Histone/Protein Deacetylase 11 Targeting Promotes Foxp3+ Treg Function.

Current interest in Foxp3+ T-regulatory (Treg) cells as therapeutic targets in transplantation is largely focused on their harvesting pre-transplant, expansion and infusion post-transplantation. An alternate strategy of pharmacologic modulation of Treg function using histone/protein deacetylase inhibitors (HDACi) may allow more titratable and longer-term dosing. However, the effects of broadly acting HDACi vary, such that HDAC isoform-selective targeting is likely required. We report data from mice with constitutive or conditional deletion of HDAC11 within Foxp3+ Treg cells, and their use, along with small molecule HDAC11 inhibitors, in allograft models. Global HDAC11 deletion had no effect on health or development, and compared to WT controls, Foxp3+ Tregs lacking HDAC11 showed increased suppressive function, and increased expression of Foxp3 and TGF-β. Likewise, compared to WT recipients, conditional deletion of HDAC11 within Tregs led to long-term survival of fully MHC-mismatched cardiac allografts, and prevented development of transplant arteriosclerosis in an MHC class II-mismatched allograft model. The translational significance of HDAC11 targeting was shown by the ability of an HDAC11i to promote long-term allograft allografts in fully MHC-disparate strains. These data are powerful stimuli for the further development and testing of HDAC11-selective pharmacologic inhibitors, and may ultimately provide new therapies for transplantation and autoimmune diseases

Transcatheter placement of a low-profile biodegradable pulmonary valve made of small intestinal submucosa: A long-term study in a swine model

ObjectiveWe sought to investigate a placement of a percutaneous low-profile prosthetic valve constructed of small intestinal submucosa in the pulmonary position in a swine model.MethodsTwelve female farm pigs were stented at the native pulmonary valve to induce pulmonary insufficiency. Once right ventricular dilation occurred, the small intestinal submucosa valve was implanted. The pigs were followed up with transthoracic echocardiographic Doppler scanning. One animal died of heart failure before valve replacement. Animals were euthanized at 1 day, 1 month, 3 months, 6 months, and 12 months after valve implantation.ResultsThe small intestinal submucosa pulmonary valve showed effective reversal of pulmonary regurgitation. There were no misplacements during deployment. There were no embolizations. One-year echocardiographic follow-up showed minimal regurgitation and no stenosis for a valve/vessel ratio of 0.78 or greater. Histologic examination demonstrated intensive remodeling of the small intestinal submucosal valve. Within 1 month, the surface was covered by endothelium, and fibroblasts invaded the interior. Over the following months, the small intestinal submucosal valve remodeled without apparent graft rejection.ConclusionThe small intestinal submucosa valve has the potential for graft longevity without the need for anticoagulation or immunosuppression. Histologic remodeling of the valve tissue provides a replacement capable of resembling a native valve that can be placed percutaneously with low-profile delivery systems

The Noninvasive Urinary Polyomavirus Haufen Test Predicts BK Virus Nephropathy in Children After Hematopoietic Cell Transplantation: A Pilot Study

After hematopoietic cell transplantation (HCT), polyoma-BK virus is associated with hemorrhagic cystitis and also with polyomavirus nephropathy (PVN). However, the true burden of post-HCT PVN is unknown because kidney biopsies are avoided due to their bleeding risk. The novel, non-invasive urinary PV-Haufen test detects PVN in kidney transplant recipients with >95% positive/negative predictive values. We hypothesized that the detection of PV-Haufen in voided urine samples–a positive PV-Haufen test–was also clinically significant after HCT

Control of disseminated intravascular coagulation in Klippel-Trenaunay-Weber syndrome using enoxaparin and recombinant activated factor VIIa: a case report

<p>Abstract</p> <p>Introduction</p> <p>Vascular malformation is associated with coagulopathies, especially when hemostasis is challenged.</p> <p>Case presentation</p> <p>We present the case of an 11-year-old Hispanic girl with Klippel-Trenaunay-Weber syndrome that developed disseminated intravascular coagulation after minor surgery, which was controlled by blood product transfusions and enoxaparin to address an ongoing consumptive coagulopathy. The patient, however, developed bacteremia and liver trauma that resulted in severe bleeding. To the best of our knowledge, we report here the first known instance of administering recombinant coagulation factor VIIa to control acute bleeding in a patient with Klippel-Trenaunay-Weber syndrome.</p> <p>Conclusions</p> <p>This case illustrates the concept of enoxaparin maintenance to suppress an ongoing consumptive coagulopathy and the use of recombinant coagulation factor VIIa to control its potentially fatal severe bleeding episodes.</p

Two Lysines in the Forkhead Domain of Foxp3 Are Key to T Regulatory Cell Function

Background: The forkhead box transcription factor, Foxp3, is master regulator of the development and function of CD4+CD25+ T regulatory (Treg) cells that limit autoimmunity and maintain immune homeostasis. The carboxyl-terminal forkhead (FKH) domain is required for the nuclear localization and DNA binding of Foxp3. We assessed how individual FKH lysines contribute to the functions of Foxp3 in Treg cells. Methodology/Principal Findings: We found that mutation of FKH lysines at position 382 (K17) and at position 393 (K18) impaired Foxp3 DNA binding and inhibited Treg suppressive function in vivo and in vitro. These lysine mutations did not affect the level of expression of Foxp3 but inhibited IL-2 promoter remodeling and had important and differing effects on Treg-associated gene expression. Conclusions/Significance: These data point to complex effects of post-translational modifications at individual lysines within the Foxp3 FKH domain that affect Treg function. Modulation of these events using small molecule inhibitors ma

Helios Expression Is a Marker of T Cell Activation and Proliferation

Foxp3+ T-regulatory cells (Tregs) normally serve to attenuate immune responses and are key to maintenance of immune homeostasis. Over the past decade, Treg cells have become a major focus of research for many groups, and various functional subsets have been characterized. Recently, the Ikaros family member, Helios, was reported as a marker to discriminate naturally occurring, thymic-derived Tregs from those peripherally induced from naïve CD4+ T cells. We investigated Helios expression in murine and human T cells under resting or activating conditions, using well-characterized molecules of naïve/effector/memory phenotypes, as well as a set of Treg-associated markers. We found that Helios-negative T cells are enriched for naïve T cell phenotypes and vice versa. Moreover, Helios can be induced during T cell activation and proliferation, but regresses in the same cells under resting conditions. We demonstrated comparable findings using human and murine CD4+Foxp3+ Tregs, as well as in CD4+ and CD8+ T cells. Since Helios expression is associated with T cell activation and cellular division, regardless of the cell subset involved, it does not appear suitable as a marker to distinguish natural and induced Treg cells

Helios+ expression correlates with Ki-67 expression <i>in vivo</i>.

<p>Murine thymic (left), lymph node (middle) and splenic (right) CD4+ gated cells were divided into Foxp3+ Tregs and Foxp3- cells (top row), and then divided into Helios+ and Helios- subsets (middle), and Ki-67 expression within each subset was analyzed (bottom).</p

IL-2 increased Helios expression in Ki-67+ Foxp3- GARP- cells.

<p>Murine MNC from lymph nodes and spleen (A) and human PBMC (B) were stimulated with CD3/CD28±IL-2 for 24 hrs and stained for CD4, Foxp3, Helios, Ki-67 and GARP. IL-2 addition led to a slight increase in expression of Helios by Ki-67+ (A, top) CD4+ and CD4- (B, top) and Foxp3- cells (A, B, middle rows). Helios expression was not associated with GARP expression in CD4+ gated (A, bottom) or Foxp3+ gated (B, bottom) cells.</p

Upregulation of Helios expression by Teff cells in suppression assays.

<p>(A) Analysis of CD4+ gated murine Tregs and Teffs after 3 days of suppression assay showed that while Tregs gradually lose Foxp3 (top) and Helios (bottom) expression, a substantial proportion of Teff cells upregulate Helios (bottom), but not Foxp3 (top) expression; CFSE-negative gates show Tregs. (B) Treg division during suppression assays correlated with Helios and Foxp3 co-expression, while non-dividing Tregs lost both markers. Upper row: gating of dividing and non-dividing Tregs, with numbers showing % of Treg divisions at 1/1 (left), ½ (middle) and ¼ (right) ratios; arrows point to Helios and Foxp3 co-expression in corresponding divided and non-divided Tregs subsets through the same 1/1 to ¼ Treg/Teff ratios. (C) Tregs do not induce Helios expression in Teffs since CFSE+ CD4+ Teffs have similar Helios expression despite differing numbers of Tregs (1/1, ½ and ¼ ratios shown). (D) Analysis of Teffs gated into Helios+ and Helios- subsets showed Helios+ Teffs have a higher division rate and greater resistance to Treg suppression than Helios- mouse Teffs. Human Helios+ CD4+ (E) and Helios+ CD8 (F) Teffs showed higher divisions than Helios- cells regardless of Treg presence or absence, and Helios+ responder cells were more resistant to suppression than Helios- responders. Data are representative of 3 experiments.</p

Helios expression was not restricted to CD4 cells, but among CD4+ cells correlated with Treg-associated markers.

<p>(A) Expression of Helios in murine lymphocytes (left column) and CD25+ Foxp3+ co-expression in CD4+ Helios+ gated subsets (right column) of blood (top), lymph nodes (middle) and spleen (bottom). (B) Expression of Helios in human lymphocytes (top) and co-expression of Helios with Treg-associated markers in cells gated for CD4 expression. Right column, bottom row shows absence of correlation between Helios and CD31, a marker of recent thymic emigrants cells. Data are representative of 5 experiments.</p