[Effect of NLRP3 mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation injury].

Objective: To investigate the effect of NACHT-LRR-PYD- containing proteins 3 (NLRP3) mediated pyroptosis in myocardial cells undergoing hypoxia/deoxygenation (H/R) injury. Methods: In order to observe whether H/R-treatment could cause pyroptosis, H9c2 cells were divided into 2 groups randomly using the lottery method: control group(without H/R-treatment) and H/R group (in which the H9c2 cells were underwent H/R-treatment). In order to clarify the role of pyroptosis in H/R-injury, H9c2 cells were divided into 4 groups randomly using the lottery method: control group(in which the H9c2 cells were cultivated with normal medium); YVAD group(in which the H9c2 cells were pretreated with z-Val-Ala-Asp(Ome)-fluoromethylketone (Z-YVAD-FMK) 20 µm for 4 hours, then replaced with normal medium); H/R group(H9c2 cells underwent H/R-treatment); YVAD+H/R group (in which the H9c2 cells were pretreated with 20 µm Z-YVAD-FMK for 4 hours before H/R-treatment). To determine whether H/R-induced cell pyroptosis is associated with NLRP3, H9c2 cells were divided into 4 groups randomly using the lottery method: control group (in which cells were transfected with a control nonspecific siRNA); si-NLRP3 group (in which cells were transfected with NLRP3-targeting siRNA); H/R group(in which cells were transfected with a control nonspecific siRNA before H/R-treatment); si-NLRP3+H/R group(in which the H9c2 cells were transfected with NLRP3-targeting siRNA before H/R-treatment). Pore formation on cell membrane was detected by propidium iodide (PI) staining. Cell viability was detected by CCK8 reagent. The protein expression of Caspase-1, interleukin-1ß (IL-1ß) and NLRP3 was detected by Western blot. Results: (1) The positive rate of PI staining ((26.46±5.15)% vs. (1.69±0.73)%,P<0.01), expression of NLRP3 (0.57±0.16 vs. 0.23±0.06,P<0.01), expression of Caspase-1 (1.07±0.13 vs. 0.37±0.08,P<0.01), and expression of IL-1ß (0.38±0.08 vs. 0.16±0.05,P<0.01) were significantly higher in H/R group than in control group. (2)The cell vitality was significantly higher in YVAD+H/R group than in H/R group ((87.31±9.05)% vs. (73.30±7.19)%, P<0.05).The positive rate of PI staining was significantly decreased in YVAD+H/R group than in H/R group ((18.12±4.36)% vs. (26.45±4.60)%, P<0.05). The expression of Caspase-1 (0.72±0.12 vs. 1.07±0.15, P<0.05) and IL-1ß(0.29±0.07 vs. 0.39±0.06, P<0.05) were significantly lower in YVAD+H/R group than in H/R group. (3) The cell vitality was significantly increased in si-NLRP3+H/R group than in H/R group ((85.46±7.71)% vs. (72.41±5.53)%, P<0.05). The positive rate of PI staining was significantly lower in si-NLRP3+H/R group than in H/R group ((18.22±4.20)% vs. (26.73±3.26)%, P<0.05). The expression of Caspase-1(0.87±0.07 vs. 1.15±0.15, P<0.05) and IL-1ß(0.41±0.07 vs. 0.58±0.10, P<0.05) were significantly decreased in si-NLRP3+H/R group than in H/R group. Conclusion: NLRP3 mediated pyroptosis is involved in H/R injury of myocardial cells.

Activating and dominant inactivating c-KIT catalytic domain mutations in distinct clinical forms of human mastocytosis.

Human mastocytosis is characterized by increased mast cells. It usually occurs as a sporadic disease that is often transient and limited in children and persistent or progressive in adults. The c-KIT protooncogene encodes KIT, a tyrosine kinase that is the receptor for mast cell growth factor. Because mutated KIT can transform cells, we examined c-KIT in skin lesions of 22 patients with sporadic mastocytosis and 3 patients with familial mastocytosis. All patients with adult sporadic mastocytosis had somatic c-KIT mutations in codon 816 causing substitution of valine for aspartate and spontaneous activation of mast cell growth factor receptor (P = 0.0001). A subset of four pediatric onset cases with clinically unusual disease also had codon 816 activating mutations substituting valine, tyrosine, or phenylalanine for aspartate. Typical pediatric patients lacked 816 mutations, but limited sequencing showed three of six had a novel dominant inactivating mutation substituting lysine for glutamic acid in position 839, the site of a potential salt bridge that is highly conserved in receptor tyrosine kinases. No c-KIT mutations were found in the entire coding region of three patients with familial mastocytosis. We conclude that c-KIT somatic mutations substituting valine in position 816 of KIT are characteristic of sporadic adult mastocytosis and may cause this disease. Similar mutations causing activation of the mast cell growth factor receptor are found in children apparently at risk for extensive or persistent disease. In contrast, typical pediatric mastocytosis patients lack these mutations and may express inactivating c-KIT mutations. Familial mastocytosis, however, may occur in the absence of c-KIT coding mutations.

Murine cutaneous mastocytosis and epidermal melanocytosis induced by keratinocyte expression of transgenic stem cell factor.

The growth and differentiation of mast cells and melanocytes require stem cell factor (SCF), the ligand for the kit receptor tyrosine kinase. SCF may exist as a membrane-bound or soluble molecule. Abnormalities of the SCF-kit signaling pathway, with increased local concentrations of soluble SCF, have been implicated in the pathogenesis of the human disease cutaneous mastocytosis, but have not yet been shown to play a causal role. To investigate both the potential of SCF to cause mastocytosis and its role in epidermal melanocyte homeostasis, we targeted the expression of SCF to epidermal keratinocytes in mice with two different transgenes controlled by the human keratin 14 promoter. The transgenes contained cDNAs that either produced SCF, which can exist in both membrane-bound and soluble forms, or SCF, which remains essentially membrane bound. Murine epidermal keratinocyte expression of membrane-bound/ soluble SCF reproduced the phenotype of human cutaneous mastocytosis, with dermal mast cell infiltrates and epidermal hyperpigmentation, and caused the maintenance of a population of melanocytes in the interadnexal epidermis, an area where melanocytes and melanin are found in human skin but where they are not typically found in murine skin. Expression of membrane-bound SCF alone resulted in epidermal melanocytosis and melanin production, but did not by itself cause mastocytosis. We conclude, first, that a phenotype matching that of human mastocytosis can be produced in mice by keratinocyte overproduction of soluble SCF, suggesting a potential cause of this disease. Second, we conclude that keratinocyte expression of membrane-bound SCF results in the postnatal maintenance of epidermal melanocytes in mice. Since the resulting animals have skin that more closely approximates human skin than do normal mice, their study may be more relevant to human melanocyte biology than the study of skin of normal mice.

Somatic c-KIT activating mutation in urticaria pigmentosa and aggressive mastocytosis: establishment of clonality in a human mast cell neoplasm.

Mastocytosis is characterized by accumulations of mast cells in various organs (1). Most cases are indolent and confined to the skin, where discrete mast cell infiltrates are associated increased epidermal melanin, a clinical picture known as urticaria pigmentosa (UP). Other forms of mastocytosis combine UP with aggressive involvement of other organs or with haemotologic abnormalities (1-4). It is not known whether all forms of mastocytosis are true neoplasms or whether some might represent reactive hyperplasias (5-7). The c-KIT proto-oncogene encodes a type III receptor tyrosine kinase (KIT) that is critical to the development and survival of mast cells and melanocytes (8-11). The ligand for KIT (KL) can stimulate mast cell development, proliferation, and mediator release (9,12-17), as well as melanocyte proliferation and pigment production (18-20). To determine the role of c-KIT in the pathogenesis of mastocytosis, we examined tissue and cells isolated from a patient with UP and aggressive systemic mastocytosis with massive splenic involvement. We found a mutation that results in constitutive activation and expression of c-KIT in mast cells of both skin and spleen. This is the first in situ demonstration of an activation c-KIT mutation in neoplastic cells. It also demonstrates the clonal and neoplastic nature of this form of mastocytes.

Malignant and normal T cells show random use of T-cell receptor alpha chain variable regions in patients with cutaneous T-cell lymphoma.

Cutaneous T-cell lymphoma (CTCL) is a malignancy of mature T lymphocytes, most of which express alpha/beta type T-cell receptors (TCRs). The cause of CTCL is unknown, but hypotheses postulating chronic stimulation of TCRs by superantigen or by a leukemogenic virus have been proposed. Either mechanism might produce bias in the TCR variable (V) region types used by the malignant cells. To determine if TCR alpha use is restricted in CTCL, we used reverse transcription and the polymerase chain reaction to determine V alpha and V beta usage by malignant cells purified from the peripheral blood of leukemic patients with CTCL. Usage of alpha chain V region segments appeared totally random; malignant lymphocytes isolated from each of six patients used different V alpha regions. As has been previously reported, no bias was found in beta chain V region usage either. In addition to productive (in frame) TCR V region mRNAs in malignant cells from each patient, we detected non-productive (out of frame) beta chain transcripts in these cells in two of six patients, and non-productive alpha chain transcripts in five of six. Residual normal peripheral blood lymphocytes from these patients showed a random, polyclonal or oligoclonal pattern of V region usage. We conclude that there is no bias in V region usage in CTCL, making it unlikely that interactions between superantigen or virus and the TCR V regions play a role in the pathogenesis of CTCL.

[Classification of hand and its significance].

Measurements of normal values in hand appearance and thumb movements were done in 102 male adult volunteers with no history of previous injury to their hands. The data showed some characteristics between the appearance and function of hand, for example, no case showed the distance of adduction of thumb was 0, but the normal function of hand was still acquired. Positive correlation appeared between the indexes of appearances of hand. Correlation analysis have been done for three pairs of indexes, that is, hand length and width, length of thumb and the distance of first web, adduction of thumb and thumb-opposition. The correlation charts and their formulae have been presented. Furthermore, hands of male chinese could be classified into five types according to the correlation chart or the formula. The significance of such classification i discussed as well.

Clinical observations and methods for identifying the existence of cultured epidermal allografts.

Thirty-two burned or plastic surgery patients were grafted with allogeneic cultured epidermis on autograft donor sites. Two techniques, the indirect enzyme conjugated Staphylococcus Protein A assay with monoclonal antibodies against A or B blood group antigens and the polymerase chain reaction to detect a Y chromosome-specific DNA sequence, were employed to identify the presence of cultured epidermal allograft based on different ABO blood grouping or sex between donor and recipient. The methods have the advantage of high sensitivity and specificity in identifying the existence of allogeneic skin cells in grafts. The results indicated that the survival time of cultured epidermal allograft was prolonged up to 35 days. In addition, the intact coverage on some grafting sites may be composed of both host and donor origin cells, after about 3 weeks postgrafting.

The T cell receptors of human gamma delta T cells reactive to Mycobacterium tuberculosis are encoded by specific V genes but diverse V-J junctions.

The observation that gamma delta T lymphocytes react to mycobacteria has provided an important model for investigation of these cells in the immune response to infection. One important question regarding human gamma delta T cells is the breadth of the T cell repertoire in response to specific pathogens. The present study was undertaken to characterize, in molecular terms, the mycobacterium-specific gamma delta TCR repertoire. Mononuclear cells were isolated from the peripheral blood and pleural fluid of patients with tuberculous pleuritis and stimulated with Mycobacterium tuberculosis in vitro. Cytofluorometric analysis of the expressed gamma delta TCR repertoire of M. tuberculosis expanded cells was performed using anti-V region antibodies. The majority of responding gamma delta T cells express a receptor composed of V delta 2 and V gamma 9 chains. Molecular analysis by PCR amplification confirmed use of the V delta 2 and V gamma 9 gene segments in these cells, and demonstrated predominant usage of J delta 1 and J gamma P gene segments. Analysis of nucleotide sequence at the V-J junctions revealed extensive diversity including nucleotide deletions of V, D, and J gene segments and nucleotide segment additions. The predicted amino acid sequences further indicates diversity in the V-J encoded region of the protein chains. The data indicate that M. tuberculosis-driven expansion of gamma delta T cells in vitro depends on specific pairing of the V delta 2 and V gamma 9 polypeptide chains, without apparent selection of explicit V-J junction regions.

Effects of aminoglutethimide on hypothalamic-pituitary-adrenal functions.

This study was performed on four groups of subjects, including 10 patients with Cushing's disease, 10 patients with simple obesity, 8 patients with hypopituitarism and 13 normal subjects. The study was conducted by measuring the sequential changes of plasma ACTH, serum cortisol, 24-h UFC, 24-h 17 KS and 24-h 17 KGS following aminoglutethimide (AG) administration. The results suggest that normal subjects showed sequential changes of hypothalamic-pituitary-adrenal hormone concentrations with normal feedback regulation of the axis following AG administration. Patients with Cushing's disease had obvious autonomy in the production of ACTH from the pituitary. Patients with simple obesity might display abnormality to some degree in the production from the pituitary. Patients with hypopituitarism lost the capacity of ACTH production in various degrees because of pituitary lesions.

Transformation of cultured human fetal gastric fibroblasts with physico-chemical agents.

Treatment of early passage human fetal gastric fibroblasts with ultraviolet (UV) light and the chemical carcinogen ethyl nitrosourea (ENU) in succession resulted in an immortally growing cell line, named GTS 8502. The cells of this line display typical transformation characteristics, such as irregularly shaped nuclei, heteroploidization of karyotype and frequent appearance of heteromorphic chromosomes, the enhanced volume ratio of nucleus to cytoplasm, multinucleoli, appearance of microvilli on the surface of the cells and agglutination reaction to lectin concanavalin A. The transformants have high growing and mitotic indices and the ability of focus-formation on monolayers and anchorage independent growth in soft agar medium. Moreover, these cells induced tumours in nude mice or in immunosuppressed new-born rats through heterotransplantation. The results of various methods including electromicroscopy and histochemical analyses indicate that GTS 8502 cells are of fibroblast origin. Our results thus indicate that synergism of two carcinogens may raise the transformation ratio of normal human cells, which is otherwise extremely low. The transformation of human cells may be utilized to detect environmental carcinogens.