Gary Kleiner

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.

The Role of Donor Bone Marrow Infusions in Withdrawal of Immunosuppression in Adult Liver Allotransplantation

We investigated the role of donor bone marrow cell (DBMC) infusions in immunosuppression withdrawal in adult liver transplantation. Patients enrolled were at least 3 years post‐transplantation, with stable graft function. Forty‐five (study group: G1) received DBMC, and 59 (control group: G2) did not. Immunosuppression was reduced by one third upon enrollment, by another third the second year of the study and was completely withdrawn the third year.

Neuron-like Differentiation of Adipose-Derived Stem Cells From Infant Piglets in Vitro

Abstract Background/Objective: Adipose-derived stem cells (ADSCs) are mesenchymal stem cells (MSCs) that can be extracted from adipose tissue and obtained by a less invasive method and in larger quantities compared with bone marrow-derived MSCs. The objective of this study was to harvest ADSCs from piglets and to explore their neuronal differentiation potential. Methods: Adipose tissue from piglet facial or abdominal fat was digested with collagenase type XI, followed by filter and centrifugation; the isolated adipose stromal cells were cultured in dishes. MSC markers were measured by flow cytometry; 2 to 5 passage cells were used for in vitro differentiation. Adipogenic, chondrogenic, osteogenic, and neuronal differentiation was induced by incubation of the ADSCs with different induction media. Results: ADSCs were easily expanded to beyond 15 passages, maintaining the undifferentiated state and exhibiting MSC characteristics and markers CD29, CD44, and CD90. ADSCs differentiated into other mesodermal cells including adipocytes, chondrocytes, and osteocytes. These cells were induced to differentiate into neuron-like cells as evidenced by neuronal morphology and the presence of neuronal markers including microtubule-associated protein 2, neuronal nuclear antigen, and β-tubulin Ill. Conclusions: ADSCs can be readily obtained from a small amount fat tissue and expanded in culture. Adipose tissue may be an alternative source of stem cell therapy for nervous system injury.

Comèl-Netherton syndrome defined as primary immunodeficiency

BACKGROUND Mutations in serine protease inhibitor Kazal-type 5 (SPINK5), encoding the serine protease inhibitor lympho-epithelial Kazal-type 5 related inhibitor (LEKTI), cause Comèl-Netherton syndrome, an autosomal-recessive disease characterized by congenital ichthyosis, bamboo hair, and atopic diathesis. Despite increased frequency of infections, the immunocompetence of patients with Comèl-Netherton syndrome has not been extensively investigated. OBJECTIVE To define Comèl-Netherton syndrome as a primary immunodeficiency disorder and to explore the benefit of intravenous immunoglobulin replacement therapy. METHODS We enrolled 9 patients with Comèl-Netherton syndrome, sequenced SPINK5, and analyzed LEKTI expression by immunohistochemistry. Immune function was assessed by measuring cognate immunity, serum cytokine levels, and natural killer cell cytotoxicity. RESULTS All patients presented with recurrent skin infections caused predominantly by Staphylococcus aureus. All but 1 reported recurrent respiratory tract infections; 78% had sepsis and/or pneumonia; 67% had recurrent gastrointestinal disease and failure to thrive. Mutations in SPINK5-including 6 novel mutations-were identified in 8 patients. LEKTI expression was decreased or absent in all patients. Immunologic evaluation revealed reduced memory B cells and defective responses to vaccination with Pneumovax and bacteriophage phiX174, characterized by impaired antibody amplification and class-switching. Immune dysregulation was suggested by a skewed T(h)1 phenotype and elevated proinflammatory cytokine levels, whereas serum concentrations of the chemokine (C-C motif) ligand 5 and natural killer cell cytotoxicity were decreased. Treatment with intravenous immunoglobulin resulted in remarkable clinical improvement and temporarily increased natural killer cell cytotoxicity. CONCLUSION These data provide new insights into the immunopathology of Comèl-Netherton syndrome and demonstrate that this multisystem disorder, characterized by lack of LEKTI expression in epithelial cells, is complicated by cognate and innate immunodeficiency that responds favorably to intravenous immunoglobulin therapy.

An Analysis of the Association between Serum Citrulline and Acute Rejection among 26 Recipients of Intestinal Transplant

Small preliminary studies suggest that serum citrulline levels may act as a marker for acute cellular rejection in small intestinal transplant recipients. The results comparing serum citrulline concentrations with biopsy‐based grades of rejection are summarized here for an expanded group of 26 isolated intestinal and multivisceral transplant recipients. Other factors considered included patient and donor age and sex, ischemia time, serum creatinine, and type of transplant. Straight‐line fits reasonably described how each patients citrulline levels changed over time. Among 21 patients who demonstrated increasing citrulline levels over time, the estimated median time‐to‐achieve normal citrulline (≥30 μmol/L) was 79 days post‐transplant. Using stepwise linear regression, two characteristics were associated with a significantly higher maximum grade of rejection after 14 d post‐transplant: longer time‐to‐achieve normal citrulline (using ranks, p < 0.00001) and the patient not receiving a multivisceral transplant (p = 0.0005). Only the latter characteristic was significantly associated with maximum grade of rejection during the first 14 d post‐transplant (p = 0.01). Clearly, time‐to‐normalization of citrulline was delayed by the incidence of rejection, and in some cases with moderate‐to‐severe rejection, normalization of citrulline levels never occurred. We plan to further examine the use of citrulline as a marker for rejection in larger prospective studies.

Blood citrulline level is an exclusionary marker for significant acute rejection after intestinal transplantation.

Background. Serum citrulline is a marker for acute cellular rejection (ACR) after intestinal transplantation; however, its clinical utility has not yet been established. The goal of this study was to determine clearcut serum levels beyond which the diagnosis of acute rejection could be supported or refuted, and predictors of citrulline levels posttransplant from which more accurate estimates of sensitivity and specificity could be obtained. Methods. Since March 2004, we obtained 2135 dried blood spot (DBS) citrulline samples from 57 intestinal transplant recipients at or beyond 3 months posttransplant. Stepwise linear regression was performed to determine the most significant multivariable predictors of the patient’s DBS citrulline level. Results. Seven characteristics were associated with a significantly lower citrulline in multivariable analysis: presence of mild, moderate, or severe ACR; presence of bacteremia or respiratory infection; pediatric age; and time from transplant to DBS sample (P<0.00001 in each case). Using a <13 vs. ≥13 &mgr;moles/L cutoff point, the sensitivity for detecting moderate or severe ACR and the negative predictive value were high (96.4% and >99% respectively). Specificity was 54% to 74% in children and 83% to 88% in adults. Conclusions. Citrulline levels <13 &mgr;moles/L should alert the clinical team that a serious problem (rejection or infection) could be looming in a previously stable intestinal recipient. Levels ≥13&mgr;moles/L practically rule out moderate or severe rejection.

Transplantation of the Spleen: Effect of Splenic Allograft in Human Multivisceral Transplantation

Objectives:To describe the effect of the splenic allograft in human multivisceral transplantation. Summary Background Data:We performed transplants of the spleen as part of a multivisceral graft in an attempt to decrease both the risk of infection from an asplenic state and the risk of rejection by a possible tolerogenic effect. To our knowledge, this is the first report of human splenic transplantation in a large series. Methods:All primary multivisceral recipients who received a donor spleen (N = 60) were compared with those who did not receive a spleen (N = 81). Results:Thirty-five of 60 (58%) are alive in the spleen group, and 39 of 81 (48%) are alive in control group (P = 0.98). In univariate analysis, splenic recipients showed superiority in freedom-from-any rejection (P = 0.02) and freedom-from-moderate or severe rejection (P = 0.007). No significant differences were observed in analyses of infectious complications between the spleen and control groups. Both platelet and leukocyte counts became normal in splenic patients, whereas these counts were significantly increased in nonsplenic recipients. Observed incidence of graft versus host disease (GVHD) was 8.25% (5 of 60) in the spleen group and 6.2% (5 of 81) in the control group (P = 0.70). Increased incidence of autoimmune hemolysis was observed in the spleen group. Conclusions:Allograft spleen can be transplanted within a multivisceral graft without significantly increasing the risk of GVHD. The allogenic spleen seems to show a protective effect on small bowel rejection. Further investigation with longitudinal follow-up is required to precisely determine the immunologic and hematologic effects of the allograft spleen.

Utilization of dried blood spot citrulline level as a noninvasive method for monitoring graft function following intestinal transplantation

Background. Citrulline concentrations have been proposed as a marker for intestinal allograft rejection. We instituted dried blood spot (DBS) specimen monitoring of citrulline to simplify sample collection posttransplant. This study demonstrates the correlation between plasma and dried blood spot specimen citrulline concentrations after intestinal transplantation. Methods. Plasma and DBS samples were analyzed by hydrophilic interaction chromatography tandem mass spectrometry. Comparison of the strength of linear correlation was made according to the type of surgery, sonication time, DBS citrulline levels, and the time interval between the blood sample collection and the assay date. Results. A very strong linear correlation exists between the plasma and DBS citrulline concentrations (r2=0.87; P<0.001). The correlation between plasma and DBS citrulline concentrations was maintained when evaluating only the intestinal transplant recipients. There was no significant difference in the strength of linear correlation according to sonication time, cirtrulline concentrations, or length of time to assay date. Conclusions. DBS citrulline monitoring will ease sample collection following intestinal transplantation and improve the ability to detect intestinal dysfunction and rejection by a noninvasive means.

FoxP3 mRNA transcripts and regulatory cells in renal transplant recipients 10 years after donor marrow infusion

Background. We update more favorable 10-year deceased donor kidney transplant survival in 63 recipients infused perioperatively with donor vertebral body bone marrow (DBMC-i) vs. 219 noninfused controls having equivalent immunosuppression and demographics. We questioned if this was associated with putatively regulatory FoxP3 mRNA and cell phenotypes (CD4+CD25+high percentages and high DC2:DC1 ratios) in DBMC-i vs. noninfused controls. Methods. Baseline studies were performed on peripheral blood lymphocytes (PBLs) vs. marrow in normal laboratory volunteers of CD4+CD25+high percentages and DC2:DC1 by flow cytometry, and FoxP3 mRNA in CD3+ cells by real-time polymerase chain reaction. Similar studies were performed on PBL of the majority of the 10-year patients remaining with graft function: 21 (of the remaining 37) DBMC-i vs. 55 (of the remaining 105) controls. Results. In normal subjects, all parameters were significantly higher in marrow than in PBL, supporting our previous reports of ex vivo DBMC immunoregulation. At 9.8±.02 years posttransplant in DBMC-i vs. controls, death-censored percent graft failure was 17.5% vs. 32.9% (P=0.02) with 247.6±24 vs. 79.9±3.1 (mean±SE) FoxP3 copies/5,000 CD3+ cells (P=0.0001). PBL CD4+CD25+high percentages were lower, but DC2:DC1 values higher in both recipient groups than in end-stage renal disease patients who had lower FoxP3 levels (40.8±5.9, P<0.0001), consistent with non-CD4+CD25+high T regulatory cells generated long-term posttransplant. Individual higher FoxP3 values correlated with higher iliac crest chimerism in DBMC-i, but not in controls (with 50-fold lower chimerism). In chronically rejecting controls, FoxP3 was further decreased. Conclusions. Peritransplant DBMC-i has higher 10-year renal transplant acceptance, chimerism, and FoxP3 mRNA in thus-far unclarified regulatory cell phenotypes.

Pediatric Contact Dermatitis Registry Inaugural Case Data

BackgroundLittle is known about the epidemiology of allergic contact dermatitis (ACD) in US children. More widespread diagnostic confirmation through epicutaneous patch testing is needed. ObjectiveThe aim was to quantify patch test results from providers evaluating US children. MethodsThe study is a retrospective analysis of deidentified patch test results of children aged 18 years or younger, entered by participating providers in the Pediatric Contact Dermatitis Registry, during the first year of data collection (2015–2016). ResultsOne thousand one hundred forty-two cases from 34 US states, entered by 84 providers, were analyzed. Sixty-five percent of cases had one or more positive patch test (PPT), with 48% of cases having 1 or more relevant positive patch test (RPPT). The most common PPT allergens were nickel (22%), fragrance mix I (11%), cobalt (9.1%), balsam of Peru (8.4%), neomycin (7.2%), propylene glycol (6.8%), cocamidopropyl betaine (6.4%), bacitracin (6.2%), formaldehyde (5.7%), and gold (5.7%). ConclusionsThis US database provides multidisciplinary information on pediatric ACD, rates of PPT, and relevant RPPT reactions, validating the high rates of pediatric ACD previously reported in the literature. The registry database is the largest comprehensive collection of US-only pediatric patch test cases on which future research can be built. Continued collaboration between patients, health care providers, manufacturers, and policy makers is needed to decrease the most common allergens in pediatric consumer products.