In vivo topical gene therapy for recessive dystrophic epidermolysis bullosa: a phase 1 and 2 trial.

Recessive dystrophic epidermolysis bullosa (RDEB) is a lifelong genodermatosis associated with blistering, wounding, and scarring caused by mutations in COL7A1, the gene encoding the anchoring fibril component, collagen VII (C7). Here, we evaluated beremagene geperpavec (B-VEC), an engineered, non-replicating COL7A1 containing herpes simplex virus type 1 (HSV-1) vector, to treat RDEB skin. B-VEC restored C7 expression in RDEB keratinocytes, fibroblasts, RDEB mice and human RDEB xenografts. Subsequently, a randomized, placebo-controlled, phase 1 and 2 clinical trial (NCT03536143) evaluated matched wounds from nine RDEB patients receiving topical B-VEC or placebo repeatedly over 12 weeks. No grade 2 or above B-VEC-related adverse events or vector shedding or tissue-bound skin immunoreactants were noted. HSV-1 and C7 antibodies sometimes presented at baseline or increased after B-VEC treatment without an apparent impact on safety or efficacy. Primary and secondary objectives of C7 expression, anchoring fibril assembly, wound surface area reduction, duration of wound closure, and time to wound closure following B-VEC treatment were met. A patient-reported pain-severity secondary outcome was not assessed given the small proportion of wounds treated. A global assessment secondary endpoint was not pursued due to redundancy with regard to other endpoints. These studies show that B-VEC is an easily administered, safely tolerated, topical molecular corrective therapy promoting wound healing in patients with RDEB.

Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection.

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

Natural Tr1-like cells do not confer long-term tolerogenic memory.

IL-10-producing Tr1 cells promote tolerance but their contributions to tolerogenic memory are unclear. Using 10BiT mice that carry a Foxp3-eGFP reporter and stably express CD90.1 following IL-10 production, we characterized the spatiotemporal dynamics of Tr1 cells in a house dust mite model of allergic airway inflammation. CD90.1+Foxp3-IL-10+ Tr1 cells arise from memory cells and rejoin the tissue-resident memory T-cell pool after cessation of IL-10 production. Persistent antigenic stimulation is necessary to sustain IL-10 production and Irf1 and Batf expression distinguishes CD90.1+Foxp3-IL-10+ Tr1 cells from CD90.1+Foxp3-IL-10- 'former' Tr1. Depletion of Tr1-like cells after primary sensitization exacerbates allergic airway inflammation. However, neither transfer nor depletion of former Tr1 cells influences either Tr1 numbers or the inflammatory response during subsequent allergen memory re-challenge weeks later. Together these data suggest that naturally-arising Tr1 cells do not necessarily give rise to more Tr1 upon allergen re-challenge or contribute to tolerogenic memory. This phenotypic instability may limit efforts to re-establish tolerance by expanding Tr1 in vivo.

4-Methylumbelliferyl glucuronide contributes to hyaluronan synthesis inhibition.

4-Methylumbelliferone (4-MU) inhibits hyaluronan (HA) synthesis and is an approved drug used for managing biliary spasm. However, rapid and efficient glucuronidation is thought to limit its utility for systemically inhibiting HA synthesis. In particular, 4-MU in mice has a short half-life, causing most of the drug to be present as the metabolite 4-methylumbelliferyl glucuronide (4-MUG), which makes it remarkable that 4-MU is effective at all. We report here that 4-MUG contributes to HA synthesis inhibition. We observed that oral administration of 4-MUG to mice inhibits HA synthesis, promotes FoxP3+ regulatory T-cell expansion, and prevents autoimmune diabetes. Mice fed either 4-MUG or 4-MU had equivalent 4-MU:4-MUG ratios in serum, liver, and pancreas, indicating that 4-MU and 4-MUG reach an equilibrium in these tissues. LC-tandem MS experiments revealed that 4-MUG is hydrolyzed to 4-MU in serum, thereby greatly increasing the effective bioavailability of 4-MU. Moreover, using intravital 2-photon microscopy, we found that 4-MUG (a nonfluorescent molecule) undergoes conversion into 4-MU (a fluorescent molecule) and that 4-MU is extensively tissue bound in the liver, fat, muscle, and pancreas of treated mice. 4-MUG also suppressed HA synthesis independently of its conversion into 4-MU and without depletion of the HA precursor UDP-glucuronic acid (GlcUA). Together, these results indicate that 4-MUG both directly and indirectly inhibits HA synthesis and that the effective bioavailability of 4-MU is higher than previously thought. These findings greatly alter the experimental and therapeutic possibilities for HA synthesis inhibition.

The bone marrow is patrolled by NK cells that are primed and expand in response to systemic viral activation.

The bone marrow hosts NK cells whose distribution, motility and response to systemic immune challenge are poorly understood. At steady state, two-photon microscopy of the bone marrow in Ncr1gfp/+ mice captured motile NK cells interacting with dendritic cells. NK cells expressed markers and effector molecules of mature cells. Following poly (I:C) injection, RNA-Seq of NK cells revealed three phases of transcription featuring immune response genes followed by posttranscriptional processes and proliferation. Functionally, poly (I:C) promoted upregulation of granzyme B, enhanced cytotoxicity in vitro and in vivo, and, in the same individual cells, triggered proliferation. Two-photon imaging revealed that the proportion of sinusoidal NK cells decreased, while at the same time parenchymal NK cells accelerated, swelled and divided within the bone marrow. MVA viremia induced similar responses. Our findings demonstrate that the bone marrow is patrolled by mature NK cells that rapidly proliferate in response to systemic viral challenge while maintaining their effector functions.

Active dissemination of cellular antigens by DCs facilitates CD8+ T-cell priming in lymph nodes.

Antigen (Ag) specific activation of naïve T cells by migrating dendritic cells (DCs) is a highly efficient process, although the chances for their colocalization in lymph nodes (LNs) appear low. Ag presentation may be delegated from Ag-donor DCs to the abundant resident DCs, but the routes of Ag transfer and how it facilitates T-cell activation remain unclear. We visualized CD8+ T cell-DC interactions to study the sites, routes, and cells mediating Ag transfer in mice. In vitro, Ag transfer from isolated ovalbumin (OVA)+ bone marrow (BM) DCs triggered widespread arrest, Ca2+ flux, and CD69 upregulation in OT-I T cells contacting recipient DCs. Intravital two-photon imaging revealed that survival of Ag-donor DCs in LNs was required for Ag dissemination among resident CD11c+ DCs. Upon interaction with recipient DCs, CD8+ T cells clustered, upregulated CD69, proliferated and differentiated into effectors. Few DCs sufficed for activation, and for efficient Ag dissemination lymphocyte function associated antigen 1 (LFA-1) expression on recipient DCs was essential. Similar findings characterized DCs infected with a replication-deficient OVA-expressing Vaccinia virus known to downregulate MHC-I. Overall, active Ag dissemination from live incoming DCs helped activate CD8+ T cells by increasing the number of effective presenting cells and salvaged T-cell priming when Ag-donor DCs could not present Ag.

Hyaluronan synthesis is necessary for autoreactive T-cell trafficking, activation, and Th1 polarization.

The extracellular matrix polysaccharide hyaluronan (HA) accumulates at sites of autoimmune inflammation, including white matter lesions in multiple sclerosis (MS), but its functional importance in pathogenesis is unclear. We have evaluated the impact of 4-methylumbelliferone (4-MU), an oral inhibitor of HA synthesis, on disease progression in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Treatment with 4-MU decreases the incidence of EAE, delays its onset, and reduces the severity of established disease. 4-MU inhibits the activation of autoreactive T cells and prevents their polarization toward a Th1 phenotype. Instead, 4-MU promotes polarization toward a Th2 phenotpye and induction of Foxp3(+) regulatory T cells. Further, 4-MU hastens trafficking of T cells through secondary lymphoid organs, impairs the infiltration of T cells into the CNS parenchyma, and limits astrogliosis. Together, these data suggest that HA synthesis is necessary for disease progression in EAE and that treatment with 4-MU may be a potential therapeutic strategy in CNS autoimmunity. Considering that 4-MU is already a therapeutic, called hymecromone, that is approved to treat biliary spasm in humans, we propose that it could be repurposed to treat MS.

Heparanase of murine effector lymphocytes and neutrophils is not required for their diapedesis into sites of inflammation.

Heparanase, the exclusive mammalian heparan sulfate-degrading enzyme, has been suggested to be utilized by leukocytes to penetrate through the dense basement membranes surrounding blood venules. Despite its established role in tumor cell invasion, heparanase function in leukocyte extravasation has never been demonstrated. We found that TH1/TC1-type effector T cells are highly enriched for this enzyme, with a 3.6-fold higher heparanase mRNA expression compared with naive lymphocytes. Using adoptive transfer of wild-type and heparanase-deficient effector T cells into inflamed mice, we show that T-cell heparanase was not required for extravasation inside inflamed lymph nodes or skin. Leukocyte extravasation through acute inflamed skin vessels was also heparanase independent. Furthermore, neutrophils emigrated to the inflamed peritoneal cavity independently of heparanase expression on either the leukocytes or on the endothelial and mesothelial barriers, and overexpression of the enzyme on neutrophils did not facilitate their emigration. However, heparanase absence significantly reduced monocyte emigration into the inflamed peritoneal cavity. These results collectively suggest that neither leukocyte nor endothelial heparanase is required for T-cell and neutrophil extravasation through inflamed vascular barriers, whereas this enzyme is required for optimal monocyte recruitment to inflamed peritoneum.

The integrin coactivator Kindlin-3 is not required for lymphocyte diapedesis.

Kindlin-3 is an integrin-binding focal adhesion adaptor absent in patients with leukocyte and platelet adhesion deficiency syndrome and is critical for firm integrin-dependent leukocyte adhesion. The role of this adaptor in leukocyte diapedesis has never been investigated. In the present study, the functions of Kindlin-3 in this process were investigated in effector T lymphocytes trafficking to various lymphoid and nonlymphoid tissues. In vitro, Kindlin-3-deficient T cells displayed severely impaired lymphocyte function antigen-1-dependent lymphocyte adhesion but partially conserved very late antigen-4 adhesiveness. In vivo, the number of adoptively transferred Kindlin-3-deficient T effectors was dramatically elevated in the circulating pool compared with normal effectors, and the Kindlin-3 mutant effectors failed to enter inflamed skin lesions. The frequency of Kindlin-3-deficient T effectors arrested on vessel walls within inflamed skin-draining lymph nodes was also reduced. Strikingly, however, Kindlin-3-deficient effector T cells accumulated inside these vessels at significantly higher numbers than their wild-type lymphocyte counterparts and successfully extravasated into inflamed lymph nodes. Nevertheless, on entering these organs, the interstitial motility of these lymphocytes was impaired. This is the first in vivo demonstration that Kindlin-3-stabilized integrin adhesions, although essential for lymphocyte arrest on blood vessels and interstitial motility, are not obligatory for leukocyte diapedesis.

Luminal bacteria recruit CD103+ dendritic cells into the intestinal epithelium to sample bacterial antigens for presentation.

CD103+ dendritic cells (DCs) carry bacteria from the small intestine and can present antigens to T cells. Yet they have not been recorded sampling luminal bacteria or presenting bacterial antigens in mesentery lymph nodes. We used 2-photon microscopy in live Cx3cr1(+/gfp) ×Cd11c-YFP mice to study these processes. At steady state, sparse CD103+ DCs occupied the epithelium. They patrolled among enterocytes while extending dendrites toward the lumen, likely using tight-junction proteins to penetrate the epithelium. Challenge with Salmonella triggered chemokine- and toll-like receptor (TLR)-dependent recruitment of additional DCs from the lamina propria (LP). The DCs efficiently phagocytosed the bacteria using intraepithelial dendrites. Noninvasive bacteria were similarly sampled. In contrast, CD103+ DCs sampled soluble luminal antigen inefficiently. In mice harboring CD103+ DCs, antigen-specific CD8 T cells were subsequently activated in MLNs. Intestinal CD103+ DCs are therefore equipped with unique mechanisms to independently complete the processes of uptake, transportation, and presentation of bacterial antigens.

Murine anti-third-party central-memory CD8(+) T cells promote hematopoietic chimerism under mild conditioning: lymph-node sequestration and deletion of anti-donor T cells.

Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders and can mediate immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. Here, we address this barrier using donorderived central memory CD8(+) T cells (Tcms), directed against third-party antigens. Our results show that fully allogeneic or (hostXdonor)F1-Tcm, support donor chimerism (> 6 months) in sublethally irradiated (5.5Gy) mice, without GVHD symptoms. Chimerism under yet lower irradiation (4.5Gy) was achieved by combining Tcm with short-term administration of low-dose Rapamycin. Importantly, this chimerism resulted in successful donor skin acceptance, whereas third-party skin was rejected. Tracking of host anti-donor T cells (HADTCs), that mediate TDBMT rejection, in a novel bioluminescence-imaging model revealed that Tcms both induce accumulation and eradicate HADTCs in the LNs,concomitant with their elimination from other organs, including the BM. Further analysis with 2-photon microcopy revealed that Tcms form conjugates with HADTCs, resulting in decelerated and confined movement of HADTCs within the LNs in an antigen-specific manner. Thus, anti-third-party Tcms support TDBMT engraftment under reduced-conditioning through lymph-node sequestration and deletion of HADTCs, offering a novel and potentially safe approach for attaining stable hematopoietic chimerism.

DC mobilization from the skin requires docking to immobilized CCL21 on lymphatic endothelium and intralymphatic crawling.

Dendritic cells (DCs) must travel through lymphatics to carry skin antigens into lymph nodes. The processes controlling their mobilization and migration have not been completely delineated. We studied how DCs in live mice respond to skin inflammation, transmigrate through lymphatic endothelium, and propagate in initial lymphatics. At steady state, dermal DCs remain sessile along blood vessels. Inflammation mobilizes them, accelerating their interstitial motility 2.5-fold. CCR7-deficient BMDCs crawl as fast as wild-type DCs but less persistently. We observed discrete depositions of CCL21 complexed with collagen-IV on the basement membrane of initial lymphatics. Activated DCs move directionally toward lymphatics, contact CCL21 puncta, and migrate through portals into the lumen. CCR7-deficient DCs arrive at lymphatics through random migration but fail to dock and transmigrate. Once inside vessels, wild-type DCs use lamellipodia to crawl along lymphatic endothelium and, sensing lymph flow, proceed downstream. DCs start drifting freely only in collecting lymphatics. These results demonstrate in vivo that the CCL21-CCR7 axis plays a dual role in DC mobilization: promoting both chemotaxis and arrest of DCs on lymphatic endothelium. Intralymphatic crawling, in which DCs combine active adhesion-based migration and directional cues from lymph flow, represents a new step in DC mobilization which may be amenable to regulation.

Profile and prognosis of febrile elderly patients with bacteremic urinary tract infection.

OBJECTIVE: To describe the profile of elderly patients with bacteremic urinary tract infections (UTI) and correlate clinical and laboratory findings with the outcome in order to identify independent predictors of mortality. METHODS: This retrospective study took place in a large community-based, geriatric hospital and included 191 patients aged 75-105 years with urine and blood cultures simultaneously positive for bacterial organisms. Records were analysed for demographic information, clinical and laboratory data over a 29 month period. Mortality was assessed and was correlated with these findings. RESULTS: Most of the patients (80.1%) had community-acquired infection. Gram-negative organisms accounted for 87.6% of bacterial isolates, with Escherichia coli accounting for 46.1% of cases. Non-Escherichia coli Gram-negative organisms were highly resistant to two common urinary tract antibiotics (gentamicin and ceftriaxone). Patients with chronic urinary catheter had Gram-negative bacteria significantly less sensitive to ciprofloxacin, gentamycin, ampicillin and ceftriaxon than patients without catheter (p<0.05). In-hospital mortality was 33%. Multiple logistic regression analysis revealed that mortality was significantly related to the number of underlying diagnoses (p<0.0203), cognitive status (p<0.0003), length of hospitalization (p<0.0397), low level of serum albumin (p<0.0021), high neutrophil count (p<0.0001) and high level of lactate dehydrogenase (p<0.0351). Fatality was not associated with advanced age in the very old. CONCLUSION: Bacteremic UTI in the elderly has a high mortality rate. In frail elderly patients with age-associated multiple severe underlying disorders and cognitive impairment, early recognition of bacteremic UTI and prompt, appropriate treatment are critical in reducing the mortality.

Risk factors for recurrence of Clostridium difficile-associated diarrhea in the elderly.

Risk factors that influence the recurrence of nosocomial Clostridium difficile-associated diarrhea (CDAD) were determined in an 18-month follow-up study in a subacute geriatric department. A case-control study was conducted, comparing 43 patients with recurrent C. difficile-associated diarrhea (RCDAD) (case patients) with 38 patients who had only 1 episode of CDAD (control patients). The mean age of patients was 81.6 +/- 7.2 y (range 64-95 y). All cases with CDAD were receiving antibiotic therapy. RCDAD occurred in 53.1% of patients. Risk factors for RCDAD included fecal incontinence (p < or = 0.04), longer duration of fever from admission until first episode of CDAD (p < or = 0.02) and H2-antagonist treatment (p < or = 0.02). This study shows that RCDAD is a common clinical event in elderly hospitalized patients and may be predictable in some groups of patients.