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BACKGROUND Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in the homeostasis of mucosal immunity; however, their role in inflammatory bowel disease (IBD) is unclear. METHODS Flow cytometry was used to enumerate peripheral blood MAIT cells in 88 patients with ulcerative colitis (UC), 68 with Crohn's disease (CD), and in 57 healthy controls. Immunohistochemistry identified MAIT cells in intestinal tissue samples from patients with UC (n = 5) and CD (n = 10), and in control colon (n = 5) and small intestine (n = 9) samples. In addition, expression of activated caspases by MAIT cells in the peripheral blood of 14 patients with UC and 15 patients with CD, and 16 healthy controls was examined. RESULTS Peripheral blood analysis revealed that patients with IBD had significantly fewer MAIT cells than healthy controls (P < 0.0001). The number of MAIT cells in the inflamed intestinal mucosae of patients with UC and CD was also lower than that in control mucosae (P = 0.0079 and 0.041, respectively). The number of activated caspase-expressing MAIT cells in the peripheral blood of patients with UC and CD was higher than that in healthy controls (P = 0.0061 and 0.0075, respectively), suggesting that the reduced MAIT cell numbers in IBD are associated with an increased level of apoptosis among these cells. CONCLUSIONS The number of MAIT cells in the peripheral blood and inflamed mucosae of patients with UC and CD was lower than that in non-IBD controls. Also, MAIT cells from patients with IBD exhibited proapoptotic features. These data suggest the pathological involvement and the potential for therapeutic manipulation of these cells in patients with IBD.

Ichthyosiform mycosis fungoides (MF) is a rare variant of MF, occurring in 3.5% of MF patients according to one study (1, 2). Patients with ichthyosiform MF exhibit clinical manifestations similar to those of acquired ichthyosis; histologically, however, they show epidermotropism of atypical lymphocytes. Ichthyosiform MF is characterized by indolent course and usually good response to phototherapy. Even among patients with conventional MF, dual CD4/CD8-positive MF is extremely rare, in contrast to a relatively high incidence of a mixture of CD4-positive neoplastic cells and inflammatory CD8positive infiltrating lymphocytes. We report here a case of ichthyosiform MF with aggressive extracutaneous involvement, including cardiac involvement, with a dual CD4/CD8-positive immunophenotype.

Erythropoiesis requires large amounts of iron for hemoglobin synthesis, which is mainly provided by macrophages and the intestines in a transferrin (Tf)-bound form. Bone marrow erythroblasts incorporate Tf through endocytosis, which is mediated by transferrin receptor 1 (TFR1). Recently, human TFR1, aside from its role as a Tf receptor, was also found to be a receptor for the H-subunit of ferritin (FTH). In humans, hematopoietic erythroid precursor cells express high levels of TFR1 and specifically take up the FTH homopolymer (H-ferritin). H-ferritin inhibits the formation of burst forming unit-erythroid colonies in vitro. TFR2, which is also a Tf receptor, is predominantly expressed in hepatocytes and erythroid precursor cells. In the liver, TFR2 forms a complex with HFE, a hereditary hemochromatosis-associated protein, and acts as an iron sensor. In mice, hepatocyte-specific knockout of the TFR2 gene has been shown to cause systemic iron-overload with decreased expression of hepcidin, the central regulator of iron homeostasis. In erythroid cells, TFR2 forms a complex with the erythropoietin receptor and facilitates its trafficking to the cell membrane. Moreover, hematopoietic cell-specific knockout of the TFR2 gene causes microcytic erythrocytosis in mice. This review focuses on the molecular evolution and functions of these TFRs and their ligands.

CD2 is an adhesion molecule present on the cell surface of T and natural killer (NK) cells, and its interaction with CD58 on antigen-presenting cells plays an important role in their immune reaction. Downregulation of CD58 is a frequent mechanism for immune escape in hematological malignancies, whereas there are very few reports that decreased CD2 expression in immune cells is associated with tumor development. We report here a patient who developed Epstein-Barr virus-associated lymphoproliferative disorder (EBV-LPD) along with diminished CD2 expression in T and NK cells. The patient exhibited severely decreased numbers of peripheral T cells and with Th2 cell-biased cytokine production. Although EBV-LPD was refractory to chemotherapy, the patient was treated successfully with allogeneic stem cell transplantation from a donor with normal CD2 expression. It is suggested that CD2-CD58 interactions play a critical role in the anti-tumor immune response, and restoration of this signaling is considered to be an important strategy for anti-tumor therapy when this signaling is blocked.

Previous reports have shown that circulating endothelial progenitor cells (CEPs) are released in response to cytotoxic chemotherapy. We investigate the relationship between the kinetics of CEPs during one cycle of chemotherapy and the response to cytotoxic chemotherapy and prognostic impacts. Previously untreated patients (n = 38) receiving cytotoxic chemotherapy for non‐small‐cell lung cancer were included. Blood sampling was carried out on day 1, day 8, and just before the second cycle of chemotherapy. The mononuclear cell fraction was analyzed for CEPs by FACS analysis. We evaluated the relationship between the kinetics of CEPs, each independent clinicopathological variable, the response to chemotherapy, and the risk factors associated with prognosis. On the eighth day after chemotherapy, a significant decrease in CEPs was observed. In contrast, CEP counts before the second cycle of chemotherapy were significantly increased. The high percentage change in CEPs between day 1 and before the second cycle of chemotherapy is an independent predictive factor for response to chemotherapy. However, the change in CEP levels did not predict progression‐free survival. These findings indicate that the late release of CEPs is a common phenomenon after chemotherapeutic treatment. The correlation with clinical response to chemotherapy provides further support for the biologic relevance of these cells in patients' prognosis and highlights the potential use of CEPs as therapeutic targets. (Cancer Sci 2012; 103: 1065–1070)

Reversion mosaicism is increasingly being reported in primary immunodeficiency diseases, but there have been few cases with clinically improved immune function. Here, a case is reported of X-linked severe combined immunodeficiency (SCID-X1) with multiple somatic reversions in T cells, which restored sufficient cell-mediated immunity to overcome viral infection. Lineage-specific analysis revealed multiple reversions in T cell receptor (TCR) αβ+ and TCRγδ+ T cells. Diversity of the TCRVβ repertoire was comparable to normal and, furthermore, mitogen-induced proliferation of the patient's T cells was minimally impaired compared to healthy controls. In vivo and in vitro varicella antigen-specific T cell responses were comparable to those of healthy controls, although a reduced level of T cell receptor excision circles suggested that recent thymic output was low. During long-term evaluation of the patient's immunologic status, both the number of CD4+ and CD8+ T cells and T cell proliferation responses were stable and the patient remained healthy. This case demonstrates that multiple but restricted somatic reversions in T cell progenitors can improve the clinical phenotype of SCID-X1.

B7-H1 is a member of the B7 family that inhibits the function of T-cells through its receptor programmed death-1 (PD-1). We examined B7-H1 expression in anaplastic large cell lymphoma (ALCL) and Hodgkin lymphoma (HL) and found that it was constitutively expressed in both clinical samples and cell lines. In anaplastic lymphoma kinase-positive (ALK(+)) ALCL cells, B7-H1 expression was suppressed by the blocking of extracellular signal-regulated kinase (ERK) signaling and upregulated by the augmentation of ERK activity by phorbol 13-myristate 12-acetate stimulation, suggesting that B7-H1 expression is regulated by ERK signaling pathway in ALCL. ERK is one of the downstream mediators of nucleophosmin (NPM)/ALK signaling in ALK(+)ALCL, and pharmacological inhibition of ALK was shown to dephosphorylate ERK and down-regulate B7-H1. The involvement of NPM/ALK in B7-H1 expression was also demonstrated by introducing the construct into human non-ALCL lymphoid cell lines, which resulted in B7-H1 expression. In the case of HL, B7-H1 expression was shown to be dependent on the ERK and p38 mitogen-activated protein kinase (MAPK) signaling pathways. These results suggest that B7-H1 expression is controlled by common ERK signaling pathways in ALCL and HL cells. Our findings provide a potentially effective immunotherapeutic strategy for these B7-H1-expressing tumors.

Programmed death-1 (PD-1)–PD-1 ligand (PD-L) signaling system is involved in the functional impairment of T cells such as in chronic viral infection or tumor immune evasion. We examined PD-L expression in lymphoid cell lines and found that they were up-regulated on Hodgkin lymphoma (HL) and several T-cell lymphomas but not on B-cell lymphomas. PD-L expression was also demonstrated in primary Hodgkin/Reed-Sternberg (H/RS) cells. On the other hand, PD-1 was elevated markedly in tumor-infiltrating T cells of HL, and was high in the peripheral T cells of HL patients as well. Blockade of the PD-1 signaling pathway inhibited SHP-2 phosphorylation and restored the IFN-γ–producing function of HL-infiltrating T cells. According to these results, deficient cellular immunity observed in HL patients can be explained by “T-cell exhaustion,” which is led by the activation of PD-1–PD-L signaling pathway. Our finding provides a potentially effective immunologic strategy for the treatment of HL.

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body's iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.

We previously identified the carbohydrate determinant sialyl 6-sulfo Lewis X (Le(x)) as the major L-selectin ligand on high endothelial venules of peripheral lymph nodes. In this study, we examined the distribution of the sialyl 6-sulfo Le(x) determinant among peripheral lymphocytes. The determinant was expressed on a subset of helper memory T and NK cells. The helper memory T cells expressing sialyl 6-sulfo Le(x) were CD45RO(bright+) PSGL-1(high+) CCR4+ L-selectin+ CCR7+ but did not express alpha4beta7 integrin or CCR9, indicating that they were the skin-homing population of central memory T cells. The T-cell subset significantly expressed mRNA for 6-sulfotransferase HEC-GlcNAc6ST and fucosyltransferase Fuc-T VII, responsible for the synthesis of sialyl 6-sulfo Le(x). Characteristics of the T-cell population were similar to those previously described for cutaneous lymphocyte-associated antigen (CLA)-positive T cells defined by the HECA-452 or 2F3 antibody. The binding of the T-cell subset with the specific anti-sialyl 6-sulfo Le(x) antibody G152 was almost completely abrogated by HECA-452 or 2F3. Binding of recombinant E-, P-, and L-selectins to the T-cell subset was significantly inhibited by G152 and by HECA-452 antibodies. We propose that CLA, which is expressed without any activation stimuli on peripheral skin-homing helper memory T cells in healthy persons, is at least partly the sialyl 6-sulfo Le(x) determinant.

Chronic infantile neurologic, cutaneous, articular syndrome (CINCA syndrome) is a severe inflammatory disease that was recently found to be associated with mutations in CIAS1. However, CIAS1 mutations have been detected in only half of CINCA syndrome patients, and it remains unclear which genes are responsible for the syndrome in the remaining patients. We describe here a patient with CINCA syndrome who exhibited CIAS1 somatic mosaicism. We genetically analyzed the CIAS1 gene in various blood cells and the buccal mucosa of the patient. The production of interleukin-1β (IL-1β) by peripheral blood mononuclear cells (PBMCs) was measured by enzyme-linked immunosorbent assay, and the ability of the mutant CIAS1 gene to enhance ASC-dependent NF-κB activation was assessed to confirm that the mutations of CIAS1 found were responsible for the patient's clinical manifestations of the CINCA syndrome. The patient had 1 heterologous single-nucleotide polymorphism, 587G>A (S196N), and 1 heterologous mutation, 1709A>G (Y570C), in exon 3 of CIAS1. The latter mutation was found to occur as somatic mosaicism. The patient's PBMCs produced a large amount of IL-1β in the absence of stimulation, unlike those from controls or from his mother, who also bore the S196N polymorphism. In addition, the Y570C mutation (with or without the S196N polymorphism) increased the ability of CIAS1 to induce ASC-dependent NF-κB activation, unlike the wild-type gene or the gene bearing the S196N polymorphism alone. The findings in this patient indicate that somatic mosaicism is one reason CIAS1 mutations have not been detected in some patients with CINCA syndrome.

This retrospective study examined the clinical significance of FCM-MRD in 36 patients with ALL and 29 patients with AML after their first allogeneic HSCT. Hematological (FCM-MRD ≥5.0%) and molecular relapse (FCM-MRD

Stevens-Johnson syndrome (SJS) is a rare but extremely severe drug-induced eruption characterised by widespread necrosis or apoptosis of the epidermis (1). The main causal factor for SJS is an aberrant activation of CD8 T cells. Although the dysfunction of the regulatory T cells (Treg) is considered a possible cause of excess CD8 T cells activation (2), the actual significance of Tregs in vivo remains unknown.

Several sialyl sulfoglycans in human cells and tissues have recently been found to serve as specific ligands for carbohydrate-recognition molecules in the selectin and siglec families and are expected to play crucial roles in the regulation of immune cell functions in health and diseases. One such sialyl sulfoglycan, sialyl 6-sulfo Lewis X, can serve as a ligand for all members of the selectin family and siglec-7. This glycan was known to be expressed in skin-homing central helper memory T-cells which exhibited bidirectional homing behavior to the skin and peripheral lymph nodes, and was recently found to be involved in the pathophysiology of atopic dermatitis. The binding activity of this glycan to selectins and siglecs is regulated by cyclization of the terminal sialic acid residue, and examination of T-cell subsets expressing sialyl 6-sulfo Lewis X with normal and cyclic sialic acid residues in peripheral blood of the patients is proposed to be clinically useful in evaluating the risk of atopic dermatitis recurrence.

We describe the establishment and characterization of a new multiple myeloma (MM) cell line, KYdelta-1, which expressed delta/kappa type immunoglobulin (Ig). The patient was a 65-year-old woman with MM, who presented extramedullary dissemination, lymphadenopathy and short survival. The KYdelta-1 cell line was derived from the pleural fluid obtained in the terminal phase of the disease. The cells expressed delta/kappa Ig in the cytoplasm, and CD10, CD29, CD33, CD38, CD44, CD54, and HLA-DR antigens on the cell surface. Chromosomal analysis revealed two independent translocations, t(3;14)(p21;q32) and t(3;11)(p21;q13), which were confirmed by fluorescence in situ hybridization using chromosome painting probes. Reverse transcriptase-mediated polymerase chain reaction (PCR) and Northern blot analyses demonstrated overexpression of the CCND1 gene, suggesting alteration of the BCL1-CCND1 locus. We thus performed long-distance inverse PCR using nested primers for the Calpha constant region of immunoglobulin heavy chain gene (IGH) and obtained a clone that encompassed the 11q13/IGH fusion. Nucleotide sequencing determined that the fusion occurred at the Salpha2 switch region and at the centromeric side of the major translocation cluster of BCL1. The other IGH allele consisted of a VDJ complex that was adjacent to the Cdelta constant gene, indicating that a class switch-like mechanism from the C(mu) to Cdelta was involved in the production of the Ig delta heavy chain. Point mutations within the P53 and N-RAS genes were presumably related to the rapidly progressive disease in this particular MM patient.

The clinicopathological features of 10 patients with primary mediastinal large B-cell lymphoma (PMLBCL) are described. The patients were aged 19 to 63 years, with a median age of 25.5 years. There were 5 men and 5 women. All patients presented with chest symptoms, and 6 presented with superior vena cava syndrome. Nine patients had bulky mediastinal tumors. The disease was confined within the thorax and contiguous lymph nodes, although multiple liver tumors were observed in 1 patient. Laboratory findings included high lactate dehydrogenase levels and elevated C-reactive protein levels. The soluble interleukin 2-receptor level was high in 6 patients tested. A comparative study of PMLBCL and nodular sclerosis-type Hodgkin's disease (NS-HD) with a mediastinal mass revealed substantial overlap in clinical features. Histopathological examination of biopsy specimens of PMLBCL revealed clusters of CD20+ large cells; however, CD30+ Hodgkin/Reed-Sternberg-like cells were occasionally seen, raising the potential to misdiagnose PMLBCL as NS-HD. The patients with PMLBCL were treated with CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisolone), biweekly CHOP, or MACOP-B (methotrexate, doxorubicin, cyclophosphamide, vincristine, prednisolone, and bleomycin) regimen, and 6 received consolidation radiotherapy to the involved field. With the exception of 1 patient who was primarily refractory to therapy, 9 patients (90%) achieved complete response and 7 (70%) remain in continuous remission with a mean follow-up of 24 months.

SUMMARYCD8 deficiency is a rare primary immunodeficiency caused by the defect of a tyrosine kinase, ZAP-70, which transduces signals from the T cell receptor. We report here a case of CD8 deficiency, having CD4+ T cells with a unique phenotype. The patient's T cells did not respond to anti-CD3 stimulation in vitro, suggesting that they were naive. However, many CD4+ T cells with activated and memory phenotypes, which expressed CD45RO+, HLA-DR+ and CD25+, were present in the peripheral blood, and these cells accumulated in the perivascular area of his infiltrative erythematous skin lesions. The patient's T cells could be activated by a high concentration of phytohaemagglutinin (PHA), indicating the presence of an alternate signalling pathway which bypasses ZAP-70 and activates CD4+ T cells in vivo. The origin and role of activated CD4+ T cells in the pathogenesis involved in the skin lesions are discussed.

Durable remission of large B-cell lymphoma transformed from lymphoplasmacytic lymphoma/Waldenstrom macroglobulinemia successfully treated with sequential immunochemotherapy followed by reduced-intensity allogeneic stem cell transplantation

Liver cirrhosis, which is associated with decreased plasma and hepatic glutathione (GSH), has been reported to cause the suppression of NK activity in peripheral blood mononuclear cells. Since low GSH levels in lymphocytes are known to alter lymphocyte function, we examined the correlation between intracellular GSH levels and the cytotoxic activity of liver-associated mononuclear cells (liver MNC). We show here that rat liver contains a highly active population of NK cells (CD3 - NKR-P1 F cells) that kill Yac-1 in vitro and that the cytotoxic activity of this NK population is directly proportional to liver MNC GSH. This proportionality is independent of the methods used to alter GSH level. Thus, in vitro treatment of liver MNC with buthionine sulfoximine to lower GSH levels lowers the cytotoxic activity. MNC from cirrhotic liver, in which implanted tumor cells grow faster, have both low GSH levels and low cytotoxicity, and supplementation of cirrhotic liver MNC with N-acetylcysteine raises GSH levels and increases cytotoxicity. These findings suggest a physiologic mechanism, i.e. decreased GSH, may be causally associated with the increased incidence of hepatoma in cirrhotic individuals and the increased growth of hepatoma cells in cirrhotic animals. Thus, we suggest that the GSH is important to the optimal functioning of the hepatic immunity that protects against hepatoma development.

We have proposed the "vascular natriuretic peptide system", in which C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, can control vascular tone and growth as an endothelium-derived relaxing peptide. We aimed at overexpression of CNP gene in vascular smooth muscle cells (SMCs) by adenovirus-mediated gene transfer to examine the growth characteristics of SMCs via the augmentation of cGMP production. Rat aortic SMCs infected with Ad.CNP, a replication-deficient adenovirus driving rat CNP cDNA, produced 162 +/- 55 fmol/mL of CNP, which was 4,000 times higher than that produced by endothelial cells. cGMP production was also augmented in Ad.CNP-infected SMCs (2200 +/- 270 fmol/10(4) cells). Accordingly, significant growth inhibition was observed in SMCs infected with Ad.CNP. The flow cytometry analysis revealed that the population of the S and G2 + M phases was reduced by 60% of the control in Ad.CNP-infected SMCs. The gene expression of ANP-B receptor, which is expressed abundantly in SMCs with the synthetic phenotype, was suppressed in Ad.CNP-infected SMCs, while the gene expression of ANP-A receptor, which is expressed predominantly in SMCs with the contractile phenotype, became detectable in Ad.CNP-infected SMCs. In addition, the gene expression of smooth muscle myosin heavy chain-2 (SM-2), which is the molecular marker of highly-differentiated SMCs, was also induced in Ad.CNP-treated SMCs. These results suggest that cGMP cascade activation induces re-differentiation of SMCs. The present study demonstrated that overexpression of CNP induced growth inhibition of SMCs at the G1 phase with possible alteration of the phenotype.

Background/Aims: We previously reported that the populations of lymphocytes and the expression of activated antigens in human sinusoidal mononuclear cells were different from those in peripheral blood mononuclear cells. Attempts to culture these cells for further study failed because they died rapidly under standard culture conditions in vitro after isolation from the liver. In this study, we evaluated the characteristics of cell death and the effects of various culture conditions on the viability of these cells. Methods: Sinusoidal mononuclear cells were isolated from University of Wisconsin solution that had been perfused through the portal veins of normal healthy human livers harvested for transplantation into living related recipients. Results: 70% of sinusoidal mononuclear cells cultured in in vitro were nonviable within 48 h after isolation, while only 10% of peripheral blood mononuclear cells died under the same conditions. Sinusoidal mononuclear cells showed DNA ladder formation of DNa on electrophoresis and characteristic morphological pattern on electron microscopic examination that suggested they had died in an apoptotic manner. The addition of human liver extracts or 2-mercaptoethanol and reduced glutathione to the cultures rescued the sinusoidal mononuclear cells from apoptosis. Furthermore, diamide, a sulfhydryl group specific oxidant negated the effect of the liver extract. Conclusion: In comparison with peripheral blood mononuclear cells, human sinusoidal mononuclear cells were more subject to death by apoptosis ex vivo , which was reversed by exogenous agents producing reducing conditions. These results suggested that hepatic sinusoidal mononuclear cells might express a different sensitivity to redox environment than peripheral blood mononuclear cells.

Adhesion of cancer cells to vascular endothelium is an important step in haematogenous metastasis of cancer. A human hepatocellular carcinoma cell line, HepG2, strongly adheres to human umbilical vein endothelial cells (HUVECs) through the interaction of E-selectin and its carbohydrate ligand sialyl Lewis X. In this study, we investigated alteration in integrin expression on HepG2 cells, which follows the selectin-mediated initial adhesion of HepG2 cells to HUVECs. Expression of alpha2beta1 integrin was markedly increased when the HepG2 cells adhered to HUVECs. Among the tested cytokines that are known to be produced by endothelial cells, recombinant hepatocyte growth factor (rHGF) could replace the effect of HUVECs, and a similar increase in integrin expression was observed by the addition of 20 ng ml-1 rHGF to HepG2. The increment of alpha2beta1 integrin expression was significantly inhibited by anti-HGF neutralizing antibody treatment. HepG2 cells expressed alpha2, alpha6, beta1, and beta4 integrin subunits, but expression of integrins other than alpha2beta1 was not affected by the rHGF treatment. The rHGF treatment of HepG2 cells resulted in augmented adhesion to immobilized collagen. This augmentation in adhesion to collagen was completely blocked by the addition of anti-alpha2- or anti-beta1-integrin antibody. In double-chamber chemoinvasion experiments, transmigration of the HepG2 cells through extracellular matrix (ECM) gel was significantly accelerated by co-cultivation with HUVECs. A similar level of enhancement in transmigration activity of the cancer cells was observed by the addition of rHGF. Our interpretation of the results described above is that the cancer cells received stimulation from cytokines, such as HGF, presented by vascular endothelial cells, following the initial adhesion of cancer cells via selectins. This resulted in the secondary increment in the expression of cell adhesion molecules, such as the alpha2beta1 integrin, and led to the augmented adhesive activities of cancer cells towards extracellular matrices at vascular walls. We suggest that this sequence of events is involved in the facilitated migration of some cancer cells to extravascular tissues.

The antisense cDNA approach was used to identify the endogenous fucosyltransferase species responsible for synthesis of the sialyl Lewis X (NeuAcalpha2--3 Galbeta1--4[Fucalpha1--3]GlcNAcbeta1--R) determinant in human lymphoid cells. The cultured human adult T-cell leukemia cell line, ED40515-N, expressed the message of alpha1--3 fucosyltransferase (Fuc-T) IV and VII, with a low level of the Fuc-T III and VI message, and manifested the sialyl Lewis X as well as Lewis X (Galbeta1--4 [Fucalpha1--3]GlcNAcbeta1--R) determinant at the cell surface. Transfection of this cell line with the pRc/CMV vector containing an antisense human Fuc-T VII construct (pRc/CMV/5'FT7AS) resulted in a significant decrease of endogenous Fuc-T VII message and a marked reduction in the cell surface expression of sialyl Lewis X determinant as well as a reduction in the enzymatic activity of alpha1--3 fucosyltransferase against sialylated type 2 chain substrate. This was accompanied by diminution of cell adhesive activity toward E-selectin on interleukin-1beta-treated endothelial cells. These results indicated that the synthesis of the sialyl Lewis X determinants that were functionally active as E-selectin ligands was mainly mediated by Fuc-T VII in these lymphoid cells. On the other hand, the message of Fuc-T IV showed no significant change in the transfectant clones, and the surface expression of the Lewis X antigen as well as the enzymatic activity of alpha1--3 fucosyltransferase against non-sialylated type 2 chain substrate was well preserved. The clear contrast between the diminished expression of sialyl Lewis X and the conserved manifestation of Lewis X in the transfectant clones suggested that the synthesis of sialyl Lewis X and that of Lewis X are independently regulated by different fucosyltransferases in human lymphoid cells. Fuc-T VII must be involved in the synthesis of sialyl Lewis X, while the synthesis of Lewis X is mediated by an enzyme other than Fuc-T VII, most probably Fuc-T IV.

Although it is well established that the addition of 1,25- dihydroxyvitamin D3 (D3) to the culture of normal human granulocyte/macrophage progenitors induces monocyte/macrophage (Mo/M phi) colonies, the target cells of D3 in the Mo/M phi differentiation have not been identified. We examined whether neutrophilic promyelocytes are the target cells. As a source of the promyelocyte fraction, we used colonies after 5 days of culture (5-day colonies) of colony-forming unit-granulocyte. The culture contained granulocyte colony-stimulating factor (G-CSF) as the growth factor and generated only neutrophilic colonies. The promyelocytic nature of the 5-day colonies was confirmed morphologically, cytochemically, and ultrastructurally. After morphological evaluation on part of the individual colonies, they were transferred into new semisolid cultures with or without D3 (10(-7) mol/L) in the presence of G-CSF, then incubated for the subsequent 7 days. With D3, the colonies were loose, and all the constituent cells were morphologically small macrophages, which were positive for alpha-naphthyl butyrate (alpha NB) esterase, strongly positive for CD14 antigen, and plastic-adherent. While without D3, the colonies were rather compact, and all the constituent cells were morphologically mature neutrophils, which were positive for naphthol ASD-chloroacetate esterase and weakly positive for CD14 antigen. Secondary culture of the 8- or 10-day colonies with D3 induced a lower number of alpha NB-positive cells, in proportion to the percentage of promyelocytes at the time of transfer in each colony. Four days of secondary culture with D3 was sufficient to induce alpha NB-positive cells. G-CSF was not an essential factor to induce alpha NB- positive cells. These findings indicate that D3 differentiates normal human neutrophilic promyelocytes into the Mo/M phi lineage in vitro.

Background and aim Cytomegalovirus (CMV) exacerbates ulcerative colitis (UC) refractory to immunosuppressive therapies. The conditions under which CMV reactivation occurs in patients with UC, however, is unclear. In addition, the diagnostic and treatment strategies for UC positive for CMV have not been established. Granulocyte and monocyte adsorptive apheresis (GMAA) is natural biological therapy for UC in which the granulocytes/macrophages producing inflammatory cytokines are removed. We investigated the rate of colonic CMV reactivation and the efficacy of GMAA in active UC patients positive for CMV without concomitant corticosteroid (CS) therapy. Methods Fifty-one active UC patients without concomitant CS therapy were enrolled. Colonic CMV reactivation was examined by real-time polymerase chain reaction (PCR) using biopsy specimen and/or histological examination. All patients were treated with intensive GMAA (twice per week). Rates of clinical remission and mucosal healing were compared between UC patients positive and negative for CMV. Results Of 51 patients, 15 (29.4%) were diagnosed as CMV positive. The clinical remission rates following intensive GMAA did not differ between UC patients positive and negative for CMV (73.3% vs 69.4%, p = 0.781). Proportion of patients achieving mucosal healing was also similar between these two groups. CMV-DNA became negative in all UC patients positive for CMV who achieved clinical remission 1 week after completion of intensive GMAA. Conclusions Intestinal inflammation might trigger CMV reactivation in a subpopulation of active UC patients without CS treatment. GMAA could be a promising option for active UC positive for CMV.

Background The molecular pathogenesis underlying recurrent exacerbations of atopic dermatitis (AD) is unclear. Some peripheral CCR4 + and CCR7 + helper memory T cells express the specific homing receptor, sialyl 6-sulfo Lewis X (G152 glycan). This glycan loses receptor activity via cyclization of its sialic acid moiety, thus becoming cyclic sialyl 6-sulfo Lewis X (G159 glycan). These findings suggest that the disordered expression of G152 and G159 glycans may be associated with recurrent exacerbations of AD. Objective To assess the possible association of G152 and G159 glycans, which are expressed on peripheral helper T (Th) cells, with frequency of exacerbations. Methods The percentage of glycan-expressing cells among peripheral blood CD4 + CD45RO + lymphocytes was determined by flow cytometry. The association of glycans with the frequency of exacerbations determined by recurrence scores as well as with current disease activity was statistically tested. Results Current disease activity was significantly associated with CCR4 + CCR7 − memory Th cells expressing CSLEX-1 glycan, the conventional skin-trafficking receptor without sialic-acid-cyclization activity. In contrast, the frequency of exacerbations was positively and negatively associated with CCR4 + CCR7 + memory Th cells expressing G152 and G159 glycans, respectively. Receiver operating characteristics analyses indicated that the ratio of the G152 + /G159 + cell percentages discriminated patients with highly recurrent AD with the best accuracy. Conclusion Flow cytometric determination of G159 and G152 glycans on peripheral helper memory T cells may be clinically useful for identifying patients with highly recurrent AD. Disordered sialic acid cyclization of G152 glycan may underlie highly recurrent AD, which may provide a novel therapeutic approach.

Cell-surface glycans play important roles in lymphocyte traffic and homing. Cell adhesion mediated by glycans, such as selectin-mediated cell adhesion, is involved in the first step of every process of extravasation of leukocytes, either in granulocytes or in lymphocytes (Fig. 1). Glycan-mediated cell adhesion is virtually an only one cell-adhesion system that tolerates a strong shear force of normal blood flow in the general circulation. This step is crucial for every leukocyte diapedesis; without glycan-mediated cell adhesion, other secondary proteinaceous cell-adhesion molecules such as integrins or the Ig superfamily do not operate effectively. Recently, it has become increasingly clear that homing processes of various subsets of T lymphocytes are elegantly regulated by different species of cell-adhesive glycans, and alterations in glycan expression are closely associated with the pathophysiology of several well-known human diseases.

Selectin-mediated adhesion to endothelial cells is the first step of extravasation of leukocytes. Neutrophils constitutively express sialyl Lewis x, the selectin ligand, and are ready to adhere to endothelial cells once the latter cells express E- or P-selectin. This leads to rapid and massive extravasation of neutrophils into acute inflammatory lesions.

Expression of two developmentally regulated carbohydrate antigens, the sialyl stage-specific embryonic antigen-1 (SSEA-1) and I-antigens, in human lymphocytes and lymphocytic leukemia cells was investigated using specific monoclonal antibodies. Sialyl SSEA-1 was expressed only on natural killer (NK) cells, and was essentially absent on resting mature T and B cells among normal peripheral lymphocytes. On the other hand, the I-antigen was strongly expressed on virtually all mature B cells, moderately expressed on most mature T cells, but not expressed on NK cells in normal donors. Expression of the two antigens on normal T and B cells was reversible; in vitro stimulation of normal lymphocytes with concanavalin A (Con A) resulted in the loss of I-antigen and appearance of sialyl SSEA-1 on CD3+ T blasts, whereas stimulation with pokeweed mitogen led to loss of I-antigen expression and appearance of sialyl SSEA-1 antigen on CD19+ B blasts. Among lymphocytic leukemia cells, sialyl SSEA-1 was detected primarily on leukemia cells having immature properties such as most common acute lymphocytic leukemia (cALL) blasts, while the I-antigen was frequently expressed on malignant cells having relatively mature properties, such as those found in adult T- cell leukemia or chronic lymphocytic leukemia, and only occasionally on cALL blasts. Among normal peripheral lymphocytes, the sialyl SSEA-1+I- antigen- NK cells selectively underwent E-selectin (ELAM-1, endothelial- leukocyte adhesion molecule-1)-dependent adhesion to endothelial cells, while the I-antigen+sialyl SSEA-1- mature T and B cells did not, in line with the recent finding that sialyl SSEA-1 serves as a specific ligand for E-selectin. Con A blasts, which are sialyl SSEA-1+I-antigen- , also exhibited significant E-selectin-dependent adhesion to endothelial cells. These results indicate that expression of the sialyl SSEA-1 and I-antigens varies alternately depending on the differentiation/activation status of the lymphocytes, and that this at least partly regulates the behavior of lymphocytes at the vessel wall.

The first step of leukocyte extravasation is adhesion of leukocytes to endothelial cells mediated by selectins and their carbohydrate ligands (Fig. 1). This cell adhesion system tethers leukocytes in the bloodstream to endothelial walls, and this in turn triggers the action of chemokines that are expressed on the endothelial surface. Signal transduction through leukocyte chemokine receptors activates leukocyte integrins, which lead to extravasation of leukocytes.

Recently the lectin-like domain on ELAM-1 (endothelial leukocyte adhesion molecule-1) was shown to recognize a carbohydrate antigen, sialyl Lewis x. In this paper we demonstrate, by a series of inhibition experiments utilizing specific monoclonal antibodies and pure glycolipid preparations, that the sialyl Lewis a antigen serves as a specific ligand for ELAM-1 as well as sialyl Lewis x and plays a significant role in the ELAM-1-mediated binding of human cancer cells to activated endothelial cells.

A 57-year-old Japanese man with tumor-stage mycosis fungoides suddenly presented multiple small papules on the right chest. Histopathology of a biopsy specimen from the papules revealed medium-to-large pleomorphic lymphoid cells throughout the entire dermis but not in the epidermis, and the large cells expressed CD30 antigen. These newly-developed papules underwent spontaneous remission in the following 3 months. We reviewed the reported cases of mycosis fungoides, which showed CD30-positive large cell transformation and those of CD30-positive lymphoproliferative disorders associated with mycosis fungoides.

Background and purpose: More than half of the retinitis pigmentosa (RP) cases are genetically simplex or multiplex. To date, 37 causative genes of RP have been identified; however, the elucidation of gene defects in simplex or multiplex RP patients/families remains problematic. The aim of our study was to identify the genetic causes of RP in patients with unknown or non-Mendelian inheritance. Methods and results: Since 2003, 52 simplex RP patients, 151 patients from 141 multiplex RP families, and six sporadic patients with retinal degeneration were studied. A total of 108 exons of 30 RP-causing genes that harboured the reported mutations were screened by an efficient denaturing high performance liquid chromatography (dHPLC) based assay. Aberrant fragments were subsequently analysed by automatic sequencing. Twenty-six mutations, including two frameshift mutations, one single amino acid deletion, and 23 missense mutations, were identified in 28 probands (14.07%). Eighteen mutations have not been reported to date. Three pairs of combined mutations in different genes were identified in two sporadic cases and one multiplex family, indicating the possibility of novel digenic patterns. Of the 23 missense mutations, 21 were predicted as deleterious mutations by computational methods using PolyPhen, SIFT, PANTHER, and PMut programs. Conclusion: We elucidated the mutation spectrum in Japanese RP patients and demonstrated the validity of the mutation detection system using dHPLC sequencing for genetic diagnosis in RP patients independent of familial incidence, which may provide a model strategy for identifying genetic causes in other diseases linked to a wide range of genes.

Several sulfated sialoglycoconjugates have recently been shown to serve as ligands for selectins and siglecs. For instance, alpha2--3 sialylated 6-sulfo-Lewis x was found to serve as a ligand for selectins on skin-homing helper memory T cells, and alpha2--6 sialylated 6-sulfo-LacNAc to be a preferred ligand for CD22/siglec-2 on human naïve B cells. Monoclonal antibodies specific to sulfated sialoglycoconjugates are effective tools to dissect these ligands on minor subsets of human leukocytes.

CD22/Siglec-2, an important inhibitory co-receptor on B-lymphocytes, is known to recognize alpha2-6-sialylated glycan as a specific ligand. Here we propose that the alpha2-6-sialylated and 6-GlcNAc-sulfated determinant serves as a preferred ligand for CD22 because the binding of a human B-cell line to CD22 was almost completely abrogated after incubating the cells with NaClO3, an inhibitor of cellular sulfate metabolism, and was also significantly inhibited by a newly generated monoclonal antibody specific to the alpha2-6-sialylated 6-sulfo-N-acetyllactosamine (LacNAc) determinant (KN343, murine IgM). The alpha2-6-sialylated 6-sulfo-LacNAc determinant defined by the antibody was significantly expressed on a majority of normal human peripheral B-lymphocytes as well as follicular B-lymphocytes in peripheral lymph nodes. The determinant was also expressed in endothelial cells of high endothelial venules of secondary lymphoid tissues, including lymph nodes, tonsils, and intestine-associated lymphoid tissues, more strongly than on B-lymphocytes, suggesting a role for CD22 in B-cell interaction with blood vessels and trafficking. These results indicate that the alpha2-6-sialylated 6-sulfo-LacNAc determinant serves as an endogenous ligand for human CD22 and suggest the possibility that 6-GlcNAc sulfation as well as alpha2-6-sialylation may regulate CD22/Siglec-2 functions in humans.

Although the effectiveness of rituximab has been reported in ABO blood group (ABO)-incompatible (ABO-I) organ transplantation, the protocol is not yet established. We studied the impact of the timing of rituximab prophylaxis and the humoral immune response of patients undergoing ABO-I living donor liver transplantation (LDLT), focusing on clinicopathological findings and the B-cell subset. From July 2003 to December 2005, 30 adult patients were treated with hepatic artery infusion (HAI) protocol without splenectomy for ABO-I LDLT. A total of 17 patients were treated only with HAI (no prophylaxis), and the other 13 were treated with rituximab prophylaxis at various times prior to transplantation. For B-cell study of the spleen, another 4 patients undergoing ABO-I LDLT both with HAI after prophylaxis and eventual splenectomy, and 3 patients with ABO-compatible LDLT with splenectomy were enrolled. The mortality of the 30 patients with HAI, without splenectomy, and with/without rituximab prophylaxis was 33% and the main cause of death was sepsis. Peripheral blood B cells were completely depleted, anti-donor blood-type antibody titer was lower, and clinical and pathological antibody-mediated rejection was not observed in patients with prophylaxis earlier than 7 days before transplantation (early prophylaxis). Early rituximab prophylaxis significantly depleted B cells and memory B cells in the spleen but not in lymph nodes. On the other hand, B cells and memory B cells increased and memory B cells became dominant during antibody-mediated rejection. In conclusion, early prophylaxis with rituximab depletes B cells, including memory B cells, in the spleen and is associated with a trend toward lower humoral rejection rates and lower peak immunoglobulin (Ig)G titers in ABO-I LDLT patients. Liver Transpl 13:579–588, 2007. © 2007 AASLD.

Expression of sialyl Lewisa is known to be increased in cancers of the digestive organs. The determinant serves as a ligand for E-selectin and mediates hematogenous metastasis of cancers. In contrast, disialyl Lewisa, which has an extra sialic acid attached at the C6-position of penultimate GlcNAc in sialyl Lewisa, is expressed preferentially on nonmalignant colonic epithelial cells, and its expression decreases significantly on malignant transformation. Introduction of the gene for an α2→6 sialyl-transferase responsible for disialyl Lewisa synthesis to colon cancer cells resulted in a marked increase in disialyl Lewisa expression and corresponding decrease in sialyl Lewisa expression. This was accompanied by the complete loss of E-selectin binding activity of the cells. In contrast, the transfected cells acquired significant binding activity to sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7)/p75/adhesion inhibitory receptor molecule-1, an inhibitory receptor expressed on lymphoid cells. These results indicate that the transition of carbohydrate determinants from disialyl Lewisa-dominant status to sialyl Lewisa-dominant status on malignant transformation has a dual functional consequence: the loss of normal cell-cell recognition between mucosal epithelial cells and lymphoid cells on one hand and the gain of E-selectin binding activity on the other. The transcription of a gene encoding the α2→6 sialyltransferase was markedly down-regulated in cancer cells compared with nonmalignant epithelial cells, which is in line with the decreased expression of disialyl Lewisa and increased expression of sialyl Lewisa in cancers. Treatment of cancer cells with butyrate or 5-azacytidine induced strongly disialyl Lewisa expression, suggesting that histone deacetylation and/or DNA methylation may be involved in the silencing of the gene in cancers.

X-linked ectodermal dysplasia and immunodeficiency (XL-EDA-ID) is an X-linked recessive disease caused by a mutation in the nuclear factor-κB (NF-κB) essential modulator (NEMO). Here we report an XL-EDA-ID patient with atypical features of very few naive-phenotype T cells and defective mitogen-induced proliferation of peripheral blood mononuclear cells (PBMCs). The patient's NEMO defect was diagnosed by flow cytometric analysis of intracellular NEMO staining. Specific cell lineages (monocytes and neutrophils) expressed reduced levels of NEMO, but 2 populations of T, B, and NK cells were detected with normal and reduced expression of NEMO. Genomic analysis revealed that duplication of a 4.4-kb sequence ranging from intron 3 to exon 6 caused the reduced expression of NEMO. Polymorphism analysis showed that the patient's B- and T-cell lines with reduced and normal expression of NEMO had the same X chromosome, indicating that the somatic mosaicism was not due to fetomaternal transfusion but was most likely due to postzygotic reversion. This XLEDA-ID case adds to our understanding of NEMO biology, indicating that NEMO is critical for T-cell development and/or survival in humans as well as in mice. (Blood. 2004;103:4565-4572)

Recently we identified sialyl 6-sulfo Le(x) as a major L-selectin ligand on high endothelial venules of human peripheral lymph nodes. In this study we investigated the ligand activity of sialyl 6-sulfo Le(x) to E- and P-selectins and compared it with the binding activity of conventional sialyl Le(x), by using cultured human lymphoid cells expressing both carbohydrate determinants. The results of the recombinant selectin binding studies and the nonstatic monolayer cell adhesion assays indicated that both sialyl 6-sulfo Le(x) and conventional sialyl Le(x) served as ligand for E- and P-selectins, while L-selectin was quite specific to sialyl 6-sulfo Le(x). Anti-PSGL-1 antibodies as well as O-sialoglycoprotein endopeptidase treatment almost completely abrogated the binding of P-selectin but barely affected the binding of E-selectin, indicating that these carbohydrate determinants carried by O-glycans of PSGL-1 selectively serves as a ligand for P-selectin, while the ligand for E-selectin is not restricted to PSGL-1 nor to O-sialoglycoprotein endopeptidase-sensitive glycans. The binding of L-selectin was markedly reduced by O-sialoglycoprotein endopeptidase treatment but only minimally affected by anti-PSGL-1 antibodies, indicating that O-glycans carrying sialyl 6-sulfo Le(x) were the major L-selectin ligands, while PSGL-1 was only a minor core protein for L-selectin in these cells. These results indicated that each member of the selectin family has a distinct ligand binding specificity.

CD8 deficiency is a rare primary immunodeficiency caused by a defect of ZAP-70, which plays a pivotal role in T cell activation. We previously reported the existence of memory phenotype-CD4 + T cells in a case of CD8 deficiency, which demonstrates that activation signals through ZAP-70 are not essential to the phenotypic conversion of T cells from “naive” to “memory.” In this study, we further characterized CD45RO + T cells in a CD8 deficient patient. We showed that the patient’s CD45RO + T cell population had a wide variety of T cell receptor Vβ-chain gene usage, and contained few clonally expanded T cells, while many clonally expanded T cells were present in the memory T cell population of age-matched healthy children. These results suggest that various kinds of antigens were involved in the differentiation of the patient’s T cells, and that the differentiation into memory T cells was not accompanied by profound T cell proliferation. Moreover, our findings confirmed that the patient’s CD45RO + CD4 + T cells had acquired effector-cytokine producing ability, indicating that there exists an alternative activation pathway which is independent of ZAP-70 for the acquisition of effector-cytokine producing ability.

We provide here evidence that supports the occurrence of a biologically dormant form of selectin ligand carbohydrate, the sialyl 6-sulfo Lewis X containing modified sialic acid, in human leukocytes. The modification of sialic acid involves first de-N-acetylation of sialic acid moiety through ubiquitous de-N-acetylation/re-N-acetylation cycle, followed by the dehydrative cyclization of de-N-acetyl sialic acid to form “cyclic sialic acid.” The enzyme involved in the dehydration of de-N-acetyl sialic acid is a calcium-dependent enzyme having neutral–alkaline pH optimum. De-N-acetyl sialyl 6-sulfo Lewis X retained selectin binding activity as well as parental sialyl 6-sulfo Lewis X, but cyclic sialyl 6-sulfo Lewis X was devoid of selectin binding activity. Sialyl 6-sulfo Lewis X carrying the cyclic sialic acid is specifically recognized by the newly generated mAb, G159. The determinant was distributed widely among normal human leukocytes, especially on monocytes and subsets of lymphocytes including NK cells, helper memory T cells, Tcr-γδ T cells, and a part of B cells. The determinant was detected also on several cultured lymphocytic leukemia cell lines andO-tetradecanoylphorbol 13-acetate-activated lymphoid cells. Cyclic sialyl 6-sulfo Lewis X is efficiently formed by the action of the partly membrane-bound calcium-dependent enzyme, tentatively called “sialic acid cyclase,” and a possible physiological significance of this reaction could be a rapid inactivation of selectin binding activity at the cell surface. Conversely, the accumulated intracellular cyclic sialyl 6-sulfo Lewis X determinant may function as a dormant pool of selectin ligands, which, on appropriate stimulation, is hydrolyzed and becomes active in selectin-dependent cell adhesion.

In August, 1992, we established a leukemic cell line (NS-Meg) from a patient in megakaryoblastic transformation of Philadelphia chromosome-positive chronic myeloid leukemia. The NS-Meg cells were positive for alpha-naphthyl acetate esterase and periodic acid-Schiff (PAS) staining and for surface CD4, CD7, CD13, CD34, CD41a, and glycophorin A antigens. Ultrastructurally, the cells had alpha-granules, demarcation membranes, and platelet peroxidase activity. The NS-Meg cells spontaneously produced platelet-like particles which contained alpha-granules, mitochondria and dense bodies, strongly suggesting platelet production. Erythropoietin (Epo), granulocyte/macrophage colony stimulating factor(GM-CSF), and interleukin 3 (IL-3) promoted the growth of NS-Meg cells. Phorbol-12-myristate-13-acetate increased the expression of both CD41a and CD61 antigens. Ten-day exposure to Epo induced mature erythroblasts and red cells. These benzidine-positive cells were positive for hemoglobin F staining. Untreated NS-Meg cells expressed mRNA for the Epo receptor (EpoR), for GATA-1, and for alpha 1, alpha 2 and gamma globin genes. These results indicate that NS-Meg cells undergo terminal differentiation of both megakaryocytic and erythroid lineages. This cell line should be a very useful tool for the investigation of both megakaryocytic and erythroid maturation.