mIHC/IF assays in immuno-oncology research and other translational studies, especially in circumstances where frozen sections are required for detection of particular markers, or for applications where frozen sections may be preferred, such as spatial transcriptomics.

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1) induces G2/M arrest and mitotic catastrophe in human leukemia HL-60 cells.

2-(3-Methoxyphenyl)-5-methyl-1,8-naphthyridin-4(1H)-one (HKL-1), a 2-phenyl-1,8-naphthyridin-4-one (2-PN) derivative, was synthesized and evaluated as an effective antimitotic agent in our laboratory. However, the molecular mechanisms are uncertain. In this study, HKL-1 was demonstrated to induce multipolar spindles, sustain mitotic arrest and generate multinucleated cells, all of which indicate mitotic catastrophe, in human leukemia HL-60 cells. Western blotting showed that HKL-1 induces mitotic catastrophe in HL-60 cells through regulating mitotic phase-specific kinases (down-regulating CDK1, cyclin B1, CENP-E, and aurora B) and regulating the expression of Bcl-2 family proteins (down-regulating Bcl-2 and up-regulating Bax and Bak), followed by caspase-9/-3 cleavage. These findings suggest that HKL-1 appears to exert its cytotoxicity toward HL-60 cells in culture by inducing mitotic catastrophe.

Mapping of IgE and IgG4 antibody-binding epitopes in Cyn d 1, the major allergen of Bermuda grass pollen.

BACKGROUND: Bermuda grass pollen (BGP) is an important seasonal aeroallergen worldwide which induces allergic disorders such as allergic rhinitis, conjunctivitis and asthma. Cyn d 1 is the major allergen of BGP. This study is aimed to map human IgE and IgG(4) antibody-binding sequential epitopes on Cyn d 1 by dot immunoblotting. METHODS: Synthetic peptides (10-mers; 5 overlapping residues) spanning the full length of Cyn d 1 were used for dot immunoblotting to map human IgE and IgG(1-4) antibody-binding regions with sera from BGP-allergic patients. Synthetic peptides with more overlapping residues were used for further mapping. Essential amino acids in each epitope were examined by single amino acid substitution with alanine. Peptides with sequence polymorphism of epitopes of Cyn d 1 were also synthesized to extrapolate their differences in binding capability. RESULTS: Four major IgE-binding epitopes (peptides 15(-1), 21, 33(-2) and 35(+1), corresponding to amino acids 70-79, 101-110, 159-167 and 172-181) and 5 major IgG(4)-binding epitopes (peptides 15(-1), 30(-2), 33(-2), 35(+1) and 39, corresponding to amino acids 70-79, 144-153, 159-167, 172-181 and 192-200) were identified. They are all located on the surface of the simulated Cyn d 1 molecule, and three of them are major epitopes for both IgE and IgG(4). Their critical amino acids were all characterized. Major epitopes for human IgG(1) to IgG(4) are almost identical. CONCLUSIONS: This is the first study to map the sequential epitopes for human IgE and IgG(4) subclasses in Cyn d 1. It will be helpful for future development in immunotherapy and diagnosis.