Chitin synthase (CHS) gene analysis of dermatophytes]

About 620-bp genomic DNA fragments of CHS1 genes were amplified from 13 species of dermatophytes by polymerase chain reaction (PCR) and sequenced. The phylogenetic analysis of CHS1 gene fragments of these dermatophyte species revealed that 3 genera of Epidermophyton, Microsporum and Trichophyton were genetically different from each other. The molecular analysis of CHS1 genes will provide useful information for the identification of dermatophytes. The species-specific primers were designed from the nucleotide sequences of CHS1 gene in 3 teleomorphs of T. mentagrophytes. Using these primers the PCR analysis identified the clinical isolates of T. mentagrophytes from rabbit as A. benhamiae. By PCR analysis with the dermatophyte specific primer pair, dermatophyte DNA could be diagnosed directly and rapidly in clinical skin samples. The full length of CHS1 and CHS2 genes of Arthroderma benhamiae (one of the teleomorphs of T. mentagrophytes) was sequenced by 5'-RACE and 3'-RACE methods using cDNA as a template. The full length cDNA sequences of CHS1 gene (3158bp) and CHS2 gene (3392bp) were proved to encode 890 and 419 amino acids, respectively. The amino acid sequences of A. benhamiae CHS1 and CHS2 in the conserved regions shared, respectively, about 70% and 80% sequence similarity with those of the other filamentous ascomycetes registered in the data base of the GeneBank. RT-PCR analysis suggested that chitin synthase inhibitors (nikkomycin Z and polyoxin D) might stimulate the expression of CHS1 mRNA in A. benhamiae, and not the expression of CHS2 mRNA.     

Two novel CHS1 (LYST) mutations: clinical correlations in an infant with Chediak-Higashi syndrome

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disease characterized by variable degrees of oculocutaneous albinism, recurrent infections, and a mild bleeding tendency, with late neurologic dysfunction. Most patients also undergo an accelerated phase of lymphohistiocytosis and die at an early age unless they receive an allogeneic hematopoietic stem cell transplant (SCT). Mutations in the CHS1 (LYST) gene result in CHS. Here, we describe an adopted infant who is compound heterozygous for two novel CHS1 gene mutations, both of which are predicted to result in truncated proteins. The two mutations are a nonsense mutation (c.1540 C>T, CGA>TGA, R514X) in exon 5 and a one base pair deletion (del c.9893T, F3298fsX3304) in exon 43, coding for part of the CHS1 protein's BEACH domain. These two newly described mutations are expected to give rise to a severe phenotype and, indeed, the patient had absolutely no cytotoxicity by natural killer cells or cytotoxic lymphocytes prior to his allogeneic SCT.     

Fusarium verticillioides chitin synthases CHS5 and CHS7 are required for normal growth and pathogenicity

Fusarium verticillioides is both an endophyte and a pathogen of maize and is a health threat in many areas of the world because it can contaminate maize with fumonisins, a toxic secondary metabolite. We identified eight putative chitin synthase (CHS) genes in F. verticillioides genomic sequence, and phylogenetic evidence shows that they group into seven established CHS gene classes. We targeted two CHSs (CHS5 and CHS7) for deletion analysis and found that both are required for normal hyphal growth and maximal disease of maize seedlings and ears. CHS5 and CHS7 encode a putative class V and class VII fungal chitin synthase, respectively; they are located adjacent to each other and are divergently transcribed. Fluorescent microscopy found that both CHS deficient strains produce balloon-shaped hyphae, while growth assays indicated that they were more sensitive to cell wall stressing compounds (e.g., the antifungal compound Nikkomycin Z) than wild type. Pathogenicity assays on maize seedlings and ears indicated that both strains were significantly reduced in their ability to cause disease. Our results demonstrate that both CHS5 and CHS7 are necessary for proper hyphal growth and pathogenicity of F. verticillioides on maize.     

Chediak-Higashi syndrome associated with maternal uniparental isodisomy of chromosome 1.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder (incidence around 1 in 106 births), characterised by a complex immunologic defects, reduced pigmentation, and presence of giant granules in many different cell types. It most likely results from defective organellar trafficking or protein sorting. The causative gene (LYST) has recently been identified and shown to be homologous to the beige locus in the mouse. CHS has always been reported associated with premature-termination-codon mutations in both alleles of LYST. We report a unique patient with CHS, who was homozygous for a stop codon in the LYST gene on chromosome 1 and who had a normal 46,XY karyotype. The mother was found to be a carrier of the mutation, whereas the father had two normal LYST alleles. Non-paternity was excluded by the analysis of microsatellite markers from different chromosomes. The results of 13 informative microsatellite markers spanning the entire chromosome 1 revealed that the proband had a maternal isodisomy of chromosome 1 encompassing the LYST mutation. The proband's clinical presentation also confirms the absence of imprinted genes on chromosome 1.     

IL-1-induced tumor necrosis factor-alpha elicits inflammatory cell infiltration in the skin by inducing IFN-gamma-inducible protein 10 in the elicitation phase of the contact hypersensitivity response

Contact hypersensitivity (CHS) is a typical inflammatory response against contact allergens. Inflammatory cytokines, including IL-1 and tumor necrosis factor (TNF)-alpha, are implicated in the reaction, although the precise roles of each cytokine have not been completely elucidated. In this report, we dissected the functional roles of IL-1 and TNF-alpha during CHS. CHS induced by 2,4,6-trinitorochlorobenzene as well as oxazolone was suppressed in both IL-1alpha/beta(-/-) and TNF-alpha(-/-) mice. Hapten-specific T cell activation, as examined by T cell proliferation, OX40 expression and IL-17 production, was reduced in IL-1alpha/beta(-/-) mice, but not in TNF-alpha(-/-) mice, suggesting that IL-1 but not TNF-alpha is required for hapten-specific T cell priming in the sensitization phase. On the other hand, TNF-alpha, induced by IL-1, was necessary for the induction of local inflammation during the elicitation phase. We also found that the expression of IFN-gamma-inducible protein 10 (IP-10) was augmented at the inflammatory site. Although IP-10 mRNA expression was abrogated in TNF-alpha(-/-) mice, both CHS development and TNF-alpha mRNA expression occurred normally in IFN-gamma(-/-) mice, indicating that the induction of IP-10 during CHS was primarily controlled by TNF-alpha. Interestingly, CHS was suppressed by treatment with anti-IP-10 mAb, suggesting a critical role for IP-10 in CHS. Reduced CHS in TNF-alpha(-/-) mice was reversed by IP-10 injection during the elicitation phase. Thus, it was shown that the roles for IL-1 and TNF-alpha are different, although both cytokines are crucial for the development of CHS.     

The Chédiak-Higashi syndrome; the nature of the giant neutrophil granules and their interactions with cytoplasm and foreign particulates. I. Progressive enlargement of the massive inclusions in mature neutrophils. II. Manifestations of cytoplasmic injury and sequestration. III. Interactions between giant organelles and foreign particulates.

Defective bactericidal functioning of polymorphonuclear leukocytes (PMNs) from patients with the Chédiak-Higashi syndrome (CHS) has been related in previous reports to a failure of the giant granules characteristics of the disorder to participate in degranulation after uptake of foreign particulates by neutrophils. However, the reason massive CHS inclusions do not fuse with and discharge their contents into phagocytic vacuoles has not been defined. The problem is particularly puzzling because it has been postulated that the hugh organelles in CHS neutophils originate by fusion of small azurophilic granules in promyelocytes and myelocytes. The present series of investigations into the cytopathology of the CHS has employed electron microscopy and ultrastructural cytochemistry to characterize the progressive enlargement of the hugh bodies in mature PMNs, their interaction with cytoplasmic constituents resulting in various manifestations of cell injury, and their response to foreign particulates. Each study clarifies abnormal features of the giant organelles essential to the understanding of their role in the defective bactericidal function of CHS neutrophils. The first report demonstrates that most of the hugh inclusions in PMNs are not primary lysosomes. The interaction and fusion of giant azurophilic granules with each other, with normal-sized primary and secondary granules, and with cytoplasmic components converts the massive primary granules into huge secondary lysosomes. Transformation to secondary hysosomes represents a critical alteration in the state of the giant granules that underlies their damaging influence on the cytoplasm and loss of reactivity wtih phagocytic vacuoles.     

Chitin Synthase 2 (CHS2) gene of Malassezia species]

Malassezia species have been recognized as members of the microbiological flora of human and animal skin; they are also considered to play an important role in the pathogenesis of folliculitis, atopic dermatitis and otitis externa. Therefore, the molecular characteristics were investigated to clarify the epidemiology and the pathogenesis of diseases associated with Malassezia species in human and animals. Molecular investigation was made of 105 clinical isolates of M. pachydermatis from dogs and cats by random amplification of polymorphic DNA (RAPD) and chitin synthase 2 (CHS2) gene sequence analyses. The RAPD analysis and CHS2 gene analysis indicated that clinical isolates of M. pachydermatis were divided into four distinct genetic types (A, B, C and D). Type A was isolated from lesions of atopic dermatitis, flea allergic dermatitis, otitis externa, pyoderma and seborrheic (dermatitidis) in dogs and cats, and might be predominant on this. The phylogenetic analysis of the nucleotide sequences of CHS2 gene fragments of standard strains of 11 Malassezia species showed 11 distinct clusters of this species.     

Clinical, molecular, and cell biological aspects of Chediak-Higashi syndrome.

Chediak-Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by variable degrees of oculocutaneous albinism, easy bruisability, and bleeding as a result of deficient platelet dense bodies, and recurrent infections, with neutropenia, impaired chemotaxis and bactericidal activity, and abnormal NK cell function. Neurologic involvement is variable, but often includes peripheral neuropathy. Most patients also undergo an "accelerated phase," which is a nonmalignant lymphohistiocytic infiltration of multiple organs resembling lymphoma. Death often occurs in the first decade from infection, bleeding, or development of the accelerated phase. The hallmark of CHS is the presence of huge cytoplasmic granules in circulating granulocytes and many other cell types. These granules are peroxidase-positive and contain lysosomal enzymes, suggesting that they are giant lysosomes or, in the case of melanocytes, giant melanosomes. The underlying defect in CHS remains elusive, but the disorder can be considered a model for defects in vesicle formation, fusion, or trafficking. Because the beige mouse demonstrates many characteristics similar to those of human CHS patients, including dilution of coat color, recurrent infections, and the presence of giant granules, it is considered the animal homologue of CHS. The beige gene, Lyst, was mapped and sequenced in 1996, prompting identification of the human LYST gene on chromosome 1q42. Lyst and LYST show 86.5% sequence homology. LYST encodes a 429 kDa protein with a function that remains unknown, but the source of extensive speculation among students of cell biology.     

The Chédiak-Higashi syndrome: ring-shaped lysomomes in circulating monocytes.

The giant inclusion anomaly of leukocytes from three patients with the Chédiak-Higashi syndrome (CHS) has been reexamined by the ultrastructural and cytochemical techniques placing emphasis on the monocyte. Contrary to previous assumptions that all CHS leukocytes shared a common form of giant granule with similar mechanisms of formation, it is shown that CHS monocytes contain ring-shaped organelles as their principle manifestation of the disease. These granules consist of a double membrane surrounding a segment of cytoplasm, which may or may not be sequestered from the remaining cytoplasm and which usually contains one or more normal-sized azurophilic granules. The material lying between the double membranes contains the specific reaction products for peroxidase and acid phosphatase. Therefore, the ring-shaped organelles share identity with the azurophilic granules and probably share a common origin with the primary lysosomes. The double-ring form of giant CHS organelle has also been found infrequently in neutrophils, eosinophils, and lymphocytes.     

Giant dense bodies in fibroblasts cultured from beige mice with the Chédiak-Higashi syndrome

Fibroblasts cultured from the skin of beige mice manifesting the Chédiak-Higashi syndrome (CHS), unlike cells derived from normal black mice, exhibited giant dense bodies in the cytoplasm. These megabodies were membrane-delimited and exhibited dense content by electron microscopy, with myelin figures, highly osmiophilic, thick membranous contours, and lucent areas. The megabodies evidenced acid phosphatase ultrastructurally. Cells of both normal and CHS mice contained smaller dense bodies. During a 2--6 hour exposure to colloidal gold, the smaller dense bodies of normal and CHS fibroblasts selectively incorporated the gold spherules and, accordingly, were identified as secondary lysosomes of heterophagic origin. With longer exposure to colloidal gold, the small dense bodies of the normal and CHS cells disclosed increased content of colloidal gold. After 24 hour exposure to colloidal gold, many giant dense bodies also exhibited gold particles, evidencing uptake of endocytosed material into the giant structures and the heterophagic origin of at least some of the content of the bodies. The gold spherules initially incorporated into the giant dense bodies were concentrated in foci along their periphery and indicated fusion of small dense bodies into the giant structures. Transformed normal and CHS cells appeared to contain more abundant myelin figures than nontransformed cells, and these were larger in transformed CHS cells and constituted a major component of their giant dense bodies. The giant inclusions of the transformed CHS cells generally contained little colloidal gold, suggesting their derivation principally through cellular autophagy.     

Chediak-Higashi Syndrome: Variable Cytochemical Reactivity of Giant Inclusions in Polymorphonuclear Leukocytes

Neutrophils from the peripheral blood of patients with the Chediak-Higashi syndrome (CHS) contain large numbers of giant granules. Previous studies have demonstrated that the huge neutrophil organelles contain hydrolytic enzymes and are, therefore, massive lysosomes. The basis for the inability of giant granules to react with phagocytic vacuoles containing foreign particulates taken up by CHS neutrophils has not been determined. In the present investigation we have examined the ultrastructural cytochemistry of the huge inclusions in neutrophils from 3 patients. Electron-dense reaction product of the acid phosphatase reaction stained the giant inclusions and a few normal-sized azurophilic granules in the process of fusion with them. The amount of reaction product deposited in the huge organelles and its distribution, however, were extremely variable, even in the same cell. Myeloperoxidase staining revealed a similar variability in the intensity and distribution of its electrondense reaction product. The findings suggest that giant primary granules in circulating CHS neutrophils retain characteristics similar to newly formed azurophilic lysosomes in promyelocytes and myelocytes. Dilution of enzyme reaction products and variability in their localization indicate that most of the giant primary granules undergo transformation or incorporation into huge secondary lysosomes, which are virtually unable to participate in the degranulation reaction after uptake of foreign particulates by CHS neutrophils.     

Chediak-Higashi Syndrome: Variable Cytochemical Reactivity of Giant Inclusions in Polymorphonuclear Leukocytes

Neutrophils from the peripheral blood of patients with the Chediak-Higashi syndrome (CHS) contain large numbers of giant granules. Previous studies have demonstrated that the huge neutrophil organelles contain hydrolytic enzymes and are, therefore, massive lysosomes. The basis for the inability of giant granules to react with phagocytic vacuoles containing foreign particulates taken up by CHS neutrophils has not been determined. In the present investigation we have examined the ultrastructural cytochemistry of the huge inclusions in neutrophils from 3 patients. Electron-dense reaction product of the acid phosphatase reaction stained the giant inclusions and a few normal-sized azurophilic granules in the process of fusion with them. The amount of reaction product deposited in the huge organelles and its distribution, however, were extremely variable, even in the same cell. Myeloperoxidase staining revealed a similar variability in the intensity and distribution of its electrondense reaction product. The findings suggest that giant primary granules in circulating CHS neutrophils retain characteristics similar to newly formed azurophilic lysosomes in promyelocytes and myelocytes. Dilution of enzyme reaction products and variability in their localization indicate that most of the giant primary granules undergo transformation or incorporation into huge secondary lysosomes, which are virtually unable to participate in the degranulation reaction after uptake of foreign particulates by CHS neutrophils.     

Chediak-Higashi lymphoblastoid cell lines: granule characteristics and expression of lysosome-associated membrane proteins.

Chediak-Higashi syndrome (CHS) is characterized morphologically by the presence of giant lysosomal granules resulting from the dysregulated fusion of primary lysosomes. Lysosome-associated membrane proteins comprise a family of highly glycosylated proteins which are postulated to facilitate many aspects of normal lysosomal function. In this study, Epstein-Barr virus-transformed lymphoblastoid cell lines derived from a patient with CHS were analyzed for the presence of giant granules and the expression of the lysosome-associated membrane proteins lamp1 and lamp2. Giant myeloperoxidase positive granules typical of CHS, which had a complex structure when examined by electron microscopy, could be demonstrated in the lymphoblastoid cell lines. In situ immunofluorescence with antibodies directed against lamp1 and lamp2 demonstrated abundant expression of each of these proteins in the giant CHS granules. Lack of expression of lysosomal cathepsin G in these granules was also noted. These observations suggest that the lymphoblastoid cell lines provide a convenient model for the study of Chediak-Higashi granules and the lysosome-associated membrane proteins and provide additional evidence that CHS is a "lysosomal" disease. Further study will be necessary to delineate whether the function of these membrane proteins is altered in Chediak-Higashi syndrome.     

Quinacrine inhibits the epidermal dendritic cell migration initiating T cell-mediated skin inflammation

Quinacrine (QC) is an anti-inflammatory drug that has been used for the treatment of malaria and rheumatoid diseases. The mechanism(s) underlying the anti-inflammatory activity of QC remains poorly understood. We recently reported the QC-mediated inhibition of the NF-kappaB pathway using an in vitro model. To test this potential mechanism in vivo, we used the contact hypersensitivity response (CHS) to chemical allergen sensitization and challenge in mice as a model of skin inflammation. The results indicated that QC treatment inhibited NF-kappaB activation in the skin during allergen sensitization. This inhibition was reflected by decreased mRNA expression and protein production of the NF-kappaB-dependent cytokines TNF-alpha and IL-1beta and the chemokine CCL21 in the skin. The decreases in these cytokines resulted in reduced migration of allergen-presenting dendritic cells from the skin into skin-draining lymph nodes and markedly decreased activation of effector CD8+ T cells for the CHS response to allergen challenge (inhibitory concentration 50% or IC50 was 55 mg/kg). These findings reveal a previously unrecognized mechanism of QC-mediated inhibition of inflammation.     

一个Chediak-Higashi综合征家系CHS1/LYST基因新变异

目的对1个旁系血亲婚配的Chediak-Higashi综合征家系的CHS1/LYST基因进行变异检测,探讨LYST基因变异与临床表型的关系。方法收集临床资料及家系调查,分析2例患者的临床资料和实验室检查结果。提取患者外周血DNA及父母等16名亲属指甲的DNA,采用外显子组测序分析患者的致病基因,根据基因变异点对亲属相应位点做Sanger测序,确认携带者。结果该家系中2例患者临床均有皮肤部分白化及免疫缺陷,表现为反复严重感染、出血倾向且发生嗜血现象,骨髓细胞及血涂片检查均出现粗大的嗜酸性包涵体颗粒。LYST基因第34外显子存在c.8782C>T(p.Gln2928*)纯合无义变异,与其表型相关,且该变异为新变异点。确认6名表型正常的家系成员的LYST基因存在与先证者相同的杂合变异,另10名表型正常的家系成员则均未检测到该变异。结论基因变异分析结果丰富了Chediak-Higashi综合征致病基因的变异谱,为临床明确病因提供了重要依据,并为家系遗传咨询和产前诊断提供了参考依据。     

Expression of cysLT1 and cysLT2 Receptor in Chronic Hyperplastic Eosinophilic Sinusitis

Elevated production of cysteinyl leukotrienes (cysLTs) from sinus tissues and abundant sinus eosinophils are characteristic features of chronic hyperplastic eosinophilic sinusitis (CHS). CysLTs exert their action through G-protein-coupled receptors named cysLTs receptor type I (cysLT1R) and type II (cysLT2R). These expressions of cysLT receptors in the sinus mucosa have yet to be clarified and the relationship between eosinophilia and the expression of these receptors remains obscure. We compared the expressions of cysLT1R and cysLT2R in the sinus mucosa in patients with CHS, non-eosinophilic chronic sinusitis (NECS), and control sinus tissues; and analyzed the correlation between the expression of CysLTRs and the presence of sinus eosinophils by immunohistochemistry and real-time PCR. A significantly higher percentage of eosinophils expressing cysLT2R protein was observed in patients with CHS compared with NECS and controls. In addition, cysLT2R mRNA expression in CHS was significantly higher than in NECS and controls. Furthermore, a positive correlation was observed between cysLT2R mRNA expression and the number of infiltrated eosinophils. In contrast, the cysLT1R mRNA expression did not differ significantly among these groups. The effect of cysLTs on sinus eosinophils may be mediated through the cysLT2R in patients with CHS. These results may suggest the therapeutic benefit of cysLT2R antagonists in CHS.     

Apparent genotype–phenotype correlation in childhood,adolescent, and adult Chediak‐Higashi syndrome

Chediak‐Higashi syndrome (CHS) is a rare autosomal recessive disorder characterized by severe immunologic defects, reduced pigmentation, bleeding tendency, and progressive neurological dysfunction. Most patients present in early childhood and die unless treated by bone marrow transplantation. About 10–15% of patients exhibit a much milder clinical phenotype and survive to adulthood, but develop progressive and often fatal neurological dysfunction. Very rare patients exhibit an intermediate adolescent CHS phenotype, presenting with severe infections in early childhood, but a milder course by adolescence, with no accelerated phase. Here, we describe the organization and genomic DNA sequence of the CHS1 gene and mutation analysis of 21 unrelated patients with the childhood, adolescent, and adult forms of CHS. In patients with severe childhood CHS, we found only functionally null mutant CHS1 alleles, whereas in patients with the adolescent and adult forms of CHS we also found missense mutant alleles that likely encode CHS1 polypeptides with partial function. Together, these results suggest an allelic genotype–phenotype relationship among the various clinical forms of CHS. © 2002 Wiley‐Liss, Inc.     

Induction and regulation of T-cell priming for contact hypersensitivity

Contact hypersensitivity (CHS) is a T-cell-mediated immune response to cutaneous sensitization and subsequent challenge with haptens such as dinitrofluorobenzene and oxazolone. Clinically, contact sensitivity, also called allergic contact dermatitis, is a frequently observed dermatosis in industrialized countries. Experimental CHS in mice has been used by many laboratories as a model of T-cell-mediated immune responses to antigens deposited onto the skin to study the priming, development, and function of effector and regulatory T-cell components during these responses. In this article we discuss the mechanism of T-cell priming by hapten-presenting Langerhans cells and how the priming environment influences the development of these hapten-specific T cells to different functional phenotypes during sensitization for the CHS response. Finally, we propose a model of negative regulation of the CHS response by T-cell components that are coincidentally primed with the effector T cells mediating the response. Overall, these aspects indicate a unique immune response mediated and regulated by specialized antigen-presenting cells and T-cell populations.     

Inflammatory and immune mechanisms in contact hypersensitivity (CHS) in rats

Contact hypersensitivity (CHS) is a T-cell-mediated skin inflammatory reaction to cutaneous exposure to small sensitizing chemicals, haptens. Majority of CHS studies were conducted in mice and there is paucity of data in other experimental animals. In this review, after a brief survey of murine CHS, hitherto known characteristics of CHS in rats were presented including inflammatory and immune mechanisms of both sensitization and elicitation phases. Survey of literature of rat CHS is presented, with our data concerning the importance of genetic background both in the induction and in the expression of reaction to dinitrochlorobenzene. The knowledge of CHS in rats, preferred animal in immunopharmacological studies, might help development of immunomodulatory intervention in contact allergy.