Stavroula A. Coulocheri

Patients with chronic idiopathic neutropenia of adults have increased serum concentrations of inflammatory cytokines and chemokines.

Serum levels of inflammatory cytokines and chemokines were measured in 132 patients with chronic idiopathic neutropenia of adults (CINA) and 34 healthy volunteers (controls) using commercially available micro‐ELISA determination kits. We found that serum interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), interleukin‐6 (IL‐6), transforming growth factor‐β1 (TGF‐β1), and soluble tumor necrosis factor receptor p55 (sTNF‐RI) were all significantly increased in CINA patients compared to controls. Individual cytokine values inversely correlated with the number of circulating neutrophils. Serum levels of interleukin‐8 (IL‐8) and RANTES, two potent chemokines for neutrophils and lymphocytes, respectively, were also significantly increased in the group of patients and they inversely correlated with the number of circulating neutrophils. Contrarily, serum levels of interleukin‐4 (IL‐4), interferon‐γ (IFN‐γ), soluble CD23 (sCD23), and soluble interleukin‐2 receptor (sIL‐2R) did not show any significant change in the patients studied. We assume that CINA patients have increased serum concentrations of inflammatory cytokines and chemokines mainly produced by activated macrophages, while they disclose normal levels of inflammatory molecules mainly released from activated lymphocytes. These findings provide further evidence for an underlying low‐grade chronic inflammatory process in CINA patients, as we previously have suggested. If this chronic inflammation is really the cause of the disorder or it simply represents the result of neutropenia remains to be elucidated. Am. J. Hematol. 65:271–277.

The oestrogen receptor codon 10 polymorphism detected in breast cancer is also present in non-malignant cells

Abstract The effect of oestrogens on oestrogen-receptive organs and cells is mediated via intracellular receptors (ERα and ERβ). Oestrogen receptor gene polymorphisms in the region encoding the N-terminal portion of the protein are reportedly associated with pathological conditions including breast cancer, hypertension, spontaneous abortion and coronary heart disease. A silent mutation in codon 10 of exon 1, detected in ER-negative and ER-positive human breast cancer cell lines, in breast tumors and blood DNA from breast cancer patients, has been recognized as a polymorphic site. In this study we examined, by denaturing gradient-gel electrophoresis and DNA sequence analysis, the possible presence of a codon 10 polymorphic site in normal oestrogen target organs and cells such as the uterus (myometrium and endometrium), in the placenta and peripheral blood mononuclear cells and in a benign uterus tumour (leiomyoma). We have detected ER codon 10 polymorphism in these samples and have compared them to those observed in breast cancer samples. All tissues and cells studied were homozygous for the wild-type gene, and were heterozygous as well as homozygous for the codon-10-variant type. These results indicate that the presence of the codon-10-variant type is not a characteristic of breast cancer. Out current findings suggest that further investigations are warranted to elucidate the possible linkage of ER codon 10 polymorphism to physiological and pathological conditions.

Mutation detection of the human glucocorticoid receptor alpha gene area coding for the hormone-binding domain by denaturing gradient gel electrophoresis.

Mutations in the hormone-binding domain of the human glucocorticoid receptor alpha (hGRalpha) gene have been detected in a variety of glucocorticoid resistance syndromes. Using the denaturing gradient gel electrophoresis technique, we developed a sensitive method for the detection of alterations in the gene area coding for the whole hormone-binding domain and part of the DNA-binding domain of the hGRalpha. This method can be applied for screening of glucocorticoid receptor gene alterations in glucocorticoid-dependent diseases.

Application of denaturing gradient gel electrophoresis (DGGE) to screen for mutations of the human glucocorticoid receptor α gene (hGRα)

Abstract Objectives In a previous publication, we had presented a sensitive method to detect mutations of the segment of the human glucocorticoid receptor alpha (hGRα) gene encoding the ligand binding domain (LBD) and part of the DNA binding domain (DBD) of hGRα, as several types of glucocorticoid resistance syndromes have been correlated with mutations in the respective nucleotide sequences. However, mutations affecting various regions covering the whole length of hGRα are increasingly reported in a variety of disease states. We now present an expanded screening methodology to detect mutations covering the whole length of hGRα. Design and methods We developed a sensitive, simple screening PCR-DGGE method to detect mutations in the aminoterminal domain and DNA-binding domain of the hGRα. Wild type hGRα cDNA and mutant samples were included in the analysis to ensure the accuracy and sensitivity of the method. Results The PCR-DGGE method identified the mutant samples and discriminated them from wild type hGRα. Conclusions The method described is accurate, sensitive, simple, cheap and fulfills the critera for a screening method which will be useful in delineating possible involvement of hGRα mutations in the aetiopathology of diseases correlated to derangements of glucocorticoid action.