4-Methylumbelliferone improves the thermogenic capacity of brown adipose tissue.

Therapeutic increase of brown adipose tissue (BAT) thermogenesis is of great interest as BAT activation counteracts obesity and insulin resistance. Hyaluronan (HA) is a glycosaminoglycan, found in the extracellular matrix, which is synthesized by HA synthases (Has1/Has2/Has3) from sugar precursors and accumulates in diabetic conditions. Its synthesis can be inhibited by the small molecule 4-methylumbelliferone (4-MU). Here, we show that the inhibition of HA-synthesis by 4-MU or genetic deletion of Has2/Has3 improves BAT`s thermogenic capacity, reduces body weight gain, and improves glucose homeostasis independently from adrenergic stimulation in mice on diabetogenic diet, as shown by a magnetic resonance T2 mapping approach. Inhibition of HA synthesis increases glycolysis, BAT respiration and uncoupling protein 1 expression. In addition, we show that 4-MU increases BAT capacity without inducing chronic stimulation and propose that 4-MU, a clinically approved prescription-free drug, could be repurposed to treat obesity and diabetes

Hyaluronan levels are increased systemically in human type 2 but not type 1 diabetes independently of glycemic control

Hyaluronan (HA), an extracellular matrix glycosaminoglycan, is implicated in the pathogenesis of both type 1 diabetes (T1D) as well as type 2 diabetes (T2D) and has been postulated to be increased in these diseases due to hyperglycemia. We have examined the serum and tissue distribution of HA in human subjects with T1D and T2D and in mouse models of these diseases and evaluated the relationship between HA levels and glycemic control. We found that serum HA levels are increased in T2D but not T1D independently of hemoglobin-A1c, C-peptide, body mass index, or time since diabetes diagnosis. HA is likewise increased in skeletal muscle in T2D subjects relative to non-diabetic controls. Analogous increases in serum and muscle HA are seen in diabetic db/db mice (T2D), but not in diabetic DORmO mice (T1D). Diabetes induced by the β-cell toxin streptozotozin (STZ) lead to an increase in blood glucose but not to an increase in serum HA. These data indicate that HA levels are increased in multiple tissue compartments in T2D but not T1D independently of glycemic control. Given that T2D but not T1D is associated with systemic inflammation, these patterns are consistent with inflammatory factors and not hyperglycemia driving increased HA. Serum HA may have value as a biomarker of systemic inflammation in T2D

The Immune Response to Chronic Pseudomonas aeruginosa Wound Infection in Immunocompetent Mice

Objective: Our goal was to develop a chronic wound model in mice that avoids implantation of foreign material or impaired immunity and to use this to characterize the local and systemic immune response associated with Pseudomonas aeruginosa infection. Approach: We generated bilateral full-thickness dermal wounds in healthy 10–12-week-old C57Bl6 mice. We waited 24 h to inoculate the developing wound eschar at these sites. We performed careful titration experiments with luminescent strains of P. aeruginosa to identify bacterial inoculation concentrations that consistently established stable infections in these animals. We performed flow cytometry-based immunophenotyping of immune cell infiltrates at the wound site, spleen, and draining lymph nodes over time. Finally, we compared inflammatory responses seen in wound inoculation with planktonic bacteria, preformed biofilm, and heat-killed (HK) P. aeruginosa. Results: Using this delayed inoculation model and 7.5 ± 2.5 × 102 CFU/mL of PAO1 we consistently established stable infections that lasted at 10 days in duration. During early infection, we detected a strong upregulation of inflammatory cytokines and neutrophil infiltration at the wound site, while natural killer (NK) cells and dendritic cells (DCs) were reduced. At the systemic level, only plasmacytoid DCs were increased early in infection. During later stages, there was systemic upregulation of B cells, T cells, and macrophages, whereas NK cells and interferon killer DCs were reduced. Infections with P. aeruginosa biofilms were not more virulent than infections with planktonic P. aeruginosa, whereas treatment with HK P. aeruginosa only induces a short-term inflammatory state. Innovation: We describe a versatile wound model of chronic P. aeruginosa infection that lasts 10 days without causing sepsis or other excessive morbidity. Conclusion: This model may facilitate the study of chronic wound infections in immunocompetent mice. Our findings also highlight the induction of early innate immune cell populations during P. aeruginosa infection

Abstract 5588: Clinical and pharmacodynamic responses to a modified whole tumor cell immunotherapy in patients with advanced breast cancer from two phase I-IIa trials

Abstract SV-BR-1-GM is a GM-CSF secreting breast cancer cell line that also expresses HLA class I & II antigens. Irradiated SV-BR-1-GM is used in a regimen including pre-dose low-dose cyclophosphamide and post-dose local interferon-α2b. The SV-BR-1-GM regimen has been used alone (“Monotherapy” study ClinicalTrials.gov NCT03066947) and in combination with immune checkpoint inhibitors (ongoing combination study ClinicalTrials.gov identifier NCT03328026). Here we report regression of metastatic breast cancer and pharmacodynamic analysis with immunologic correlates. 23 patients with advanced breast cancer refractory to standard therapies were treated with the SV-BR-1-GM regimen in the monotherapy trial with cycles every 2 weeks for the first month and then monthly. The combination study is evaluating the SV-BR-1-GM regimen with checkpoint inhibitors (PD-1 inhibitors pembrolizumab or INCMGA00012) with cycles every 3 weeks (11 patients have been dosed to date). Pharmacodynamic analyses include delayed-type hypersensitivity (DTH), antibodies against SV-BR-1 (precursor of SV-BR-1-GM), blood lymphocyte proliferation (determined using flow cytometry), circulating cytokines in sera and cytokine secretion (Luminex based assays) following stimulation with peptides of antigens expressed in SV-BR-1-GM cells (HER2 and PRAME). In the monotherapy study, tumor regression was seen in 3 patients. 21 patients developed measurable DTH signifying cellular immunity. Blood lymphocytes from responders after treatment showed increased proliferation and cytokine secretion (GM-CSF, IL-2, IL-21) - following stimulation with HER2 and PRAME peptides. Differential serum cytokine levels were observed (CD40L, MCP-1, IL-1RA) in 5 patients. Increased antibody levels compared to baseline were observed in 6 of the 12 patients assessed. Patients with objective tumor regression had the most pronounced responses. In the combination therapy study, 2 patients have shown objective evidence of tumor regression, including one patient with liver metastases, which decreased by 25%, and one patient with adrenal and dural metastases (29% reduction in target lesion). Both patients had Grade II tumors, similar to the tumor from which SV-BR-1-GM was derived. These observations confirm the ability of the SV-BR-1-GM regimen to elicit regression of far advanced refractory metastatic breast cancer. No serious toxicities clearly attributed to the SV-BR-1-GM regimen were observed. Pharmacodynamic analysis of humoral and cell-mediated immune responses showed notable upregulation, the strongest responses being seen in those with measurable clinical regression. Patients with Grade I or II tumors appeared more likely to respond. Citation Format: Vivekananda G. Sunkari, Jacqueline Galeas, Shaker R. Dakhil, Jarrod Holmes, Saveri Bhattacharya, Carmen J. Calfa, Ajay Kundra, Daniel L. Adams, Diane DaSilva, George E. Peoples, Charles L. Wiseman, William V. Williams, Markus D. Lacher. Clinical and pharmacodynamic responses to a modified whole tumor cell immunotherapy in patients with advanced breast cancer from two phase I-IIa trials [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5588

Inhibition of hyaluronan synthesis restores immune tolerance during autoimmune insulitis.

We recently reported that abundant deposits of the extracellular matrix polysaccharide hyaluronan (HA) are characteristic of autoimmune insulitis in patients with type 1 diabetes (T1D), but the relevance of these deposits to disease was unclear. Here, we have demonstrated that HA is critical for the pathogenesis of autoimmune diabetes. Using the DO11.10xRIPmOVA mouse model of T1D, we determined that HA deposits are temporally and anatomically associated with the development of insulitis. Moreover, treatment with an inhibitor of HA synthesis, 4-methylumbelliferone (4-MU), halted progression to diabetes even after the onset of insulitis. Similar effects were seen in the NOD mouse model, and in these mice, 1 week of treatment was sufficient to prevent subsequent diabetes. 4-MU reduced HA accumulation, constrained effector T cells to nondestructive insulitis, and increased numbers of intraislet FOXP3(+) Tregs. Consistent with the observed effects of 4-MU treatment, Treg differentiation was inhibited by HA and anti-CD44 antibodies and rescued by 4-MU in an ERK1/2-dependent manner. These data may explain how peripheral immune tolerance is impaired in tissues under autoimmune attack, including islets in T1D. We propose that 4-MU, already an approved drug used to treat biliary spasm, could be repurposed to prevent, and possibly treat, T1D in at-risk individuals

Filamentous bacteriophage delays healing of Pseudomonas-infected wounds

Chronic wounds infected by Pseudomonas aeruginosa (Pa) are characterized by disease progression and increased mortality. We reveal Pf, a bacteriophage produced by Pa that delays healing of chronically infected wounds in human subjects and animal models of disease. Interestingly, impairment of wound closure by Pf is independent of its effects on Pa pathogenesis. Rather, Pf impedes keratinocyte migration, which is essential for wound healing, through direct inhibition of CXCL1 signaling. In support of these findings, a prospective cohort study of 36 human patients with chronic Pa wound infections reveals that wounds infected with Pf-positive strains of Pa are more likely to progress in size compared with wounds infected with Pf-negative strains. Together, these data implicate Pf phage in the delayed wound healing associated with Pa infection through direct manipulation of mammalian cells. These findings suggest Pf may have potential as a biomarker and therapeutic target in chronic wounds