The AMPK-activator AICAR in thyroid cancer: effects on CXCL8 secretion and on CXCL8-induced neoplastic cell migration.

PURPOSE: The AMPK-activator AICAR recently raised great interest for its anti-cancer properties. With specific regard to thyroid cancer, AICAR reduces cancer cell growth, invasion and metastasis. CXCL8, a chemokine with several recognized tumorigenic effects, is abundantly secreted in thyroid cancer microenvironment. The aim of this study was to investigate if AICAR could inhibit the basal and the TNFα-induced CXCL8 secretion in normal human thyroid cells (NHT) and in thyroid cancer cell lines TPC-1 and BCPAP (RET/PTC and BRAFV600e mutated, respectively). METHODS: The effect of AICAR on basal and CXCL8-induced cell migration was assessed. Cells were incubated with AICAR (0.05, 0.5, 1, 2 mM) alone or in combination with TNF-α (10 ng/ml) for 24 h. CXCL8 concentrations were measured in cell supernatants. Transwell migration assays were performed in NHT, TPC-1 and BCPAP, basally and after treatment with AICAR (2 mM) and rh-CXCL8 (50 ng/ml) alone or in combination. RESULTS: AICAR dose dependently inhibited the basal secretion of CXCL8 in TPC-1 (F = 4.26; p < 0.007) and BCPAP (F = 6.75; p < 0.0001) but not in NHT. TNFα-induced CXCL8 secretion was dose dependently reduced by AICAR in NHT (F = 9.99; p < 0.0001), TPC-1 (F = 9.25; p < 0.0001) and BCPAP (F = 6.82; p < 0.0001). AICAR significantly reduced the basal migration of TPC-1 and BCPAP but not of NHT. CONCLUSIONS: CXCL8-induced cell migration was inhibited in NHT, TPC-1 and BCPAP. This is the first demonstration of the inhibition of CXCL8 secretion exerted by AICAR in TPC-1 and BCPAP indicating that the anti-cancer properties of AICAR are, at least in part, mediated by its ability to reduce the pro-tumorigenic effects of CXCL8.

In vitro generation of platelets: Where do we stand?

Millions of platelets, specialized cells that participate in haemostatic and inflammatory functions, are transfused each year worldwide, but their supply is limited. Platelets are produced by megakaryocytes by extending proplatelets, directly into the bloodstream. Bone marrow structure and extracellular matrix composition together with soluble factors (e.g. Thrombopoietin) are key regulators of megakaryopoiesis by supporting cell differentiation and platelet release. Despite this knowledge, the scarcity of clinical cures for life threatening platelet diseases is in a large part due to limited insight into the mechanisms that control the developmental process of megakaryocytes and the mechanisms that govern the production of platelets within the bone marrow. To overcome these limitations, functional human tissue models have been developed and studied to extrapolate ex vivo outcomes for new insight on bone marrow functions in vivo. There are many challenges that these models must overcome, from faithfully mimicking the physiological composition and functions of bone marrow, to the collection of the platelets generated and validation of their viability and function for human use. The overall goal is to identify innovative instruments to study mechanisms of platelet release, diseases related to platelet production and new therapeutic targets starting from human progenitor cells.

The role of the extracellular matrix in primary myelofibrosis.

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm that arises from clonal proliferation of hematopoietic stem cells and leads to progressive bone marrow (BM) fibrosis. While cellular mutations involved in the development of PMF have been heavily investigated, noteworthy is the important role the extracellular matrix (ECM) plays in the progression of BM fibrosis. This review surveys ECM proteins contributors of PMF, and highlights how better understanding of the control of the ECM within the BM niche may lead to combined therapeutic options in PMF.

Differential clinical effects of different mutation subtypes in CALR-mutant myeloproliferative neoplasms.

A quarter of patients with essential thrombocythemia or primary myelofibrosis carry a driver mutation of CALR, the calreticulin gene. A 52-bp deletion (type 1) and a 5-bp insertion (type 2 mutation) are the most frequent variants. These indels might differentially impair the calcium binding activity of mutant calreticulin. We studied the relationship between mutation subtype and biological/clinical features of the disease. Thirty-two different types of CALR variants were identified in 311 patients. Based on their predicted effect on calreticulin C-terminal, mutations were classified as: (i) type 1-like (65%); (ii) type 2-like (32%); and (iii) other types (3%). Corresponding CALR mutants had significantly different estimated isoelectric points. Patients with type 1 mutation, but not those with type 2, showed abnormal cytosolic calcium signals in cultured megakaryocytes. Type 1-like mutations were mainly associated with a myelofibrosis phenotype and a significantly higher risk of myelofibrotic transformation in essential thrombocythemia. Type 2-like CALR mutations were preferentially associated with an essential thrombocythemia phenotype, low risk of thrombosis despite very-high platelet counts and indolent clinical course. Thus, mutation subtype contributes to determining clinical phenotype and outcomes in CALR-mutant myeloproliferative neoplasms. CALR variants that markedly impair the calcium binding activity of mutant calreticulin are mainly associated with a myelofibrosis phenotype.

The genomic and proteomic blueprint of mouse megakaryocytes derived from embryonic stem cells.

BACKGROUND: Platelets are specialized cells, produced by megakaryocytes (MKs) in the bone marrow, which represent the first defense against hemorrhage. There are many diseases where platelet production or function is impaired, with severe consequences for patients. Therefore, new insights into the process of MK differentiation and platelet formation would have a major impact on both basic and clinical research. OBJECTIVES: Embryonic stem (ES) cells represent a good in vitro model to study the differentiation of MKs, with the possibility of being genetically engineered and constituting an unlimited source of MKs. However, lack of knowledge about the molecular identity of ES-derived MKs (ES-MKs) may prevent any further development and application of this model. METHODS: This paper presents the first comprehensive transcriptional and proteome profile analyses of mouse ES-MKs in comparison with MKs derived from mouse fetal liver progenitors (FL-MKs). RESULTS: In ES-MKs we found a down-regulation of cytoskeleton proteins, specific transcription factors and membrane receptors at both transcriptional and protein levels. At the phenotypic level, this molecular blueprint was displayed by ES-MKs' lower polyploidy, lower nuclear/cytoplasm ratio and reduced capacity to form proplatelets and releasing platelets. CONCLUSIONS: Overall our data demonstrate that ES-MKs represent a useful model to clarify many aspects of both MK physiology and pathological conditions where impaired MK functions are related to defective MK development, as in inherited thrombocytopenias and primary myelofibrosis.

Proplatelet formation in heterozygous Bernard-Soulier syndrome type Bolzano.

BACKGROUND: Although mutations of GPIb alpha are among the most frequent causes of inherited platelet disorders, the mechanisms for the onset of thrombocytopenia and platelet macrocytosis are still poorly defined. OBJECTIVE: In this work we analyzed in vitro megakaryocyte differentiation and proplatelet formation in six subjects heterozygous for the Ala156Val mutation in the GPIb alpha (Bolzano mutation). METHODS: Human megakaryocytes were obtained by differentiation of patient cord blood-derived CD34(+) cells and peripheral blood-derived CD45(+) cells. Proplatelet formation was evaluated by phase contrast and fluorescence microscopy. RESULTS: Megakaryocyte differentiation from both cord blood (one patient) and peripheral blood (five patients) was comparable to controls. However, proplatelet formation was reduced by about 50% with respect to controls. An identical defect of proplatelet formation was observed when megakaryocytes were plated on fibrinogen, von Willebrand factor or grown in suspension. Morphological evaluation of proplatelet formation revealed an increased size of proplatelet tips, which was consistent with the increased diameters of patients' blood platelets. Moreover, alpha-tubulin distribution within proplatelets was severely deranged. CONCLUSIONS: Megakaryocytes from patients carrying a Bolzano allele of GPIb alpha display both quantitative and qualitative abnormalities of proplatelet formation in vitro. These results suggest that a defect of platelet formation contributes to macrothrombocytopenia associated to the Bolzano mutation, and indicate a key role for GPIb alpha in proplatelet formation.

Adhesive receptors, extracellular proteins and myosin IIA orchestrate proplatelet formation by human megakaryocytes.

BACKGROUND: Megakaryocytes release platelets from the tips of cytoplasmic extensions, called proplatelets. In humans, the regulation of this process is still poorly characterized. OBJECTIVE: To analyse the regulation of proplatelet formation by megakaryocyte adhesion to extracellular adhesive proteins through different membrane receptors. METHODS: Human megakaryocytes were obtained by differentiation of cord blood-derived CD34(+) cells, and proplatelet formation was evaluated by phase contrast and fluorescence microscopy. RESULTS: We found that human megakaryocytes extended proplatelets in a time-dependent manner. Adhesion to fibrinogen, fibronectin or von Willebrand factor (VWF) anticipated the development of proplatelets, but dramatically limited both amplitude and duration of the process. Type I, but not type III or type IV, collagen totally suppressed proplatelet extension, and this effect was overcome by the myosin IIA antagonist blebbistatin. Integrin alphaIIbbeta3 was essential for megakaryocyte spreading on fibrinogen or VWF, but was not required for proplatelet formation. In contrast, proplatelet formation was prevented by blockade of GPIb-IX-V, or upon cleavage of GPIbalpha by the metalloproteinase mocarhagin. Membrane-associated VWF was detected exclusively on proplatelet-forming megakaryocytes, but not on round mature cells that do not extend proplatelets. CONCLUSIONS: Our findings show that proplatelet formation in human megakaryocytes undergoes a complex spatio-temporal regulation orchestrated by adhesive proteins, GPIb-IX-V and myosin IIA.

Cord blood in vitro expanded CD41 cells: identification of novel components of megakaryocytopoiesis.

BACKGROUND: Megakaryopoiesis represents a multi-step, often unclear, process leading to commitment, differentiation, and maturation of megakaryocytes (MKs) that release platelets. AIM: To identify the novel genes that might help to clarify the molecular mechanisms of megakaryocytopoiesis and be regarded as potential candidates of inherited platelet defects, global gene expression of hematopoietic lineages was carried out. METHODS: Human cord blood was used to purify CD34+ stem cells and in vitro expand CD41+ cells and burst-forming unit-erythroid (BFU-E). We investigated the expression profiles of these three hematopoietic lineages in the Affymetrix system and selected genes specifically expressed in MKs by comparing transcripts of the different lineages using the dchip and pam algorithms. RESULTS: A detailed characterization of MK population showed that 99% of cells expressed the CD41 antigen whereas 73% were recognizable as terminally differentiated fetal MKs. The profile of these cells was compared with that of CD34+ cells and BFU-E allowing us to select 70 transcripts (MK-core), which represent not only the genes with a well-known function in MKs, but also novel genes never detected or characterized in these cells. Moreover, the specific expression was confirmed at both RNA and protein levels, thus validating the 'MK-core' isolated by informatics tools. CONCLUSIONS: This is a global gene expression that for the first time depicts a well-characterized population of cord blood-derived fetal MKs. Novel genes have been detected, such as those encoding components of the extracellular matrix and basal membrane, which have been found in the cytoplasm of Mks, suggesting that new physiological aspects of MKs should be studied.

Altered cytoskeleton organization in platelets from patients with MYH9-related disease.

MYH9-related disease (MYH9-RD) is an autosomal dominant disorder deriving from mutations in the MYH9 gene encoding for the heavy chain of non-muscle myosin IIA, and characterized by thrombocytopenia and giant platelets. Isoform IIA of myosin is the only one expressed in platelets, but the possibility that MYH9 mutations affect the organization of contractile structures in these blood elements has never been investigated. In this work we have analyzed the composition and the agonist-induced reorganization of the platelet cytoskeleton from seven MYH9-RD patients belonging to four different families. We found that an increased amount of myosin was constitutively associated with actin in the cytoskeleton of resting MYH9-RD platelets. Upon platelet stimulation, an impaired increase in the total cytoskeletal proteins was observed. Moreover, selected membrane glycoproteins, tyrosine kinases, and small GTPases failed to interact with the cytoskeleton in agonist-stimulated MYH9-RD platelets. These results demonstrate for the first time that mutations of MYH9 result in an alteration of the composition and agonist-induced reorganization of the platelet cytoskeleton. We suggest that these abnormalities may represent the biochemical basis for the previously reported functional alterations of MYH9-RD platelets, and for the abnormal platelet formation from megakaryocytes, resulting in thrombocytopenia and giant platelets.

Utility of biochemical markers in the follow-up of heart transplant recipients.

Endomyocardial biopsy (EMB) is currently the standard method to diagnose acute graft rejection. However, considering the potential complications of this procedure, a noninvasive marker of rejection would be an ideal alternative or at least a helpful adjunct to posttransplant management. We measured myoglobin (Myo), creatine kinase MB mass (CK-MBm), troponin T (cTnT), serum amyloid A (SAA), and C-reactive protein (CRP) in 57 patients (mean age 37.5 years) who underwent orthotopic heart transplantation for end-stage cardiac failure between January and December 2001.Endomyocardial biopsies were performed routinely after surgery and histologically diagnosed rejection was graded according to the criteria of the International Society of Heart and Lung Transplantation. Concomittant with the biopsies, blood samples were drawn from the coronary sinus (central blood samples) and from a peripheral vein (peripheral blood samples) to assay biochemical markers. Among 149 EMB evaluated, 87 were negative (grade 0); 28 showed grade 1a rejection; 26 showed grade 1b; and 8 showed grade > 1b (2 were grade 2, 6 were grade 3a). Grades 0 and 1a were considered to be negative, while grades 1b and >1b were considered positive indicating potential acute graft rejection. cTnT, Myo, CK-MBm, SAA, and CRP levels were measured in 149 central blood samples and 149 peripheral blood samples. Myo and CK-MBm did not show significant changes. cTnT seems to be a potentially useful addition to the EMB results, while SAA and CRP showed variations with respect to EMB grade both in central and peripheral samples.

Comparative effects of retroviral-mediated gene transfer into primary human stromal cells of Flt3-ligand, interleukin 3 and GM-CSF on production of cord blood progenitor cells in long-term culture.

The effects of different cytokines on growth of human cord blood CD34 cells was studied by performing long-term culture (LTC) with primary human stromal cells transduced with genes for either Flt3-ligand (L) (human transmembrane, murine soluble or murine membrane-bound forms), human interleukin 3 (IL-3) or human GM-CSF. Molecular analysis of genomic DNA from transduced stromal cells using neo-specific polymerase chain reaction demonstrated gene transfer of G418-selected stromal cell populations. Enzyme-linked immunosorbent assay and biological assays of conditioned media from transduced stromal cells indicated expression and release of soluble cytokines. Numbers of both immature and more mature progenitors (colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte; CFU-GEMM, BFU-E, CFU-GM) were increased threefold compared to control in the Flt3-L (transmembrane) LTC throughout five weeks of culture. IL-3 and GM-CSF feeders increased progenitor cell output also, but these effects were significantly lower than Flt3-L feeders. The two Flt3-L isoform engineered feeders, Ex6 (soluble isoform) and 5H (membrane-bound isoform), showed a decreased effect compared to the transmembrane Flt3-L feeders and, in particular, Ex6 feeders were similar to control feeders and 5H feeders were comparable to Flt3-L feeders only in the first two weeks of LTC. These results were apparent also by limiting dilution assays that showed a higher frequency of pre-CFU in the transmembrane Flt3-L feeders compared to control and the other cytokine feeders. Exogenous addition of soluble growth factors to suspension cultures without feeder layers, while superior to stromal feeders for short-term expansion of early progenitors, were inferior to the long-term maintenance/output on stromal feeders. Pre-CFU analysis supported these data. These results may be of some significance to understanding the actions of Flt3-L on blood cell production.

Growth factors in the therapy of myelodysplasia: biological aspects.

Growth factors (GF) are reported to play an important role in the therapy of myelodisplastic syndromes (MDS). After in vitro administration a consistent group of MDS may respond to GF but the possibility of differentiation, regulation or expansion of myelodisplastic clones following GF therapy is still a question to be answered as their optimum dose and combinations. To validate if in vivo treatment with GF, may promote the regulation or the recovery of myelopoiesis and/or modify the clonality of the responses, we gave G-CSF after intensive chemotherapy in high risk MDS and acute leukemia evolving from MDS patients. According to our data the use of G-CSF after intensive chemotherapy may improve the CR rate without increase of leukemic transformation. However the answer were clonal and the remission duration remained very short so we suggest to utilize this time to perform other therapeutic strategies such as, when possible, the BMT.

[Capillary networks and osmotic uptake in fungiform papillae]. / Rete capillare e richiamo osmotico delle papille fungiformi.

In the research it has been tested the ability of convoluted capillary network of the fungiform papillae to act as countercurrent ionic exchanger and therefore to give rise a water uptake from the external environment. Manometric determinations of the water inflow during immersion of the tongue in solutions of different osmolality have shown a conspicuous water uptake, osmotic in nature and largely supported by the Na+ absorbed from the external solutions. The Na+ inhibits also the receptor electrical response to the stimulating Ca++ solutions. These observations show that the papillae can be considered as osmotic devices and emphasise also that the Na+ is directly involved in the papillary functional activity.

[Effect of metavanadate on papillary water uptake]. / Influenza del metavanadato sul richiamo idrico papillare.

It has been examined the influence of vanadium on the papillary osmotic water-salts uptake in order to differentiate it from water flow of other biological substrates as amphibia epithelia. Between the different vanadium compounds only the metavanadate is active and only after administration in the abdominal vein. The general influence of metavanadate is a facilitating one and concerns: electrical receptor afferent discharge, osmotic water uptake and ciliary motility. At the papillary level therefore vanadium is not at all an inhibitory agent as observed in many biological substrates. This observation rule out any analogy in the processes of water inflow operating respectively at the fungiform papillae and amphibia epithelia.

[Quantitative aspects of ciliary apparatus motility of fungiform papillae]. / Aspetti quantitativi della motilità dell'apparato ciliare delle papille fungiformi.

The present research is intended to evaluate the frequency of the ciliary beat in different formations of the frog's oral cavity. The phenomenon has been quantitatively evaluated on the television images of mucus-ciliary waves by means of a photometric method, in order to obtain an electrical indication of the beat frequency. The results show that frequency of the ciliary beat is not uniform in all formations examinated. Differences in the beat frequency were evidenced in the single coronae ciliatae, further the frequencies of the beat decreases sistematically from the base to the tip of the tongue. The cilia on the lingual edge and on the palat exhibit a similar behaviour. The uniformity of the ciliary behaviour in the whole oral cavity is stressed and the factors contributing to a particular ciliary frequency in the fungiform papillae are discussed.

[Facilitating effects of axotomy of the glossopharyngeal nerve on the motor reflex response of the frog medulla oblongata]. / Effetti facilitanti dell'assotomia del glossofaringeo sulla risposta riflessa motoria del midollo allungato di rana.

In the previous papers we have reported that the innervation of the frog lingual mucosa exercises a presynaptic inhibition at the bulbar region in order to integrate the mechanoreceptive information coming from the tongue. The present investigation was undertaken to establish if also the complementary tactile innervation of fungiform papillae has a similar ability. First of all it has been assayed the evocation of PAD tests by papillary innervation; then the facilitating effects, induced by the glossopharyngeal axotomy, on the afferents mass discharges recorded from hypoglossal nerve have been examined. Electrical stimulation of mechanical neurons at the root of IX produced DRP and DRR both at glossopharyngeal and hypoglossal nerves. After 5 and 10 days from glossopharyngeal axotomy becomes evident a considerable enhancement of efferent responses in many diastaltic arches of bulb. These effects strongly suggest a very evident inhibitory activity at the bulbar projection of papillary innervation. From all these observations it follows that presynaptic inhibition is joint to all the lingual mechanoreceptive afferences.

[Inhibitory effect on crossed bands in the tongue mechanoreceptor innervation]. / Influenza inibitoria dei fasci crociati cell'innervazione meccanocettiva linguale.

The inhibitory influence exercised at the bulbar level by crossed afferent innervation of the lingual mechanoreceptors has been examined in the frog. To reach this aim it has been electrically stimulated the central stump of both glossopharyngeal and hypoglossal unilaterally axotomized nerves and recorded the crossed reflex effects in the controlateral hypoglossal motoneurones. By comparison a similar reflex has been evoked by means of stimulation of intact glossopharyngeal and hypoglossal nerves in the same preparation. As regards the glossopharyngeal crossed afferences it has been observed that the stimulation of axotomized nerves intensifies controlateral reflex response, while stimulated intact afference produces an increased reflex only after controlateral hypoglossal section. This last kind of effect has been observed also in the case of a crossed innervation of the hypoglossal lingual afference. These results show a notable extension of lingual crossed mechanoreceptors innervation and indicate its ability to obtain complex contralateral inhibitory effects. This ability however is more evident as regards the glossopharyngeal nerve. These observations are useful in order to explain the properties of the same inhibitory influences exercised homolaterally by the mechanoreceptors afference.