Suppressor T helper type 17 cell responses in intestinal transplant recipients with allograft rejection.

BACKGROUND: Intestinal transplant (ITx) rejection is associated with memory T helper type 17 cell (Th17) infiltration of grafted tissues. Modulation of Th17 effector cell response is facilitated by T regulatory (Treg) cells, but a phenotypic characterization of this process is lacking in the context of allograft rejection. METHODS: Flow cytometry was performed to examine the expression of surface receptors, cytokines, and transcription factors in Th17 and Treg cells in ITx control (n = 34) and rejection patients (n = 23). To elucidate key pathways guiding the rejection biology, we utilized RNA sequencing (RNAseq) and assessed epigenetic stability through pyrosequencing of the Treg-specific demethylated region (TSDR). RESULTS: We found that intestinal allograft rejection is characterized by Treg cellular infiltrates, which are polarized toward Th17-type chemokine receptor, ROR-γt transcription factor expression, and cytokine production. These Treg cell subsets have maintained epigenetic stability, as defined by FoxP3-TSDR methylation status, but displayed upregulation of functional Treg and purinergic signaling genes by RNAseq analysis such as CD39, in keeping with suppressor Th17 properties. CONCLUSION: We show that ITx rejection is associated with increased polarized cells that express a Th17-like phenotype concurrent with regulatory purinergic markers.

Robot-assisted versus video-assisted thoraco-laparoscopic McKeown's esophagectomy for esophageal cancer: a propensity score-matched analysis of minimally invasive approaches.

Minimally invasive esophagectomy for esophageal cancer decreases overall complication rate and leads to faster postoperative recovery. Robot-assisted minimally invasive esophagectomy is becoming more common. Its three-dimensional view and wristed instruments may provide advantages over traditional thoraco-laparoscopic techniques. There are limited studies comparing robotic and conventional thoraco-laparoscopic esophagectomy. This study aimed to evaluate short-term outcomes of robot-assisted McKeown esophagectomy (RAME) and video-assisted McKeown esophagectomy (VAME). All consecutive patients undergoing minimally invasive McKeown esophagectomy for middle and distal third esophageal cancer between January 2016 and December 2018 at our center were included in this study. Data on baseline characteristics, pathological data and short-term outcomes were collected in a dedicated database. Postoperative complications were defined as per recommendations of Esophagectomy Complications Consensus Group. Histopathologic assessment was performed as per College of American Pathologists guidelines. Propensity score matching was performed for comparison between RAME and VAME groups using age, gender, performance status, American Society of Anesthesiologists grade, body mass index, Charlson Index, tumor location, clinical tumor stage, and neoadjuvant treatment as covariates. A total of 74 patients were included, 25 of whom underwent RAME and 49 underwent VAME. Propensity score matching on 1:1 basis produced 25 pairs of patients, comparable in terms of baseline characteristics. Total operative time and estimated blood loss was similar between the two groups. Length of hospital stay was significantly lower in RAME group. Major postoperative complications (Clavien-Dindo grade ≥ 3A) were more common in VAME group, but not statistically significant. Median number of harvested lymph nodes and R0 resection rate did not differ in between the two groups. In our experience, robot-assisted McKeown esophagectomy was comparable to video-assisted McKeown esophagectomy in terms of safety, feasibility and oncologic adequacy. Use of the robot was associated with reduced hospital stay. Further randomized controlled studies with larger patient samples are needed to compare the two.

Rejection of intestinal allotransplants is driven by memory T helper type 17 immunity and responds to infliximab.

Intestinal transplantation (ITx) can be life-saving for patients with advanced intestinal failure experiencing complications of parenteral nutrition. New surgical techniques and conventional immunosuppression have enabled some success, but outcomes post-ITx remain disappointing. Refractory cellular immune responses, immunosuppression-linked infections, and posttransplant malignancies have precluded widespread ITx application. To shed light on the dynamics of ITx allograft rejection and treatment resistance, peripheral blood samples and intestinal allograft biopsies from 51 ITx patients with severe rejection, alongside 37 stable controls, were analyzed using immunohistochemistry, polychromatic flow cytometry, and reverse transcription-PCR. Our findings inform both immunomonitoring and treatment. In terms of immunomonitoring, we found that while ITx rejection is associated with proinflammatory and activated effector memory T cells in the blood, evidence of treatment efficacy can only be found in the allograft itself, meaning that blood-based monitoring may be insufficient. In terms of treatment, we found that the prominence of intra-graft memory TNF-α and IL-17 double-positive T helper type 17 (Th17) cells is a leading feature of refractory rejection. Anti-TNF-α therapies appear to provide novel and safer treatment strategies for refractory ITx rejection; with responses in 14 of 14 patients. Clinical protocols targeting TNF-α, IL-17, and Th17 warrant further testing.

CD69+ resident memory T cells are associated with graft-versus-host disease in intestinal transplantation.

Graft-versus-host disease (GvHD) is a common, morbid complication after intestinal transplantation (ITx) with poorly understood pathophysiology. Resident memory T cells (TRM ) are a recently described CD69+ memory T cell subset localizing to peripheral tissue. We observed that T effector memory cells (TEM ) in the blood increase during GvHD and hypothesized that they derive from donor graft CD69+TRM migrating into host blood and tissue. To probe this hypothesis, graft and blood lymphocytes from 10 ITx patients with overt GvHD and 34 without were longitudinally analyzed using flow cytometry. As hypothesized, CD4+ and CD8+CD69+TRM were significantly increased in blood and grafts of GvHD patients, alongside higher cytokine and activation marker expression. The majority of CD69+TRM were donor derived as determined by multiplex immunostaining. Notably, CD8/PD-1 was significantly elevated in blood prior to transplantation in patients who later had GvHD, and percentages of HLA-DR, CD57, PD-1, and naïve T cells differed significantly between GvHD patients who died vs. those who survived. Overall, we demonstrate that (1) there were significant increases in TEM at the time of GvHD, possibly of donor derivation; (2) donor TRM in the graft are a possible source; and (3) potential biomarkers for the development and prognosis of GvHD exist.

Type 3 innate lymphoid cells are associated with a successful intestinal transplant.

Although innate lymphoid cells (ILCs) play fundamental roles in mucosal barrier functionality and tissue homeostasis, ILC-related mechanisms underlying intestinal barrier function, homeostatic regulation, and graft rejection in intestinal transplantation (ITx) patients have yet to be thoroughly defined. We found protective type 3 NKp44+ ILCs (ILC3s) to be significantly diminished in newly transplanted allografts, compared to allografts at 6 months, whereas proinflammatory type 1 NKp44- ILCs (ILC1s) were higher. Moreover, serial immunomonitoring revealed that in healthy allografts, protective ILC3s repopulate by 2-4 weeks postoperatively, but in rejecting allografts they remain diminished. Intracellular cytokine staining confirmed that NKp44+ ILC3 produced protective interleukin-22 (IL-22), whereas ILC1s produced proinflammatory interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α). Our findings about the paucity of protective ILC3s immediately following transplant and their repopulation in healthy allografts during the first month following transplant were confirmed by RNA-sequencing analyses of serial ITx biopsies. Overall, our findings show that ILCs may play a key role in regulating ITx graft homeostasis and could serve as sentinels for early recognition of allograft rejection and be targets for future therapies.

From bench to bedside: preclinical evaluation of a self-inactivating gammaretroviral vector for the gene therapy of X-linked chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.

Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells.

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.

Newly diagnosed chronic granulomatous disease in a 53-year-old woman with Crohn disease.

BACKGROUND: Chronic granulomatous disease (CGD) is characterized by defective bactericidal activity of white blood cells, specifically, a defect in superoxide production. Patients experience infections, predominantly caused by catalase-positive bacteria and fungal organisms, that may be severe and life-threatening. Most cases of CGD are diagnosed in children; however, it may rarely go undiagnosed until adulthood in individuals with unexplained infections and granulomatous inflammation. OBJECTIVE: To describe an adult with Crohn disease and recurrent infections who was newly diagnosed as having CGD. METHODS: A 53-year-old woman with a history of liver abscesses and Crohn disease presented with Burkholderia cepacia pneumonia and required a right middle lobe resection. Nitroblue tetrazolium test results confirmed the diagnosis of CGD, and Western blot analysis revealed the absence of the 47-phagocyte oxidase protein. Levels of Crohn-associated specific antibodies to Saccharomyces cerevisiae and Escherichia coli outer membrane porin C were elevated. RESULTS: The patient, newly diagnosed as having CGD, was given intravenous trimethoprim-sulfamethoxazole, after which she improved clinically and was discharged from the hospital in stable condition to receive daily oral trimethoprim-sulfamethoxazole treatment. CONCLUSIONS: The concomitant occurrence of Crohn disease and CGD, both characterized by granulomatous inflammation, is noteworthy. This case study demonstrates that CGD should be considered in adults with recurrent infections, especially those caused by catalase-positive organisms, such as B cepacia.

Real-Time PCR: an Effective Tool for Measuring Transduction Efficiency in Human Hematopoietic Progenitor Cells.

Accurate measurement of gene transfer into hematopoietic progenitor cells is an essential prerequisite for assessing the utility of gene therapy approaches designed to correct hematologic defects. We developed a reliable method to measure transduction efficiency at the level of the progenitor cell with real-time polymerase chain reaction (PCR) analysis of individual progenitor-derived colonies. We hypothesized that this method would demonstrate better sensitivity and specificity than are currently achievable with conventional PCR. An oncoretroviral vector containing the enhanced green fluorescent protein was used to transduce human CD34+ cells derived from bone marrow or granulocyte-colony-stimulating factor-mobilized peripheral blood. Progenitor assays were set up and colonies plucked after visualization by fluorescence microscopy. By analyzing microscopically identified fluorescent samples and nontransduced samples, we calculated an overall sensitivity and specificity of 90.2 and 95.0%, respectively. Real-time PCR had higher specificity and sensitivity than conventional PCR as analyzed by generalized linear models (P = 0.002 and P = 0.019, respectively). In conclusion, we found real-time PCR to have superior sensitivity and specificity compared to conventional PCR in determining transduction efficiency of hematopoietic progenitor cells.

Long-term high-level reconstitution of NADPH oxidase activity in murine X-linked chronic granulomatous disease using a bicistronic vector expressing gp91phox and a Delta LNGFR cell surface marker.

A murine model of X-linked chronic granulomatous disease (X-CGD), an inherited immune deficiency with absent phagocyte NADPH oxidase activity caused by defects in the gp91(phox) gene, was used to evaluate a bicistronic retroviral vector in which expression of human gp91(phox) and a linked gene for Delta LNGFR, a truncated form of human low-affinity nerve growth factor receptor, are under the control of a spleen focus-forming virus long-terminal repeat (LTR). Four independent cohorts of 11-Gy irradiated X-CGD mice (total, 22 mice) were transplanted with or without preselection of transduced X-CGD bone marrow (BM). Transplanted mice had high-level correction of neutrophil gp91(phox) expression and reconstitution of NADPH oxidase activity. Expression lasted for at least 14 months in primary transplants, and persisted in secondary and tertiary transplants. Both gp91(phox) and Delta LNGFR were detected on circulating granulocytes, lymphocytes, lymphoid, and (for Delta LNGFR) red blood cells. Mice receiving transduced bone marrow [BM] preselected ex vivo for Delta LNGFR expression had high-level (= 80%) reconstitution with transduced cells, with an improved fraction of oxidase-corrected neutrophils posttransplant. Analysis of secondary and tertiary CFU-S showed that silencing of individual provirus integrants can occur even after preselection for Delta LNGFR prior to transplantation, and that persistent provirus expression was associated with multiple integration sites in most cases. No obvious adverse consequences of transgenic protein expression were observed.