The immunosuppressive cytokine interleukin-4 increases the clonogenic potential of prostate stem-like cells by activation of STAT6 signalling.

Interleukin-4 plays a critical role in the regulation of immune responses and has been detected at high levels in the tumour microenvironment of cancer patients, where concentrations correlate with the grade of malignancy. In prostate cancer, interleukin-4 has been associated with activation of the androgen receptor, increased proliferation and activation of survival pathways such as Akt and NF-κB. However, its role in therapy resistance has not yet been determined. Here we investigate the influence of interleukin-4 on primary epithelial cells from prostate cancer patients. Our data demonstrate an increase in the clonogenic potential of these cells when cultured in the presence of interleukin-4. In addition, a Phospho-Kinase Array revealed that in contrast to previously published work, signal transducer and activator of transcription6 (STAT6) is the only signalling molecule activated after interleukin-4 treatment. Using the STAT6-specific inhibitor AS1517499 we could confirm the role of STAT6 in increasing colony-forming frequency. However, clonogenic recovery assays revealed that interleukin-4 does not rescue the effects of either irradiation or docetaxel treatment. We therefore propose that although the interleukin-4/STAT6 axis does not appear to be involved in therapy resistance, it does play a crucial role in the colony-forming abilities of the basal cell population in prostate cancer. IL-4 may therefore contribute to disease relapse by providing a niche that is favourable for the clonogenic growth of prostate cancer stem cells.

The AAA+ ATPase RUVBL2 is a critical mediator of MLL-AF9 oncogenesis.

The most frequent chromosomal translocations in pediatric acute myeloid leukemia affect the 11q23 locus and give rise to mixed lineage leukemia (MLL) fusion genes, MLL-AF9 being the most prevalent. The MLL-AF9 fusion gene has been shown to induce leukemia in both mouse and human models. In this study, we demonstrate that leukemogenic activity of MLL-AF9 requires RUVBL2 (RuvB-like 2), an AAA+ ATPase family member that functions in a wide range of cellular processes, including chromatin remodeling and transcriptional regulation. Expression of RUVBL2 was dependent on MLL-AF9, as it increased upon immortalization of human cord blood-derived hematopoietic progenitor cells with the fusion gene and decreased following loss of fusion gene expression in conditionally immortalized mouse cells. Short hairpin RNA-mediated silencing experiments demonstrated that both the immortalized human cells and the MLL-AF9-expressing human leukemia cell line THP-1 required RUVBL2 expression for proliferation and survival. Furthermore, inhibition of RUVBL2 expression in THP-1 cells led to reduced telomerase activity and clonogenic potential. These data were confirmed with a dominant-negative Walker B-mutated RUVBL2 construct. Taken together, these data suggest the possibility of targeting RUVBL2 as a potential therapeutic strategy for MLL-AF9-associated leukemia.

MLL-AF9-mediated immortalization of human hematopoietic cells along different lineages changes during ontogeny.

The MLL-AF9 fusion gene is associated with aggressive leukemias of both the myeloid and lymphoid lineage in infants, whereas in adults, this translocation is mainly associated with acute myeloid leukemia. These observations suggest that differences exist between fetal and adult tissues in terms of the 'cell of origin' from which the leukemia develops. Here we show that depending on extrinsic cues, human neonatal CD34(+) cells are readily immortalized along either the myeloid or lymphoid lineage upon MLL-AF9 expression and give rise to mainly lymphoid leukemia in immunocompromised mice. In contrast, immortalization of adult bone marrow CD34(+) cells is more difficult to achieve and is myeloid-biased, even when MLL-AF9 is expressed in purified hematopoietic stem cells (HSCs). Transcriptome analysis identified enrichment of HSC but not progenitor gene signatures in MLL-AF9-expressing cells. Although not observed in adult cells, neonatal cells expressing MLL-AF9 were enriched for gene signatures associated with poor prognosis, resistance to chemotherapeutic agents and MYC signaling. These results indicate that neonatal cells are inherently more prone to MLL-AF9-mediated immortalization than adult cells and suggest that intrinsic properties of the cell of origin, in addition to extrinsic cues, dictate lineage of the immortalized cell.

[Antiphospholipid antibodies and recurrent abortions: possible pathogenetic role of annexin A5 investigated by confocal microscopy]. / Anticorpi antifosfolipidi e aborti ricorrenti: possibile ruolo patogenetico dell'annessina A5 indagato al microscopio confocale.

AIM: It has been established that antiphospholipid antibodies (aPL) are associated with recurrent abortions, but the pathophysiologic mechanisms that characterize thrombosis and recurrent pregnancy losses are still not clear.However, it is known that they are associated with the presence of antibodies directed against anionic phospholipids and putative cofactors. In this study the pathogenetic role of annexin A5, a potent anticoagulant cofactor protein for its anticoagulant property in recurrent abortions, was investigated. METHODS: Endothelial cells of human umbilical veins ''EAHY2936 Line'' in culture were used, incubated with antiphospholipid anticardiolipin (ACA) antibodies purified from plasma of patients with recurrent abortions. The expression of annexin A5 on the cells with ACA was investigated by immunofluorescence and by confocal microscope. The negative control was also carried out: EAHY cells in cultivation medium without ACA. RESULTS: Confocal analysis revealed a uniform distribution of annexin A5 on the cellular membranes in the negative control. Instead, in EAHY cells with ACA, the annexin A5 appears distributed in irregular manners on the cellular membranes (cytoplasmic and nuclear). CONCLUSION: The results of an irregular ''cluster'' distribution of annexin A5 on the EAHY cells in presence of aPL, and in agreement with the literature, demonstrated that aPL, inhibiting annexin A5 ability to protect anionic phospholipid, promote the coagulation factors to diffuse laterally against phospholipids.

NUP98 dysregulation in myeloid leukemogenesis.

Nucleoporin 98 (NUP98) is a component of the nuclear pore complex that facilitates mRNA export from the nucleus. It is mapped to 11p15.5 and is fused to a number of distinct partners, including nine members of the homeobox family as a consequence of leukemia-associated chromosomal translocations. NUP98-HOXA9 is associated with the t(7;11)(p15;p15) translocation in acute myeloid leukemia (AML), myelodysplastic syndrome, and blastic crisis of chronic myeloid leukemia. Expression of NUP98-HOXA9 in murine bone marrow resulted in a myeloproliferative disease progressing to AML by 7-8 months. Transduction of NUP98 fusion genes into human CD34(+) cells confers a proliferative advantage in long-term cytokine-stimulated and stromal cocultures and in NOD-SCID engrafted mice, associated with a five- to eight-fold increase in hematopoietic stem cells. NUP98-HOXA9 expression inhibited erythroid and myeloid differentiation but enhanced serial progenitor replating. NUP98-HOXA9 upregulated a number of homeobox genes of the A and B cluster as well as MEIS1 and Pim-1, and downmodulated globin genes and C/EBPalpha. The HOXA9 component of the NUP98-HOXA9 fusion protein was protected from cullin-4A-mediated ubiquitination and subsequent proteasome-dependent degradation. In NUP98-HOX-transduced CD34(+) cells and cells from AML patients with t(7;11)(p15;p15) NUP98 was no longer associated with the nuclear pore complex but formed intranuclear aggregation bodies. Analysis of NUP98 allelic expression in AML and myelodysplastic syndrome showed loss of heterozygosity observed in 29% of the former and 8% of the latter. This was associated with poor prognosis.

Expression of constitutively active Notch4 (Int-3) modulates myeloid proliferation and differentiation and promotes expansion of hematopoietic progenitors.

The Notch family of transmembrane receptors has been implicated in the regulation of many developmental processes. In this study, we evaluated the role of Notch4 in immature hematopoietic progenitors by inducing, with retroviral transduction, enforced expression of Int-3, the oncogenic and constitutively active form of mouse Notch4. Int-3-transduced human myeloid leukemia (HL-60) cells demonstrated significantly delayed expression of differentiation markers following retinoic acid and 12-0-tetradecanoylphorbol 13-acetate treatment. Furthermore, HL-60 cells expressing Int-3 displayed a slower growth rate than cells infected with void virus, and accumulation in the G0/G1 phases of cell cycle. Transduction with deletion mutants of Int-3 defined the importance of individual domains of the protein (in particular, the ANK domain and the C-terminal domain) in the inhibition of differentiation and growth arrest of HL-60 cells. When mouse bone marrow enriched for stem cells (5-fluorouracil-resistant, lineage negative) was transduced and cultured for two weeks, the Int-3-transduced population displayed a lower expression of differentiation markers and a three- to five-fold higher frequency of colony-forming cells (CFU-GM/BFU-E) than control cultures. These results strongly support the notion that Notch signaling inhibits differentiation and promotes expansion of hematopoietic stem/progenitor cells.

Low-power diode laser stimulation of surgical osteochondral defects: results after 24 weeks.

The purpose of this study was to evaluate osteochondral lesions of the knee, treated intraoperatively with low-power laser stimulation, and assess results at 24 weeks. Surgery was performed under general anesthesia on six rabbits; a bilateral osteochondral lesion was created in the femoral medial condyles with a drill. All of the left lesions underwent immediate stimulation using the diode Ga-Al-As laser (780nm), whereas the right knees were left untreated as control group. After 24 weeks, the explants from the femoral condyles, either treated employing laser energy or left untreated, were examined histomorphometrically. Results obtained on the lased condyles showed good cell morphology and a regular aspect of the repaired osteocartilaginous tissue.

Laser biostimulation of cartilage: in vitro evaluation.

An in vitro study was performed to evaluate the laser biostimulation effect on cartilage using a new gallium-aluminium-arsenic diode laser. Chondrocyte cultures were derived from rabbit and human cartilage. These cells were exposed to laser treatment for 5 days, using the following parameters: 300 joules, 1 watt, 100 (treatment A) or 300 (treatment B) hertz, pulsating emission for 10 minutes, under a sterile laminar flow. Control cultures (no treatment) received the same treatment with the laser device off. Cell viability was measured by MTT assay at the end of the laser treatment and then after 5 days. Neither rabbit nor human cultured chondrocytes showed any damage under a light microscope and immunostaining control following laser treatment. The MTT test results indicated a positive biostimulation effect on cell proliferation with respect to the control group. The increase in viability of irradiated chondrocytes was maintained for five days following the end of the laser treatment. The results obtained with the Ga-Al-As diode laser using the above tested parameters for in vitro biostimulation of cartilage tissues provide a basis for a rational approach to the experimental and clinical use of this device.

Assessment of low-power laser biostimulation on chondral lesions: an "in vivo" experimental study.

The purpose of this study was to evaluate whether intraoperative laser biostimulation can enhance healing of cartilaginous lesions of the knee. Surgery was performed on eighteen rabbits: a bilateral chondral lesion of 1.25 +/- 0.2 mm in length and 0.8 +/- 0.2 mm in width was created in the femoral medial condyle with a scalpel. The lesion in the left knee of each animal was treated intraoperatively using the diode Ga-Al-As 780nm. laser (300 Joules/cm2, 1 Watt, 300 Hertz, 10 minutes), while the right knee was left untreated, as control group. The animals were divided into three groups, A, B and C, according to the survival time after surgery, two, six and twelve weeks, respectively. The explants from the femoral condyles, both treated employing laser energy and left untreated, were examined histologically. Results showed a progressive filling with fibrous tissue of the cartilaginous lesion treated with laser irradiation, while no changes in the original lesion of the untreated group were observed at the end of the study. Maybe, in this experimental research, underexposure to laser irradiation was the cause for the absence of the necessary conditions for biostimulation.

Osteochondral lesion repair of the knee in the rabbit after low-power diode Ga-Al-As laser biostimulation: an experimental study.

The purpose of this study was to evaluate whether low-power laser biostimulation of the osteo-chondral lesions of the knee could by itself reduce repair healing time. Surgery was performed on eighteen rabbits; a bilateral osteo-chondral lesion of 2.5mm in diameter and 2mm depth was created in the femoral medial condyle with a drill. The left knee of each animal was treated intraoperatively using the diode Ga-Al-As laser (780nm) with the following parameters: 300 Joules/cm2, 1 Watts, 300 Hertz, 10 minutes; the right knee was left untreated, as control group. The animals were divided into three groups, A, B and C, according to the survival time after surgery, two, six and twelve weeks, respectively. The explants from the femoral condyles, either treated employing laser energy or left untreated, were examined histomorphometrically. Results after laser treatment showed faster healing of the lesion at week 2 (p=0.043) and an overall improvement in cellular morphology (p=0.044), while a more regular aspect of the osteocartilaginous tissue was observed at week 12 (p=0.004). A relationship between laser biostimulation properties and healing of the osteo-chondral defect has been demonstrated.

Growth inhibition and synergistic induction of apoptosis by zoledronate and dexamethasone in human myeloma cell lines.

Bisphosphonates (BPs) are commonly used in the treatment of myeloma-associated osteolytic lesions. Recent reports have suggested that BPs may also exert direct antitumor effects on myeloma cells. Here, we show that the treatment of myeloma cell lines with the combination of the potent BP zoledronate and dexamethasone inhibits cell growth and synergistically induces apoptotic cell death, providing a rationale for potential applications in vivo.

Biostimulation of human chondrocytes with Ga-Al-As diode laser: 'in vitro' research.

The aim of this study was to verify the effects of laser therapy performed with Ga-Al-As Diode Lasers (780 nm, 2500 mW) on human cartilage cells in vitro. The cartilage sample used for the biostimulation treatment was taken from the right knee of a 19-year-old patient. After the chondrocytes were isolated and suspended for cultivation, the cultures were incubated for 10 days. The cultures were divided into four groups. Groups I, II, III were subject to biostimulation with the following laser parameters: 300 J, 1 W, 100 Hz, 10 min. exposure, pulsating emission; 300 J, 1 W, 300 Hz, 10 min. exposure, pulsating emission; and 300 J, 1 W, 500 Hz, 10 min. exposure, pulsating emission, respectively. Group IV did not receive any treatment. The laser biostimulation was conducted for five consecutive days. At the end of the treatment, the Calcium, Alkaline Phosphate, MTT tests and proteoglycan were performed to assess cell metabolism and toxicity level. The data showed good results in terms of cell viability and levels of Ca and Alkaline Phosphate in the groups treated with laser biostimulation compared to the untreated group. The results obtained confirm our previous positive in vitro results that the Ga-Al-As Laser provides biostimulation without cell damage.

The effect of osteopenia on the osteointegration of different biomaterials: histomorphometric study in rats.

The osteointegration of Hydroxyapatite (HA), Titanium (Ti-6Al-4V: Ti), Zirconia (ZrO2), Alumina (Al2O3) and 2 biological glasses (AP40 and RKKP) was comparatively investigated in normal and osteopenic rats by means of histomorphometry. Thirty-six Sprague Dawley female rats were left intact (Group C) while 36 were ovariectomized (Group OVX). Group C and OVX were further divided into 6 subgroups. After 16 weeks all animals were submitted to the femoral implant of nails made of the above-mentioned materials. Eight weeks after implantation the animals were euthanized, the femurs were harvested for histomorphometric analysis. The data showed that: (1) all the tested materials were biocompatible in vitro; (2) no significant differences existed in Affinity Index (AI) of Group C; and (3) results from paired comparison applied to the AI showed significant differences among the Groups C and OVX. The AI did not significantly change among intact groups, while it significantly decreased when some materials were implanted in OVX subgroups (AP40, ZrO2 and Ti-6Al-4V: p < 0.0005, p < 0.05 and p < 0.01). It is confirmed that bone mineral density is a strong predictor of the osteointegration of an orthopedic implant and that the use of pathological animal models is necessary to completely characterize biomaterials.

[Cartilage cell stimulation with low-power laser: experimental assessment]. / Stimolazione delle cellule cartilaginee con low-power laser: valutazione sperimentale.

The aim of this study was to verify the effects of laser therapy performed with Ga-Al-As diode laser (780 nm, 2500 mW) on cartilage cells in vitro. The cartilage sample used for biostimulation was taken from the knee of an adult patient. The cultures were divided into four groups: Groups I, II, III were subjected to biostimulation with different laser parameters; Group IV did not received any treatment. The laser biostimulation was conducted for five consecutive days. At the end of the treatment, cell count and MTT tests were performed to assess cell metabolism. The data showed good results in terms of cell viability in the groups treated with laser biostimulation compared to the untreated group. The results obtained with the use of this new low-power diode laser Ga-Al-As device in the biostimulation of the cartilage tissue, permits us to consider the use of this device clinically.

Purification and characterization of a human sialoglycoprotein antigen expressed in immature thymocytes and fetal tissues.

The monoclonal antibody UN1 was previously produced in our laboratory on the basis of selective reactivity with human thymocytes and has been classified as unclustered by the 5th and 6th International Workshop and Conference on Human Leukocyte Differentiation Antigens. The antigen recognized by mAb UN1 was found to be expressed on the cell surface of immature human thymocytes, a subpopulation of peripheral T lymphocytes and on several fetal tissues including thymus. The UN1 antigen is purified from children's thymus by ion-exchange and affinity chromatography. Two-dimensional electrophoresis shows that the purified antigen displays microheterogeneity appearing as multiple spots over a pI range 4.4-5.0 at 100-120 kDa. Treatment with neuraminidase results in a retarded migration in SDS-PAGE, an increase in isoelectric point and a reduction in carbohydrate content, indicating a substantial content of sialic acid. Glycosidase digestion and lectin-binding analysis indicate that the carbohydrate residues are essentially O-linked. A preliminary analysis has detected the UN1 antigen in human breast carcinoma tissues but not in normal breast. The biochemical features and the pattern of expression of the UN1 antigen indicate that this molecule may have the characteristics typical of the family of cell-membrane-associated mucin-like glycoproteins; a number of these molecules are thought to have a role in cell-cell interaction, tumor progression and metastasis.

In vitro experimental research of rabbit condrocytes biostimulation with diode laser Ga-Al-As: a preliminary study.

The scope of our study was to verify the effects of a new diode laser device with active material composed of Gallium, Aluminum and Arsenic (Ga-Al-As) configured as MOCVD (780 nm., 3000 mW) for the biostimulation of the cartilage cells in vitro. The condrocytes cells, withdrawn from the cartilage of the medial condyle of the femur of the rabbit, were cultivated, incubated and subject to biostimulation treatment with the laser. The condrocytes cells were placed in 24 Petri dishes at the concentration of 0.25 x 10(5)/ml and divided into 4 groups: 3 group (I, II, III) were treated with the laser and the fourth group (IV) was used as the control group. At the end of the treatment, all four groups, were evaluated with a MTT test and a cell count of the condrocytes cells. Group III (300 J, 1 Watt, 300 Hz, 10' of exposure time with a pulsating emission) provided the best results in terms of cell viability (MTT test) and for the number of cells found in the dishes when compared to the other treated groups and the control group. The results obtained with the use of this new diode laser Ga-Al-As device in the biostimulation of the cartilage tissue, permits us to consider the use of this device clinically.

A 5' regulatory sequence containing two Ets motifs controls the expression of the Wiskott-Aldrich syndrome protein (WASP) gene in human hematopoietic cells.

The recently-identified Wiskott-Aldrich syndrome protein gene (WASP) is responsible for the Wiskott-Aldrich X-linked immunodeficiency as well as for isolated X-linked thrombocytopenia (XLT). To characterize the regulatory sequences of the WASP gene, we have isolated, sequenced and functionally analyzed a 1.6-Kb DNA fragment upstream of the WASP coding sequence. Transfection experiments showed that this fragment is capable of directing efficient expression of the reporter chloramphenicol acetyltransferase (CAT) gene in all human hematopoietic cell lines tested. Progressive 5' deletions showed that the minimal sequence required for hematopoietic-specific expression consists of 137 bp upstream of the transcription start site. This contains potential binding sites for several hematopoietic transcription factors and, in particular, two Ets-1 consensus that proved able to specifically bind to proteins present in nuclear extracts of Jurkat cells. Overexpression of Ets-1 in HeLa resulted in transactivation of the CAT reporter gene under the control of WASP regulatory sequences. Disruption of the Ets-binding sequences by side-directed mutagenesis abolished CAT expression in Jurkat cells, indicating that transcription factors of the Ets family play a key role in the control of WASP transcription.

Identification by differential display of transcripts regulated during hematopoietic differentiation.

The polymerase chain reaction-based differential display method (DDRT-PCR) was used to identify mRNAs differentially expressed during the maturation of human CD34+ progenitor cells stimulated to differentiate in vitro towards granulomonocytic or erythroid lineages with a mixture of hemopoietins (kit ligand + interleukin 3 + GM-CSF in the absence or presence of erythropoietin, respectively). Three cDNA transcripts (B32, B41, and B56) display differential expression during cytokine-induced maturation of CD34+ cells. These clones have no homology with already-described sequences. Primer extension cofirmed the presence of the corresponding mRNA. The levels of mRNA corresponding to B32 are enhanced in the later phases of the granulomonocytic as well as in the erythroid differentiation of CD34+ cells. The mRNA identified by B41 was induced by a late stage in only granulomonocytic differentiation of CD34+ cells. The mRNA corresponding to B56 was instead present in nonstimulated CD34+ cells, declined in the early stages of differentiation, and reappeared at later stages in cells treated with both combinations of cytokines. Expression of these genes was detected in a number of acute myelogenous leukemias, as well as in some leukemic cell lines. B32 and B41 were downregulated in KG-1 cells induced to differentiate towards the monocytic lineage, whereas the levels of B56 were unchanged. In K562 cells, clones B41 and B56 were downregulated only in the late phases of PMA-induced megakaryocytic differentiation and during erythroid differentiation. B32 was rapidly downregulated when K562 cells were induced to differentiate towards either megakaryocytic or erythroid phenotypes. These transcripts represent novel hematopoietic cDNAs that should prove of value for the study of human blood cells and their disorders.