Differentiation of mouse embryonic stem cells into hepatocytes in vitro and in vivo

OBJECTIVE: Embryonic stem (ES) cells have a pluripotent ability to differentiate into a variety of cell lineages. Cell‐to‐cell contact is important for cell differentiation. Mouse ES cells were cocultured with mouse fetal liver cells and the green fluorescent protein (GFP) positive ES cells were transplanted into rats liver through the portal vein in order to investigate their potential to differentiate into hepatocytes. METHODS: Mouse ES cells were cocultured with the mouse fetal liver cell line, BNL.CL₂. They did not make direct contact; instead the culture media was exchanged freely. After coculture for 48 h, albumin, transthyretin, glucose 6 phosphates, hepatic nuclear factor 4 and SEK1 mRNA were assayed by RT‐PCR, and alpha‐fetoprotein by immunohistochemistry. The morphology was investigated by microscopy. After transplantion of the GFP‐positive ES cells, the whole liver was removed from a rat every four days. The liver slices were examined under a fluorescent microscope to detect the GFP‐positive cells. Albumin was detected on the same slices by immunohistochemistry. RESULTS: After coculture with BNL.CL₂ cells, the differentiated ES cells had the same morphology as the BNL.CL₂ cells, and albumin, transthyretin, glucose 6 phosphates and SEK‐1 mRNA were found by RT‐PCR, and alpha‐fetoprotein was detected immuno­histochemically. The transplanted GFP‐positive ES cells were found in the rats’ liver slices by GFP fluorescence, and development of teratomas was not observed. The immunohistochemistry results indicated that the transplanted GFP‐positive ES cells retained an albumin‐producing ability. CONCLUSIONS: Cell‐to‐cell contact is important for the differentiation of ES cells. Mouse embryonic stem cells can differentiate into hepatocytes directly either in vitro or in vivo.     

High incidence of engraftment syndrome after haploidentical allogeneic stem cell transplantation

We determined the incidence, clinical manifestations, and outcomes of engraftment syndrome (ES) in haploidentical stem cell transplantation (SCT) recipients. We compared the incidence of ES between the patient group that received haploidentical SCT (n = 516) and the patient group that received HLA‐identical sibling SCT (n = 393). The transplantations were performed in the Peking University People's Hospital in the period between October 2001 and October 2012. The ES incidence data were collected retrospectively. Patients that presented non‐infectious fever or skin rash within the 24‐h window before or after the beginning of neutrophil recovery were diagnosed with ES in accordance with the Maiolino criteria. ES incidence in haploidentical SCT recipients (21.9%) was significantly higher than that in HLA‐identical sibling SCT recipients (2.0%; P < 0.001). Major symptoms included fever (119/121, 98.3%), skin rash (98/121, 81.0%), and diarrhea (51/121, 42.1%), with the median time of +10 d (range: 6–20 d). The median C‐reactive protein level of the ES group (99.0 mg/L; n = 13) was significantly higher than that of the non‐ES group (13.9 mg/L; n = 38; P < 0.001). Similarly, the results showed that the median C3 plasma concentration of the ES group (1.30 g/L) was higher than that of the non‐ES group (1.16 g/L, P = 0.003). ES was not associated with non‐relapse mortality or overall survival. High incidence of ES was observed in haploidentical SCT recipients; however, ES did not predict poor clinical outcomes.     

Influence of ex vivo purging with CliniMACS CD34+ selection on outcome after autologous stem cell transplantation in non‐Hodgkin lymphoma

The major limitation of autologous stem cell transplantation (auto‐SCT) in non‐Hodgkin lymphoma (NHL) is relapse. Although autologous graft contamination may be a potential cause, prior purging of the autograft remains controversial. Therefore, we retrospectively analysed 56 consecutive patients with NHL receiving auto‐SCT at complete (n = 41) or partial remission (n = 15). Among them, 24 patients underwent autograft manipulation with positive selection of CD34⁺ cells using a CliniMACS device (purged group). Twenty‐five patients had received ≥2 previous chemotherapy regimens before auto‐SCT. After a median follow‐up of 41·4 months, transplant‐related mortality was observed only in unpurged group (n = 2; 3·6%). The 3‐year overall survival (91·7% vs. 56·1%, P = 0·009) and progression‐free survival (78·7% vs. 53·1%, P = 0·034) favoured CD34⁺ purification. While neutrophil recovery was similar, platelet recovery was delayed in the purged group. Cytomegalovirus reactivation was predominantly observed in the purged group, although no other clinically unmanageable infectious complications occurred. Although this study has the inevitable limitations of heterogeneity in previous treatment and NHL subtypes, and a small number of patients analysed, the high survival rate in the purged group may suggest the need for prospective randomized trials to determine the role of CD34⁺ purification in auto‐SCT for NHL.     

Combined cord blood and bone marrow transplantation from the same human leucocyte antigen‐identical sibling donor for children with malignant and non‐malignant diseases

Umbilical cord blood (UCB) from an human leucocyte antigen (HLA)‐identical sibling can be used for transplantation of patients with malignant and non‐malignant diseases. However, the low cellular content of most UCB units represents a limitation to this approach. An option to increase cell dose is to harvest bone marrow (BM) cells from the same donor and infuse them along with the UCB. We studied 156 children who received such a combined graft between 1992 and 2011. Median age was 7 years and 78% of patients (n = 122) were transplanted for non‐malignant diseases, mainly haemoglobinopathies. Acute leukaemia (n = 26) was the most frequent malignant diagnosis. Most patients (91%) received myeloablative conditioning. Median donor age was 1·7 years, median infused nucleated cell dose was 24·4 × 10⁷/kg and median follow‐up was 41 months. Sixty‐days neutrophil recovery occurred in 96% of patients at a median of 17 d. The probabilities of grade‐II‐IV acute and chronic graft‐versus‐host disease (GVHD) were 19% and 10%, respectively. Four‐year overall survival was 90% (68% malignant; 97% non‐malignant diseases) with 3% probability of death. In conclusion, combined UCB and BM transplantation from an HLA‐identical sibling donor is an effective treatment for children with malignant and non‐malignant disorders with high overall survival and low incidence of GVHD.     

Oral anti‐CD3 immunotherapy for HCV‐nonresponders is safe,promotes regulatory T cells and decreases viral load and liver enzyme levels: results of a phase‐2a placebo‐controlled trial

Orally administered anti‐CD3 antibodies are biologically active in the gut through induction of regulatory T cells, exert an immune‐modulatory effect, and alleviate insulin resistance and liver damage in patients with NASH. Aims: To determine the safety of oral anti‐CD3 monoclonal antibody (MAb) immunotherapy in chronic HCV patients with associated immune dysfunction. Methods: Four groups (n = 9) of chronic HCV patients who were nonresponders to interferon plus ribavirin therapy received oral placebo (group A) or anti‐CD3 MAb at one of three dosage levels for 30 days. Patients were followed for safety parameters and serum levels of liver enzymes, virus, cytokines and regulatory T cells. Results: Oral anti‐CD3 immunotherapy was safe and well tolerated; no treatment‐related adverse events were noted. The following improvements were noted relative to pretreatment levels: HCV viral load and AST and ALT levels decreased in the low‐ and high‐dose groups following 30 days of therapy. In two of the treated groups, an increase in regulatory T cells (CD4⁺ CD25⁺) was noted. The positive effects were somewhat more apparent in subjects with initially elevated liver enzyme levels. Conclusions: Oral anti‐CD3 MAb immunotherapy for nonresponder HCV patients was safe and well tolerated. Trends and statistically significant improvements were observed as reductions in viral load and liver enzyme levels, along with an increase in regulatory T‐cell levels. These data support a role for the immune system in the pathogenesis of HCV infection and suggest that this immunotherapy is worthy of evaluation in combination with HCV antiviral drugs.     

Apolipoprotein M can discriminate HNF1A‐MODY from Type 1 diabetes

Aims

Missed diagnosis of maturity‐onset diabetes of the young (MODY) has led to an interest in biomarkers that enable efficient prioritization of patients for definitive molecular testing. Apolipoprotein M (apoM) was suggested as a biomarker for hepatocyte nuclear factor 1 alpha (HNF1A)‐MODY because of its reduced expression in Hnf1a^–/– mice. However, subsequent human studies examining apoM as a biomarker have yielded conflicting results. We aimed to evaluate apoM as a biomarker for HNF1A‐MODY using a highly specific and sensitive ELISA.

Methods

ApoM concentration was measured in subjects with HNF1A‐MODY (n = 69), Type 1 diabetes (n = 50), Type 2 diabetes (n = 120) and healthy control subjects (n = 100). The discriminative accuracy of apoM and of the apoM/HDL ratio for diabetes aetiology was evaluated.

Results

Mean (standard deviation) serum apoM concentration (μmol/l) was significantly lower for subjects with HNF1A‐MODY [0.86 (0.29)], than for those with Type 1 diabetes [1.37 (0.26), P = 3.1 × 10^–18) and control subjects [1.34 (0.22), P = 7.2 × 10^–19). There was no significant difference in apoM concentration between subjects with HNF1A‐MODY and Type 2 diabetes [0.89 (0.28), P = 0.13]. The C‐statistic measure of discriminative accuracy for apoM was 0.91 for HNF1A‐MODY vs. Type 1 diabetes, indicating high discriminative accuracy. The apoM/HDL ratio was significantly lower in HNF1A‐MODY than other study groups. However, this ratio did not perform well in discriminating HNF1A‐MODY from either Type 1 diabetes (C‐statistic = 0.79) or Type 2 diabetes (C‐statistic = 0.68).

Conclusions

We confirm an earlier report that serum apoM levels are lower in HNF1A‐MODY than in controls. Serum apoM provides good discrimination between HNF1A‐MODY and Type 1 diabetes and warrants further investigation for clinical utility in diabetes diagnostics.     

Haematopoietic stem cell transplantation for severe sickle cell disease in childhood: a single centre experience of 50 patients

Despite improvements in medical management, sickle cell disease (SCD) remains associated with severe morbidity and decreased survival. Allogeneic haematopoietic stem cell transplantation (HSCT) remains the only curative approach. We report the outcome of 50 consecutive children with severe SCD that received HSCT in our unit between November 1988 and April 2013. The stem cell source was bone marrow (n = 39), cord blood (n = 3), bone marrow and cord blood (n = 7) and peripheral blood stem cells (n = 1). All patients had ≥1 severe manifestation: 37 presented with recurrent vaso‐occlusive crises/acute chest syndrome, 27 cerebral vasculopathy and 1 nephropathy. The conditioning regimen consisted of busulfan + cyclophosphamide (BuCy) before November 1991 and BuCy + rabbit antithymocyte globulin after that date. Since 1995, all patients have been treated with hydroxycarbamide (HC) prior to transplantation for a median duration of 2·7 years. Median age at transplantation and median follow‐up was 8·3 and 7·7 years, respectively. Acute graft‐versus‐host disease (GVHD) and chronic GVHD were observed in 11 and 10 patients, respectively. An excellent outcome was achieved, with 8‐year overall survival and event‐free survival (EFS) rates of 94·1% and 85·6%, respectively. Since HC introduction, no graft failure occurred and EFS reached 97·4%. Prior treatment with HC may have contributed to successful engraftment.     

Rapamycin combined with donor immature dendritic cells promotes liver allograft survival in association with CD4(+) CD25(+) Foxp3(+) regulatory T cell expansion

Aim: To determine whether donor immature dendritic cells (imDCs) combined with a short postoperative course of rapamycin (Rapa) has the ability to expand the CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and prolong liver allograft survival. Methods: Orthotopic liver transplantation (OLT) was performed from Lewis rats to Brown Norway recipients. Three days before transplantation, animals were injected intravenously with 2 × 10⁶ donor bone marrow‐derived imDCs. Recipient rats (the combined treated group) also received Rapa for 7 d after liver transplantation. Additional groups received either imDCs alone, Rapa alone, or saline alone. Every six recipients from each group were killed at 14 days, 28 days after OLT. The changes of CD4⁺CD25⁺Foxp3⁺ Treg cells in peripheral blood and spleen, histological changes of liver grafts, and serum cytokine levels were investigated. The other six recipients were left in each group to observe the animal survival. Results: Donor imDCs followed by a short postoperative course of Rapa induced long‐term allograft survival. The percentage of CD4⁺CD25⁺Foxp3⁺ Treg cells in CD4⁺ T cells in the combination treatment group were significantly higher compared with the acute rejection group. Moreover, within the CD4⁺CD25⁺ T cell population the combination treatment recipients maintained a higher incidence of Foxp3⁺ T cells compared with the other groups. Despite the lower serum levels of interleukin (IL)‐2, IL‐12, and interferon‐γ in the combined treated group, the cytokine levels in the combined treated group at 7 days after OLT was nearly twice that at 3 days after OLT but decreased significantly compared with the other groups at 28 days after OLT. Serum IL‐10 level in the combined treated group was higher than the other groups. Conclusions: A single imDC infusion followed by a short postoperative course of Rapa prolongs liver allograft survival and enhances the expansion of Treg cells. This optimal protocol may be a promising administration protocol for the peritransplant tolerance induction.     

CD34+ selected stem cell boosts can improve poor graft function after paediatric allogeneic stem cell transplantation

Poor graft function (PGF) is a severe complication of haematopoietic stem cell transplantation (HSCT) and administration of donor stem cell boosts (SCBs) represents a therapeutic option. We report 50 paediatric patients with PGF who received 61 boosts with CD34⁺ selected peripheral blood stem cells (PBSC) after transplantation from matched unrelated (n = 25) or mismatched related (n = 25) donors. Within 8 weeks, a significant increase in median neutrophil counts (0·6 vs. 1·516 × 10⁹/l, P < 0·05) and a decrease in red blood cell and platelet transfusion requirement (median frequencies 1 and 7 vs. 0, P < 0·0001 and <0·001), were observed, and 78·8% of patients resolved one or two of their cytopenias. 36·5% had a complete haematological response. Median lymphocyte counts for CD3⁺, CD3⁺CD4⁺, CD19⁺ and CD56⁺ increased 8·3‐, 14·2‐, 22.‐ and 1·6‐fold. The rate of de novo acute graft‐versus‐host disease (GvHD) grade I–III was only 6% and resolved completely. No GvHD grade IV or chronic GvHD occurred. Patients who responded to SCB displayed a trend toward better overall survival (OS) (P = 0·07). Thus, administration of CD34⁺ selected SCBs from alternative donors is safe and effective. Further studies are warranted to clarify the impact on immune reconstitution and survival.     

Donor lymphocyte infusions and second transplantation as salvage treatment for relapsed myelofibrosis after reduced‐intensity allografting

Thirty myelofibrosis patients (21 males, nine females) with relapse (n = 27) or graft‐rejection (n = 3) after dose‐reduced allografting underwent a salvage strategy including donor lymphocyte infusions (DLIs) and/or second allogeneic haematopoietic stem cell transplantation (HSCT). Twenty‐six patients received a median number of three (range, 1–5) DLIs in a dose‐escalated mode starting with a median dose of 1·2 × 10⁶ (range, 0·003–8 × 10⁶) up to median dose of 40 × 10⁶ T‐cells/kg (range, 10–130 × 10⁶). 10/26 patients (39%) achieved complete response (CR) to DLIs. Acute (grade II‐IV) and chronic graft‐versus‐host (GvHD) disease occurred in 12% and 36% cases. Thirteen non‐responders to DLI and four patients who did not receive DLI due to graft‐rejection or acute transformation of the blast phase underwent a second allogeneic HSCT from alternative (n = 15) or the same (n = 2) donor. One patient (6%) experienced primary graft‐failure and died. Acute (II‐IV) and chronic GvHD were observed in 47% and 46% of patients. Overall responses after second HSCT were seen in 12/15 patients (80%: CR: n = 9, partial response: n = 3). The 1‐year cumulative incidence of non‐relapse mortality for recipients of a second allograft was 6%, and the cumulative incidence of relapse was 24%. After a median follow‐up of 27 months, the 2‐year overall survival and progression‐free survival for all 30 patients was 70% and 67%, respectively. In conclusion, our two‐step strategy, including DLI and second HSCT for non‐responding or ineligible patients, is an effective and well‐tolerated salvage approach for patients relapsing after reduced‐intensity allograft after myelofibrosis.     

Indicators and outcome of liver transplantation in acute liver decompensation after flares of hepatitis B

Summary. Non‐cirrhotic patients having acute liver decompensation in flares of hepatitis B can recover spontaneously or die without liver transplantation. Criteria for identifying patients in need of liver transplantation are lacking. Fifty‐one non‐cirrhotic patients having acute liver decompensation in flares of hepatitis B were retrospectively reviewed. The patients were divided into three groups: group A patients (n = 18) recovered from acute liver decompensation spontaneously; group B patients (n = 22) died of acute liver failure; and group C patients (n = 11) had liver transplantation. Model of end‐stage liver disease (MELD) scores were evaluated to identify the criteria for liver transplantation. The cut‐off point of MELD scores for liver transplantation was evaluated by receiver operating characteristic (ROC) curve. Comparing group A and B patients, MELD score was an independent factor to predict prognosis. By analysing ROC curve, a MELD score > 30 was the most optimal cut‐off point to indicate liver transplantation; however, the false positive rate was 11.1%. By weekly measurement of MELD scores, subsequent increase in MELD scores could help to avoid false positives. Moreover, a MELD score > 34 yielded 0% false positive rate and indicated the necessity of definite liver transplantation. For group C patients, ten of 11 patients were saved by liver transplantation. In conclusion, for the patients having acute liver decompensation in flares of hepatitis B, liver transplantation is definitely indicated by MELD scores > 34. Liver transplantation is also indicated if the MELD score increases in the subsequent 1–2 weeks. Liver transplantation has a good outcome if performed on time.     

Early lymphocyte recovery at 28 d post‐transplant is predictive of reduced risk of relapse in patients with acute myeloid leukemia transplanted with peripheral blood stem cell grafts

Allogeneic hematopoietic cell transplantation (HCT) is potentially curative for acute myeloid leukemia (AML). Impact of lymphocyte recovery on post‐transplant outcomes has been suggested but reports are conflicting. We evaluated the impact of lymphocyte recovery at 28 d post‐HCT in 191 AML patients using peripheral blood stem cells as graft. Patients were divided into those with absolute lymphocyte count (ALC) ≥0.5 × 10⁹/L (n = 111, 58%; high ALC group) and those with ALC <0.5 × 10⁹/L (n = 80, 42%; low ALC group), at day 28 post‐transplant. With a median follow‐up of 49 months, overall survival (OS) was significantly improved in the high ALC group (59% at 3 yr) vs. patients with low ALC (40% at 3 yr, P = 0.03). Cumulative incidence of relapse (CIR) was significantly lower in the high ALC group (16% at 3 yr) vs. low ALC group (36% at 3 yr, P = 0.001). Multivariable analysis for CIR demonstrated high ALC group as an independent factor decreasing relapse risk (P = 0.03, HR = 0.49, 95% CI = 0.26–0.92). Multivariable analysis for OS and non‐relapse mortality did not demonstrate ALC ≥0.5 × 10⁹/L at 28 d post‐transplant to be predictive. We conclude that lymphocyte recovery with ALC ≥0.5 × 10⁹/L at day 28 post‐transplant is associated with less relapse in AML patients undergoing allogeneic peripheral blood HCT, but without survival benefit.     

Treatment of graft failure with TNI‐based reconditioning and haploidentical stem cells in paediatric patients

Graft failure is a life‐threatening complication after allogeneic haematopoietic stem cell transplantation (HSCT). We report a cohort of 19 consecutive patients (median age: 8·5 years) with acute leukaemias (n = 14) and non‐malignant diseases (n = 5) who experienced graft failure after previous HSCT from matched (n = 3) or haploidentical donors (n = 16) between 2003 and 2012. After total nodal irradiation (TNI)‐based reconditioning combined with fludarabine, thiotepa and anti‐T cell serotherapy, all patients received T cell‐depleted peripheral blood stem cell grafts from a second, haploidentical donor. Median time between graft failure and retransplantation was 14 d (range 7–40). Sustained engraftment (median: 10 d, range 9–32) and complete donor chimerism was observed in all evaluable patients. 5 patients additionally received donor lymphocyte infusions. Graft‐versus‐host disease (GvHD) grade II and III occurred in 1 patient each (22%); no GvHD grade IV was observed. 2 patients had transient chronic GvHD. The regimen was well tolerated with transient interstitial pneumonitis in one patient. Treatment‐related mortality after one year was 11%. Event‐free survival and overall survival 3 years after retransplantation were 63% and 68%. Thus, a TNI‐based reconditioning regimen followed by transplantation of haploidentical stem cells is an option to rescue patients with graft failure within a short time span and with low toxicity.     

Administration of alemtuzumab and G‐CSF to adults with relapsed or refractory acute lymphoblastic leukemia: results of a phase II study

The outlook for adults with refractory and relapsed acute lymphocytic leukemia (ALL) is poor. CD52 is expressed in most patients with ALL. Alemtuzumab is an anti‐CD52 humanized monoclonal antibody. This phase II study assessed the efficacy of alemtuzumab combined with granulocyte‐colony stimulating factor (G‐CSF) to boost antibody‐dependent cell cytotoxicity mediated by neutrophils. Twelve patients with relapsed (n = 11) or refractory (n = 1) ALL, including four relapses postallogeneic stem cell transplantation, were treated and monitored between October 2006 and January 2011. Patients received 1 wk of alemtuzumab every other day at increasing doses of 3, 10, and 30 mg to test tolerance and 30 mg three times a week for 12–18 infusions. If in complete remission (CR), patients received maintenance therapy for 1 wk, every 2 months. G‐CSF was administered at 5 μg/kg per day during alemtuzumab administration. The primary endpoint was disappearance of blast cells on a marrow aspirate. CD52 was expressed in all patients. Four patients reached CR. In one additional patient, clearance of blast cells was observed in peripheral blood but not in the marrow. The most frequent adverse events during course 1 of treatment were fever and chills (n = 3), skin rash (n = 3), and bronchospasm (n = 2). Tumor lysis syndrome was observed at treatment initiation in one patient who reached CR. All patients progressed within a few months and all but one died. The surviving patient is still alive after relapse and a second allogeneic stem cell transplantation. This study shows that in relapse/refractory ALL, alemtuzumab with G‐CSF can produce good responses of short duration.     

Effects of high‐intensity focused ultrasound and anti‐angiogenic agents on the ablation of experimental liver cancers

OBJECTIVE : To assess the efficacy of high‐intensity focused ultrasound (HIFU) when combined with heparin and cortisone acetate in treating experimental liver cancer LTNM₄ implanted subcutaneously in nude mice. METHODS : High‐intensity focused ultrasound treatment was administered with a focused 1.1 MHz transducer at a peak intensity of 750 W/cm² for a continuous exposure of 20 s and, 1 day later, heparin (300 U/mL) was given orally in drinking water and an initial dose of 250 mg/kg cortisone acetate was injected subcutaneously into each mouse. Forty‐eight mice with tumors were randomly divided into four groups. Group I (n = 12) were controls; Group II (n = 12) received heparin and cortisone therapy; Group III (n = 12) received HIFU therapy only; Group IV (n = 12) received both HIFU, heparin and cortisone therapy. RESULTS : Significant inhibition of tumor growth was seen in groups II, III and IV as compared with group I (P < 0.05), with the tumor growth inhibition rate on the 28th day being 86, 90 and 99%, respectively. All mice in group IV were cured, with no evidence of tumors by the 35th day and, histologically, no tumor cells remained. CONCLUSIONS : The data suggest that HIFU can destroy liver cancer effectively and that HIFU combined with treatment using the anti‐angiogenic agents heparin and cortisone may constitute a comprehensive treatment to ablate liver cancer.     

Over-expression of CXCR4 on mesenchymal stem cells augments myoangiogenesis in the infarcted myocardium

Bone marrow mesenchymal stem cells (MSCs) participate in myocardial repair following myocardial infarction. However, their in vivo reparative capability is limited due to lack of their survival in the infarcted myocardium. To overcome this limitation, we genetically engineered male rat MSCs overexpressing CXCR4 in order to maximize the effect of stromal cell-derived factor-1α (SDF-1α) for cell migration and regeneration. MSCs were isolated from adult male rats and cultured. Adenoviral transduction was carried out to over-express either CXCR4/green fluorescent protein (Ad-CXCR4/GFP) or Ad-null/GFP alone (control). Flow cytometry was used to identify and isolate GFP/CXCR4 over-expressing MSCs for transplantation. Female rats were assigned to one of four groups (n = 8 each) to receive GFP-transduced male MSCs (2 × 10⁶) via tail vein injection 3 days after ligation of the left anterior descending (LAD) coronary artery: GFP-transduced MSCs (Ad-null/GFP-MSCs, group 1) or MSCs over-expressing CXCR4/GFP (Ad-CXCR4/GFP-MSCs, group 2), or Ad-CXCR4/GFP-MSCs plus SDF-1α (50 ng/μl) (Ad-CXCR4/GFP-MSCs/SDF-1α, group 3), or Ad-miRNA targeting CXCR4 plus SDF-1α (Ad-miRNA/GFP-MSCs + SDF-1α treatment, group 4). Cardiodynamic data were obtained 4 weeks after induction of regional myocardial infarction (MI) using echocardiography after which hearts were harvested for immunohistochemical studies. The migration of GFP and Y-chromosome positive cells increased significantly in the peri- and infarct areas of groups 2 and 3 compared to control group (p < 0.05), or miRNA-CXCR4 group (p < 0.01). The number of CXCR4 positive cells in groups 2, 3 was intimately associated with angiogenesis and myogenesis. MSCs engraftment was blocked by pretreatment with miRNA (group 4). Cardiac function was significantly improved in rats receiving MSCs over-expressing CXCR4 alone or with SDF-1α. The up-regulation of matrix metalloproteinases (MMPs) by CXCR4 overexpressing MSCs perhaps facilitated their engraftment in the collagenous tissue of the infarcted area. CXCR4 over-expression led to enhance in vivo mobilization and engraftment of MSCs into ischemic area where these cells promoted neomyoangiogenesis and alleviated early signs of left ventricular remodeling.     

Hepatic compartmentalization of exhausted and regulatory cells in HIV/HCV‐coinfected patients

Accelerated intrahepatic hepatitis C virus (HCV) pathogenesis is likely the result of dysregulation within both the innate and adaptive immune compartments, but the exact contribution of peripheral blood and liver lymphocyte subsets remains unclear. Prolonged activation and expansion of immunoregulatory cells have been thought to play a role. We determined immune cell subset frequency in contemporaneous liver and peripheral blood samples from chronic HCV‐infected and HIV/HCV‐coinfected individuals. Peripheral blood mononuclear cells (PBMC) and biopsy‐derived liver‐infiltrating lymphocytes from 26 HIV/HCV‐coinfected, 10 chronic HCV‐infected and 10 HIV‐infected individuals were assessed for various subsets of T and B lymphocytes, dendritic cell, natural killer (NK) cell and NK T‐cell frequency by flow cytometry. CD8⁺ T cells expressing the exhaustion marker PD‐1 were increased in HCV‐infected individuals compared with uninfected individuals (P = 0.02), and HIV coinfection enhanced this effect (P = 0.005). In the liver, regulatory CD4⁺CD25⁺Foxp3⁺ T cells, as well as CD4⁺CD25⁺PD1⁺ T cells, were more frequent in HIV/HCV‐coinfected than in HCV‐monoinfected samples (P < 0.001). HCV was associated with increased regulatory T cells, PD‐1⁺ T cells and decreased memory B cells, regardless of HIV infection (P ≤ 0.005 for all). Low CD8⁺ expression was observed only in PD‐1⁺CD8⁺ T cells from HCV‐infected individuals and healthy controls (P = 0.002) and was associated with enhanced expansion of exhausted CD8⁺ T cells when exposed in vitro to PHA or CMV peptides. In conclusion, in HIV/HCV coinfection, ongoing HCV replication is associated with increased regulatory and exhausted T cells in the periphery and liver that may impact control of HCV. Simultaneous characterization of liver and peripheral blood highlights the disproportionate intrahepatic compartmentalization of immunoregulatory T cells, which may contribute to establishment of chronicity and hepatic fibrogenesis in HIV coinfection.     

Expansion of CD4+CD25+FoxP3+ regulatory T cells in hepatitis C virus‐related chronic hepatitis,cirrhosis and hepatocellular carcinoma

Aim: Regulatory T (Treg) cells may play a pivotal role in the persistence of hepatitis C virus (HCV) infection and the development of hepatocellular carcinoma (HCC). Therefore, we examined their frequency in peripheral blood from patients with HCV‐positive chronic hepatitis (CH), cirrhosis (LC) and HCC. Methods: Treg cells were identified as CD4⁺, CD25⁺ and FoxP3⁺ T lymphocytes using three‐color FACS. The frequency of Treg cells was expressed as a percentage of the total CD4⁺ T lymphocytes, and the phenotype of Treg cells was examined using CD45RA. Results: Treg cells were significantly increased in CH (5.88 ± 0.19%, n = 76; P < 0.01), LC (6.10 ± 0.28%, n = 40; P < 0.001) and HCC (6.80 ± 0.30%, n = 57; P < 0.0001) compared to healthy control (5.13 ± 0.25%, n = 31). However, Treg cells were not increased with the progression of fibrosis or the grade of inflammations. Treg cells were slightly increased in early‐stage HCC (6.91 ± 0.40%) compared with advanced‐stage HCC (6.58 ± 0.39%), but these results were not statistically significant. In a serial examination, a distinct increase in Treg cells after local therapy for early‐stage HCC was a hallmark of early recurrence. Most expanded Treg cells in HCC were CD45RA^‐, suggesting that a memory‐type Treg population had differentiated in the periphery and not in the thymus. Conclusion: We observed an increase in Treg cells in HCV‐related chronic liver disease, particularly in HCC, and these cells were shown to be memory‐type Treg cells.