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.

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.

Characterization of GMP-140 (P-selectin) as a circulating plasma protein.

GMP-140 is a 140-kD granule membrane protein, found in the alpha granules of platelets and the Weibel-Palade bodies of endothelial cells, that is surface expressed on cell activation and mediates neutrophil attachment. Cloning data for GMP-140 from an endothelial library predict a soluble form of the protein, the transcription message for which is also found in platelets. In this study, we report the detection by enzyme-linked immunosorbent assay of soluble GMP-140 in plasma centrifuged for 3 h at 100,000 g (to remove platelet microparticles) and confirm its identity by purification from plasma. Plasma concentrations were found to be 0.251 +/- 0.043 micrograms/ml (means +/- SD, n = 10) in normal male controls and 0.175 +/- 0.063 micrograms/ml (means +/- SD, n = 10) in normal female controls. The purified protein had an identical molecular mass (nonreduced) to platelet membrane GMP-140 (approximately 3 kD lower, reduced) and was immunoblotted by polyclonal anti-GMP-140, and the anti-GMP-140 monoclonal antibodies AK4 and AK6. Analytical gel filtration studies indicated that the plasma GMP-140 eluted as a monomer whereas detergent-free, platelet membrane GMP-140 eluted as a tetramer consistent with plasma GMP-140 lacking a transmembrane domain. Purified plasma GMP-140 bound to the same neutrophil receptor as the membrane-bound form, and when immobilized on plastic, bound neutrophils equivalently to immobilized platelet membrane GMP-140. Since it has been shown that fluid-phase GMP-140 is antiinflammatory and downregulates CD18-dependent neutrophil adhesion and respiratory burst, its presence in plasma may be of major importance in preventing the inadvertent activation of neutrophils in the circulation.

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.

Activation-dependent changes in human platelet PECAM-1: phosphorylation, cytoskeletal association, and surface membrane redistribution.

PECAM-1 is a recently described member of the immunoglobulin gene (Ig) superfamily that is expressed on the surface on platelets, several leukocyte subsets, and at the endothelial cell intracellular junction. Recent studies have shown that the extracellular domain of PECAM-1, which is comprised of 6 Ig-like homology units, participates in mediating cell-cell adhesion, plays a role in initiating endothelial cell contact, and may later serve to stabilize the endothelial cell monolayer. PECAM-1 also has a relatively large 108 amino acid cytoplasmic domain, with potential sites for phosphorylation, lipid modification, and other posttranslational events that could potentially modulate its adhesive function or regulate its subcellular distribution. Virtually nothing is known about the contribution of the intracellular region of the PECAM-1 molecule to either of these cellular processes. Using human platelets as a model, we now demonstrate that PECAM-1 becomes highly phosphorylated in response to cellular activation, and coincident with phosphorylation associates with the cytoskeleton of activated, but not resting, platelets. The engagement of PECAM-1 with the platelet cytoskeleton enables it to move large distances within the plane of the membrane of fully-spread, adherent platelets. This redistribution may similarly account for the ability of PECAM-1 to localize to the intracellular borders of endothelial cells once cell-cell contact has been achieved.

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.

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.

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.

Reduction of antigen-induced contraction of sensitized human bronchus in vitro by indomethacin.

1. Contraction of passively sensitized human bronchus induced by antigen challenge in vitro, was reduced by the prostaglandin F2alpha synthetase inhibitor, indomethacin. 2. Prostaglandin F2alpha was released during challenge and this release was inhibited by indomethacin. 3. There was a significant correlation between prostaglandin F2alpha release and antigen challenge-induced contraction, suggesting that this substance may contribute to the bronchoconstrictor response of sensitized human bronchus to antigen.

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.

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.

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.

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.

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).

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.

The magnitude of reproductive wastage to lamb marking in 30 Merino flocks in south-west Queensland.

Thirty groups of Merino ewes in 6 districts of south-west Queensland were studied between 1976 and 1985 to determine the magnitude of reproductive wastage to lamb making. Relatively high pregnancy rates (77 to 100%, mean 93%) and a wide range of lamb marking percentages (10 to 115%, mean 78%) were recorded. The mean reproductive wastage due to failure to mate and failure to lamb was low (1.6 and 3.4% respectively) in 8 flocks where harnessed rams were used. This indicated that loss of lambs from birth to lamb making was the major cause of reproductive wastage in most years.

Prostitution and risk of HIV: female prostitutes in London.

OBJECTIVE: To measure the prevalence of HIV and to describe established risk factors in female prostitutes. DESIGN: A cross sectional survey. SETTING: A genitourinary medicine clinic, streets, and magistrates' courts in London. SUBJECTS: 280 female prostitutes recruited between April 1989 and August 1991. MAIN OUTCOME MEASURES: Infection with HIV-1, reported risk behaviours, and prevalence of sexually transmitted infections. RESULTS: 228 of the women had HIV tests, and two (0.9% (95% confidence interval 0% to 2.1%)) were infected with HIV-1. Reported use of condoms was high for commercial clients and low for non-paying partners: 98% (251/255) of women used condoms with all clients and 12% (25/207) with non-paying partners for vaginal intercourse. Twenty two women were current or past injecting drug users. Of the 193 women examined for sexually transmitted infections, 27 had an acute infection (gonorrhoea, chlamydia, trichomonas, or primary genital herpes) at the time of interview. Infection was associated with younger age and increasing numbers of non-paying sexual partners, but not with duration of prostitution, numbers of clients, or reports of condom failures. When age and numbers of non-paying partners were analysed by logistic regression they remained significantly associated with sexually transmitted infections. CONCLUSIONS: A large and diverse sample of prostitutes had a low prevalence of infection with HIV and high levels of use of condoms in commercial sex. There was a significant risk of other sexually transmitted infections associated with prostitutes' non-commercial sexual relationships, in which unprotected sex is common. Interventions to reduce the risk of sexually transmitted infections in prostitutes should address both commercial and non-commercial sexual partnerships.

PIP: In a cross sectional survey, 280 female prostitutes were recruited between April 1989 and August 1991 by referral from health workers in the genitourinary medicine clinic at St. Mary;s Hospital, London, England, and referral from friends and colleagues of prostitutes, fieldwork (visiting streets, magistrates' courts, flats, agencies, and saunas), and telephone contacts. The objective was to measure the prevalence of HIV and to describe established risk factors in female prostitutes. 228 of the women had HIV tests, and 2 (09%) were infected with HIV-1. A high 98% (251/255) of women used condoms with all clients, while 12% (25/207) did with nonpaying partners for vaginal intercourse, 22 of the women had a history of blood transfusion; 22 women were current or past iv drug users; 53 reported use of injected drugs either by themselves or by their sexual partners; and 58 reported having sex with bisexual men and 4 with men known to be infected with HIV. Women recruited through fieldwork were more likely to report use of injected drugs than those interviewed at the clinic (11/87 (13%) vs. 11/193 (6%). 193 women were examined for sexually transmitted infections (STDs) on the day of their interview or within a week later, and 27 had one or more current, acute infections; 9 had gonorrhoea, 12 chlamydia, 7 trichomonas, and 4 primary genital herpes. Infection was related to younger age and increasing numbers of nonpaying sexual partners but not to duration of prostitution, numbers of clients, or reports of condom failures. The age and numbers of nonpaying partners remained significantly associated with STDs when analyzed by logistic regression. There was a significant risk of other sexually transmitted infections associated with the prostitutes frequently unprotected, noncommercial sexual relationships. Interventions should consider both commercial and noncommercial sexual partnerships in order to reduce the risk of sexually transmitted infections in prostitutes.