A novel signalling screen demonstrates that CALR mutations activate essential MAPK signalling and facilitate megakaryocyte differentiation.

Most myeloproliferative neoplasm (MPN) patients lacking JAK2 mutations harbour somatic CALR mutations that are thought to activate cytokine signalling although the mechanism is unclear. To identify kinases important for survival of CALR-mutant cells, we developed a novel strategy (KISMET) that utilizes the full range of kinase selectivity data available from each inhibitor and thus takes advantage of off-target noise that limits conventional small-interfering RNA or inhibitor screens. KISMET successfully identified known essential kinases in haematopoietic and non-haematopoietic cell lines and identified the mitogen activated protein kinase (MAPK) pathway as required for growth of the CALR-mutated MARIMO cells. Expression of mutant CALR in murine or human haematopoietic cell lines was accompanied by myeloproliferative leukemia protein (MPL)-dependent activation of MAPK signalling, and MPN patients with CALR mutations showed increased MAPK activity in CD34 cells, platelets and megakaryocytes. Although CALR mutations resulted in protein instability and proteosomal degradation, mutant CALR was able to enhance megakaryopoiesis and pro-platelet production from human CD34+ progenitors. These data link aberrant MAPK activation to the MPN phenotype and identify it as a potential therapeutic target in CALR-mutant positive MPNs.

Toxoplasma gondii meningoencephalitis without cerebral MRI findings in a patient with ulcerative colitis under immunosuppressive treatment.

Toxoplasmosis is the most common opportunistic infection of the central nervous system in immunosupressed patients. It is usually presented as a space-occupying lesion detected by cerebral computerized tomography or magnetic resonance imaging. The diffuse form of the disease (diffuse toxoplasmic meningoencephalitis) lacks the characteristic cerebral radiologic findings rendering pre-mortem diagnosis much more difficult. Herein, we describe a case of toxoplasmic menincoencephalitis, without evidence of cerebral space-occupying lesions, in a patient with ulcerative colitis under combined therapy with systemic glucocorticoids and azathioprine. Diagnosis was based on microscopic examination of cerebrospinal fluid (CSF) for the parasite, whereas, RT-PCR for Toxoplasma gondii was negative. Taking into consideration the limitations of molecular methods, investigation of the etiology of meningeal involvement in patients under immunosuppressive therapy presenting positive serology of previous T. gondii infection, should include microscopic examination of CSF for parasite presence.

Intestinal barrier dysfunction in HIV infection: pathophysiology, clinical implications and potential therapies.

INTRODUCTION: Current pathogenetic aspects on HIV infection highlight the importance of a chronic immune activation ultimately leading to T lymphocyte homeostasis disruption and immune deregulation associated with disease manifestations and progression. It is widely accepted that this continuous immune activation in HIV infection is principally driven by the phenomenon of pathological microbial translocation (MT). METHODS: Review of the literature on the role of intestinal barrier dysfunction in HIV infection, with emphasis on the implicated pathophysiological mechanisms, clinical implications and potentially effective therapeutic interventions. FINDINGS: MT in HIV infection is promoted by a multifactorial disruption of all major levels comprising the intestinal barrier defense. Specifically, HIV infection disrupts the integrity of the intestinal biological (quantitative and qualitative alterations of gut microecology, overgrowth of pathogenic bacteria), immune (depletion of CD4(+) T cells, especially Th17 cells, increased CD4+ FoxP3+ Tregs, decreased mucosal macrophages phagocytic capacity, development of intestinal proinflammatory milieu) and mechanical barrier (enterocytes' apoptosis, disruption of tight junctions). Intestinal barrier dysfunction allows the passage of microbes and immunostimulatory bioproducts from the gut lumen first in the lamina propria and thereafter in the systemic circulation, thus continuously promoting a local and systemic inflammatory response. This chronic immune activation is associated with HIV disease progression, suboptimal response to HAART and development of non-AIDS comorbidities. CONCLUSIONS: We have reached a point where the effective control of HIV viremia by HAART should be combined with emerging pharmacological approaches aiming at the restoration of the intestinal barrier, targeting its diverse levels of structure and function. Elimination of the MT phenomenon would mitigate its effect on immune homeostasis, which might improve the prognosis of the HIV-infected patient in terms of morbidity and mortality.

Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2.

BACKGROUND: Somatic mutations in the Janus kinase 2 gene (JAK2) occur in many myeloproliferative neoplasms, but the molecular pathogenesis of myeloproliferative neoplasms with nonmutated JAK2 is obscure, and the diagnosis of these neoplasms remains a challenge. METHODS: We performed exome sequencing of samples obtained from 151 patients with myeloproliferative neoplasms. The mutation status of the gene encoding calreticulin (CALR) was assessed in an additional 1345 hematologic cancers, 1517 other cancers, and 550 controls. We established phylogenetic trees using hematopoietic colonies. We assessed calreticulin subcellular localization using immunofluorescence and flow cytometry. RESULTS: Exome sequencing identified 1498 mutations in 151 patients, with medians of 6.5, 6.5, and 13.0 mutations per patient in samples of polycythemia vera, essential thrombocythemia, and myelofibrosis, respectively. Somatic CALR mutations were found in 70 to 84% of samples of myeloproliferative neoplasms with nonmutated JAK2, in 8% of myelodysplasia samples, in occasional samples of other myeloid cancers, and in none of the other cancers. A total of 148 CALR mutations were identified with 19 distinct variants. Mutations were located in exon 9 and generated a +1 base-pair frameshift, which would result in a mutant protein with a novel C-terminal. Mutant calreticulin was observed in the endoplasmic reticulum without increased cell-surface or Golgi accumulation. Patients with myeloproliferative neoplasms carrying CALR mutations presented with higher platelet counts and lower hemoglobin levels than patients with mutated JAK2. Mutation of CALR was detected in hematopoietic stem and progenitor cells. Clonal analyses showed CALR mutations in the earliest phylogenetic node, a finding consistent with its role as an initiating mutation in some patients. CONCLUSIONS: Somatic mutations in the endoplasmic reticulum chaperone CALR were found in a majority of patients with myeloproliferative neoplasms with nonmutated JAK2. (Funded by the Kay Kendall Leukaemia Fund and others.).

Septic Thrombophlebitis Caused by Fusobacterium necrophorum in an Intravenous Drug User.

Septic thrombophlebitis is characterized by venous thrombosis, inflammation and bacteremia, that can lead to fatal complications such as sepsis, septic emboli and even death. Though most commonly caused by indwelling catheters, it is also related to intravenous drug users (IVDU) especially those who attempt to inject drugs into more proximal and central veins. Lemierre's syndrome, also referred to as post-anginal sepsis or necrobacillosis, is a suppurative thrombophlebitis of the internal jugular vein. Primary infection is associated with oropharyngeal and dental infections and the most common causative organism is Fusobacterium necrophorum. We report a case of Lemierre's syndrome in an IVDU, caused by Fusobacterium necrophorum, which was inoculated at the site of injection, without a history of sore throat or pharyngitis.

Severe sepsis and diabetes mellitus have additive effects on red blood cell deformability.

OBJECTIVES: Diabetes mellitus is accompanied by microvascular complications leading to organ dysfunction, while sepsis is a major cause of morbidity and mortality in diabetics. We addressed the hypothesis that red blood cell (RBC) deformability may be additively compromised in septic diabetic patients, leading to a further impairment of microcirculation. METHODS: Forty patients suffering from severe sepsis, 12 patients suffering from diabetes and 24 diabetic patients with severe sepsis were enrolled. A filtration method and a hemorheometer were used to measure the RBCs' index of rigidity (IR). RESULTS: We observed no differences in severity, organ dysfunction and outcome between diabetic and non-diabetic septic patients. Mean SAPS II score was 23.5% vs 26.8% in non-diabetic and diabetic septic patients, respectively. The mortality in non-diabetic septic patients was 22.5% and in septic diabetics was 34.3%, while septic shock occurred in 15.0% and 20.8%, respectively. We detected higher IR (17.72+/-6.31) in septic diabetics than in patients with diabetes and no sepsis (12.26+/-2.28, p< or =0.001) and in patients with sepsis and no diabetes (13.9+/-2.86, p< or =0.01). CONCLUSION: The presence of diabetes mellitus seems to affect the already compromised RBC deformability of septic patients, probably leading to serious microcirculatory functional impairments in septic diabetic patients.