SIGLEC-G deficiency increases susceptibility to develop B-cell lymphoproliferative disorders.

The sialic-acid-binding immunoglobulin-like lectin SIGLEC-G is a negative regulator of B-cell receptor-mediated calcium signaling. Its deficiency leads to reduced turnover and increased proliferation and survival of murine B-1a cells. Siglecg(-/-) mice show a premature expansion of polyclonal CD5(+) B cells in the spleen and the peritoneal cavity. Here we studied the fate of B lymphocytes in Siglecg(-/-) mice over time. We demonstrate that in aging animals SIGLEC-G deficiency promotes progressive accumulation of monoclonal B lymphocytes and increases the susceptibility to develop B-cell lymphoproliferative disorders. Lymphoid tumors arising in aged Siglecg(-/-) mice are monoclonal and histologically heterogeneous as they include diffuse large B-cell lymphoma, follicular lymphoma, and medium-to-large B-cell monomorphic lymphoma but surprisingly not chronic lymphocytic leukemia. The tumors express high levels of BCL-2 and are transplantable. In keeping with these findings we have also observed a remarkable down-regulation of the human ortholog SIGLEC10 in human B-cell lymphoma and leukemia cell lines. Taken together, these observations indicate that the down-regulation of negative B-cell receptor regulators such as SIGLEC-G/SIGLEC10 may represent another mechanism relevant to the pathogenesis of B-cell lymphomas.

Pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.

Cells derived from blood vessels of human skeletal muscle can regenerate skeletal muscle, similarly to embryonic mesoangioblasts. However, adult cells do not express endothelial markers, but instead express markers of pericytes, such as NG2 proteoglycan and alkaline phosphatase (ALP), and can be prospectively isolated from freshly dissociated ALP(+) cells. Unlike canonical myogenic precursors (satellite cells), pericyte-derived cells express myogenic markers only in differentiated myotubes, which they form spontaneously with high efficiency. When transplanted into severe combined immune deficient-X-linked, mouse muscular dystrophy (scid-mdx) mice, pericyte-derived cells colonize host muscle and generate numerous fibres expressing human dystrophin. Similar cells isolated from Duchenne patients, and engineered to express human mini-dystrophin, also give rise to many dystrophin-positive fibres in vivo. These data show that myogenic precursors, distinct from satellite cells, are associated with microvascular walls in the human skeletal muscle, may represent a correlate of embryonic 'mesoangioblasts' present after birth and may be a promising candidate for future cell-therapy protocols in patients.

An evolutionarily acquired genotoxic response discriminates MyoD from Myf5, and differentially regulates hypaxial and epaxial myogenesis.

Despite having distinct expression patterns and phenotypes in mutant mice, the myogenic regulatory factors Myf5 and MyoD have been considered to be functionally equivalent. Here, we report that these factors have a different response to DNA damage, due to the presence in MyoD and absence in Myf5 of a consensus site for Abl-mediated tyrosine phosphorylation that inhibits MyoD activity in response to DNA damage. Genotoxins failed to repress skeletal myogenesis in MyoD-null embryos; reintroduction of wild-type MyoD, but not mutant Abl phosphorylation-resistant MyoD, restored the DNA-damage-dependent inhibition of muscle differentiation. Conversely, introduction of the Abl-responsive phosphorylation motif converts Myf5 into a DNA-damage-sensitive transcription factor. Gene-dosage-dependent reduction of Abl kinase activity in MyoD-expressing cells attenuated the DNA-damage-dependent inhibition of myogenesis. The presence of a DNA-damage-responsive phosphorylation motif in vertebrate, but not in invertebrate MyoD suggests an evolved response to environmental stress, originated from basic helix-loop-helix gene duplication in vertebrate myogenesis.

Necdin mediates skeletal muscle regeneration by promoting myoblast survival and differentiation.

Regeneration of muscle fibers that are lost during pathological muscle degeneration or after injuries is sustained by the production of new myofibers. An important cell type involved in muscle regeneration is the satellite cell. Necdin is a protein expressed in satellite cell-derived myogenic precursors during perinatal growth. However, its function in myogenesis is not known. We compare transgenic mice that overexpress necdin in skeletal muscle with both wild-type and necdin null mice. After muscle injury the necdin null mice show a considerable defect in muscle healing, whereas mice that overexpress necdin show a substantial increase in myofiber regeneration. We also find that in muscle, necdin increases myogenin expression, accelerates differentiation, and counteracts myoblast apoptosis. Collectively, these data clarify the function and mechanism of necdin in skeletal muscle and show the importance of necdin in muscle regeneration.

Mesoangioblast stem cells ameliorate muscle function in dystrophic dogs.

Duchenne muscular dystrophy remains an untreatable genetic disease that severely limits motility and life expectancy in affected children. The only animal model specifically reproducing the alterations in the dystrophin gene and the full spectrum of human pathology is the golden retriever dog model. Affected animals present a single mutation in intron 6, resulting in complete absence of the dystrophin protein, and early and severe muscle degeneration with nearly complete loss of motility and walking ability. Death usually occurs at about 1 year of age as a result of failure of respiratory muscles. Here we report that intra-arterial delivery of wild-type canine mesoangioblasts (vessel-associated stem cells) results in an extensive recovery of dystrophin expression, normal muscle morphology and function (confirmed by measurement of contraction force on single fibres). The outcome is a remarkable clinical amelioration and preservation of active motility. These data qualify mesoangioblasts as candidates for future stem cell therapy for Duchenne patients.

Mesoangioblasts at 20: From the embryonic aorta to the patient bed.

In 2002 we published an article describing a population of vessel-associated progenitors that we termed mesoangioblasts (MABs). During the past decade evidence had accumulated that during muscle development and regeneration things may be more complex than a simple sequence of binary choices (e.g., dorsal vs. ventral somite). LacZ expressing fibroblasts could fuse with unlabelled myoblasts but not among themselves or with other cell types. Bone marrow derived, circulating progenitors were able to participate in muscle regeneration, though in very small percentage. Searching for the embryonic origin of these progenitors, we identified them as originating at least in part from the embryonic aorta and, at later stages, from the microvasculature of skeletal muscle. While continuing to investigate origin and fate of MABs, the fact that they could be expanded in vitro (also from human muscle) and cross the vessel wall, suggested a protocol for the cell therapy of muscular dystrophies. We tested this protocol in mice and dogs before proceeding to the first clinical trial on Duchenne Muscular Dystrophy patients that showed safety but minimal efficacy. In the last years, we have worked to overcome the problem of low engraftment and tried to understand their role as auxiliary myogenic progenitors during development and regeneration.

IL1R8 Deficiency Drives Autoimmunity-Associated Lymphoma Development.

Chronic inflammation, including that driven by autoimmunity, is associated with the development of B-cell lymphomas. IL1R8 is a regulatory receptor belonging to the IL1R family, which negatively regulates NF-κB activation following stimulation of IL1R or Toll-like receptor family members. IL1R8 deficiency is associated with the development of severe autoimmune lupus-like disease in lpr mice. We herein investigated whether concomitant exacerbated inflammation and autoimmunity caused by the deficiency of IL1R8 could recapitulate autoimmunity-associated lymphomagenesis. We thus monitored B-cell lymphoma development during the aging of IL1R8-deficient lpr mice, observing an increased lymphoid cell expansion that evolved to diffuse large B-cell lymphoma (DLBCL). Molecular and gene-expression analyses showed that the NF-κB pathway was constitutively activated in Il1r8 -/-/lpr B splenocytes. In human DLBCL, IL1R8 had reduced expression compared with normal B cells, and higher IL1R8 expression was associated with a better outcome. Thus, IL1R8 silencing is associated with increased lymphoproliferation and transformation in the pathogenesis of B-cell lymphomas associated with autoimmunity.

Acid-base balance and oxygen tension during dialysis in uremic patients with chronic obstructive pulmonary disease.

Recent reports on the effects of dialysis on acid-base balance and metabolic acidosis correction in end-stage renal disease (ESRD) patients with chronic obstructive pulmonary disease (COPD) are lacking. Here, we compared acid-base balance and blood gasses among 14 patients with established COPD (group A) and eight patients with normal respiratory function (group B). The two groups were homogeneous for age, time on dialysis, and male/female ratio. At the beginning of dialysis, acid-base balance and blood gasses were comparable between patients of groups A and B. A significant difference between groups was observed only in pCO(2) at 20 min, together with a delay in pH increase. Effective correction of acidosis was reported at the end of dialysis and is not significantly affected by COPD. Nevertheless, weight loss must be carefully monitored in these patients in order to prevent hyperhydration and worsening of respiratory function.

Silencing Nfix rescues muscular dystrophy by delaying muscle regeneration.

Muscular dystrophies are severe disorders due to mutations in structural genes, and are characterized by skeletal muscle wasting, compromised patient mobility, and respiratory functions. Although previous works suggested enhancing regeneration and muscle mass as therapeutic strategies, these led to no long-term benefits in humans. Mice lacking the transcription factor Nfix have delayed regeneration and a shift toward an oxidative fiber type. Here, we show that ablating or silencing the transcription factor Nfix ameliorates pathology in several forms of muscular dystrophy. Silencing Nfix in postnatal dystrophic mice, when the first signs of the disease already occurred, rescues the pathology and, conversely, Nfix overexpression in dystrophic muscles increases regeneration and markedly exacerbates the pathology. We therefore offer a proof of principle for a novel therapeutic approach for muscular dystrophies based on delaying muscle regeneration.

Mesoangioblasts, vessel-associated multipotent stem cells, repair the infarcted heart by multiple cellular mechanisms: a comparison with bone marrow progenitors, fibroblasts, and endothelial cells.

OBJECTIVE: To test the potential of mesoangioblasts (Mabs) in reducing postischemic injury in comparison with bone marrow progenitor cells (BMPCs), fibroblasts (Fbs), and embryonic stem cell-derived endothelial cells (ECs), and to identify putative cellular protective mechanisms. METHODS AND RESULTS: Cells were injected percutaneously in the left ventricular (LV) chamber of C57BL/6 mice, 3 to 6 hours after coronary ligation, and detected in the hearts 2 days and 6 weeks later. Echocardiographic examinations were performed at 6 weeks. LV dilation was reduced and LV shortening fraction was improved with Mabs and BMPCs but not with ECs and Fbs. Donor cell colonization of the host myocardium was modest and predominantly in the smooth muscle layer of vessels. Capillary density was higher in the peripheral infarct area and apoptotic cardiomyocytes were fewer with Mabs and BMPCs. Mabs and BMPCs, but not Fbs or ECs, promoted survival of cultured cardiocytes under low-oxygen in culture. This activity was present in Mab-conditioned medium and could be replaced by a combination of basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-1, and hepatocyte growth factor (HGF), all of which are produced by these cells. Conditioned medium from Mabs, but not from Fbs, stimulated proliferation of smooth muscle cells in vitro. CONCLUSIONS: Mabs appear as effective as BMPCs in reducing postinfarction LV dysfunction, likely through production of antiapoptotic and angiogenic factors.

Serological immune response against ADAM10 pro-domain is associated with favourable prognosis in stage III colorectal cancer patients.

A humoral immune response against aberrant tumor proteins can be elicited in cancer patients, resulting in the production of auto-antibodies (Abs). By serological proteome analysis we identified the surface membrane protein ADAM10, a metalloproteinase that has a role in epithelial-tumor progression and invasion, as a target of the immune response in colorectal cancer (Crc). A screening carried out on the purified protein using testing cohorts of sera (Crc patients n = 57; control subjects n = 39) and validation cohorts of sera (Crc patients n = 49; control subjects n = 52) indicated that anti-ADAM10 auto-Abs were significantly induced in a large group (74%) of colon cancer patients, in particular in patients at stage II and III of the disease. Interestingly, in Crc patients classified as stage III disease, the presence of anti-ADAM10 auto-Abs in the sera was associated with a favourable follow-up with a significant shifting of the recurrence-free survival median time from 23 to 55 months. Even though the ADAM10 protein was expressed in Crc regardless the presence of auto-Abs, the immature/non-functional isoform of ADAM10 was highly expressed in the tumor of anti-ADAM10-positive patients and was the isoform targeted by the auto-Abs. In conclusion, the presence of anti-ADAM10 auto-Abs seems to reflect the increased tumor expression of the immunogenic immature-ADAM10 in a group of Crc patients, and is associated with a favourable prognosis in patients at stage III of the disease.

Bhlhb5 is expressed in the CNS and sensory organs during mouse embryonic development.

Basic loop helix transcription factors have been shown to be implicated in the specification of various cell types, and many are involved in different aspects of neural development. Here we describe the expression of Bhlhb5, belonging to the beta3 subfamily, in mouse embryos ranging from 9 to 16.5dpc. Bhlhb5 is initially detected at 9.5dpc in the neural tube, restricted to longitudinal ventral columns of neurons, extending from the hindbrain to the caudal spinal cord. At later stages the expression is maintained in the central nervous system, in particular in the brain, where it is detected in the outer and more mature layer of the forming cerebral cortex and in the spinal cord. In addition Bhlhb5 is expressed in the developing eye and hair follicles, in the epithelial layer of the cochlea in the developing inner, and in the nasal epithelium.

Dll4 and PDGF-BB convert committed skeletal myoblasts to pericytes without erasing their myogenic memory.

Pericytes are endothelial-associated cells that contribute to vessel wall. Here, we report that pericytes may derive from direct conversion of committed skeletal myoblasts. When exposed to Dll4 and PDGF-BB, but not Dll1, skeletal myoblasts downregulate myogenic genes, except Myf5, and upregulate pericyte markers, whereas inhibition of Notch signaling restores myogenesis. Moreover, when cocultured with endothelial cells, skeletal myoblasts, previously treated with Dll4 and PDGF-BB, adopt a perithelial position stabilizing newly formed vessel-like networks in vitro and in vivo. In a transgenic mouse model in which cells expressing MyoD activate Notch, skeletal myogenesis is abolished and pericyte genes are activated. Even if overexpressed, Myf5 does not trigger myogenesis because Notch induces Id3, partially sequestering Myf5 and inhibiting MEF2 expression. Myf5-expressing cells adopt a perithelial position, as occasionally also observed in wild-type (WT) embryos. These data indicate that endothelium, via Dll4 and PDGF-BB, induces a fate switch in adjacent skeletal myoblasts.

Nitric oxide release combined with nonsteroidal antiinflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy.

Duchenne muscular dystrophy is a relatively common disease that affects skeletal muscle, leading to progressive paralysis and death. There is currently no resolutive therapy. We have developed a treatment in which we combined the effects of nitric oxide with nonsteroidal antiinflammatory activity by using HCT 1026, a nitric oxide-releasing derivative of flurbiprofen. Here, we report the results of long-term (1-year) oral treatment with HCT 1026 of two murine models for limb girdle and Duchenne muscular dystrophies (alpha-sarcoglycan-null and mdx mice). In both models, HCT 1026 significantly ameliorated the morphological, biochemical, and functional phenotype in the absence of secondary effects, efficiently slowing down disease progression. HCT 1026 acted by reducing inflammation, preventing muscle damage, and preserving the number and function of satellite cells. HCT 1026 was significantly more effective than the corticosteroid prednisolone, which was analyzed in parallel. As an additional beneficial effect, HCT 1026 enhanced the therapeutic efficacy of arterially delivered donor stem cells, by increasing 4-fold their ability to migrate and reconstitute muscle fibers. The therapeutic strategy we propose is not selective for a subset of mutations; it provides ground for immediate clinical experimentation with HCT 1026 alone, which is approved for use in humans; and it sets the stage for combined therapies with donor or autologous, genetically corrected stem cells.

The meso-angioblast: a multipotent, self-renewing cell that originates from the dorsal aorta and differentiates into most mesodermal tissues.

We have previously reported the origin of a class of skeletal myogenic cells from explants of dorsal aorta. This finding disagrees with the known origin of all skeletal muscle from somites and has therefore led us to investigate the in vivo origin of these cells and, moreover, whether their fate is restricted to skeletal muscle, as observed in vitro under the experimental conditions used. To address these issues, we grafted quail or mouse embryonic aorta into host chick embryos. Donor cells, initially incorporated into the host vessels, were later integrated into mesodermal tissues, including blood, cartilage, bone, smooth, skeletal and cardiac muscle. When expanded on a feeder layer of embryonic fibroblasts, the clonal progeny of a single cell from the mouse dorsal aorta acquired unlimited lifespan, expressed hemo-angioblastic markers (CD34, Flk1 and Kit) at both early and late passages, and maintained multipotency in culture or when transplanted into a chick embryo. We conclude that these newly identified vessel-associated stem cells, the meso-angioblasts, participate in postembryonic development of the mesoderm, and we speculate that postnatal mesodermal stem cells may be derived from a vascular developmental origin.

Cell therapy of alpha-sarcoglycan null dystrophic mice through intra-arterial delivery of mesoangioblasts.

Preclinical or clinical trials for muscular dystrophies have met with modest success, mainly because of inefficient delivery of viral vectors or donor cells to dystrophic muscles. We report here that intra-arterial delivery of wild-type mesoangioblasts, a class of vessel-associated stem cells, corrects morphologically and functionally the dystrophic phenotype of virtually all downstream muscles in adult immunocompetent alpha-sarcoglycan (alpha-SG) null mice, a model organism for limb-girdle muscular dystrophy. When mesoangioblasts isolated from juvenile dystrophic mice and transduced with a lentiviral vector expressing alpha-SG were injected into the femoral artery of dystrophic mice, they reconstituted skeletal muscle in a manner similar to that seen in wild-type cells. The success of this protocol was mainly due to widespread distribution of donor stem cells through the capillary network, a distinct advantage of this strategy over previous approaches.