Lentiviral Gene Therapy for Artemis-Deficient SCID.

BACKGROUND: The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation. METHODS: We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months. RESULTS: Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor ß chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report. CONCLUSIONS: Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).

The effect of granulocyte colony-stimulating factor dose and administration interval after allogeneic hematopoietic cell transplantation on early engraftment of neutrophil and platelet.

BACKGROUND: Hematopoietic stem cell transplantation (HSCT) is one of the treatments for hematologic malignancies. Numerous factors affect the HSCT outcome. The purpose of this study was to investigate the effect of post-HSCT administration of granulocyte colony-stimulating factor (post-G-CSF) on early neutrophil and platelet engraftment in allogeneic HSCT (allo-HSCT). MATERIAL & METHODS: The study was performed on 76 patients diagnosed with AML and ALL. All patients underwent allo-HSCT at Taleghani stem cell transplantation center, Tehran, Iran, from February 2016 to December 2018. Chemotherapy regimens based on patients' conditions were selected between myeloablative and reduced-intensity regimens. RESULTS: Statistical analysis revealed that the number of administered G-CSF units after HSCT was a time-dependent variable. Statistical analysis before day +11 reported that patients who received G-CSF <14 units had three times better early neutrophil engraftment than those with G-CSF ≥14 (CI 95%, AHR = 3.03, p:0.002). CD3+ cells count <318.5 × 106 /kg was associated with fast platelet engraftment (CI 95%, AHR 2.28, p:0.01). CONCLUSION: In this study, post-G-CSF stimulation was associated with early engraftment in a time- and dose-dependent manner. Administration of G-CSF beyond 14 units resulted in adverse effects on neutrophil early engraftment. It also appeared that with a reduction in CD3+ cell counts, the likelihood of GVHD decreases, and platelet engraftment occurs earlier. Further investigations in the future are required to determine the factors affecting the process of early engraftment.

Conjunctival myxoid stromal tumour: a distinctive clinicopathological and immunohistochemical study.

BACKGROUND/AIMS: To describe the clinicopathological and immunohistochemical characteristics of 10 patients representing a new entity of benign conjunctival myxoid stromal tumours. METHODS: Retrospective review of clinical findings, histopathological and immunohistochemical studies identified 10 cases of low-grade conjunctival myxoid stromal tumours. Specimens were routinely processed and stained with H&E. Immunohistochemical stains for CD34, CD68, vimentin, S100, smooth muscle actin (SMA), myosin, desmin, actin, Bcl-2 and Ki-67 were performed. Specific stains for Alcian-blue periodic acid-Schiff (AB-PAS) and aldehyde fuchsin stains were also performed. RESULTS: Ten patients with an average age of 45.6±11.1 years had a tender white or faint yellow to red mass on the bulbar conjunctiva. All the lesions were completely removed, and none of the patients relapsed. Histologically, all neoplasms consisted of spindle-shaped cells that showed signs of pseudonuclear inclusions, multinuclear cells and had no atypia. The stroma consisted of a large amount of mucus and was infiltrated with delicate to ropey collagens, a few mast cells and new vessels. Immunohistochemical stains were positive for CD34, vimentin and Bcl-2; partial positive for CD68; very low for Ki-67; and negative for S100, SMA, myosin, desmin and actin. AB-PAS suggested that the stroma was mucinous. CONCLUSIONS: These rare benign mesenchymal conjunctival tumours are mostly unilateral and occur in the bulbar conjunctiva. Complete resection is the radical treatment. These lesions are characterised by multiple spindle cells, a large amount of mucus, and sharing similar basic histopathological features with conjunctival myxoma and conjunctival stromal tumour. We suggest naming these lesions 'conjunctival myxoid stromal tumours'.

Does Reinfusion of Stem Cell Products on Multiple Days Affect Engraftment?

Objective: High-doses of melphalan treatment with autologous stem cell transplantation in multiple myeloma (MM) remains a major treatment modality in suitable patients. A minimal dose of 2x106/kg CD34+ cells is preferred to achieve engraftment. Some patients need multiple leukapheresis procedures to achieve the necessary number of CD34+ cells, but this can cause a high volume of stem cell product that cannot be given in a single day. Whether or not the number of infusion days affects engraftment has not been studied before. We aimed to evaluate the impact of reinfusion of stem cells on multiple days on engraftment results. Materials and Methods: Demographic features, CD34+ cell doses, neutrophil and platelet engraftment days, hospitalization days, and number of infusion days of 149 autologous transplantations of 143 MM patients were evaluated retrospectively. Results: The data of 143 MM patients who were transplanted were analyzed retrospectively. Median age was 55±8.5 (range: 26-70) years with a male/female ratio of 91/58. Hospitalization days for all patients were 24±6 (range: 14-50) days. Mean CD34+ cell number was (7.5±5.3)x106/kg (range: 1.5-31x106/kg). CD34+ cells were reinfused in 1 day in 80.5% (n=120) of the patients, 2 days in 18.2% of the patients (n=27), and 3 days in 1.3% of the patients (n=2). For 29 patients, reinfusion was applied in more than 1 day because of the high volume of stem cell product. We did not see any dimethyl sulfoxide toxicity, cardiac arrhythmia, or volume overload complications. Hypertensive attacks during infusion were easily controlled by furosemide treatment. In the group with multiple infusions, the infused CD34+ cell numbers had a mean of (4.8±2.8)x106/kg, and in the single infusion group the mean was (8.1±5.5)x106/kg. There were no statistical differences between the two groups regarding platelet and neutrophil engraftment days (p=0.850, r=0.820 and p=0.500, r=0.440). There was no statistical difference between the two groups for hospitalization days (p=0.060, r=0.050). Conclusion: In cases with a high volume of stem cell product to acquire adequate stem cells, reinfusion can be safely applied across multiple days without any delay in engraftment.

Effects of umbilical cord blood stem cells on healing factors for diabetic foot injuries.

The use of stem or progenitor cells from bone marrow, or peripheral or umbilical cord blood is becoming more common for treatment of diabetic foot problems. These cells promote neovascularization by angiogenic factors and they promote epithelium formation by stimulating cell replication and migration under certain pathological conditions. We investigated the role of CD34 + stem cells from human umbilical cord blood in wound healing using a rat model. Rats were randomly divided into a control group and two groups with diabetes induced by a single dose of 55 mg/kg intraperitoneal streptozocin. Scarred areas 5 mm in diameter were created on the feet of all rats. The diabetic rats constituted the diabetes control group and a diabetes + stem cell group with local injection into the wound site of 0.5 × 106 CD34 + stem cells from human umbilical cord blood. The newly formed skin in the foot wounds following CD34 + stem cell treatment showed significantly improvement by immunohistochemistry and TUNEL staining, and were closer to the wound healing of the control group than the untreated diabetic animals. The increase in FGF expression that accompanied the local injection of CD34 + stem cells indicates that FGF stimulation helped prevent apoptosis. Our findings suggest a promising new treatment approach to diabetic wound healing.

Electroanatomic Properties of the Myocardium Predict Response to CD34+ Cell Therapy in Patients With Ischemic and Nonischemic Heart Failure.

BACKGROUND: We investigated a correlation between electromechanical properties of the myocardium and response to CD34+ cell therapy in patients with chronic heart failure. METHODS AND RESULTS: We enrolled 40 patients with ischemic cardiomyopathy (ICM) and 40 with nonischemic dilated cardiomyopathy (DCM). All patients were in New York Heart Association functional class III and had a left ventricular ejection fraction (LVEF) <40%. CD34+ cells were mobilized by granulocyte colony-stimulating factor and collected via apheresis. Electroanatomic mapping was performed to define areas of myocardial scar and hibernation, and CD34+ cells were injected transendocardially in the hibernating areas. Patient were followed for 6 months; responders were defined as patients with LVEF increase of >5%. At baseline, the groups did not differ in sex, LVEF, creatinine, N-terminal pro-B-type natriuretic peptide or electroanatomic parameters (scar area: 53 ± 18% in ICM vs 55 ± 23% in DCM [P = .83]; hibernating area: 23 ± 13% vs 22 ± 12% [P = .56]). At 6 months we found similar rates of responders in both groups (60% in ICM vs 65% in DCM [P = .95]). When compared with nonresponders, responders had less myocardial scar (47 ± 17% vs 58 ± 15% [P = .003]). CONCLUSIONS: In patients with chronic heart failure due to ICM and DCM we observed similar electroanatomic properties of the myocardium. In both groups, lower myocardial scar burden was associated with better clinical response to CD34+ cell therapy.

PreSERVE-AMI: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial of Intracoronary Administration of Autologous CD34+ Cells in Patients With Left Ventricular Dysfunction Post STEMI.

RATIONALE: Despite direct immediate intervention and therapy, ST-segment-elevation myocardial infarction (STEMI) victims remain at risk for infarct expansion, heart failure, reinfarction, repeat revascularization, and death. OBJECTIVE: To evaluate the safety and bioactivity of autologous CD34+ cell (CLBS10) intracoronary infusion in patients with left ventricular dysfunction post STEMI. METHODS AND RESULTS: Patients who underwent successful stenting for STEMI and had left ventricular dysfunction (ejection fraction≤48%) ≥4 days poststent were eligible for enrollment. Subjects (N=161) underwent mini bone marrow harvest and were randomized 1:1 to receive (1) autologous CD34+ cells (minimum 10 mol/L±20% cells; N=78) or (2) diluent alone (N=83), via intracoronary infusion. The primary safety end point was adverse events, serious adverse events, and major adverse cardiac event. The primary efficacy end point was change in resting myocardial perfusion over 6 months. No differences in myocardial perfusion or adverse events were observed between the control and treatment groups, although increased perfusion was observed within each group from baseline to 6 months (P<0.001). In secondary analyses, when adjusted for time of ischemia, a consistently favorable cell dose-dependent effect was observed in the change in left ventricular ejection fraction and infarct size, and the duration of time subjects was alive and out of hospital (P=0.05). At 1 year, 3.6% (N=3) and 0% deaths were observed in the control and treatment group, respectively. CONCLUSIONS: This PreSERVE-AMI (Phase 2, randomized, double-blind, placebo-controlled trial) represents the largest study of cell-based therapy for STEMI completed in the United States and provides evidence supporting safety and potential efficacy in patients with left ventricular dysfunction post STEMI who are at risk for death and major morbidity. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01495364.

Tumor de Bednar (dermatofibrosarcoma protuberans pigmentado) / Bednar Tumor (Pigmented Dermatofibrosarcoma Protuberans)

No disponible

Comparison of transendocardial and intracoronary CD34+ cell transplantation in patients with nonischemic dilated cardiomyopathy.

BACKGROUND: In an open-label blinded study, we compared intracoronary and transendocardial CD34(+) cell transplantation in patients with nonischemic dilated cardiomyopathy. METHODS AND RESULTS: Of the 40 patients with dilated cardiomyopathy, 20 were randomized to receive intracoronary injection and 20 received transendocardial CD34(+) cell delivery. In both groups, CD34(+) cells were mobilized by filgrastim, collected via apheresis, and labeled with technetium-99m radioisotope for single-photon emission computed tomographic imaging. In the intracoronary group, cells were injected intracoronarily in the artery supplying segments of greater perfusion defect on myocardial perfusion scintigraphy. In the transendocardial group, electroanatomic mapping was used to identify viable but dysfunctional myocardium, and transendocardial cell injections were performed. Nuclear single-photon emission computed tomographic imaging for quantification of myocardial retention was performed 18 hours thereafter. At baseline, groups did not differ in age, sex, left ventricular ejection fraction, or N-terminal pro-brain natriuretic peptide levels. The number of CD34(+) cells was also comparable (105 ± 31 × 10(6) in the transendocardial group versus 103 ± 27 × 10(6) in the intracoronary group, P=0.62). At 18 hours after procedure, myocardial retention was higher in the transendocardial group (19.2 ± 4.8%) than in the intracoronary group (4.4 ± 1.2%, P<0.01). At 6 months, left ventricular ejection fraction improved more in the transendocardial group (+8.1 ± 4.3%) than in the intracoronary group (+4.2 ± 2.3%, P=0.03). The same pattern was observed for the 6-minute walk test distance (+125 ± 33 m in the transendocardial group versus +86 ± 13 m in the intracoronary group, P=0.03) and N-terminal pro-brain natriuretic peptide (-628 ± 211 versus -315 ± 133 pg/mL, P=0.04). CONCLUSIONS: In patients with dilated cardiomyopathy, transendocardial CD34(+) cell transplantation is associated with higher myocardial retention rates and greater improvement in ventricular function, N-terminal pro-brain natriuretic peptide, and exercise capacity compared with intracoronary route. CLINICAL TRIAL REGISTRATION URL: http://www.clinicaltrials.gov. Unique identifier: NCT01350310.

Sonic hedgehog-modified human CD34+ cells preserve cardiac function after acute myocardial infarction.

RATIONALE: Ischemic cardiovascular disease represents one of the largest epidemics currently facing the aging population. Current literature has illustrated the efficacy of autologous, stem cell therapies as novel strategies for treating these disorders. The CD34+ hematopoetic stem cell has shown significant promise in addressing myocardial ischemia by promoting angiogenesis that helps preserve the functionality of ischemic myocardium. Unfortunately, both viability and angiogenic quality of autologous CD34+ cells decline with advanced age and diminished cardiovascular health. OBJECTIVE: To offset age- and health-related angiogenic declines in CD34+ cells, we explored whether the therapeutic efficacy of human CD34+ cells could be enhanced by augmenting their secretion of the known angiogenic factor, sonic hedgehog (Shh). METHODS AND RESULTS: When injected into the border zone of mice after acute myocardial infarction, Shh-modified CD34+ cells (CD34(Shh)) protected against ventricular dilation and cardiac functional declines associated with acute myocardial infarction. Treatment with CD34(Shh) also reduced infarct size and increased border zone capillary density compared with unmodified CD34 cells or cells transfected with the empty vector. CD34(Shh) primarily store and secrete Shh protein in exosomes and this storage process appears to be cell-type specific. In vitro analysis of exosomes derived from CD34(Shh) revealed that (1) exosomes transfer Shh protein to other cell types, and (2) exosomal transfer of functional Shh elicits induction of the canonical Shh signaling pathway in recipient cells. CONCLUSIONS: Exosome-mediated delivery of Shh to ischemic myocardium represents a major mechanism explaining the observed preservation of cardiac function in mice treated with CD34(Shh) cells.

In situ induction of dendritic cell-based T cell tolerance in humanized mice and nonhuman primates.

Induction of antigen-specific T cell tolerance would aid treatment of diverse immunological disorders and help prevent allograft rejection and graft versus host disease. In this study, we establish a method of inducing antigen-specific T cell tolerance in situ in diabetic humanized mice and Rhesus monkeys receiving porcine islet xenografts. Antigen-specific T cell tolerance is induced by administration of an antibody ligating a particular epitope on ICAM-1 (intercellular adhesion molecule 1). Antibody-mediated ligation of ICAM-1 on dendritic cells (DCs) led to the arrest of DCs in a semimature stage in vitro and in vivo. Ablation of DCs from mice completely abrogated anti-ICAM-1-induced antigen-specific T cell tolerance. T cell responses to unrelated antigens remained unaffected. In situ induction of DC-mediated T cell tolerance using this method may represent a potent therapeutic tool for preventing graft rejection.

Intramedullary spinal cord implantation of human CD34+ umbilical cord-derived cells in ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with marginal therapeutic options. Degeneration of motor neurons in the primary motor cortex, brainstem and spinal cord lead to rapidly progressive paralysis and finally to death due to respiratory failure. As pharmacological therapies have failed to provide sufficient neuroprotective effects in ALS, transplantation of stem or progenitor cells is considered a promising treatment strategy. Cell transplantation approaches in ALS mainly aim to generate a neuroprotective environment for degenerating motor neurons by transplantation of non-neuronal cells, rather than to replace lost motor neurons. We present a 63-year-old male patient suffering from ALS who underwent intramedullary thoracic spinal cord implantation of human CD34(+) umbilical cord-derived haematopoietic progenitor cells with a three-year follow up after transplantation.

Loss of neuronal integrity during progressive HIV-1 infection of humanized mice.

Neuronal damage induced by ongoing human immunodeficiency virus type 1 (HIV-1) infection was investigated in humanized NOD/scid-IL-2Rγ(c)(null) mice transplanted at birth with human CD34-positive hematopoietic stem cells. Mice infected at 5 months of age and followed for up to 15 weeks maintained significant plasma viral loads and showed reduced numbers of CD4(+) T-cells. Prospective serial proton magnetic resonance spectroscopy tests showed selective reductions in cortical N-acetyl aspartate in infected animals. Diffusion tensor imaging revealed structural changes in cortical gray matter. Postmortem immunofluorescence brain tissue examinations for neuronal and glial markers, captured by multispectral imaging microscopy and quantified by morphometric and fluorescence emission, showed regional reduction of neuronal soma and synaptic architectures. This was evidenced by loss of microtubule-associated protein 2, synaptophysin, and neurofilament antigens. This study is the first, to our knowledge, demonstrating lost neuronal integrity after HIV-1 infection in humanized mice. As such, the model permits studies of the relationships between ongoing viral replication and virus-associated neurodegeneration.

Vasoprotective effects of human CD34+ cells: towards clinical applications.

BACKGROUND: The development of cell-based therapeutics for humans requires preclinical testing in animal models. The use of autologous animal products fails to address the efficacy of similar products derived from humans. We used a novel immunodeficient rat carotid injury model in order to determine whether human cells could improve vascular remodelling following acute injury. METHODS: Human CD34+ cells were separated from peripheral buffy coats using automatic magnetic cell separation. Carotid arterial injury was performed in male Sprague-Dawley nude rats using a 2F Fogarty balloon catheter. Freshly harvested CD34+ cells or saline alone was administered locally for 20 minutes by endoluminal instillation. Structural and functional analysis of the arteries was performed 28 days later. RESULTS: Morphometric analysis demonstrated that human CD34+ cell delivery was associated with a significant reduction in intimal formation 4 weeks following balloon injury as compared with saline (I/M ratio 0.79 +/- 0.18, and 1.71 +/- 0.18 for CD34, and saline-treated vessels, respectively P < 0.05). Vasoreactivity studies showed that maximal relaxation of vessel rings from human CD34+ treated animals was significantly enhanced compared with saline-treated counterparts (74.1 +/- 10.2, and 36.8 +/- 12.1% relaxation for CD34+ cells and saline, respectively, P < 0.05) CONCLUSION: Delivery of human CD34+ cells limits neointima formation and improves arterial reactivity after vascular injury. These studies advance the concept of cell delivery to effect vascular remodeling toward a potential human cellular product.

Circulating human CD34+ progenitor cells modulate neovascularization and inflammation in a nude mouse model.

CD34+ progenitor cells hold promise for therapeutic neovascularization in various settings. In this study, the role of human peripheral blood CD34+ cells in neovascularization and inflammatory cell recruitment was longitudinally studied in vivo. Human CD34+ cells were incorporated in Matrigel, implanted subcutaneously in nude mice, and explanted after 2, 4, 7, or 14 days. Cell-free Matrigels served as controls. Histochemical analyses demonstrated that neovascularization occurred almost exclusively in CD34+ implants. Cellular and capillary density were increased in cell-loaded Matrigels after 2 days and further increased at 14 days. Human CD34+ cells did not incorporate in neovessels, but formed vWF+/CD31+/VEGF+ cell clusters that were present up to day 14. However, CD34+ cells induced host neovascularization, as demonstrated by increased presence of murine CD31+ and vWF+ vasculature from day 7 to 14. Moreover, recruitment of murine monocytes/macrophages was significantly enhanced in CD34+ implants at all time points. Gene expression of chemotactic cytokines MCP-1 and IL-8 was detected on CD34+ cells in vitro and confirmed immunohistochemically in cell-loaded explants at all time points. Our data indicate that human CD34+ cells, implanted in a hypoxic environment, generate an angiogenic niche by secreting chemotactic and angiogenic factors, enabling rapid neovascularization, possibly via recruitment of monocytes/macrophages.

C34, a membrane fusion inhibitor, blocks HIV infection of langerhans cells and viral transmission to T cells.

Development of topical microbicides that prevent sexual transmission of HIV is an active area of investigation. The purpose of this study was to test the ability of the potent membrane fusion inhibitor C34, an HIV gp41 antagonist, to block HIV infection of human Langerhans cells (LCs) in an epithelial environment that mimics a major route of HIV infection. We incubated freshly isolated epidermal explants containing LCs with various doses of C34 before, during, and after exposing explants to HIV. Although C34 only partially blocked HIV infection of LCs when pre-incubated with skin, it displayed full, dose-dependent inhibition when present during and after viral exposure. The poor protection from HIV infectivity in pre-incubated samples is consistent with mechanism of C34 inhibition and starkly contrasts to the full protection provided by PSC-RANTES, an entry inhibitor that prevents HIV gp120 interaction with its co-receptor CCR5. Real-time PCR confirmed that C34 blocked HIV infection of LCs before reverse transcription and inhibited LC-mediated transfer of virus to T cells. We conclude that C34, if used topically at susceptible mucosal sites, and if continually present, has the potential to block sexual transmission of HIV.