Correction of T-Cell Repertoire and Autoimmune Diabetes in NOD Mice by Non-myeloablative T-Cell Depleted Allogeneic HSCT.

The induction of partial tolerance toward pancreatic autoantigens in the treatment of type 1 diabetes mellitus (T1DM) can be attained by autologous hematopoietic stem cell transplantation (HSCT). However, most patients treated by autologous HSCT eventually relapse. Furthermore, allogeneic HSCT which could potentially provide a durable non-autoimmune T-cell receptor (TCR) repertoire is associated with a substantial risk for transplant-related mortality. We have previously demonstrated an effective approach for attaining engraftment without graft versus host disease (GVHD) of allogeneic T-cell depleted HSCT, following non-myeloablative conditioning, using donor-derived anti-3rd party central memory CD8 veto T cells (Tcm). In the present study, we investigated the ability of this relatively safe transplant modality to eliminate autoimmune T-cell clones in the NOD mouse model which spontaneously develop T1DM. Our results demonstrate that using this approach, marked durable chimerism is attained, without any transplant-related mortality, and with a very high rate of diabetes prevention. TCR sequencing of transplanted mice showed profound changes in the T-cell repertoire and decrease in the prevalence of specific autoimmune T-cell clones directed against pancreatic antigens. This approach could be considered as strategy to treat people destined to develop T1DM but with residual beta cell function, or as a platform for prevention of beta cell destruction after transplantation of allogenic beta cells.

Lung Regeneration by Transplantation of Allogeneic Lung Progenitors Using a Safer Conditioning Regimen and Clinical-grade Reagents.

Over the last decades, several studies demonstrated the possibility of lung regeneration through transplantation of various lung progenitor populations. Recently, we showed in mice that fetal or adult lung progenitors could potentially provide donor cells for transplantation, provided that the lung stem cell niche in the recipient is vacated of endogenous lung progenitors by adequate conditioning. Accordingly, marked lung regeneration could be attained following i.v. infusion of a single cell suspension of lung cells into recipient mice conditioned with naphthalene (NA) and 6Gy total body irradiation (TBI). As clinical translation of this approach requires the use of allogenic donors, we more recently developed a novel transplantation modality based on co-infusion of hematopoietic and lung progenitors from the same donor. Thus, by virtue of hematopoietic chimerism, which leads to immune tolerance toward donor antigens, the lung progenitors can be successfully engrafted without any need for post-transplant immune suppression. In the present study, we demonstrate that it is possible to replace NA in the conditioning regimen with Cyclophosphamide (CY), approved for the treatment of many diseases and that a lower dose of 2 GY TBI can successfully enable engraftment of donor-derived hematopoietic and lung progenitors when CY is administered in 2 doses after the stem cell infusion. Taken together, our results suggest a feasible and relatively safe protocol that could potentially be translated to clinical transplantation of lung progenitors across major MHC barriers in patients with terminal lung diseases.

Caffeine citrate for apnea of prematurity-One dose does not fit all a prospective study.

BACKGROUND: Caffeine citrate is the most frequently used medication in preterm neonates for the prevention of apnea of prematurity. There is no accepted consensus regarding the optimal caffeine citrate dosing. In this study, we evaluate clinical responses of premature neonates to standard-dose caffeine citrate treatment. METHODS: A prospective observational study conducted at the NICU at Sheba Medical Center (3/2016-2/2017). The study population included preterm neonates born at a gestational age (GA) < 33 weeks and treated with caffeine citrate according to the local NICU protocol. RESULTS: The study cohort included 66 preterm neonates of GA < 33 weeks. Thirty infants were defined as responders and 36 as nonresponders to 7.5 mg/kg caffeine citrate treatment, and they required a further dose increase to 10 mg/kg. Infants in the nonresponders group were born at earlier GA than responders (29 vs. 31 weeks, respectively, P = 0.004). The nonresponders required a significantly longer hospital stay (56 vs. 46 days, P = 0.014), and longer supplemental oxygen support (18 vs 2 days, P = 0.008). CONCLUSIONS: Caffeine citrate initiation at higher doses is safe and does not require routine serum levels monitoring. It might be more effective for controlling apnea of prematurity in preterm neonates born ≤29 weeks of gestation.

Increased risk for early hypertriglyceridemia in small for gestational age preterm infants.

Small for gestational age preterm are at increased risk for future metabolic syndrome. Early indication for the disrupted metabolism may be found in the perinatal period. We aimed to evaluate whether small for gestational age preterm infants are at increased risk for hypertriglyceridemia when treated with lipid emulsions, and to investigate the association between triglyceride levels and morbidity. Small for gestational age infants ≤ 34 weeks' gestation age born during 2013-2016 were matched and compared with appropriate for gestational age counterparts. Triglyceride concentration > 250 mg/dL during treatment with parenteral nutrition was considered high. The study included 71 pairs of preterm infants. Hypertriglyceridemia was documented among 22.5% of the small for gestational age infants vs. 5.6% of the appropriate for gestational age infants (p = 0.007). Mean triglyceride levels were 194.4 ± 192.3 mg/dL and 99.9 ± 82.8 mg/dL, respectively (p < 0.001). Small for gestational age was predictive of hypertriglyceridemia (OR = 6.41; 95% CI 1.8-22.9). No significant association was found between triglyceride levels and morbidities in multivariate analysis.Conclusion: Small for gestational age preterm infants receiving lipid emulsions might be at a higher risk for hypertriglyceridemia. Routine monitoring of triglyceride levels will enable identification of the necessity for a slower increase in lipid emulsion therapy. What is Known: • Moderate and very preterm infants are routinely treated with lipid emulsions. • Small for gestational age (SGA) infants may have different metabolism, as they demonstrate higher risk for metabolic syndrome. What is New: • • SGA infants had a higher mean triglyceride level and more commonly had early hypertriglyceridemia (triglycerides > 250 mg/dL) compared with appropriate for gestational age infants treated with the same intravenous lipid dose. Small for gestational age was predictive of hypertriglyceridemia. • No significant association was found between triglyceride levels and morbidities in multivariate analysis.

Multi-lineage Lung Regeneration by Stem Cell Transplantation across Major Genetic Barriers.

Induction of lung regeneration by transplantation of lung progenitor cells is a critical preclinical challenge. Recently, we demonstrated that robust lung regeneration can be achieved if the endogenous stem cell niches in the recipient's lung are vacated by sub-lethal pre-conditioning. However, overcoming MHC barriers is an additional requirement for clinical application of this attractive approach. We demonstrate here that durable tolerance toward mis-matched lung progenitors and their derivatives can be achieved without any chronic immune suppression, by virtue of co-transplantation with hematopoietic progenitors from the same donor. Initial proof of concept of this approach was attained by transplantation of fetal lung cells comprising both hematopoietic and non-hematopoietic progenitors. Furthermore, an even higher rate of blood and epithelial lung chimerism was attained by using adult lung cells supplemented with bone marrow hematopoietic progenitors. These results lay the foundation for repair of lung injury through a procedure akin to bone marrow transplantation.

Induction of Nitric-Oxide Metabolism in Enterocytes Alleviates Colitis and Inflammation-Associated Colon Cancer.

Nitric oxide (NO) plays an established role in numerous physiological and pathological processes, but the specific cellular sources of NO in disease pathogenesis remain unclear, preventing the implementation of NO-related therapy. Argininosuccinate lyase (ASL) is the only enzyme able to produce arginine, the substrate for NO generation by nitric oxide synthase (NOS) isoforms. Here, we generated cell-specific conditional ASL knockout mice in combination with genetic and chemical colitis models. We demonstrate that NO derived from enterocytes alleviates colitis by decreasing macrophage infiltration and tissue damage, whereas immune cell-derived NO is associated with macrophage activation, resulting in increased severity of inflammation. We find that induction of endogenous NO production by enterocytes with supplements that upregulate ASL expression and complement its substrates results in improved epithelial integrity and alleviation of colitis and of inflammation-associated colon cancer.

Lung Injury Repair by Transplantation of Adult Lung Cells Following Preconditioning of Recipient Mice.

Repair of injured lungs represents a longstanding therapeutic challenge. We recently demonstrated that human and mouse embryonic lung tissue from the canalicular stage of development are enriched with lung progenitors, and that a single cell suspension of canalicular lungs can be used for transplantation, provided that lung progenitor niches in the recipient mice are vacated by strategies similar to those used in bone marrow transplantation. Considering the ethical limitations associated with the use of fetal cells, we investigated here whether adult lungs could offer an alternative source of lung progenitors for transplantation. We show that intravenous infusion of a single cell suspension of adult mouse lungs from GFP+ donors, following conditioning of recipient mice with naphthalene and subsequent sublethal irradiation, led to marked colonization of the recipient lungs, at 6-8 weeks post-transplant, with donor derived structures including epithelial, endothelial, and mesenchymal cells. Epithelial cells within these donor-derived colonies expressed markers of functionally distinct lung cell types, and lung function, which is significantly compromised in mice treated with naphthalene and radiation, was found to be corrected following transplantation. Dose response analysis suggests that the frequency of patch forming cells in adult lungs was about threefold lower compared to that found in E16 fetal lungs. However, as adult lungs are much larger, the total number of patch forming cells that can be collected from this source is significantly greater. Our study provides proof of concept for lung regeneration by adult lung cells after preconditioning to vacate the pulmonary niche. Stem Cells Translational Medicine 2018;7:68-77.

Assessing remyelination - metabolic labeling of myelin in an animal model of multiple sclerosis.

The lack of biomarkers is a major obstacle for investigating myelin repair. We used metabolic incorporation of the choline analog - propargyl-choline (P-Cho) to label and visualize newly synthesized myelin in the CNS of mice induced with experimental autoimmune encephalomyelitis (EAE). We further developed unbiased colocalization analysis to quantify P-Cho incorporation specifically into the myelin. Our findings indicate that P-Cho injection to mice recovering from EAE, either spontaneously or following glatiramer acetate treatment, results in significant elevation of its incorporation into the myelin, offering a novel strategy for assessing remyelination in animal models and the remyelination potential of candidate drugs.

Perforin-Positive Dendritic Cells Exhibit an Immuno-regulatory Role in Metabolic Syndrome and Autoimmunity.

Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.

Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice.

Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche.

A novel role for factor VIII and thrombin/PAR1 in regulating hematopoiesis and its interplay with the bone structure.

Analysis of hematopoietic stem cells (HSCs) in factor VIII knockout (FVIIIKO) mice revealed a novel regulatory role for the coagulation cascade in hematopoiesis. Thus, HSCs in FVIIIKO mice had reduced proportions of CD34(low) cells within Lin(-)Sca(+)Kit(+) progenitors, and exhibited reduced long-term repopulating capacity as well as hyper granulocyte-colony-stimulating factor (G-CSF)-induced mobilization. This disregulation of HSCs is likely caused by reduced levels of thrombin, and is associated with altered protease-activated receptor 1 (PAR1) signaling, as PAR1 KO mice also exhibited enhanced G-CSF-induced mobilization. Analysis of reciprocal bone marrow (BM) chimera (FVIIIKO BM into wild-type recipients and vice versa) and the detection of PAR1 expression on stromal elements indicates that this phenotype is likely controlled by stromal elements. Micro-computed tomography analysis of distal tibia metaphyses also revealed for the first time a major impact of the FVIII/thrombin/PAR1 axis on the dynamic bone structure, showing reduced bone:tissue volume ratio and trabecular number in FVIIIKO and PAR1KO mice. Taken together, these results show a critical and novel role for the coagulation cascade, mediated in part by thrombin-PAR1 interaction, and regulates HSC maintenance and a reciprocal interplay between HSCs and the dynamic bone structure.

Embryonic pig pancreatic tissue for the treatment of diabetes: potential role of immune suppression with "off-the-shelf" third-party regulatory T cells.

BACKGROUND: Xenogeneic embryonic pancreatic tissue can provide an attractive alternative for organ replacement therapy. However, immunological rejection represents a major obstacle. This study examines the potential of regulatory T cells (Tregs) in the prevention of E42 pancreas rejection. METHODS: To develop new approaches to combat rejection, we evaluated engraftment, growth, and development of E42 pig pancreatic tissue in mice treated with ex vivo expanded Tregs in combination with T-cell debulking and the conventional immunosuppressive drugs, rapamycin and FTY720. RESULTS: Transplantation of E42 pig pancreas into C57BL/6 mice immunosuppressed by this protocol resulted in complete rejection within less than 6 weeks. In contrast, additional treatment with a single infusion of ex vivo expanded third-party Tregs markedly delayed the onset of graft rejection to 10 weeks. The infusion of Tregs was associated with a significant reduction in CD4 and CD8 expansion in the lymph nodes and other peripheral organs at the priming stages after implantation. Freezing and thawing of the Tregs did not affect their efficacy, indicating the potential of Tregs banking. CONCLUSION: Considering the technical difficulties encountered in the generation of Tregs from patients or from specific donors, our results demonstrate the feasibility of using "off-the-shelf" fresh or frozen third-party Tregs to control rejection in organ transplantation.

Treatment of patent ductus arteriosus: indomethacin or ibuprofen?

We compared ibuprofen and indomethacin for the treatment of patent ductus arteriosus (PDA) in preterm infants. A retrospective comparative study was conducted at a pediatric tertiary center in preterm infants diagnosed with PDA. Infants born from January 2000 to June 2003 were treated with indomethacin, whereas infants born from July 2003 to November 2005 were treated with ibuprofen. The two treatment groups were compared. Demographic data and clinical, laboratory, and outcome data were collected from the medical files. Seventy-three infants were included in the ibuprofen group and 46 in the indomethacin group. No significant difference in efficacy was found between indomethacin and ibuprofen. Compared with ibuprofen, indomethacin treatment was associated with significantly higher mean creatinine levels and a higher percent of infants with creatinine >1.2 mg/dL, hyponatremia <120 mmol/L, and platelet level <100,000 platelets/mL(3). There were no significant differences in bilirubin levels, incidence and grade of intraventricular hemorrhage, necrotizing enterocolitis, retinopathy of prematurity, rate of surgical duct ligation, sepsis, length of hospital stay, or mortality. Indomethacin and ibuprofen are equally effective for PDA closure in premature infants. Treatment with ibuprofen is safer, decreasing the risk of renal failure, thrombocytopenia, and hyponatremia.

Enhancement of pig embryonic implants in factor VIII KO mice: a novel role for the coagulation cascade in organ size control.

Very little is known about the mechanisms that contribute to organ size differences between species. In the present study, we used a mouse model of embryonic pig tissue implantation to define the role of host Factor VIII in controlling the final size attained by the implant. We show here that pig embryonic spleen, pancreas, and liver all grow to an increased size in mice that are deficient in the Factor VIII clotting cascade. Similar results were obtained using the transplantation model after treatment with the low molecular weight heparin derivative Clexane which markedly enhanced transplant size. Likewise, enhanced size was found upon treatment with the direct thrombin inhibitor Dabigatran, suggesting that organ size regulation might be mediated by thrombin, downstream of Factor VIII. Considering that thrombin was shown to mediate various functions unrelated to blood clotting, either directly by cleavage of protease-activated receptors (PARs) or indirectly by cleaving osteopontin (OPN) on stroma cells, the role of PAR1 and PAR4 antagonists as well as treatment with cleaved form of OPN (tcOPN) were tested. While the former was not found to have an impact on overgrowth of embryonic pig spleen implants, marked reduction of size was noted upon treatment with the (tcOPN). Collectively, our surprising set of observations suggests that factors of the coagulation cascade have a novel role in organ size control.

Embryonic pig pancreatic tissue for the treatment of diabetes in a nonhuman primate model.

Xenotransplantation of pig tissues has great potential to overcome the shortage of organ donors. One approach to address the vigorous immune rejection associated with xenotransplants is the use of embryonic precursor tissue, which induces and utilizes host vasculature upon its growth and development. Recently, we showed in mice that embryonic pig pancreatic tissue from embryonic day 42 (E42) exhibits optimal properties as a beta cell replacement therapy. We now demonstrate the proof of concept in 2 diabetic Cynomolgus monkeys, followed for 393 and 280 days, respectively. A marked reduction of exogenous insulin requirement was noted by the fourth month after transplantation, reaching complete independence from exogenous insulin during the fifth month after transplantation, with full physiological control of blood glucose levels. The porcine origin of insulin was documented by a radioimmunoassay specific for porcine C-peptide. Furthermore, the growing tissue was found to be predominantly vascularized with host blood vessels, thereby evading hyperacute or acute rejection, which could potentially be mediated by preexisting anti-pig antibodies. Durable graft protection was achieved, and most of the late complications could be attributed to the immunosuppressive protocol. While fine tuning of immune suppression, tissue dose, and implantation techniques are still required, our results demonstrate that porcine E-42 embryonic pancreatic tissue can normalize blood glucose levels in primates. Its long-term proliferative capacity, its revascularization by host endothelium, and its reduced immunogenicity, strongly suggest that this approach could offer an attractive replacement therapy for diabetes.

Pig embryonic pancreatic tissue as a source for transplantation in diabetes: transient treatment with anti-LFA1, anti-CD48, and FTY720 enables long-term graft maintenance in mice with only mild ongoing immunosuppression.

OBJECTIVE: Defining an optimal costimulatory blockade-based immune suppression protocol enabling engraftment and functional development of E42 pig embryonic pancreatic tissue in mice. RESEARCH DESIGN AND METHODS: Considering that anti-CD40L was found to be thrombotic in humans, we sought to test alternative costimulatory blockade agents already in clinical use, including CTLA4-Ig, anti-LFA1, and anti-CD48. These agents were tested in conjunction with T-cell debulking by anti-CD4 and anti-CD8 antibodies or with conventional immunosuppressive drugs. Engraftment and functional development of E42 pig pancreatic tissue was monitored by immunohistology and by measuring pig insulin blood levels. RESULTS: Fetal pig pancreatic tissue harvested at E42, or even as early as at E28, was fiercely rejected in C57BL/6 mice and in Lewis rats. A novel immune suppression comprising anti-LFA1, anti-CD48, and FTY720 afforded optimal growth and functional development. Cessation of treatment with anti-LFA1 and anti-CD48 at 3 months posttransplant did not lead to graft rejection, and graft maintenance could be achieved for >8 months with twice-weekly low-dose FTY720 treatment. These grafts exhibited normal morphology and were functional, as revealed by the high pig insulin blood levels in the transplanted mice and by the ability of the recipients to resist alloxan induced diabetes. CONCLUSIONS: This novel protocol, comprising agents that simulate those approved for clinical use, offer an attractive approach for embryonic xenogeneic transplantation. Further studies in nonhuman primates are warranted.