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.

Extracellular thermostable proteolytic activity of the milk spoilage bacterium Pseudomonas fluorescens PS19 on bovine caseins.

We studied the thermostable proteolytic activity of Pseudomonas fluorescens PS19 isolated from raw bovine milk. The heat-treated cell-free supernatant (HT-CFS) contained a thermostable protease of approximately 45 kDa, as revealed by casein zymography. We assigned this enzyme to P. fluorescens AprX metalloprotease (UniProtKB Acc. No. C9WKP6). After concentration by ultrafiltration at 10 kDa, the HT-CFS showed 2 other thermostable proteolytic bands on zymogram, with molecular masses of approximately 15 and 25 kDa. The former resulted a fragment of the AprX protease, whereas the 25-kDa protease was not homologous to any known protein of Pseudomonas spp. Subsequently, we assessed the proteolytic activity of the HT-CFS on bovine αS-, ß-, and κ-casein during in vitro incubation at 7 or 22°C. By means of ultra-performance liquid chromatography-tandem mass spectrometry we identified the released peptides (n=591). Some of them resisted proteolysis during the whole incubation period at both incubation temperatures and, therefore, they could be assumed as indicators of the proteolytic action of P. fluorescens PS19 on bovine caseins.

Metabolic labeling and protein linearization technology allow the study of proteins secreted by cultured cells in serum-containing media.

Supernatants from cell cultures (also called conditioned media, CMs) are commonly analyzed to study the pool of secreted proteins (secretome). To reduce the exogenous protein background, serum-free media are often used to obtain CMs. Serum deprivation, however, can severely affect cell viability and phenotype, including protein secretion. We present a strategy to analyze the proteins secreted by cells in fetal bovine serum-containing CMs, which combines the advantage of metabolic labeling and protein concentration linearization techniques. Incubation of CMs with a hexapeptide ligand library was used to reduce the dynamic range of the samples and led to the identification of 3 times more proteins than in untreated CM samples. Labeling with a deuterated amino acid was used to distinguish between cellular proteins and homologous bovine proteins contained in the medium. Application of the strategy to two breast cancer cell lines led to the identification of proteins secreted in different amounts and which could correlate with their varying degree of aggressiveness. Selected reaction monitoring (SRM)-based quantitation of three proteins of interest in the crude samples yielded data in good agreement with the results from concentration-equalized samples.

[Carbamazepine-induced agranulocytosis. Apropos of 2 cases]. / Agranulocitosi indotta da carbamazepina. A proposito di due casi.

Carbamazepine-induced agranulocytosis (CIA) is a rare event. We report on two cases, highlighting the wide variability of the bone marrow, which may show pseudohypercellularity with disappearance of neutrophils and excess of immature cells (myeloblasts and promyelocytes), thus mimicking the features of acute myeloid leukemia. Although its pathogenesis is still unclear, CIA must be considered as an idiosyncratic hemopathy and moreover it appears to be an unpredictable complication among patients taking the drug. It should be clearly distinguished from the benign neutropenia frequently associated with carbamazepine therapy and often self-limiting. Anyhow a careful clinical and hematological monitoring is the only mean to recognize promptly this life-threatening disease and to treat it with the withdrawal of the drug and the administration of an adequate anti-infectious therapy.

Chronic-relapsing polyneuropathy in the course of cryoglobulinemia. Clinical aspects and plasmapheretic treatment.

During the last 3 years we have observed 7 cases (4.1%) of cryoglobulinemia-associated polyneuropathy out of 167 patients with demonstrated cryoglobulinemia. Nerve involvement in the course of cryoglobulinemia is usually symmetrical, affecting the distal portions of the limbs and has a chronic or chronic-relapsing evolution. In our experience, the combined treatment by plasmapheresis and glucocorticoids and/or cytostatic drugs has proved to be quite effective, particularly when instituted during the symptomatic exacerbations. Therefore, we suggest to perform a systematic investigation of the cryoglobulinemic phenomenon in every patient presenting with symptoms and/or signs of an apparently idiopathic polyneuropathy.

Heart involvement in essential mixed cryoglobulinemia.

Cardiovascular involvement has been investigated in the course of essential mixed cryoglobulinemia (EMC). A direct pathogenetic role of cryoglobulins, due to coexisting coronary risk factors, is difficult to demonstrate. Nevertheless, a clear role of cryoglobulins in determining heart damage was hypothesized. A significant statistical incidence (p less than 0.05) of heart impairment was found in patients affected by EMC compared with an age- and sex-matched control group. Cryoglobulinemic coronary vasculitis found at post-mortem examination further supports this point of view.