Dongmei Han

Mesenchymal Stem Cells Enhance Allogeneic Islet Engraftment in Nonhuman Primates

OBJECTIVE To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation. RESEARCH DESIGN AND METHODS Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype, gene expression, differentiation potential, and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels, reduction of exogenous insulin requirement (EIR), C-peptide levels, changes in peripheral blood T regulatory cells, and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection. RESULTS MSC phenotype and a normal karyotype were observed through passage 11. IL-6, IL-10, vascular endothelial growth factor, TGF-β, hepatocyte growth factor, and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8), as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood. CONCLUSIONS MSCs may provide an important approach for enhancement of islet engraftment, thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore, MSCs may serve as a new, safe, and effective antirejection therapy.

Mesenchymal stem cell labeling and in vitro MR characterization at 1.5 T of new SPIO contrast agent: Molday ION Rhodamine-B™

In vivo detection of transplanted stem cells is requisite for improving stem cell-based treatments by developing a thorough understanding of their therapeutic mechanisms. MRI tracking of magnetically labeled cells is non-invasive and is suitable for longitudinal studies. Molday ION Rhodamine-B™ (MIRB) is a new superparamagnetic iron oxide (SPIO) contrast agent specifically formulated for cell labeling and is readily internalized by non-phagocytic cells. This investigation characterizes mesenchymal stem cell (MSC) labeling and MR imaging properties of this new SPIO agent. Effects of MIRB on MSC viability and differentiation as well as cellular loading properties were assessed for MSC labeled with MIRB at concentrations from 5 to 100 µg Fe/ml. Labeled MSC were evaluated, in vitro, on a clinical 1.5 T MRI. Optimal scanning sequences and imaging parameters were determined based on contrast-to-noise ratio and contrast modulation. Relaxation rates (1/T(2)*) for gradient-echo sequences were approximated and an idealized limit of detection was established. MIRB labeling did not affect MSC viability or the ability to differentiate into either bone or fat. Labeling efficiency was found to be approximately 95% for labeling concentrations at or above 20 µg Fe/ml. Average MIRB per MSC ranged from 0.7 pg Fe for labeling MIRB concentration of 5 µg Fe/ml and asymptotically approached a value of 20-25 pg Fe/MSC as labeling concentration increased to 100 µg Fe/ml. MRI analysis of MIRB MSC revealed long echo time, gradient echo sequences to provide the most sensitivity. Limit of detection for gradient echo sequences was determined to be less than 1000 MSC, with approximately 15 pg Fe/MSC (labeled at 20 µg Fe/ml). These investigations have laid the groundwork and established feasibility for the use of this contrast agent for in vivo MRI detection of MSC. Properties evaluated in this study will be used as a reference for tracking labeled MSC for in vivo studies.

Combined islet and hematopoietic stem cell allotransplantation: a clinical pilot trial to induce chimerism and graft tolerance.

To prevent graft rejection and avoid immunosuppression‐related side‐effects, we attempted to induce recipient chimerism and graft tolerance in islet transplantation by donor CD34+hematopoietic stem cell (HSC) infusion. Six patients with brittle type 1 Diabetes Mellitus received a single‐donor allogeneic islet transplant (8611 ± 2113 IEQ/kg) followed by high doses of donor HSC (4.3 ± 1.9 × 106 HSC/kg), at days 5 and 11 posttransplant, without ablative conditioning. An ‘Edmonton‐like’ immunosuppression was administered, with a single dose of anti‐TNFα antibody (Infliximab) added to induction. Immunosuppression was weaned per protocol starting 12 months posttransplant. After transplantation, glucose control significantly improved, with 3 recipients achieving insulin‐independence for a short time (24 ± 23 days). No severe hypoglycemia or protocol‐related adverse events occurred. Graft function was maximal at 3 months then declined. Two recipients rejected within 6 months due to low immunosuppressive trough levels, whereas 4 completed 1‐year follow‐up with functioning grafts. Graft failure occurred within 4 months from weaning (478 ± 25 days posttransplant). Peripheral chimerism, as donor leukocytes, was maximal at 1‐month (5.92 ± 0.48%), highly reduced at 1‐year (0.20 ± 0.08%), and was undetectable at graft failure. CD25+T‐lymphocytes significantly decreased at 3 months, but partially recovered thereafter. Combined islet and HSC allotransplantation using an ‘Edmonton‐like’ immunosuppression, without ablative conditioning, did not lead to stable chimerism and graft tolerance.

Immune profiling by multiple gene expression analysis in patients at-risk and with type 1 diabetes.

There is a need for biomarkers to monitor the development and progression of type 1 DM. We analyzed mRNA expression levels for granzyme B, perforin, fas ligand, TNF-α, IFN-γ, Foxp3, IL-10, TGF-β, IL-4, IL-6, IL-17, Activation-induced cytidine deaminase (AID) and Immunoglobulin G gamma chain (IgG) genes in peripheral blood of at-risk, new-onset and long-term type 1 DM , and healthy controls. The majority of the genes were suppressed in long-term type 1 DM compared to controls and new-onset patients. IFN-γ, IL-4 and IL-10 mRNA levels were significantly higher in new-onset compared to at-risk and long-term groups. There was decreased mRNA expression for AID and IgG and up-regulation of IFN-γ with age in controls. Data suggest an overall depressed immunity in long-term type 1 DM. Increased gene expression levels for IFN-γ, IL-4 and IL-10 in new-onset patients from at-risk patients might be used as potential markers for progression of the disease.

Innate and adaptive immune gene expression profiles as biomarkers in human type 1 diabetes

The mRNA levels of a set of immune‐related genes were analysed with peripheral blood samples from at‐risk, new‐onset and long‐term type 1 diabetes (T1D) patients, in comparison to those from healthy controls. The selected set includes T lymphocyte genes [CD3G and cytotoxic T lymphocyte‐associated antigen 4 (CTLA4)], B lymphocyte genes (CD19 and CD20) and myeloid cell‐related genes [CD11b, Toll‐like receptor (TLR)‐9, arginase (ARG1)]. Also included is a subset of the S100 family members that has been documented recently as regulatory elements of innate immunity. Samples from patients with long‐term T1D had a reduced level of mRNA for most of selected innate and adaptive immune genes. No such reduction was detected in samples collected from at‐risk or new‐onset T1D patients. Analyses of regulatory gene expression ratios revealed a dynamic disproportion of CTLA4 versus CD3G expression in samples from at‐risk, new‐onset and long‐term T1D patients. These changes could serve as immunological biomarkers for the status of the immune system during T1D progression and therapeutic interventions.

Choice of immunosuppression influences cytomegalovirus DNAemia in cynomolgus monkey (Macaca fascicularis) islet allograft recipients.

This retrospective study reviews the results of our experience with the occurrence of CMV DNAemia in islet cell transplanted cynomolgus monkeys subjected to different immunosuppressive protocols, including induction treatment with thymoglobulin (TMG), with a combination of thymoglobulin and fludarabine (FLUD), with cyclophosphamide, or with daclizumab. CMV DNA in the peripheral blood (CMV DNAemia) of 47 monkeys was quantified by real-time PCR on a weekly to biweekly basis. As compared to other immunosuppressive regimens, and in association with greater decreases in WBC, lymphocyte, CD3+CD4+, and CD3+CD8+ lymphocyte counts, frequent CMV DNAemia occurred earlier (within the first month posttransplant), and was of greater severity and duration in recipients of TMG ± FLUD. Treatment of recipients with alternative induction agents that resulted in less dramatic reductions in WBC and lymphocyte counts, however, resulted in occurrence of CMV DNAemia after postoperative day 60. The frequency, average intensity, duration, and area under the curve (AUC) for CMV DNAemia in animals receiving TMG ± FLUD were 75–100%, 4.02 ± 1.75 copies/ng DNA, 23.0 ± 5.3 days, and 367.0 ± 121.1 days x copies/ng DNA, respectively; corresponding values in animals receiving other treatments (0–44%, 0.19 ± 0.10 copies/ng DNA, 0.5 ± 0.3 days, and 75.4 ± 40.2 days x copies/ng DNA, respectively) were significantly different. The value of WBC, T and B cells at the nadir of cell depletion greatly affects the occurrence of CMV DNAemia. No animals developed CMV DNAemia within the next 3 weeks when the lowest value of WBC, lymphocyte, CD3+, CD3+CD4+, CD3+CD8+, or CD20+ cells was above 4500, 1800, 300, 200, 150, or 300 cells/μl, respectively. Oral valganciclovir prophylaxis did not completely prevent the appearance of CMV DNAemia.

Peripheral blood cytotoxic lymphocyte gene transcript levels differ in patients with long-term type 1 diabetes compared to normal controls

The purpose of this study was to compare mRNA levels of the cytotoxic lymphocyte (CL) gene products: granzyme B (GB), perforin (P), and fas ligand (FasL) in patients with long-term type 1 diabetes and healthy controls. The objective was to utilize this information to follow patients as they undergo islet cell transplantation at our center and to determine if changes in CL gene transcript levels correlate with graft status. We have measured mRNA levels for CL genes in peripheral blood samples from 65 long-term (>5 years) type 1 diabetes patients and 29 healthy controls. Total RNA was extracted from EDTA anticoagulated peripheral blood samples and reverse transcribed into first-strand cDNA using SuperScript II reverse Transcriptase. Quantitative, real-time PCR was utilized to determine CL gene transcript levels. mRNA levels of P and FasL genes were found to be significantly lower for patients with type 1 diabetes compared to normal controls (p < 0.05). However, there was no significant difference for GB mRNA levels between patients and controls (p > 0.05). The decreased expression of P and FasL in patients with long-term type 1 diabetes might contribute to the inability to maintain normal levels of peripheral tolerance, which is essential for protection from autoimmune disease.

From biomarkers to a clue of biology: a computation-aided perspective of immune gene expression profiles in human type 1 diabetes

Dysregulated expression of key immune genes may cause breakdown of immunological tolerance and development of autoimmune disorders such as type 1 diabetes (T1D). General immune insufficiencies have also been implicated as a trigger of autoimmunity, due to their potential impact on immune homeostasis. Recent studies have detected evidence of systemic reduction in immune gene expression in long-term diabetic patients but the changes were not present before or at T1D onset. The changes could not be merely correlated with alteration in metabolic parameters. The studies also identified a dynamic expression pattern of several well-known as well as little-studied, immune-related genes during the course of T1D. An intriguing “ratio profile” of immune regulatory genes, such as CTLA4 and members of the S100 family, versus “baseline” immune genes, such as CD3G, prompted us to further examine immune gene expression relationships for a set of molecules representing T cells, B cells, and myeloid cells. No evidence was found to suggest an overall breach of tolerance equilibrium in T1D. Perplexingly, patients with long-term T1D presented a gene expression profile that was surprisingly more coordinated in analyses of “networking” relationship. Computational analyses of the “ratio profiles” or “relationship profiles” of immune gene expression might provide a clue for further studies of immunobiology in human T1D and other autoimmune diseases, as to how the profiles may be related to the pathogenic cause of the disease, to the effect of the diseases on immune homeostasis, or to an immunological process associated with the course of the diseases but is neither a direct cause nor a direct effect of the diseases.

Quantitative polymerase chain reaction assessment of chimerism in non-human primates after sex-mismatched islet and bone marrow transplantation.

BACKGROUND Accurate assessment of chimerism in recipients of islet and bone marrow transplantation (BMT) may allow for a clearer assessment of the role of chimerism in islet engraftment or rejection. A quantitative polymerase chain reaction (PCR) assay was developed for the detection of the sex-determining region of the Y chromosome (SRY) in peripheral blood samples from female non-human primate recipients of allogeneic male islets and vertebral body marrow (VBM) from the same donor. METHODS The assay incorporates a synthetic internal standard (IS) containing the same primer template sequences as the target to compete for primer annealing and amplification. Each DNA sample was coamplified with a constant amount of IS. The concentration of male DNA in the test samples was calculated from the regression equation of a standard curve that was generated by plotting the logarithm of the ratio of the intensities of SRY to IS PCR products versus the logarithm of known percentages of input male DNA. RESULTS This method allows for a correction of the variability of efficiency of the PCR technique and also overcomes the drawback of time-consuming competitive PCR. Using this assay, we quantitated the amount of male DNA in samples taken from female baboon recipients of male islets and VBM. There was detectable male donor DNA in the samples taken one day after BMT; pre-BMT samples were negative. This technique works well for samples obtained from rhesus and cynomogus monkeys as well. CONCLUSIONS It is a practical method for accurately evaluation of chimerism after sex-mismatched allogeneic BMT in non-human primate models.

Sequence-specific analysis of microchimerism by real-time quantitative polymerase chain reaction in same-sex nonhuman primates after islet and bone marrow transplantation.

Background. Accurate and sensitive detection of microchimerism in nonhuman primates (NHPs) after hematopoietic cell transplantation is essential for monitoring cell engraftment, for evaluating the success of transplant protocols, and for expanding the utility of NHP in transplantation studies. Because limited sequences are available for NHP major histocompatibility complex polymorphic loci, methods that can accurately determine low levels of donor cells in recipients with same-sex bone marrow transplantation are essential. Methods. Thirty-seven pairs of primers, 16 from monkey and 21 from human, were screened with cynomolgus DNA samples. Real-time quantitative polymerase chain reaction was developed for accurately determining low levels of donor-specific DNA in the peripheral blood of islet/bone marrow transplant recipients of same sex cynomolgus monkeys. Results. A total of six sets of primer and Taqman® probe combinations were included in this study, which are the most informative primer and probe sets ever reported for cynomolgus monkeys. Three pairs of primers were chosen from exon 2 of the Macaca DRB1 gene and another three pairs were chosen from human HLA DRB1 and DRB3 loci. Three of the six primer-probe sets were also found to work well for baboon (Papio hamadryas) and rhesus monkeys (Macaca mulatta). Sensitivity of the assay ranged from 0.03% to 0.1%, depending on the primer-probe set and donor-recipient pair. The methods are reproducible with relatively low standard error and coefficient of variation. Conclusions. This method is an informative, practical and sensitive method for the determination of donor-specific cells in the peripheral blood of NHP recipients of bone marrow transplant.