Thrombocytopenia and CD34 expression is decoupled from α-granule deficiency with mutation of the first growth factor-independent 1B zinc finger.

Essentials The phenotypes of different growth factor-independent 1B (GFI1B) variants are not established. GFI1B variants produce heterogeneous clinical phenotypes dependent on the site of mutation. Mutation of the first non-DNA-binding zinc-finger causes a mild platelet and clinical phenotype. GFI1B regulates the CD34 promoter; platelet CD34 expression is an indicator of GFI1B mutation. SUMMARY: Background Mutation of the growth factor-independent 1B (GFI1B) fifth DNA-binding zinc-finger domain causes macrothrombocytopenia and α-granule deficiency leading to clinical bleeding. The phenotypes associated with GFI1B variants disrupting non-DNA-binding zinc-fingers remain uncharacterized. Objectives To determine the functional and phenotypic consequences of GFI1B variants disrupting non-DNA-binding zinc-finger domains. Methods The GFI1B C168F variant and a novel GFI1B c.2520 + 1_2520 + 8delGTGGGCAC splice variant were identified in four unrelated families. Phenotypic features, DNA-binding properties and transcriptional effects were determined and compared with those in individuals with a GFI1B H294 fs mutation of the fifth DNA-binding zinc-finger. Patient-specific induced pluripotent stem cell (iPSC)-derived megakaryocytes were generated to facilitate disease modeling. Results The DNA-binding GFI1B variant C168F, which is predicted to disrupt the first non-DNA-binding zinc-finger domain, is associated with macrothrombocytopenia without α-granule deficiency or bleeding symptoms. A GFI1B splice variant, c.2520 + 1_2520 + 8delGTGGGCAC, which generates a short GFI1B isoform that lacks non-DNA-binding zinc-fingers 1 and 2, is associated with increased platelet CD34 expression only, without quantitative or morphologic platelet abnormalities. GFI1B represses the CD34 promoter, and this repression is attenuated by different GFI1B zinc-finger mutations, suggesting that deregulation of CD34 expression occurs at a direct transcriptional level. Patient-specific iPSC-derived megakaryocytes phenocopy these observations. Conclusions Disruption of GFI1B non-DNA-binding zinc-finger 1 is associated with mild to moderate thrombocytopenia without α-granule deficiency or bleeding symptomatology, indicating that the site of GFI1B mutation has important phenotypic implications. Platelet CD34 expression appears to be a common feature of perturbed GFI1B function, and may have diagnostic utility.

Practical management of myelofibrosis with ruxolitinib.

Treatment for the majority of patients with myelofibrosis is primarily based on symptom control as curative allogeneic stem cell transplantation is typically offered only to younger patients, especially those with poor prognosis disease. Around 50% of patients with myelofibrosis have the JAK2(V617F) mutation, but almost all patients have aberrant activation of the JAK-STAT signalling pathway. Recent efforts have focussed on the clinical use of JAK2 inhibitors to treat myelofibrosis. In this article, we present our recommendations for the practical management of myelofibrosis with ruxolitinib, a selective inhibitor of both JAK1 and JAK2. Ruxolitinib can significantly improve the quality of life of patients with myelofibrosis. There is also increasing evidence of a positive impact on survival. Consistent with the physiological role of JAK signalling the major toxicity of ruxolitinib is cytopenia. Managing cytopenia is key to maximising the therapeutic benefit of ruxolitinib. Further research into the safety of ruxolitinib in patients with thrombocytopenia is warranted, as is its role in special subgroups of patients, such as those undergoing stem cell transplantation and those experiencing thrombosis as a major manifestation of myelofibrosis.

Common genetic variants at the MC4R locus are associated with obesity, but not with dietary energy intake or colorectal cancer in the Scottish population.

BACKGROUND: Common single-nucleotide polymorphism (SNP) variants around the melanocortin 4 receptor (MC4R) gene have recently been associated with obesity risk and insulin resistance. Obesity is a known risk factor for colorectal cancer (CRC) and we hypothesized that there might be a common inherited genetic component. METHODS AND RESULTS: Four of the variants reported earlier were genotyped and tested for association with body mass index (BMI), waist circumference (WC), dietary energy intake (DEI) and CRC. Using a case-control genetic association study, we replicated the association with BMI (P=0.0001, additive genetic effect=0.37 kg/m(2)) and WC (P=0.005, additive genetic effect=0.70 cm) using over 3800 individuals. However, there was no association between these variants and CRC risk. Rare (highly penetrant) variants within the MC4R gene have been shown to influence eating behaviour and hyperphagia. We hypothesized that the newly identified common variants might also influence hyperphagia. Using DEI data recorded from a validated food frequency questionnaire, we found no significant genetic association between MC4R SNPs and DEI. CONCLUSIONS: As the MC4R locus explains only 0.28% of the BMI and 0.14% of the WC phenotypic variance in the Scottish population, most of the genetic contribution to obesity remains to be identified.

The immature reticulocyte fraction: a negative predictor of the harvesting of CD34 cells for autologous peripheral blood stem cell transplantation.

The optimal time for the harvesting of peripheral blood stem cells following chemotherapy and growth factors for autologous transplantation is based on the CD34 cell count. In this study, 51 patients having 59 stem cell mobilizations were assessed for the timing of the harvest by a CD34 cell count and an immature reticulocyte fraction (IRF). Results from 272 preharvest tests showed that when the CD34 cells were not harvestable, defined as a CD34 cell count of < 15 cells/microl, the IRF was always < or = 0.2. A low IRF resulted in a negative predictive value of 1 for the harvesting of stem cells. The IRF is therefore a valuable negative predictor of the timing of autologous stem cell harvesting.

Systemic capillary leak syndrome associated with compartment syndrome and rhabdomyolysis.

Systemic capillary leak syndrome (SCLS) is a rare disorder characterized by recurrent spontaneous episodes of hypovolaemic shock due to marked plasma shifts from the intravascular to the extravascular space. This presents as the characteristic triad of hypotension, haemoconcentration and hypoalbuminemia often with an associated monoclonal gammopathy. We describe a patient with SCLS who required aggressive fluid resuscitation and emergency fasciotomies for compartment syndrome with rhabdomyolysis. At presentation the patient was considered to have severe erythrocytosis and was therefore initially referred to a haematologist, which appears to be a frequent sequence of presentation for patients with SCLS. This patient also highlights the importance of muscle compartment pressure monitoring during volume resuscitation in patients with SCLS.

Routine fluorescence in situ hybridization with the MLL probe does not reliably detect two separate signals on one chromosome 11 in patients with trisomy 11.

Trisomy 11 is considered to be a rare cytogenetic abnormality in myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML). Duplication of the MLL gene (localized to 11q23) has been found on one chromosome 11 in patients with trisomy 11, detected by DNA techniques. We investigated copy number of MLL in seven patients with trisomy 11 to see if duplication could be assessed by the detection of two separate signals on fluorescence in situ hybridization (FISH). If so, FISH could provide a quick easy screen of MLL status in routine referrals. The diagnostic bone marrow aspirate showed trisomy 11 in five adult patients with MDS/AML as part of a complex karyotype and in two children with acute lymphoblastic leukemia (ALL) as part of a hyperdiploid karyotype. Fluorescence in situ hybridization utilized the suspensions remaining after the cytogenetic harvest. Two FISH probes were used on the adult patients (MLL - Oncor and Vysis), and one (Vysis) for the two children with ALL. Analysis showed that the proximity of the two putative hybridization signals made it very difficult to unambiguously see two separate signals. The hybridisations (Oncor probe) were convincing of MLL duplication (namely two distinct signals) in only one patient, but this was not borne out with the other MLL probe (Vysis). We conclude that conventional FISH with MLL probe is not suited to act as a screen for MLL duplication in patients with trisomy 11.

Effects of glycosylated recombinant human granulocyte colony-stimulating factor after high-dose cytarabine-based induction chemotherapy for adult acute myeloid leukaemia.

The Australian Leukaemia Study Group (ALSG) investigated whether G-CSF would accelerate haemopoietic recovery after induction treatment for acute myeloid leukaemia (AML) intensified with high-dose cytarabine, and therefore improve response rates and survival. Patients were randomised to receive lenograstim (glycosylated recombinant human G-CSF) 5 microg per kg body weight subcutaneously daily from day 8 after starting chemotherapy, or no cytokine, following chemotherapy with cytarabine 3 g/m2 every 12 h on days 1, 3, 5, and 7, together with idarubicin 9 or 12 mg/m2 on days 1, 2, and 3, plus etoposide 75 mg/m2 on days 1 to 7 inclusive. Patients had untreated AML, and were aged 16 to 60 years. Overall, 54 evaluable patients were randomised to receive lenograstim and 58 to no cytokine. Patients in the lenograstim arm had a significantly shorter duration of neutropenia <0.5 x 10(9)/l compared to patients in the no cytokine arm (median 18 vs 22 days; P = 0.0005), and also shorter duration of total leucopenia <1.0 x 10(9)/l (17 vs 19 days; P = 0.0002), as well as a reduction in duration of treatment with therapeutic intravenous antibiotics (20 vs 24 days; P= 0.015) and a trend to reduced number of days with fever >38.0 degrees C (9 vs 12 days; P = 0.18). There were no differences between the two groups in platelet recovery, red cell or platelet transfusions, or non-haematological toxicities. For patients achieving CR after their first induction course, a reduction in the time to the start of the next course of therapy was observed in the lenograstim arm, from a median of 40.5 days to a median of 36 days (P = 0.082). The overall complete response rates to chemotherapy were similar, 81% in the lenograstim arm vs 75% for the no cytokine arm (P = 0.5), and there was no significant difference in the survival durations. We conclude that the granulopoietic stimulating effect of G-CSF is observed after induction therapy for AML intensified by high-dose cytarabine, resulting in an improvement in a number of clinically important parameters with no major adverse effects.

17p- syndrome arising from a novel dicentric translocation in a patient with acute myeloid leukemia.

The cytogenetic contribution to the poor prognosis when myelodysplastic syndrome (MDS) progresses to acute myeloid leukemia (AML) is not well understood. We present a 66-year-old male who had thrombocytopenia with dysplastic features in peripheral blood neutrophils (hypogranular, hyposegmented neutrophils) comprising the Pelger-Huet anomaly, increased blasts in the marrow, and markers consistent with AML. Diagnostic marrow cytogenetics showed a complex karyotype including del(5q), a novel unbalanced dicentric translocation, t(17;20), resulting in both del(20q) and del(17p). Fluorescence in situ hybridization (with probe TP53) showed deletion of 17p13 on the dicentric chromosome, completing the criteria for the 17p- syndrome. Fluorescence in situ hybridization with probes for two tumor suppressor genes on chromosome 5q also showed deletion (CSF1R [at 5(q33.2-q33.4) and EGR-1 [5(q31-q32)]). Remission was difficult to achieve and cytogenetic relapse occurred 6 months postdiagnosis, and clinical relapse approximately one month later. Our case provides a novel mechanism for the 17p- syndrome, and highlights the difficulty of attributing prognostic significance to a particular cytogenetic abnormality in AML.

Inhibition of beta1-integrin expression reduces clone size during early retinogenesis.

We have used an antisense retrovirus strategy to test the role of beta1-integrin in the early period of clone development in the embryonic chick neural retina. Analyses of clone size and dispersion demonstrated a marked effect of reducing the levels of expressed beta1-integrin on this critical phase of retina cell and tissue development.

Pubertal timing and self-reported delinquency among male adolescents.

Pubertal timing has consequences for adolescent adaptation, and Moffitt has theorized that puberty is a motivating factor for delinquency. Pubertal timing and self-reported delinquency were examined in a questionnaire-based survey of 14-year-old boys (n=99). The questionnaire was completed anonymously, under test conditions, in the school classroom. The results showed that offtime maturers (those early or late) reported a wider range of delinquency, including higher levels of crime and school opposition behaviours. Offtimers also reported a greater frequency of particular delinquent acts over a 12-month period. Overall, the results lend support to the "deviance hypothesis" of pubertal timing.

Sequential development of myelodysplasia and acute myeloid leukemia but with no karyotypic evolution after autografting in a patient with Philadelphia positive acute lymphoblastic leukemia.

A woman with Philadelphia chromosome-positive c-ALL with +8 and i17q in addition underwent an unpurged blood stem cell autograft after 200mg/m2 melphalan in first relapse. Maintenance therapy with 6-mercatopurine was started following the autograft. Moderate pancytopenia developed after 4 months, and myelodysplasia (refractory anemia) was diagnosed which rapidly evolved into AML. The cytogenetic findings remained unchanged. She also developed CNS disease, but the blasts in the cerebrospinal fluid were lymphoid in character on immunophenotyping. She then received palliative treatment until death. The remarkable features here are the evolution into myelodysplasia and AML with retention of the original complex karyotype, and subsequent coexistence of lymphoid disease in the CNS and myeloid disease systemically. It is possible that the lineage switch and development of myelodysplasia in this case may have been secondary to treatment, but persistence of the original cytogenetic clone makes this unlikely. This may have been the result of some unusual effect of the treatment on the original clone, or expansion of a small unidentified myeloid clone present originally which gained a proliferative advantage due to the ALL-type treatment. This case confirms the aggressive and polymorphic nature of Ph+ ALL which may be the result of origin from an early progenitor cell (stem cell disease).

Campath-1G in vivo confers a low incidence of graft-versus-host disease associated with a high incidence of mixed chimaerism after bone marrow transplantation for severe aplastic anaemia using HLA-identical sibling donors.

We have evaluated the effect of in vivo Campath-1G on engraftment and GVHD in 23 patients with severe aplastic anaemia transplanted from HLA-identical sibling donors. In 14 patients Campath 1g was given pre-transplant for up to 9 days in an attempt to overcome graft rejection (group 1). In nine patients Campath-1G was given pre-transplant, but also continued post-transplant until day +5 to reduce GVHD (group 2). There were three patients with late graft failure in group I following initial neutrophil engraftment, and four cases of grade II+ GVHD. In group II, two patients had early graft failure (no take), and there were no cases of acute GVHD out of seven evaluable patients. One patient in group I developed chronic GVHD of the liver, and two patients (one in each group) had transient localised chronic GVHD. PCR of short tandem repeats was used to evaluate chimaeric status in 13 patients. Of 11 patients with initial neutrophil engraftment, only one had 100% donor haemopoiesis at all times. The remaining patients had either transient mixed chimaerism or persistence of recipient (< 20%) cells. We conclude that in vivo Campath-1G is associated with a high incidence of mixed chimaerism which tips the balance away from GVHD but towards graft rejection.

Bone marrow transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia.

Between 1986 and 1995, 19 patients with Philadelphia chromosome-positive (Ph + ) acute lymphoblastic leukemia underwent 20 autologous (n = 9) or allogeneic (n = 11) blood or marrow transplant procedures in first (n = 12) or second (n = 3) remission, or in relapse (n = 5). Four patients died due to transplant-related causes, 11 relapsed at 3-39 months, one survives with disease which did not remit after transplant, and three are alive in continuous remission at 1, 26 and 65 months. Two of the relapsing patients are alive; one autografted patient after an allograft in second remission and one allografted patient after a donor leukocyte infusion. The projected overall survival is 37.5% at 3 years and 12.5% at 5 years. The 3-year probabilities of relapse and disease-free survival for autografted patients are 65.9% and 25.6% respectively, and for allografted patients, 63.4% and 21.8% respectively. The stage of the disease at the time of transplant or the type of transplant did not affect the outcome significantly, and late relapses beyond 3 years were seen after allogeneic as well as autologous transplantation. In our experience, the outcome of patients with Ph + acute lymphoblastic leukemia continues to be poor despite high-dose therapy due to high relapse rates, and the development of additional measures to enhance the antileukemic efficacy of bone marrow transplantation is necessary.

A novel cobra venom metalloproteinase, mocarhagin, cleaves a 10-amino acid peptide from the mature N terminus of P-selectin glycoprotein ligand receptor, PSGL-1, and abolishes P-selectin binding.

Initial rolling of circulating neutrophils on a blood vessel wall prior to adhesion and transmigration to damaged tissue is dependent upon P-selectin expressed on endothelial cells and its specific neutrophil receptor, the P-selectin glycoprotein ligand-1 (PSGL-1). Pretreatment of neutrophils, HL60 cells, or a recombinant fucosylated soluble form of PSGL-1 (sPSGL-1.T7) with the cobra venom metalloproteinase, mocarhagin, completely abolished binding to purified P-selectin in a time-dependent and EDTA- and diisopropyl fluorophosphate-inhibitable manner consistent with mocarhagin selectively cleaving PSGL-1. A polyclonal antibody against the N-terminal peptide Gln-1-Glu-15 of mature PSGL-1 immunoprecipitated sPSGL-1.T7 but not sPSGL-1.T7 treated with mocarhagin, indicating that the mocarhagin cleavage site was near the N terminus. A single mocarhagin cleavage site between Tyr-10 and Asp-11 of mature PSGL-1 was determined by N-terminal sequencing of mocarhagin fragments of sPSGL-1.T7 and is within a highly negatively charged amino acid sequence 1-QATEYEYLDY decreases DFLPETEPPE, containing three tyrosine residues that are consensus sulfation sites. Consistent with a functional role of this region of PSGL-1 in binding P-selectin, an affinity-purified polyclonal antibody against residues Gln-1-Glu-15 of PSGL-1 strongly inhibited P-selectin binding to neutrophils, whereas an antibody against residues Asp-9-Arg-23 was noninhibitory. These combined data strongly suggest that the N-terminal anionic/sulfated tyrosine motif of PSGL-1 as well as downstream sialylated carbohydrate is essential for binding of P-selectin by neutrophils.

Relationships between cellular condensation, preosteoblast formation and epithelial-mesenchymal interactions in initiation of osteogenesis.

Initiation of osteogenesis or bone formation is dependent on cell and tissue interactions. We investigated the events between 4 and 7 days of incubation that translate epithelial-mesenchymal signalling into overt differentiation of osteoblasts and deposition of bone in the mandibles of chick embryos. Condensation of mandibular mesenchyme (the membranous skeleton), visualized with PNA-lectin, occurred at H.H. mid-26 (5.75 days), lasted 12 h and preceded osteoblast differentiation by 1.5 days. As determined from 3D-reconstruction all mandibular membrane bones arose from a single condensation closely associated with the stomodeal epithelium. The finding that the osteogenic condensation in the mandibular arch is a major branch of a common condensation that provides osteogenic mesenchyme to both maxillary and mandibular arches establishes a closer link between mechanisms controlling development of the skeleton in these two arches than previously suspected. Preosteoblasts (alkaline phosphatase-positive cells) form in the mandible at H.H. early 25, which is before condensation but after the epithelial-mesenchymal interaction upon which preosteoblast formation and condensation depend--neither form in isolated mesenchyme, whereas both form after recombination of mesenchyme and epithelium. Tenascin was present in the mandibular epithelium only at H.H. stage 19 but not in the mesenchyme at any age. Therefore, the epithelial-mesenchymal interaction controls initiation of osteogenesis at the preosteoblast stage. Preosteoblasts then condense, transform into osteoblasts and deposit bone matrix. Differentiation of preosteoblasts precedes condensation which amplifies their number. This is in contrast with chondrogenesis where condensation triggers prechondroblast differentiation.

Plasma P-selectin is increased in thrombotic consumptive platelet disorders.

P-selectin is a 140-kD protein found in the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells that on cell activation is expressed on the cell surface and also secreted into the plasma. The secreted form of P-selectin, like plasma P-selectin, differed from platelet membrane P-selectin in that its molecular mass was approximately 3 kD lower under reducing conditions. Both the secreted and plasma forms of P-selectin contained cytoplasmic sequence as determined by Western blot analysis with an affinity-purified rabbit anti-P-selectin cytoplasmic peptide antibody. We have measured plasma P-selectin and beta-thromboglobulin (beta TG) concurrently in (1) patients with consumptive thrombotic disorders, including disseminated intravascular coagulation (DIC), heparin-induced thrombocytopenia (HIT), and thrombotic thrombocytopenic purpura (TTP)/haemolytic uremic syndrome (HUS); (2) patients with idiopathic thrombocytopenic purpura (ITP); and (3) healthy controls. Patients with DIC, HIT, and TTP/HUS, but not ITP, had significantly elevated plasma P-selectin and beta TG levels when compared with their age-matched healthy controls. The increased plasma P-selectin and beta TG in patients with thrombotic disorders were likely to be the result of in vivo platelet and endothelial cell damage or activation. We also found that avoidance of veno-occlusion and other tedious measures customarily taken during blood collection and sample preparation to prevent in vitro platelet activation did not affect plasma P-selectin assay results. In addition, plasma P-selectin levels were not influenced by the presence of renal failure or heparin administration. These results indicate that plasma P-selectin may be a useful new marker for thrombotic diseases.

P-selectin and platelet-activating factor mediate initial endotoxin-induced neutropenia.

Polymorphonuclear neutrophil (PMN) accumulation within damaged tissues, a hallmark of acute inflammation, is dependent upon initial adhesion to endothelial cells. In vitro studies suggest that P-selectin and platelet activating factor (PAF) are key molecules in this process by promoting the initial adhesion of PMN to endothelial cells. We report in vivo studies in which intravenous administration of lipopolysaccharide (LPS) to anesthetized rats caused a very rapid onset (< 5 min) of neutropenia, in association with induction of surface expression of P-selectin on microvascular endothelial cells in kidney, liver and lung; analogous induction of P-selectin expression by cultured endothelial cells was observed in response to LPS stimulation in vitro. In addition, treatment with an antibody (Ab) to P-selectin (or use of a PAF antagonist) blocked development of neutropenia in vivo for at least 15 min post-LPS injection, and Ab treatment was shown to block PMN accumulation in tissues. These studies document roles for P-selectin and PAF in the early adhesion of PMN to endothelial cells in vivo.

Influence of the antigen delivery system on immunoglobulin isotype selection and cytokine production in response to influenza A nucleoprotein.

The influence of different antigen delivery systems on antibody isotype and lymphokine profile has been investigated using influenza nucleoprotein as a model antigen system. Mice exposed to live or inactivated influenza virus produced antibody against whole virus or recombinant nucleoprotein (rNP), which was predominantly of the IgG2a isotype. Spleen or lymph node cells from these mice rapidly produced large amounts of interferon-gamma (IFN-gamma), but no detectable interleukin-5 (IL-5) when stimulated in vitro with specific antigen. In contrast, after primary immunization with rNP or p206-229 in different adjuvants (CFA, quil A or alhydrogel), specific antibody was predominantly of the IgG1 isotype and relatively lower amounts of IFN-gamma but no IL-5 were detected following in vitro antigenic stimulation. Secondary immunization, however, resulted in detection of IgG2a antibodies and increased levels of IFN-gamma. IL-5 was only detected after secondary immunization with peptide in adjuvant. Mice infected with aro A- Salmonella typhimurium expressing NP produced antibody of both IgG1 and IgG2a isotypes and large amounts of IFN-gamma and no IL-5, following in vitro antigenic stimulation, and therefore parallelled the pattern seen with whole virus more closely than that seen following primary immunization with protein or peptide in conventional adjuvants. The results suggest that the antigen delivery vehicle influences both quantitative and qualitative differences in the type of immune response elicited, which may be important in determining the potency of protective immunity induced.