Background and Purpose-In a murine model of stroke, we identified a population of very small embryonic-like (VSEL) stem cells (SCs) in adult murine bone marrow that could be mobilized into peripheral blood (PB). This raised the question of whether a similar population of cells is mobilized in human stroke patients. Methods-We evaluated a number of cells that corresponded to VSEL SCs in the PB of 44 stroke patients and 22age-matched controls. After each patient's stroke, PB samples were harvested during the first 24 hours, on day ϩ3, and on day ϩ7 and then compared with normal controls. The circulating human cells with the phenotype of VSEL SCs were evaluated in PB by real-time quantitative polymerase chain reaction, fluorescence-activated cell sorting analysis, and direct immunofluorescence staining. In parallel, we also measured the serum concentration of stromal derived factor-1 by ELISA. Results-In stroke patients, we found an increase in the number of circulating cells expressing SC-associated antigens, such as CD133, CD34, and CXCR4. More important, we found an increase in the number of circulating primitive cells expressing the VSEL phenotype (CXCR4

Transcriptional control of marginal zone (MZ) and follicular (FO) B cell development remains incompletely understood. The transcription factor, IFN regulatory factor (IRF)8, is known to play important roles in the differentiation of early B cells. In this article, we demonstrate that IRF8 is also required for normal development of MZ and FO B cells. Mice with a conventional knockout of Irf8 (IRF8–/–) or a point mutation in the IRF association domain of IRF8 had increased numbers of MZ B cells. To determine the B cell-intrinsic effects of IRF8 deficiency, we generated mice with a conditional allele of Irf8 crossed with CD19-Cre mice (designated IRF8-conditional knockout [CKO]). These mice had enlarged MZ and increased numbers of MZ and FO B cells compared with controls. The FO B cells of CKO mice exhibited reduced expression of CD23 and moderately increased expression of CD21. Gene-expression profiling showed that increased B cell production in IRF8-CKO mice was associated with changes in expression of genes involved in regulation of transcription, signaling, and inflammation. Functional studies showed that IRF8-CKO mice generated normal Ab responses to T-independent and T-dependent Ags. Thus, IRF8 controls the expansion and maturation of MZ and FO B cells but has little effect on B cell function.

IntroductionLocalization and retention of B cells in the marginal zone (MZ) depends on antigenic specificity, 1,2 interactions between integrins and their receptors, 3 and signaling by the lysophospholipid, S1P, through the S1P 1 receptor. 4 Although MZ B cells are considered nonrecirculatory, exposure to cognate Ag or bacterial products causes them to relocalize from the MZ to the splenic white pulp, 4 providing for efficient delivery of captured Ags to follicular dendritic cells. 5 Although no direct evidence links chemokine involvement to MZ B-cell localization and retention, the MZs are strikingly reduced or absent in mice deficient in a series of factors that may act downstream of chemokine receptors. 6 Recent studies, however, showed that transcripts for the chemokine receptor, CXCR7, were expressed at much higher levels by mouse MZ than follicular (FO) B cells. 7-9 CXCR7 has variously been reported to function as the primary receptor and scavenger for CXCL12, 8,10,11 a modulator of CXCR4 activity, or to act directly on different tumor types. 11,12 This prompted us to determine whether CXCR7 and its ligands, CXCL11 and CXCL12, 11,13 might influence MZ B-cell biology by taking advantage of recently developed CXCR7-specific mAbs and small molecule inhibitors. We found that CXCR7 was expressed on MZ but not on FO B cells and that treatment with inhibitors disrupted the normal organization of the MZ, altered MZ B-cell numbers, and increased serum levels of CXCL12, resulting in altered homeostasis of granulocytes. Methods Flow cytometrySplenic single-cell suspensions were blocked with a Fc receptor-blocking mAb, 2.4G2, and stained with fluorochrome-labeled Abs specific for B220, CD19, CD23, CD21, Gr-1, CD3, CD11b, ␣L, ␣4, ␤1, and ␣4␤7 (purchased from BD Biosciences or eBioscience). In some experiments, cells were stained with anti-CXCR7 mAb (11G8; provided by ChemoCentryx) followed by FITC-conjugated secondary Ab. An isotype control Ab was included as a negative control. The cells were analyzed by FACSCalibur or LSRII (BD Biosciences), and the data were analyzed by FlowJo Version 7.5.5 software (TreeStar). Animals, treatment, serum CXCL12, and histologic analysisFVB and B6 mice (2-6 months old) were purchased from The Jackson Laboratory. FVB mice have unusually large MZs, 14 whereas B6 mice are functionally deficient in CXCL11 because of a deletion in the coding sequence. 15 Mice were injected subcutaneously with CXCR7 antagonists CCX754 (100 mg/kg twice daily) or CCX771 (30 mg/kg daily; provided by ChemoCentryx) for 2-7 days. The specificity, half-life, and effect of the inhibitors have been reported previously. 11,16,17 Control mice were injected with equal volumes of vehicles (10% Captisol). The spleens were analyzed by flow cytometry and confocal microscopy, as described previously, with the use of labeled mAb to IgM, metallophilic macrophages (MOMA-1), and marginal sinus epithelial cells (MAdCAM-1). 18 The slides were imaged at 20°C with the use of ϫ10 and ϫ20 oil-immersion lenses on a Leica SP2 4-chanel c...

Primary malignant melanoma of esophagus is very rare, and its clinicopathologic and genetic features have not been extensively investigated. In this study, 20 tumors from 14 male and 6 female patients (40-79 years old) were evaluated. Dysphagia, chest pain, and weight loss were frequent symptoms. Thirteen melanomas, including two with multiple lesions, involved the distal third of esophagus. The median tumor diameter was 6 cm. Epithelioid morphology, moderate atypia, and pigmentation were typical findings. None of the patients had melanoma elsewhere, and all tumors exhibited a junctional periepithelial component consistent with a primary lesion. The median mitotic activity was 11 per 10 high-power fields (range, 0-31). Nine patients died of tumor within 4-22 months, however, two showed long-term (96 and 104 months) survival. In 15 cases, tissue for further immunohistochemical and molecular studies were available. BRAF, KIT, and NRAS mutation status was assessed by Sanger sequencing in all 15 tumors. The next-generation sequencing of 50 or 409 genes was performed in five and three cases, respectively. IGF1R expression indicating activation of the IGF axis was seen in 82% (9/11) of tumors. However, no BRAF mutations were identified. In 33% (5/15) of tumors, NRAS mutations were detected. KIT expression was seen in 50% (7/14) of melanomas including single KIT mutant. Two of three tumors evaluated with 409 genes panel revealed multiple driver mutations indicating sub-clonal expansion, whereas a single mutation (TSC1 p.H371Q) was the sole change in the third case. SF3B1 p.K666T and p.R625C mutations were detected in two cases. However, no cooccurrence of SF3B1 and GNAQ or GNA11 mutations, seen in uveal melanoma, was detected. FBXW7 p.R465C and p. R479G mutations, linked to cancer progression, were found in two of eight tumors. In summary, esophageal melanoma mutation profile indicates complexity of molecular mechanisms underlying its pathogenesis.

(Print ISSN 1478-6354; Online ISSN 1478-6362). This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. AbstractClonal expansion of CD4 + CD28 -T cells is a characteristic finding in patients with rheumatoid arthritis (RA). Expanded CD4 + clonotypes are present in the peripheral blood, infiltrate into the joints, and persist for years. CD4 + CD28 -T cells are oligoclonal lymphocytes that are rare in healthy individuals but are found in high percentages in patients with chronic inflammatory diseases. The size of the peripheral blood CD4 + CD28 -T-cell compartment was determined in 42 patients with RA and 24 healthy subjects by two-color FACS analysis. The frequency of CD4 + CD28 -T cells was significantly higher in RA patients than in healthy subjects. Additionally, the number of these cells was significantly higher in patients with extra-articular manifestations and advanced joint destruction than in patients with limited joint manifestations. The results suggest that the frequency of CD4 + CD28 -T cells may be a marker correlating with extra-articular manifestations and joint involvement.Keywords: arthritis, CD4 + CD28 -, lymphocytes Open AccessAvailable online http://arthritis-research.com/content/5/4/R210 R211In the present study we evaluated the correlation between the CD4 + CD28 -T-cell subset and extra-articular manifestations, magnitude of joint involvement, and presence of rheumatoid factor. Material and methods PatientsForty-two patients (26 women, 16 men, age 24-74 years, mean 51.7 years) with rheumatoid arthritis diagnosed according to the criteria of the American College of Rheumatology were included in the study. The disease duration was 4-19 years (mean 12.8 years). Patients were recruited from the outpatient and inpatient population of the Department of Rheumatology, University Hospital, Szczecin, Poland. All subjects were white and were from the Pomeranian region of Poland.

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The rarity of the coexistence of these two diseases results in a lack of effective therapy. In such cases sulfone derivatives are still effective and provide an alternative to standard immunosuppression methods. Hyperbaric therapy and plasmapheresis can also play an important complementary role.

The signal transducers and activators of transcription--STAT5A and STAT5B--take part in the regulation of many essential physiopathological processes. They influence the cell cycle, apoptosis and the proliferation of different types of cell lines. The STAT5 proteins are induced in response to multiple haematopoietic cytokines. Because they are constitutively active in certain haemato-oncologic diseases, it is also suggested that they play an important role in leukaemogenesis. However, function of these proteins in haematopoietic cell transformation and proliferation is not clear. The aim of this study was to evaluate the impact of perturbation of STAT5 expression [using oligodeoxynucleotide (ODN) against STAT5 mRNA], on the clonogenicity and survival of selected human leukaemic cell lines, HEL, HL-60, K562, TF-1. We analysed the effect of ODN pre-treatment on the cell clonogenicity in methylcellulose cultures according to the time and the temperature of exposure. Moreover, we attempted to estimate apoptosis induced in examined cells, by flow cytometry using combined Annexin V-PI staining and the TUNEL method. We also applied the RT-PCR method to analyse Bax and Bcl-xL gene expression. We found that the perturbation of STAT5 expression with antisense oligonucleotides caused a decrease in the proliferative potential of human K562 and TF-1 cell lines. Also, we observed higher induction of apoptotic cell death in the K562 and TF-1 cells incubated with the antisense STAT5A ODNs. We did not notice any impact of ODNs on the HL-60 and HEL cells. Our studies using STAT5 antisense oligonucleotides showed that these proteins may be critical in the regulation of growth and apoptosis of some types of leukaemic blasts.