Climate change-induced ecosystem disturbance: a review on sclerophyllous and semi-deciduous forests in Tunisia.

According to the sixth assessment report of the Intergovernmental Panel on Climate Change (IPCC), global climate change is now unequivocal. Tunisia, like many other countries, has been affected by climate changes, including rising temperatures, intense heatwaves, and altered precipitation regimes. Tunisia's mean annual temperatures has risen about +1.4 °C in the twentieth century, with the most rapid warming taking place since the 1970s. Drought represents a primary contributing factor to tree decline and dieback. Long-term drought can result in reduced growth and health of trees, thereby increasing their susceptibility to insect pests and pathogens. Reported increases in tree mortality point toward accelerating global forest vulnerability under hotter temperatures and longer, more intense droughts. In order to assess the effect of these climate changes on the current state of forest ecosystems in Tunisia and their evolution, an investigative study was required. Here, we review the current state of knowledge on the effects of climate change on sclerophyllous and semi-deciduous forest ecosystems in Tunisia. Natural disturbance during recent years, as well as the adaptability and resilience of some forest species to climate change, were surveyed. The Standardized Precipitation Evapotranspiration Index (SPEI) is a multi-scalar drought index based on climate data that has been used to analyse drought variability. The SPEI time scale analysis showed a negative trend over the 1955-2021 period in Tunisian forest regions. In 2021, Tunisia lost 280 km2 of tree cover to fires, which is equivalent to 26% of the total lost area between 2008 and 2021. Changing climate conditions have also affected phenological parameters, with an advance in the start of the green season (SOS) of 9.4 days, a delay at the end of the green season (EOS) of 5 days, with a consequent extended duration of the green season (LOS) by an average of 14.2 days. All of these alarming findings invite us to seek adaptation strategies for forest ecosystems. Adapting forests to climate change is therefore a challenge for scientists as well as policymakers and managers.

Kinetics of growth, development and absorption of nutrients in stone pine (Pinus pinea L.) cultivated in the presence of NaCl.

The aim of the present work was to evaluate and analyze the growth and mineral nutrition response of stone pine (Pinus pinea L.) seedlings, an economically important forest species. We analyzed the salinity effects on the kinetics of growth, development, and absorption of nutrients of plants cultivated under controlled conditions on a solid organic substrate. Pinus pinea plants were able to tolerate 25 mM NaCl concentration without reduced growth compared to the non-saline control. However, the salt concentration of 50 mM significantly affected the seedling growth after two weeks of treatment. Root growth activity was decreased more than the aerial parts at applied NaCl concentrations. On the other hand, seedlings restricted the transport of Na+ ions to the aerial parts and were strongly selective in favour of K+ ions. The presence of NaCl in the culture medium decreased the absorption rate and the export of K+ and Na+ ions to the aerial parts. This was reflected in the accumulation way of these two ions in the whole plant.

A laser emitting contact lens for eye tracking.

In this paper, we present the first realisation and experimentation of a new eye tracking system using an infrared (iR) laser pointer embedded into a wireless smart contact lens. We denote this contact lens prototype as the cyclops lens, in reference to the famous hero of the X-Men comics. The full eye tracker device combines the smart contact lens and its eyewear, which provides a primary source of energy and the beam detection system. We detail the assembling and encapsulation process of the main functionalities into the contact lens and present how a gaze tracking system is achieved, compared to existing conventional eye-tracking ones. Finally, we discuss future technical improvements.

First field evidence infection of Culex perexiguus by West Nile virus in Sahara Oasis of Algeria.

BACKGROUND & OBJECTIVES: West Nile virus (WNV) is considered one of the most widely distributed arboviruses in the world which is transmitted by several mosquito species including the Culex genus. Culex pipiens is the major vector of this virus in Europe and USA whereas in African countries, other species such as Cx. perexiguus is considered as an important vector. This paper aimed to study the mosquito species involved in WNV transmission in Aougrout, one of the highly populated Oasis of Timimoun Province in Algeria where human WNV neuroinvasive diseases are prevalent. METHODS: CDC light-traps were installed in animal and human shelters for three nights. Collected mosquitoes were pooled and real-time PCR was performed to detect and identify WNV lineages 1 and 2 in the samples. Results: CDC light-traps collected 270 mosquitoes belonging to three genera. Culex genus was predominant with Cx. perexiguus as main species followed by Aedes and Anopheles genus. A total of 33 pools were tested; one pool containing Cx. perexiguus was found positive for WNV lineage 1. INTERPRETATION & CONCLUSION: This study reports for the first time a WNV natural infection of Culex perexiguus in the study region indicating that species other than Cx. pipiens should be taken into consideration in WNV surveillance, especially in specific environments like Saharan Oasis ecosystem.

Phenotypic and Functional Characterization of Mesenchymal Stem/Multipotent Stromal Cells from Decidua Basalis of Human Term Placenta.

Mesenchymal stem cell (MSC) therapies for the treatment of diseases associated with inflammation and oxidative stress employ primarily bone marrow MSCs (BMMSCs) and other MSC types such as MSC from the chorionic villi of human term placentae (pMSCs). These MSCs are not derived from microenvironments associated with inflammation and oxidative stress, unlike MSCs from the decidua basalis of the human term placenta (DBMSCs). DBMSCs were isolated and then extensively characterized. Differentiation of DBMSCs into three mesenchymal lineages (adipocytes, osteocytes, and chondrocytes) was performed. Real-time polymerase chain reaction (PCR) and flow cytometry techniques were also used to characterize the gene and protein expression profiles of DBMSCs, respectively. In addition, sandwich enzyme-linked immunosorbent assay (ELISA) was performed to detect proteins secreted by DBMSCs. Finally, the migration and proliferation abilities of DBMSCs were also determined. DBMSCs were positive for MSC markers and HLA-ABC. DBMSCs were negative for hematopoietic and endothelial markers, costimulatory molecules, and HLA-DR. Functionally, DBMSCs differentiated into three mesenchymal lineages, proliferated, and migrated in response to a number of stimuli. Most importantly, these cells express and secrete a distinct combination of cytokines, growth factors, and immune molecules that reflect their unique microenvironment. Therefore, DBMSCs could be attractive, alternative candidates for MSC-based therapies that treat diseases associated with inflammation and oxidative stress.

Human Chorionic Villous Mesenchymal Stem Cells Modify the Functions of Human Dendritic Cells, and Induce an Anti-Inflammatory Phenotype in CD1+ Dendritic Cells.

BACKGROUND: Mesenchymal stem cells derived from the chorionic villi of human term placenta (pMSCs) have drawn considerable interest because of their multipotent differentiation potential and their immunomodulatory capacity. These properties are the foundation for their clinical application in the fields of stem cell transplantation and regenerative medicine. Previously, we showed that pMSCs induce an anti-inflammatory phenotype in human macrophages. In this study, we determined whether pMSCs modify the differentiation and maturation of human monocytes into dendritic cells (DCs). The consequences on dendritic function and on T cell proliferation were also investigated. METHODS: Interleukin-4 (IL-4) and granulocyte-macrophage colony stimulating factor (GM-CSF) were used to stimulate the differentiation of monocytes into immature dendritic cells (iDCs), which were subsequently co-cultured with pMSCs. Lipopolysaccharide (LPS) was used to induce maturation of iDCs into mature dendritic cells (mDCs). Flow cytometry and enzyme-linked immunosorbent assays (ELISA) were used to quantify the effect pMSC co-culturing on DC differentiation using CD1a, a distinctive marker of DCs, as well as other molecules important in the immune functions of DCs. The phagocytic activity of iDCs co-cultured with pMSCs, and the effects of iDCs and mDC stimulation on T cell proliferation, were also investigated. RESULTS: Monocyte differentiation into iDCs was inhibited when co-cultured with pMSCs and maturation of iDCs by LPS treatment was also prevented in the presence of pMSCs as demonstrated by reduced expression of CD1a and CD83, respectively. The inhibitory effect of pMSCs on iDC differentiation was dose dependent. In addition, pMSC co-culture with iDCs and mDCs resulted in both phenotypic and functional changes as shown by reduced expression of costimulatory molecules (CD40, CD80, CD83 and CD86) and reduced capacity to stimulate CD4(+) T cell proliferation. In addition, pMSC co-culture increased the surface expression of major histocompatibility complex (MHC-II) molecules on iDCs but decreased MHC-II expression on mDCs. Moreover, pMSC co-culture with iDCs or mDCs increased the expression of immunosuppressive molecules [B7H3, B7H4, CD273, CD274 and indoleamine-pyrrole 2,3-dioxygenase (IDO). Additionally, the secretion of IL-12 and IL-23 by iDCs and mDCs co-cultured with pMSCs was decreased. Furthermore, pMSC co-culture with mDCs decreased the secretion of IL-12 and INF-γ whilst increasing the secretion of IL-10 in a T cell proliferation experiment. Finally, pMSC co-culture with iDCs induced the phagocytic activity of iDCs. CONCLUSIONS: We have shown that pMSCs have an inhibitory effect on the differentiation, maturation and function of DCs, as well as on the proliferation of T cells, suggesting that pMSCs can control the immune responses at multiple levels.

In vitro antimicrobial activity of Pistacia lentiscus L. edible oil and phenolic extract.

Pistacia lentiscus L. is known in some Tunisian forest area by its fixed oil used in traditional medicine as an antiseptic product. This investigation is the first to study the antimicrobial activity of P.lentiscus edible oil and its phenolic extract. Oil was extracted from fruits harvested from six provenances located in Tunisia. The antimicrobial activity was tested using disc diffusion assay and the broth dilution method. Kbouch and Sidi Zid oils were most efficient (p < 0.003) against, respectively, Staphylococcus aureus and Aspergillus niger with an inhibition zone of 9.33 mm. The phenolic extract had the largest spectrum of sensitive microorganisms. The minimum inhibitory concentration and minimum bactericidal concentration results showed that all strains were inhibited by both oil and extract.

Effect of growing area on tocopherols, carotenoids and fatty acid composition of Pistacia lentiscus edible oil.

In this investigation, we aim to study, for the first time, the effect of the growing area on tocopherols, carotenoids and fatty acid content of Pistacia lentiscus fixed oil. Fruits were harvested from eight different sites located in the north and the centre of Tunisia. Tocopherols, carotenoids and fatty acid content of the fixed oils were determined. The highest carotenoid content was exhibited by Feija oil (10.57 mg/kg of oil). Oueslatia and Tabarka oils displayed the highest α-tocopherol content (96.79 and 92.79 mg/kg of oil, respectively). Three major fatty acids were determined: oleic, palmitic and linoleic acids. Oleic acid was the main fatty acid presenting more than 50% of the total fatty acid content. Kebouche oil presented the highest oleic acid content (55.66%). All these results highlight the richness of carotenoids, tocopherols and unsaturated fatty acids in P. lentiscus seed oil and underscore the nutritional value of this natural product.

Human placental mesenchymal stem cells (pMSCs) play a role as immune suppressive cells by shifting macrophage differentiation from inflammatory M1 to anti-inflammatory M2 macrophages.

BACKGROUND: Mesenchymal stem cells (MSCs) have a therapeutic potential in tissue repair because of capacity for multipotent differentiation and their ability to modulate the immune response. In this study, we examined the ability of human placental MSCs (pMSCs) to modify the differentiation of human monocytes into macrophages and assessed the influence of pMSCs on important macrophage functions. METHODS: We used GM-CSF to stimulate the differentiation of monocytes into the M1 macrophage pathway and then co-cultured these cells with pMSCs in the early stages of macrophage differentiation. We then evaluated the effect on differentiation by microscopic examination and by quantification of molecules important in the differentiation and immune functions of macrophages using flow cytometry and ELISA. The mechanism by which pMSCs could mediate their effects on macrophage differentiation was also studied. RESULTS: The co-culture of pMSCs with monocytes stimulated to follow the inflammatory M1 macrophage differentiation pathway resulted in a shift to anti-inflammatory M2-like macrophage differentiation. This transition was characterized by morphological of changes typical of M2 macrophages, and by changes in cell surface marker expression including CD14, CD36, CD163, CD204, CD206, B7-H4 and CD11b, which are distinctive of M2 macrophages. Co-culture with pMSCs reduced the expression of the costimulatory molecules (CD40, CD80 and CD86) and increased the expression of co-inhibitory molecules (CD273, CD274 and B7-H4) as well as the surface expression of major histocompatibility complex (MHC-II) molecules. Furthermore, the secretion of IL-10 was increased while the secretion of IL-1ß, IL-12 (p70) and MIP-1α was decreased; a profile typical of M2 macrophages. Finally, pMSCs induced the phagocytic activity and the phagocytosis of apoptotic cells associated with M2- like macrophages; again a profile typical of M2 macrophages. We found that the immunoregulatory effect of pMSCs on macrophage differentiation was mediated by soluble molecules acting partially via glucocorticoid and progesterone receptors. CONCLUSIONS: We have shown that pMSCs can transition macrophages from an inflammatory M1 into an anti-inflammatory M2 phenotype. Our findings suggest a new immunosuppressive property of pMSCs that may be employed in the resolution of inflammation associated with inflammatory diseases and in tissue repair.

Phenotypic and functional characterization of mesenchymal stem cells from chorionic villi of human term placenta.

BACKGROUND: Bone marrow derived mesenchymal stem cells (BM-MSCs) are used extensively in transplantation but their use is associated with many problems including low abundance in BM, low overall number, decreased differentiation potential with age and the invasive isolation procedures needed to obtain BM. We report a novel method of isolating placental MSCs (pMSCs) from chorionic villi, which exhibit the phenotypic and functional characteristics that will make them an attractive source of MSCs for cell-based therapy. METHODS: A novel explant approach was used to isolate pMSCs from chorionic villi of human placentae. These pMSCs were characterized by flow cytometry and were differentiated into adipocytes, osteocytes and chondrocytes using differentiation medium as demonstrated by cytochemical staining. The gene and protein expression profiles of pMSCs were also characterized using real time polymerase chain reaction (PCR) and flow cytometry, respectively. In addition, cytokine secretion by pMSCs was also analysed using sandwich enzyme-linked immunosorbent assay (ELISA) technique. Moreover, the migration and proliferation potentials of pMSCs were also determined. RESULTS: pMSCs were isolated from fetal part of the chorionic villi and these pMSCs expressed CD44, CD90, CD105, CD146, CD166 and HLA-ABC but not CD14, CD19, CD40, CD45, CD80, CD83, CD86 and HLA-DR. In addition, these pMSCs differentiated into osteocytes, chondrocytes and adipocytes and they also expressed several adhesion molecules, chemokines/receptors, growth factor receptors and cytokines/receptors. Moreover, they secreted many cytokines (IL-1Ra, IL6, IL8, IL10, IL11 and IL15) and they were able to proliferate. Furthermore, they migrated in response to chemotactic factors including stromal cell-derived factor-1 (SDF-1), platelet derived growth factor (PDGF), hepatocyte growth factor (HGF), and monocyte chemotactic protein-1 (MCP-1). CONCLUSIONS: We devised a novel explant method of isolating pMSCs that expressed many biological factors responsible for mediating cellular processes such as migration/homing, immune modulation and angiogenesis. Therefore, we suggest that pMSCs prepared from human term placental chorionic villous explants are an attractive source of MSCs for cell therapy.

Axium MicroFX coil for the completing endovascular aneurysm surgery study (ACCESS). A prospective evaluation of the safety and durability of Axium MicroFX PGLA coils.

Recanalization of previously coiled aneurysms remains a major drawback of endovascular aneurysm therapy. We performed a prospective single arm trial to provide early initial data regarding the safety and angiographic durability of a new coil technology, the Axium MicroFX Polyglycolic/polylactic acid (PGLA) coil, which was designed to lower recanalization rates. Fifteen patients (16 aneurysms) were prospectively enrolled. Demographic and peri-procedural data were collected. Angiographic images of the initial coil embolization and three to six month follow-up angiographic images underwent blinded evaluation. Seven (47%) SAH and eight (53%) elective patients were enrolled. Blinded evaluation of the initial embolization demonstrated that 5/16 (31%) aneurysms achieved Raymond grade 1, 5/16 (31%) grade 2 and 6/16 (38%) grade 3. Three to six month angiography was obtained in 12/15 patients (80%); two patients expired (1 SAH, 1 elective) and one was lost to follow-up (SAH). All patients who underwent follow-up angiography had a mRS ≤1. Blinded evaluation of embolization demonstrated 7/13 aneurysms (54%) improved in Raymond grading, five (38%) were stable and one aneurysm (8%) worsened. One patient developed an asymptomatic peri-aneurysmal parent vessel stenosis. Axium MicroFX coils appear to be safe, though the small number of patients in this series obviates comparative analysis with other series. Further studies are needed with more patients to compare the angiographic durability of Axium MicroFX coils to other coils.

[Pertussis infection and fatal pulmonary hypertension]. / Coqueluche et hypertension artérielle pulmonaire fatale.

Pertussis is ranked among the leading causes of childhood mortality. The most catastrophic clinical complication of pertussis in infants, intractable pulmonary hypertension with shock, is not very well known. We describe the clinical course of a fatal case of severe pertussis complicated by refractory pulmonary hypertension and shock in a 2-month-old infant.

[Malignant pertussis: an underdiagnosed illness]. / La coqueluche maligne: une maladie sous diagnostiquée.

PURPOSE: Malignant pertussis is a rare life-threatening illness characterized by severe respiratory failure, severe leukocytosis, and pulmonary hypertension. The purpose of this study was to determine the prevalence of malignant pertussis in infants admitted to a pediatric intensive care unit (PICU) for severe acute respiratory failure associated with severe leukocytosis. METHODS: This retrospective study was based on review of the medical charts of infants aged less than 3 months admitted to the PICU between 2006 and 2008 for severe acute respiratory failure requiring mechanical ventilation with leukocytosis greater than 50,000/mm3. Clinical and laboratory data were collected. Polymerase chain reaction (PCR) for detection of Bordetella pertussis was performed on nasopharyngeal washes (NPW) stored at -70 degrees C. RESULTS: Ten patients meeting inclusion criteria were identified. Median age was 2.1 months (range, 0.6 - 3). None of the infants had been vaccinated against pertussis. Although PCR for pertussis was positive in all ten cases, presumptive diagnosis was made in only 3 patients during hospitalization. Nine patients died within a mean of 4.7 +/- 3.3 days after admission. The cause of death was refractory shock and hypoxemia in all cases. Only one patient survived. CONCLUSION: Malignant pertussis is a severe disease that is almost always fatal. It was underdiagnosed in our PICU. Use of PCR for detection of B. pertussis, i.e., the reference method, should be promoted in developing countries.

[Mortality rates in pediatric septic shock secondary to community-acquired infection: about 70 cases]. / Mortalité au cours du choc septique de l'enfant secondaire à une infection communautaire: à propos de 70 cas.

GOAL: This study had for aim to determine the mortality rate and the factors affecting mortality among 70 children admitted for septic shock secondary to a community acquired infection. PATIENTS AND METHODS: A retrospective analysis was made of patients admitted between January 1998 and August 2005, in a pediatric ICU for septic shock secondary to a community-acquired infection. Neonates under 7 days of age were excluded from the study. RESULTS: Seventy cases were included and 32 (45.7 %) of them died. Their average age was 3.8+/-4.2 years and their PRISM during the first 24 hours was 19.2+/-8.4. Sixty-nine children (98.6 %) presented with multivisceral failure and 60 (85.7 %) with more than two deficient organs. The average time between the observation of first hemodynamic disorders and admission to ICU was 9.4+/-11.3 hours. Three independent mortality risk factors were identified: failure of more than two organs on admission (OR, 4.4; 95 % CI [2.1-9.4]), an infusion volume superior to 20ml/kg on the second day of resuscitation (OR, 3.4; 95 CI % [1.1-10.3]), and the use of more than two vasoactive drugs (OR, 3.3; 95 CI % [1.2-9]).

[Epidemiology of nosocomial bacterial infections in a neonatal and pediatric Tunisian intensive care unit]. / Epidémiologie des infections bactériennes nosocomiales dans une unité de réanimation néonatale et pédiatrique tunisienne.

OBJECTIVE: The authors had for aim to describe the epidemiology of nosocomial bacterial infections in the neonatal and pediatric intensive care unit of the Tunis children's hospital. DESIGN: A prospective surveillance study was made from January 2004 to December 2004. All patients remaining in the intensive care unit for more than 48 h were included. CDC criteria were applied for the diagnosis of nosocomial infections. RESULTS: 340 patients including 249 (73%) neonates were included. 22 patients presented with 22 nosocomial bacterial infections. The incidence and the density incidence rates of nosocomial bacterial infections were 6.5% and 7.8 per 1,000 patient-days, respectively. Two types of infection were found: bloodstream infections (68.2%) and pneumonias (22.7%). Bloodstream infections had an incidence and a density incidence rate of 4.4% and 15.3 per 1,000 catheter-days, respectively. Pneumonia had an incidence and a density incidence rate of 2% and 4.4 per 1,000 mechanical ventilation-days, respectively. The most frequently isolated pathogens were Gram-negative bacteria (68%) with Klebsiella pneumoniae isolates accounting for 22.7%. The most common isolate in bloodstream infections was K. Pneumoniae (26.7%), which was multiple drug-resistant in 85% of the cases, followed by Staphylococcus aureus (20%). Pseudomonas aeruginosa was the most common isolate in pneumonia (28.6%). Associated factors of nosocomial infection were invasive devices and colonization with multiple drug-resistant Gram-negative bacteria. CONCLUSIONS: The major type of nosocomial bacterial infections in our unit was bloodstream infection and the majority of infections resulted from Gram-negative bacteria. Factors associated with nosocomial bacterial infections were identified in our unit.

Wound healing complications with de novo sirolimus versus mycophenolate mofetil-based regimen in cardiac transplant recipients.

Sirolimus was introduced in de novo immunosuppression at Stanford University in view of its favorable effects on reduced rejection and cardiac allograft vasculopathy. After an apparent increase in the incidence of post-surgical wound complications as well as symptomatic pleural and pericardial effusions, we reverted to a mycophenolate mofetil (MMF)-based regimen. This retrospective study compared the outcome in heart transplant recipients on sirolimus (48 patients) with those on MMF (46 patients) in de novo immunosuppressive regimen. The incidence of any post-surgical wound complication (52% vs. 28%, p=0.019) and deep surgical wound complication (35% vs. 13%, p=0.012) was significantly higher in patients on sirolimus than on MMF. More patients on sirolimus also had symptomatic pleural (p=0.035) and large pericardial effusions (p=0.033) requiring intervention. Logistic regression analysis showed sirolimus (p=0.027) and longer cardiac bypass time (OR=1.011; p=0.048) as risk factors for any wound complication. Sirolimus in de novo immunosuppression after cardiac transplantation was associated with a significant increase in the incidence of post-surgical wound healing complications as well as symptomatic pleural and pericardial effusions.

Proliferation and neuronal differentiation of mitotically active cells following traumatic brain injury.

Recent studies have identified endogenous neural stem cells in adult rodent brains. The present study characterizes the early response of mitotically active cells in the brain to traumatic brain injury. Animals were subjected to lateral fluid percussion injury and sacrificed at various times after injury. To examine increases in cell proliferation animals were injected with the mitotic marker bromodeoxyuridine (BrdU) 24 h before sacrifice. Increased numbers of mitotically active cells were observed at 2 days in the subgranular zone (SGZ) and the subependymal zone (SEZ) under the injury site. To characterize the differentiation potential of these cells, animals were injected with BrdU 18 and 20 h after injury, then sacrificed at multiple time points after injury. Histologically, co-localization with betaIII-tubulin (neuronal marker) and BrdU was evident at 10 and 15 days postinjury in the SGZ. Flow cytometry analysis was used to quantitatively assess neurogenesis in the SEZ. Animals were sacrificed 1, 5, or 10 days after injury and tissue sections extracted, grown in tissue culture for 24 h, fixed, and stained for nestin and betaIII-tubulin to identify newly formed neurons. The percentage of cells expressing both markers was determined using flow cytometry analysis. There was a significant increase in newly differentiated neurons by 10 days postinjury in the SEZ. Thus, we conclude that traumatic brain injury stimulates an increase in proliferation of endogenous neural stem/progenitor cells and that a significant number of these express a neuronal marker. This response may be the brain's way of trying to heal itself after injury.

Postnatal bone marrow stromal cells elicit a potent VEGF-dependent neoangiogenic response in vivo.

Bone marrow stromal cells (MSCs) are pluripotent cells capable of differentiation into several tissue types. This present study was performed to determine their functional neoangiogenic potential in vivo. Whole bone marrow was harvested from C57Bl/6 mice, and the adherent cellular fraction was culture expanded for 14 doublings. These MSCs were resuspended in Matrigel and their angiogenic effect assessed in isogenic recipients. At 2 weeks postimplantation, the mean vascular density in Matrigel plugs containing 2 x 10(6) MSCs/ml was 41+/-5.0 blood vessels (BVs)/mm(2) compared to 0.5+/-0.7 for empty Matrigel (P<0.001). In comparison, Matrigel plugs containing either recombinant murine VEGF 165 at 50 ng/ml or bovine bFGF at 1000 ng/ml generated 21+/-5 and 11+/-2.0 BV/mm(2), respectively. Arteriogenesis was observed only in the MSC-containing implants. With the use of LacZ retroviral labeling of ex vivo expanded MSCs, we show that approximately 10% of LacZ(+)MSCs differentiated into CD31(+) and VEGF(+) endothelial cells. More than 99% of the neoangiogenic phenomena arose from recruitment of host-derived LacZ(null) vascular structures. Neutralizing anti-VEGF antibodies inhibited the MSC-initiated angiogenic response in vivo by 85% (P<0.001). In conclusion, MSCs have the ability to effectively recruit and participate in angiogenesis and arteriogenesis de novo and VEGF plays a central role in the observed host-derived angiogenic response. We propose that ex vivo expanded autologous MSCs may serve as cell therapy to promote therapeutic angiogenesis.

A neovascularized organoid derived from retrovirally engineered bone marrow stroma leads to prolonged in vivo systemic delivery of erythropoietin in nonmyeloablated, immunocompetent mice.

Marrow stromal cells (MSCs) are postnatal progenitor cells that can be easily cultured ex vivo to large amounts. This feature is attractive for cell therapy applications where genetically engineered MSCs could serve as an autologous cellular vehicle for the delivery of therapeutic proteins. The usefulness of MSCs in transgenic cell therapy will rely upon their potential to engraft in nonmyeloablated, immunocompetent recipients. Further, the ability to deliver MSCs subcutaneously - as opposed to intravenous or intraperitoneal infusions - would enhance safety by providing an easily accessible, and retrievable, artificial subcutaneous implant in a clinical setting. To test this hypothesis, MSCs were retrovirally engineered to secrete mouse erythropoietin (Epo) and their effect was ascertained in nonmyeloablated syngeneic mice. Epo-secreting MSCs when administered as 'free' cells by subcutaneous or intraperitoneal injection, at the same cell dose, led to a significant - yet temporary - hematocrit increase to over 70% for 55+/-13 days. In contrast, in mice implanted subcutaneously with Matrigel trade mark -embedded MSCs, the hematocrit persisted at levels >80% for over 110 days in four of six mice (P<0.05 logrank). Moreover, Epo-secreting MSCs mixed in Matrigel elicited and directly participated in blood vessel formation de novo reflecting their mesenchymal plasticity. MSCs embedded in human-compatible bovine collagen matrix also led to a hematocrit >70% for 75+/-8.9 days. In conclusion, matrix-embedded MSCs will spontaneously form a neovascularized organoid that supports the release of a soluble plasma protein directly into the bloodstream for a sustained pharmacological effect in nonmyeloablated recipients.

Human cytidine deaminase as an ex vivo drug selectable marker in gene-modified primary bone marrow stromal cells.

Naturally occurring drug resistance genes of human origin can be exploited for selection of genetically engineered cells co-expressing a desired therapeutic transgene. Their non-immunogenicity in clinical applications would be a major asset. Human cytidine deaminase (hCD) is a chemoresistance gene that inactivates cytotoxic cytosine nucleoside analogs, such as cytosine arabinoside (Ara-C). The aim of this study was to establish if the hCD gene can serve as an ex vivo dominant selectable marker in engineered bone marrow stromal cells (MSCs). A bicistronic retrovector comprising the hCD cDNA and the green fluorescent protein (GFP) reporter gene was generated and used for transduction of A549 cells and primary murine MSCs. Analysis of transduced cells demonstrated stable integration of proviral DNA, more than 1000-fold increase in CD enzyme activity, and drug resistance to cytosine nucleoside analogs. In a mixture of transduced and untransduced MSCs, the percentage of retrovector-expressing cells could be increased to virtual purity (>99.5%) through in vitro drug selection with 1 microM Ara-C. Increased selective pressure with 2.5 microM Ara-C allowed for enrichment of a mixed population of MSCs expressing approximately six-fold higher levels of GFP and of CD activity when compared with unmanipulated engineered MSCs. Moreover, engraftment and endothelial differentiation of these in vitro selected and enriched gene-modified marrow stromal cells was demonstrated by Matrigel assay in vivo. In conclusion, these findings outline the potential of human CD as an ex vivo selection and enrichment marker of genetically engineered MSCs for transgenic cell therapy applications.