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Crypt base intestinal stem cells (ISCs) maintain the epithelial lining of the intestines, but their frequencies are reduced in experimental models of graft vs. host disease (GVHD). Paneth cells provide an epithelial contribution to the stem cell niche, but their frequencies are also reduced in GVHD mouse models and in patients with GVHD. Mechanisms regulating ISCs and their niche after damage remain poorly understood. Interleukin-33 (IL-33) is an immunomodulatory alarmin typically thought to target lymphocytes such as T cells and innate lymphoid cells. In experimental models of allogeneic bone marrow transplantation, IL-33 can promote or attenuate GVHD, depending on the cellular target and the timing of the exposure. A secreted isoform of the IL-33 receptor, termed soluble ST2 (sST2), acts as a negative regulator by blocking IL-33 from binding to the membrane-bound isoform that mediates intracellular signaling. sST2 also has diagnostic and prognostic utility, serving as a biomarker for GVHD severity. While IL-33 has well-described effects on lymphocytes populations, there is limited understanding of the role of IL-33 signaling within the intestinal mucosa after damage.

Locatelli: Jazz Pharmaceeutical: Speakers Bureau; Medac: Speakers Bureau; Miltenyi: Speakers Bureau; Bellicum Pharmaceutical: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Niemeyer:Celgene: Consultancy; Novartis: Consultancy.

Neurofibromatosis type 1 (NF-1) predisposes to juvenile myelomonocytic leukemia (JMML) via loss of function of the NF1tumor suppressor gene and consecutive deregulation of Ras signal transduction. Affected individuals usually carry one defective NF1allele in the germline; somatic inactivation of the second NF1allele in hematopoietic cells is associated with transformation to leukemia. We previously demonstrated that a major mechanism for biallelic loss of NF1function in patients with JMML/NF-1 is mitotic recombination leading to uniparental disomy (UPD) of the 17q chromosome arm (Flotho, 2007; Steinemann, 2010). Using contemporary resequencing and microarray technology, we have now revisited the genetics of NF1inactivation in JMML. Specifically, we addressed two questions: 1) Are genetic findings in leukemic cells of JMML/NF-1 patients consistent with the clinical diagnosis and the two-hit concept? 2) Does the quintuple-negative (QN) group of JMML (patients without clinical evidence of NF-1 and negative for mutations in PTPN11, KRAS, NRAS, or CBL) contain unrecognized cases likely driven by NF1?

Corticosteroids (CS) represent first-line treatment for gastrointestinal graft vs host disease (GI GVHD), and CS failure is associated with severe morbidity and mortality. While the immune system is the intended target of CS treatment, the glucocorticoid receptor (GR) is widely expressed, and there is limited understanding of the direct effects of CS on intestinal epithelium following immune-mediated damage.

We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (less than 1%), play the same role. HNK1-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E- C at a ratio of 1:10 (HNK1+ E+C/E-C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+ DR+- sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+ E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK1 MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK1+ T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+, and DR+ T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co- culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haplo-identical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR, anti-DP, and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+ HNK1+, or CD8+ HNK1+ E+C. In conclusion, the CD3+, HNK1+, CD8+, DR+ cells may be the T- cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.

We performed a longitudinal study of the phenotype and functions of granular lymphoid cells from seven patients with T8 hyperlymphocytosis and neutropenia. Whereas cells retained a T3+ T8+ (six cases) or T3- T8+ (one case) phenotype at different examinations, the expression of natural killer (NK)-related antigens (HNK1- and Leu11-defined antigens) exhibited striking variations, some of which were also observed after in vitro culture. Similarly, natural or antibody-mediated cytotoxic activities fluctuated in vivo and in vitro. Cells from the patient with T3- T8+ proliferation were able to inhibit directly the growth of early CFU-GM, CFU-E, and BFU-E and to a lesser extent of late CFU-GM, as shown by cultures of autologous blood or marrow progenitors after depletion (and subsequent addition) of granular cells. In the other six patients with T3+ T8+ cells, no such effect was found. However, after a 24-hour incubation of the progenitors with the granular cells, CFU-GM growth was clearly inhibited; this was not observed in all experiments, a finding which may be related to the spontaneous variations of cell killer functions. Finally, no correlation was noted between the clinical course or extent of lymphoid proliferation and cell function or phenotype or with the monoclonal (two cases), polyclonal (three cases) or germ-line (one case) patterns of T cell receptor beta gene configuration.

We have used DNA sequence polymorphism analysis to document engraftment after T cell-depleted bone marrow transplantation (BMT), with a selected panel of four DNA probes. In contrast to nondepleted BMT recipients, the patients who received T cell-depleted marrow exhibited a mixed blood chimerism. This mosaicism was observed before graft failure or relapse in six patients. However, in five other patients, this mixed chimerism was not followed by these complications with a follow-up of 9 to 31 months after transplantation. Our results support the hypothesis that transplanted bone marrow T cells may help to maintain engraftment by eliminating host cells that can cause graft failure.

An immunogold method was used for investigating the subcellular localization of von Willebrand factor (vWF) and fibrinogen (Fg) in platelets and cultured megakaryocytes from normal subjects and from three patients with the gray platelet syndrome (GPS), a rare congenital disorder characterized by the absence of alpha-granules. In normal platelets at rest, vWF was detected exclusively in alpha-granules, with a characteristic distribution: gold particles were localized at one pole of each labeled granule, outlining the inner face of its membrane. vWF was distributed similarly in the alpha-granules of megakaryocytes at day 12 of culture, where it was also found in small vesicles near the Golgi complex. In contrast, Fg was observed in the whole matrix of all platelet alpha-granules but not in the nucleoids. In platelets from three patients with GPS, vWF and Fg were distributed homogeneously in the rare normal alpha-granules, which could be recognized by their size, and also in small granules identified as abnormal alpha-granules, which were similar in size to the small, possibly immature granules present in normal megakaryocytes. In addition, in some unstimulated platelets, Fg labeling was associated with dense material in the lumen of the surface-connected canalicular system (SCCS). At day 12 of culture, megakaryocytes from the patients with GPS contained some small alpha-granules labeled for Fg and vWF identical to those found in mature platelets. The majority of alpha-granules of normal size appeared partially or completely empty. Thus, we conclude that vWF is distributed differently from Fg in normal alpha-granules, and that unstimulated platelets from patients with GPS contain Fg and vWF in a population of small granules identifiable as abnormal alpha-granules only by immunoelectron microscopy. In addition, the presence of Fg in the SCCS of gray platelets suggests a spontaneous release of the alpha- granule content.

A murine monoclonal antibody (MoAb) designated FA6–152 has been obtained by immunizing mice with fetal erythrocytes. This antibody agglutinates fetal but not adult erythrocytes. Among blood cells, this antibody bound to both adult and fetal monocytes, platelets, and reticulocytes, but did not react with lymphocytes and granulocytes. Fluorescent labeling of marrow cells and of in vitro BFU-E, CFU-GM, and CFU-MK-derived colonies has shown that the antigen defined by FA6–152 MoAb was absent from the granulocytic precursors and was detected on the megakaryocytic lineage at a later stage of differentiation than the platelet-specific markers. In contrast, the antigen appeared as a very early marker of the erythroid differentiation since all erythroblasts, including proerythroblasts, were labeled even before the expression of glycophorin A. Cells from adult marrow and fetal liver were sorted with the FA6–152 MoAb and studied by electron microscopy and cell culture. The negative fraction contained granulocytic, monocytic, and megakaryocytic precursors, whereas the positive fraction was devoid of these precursors and contained monocytes, erythroblasts at all stages of maturation, and a homogeneous population of blasts. Cultures have shown that the only hematopoietic progenitors present in this positive fraction were CFU-E and some BFU-E. The antigenic density was related to the differentiation stage of the erythroid progenitors. In conclusion, this antibody is similar to the previously described 5F1 MoAb (Bernstein and Andrews, J Immunol 128:876, 1982; and Andrews et al, Blood 62:124, 1983) and provides a useful probe for studies leading to improved understanding of normal and malignant erythroid differentiation.

The expression of carbonic anhydrase (CA) as a marker of erythroid differentiation was investigated by immunologic and enzymatic procedures. A polyclonal anti-CA antibody was obtained by immunizing rabbits with purified CA I isozyme. This antibody is reactive with CA I but not with CA II. Within blood cells, CA I was only present in erythrocytes, whereas CA II was also detected in platelet lysates by enzymatic assay. Concerning marrow cells, identifiable erythroblasts and some blast cells expressed CA I. Most of the glycophorin A-positive marrow cells were clearly labeled by the anti-CA I antibody. However, rare CA I-positive cells were not reactive with anti-glycophorin A antibodies. We therefore investigated whether these cells were erythroid precursors or progenitors. In cell sorting experiments of marrow cells with the FA6 152 monoclonal antibody, which among hematopoietic progenitors is reactive only with CFU-E and a part of BFU- E, was performed, CA I+ cells were found mainly in the positive fraction. The percentage of CA I+ cells nonreactive with anti- glycophorin A antibodies contained in the two fractions was in the same range as the percentage of erythroid progenitors identified by their capacity to form colonies. In addition, the anti-CA I antibody labeled blood BFU-E-derived colonies as early as day 6 of culture, whereas in similar experiments with the anti-glycophorin A antibodies, they were stained three or four days later. No labeling was observed in CFU-GM- or CFU-MK-derived colonies. The phenotype of the day 6 cells expressing CA I was similar to that of erythroid progenitors (CFU-E or BFU-E): negative for glycophorin A and hemoglobin, and positive for HLA-DR antigen, the antigen identified by FA6 152, and blood group A antigen. Among the cell lines tested, only HEL cells expressed CA I, while K562 was unlabeled by the anti-CA I antibody. In contrast, HEL and K562 cells expressed CA II as detected by a biochemical technique. Synthesis of CA I, as with other erythroid markers such as glycophorin A and hemoglobin, was almost abolished after 12-O-tetradecanoyl-phorbol-13 acetate treatment of HEL cells. In conclusion, CA I appears to be an early specific marker of the erythroid differentiation, expressed by a cell with a similar phenotype as an erythroid progenitor.

We previously demonstrated that after allogeneic bone marrow transplantation (BMT) a subset of CD8, HNK1, and DR-positive T lymphocytes are able to inhibit CFU-GM and BFU-E growth with an HLA-DR restriction. In this study we investigated whether these cells, present in normal marrow in low concentration (<1%), play the same role. HNK,-positive sorted marrow cells forming rosettes (E+C) were able to inhibit BFU-E and CFU-GM growth when added back to the marrow E–C at a ratio of 1:10 (HNK1+E+C/E–C) in a range from 40% to 60%. This inhibitory effect was also detected for a cellular ratio of 1:100, which is the normal marrow value for this subset of T cell. HNK1+DR+-sorted E+C after double-immunofluorescent labeling also showed the same inhibitory activity as the HNK1+E+C, whereas the negative fraction including all the other E+C had no detectable inhibitory activity. CD3 and CD8 antigens were also present on the membrane of these cells, as demonstrated in two cases by double-immunofluorescent labeling performed with anti-CD3 or anti-CD8 monoclonal antibodies (MoAbs) and HNK, MoAb, respectively, and subsequent cell sorting. Blocking experiments, performed by adding in culture anti-CD4 and anti-CD8 MoAbs to HNK,+T cells showed that only the last MoAb was able to prevent inhibition of hematopoietic colony growth. These results confirmed that one subset of CD3+, CD8+, HNK1+. and DR+T cells was responsible for in vitro inhibition of normal hematopoiesis. In addition, this inhibition was genetically restricted to HLA-class II antigens, since in three co-culture experiments with unrelated bone marrow cells inhibition occurred only when cells with one haploidentical HLA-DR antigen was added back to the culture. Indeed, this effect was really HLA-DR restricted, since in blocking experiments with different anti-HLA class II MoAbs (anti-DR. anti-DP. and anti-DQ MoAbs) only an anti-HLA-DR MoAb was able to prevent the colony growth inhibition by CD3+HNK1+, or CD8+HNK1+E +C. In conclusion, the CD3+, HNK1+. CD8+, DR+cells may be the T-cell subset able to inhibit normal hematopoiesis with an HLA-DR restriction.

A potential role for endogenous innate lymphoid cells (ILCs) in prevention of gastrointestinal (GI) GVHD has recently been described in murine and clinical models, and experimental approaches for adoptive cellular therapy with ILCs have recently been described as well. However, the specific in vivoroles of tissue-resident ILCs and their capacity for reconstitution post-transplant remain unclear. We thus sought to characterize the significance and tissue distribution of ILCs after allogeneic bone marrow transplant (allo-BMT) in mouse models and in patients.

No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Pepe: Chiesi: Speakers Bureau; ApoPharma Inc: Speakers Bureau; Novartis: Speakers Bureau.

Allogeneic haematopoietic-stem-cell transplantation (HSCT) is the treatment of choice for many malignant and non-malignant disorders. However, Graft-versus-Host Disease (GvHD), the major complication of allogeneic HSCT, limits its wider application. Among different sites that can be involved, gastrointestinal GvHD represents the major cause of patients morbidity and mortality.

No relevant conflicts of interest to declare.

Left ventricular (LV) and right ventricular (RV) wall motion abnormalities can be detected through a qualitative analysis of cine magnetic resonance (MR) images. Moreover, MR is the gold standard technique for the evaluation of myocardial iron overload (MIO), biventricular global systolic function and myocardial fibrosis. We investigated the relationships between LV movement abnormalities and MIO, LV function and myocardial fibrosis as well as between RV motion and function in thalassemia major (TM) patients.CMR was performed in 1092 TM patients (537 male; 30.6±8.5 years) enrolled in the Myocardial Iron Overload in Thalassemia Network. Cine images were acquired to evaluate wall motion and to quantify biventricular volumes and ejection fraction (EF). For MIO assessment, a T2* multislice approach was used. To detect myocardial fibrosis, late gadolinium enhanced (LGE) images were acquired. For the LV the 16-segment model of the AHA/ACC was taken into account during image analysis: wall motion, T2* value and presence/absence of enhancing area were evaluated for each segment.Abnormal motion of LV was found in 66 (6%) patients (60 hypokinetic and 6 dyskinetic). Table 1 shows the comparison between TM patients with normal and abnormal LV motion. Patients with abnormal LV motion were older and had significantly lower global T2* value and significantly higher number of segments with T2*<20 ms. Left volumes and mass indexed by body surface area were significantly higher in patients with abnormal LV motion while the EF was significantly lower. LGE areas were detected in 196 patients (18%) and were predominantly located in the mid-ventricular septum. There was a significant correlation between LGE and abnormal LV motion.Abnormal motion of the RV was found in 35 (3.2%) patients (29 hypokinetic, 5 dyskinetic and 1 akynetic). Table 2 shows the comparison between TM patients with normal and abnormal RV motion. Patients with abnormal RV motion were older and they were more frequently males. Right volumes were significantly higher in patients with abnormal RV motion while the EF was significantly lower.Abnormal LV motion was not correlated with abnormal RV motion. Seventeen patients showed movement abnormalities in both ventricles.Movement abnormalities in the left ventricle were not really frequent in TM patients but were associated with age, MIO, LV dilation and dysfunction, and myocardial fibrosis. Movement abnormalities in the right ventricle were less frequent compared to the left ventricle, but were associated with age, sex , RV dilation and dysfunction.No relevant conflicts of interest to declare.

No relevant conflicts of interest to declare.

Allogeneic haemopoietic-stem-cell transplantation (HSCT) is the treatment of choice for many malignant and non-malignant disorders. Despite the recent advances in post-transplantat immunosuppressive therapy, Graft-versus-Host Disease (GVHD) still represents the major life-threatening complication, developing in a substantial number of HSCT patients and resulting in poor outcome. The basis of GVHD pathophysiology are still poorly understood and its current diagnosis depends mainly on clinical manifestations and invasive biopsies. Specific biomarkers for GVHD would facilitate the early and accurate recognition of this invalidating disease as well as the monitoring of patient response to adopted anti-GVHD pharmacological treatment. With the aim to explore new reliable markers for predicting and monitoring GVHD course, we focused on pentraxin-3 (PTX-3), an acute-phase protein, that has been shown to play a crucial role in orchestrating inflammatory immune responses.Having obtained an informed consent, we collected plasma samples from 46 patients who received unmanipulated HSCT and from 9 healthy donors (HD) volunteers. After HSCT, 25/46 patients developed skin GVHD (18 acute GVHD and 7 chronic GVHD), while 21/46 never experienced it. Concerning GVHD patients, blood samples were collected at the day of GVHD onset/ flare, before the beginning of GVHD-specific drug therapy. PTX-3 plasma levels were monitored by ELISA assays.Patients who did not develop GVHD after HSCT showed augmented PTX-3 plasma levels (mean=3.3 ng/ml, range=1.1-8.6 ng/ml) if compared to HD (mean=1.2 ng/ml, range=0.3-2.5 ng/ml, p<0.01). Interestingly, we observed a strong increase of PTX-3 plasma levels in patients with acute GVHD (mean=42.2 ng/ml, range=6.7-218.2 ng/ml) or with flair-ups of chronic GVHD (mean=15.8 ng/ml, range=9-44.3 ng/ml). The increase of PTX-3 levels in patients with acute and active chronic GVHD was statistically significant (p<0.01 and p<0.05 respectively) when compared to both HD and HSCT patients without GVHD.These preliminary results suggest that PTX-3 plasma levels increase very rapidly in patients experiencing active GVHD, thus candidating PTX-3 as an easily measurable soluble factor useful to corroborate clinical observations in a disease in which signs and symptoms are often protean. Further studies are needed to clarify if PTX-3 could represent a good diagnostic and/or prognostic factor rapidly indicating therapy responsiveness.No relevant conflicts of interest to declare.

Background. The stomach is the most frequent site of intestinal lymphomas. However, few data are available on the clinical-endoscopic presentation of gastric lymphoma as well as on possible differences in clinical pattern and endoscopic features between low-grade (LG) and high-grade (HG) lymphomas. In this study, we evaluated such aspects on consecutive primary gastric lymphoma patients observed in the last 12 years (1993–2004) in four Italian Hospitals (1 North, 2 Centre, 2 South). Methods. Clinical, histological, and endocospic records of consecutive patients diagnosed with LG or HG gastric lymphoma were retrieved and accurately evaluated. Symptoms were categorized as “alarm” (anaemia/melaena/heamorrage, persistent vomiting, weight loss) or “no alarm” (epigastric/abdominal pain, heartburn, dyspepsia/bloating). The endoscopic findings were classified as “normal” (no macroscopic lesions) or “abnormal” (ulcer, erosions, nodular pattern, hypertrophic folds, polypoid mass). Statistical analysis was carried out by using the Chi squared test. Results. During the study period, 143 patients with primary gastric lymphoma were detected. Overall, 61 patients were observed in the first 6 years and 82 in the last 6 years. The main results of the study are summarized in the table 1. Conclusions. The incidence of primary gastric lymphoma seems to be increasing. The overall prevalence of alarm symptoms is quite low, and they may be absent in near 75% of LG lymphoma patients. Moreover, contrarily to HG, LG lymphoma may present as a normal endoscopic finding and it is more frequently associated with H. pylori infection. At diagnosis, HG lymphoma is more frequently detected in an advanced stage as compared to LG lymphoma. Overall (143 patients) LG lymphoma (73 patients) HG lymphoma (70 patients) P value Age (mean ± SD) yrs 59.5 ± 14.2 59.4 ± 13.3 59.7 ± 15.1 0.4 Sex (M /F) 83/60 44/29 39/31 0.6 Alarm symptoms 57 (40%) 19 (26%) 38 (54%) 0.0009 Normal endoscopy 15 (10%) 15 (20%) 0 (0%) 0.0004 H. pylori infection 66 (73%) 47 (86%) 27 (39%) <0.0001 Stage (IA />IA) 78/65 58/15 20/50 <0.0001