Efficacy and Safety of Treatment of Complex Idiopathic Fistula-in-Ano Using Autologous Centrifuged Adipose Tissue Containing Progenitor Cells: A Randomized Controlled Trial.

BACKGROUND: Mesenchymal stem cells derived from adipose tissue have been successfully used to promote sphincter-saving anal fistula healing. OBJECTIVE: The aim of this study was to evaluate the efficacy and safety of the use of autologous centrifuged adipose tissue in the healing process of cryptoglandular complex anal fistulas. DESIGN: This is a randomized controlled trial. SETTINGS: This study was conducted at a single center. PATIENTS: Patients with complex perianal fistulas not associated with Crohn's disease were included. Rectovaginal fistulas were not included. INTERVENTIONS: Patients were randomly allocated to receive treatment with centrifuged adipose tissue injection (experimental group) and without injection (control group) in combination with fistula surgery. MAIN OUTCOME MEASURES: The primary outcome was defined as the proportion of patients with complete fistula closure at 4 weeks (short-term outcome) and 6 months after surgery (long-term outcome). Healing was defined as when the external opening was closed with no perianal discharge on clinical assessment. The secondary outcome was safety that was evaluated by the analysis of adverse events up to 3 months after surgery. Pelvic MRI was performed at 3 months to assure safety and the accuracy of the clinical determination of healing. Postoperative pain, return to work/daily activities, persistent closure at 6 months, fecal incontinence, and patient satisfaction were evaluated. RESULTS: Fifty-eight patients who received centrifuged adipose tissue injection and 58 patients who did not receive centrifuged adipose tissue injection were included in the safety and efficacy analysis. After 4 weeks, the healing rate was 63.8% in the experimental group compared with 15.5% in the control group (p < 0.001). No major adverse events were recorded. Postoperative anal pain was significantly lower in the injection group. Time taken to return to work/daily activities was significantly shorter in the experimental group (3 days) than in the control group (17 days). At 6 months, persistent closure was similar in the 2 groups (86.2% vs 81%). Fecal Incontinence Score at 6 months after surgery was identical to the preoperative score. Patient satisfaction was high in both groups. LIMITATIONS: The absence of blinding, the lack of correlation between stem cell content, and the clinical outcome were limitations of the study. CONCLUSIONS: Autologous centrifuged adipose tissue injection may represent a safe, efficacious, and inexpensive option for the treatment of complex fistula-in-ano. See Video Abstract at http://links.lww.com/DCR/B607. CLINICAL TRIALS REGISTRATION: URL: https://www.clinicaltrials.gov. Identifier: NCT04326907. EFICACIA Y SEGURIDAD DEL TRATAMIENTO DE LA FSTULA ANAL COMPLEJA IDIOPTICA UTILIZANDO TEJIDO ADIPOSO CENTRIFUGADO AUTLOGO QUE CONTIENE CLULAS PROGENITORAS UN ENSAYO CONTROLADO ALEATORIO: ANTECEDENTES:Las células madre mesenquimales derivadas del tejido adiposo se han utilizado con éxito para promover la curación de la fístula anal con preservación de esfínter.OBJETIVO:El objetivo de este estudio fue evaluar la eficacia y seguridad del uso de tejido adiposo autólogo centrifugado en el proceso de cicatrización de fístulas anales complejas de origen criptoglandular.DISEÑO:Ensayo controlado aleatorio.ENTORNO CLÍNICO:Estudio unicéntrico.PACIENTES:Se incluyeron pacientes con fístulas perianales complejas no asociadas a Enfermedad de Crohn. No se incluyeron las fístulas rectovaginales.INTERVENCIONES:Los pacientes fueron asignados aleatoriamente para recibir tratamiento con inyección de tejido adiposo centrifugado (grupo experimental) y sin inyección (grupo de control) en combinación con cirugía de fístula.PRINCIPALES MEDIDAS DE VALORACIÓN:El resultado primario se definió como la proporción de pacientes con cierre completo de la fístula a las 4 semanas (resultado a corto plazo) y 6 meses después de la cirugía (resultado a largo plazo). La curación se definió cuando orificio externo se cerró sin secreción perianal en la valoración clínica. El resultado secundario fue la seguridad que se evaluó mediante el análisis de los eventos adversos (EA) hasta 3 meses después de la cirugía. La resonancia magnética pélvica se realizó a los 3 meses para garantizar la seguridad y la precisión clínica de la curación. Se evaluó el dolor postoperatorio, el regreso al trabajo / actividades diarias, el cierre persistente a los 6 meses, la incontinencia fecal y la satisfacción del paciente.RESULTADOS:Cincuenta y ocho pacientes que recibieron inyección de tejido adiposo centrifugado y 58 pacientes que no recibieron inyección de tejido adiposo centrifugado se incluyeron en el análisis de seguridad y eficacia. Después de 4 semanas, la tasa de curación fue del 63,8% en el grupo experimental en comparación con el 15,5% en el grupo de control (p <0,001). No se registraron eventos adversos importantes. El dolor anal posoperatorio fue significativamente menor en el grupo de inyección. El tiempo necesario para volver al trabajo / actividades diarias fue significativamente menor en el grupo experimental (3 días) con respecto al grupo de control (17 días). A los 6 meses, el cierre persistente fue similar en los dos grupos (86,2% vs 81%). La puntuación de incontinencia fecal a los 6 meses después de la cirugía fue idéntica a la puntuación preoperatoria. La satisfacción del paciente fue muy alta en ambos grupos.LIMITACIONES:Ausencia de cegamiento, falta de correlación entre el contenido de células madre y el resultado clínico.CONCLUSIONES:La inyección de tejido adiposo centrifugado autólogo puede representar una opción segura, eficaz y económica para el tratamiento de la fístula anal compleja.Registro de ensayos clínicos: www.clinicaltrials.gov, identificador NCT04326907; No patrocinado.Consulte Video Resumen en http://links.lww.com/DCR/B607.

A phase II randomized clinical trial for the treatment of recalcitrant chronic leg ulcers using centrifuged adipose tissue containing progenitor cells.

BACKGROUND AIMS: Preclinical and observational reports indicate that adipose tissue (AT) is a safe and promising tool to treat non-healing venous leg ulcers (VLUs). METHODS: From an initial cohort of 38 patients, 16 patients affected by non-healing VLUs were randomly allocated to the experimental arm (5 men and 3 women) and control arm (5 men and 3 women). In the experimental arm, wounds were treated by debridement, centrifuged adipose tissue (CAT), advanced dressings and compression. No experimental treatment (CAT) was administered to the control arm. We investigated the functional and the immunophenotypical features of the harvested CAT-derived stem cells. The primary outcome measures were healing time and safety of the cell treatment. Secondary outcomes were pain evaluated by numeric rating scale (NRS), complete wound healing at 24 weeks by Margolis Index and wound-healing process expressed in square centimeters per week. The various immunophenotypic and functional characteristics of CAT-derived stem cells were then correlated with the clinical outcomes. RESULTS: No major adverse events were recorded. The healing time was significantly faster by applying CAT, 17.5 ± 7.0 weeks versus 24.5 ± 4.9 weeks recorded in the control arm (P < 0.036). NRS dropped after the first week to 2.7 ± 2.0 in the experimental arm versus 6.6 ± 3.0 in the control group (P < 0.01). The rate of healing at the 24th week was not significantly different between arms. Interestingly, we found a strong reverse correlation between the percent of CD34+/CD45- non-hematopoietic cells, respectively, with the healing time (r = -0.894, P < 0.041) and NRS (r = -0.934, P < 0.020). CONCLUSIONS: CAT is safe and may accelerate healing time in VLUs as well as reduce wound pain. The percentage of CD34+/CD45- cells in stromal vascular fraction (SVF) seems to be a predictive biomarker of successful CAT treatment in these patients.

Human Herpes simplex 1 virus infection of endometrial decidual tissue-derived MSC alters HLA-G expression and immunosuppressive functions.

OBJECTIVES: Mesenchymal stromal/stem cells have immunosuppressive functions. Our previous results demonstrated that one of the players of this immunomodulation can be ascribed to the Human Leukocyte Antigen-G. HLA-G, a non classical HLA class I antigen, is involved in immune tolerance during pregnancy, organ transplantation, autoimmune and inflammatory diseases. In this study we wanted to verify whether human endometrial decidual tissue derived (EDT)-MSC could express HLA-G. Additionally we assessed the permissivity to Human Herpesvirus infections, using HSV-1 as a model, and the possible effect on EDT-MSC immunosuppressive functions towards peripheral blood mononuclear cell (PBMC) proliferation. METHODS: We analyzed immune-inhibitory functions and HLA-G expression in human EDT-MSC before and after HSV-1 infection. RESULTS: We observed that EDT-MSC express HLA-G molecules, that partly are responsible for the immune-inhibitory functions of EDT-MSC towards PBMC proliferation. EDT-MSC are permissive for a productive infection by HSV-1, that decreases HLA-G expression and affects EDT-MSC immune-inhibitory functions. CONCLUSIONS: We demonstrate that EDT-MSC are susceptible to HSV-1 infection, that reduces HLA-G expression and their immune-inhibitory function. These data could have a clinical implication in the use of EDT-MSC as an immunosuppressant, in particular in steroid-refractory GvHD after allogeneic hematopoietic stem cell transplantation and in autoimmune diseases.

Detection of inherited chromosomally integrated HHV-6 (ciHHV-6) in a marker chromosome.

OBJECTIVES: Inherited chromosomally integrated human herpesvirus-6 (ciHHV-6) is characterised by the complete HHV-6 genome integration into the host germ line genome and is vertically transmitted with a Mendelian inheritance. By now, the only relationship between ciHHV-6 and diseases seems to be with angina pectoris. METHODS: We report a case of an 82-year-old man diagnosed with diffuse large B-cell lymphoma (DLBCL) on October 2014. To substantiate the suspicion of ciHHV-6, we analysed peripheral blood mononuclear cells, bone marrow biopsy and pleural effusion-derived mesothelial cells with PCR, RT-PCR and FISH. RESULTS: Virological routine screening by PCR showed the absence of HHV-8 and EBV infections, while the presence of HHV-6 DNA (ie, U22, U42 and U94 HHV-6 genes), with a viral load of about 1.0 genome per cell, strongly suggests ciHHV-6. The RT-PCR showed the positivity only for the immediate-early U94, at low levels of transcription (100±15 transcripts/1 µg RNA). FISH analysis reported a case of inherited ciHHV-6 in 17p chromosome region and, for the first time, in a marker chromosome. CONCLUSIONS: This is the first case of inherited ciHHV-6 in a marker chromosome, possibly elucidating the role of this abnormality in the biology of DLBCL.

Expression of the immunoglobulin superfamily cell membrane adhesion molecule Cd146 in acute leukemia.

BACKGROUND: The expression of the immunoglobulin superfamily cell membrane adhesion molecule CD146 has been reported on several normal and pathological cell types in human. The aim of this study was to investigate CD146 expression in acute leukemia using a multiparametric cytofluorimetric approach. METHODS: Cytofluorimetric and cytogenetic studies were performed on peripheral blood and bone marrow samples from 162 patients with acute myeloid leukemia (AML, n = 121) and acute lymphoblastic leukemia (ALL, n = 41). ALL patients were subdivided in B-ALL (n = 38) and T-ALL (n = 3). Adult (n = 18) and pediatric (n = 20) B-ALL were considered as a whole group. RESULTS: Four out of 121 (3.3%) AML cases, 14/38 (36.8%) B-ALL, and 2/3 (66.6%) T-ALL expressed CD146 on 12-98% of blasts (p < 0.001). CD146 expression was not observed in 10 healthy controls. Among B-ALL CD146-positive cases, 78.6% were associated with a "common"/BII-ALL and 21.4% with a pre-B/BIII-ALL immunophenotype while pro-B/BI-ALL and mature-B/BIV-ALL cases were CD146-negative. Statistical analysis showed CD146 expression strongly associated with Ph+ positivity in B-ALL with the highest percentage of CD146-positive blasts in all Ph-positive B-ALL cases (84 ± 22% Ph-positive B-ALL SD vs. 40 ± 24% SD in Ph-negative B-ALL; p < 0,001). CONCLUSION: In our series, CD146 was expressed in all cases of Ph-positive B-ALL and in the vast majority of T-ALL, whereas it was rarely expressed by AML blasts. We suggest that CD146 may be considered as an additional marker for acute lymphoblastic leukemia diagnosis and monitoring of minimal residual disease in those cases which are CD146-positive at diagnosis. © 2015 International Clinical Cytometry Society.

Acute human herpesvirus-6A infection of human mesothelial cells modulates HLA molecules.

Human herpesvirus 6A (HHV-6A) causes ubiquitous infections and has been associated with several diseases in immunosuppressed and immune dysregulated individuals. Although considered a lymphotropic virus, HHV-6A has the potential to infect many cell types, inducing important alterations in the infected cell. In our search for additional potential targets for HHV-6A infection, we analyzed the susceptibility of human mesothelial cells to viral infection. HHV-6A infection was performed and analyzed on primary human mesothelial cells isolated from serous cavity fluid, infected in vitro with a cell-free HHV-6A inoculum. The results demonstrated that mesothelial cells are susceptible to in vitro HHV-6A infection, and more importantly, that the virus induces an alteration of HLA expression on the cell surface, inducing HLA class II and HLA-G de novo expression. Since mesothelial cells play a pivotal role in many processes, including inflammation and antigen presentation, we speculate that, in vivo, this virus-induced perturbation might be correlated to alterations in mesothelium functions.

Heterogeneity of mesenchymal stromal cells in lymphoproliferative disorders.

Accumulating evidence indicates that bone marrow microenvironment plays an important role in the pathogenesis of some myeloid and lymphoid hematological malignancies (HM). Among different environmental associated parameters, those related to functional, cytogenetic and immunological integrity of mesenchymal stromal cells (MSC) are particularly relevant. Functional alterations and immunophenotypic abnormalities have been described in MSC obtained from HM patients. These data seem to confirm the defective biological pattern of MSC especially in myeloid diseases, while MSC cytogenetic profile in HM is still an open question, because it is not clear whether BM stromal cells are "culprit or bystander" displaying or not an abnormal karyotype. Contradictory findings were reported in different HM but the functional implications of altered MSC karyotype need to be further addressed also in light of a clinical use of MSC. A "pathological" in vivo supportive function of endogenous MSC, which provide important survival and drug resistance signals to leukemic cells especially in lymphoproliferative disorders, is suggested. Thus, the mechanisms underlying these protective versus cytotoxic effects exerted by MSC on leukemic cells need further investigations.

Individual quality assessment of autografting by probability estimation for clinical endpoints: a prospective validation study from the European group for blood and marrow transplantation.

The aim of supportive autografting is to reduce the side effects from stem cell transplantation and avoid procedure-related health disadvantages for patients at the lowest possible cost and resource expenditure. Economic evaluation of health care is becoming increasingly important. We report clinical and laboratory data collected from 397 consecutive adult patients (173 non-Hodgkin lymphoma, 30 Hodgkin lymphoma, 160 multiple myeloma, 7 autoimmune diseases, and 28 acute leukemia) who underwent their first autologous peripheral blood stem cell transplantation (PBSCT). We considered primary endpoints evaluating health economic efficacy (eg, antibiotic administration, transfusion of blood components, and time in hospital), secondary endpoints evaluating toxicity (in accordance with Common Toxicity Criteria), and tertiary endpoints evaluating safety (ie, the risk of regimen-related death or disease progression within the first year after PBSCT). A time-dependent grading of efficacy is proposed with day 21 for multiple myeloma and day 25 for the other disease categories (depending on the length of the conditioning regimen) as the acceptable maximum time in hospital, which together with antibiotics, antifungal, or transfusion therapy delineates four groups: favorable (≤7 days on antibiotics and no transfusions; ≤21 [25] days in hospital), intermediate (from 7 to 10 days on antibiotics and <3 transfusions, ≤21 to 25 days in hospital or ≥7 days on antibiotics and no transfusions; from 21 to 30 days [25 to 34] in hospital), unfavorable (>7 days on antibiotics, >3 but <6 transfusions; >30/34 days in hospital after transplantation), and very unfavorable (>10 days on antibiotics, >6 transfusions; >30 to 34 days in hospital). The multivariate analysis showed that (1) PBSC harvests of ≥4 × 10(6)/kg CD34 + cells in 1 apheresis procedure were associated with a favorable outcome in all patient categories except acute myelogenous leukemia and acute lymphoblastic leukemia (P = .001), (2) ≥5 × 10(6)/kg CD34 + cells infused predicted better transplantation outcome in all patient categories (P < .0001) except acute myelogenous leukemia and acute lymphoblastic leukemia, (3) 1 or 2 aphereses (P = .001) predicted good outcome, (4) toxicity increased with higher graft volume reinfused (>500 mL) (P = .002), and (5) patients with a central venous catheter during both collection and infusion of PBSC had a more favorable outcome post-PBSCT than peripheral access (P = .007). The type of mobilization regimen did not affect the outcome of auto-PBSCT. The present study identified predictive variables, which may be useful in future individual pretransplantation probability evaluations with the goal to improve supportive care.

In vitro characterization of circulating endothelial progenitor cells isolated from patients with acute coronary syndrome.

BACKGROUND: The current understanding of the functional characteristics of circulating endothelial progenitor cells (EPC) is limited, especially in patients affected by cardiovascular diseases. In this study, we have analyzed the in vitro clonogenic capacity of circulating EPC, also known as endothelial colony-forming cells (ECFC), in patients with acute coronary syndrome (ACS), in comparison to the colony forming unit-endothelial-like cells (CFU-EC) of hematopoietic/monocytic origin. METHODOLOGY/PRINCIPAL FINDINGS: By culturing peripheral blood mononuclear cells (PBMC) of patients with ACS (n = 70), CFU-EC were frequently isolated (from 77% of ACS patients), while EPC/ECFC were obtained only in a small subset (13%) of PBMC samples, all harvested between 7-14 days after the acute cardiovascular event. Notably, ex-vivo generation of EPC/ECFC was correlated to a higher in vitro release of PDGF-AA by the corresponding ACS patient PBMC. By using specific endothelial culture media, EPC/ECFC displayed in vitro expansion capacity, allowing the phenotypic and functional characterization of the cells. Indeed, after expansion, EPC/ECFC exhibited a normal diploid chromosomal setting by FISH analysis and an immunophenotype characterized by: i) uniform positivity for the expression of CD105, CD31, CD146 and Factor VIII, i) variable expression of the CD34, CD106 and CD184 markers, and iii) negativity for CD45, CD90, CD117 and CD133. Of interest, in single-cell replanting assays EPC/ECFC exhibited clonogenic expansion capacity, forming secondary colonies characterized by variable proliferation capacities. CONCLUSION/SIGNIFICANCE: Our data indicate that a careful characterization of true EPC is needed in order to design future studies in the clinical autologous setting of patients with ACS.

Cytogenetic and molecular cytogenetic profile of bone marrow-derived mesenchymal stromal cells in chronic and acute lymphoproliferative disorders.

The possibility that human mesenchymal stromal cells (hMSC) may derive from the malignant clone in hematological malignancies (HM) is a controversial issue. In order to clarify hMSC origin and disclose possible cytogenetic heterogeneity in hMSC belonging to different patients, bone marrow (BM)-derived hMSC samples from chronic lymphocytic leukemia (CLL) and acute lymphoblastic leukemia (ALL) were expanded in culture, characterized by flow cytometry, and screened by conventional cytogenetic analysis and fluorescent in situ hybridization for the presence of possible cytogenetic aberrations, related or not to the hematopoietic neoplastic clone. Our data showed that the presence of cytogenetic aberrations in successfully expanded HM-MSC stromal layers derives from the persistence of contaminating hemopoietic cells (HC), which is greatly supported by in vitro culture conditions that could mimic in vivo microenvironmental niche. Interestingly, the presence of binucleated HM-MSC maintaining a diploid numerical setting has been also detected, while aneuploidies were observed more frequently in mononucleated HM-MSC from patients with an altered karyotype than in patients with a normal karyotype and controls. In conclusion, here, we showed that in ALL and in CLL, the BM-MSC has a normal karyotype, thus supporting a distinct origin from hematopoietic cells (HC). The presence of in vitro hMSC aneuploidy is associated with lymphoid neoplasias carrying chromosome abnormalities, suggesting that hMSC should be characterized before clinical application. The adequacy of hMSC cytogenetic characterization here proposed could represent a "prerequisite" to standardize the hMSC analysis before their use in the autologous setting and cellular therapy.

Umbilical cord blood CD34(+)cell-derived progeny produces human leukocyte antigen-G molecules with immuno-modulatory functions.

Human umbilical cord blood units (UCBs) are an alternative source in allogeneic-stem-cell transplantation. Human leukocyte antigen (HLA)-G is a tolerogenic molecule with a possible implication in UCB immunoregulatory effect. HLA-G expression was observed in UCB myeloid and plasmacytoid dendritic cells; in contrast, CD34(+) cells did not produce this molecule. CD34(+) cells are primitive hematopoietic progenitor cells that are present in UCB and are necessary for long-term engraftment via production of immunoregulatory molecules and a hematopoietic progeny that supports cellular recovery. The role of these cells in UCB transplantation needs further evaluation of HLA-G expression in CD34(+) cells and their hematopoietic progeny. We confirmed the absence of HLA-G expression in CD34(+) cells, whereas CD34(+)-derived progeny secreted HLA-G molecules and expressed HLA-G mRNA in in vitro cultures. Furthermore, soluble HLA (sHLA)-G molecules purified from the culture supernatants of CD34(+)-derived progeny were able to suppress lymphoproliferative response in an HLA-G dose-dependent manner. Overall these results identify CD34(+)-derived hematopoietic progeny as producers of HLA-G molecules and support a role of this antigen as an immuno-modulatory factor in UCB.

A simple method for identifying bone marrow mesenchymal stromal cells with a high immunosuppressive potential.

BACKGROUND AIMS: The beneficial activity of mesenchymal stromal cells (MSC) in allogeneic hematopietic stem cell transplantation requires correct use in terms of cell dose and timing of infusion and the identification of biomarkers for selection. The immunosuppressive bone marrow (BM)-derived MSC (BM-MSC) functions have been associated with the production of soluble HLA-G molecules (sHLA-G) via interleukin (IL)-10. We have established a reliable method for evaluating BM-MSC HLA-G expression without the influence of peripheral blood mononuclear cells (PBMC). METHODS: Thirteen BM-MSC from donors were activated with recombinant IL-10 or co-cultured with 10 different phytohemagglutinin (PHA)-treated PBMC (PHA-PBMC). Membrane-bound and sHLA-G expression was evaluated by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively; lymphoproliferation was measured by (methyl-(3)H)thymidine. RESULTS: The results demonstrated the ability of IL-10 to stimulate both membrane-bound and sHLA-G production by BM-MSC. The levels of HLA-G expression induced by IL-10 in BM-MSC were associated with the inhibition of PHA-PBMC proliferation (sHLA-G, P = 0.0008, r = 0.9308; membrane HLA-G, P = 0.0005, r = 0.9502). CONCLUSIONS: We propose the evaluation of sHLA-G production in IL-10-treated BM-MSC cultures as a possible marker of immunoregulatory function.

A decreased positivity for CD90 on human mesenchymal stromal cells (MSCs) is associated with a loss of immunosuppressive activity by MSCs.

Biologic and clinical interest in human mesenchymal stromal cells (hMSC) has risen over the last years, mainly due to their immunosuppressive properties. In this study, we investigated the basis of immunomodulant possible variability using hMSC from different sources (amniotic membrane, chorion, and bone marrow from either healthy subjects or patients with hematological malignancies, HM) and having discordant positivity for several immunological markers. The CD90+ hMSC reduced lymphoproliferative response in phytohemagglutinin (PHA) activated peripheral blood mononuclear cells (PBMC) via sHLA-G and IL-10 up-modulation. On the contrary, hMSC showing a significantly lower expression for CD90 antigen, elicited a lymphoproliferative allogeneic response in PHA/PBMCs without any increase in soluble HLA-G and IL-10 levels. These data seems to suggest that CD90 molecule may be considered a novel predictive marker for hMSC inhibitory ability, and might cooperate with HLA-G molecule in regulating suppressive versus stimulatory properties of hMSC. These results may have clinical implication in either transplantation or in regenerative medicine fields.

CXCR4 pos circulating progenitor cells coexpressing monocytic and endothelial markers correlating with fibrotic clinical features are present in the peripheral blood of patients affected by systemic sclerosis.

There is still controversy regarding the role of circulating endothelial and progenitor cells (CECs/CEPs) in the pathogenesis of systemic sclerosis (SSc). Using a sequential Boolean gating strategy based on a 4-color flow cytometric protocol, an increased number of CD31(pos)/CD184(pos)(CXCR4)/CD34(pos)/CD45(pos) and CD31(pos)/CD117(pos) (c-kit-R) /CD34(pos)/ CD45(pos) hematopoietic circulating progenitor cells (HCPCs) was detected in SSc patients compared with healthy subjects. In SSc, no circulating mature and progenitor endothelial cells were observed, while an enhanced generation of erythroid progenitor cells was found to be correlated with the presence of CD117+ HCPCs. The presence of freshly detected CXCR4posHCPC was correlated either to the in vitro cultured spindle-shaped endothelial like cells (SELC) with an endo/myelomonocytic profile or to SDF-1 and VEGF serum level. These data are related to more fibrotic clinical features of the disease, thus supporting a possible role of these cells in fibrosis.

The MDM-2 antagonist nutlin-3 promotes the maturation of acute myeloid leukemic blasts.

The small-molecule inhibitor of murine double minute (MDM-2), Nutlin-3, induced variable apoptosis in primary acute myeloid leukemia (AML) blasts and promoted myeloid maturation of surviving cells, as demonstrated by analysis of CD11b and CD14 surface antigens and by morphologic examination. Although the best-characterized activity of Nutlin-3 is activation of the p53 pathway, Nutlin-3 induced maturation also in one AML sample characterized by p53 deletion, as well as in the p53(-/-) human myeloblastic HL-60 cell line. At the molecular level, the maturational activity of Nutlin-3 in HL-60 cells was accompanied by the induction of E2F1 transcription factor, and it was significantly counteracted by specific gene knockdown with small interfering RNA for E2F1. Moreover, Nutlin-3, as well as tumor necrosis factor (TNF) alpha, potentiated the maturational activity of recombinant TNF-related apoptosis-inducing ligand (TRAIL) in HL-60 cells. However, although TNF-alpha significantly counteracted the proapoptotic activity of TRAIL, Nutlin-3 did not interfere with the proapoptotic activity of TRAIL. Taken together, these data disclose a novel, potentially relevant therapeutic role for Nutlin-3 in the treatment of both p53 wild-type and p53(-/-) AML, possibly in association with recombinant TRAIL.

Methylenetetrahydrofolate reductase C677T and A1298C gene variants in adult non-Hodgkin's lymphoma patients: association with toxicity and survival.

BACKGROUND AND OBJECTIVES: Common methylenetetrahydrofolate reductase gene variants (MTHFR C677T and A1298C) have been described to have opposite effects on cancer patients. They may reduce cancer susceptibility and increase drug-related toxicity when folate antagonists (e.g. methotrexate) are utilized. We analyzed 110 patients with high-grade non-Hodgkin's lymphoma (NHL), 68 of whom were eligible for a chemotherapy combination containing methotrexate (MACOP-B) and 42 for chemotherapy without methotrexate (CHOP). DESIGN AND METHODS: Patients were genotyped by polymerase chain reaction and stratified by MTHFR variants. These data were related to the toxicity (WHO grade GO-4) that the patients suffered and their survival. Overall 64 cases (58.2%) developed some form of toxicity and 23 (20.9%) had grade 3/4 toxicity. RESULTS: When considering toxicity of any grade (grade 1-4), the 677TT genotype was significantly over-represented among cases with mucositis (OR=4.85; 95% CI, 1.47-15.97; p=0.009) and those with hepatic toxicity (OR=3.43; 95% CI, 0.99-11.86; p=0.052). Sub-analyses in the group treated with MACOP-B showed a slight increase in the risk of developing mucositis (OR=5.22; 95% CI, 1.20-27.27; p=0.03), and a strong increase in the risk of hepatic toxicity (OR=7.08; 95% CI, 1.38-36.2; p=0.019) and thrombocytopenia (OR=7.69, 95% CI 1.0-58.94; p=0.05). Interestingly, compared to the risk of developing toxicity of any grade, the risk of developing severe (grade 3/4) mucositis was almost doubled in the whole group of cases with 677TT (OR=8.13; 95% CI 1.61-41.04; p=0.011) and dramatically increased in the MACOP-B-treated cases with this gene variant (OR=24.6; 95% CI 2.49-87.41; p=0.001). There were significant results for 1298CC cases exclusively for mucositis (any grade, OR=5.33; 95% CI, 1.25-22.70; p=0.023 and OR=9.15; 95% CI, 1.14-73.41; p=0.037; for the whole group and the MACOP-B-treated group, respectively). Similarly, the risk of 1298CC patients developing severe mucositis increased (OR=9.24; 95% CI, 1.47-58.0; p=0.017 and OR=11.53; 0.93-143.18; p=0.057; in the whole group and in the MACOP-B-treated group, respectively). Event-free survival analysis revealed a lower probability of event-free survival at 5 years for 677T-carriers (log-ranks, p=0.05 and p=0.07 in the whole group and in the MACOP-B-treated group, respectively). More significant results were obtained when 1298CC cases were excluded from the reference group (log-ranks, p=0.03 and p=0.04, respectively). No significant associations were found in the CHOP-treated group. INTERPRETATION AND CONCLUSIONS: Our data suggest that MTHFR gene variants play a critical role in NHL outcome, possibly by interfering with the action of methotrexate with significant effects on toxicity and survival. Genotyping of folate pathway gene variants might be useful to enable reduction of chemotherapy toxicity and/or to improve survival by indicating when dose adjustments or alternative treatments are necessary.

Immunophenotypic heterogeneity of bone marrow-derived mesenchymal stromal cells from patients with hematologic disorders: correlation with bone marrow microenvironment.

The immunophenotypic analysis of ex vivo-expanded mesenchymal stromal cells (MSC) has so far been confined to single or dual staining analysis in normal subjects. In this study, using a four-color cytofluorimetric protocol, we demonstrated that cultured MSC derived from the bone marrow of patients with hematologic malignancies showed alterations in the expression of CD105, CD90, CD184, and HLA-DR molecules. The decrease in the percentage of CD105+ and CD90+ MSC correlated with an increased bone marrow angiogenesis. This paper provides evidence that multiparametric flow cytometry is essential for the establishment of a standardized protocol to identify various MSCs subsets and aberrant phenotypes.

Functional integrity of the p53-mediated apoptotic pathway induced by the nongenotoxic agent nutlin-3 in B-cell chronic lymphocytic leukemia (B-CLL).

Deletions and/or mutations of p53 are relatively rare and late events in the natural history of B-cell chronic lymphocytic leukemia (B-CLL). However, it is unknown whether p53 signaling is functional in B-CLL and if targeted nongenotoxic activation of the p53 pathway by using nutlin-3, a small molecule inhibitor of the p53/MDM2 interaction, is sufficient to kill B-CLL cells. In vitro treatment with nutlin-3 induced a significant cytotoxicity on primary CD19(+) B-CLL cells, but not on normal CD19(+) B lymphocytes, peripheral-blood mononuclear cells, or bone marrow hematopoietic progenitors. Among 29 B-CLL samples examined, only one was resistant to nutlin-3-mediated cytotoxicity. The induction of p53 by nutlin-3 in B-CLL samples was accompanied by alterations of the mitochondrial potential and activation of the caspase-dependent apoptotic pathway. Among several genes related to the p53 pathway, nutlin-3 up-regulated the steady-state mRNA levels of PCNA, CDKN1A/p21, GDF15, TNFRSF10B/TRAIL-R2, TP53I3/PIG3, and GADD45. This profile of gene activation showed a partial overlapping with that induced by the genotoxic drug fludarabine. Moreover, nutlin-3 synergized with both fludarabine and chlorambucil in inducing B-CLL apoptosis. Our data strongly suggest that nutlin-3 should be further investigated for clinical applications in the treatment of B-CLL.

Functional expression of TRAIL and TRAIL-R2 during human megakaryocytic development.

The expression and function of surface TRAIL and TRAIL receptors were investigated in primary megakaryocytic cells, generated in serum-free liquid phase from peripheral human CD34(+) cells. The surface expression of both TRAIL and "death receptor" TRAIL-R2 became detectable starting from the early phase of megakaryocytic differentiation (day 6 of culture) and persisted at later (days10-14) culture times. On the other hand, "death receptor" TRAIL-R1, "decoy receptors" TRAIL-R3, and TRAIL-R4 were barely detectable or undetectable at any time point examined. Addition of recombinant TRAIL at day 6 of culture increased the rate of spontaneous apoptosis of CD34(+)/CD41(dim) megakaryoblasts and it significantly decreased the total output of mature megakaryocytic cells evaluated after additional 4-8 days of culture. Conversely, addition in culture of TRAIL-R2-Fc chimera, which blocked the interaction between endogenous TRAIL and TRAIL-R2 on the surface of cultured megakaryocytic cells, increased the total megakaryocytic cell count. In addition, recombinant TRAIL promoted a small but reproducible increase of maturation in the surviving megakaryocytic cell population, evaluated by both phenotypic analysis and morphology. A similar pro-maturation effect was observed when TRAIL was added to bone marrow-derived CD61(+) megakaryocytic cells. Thus, our data suggest a role of TRAIL as a regulator of megakaryocytopoiesis.