A novel approach for emerging and antibiotic resistant infections: Innate defense regulators as an agnostic therapy.

Innate Defense Regulators (IDRs) are short synthetic peptides that target the host innate immune system via an intracellular adaptor protein which functions at key signaling nodes. In this work, further details of the mechanism of action of IDRs have been discovered. The studies reported here show that the lead clinical IDR, SGX94, has broad-spectrum activity against Gram-negative and Gram-positive bacterial infections caused by intracellular or extracellular bacteria and also complements the actions of standard of care antibiotics. Based on in vivo and primary cell culture studies, this activity is shown to result from the primary action of SGX94 on tissue-resident cells and subsequent secondary signaling to activate myeloid-derived cells, resulting in enhanced bacterial clearance and increased survival. Data from non-clinical and clinical studies also show that SGX94 treatment modulates pro-inflammatory and anti-inflammatory cytokine levels, thereby mitigating the deleterious inflammatory consequences of innate immune activation. Since they act through host pathways to provide both broad-spectrum anti-infective capability as well as control of inflammation, IDRs are unlikely to be impacted by resistance mechanisms and offer potential clinical advantages in the fight against emerging and antibiotic resistant bacterial infections.

Efficacy of a Gluten-Free Diet in Subjects With Irritable Bowel Syndrome-Diarrhea Unaware of Their HLA-DQ2/8 Genotype.

BACKGROUND & AIMS: A gluten-containing diet alters bowel barrier function in patients with irritable bowel syndrome with diarrhea (IBS-D), particularly those who are positive for HLA allele DQ2/8. We studied the effects of a gluten-free diet (GFD) in patients with IBS-D who have not previously considered the effects of gluten in their diet and were unaware of their HLA-DQ2/8 genotype. METHODS: We performed a prospective study of 41 patients with IBS-D (20 HLA-DQ2/8-positive and 21 HLA-DQ2/8-negative) at the Royal Hallamshire Hospital in Sheffield, United Kingdom, from September 2012 through July 2015. All subjects were placed on a 6-week GFD following evaluation by a dietician. Subjects completed validated questionnaires at baseline and Week 6 of the GFD. The primary endpoint was mean change in IBS Symptom Severity Score; a 50-point reduction was considered to indicate a clinical response. Secondary endpoints were changes in hospital anxiety and depression score, fatigue impact score, and Short Form-36 results. Clinical responders who chose to continue a GFD after the study period were evaluated on average 18 months later to assess diet durability, symptom scores, and anthropometric and biochemical status. RESULTS: A 6-week GFD reduced IBS Symptom Severity Score by ≥50 points in 29 patients overall (71%). The mean total IBS Symptom Severity Score decreased from 286 before the diet to 131 points after 6 weeks on the diet (P < .001); the reduction was similar in each HLA-DQ group. However, HLA-DQ2/8-negative subjects had a greater reduction in abdominal distention (P = .04). Both groups had marked mean improvements in hospital anxiety and depression scores, fatigue impact score, and Short Form-36 results, although HLA-DQ2/8-positive subjects had a greater reduction in depression score and increase in vitality score than HLA-DQ2/8-negative subjects (P = .02 and P = .03, respectively). Twenty-one of the 29 subjects with a clinical response (72%) planned to continue the GFD long term; 18 months after the study they were still on a GFD, with maintained symptom reductions, and demonstrated similar anthropometric and biochemical features compared with baseline. CONCLUSIONS: A dietitian-led GFD provided sustained benefit to patients with IBS-D. The symptoms that improved differed in magnitude according to HLA-DQ status. Clinical trials.gov no: NCT02528929.

Sequestosome-1/p62 is the key intracellular target of innate defense regulator peptide.

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor alpha-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor alpha-induced p62-protein kinase zeta complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein beta activity, whereas NF-kappaB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.

An anti-infective peptide that selectively modulates the innate immune response.

We show that an innate defense-regulator peptide (IDR-1) was protective in mouse models of infection with important Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Salmonella enterica serovar Typhimurium. When given from 48 h before to 6 h after infection, the peptide was effective by both local and systemic administration. Because protection by IDR-1 was prevented by in vivo depletion of monocytes and macrophages, but not neutrophils or B- and T-lymphocytes, we conclude that monocytes and macrophages are key effector cells. IDR-1 was not directly antimicrobial: gene and protein expression analysis in human and mouse monocytes and macrophages indicated that IDR-1, acting through mitogen-activated protein kinase and other signaling pathways, enhanced the levels of monocyte chemokines while reducing pro-inflammatory cytokine responses. To our knowledge, an innate defense regulator that counters infection by selective modulation of innate immunity without obvious toxicities has not been reported previously.

Cellular and antitumor activity of a new diethylene glycol benzoporphyrin derivative (lemuteporfin).

A newly synthesized diethylene glycol functionalized chlorin-type photosensitizer, lemuteporfin, was characterized for use in photodynamic therapy (PDT) in a panel of in vitro and in vivo test systems. The photosensitizer was highly potent, killing cells at low nanomolar concentrations upon exposure to activating light. The cellular uptake of lemuteporfin was rapid, with maximum levels reached within 20 min. Mitogen-activated lymphoid cells accumulated more of the lemuteporfin than their quiescent equivalents, supporting selectivity. Photosensitizer fluorescence in the skin increased rapidly within the first few minutes following intravenous administration to mice, then decreased over the next 24 h. Skin photosensitivity reactions indicated rapid clearance of the photosensitizer. Intravenous doses as low as 1.4 micromol/kg combined with exposure to 50 J/cm2 red light suppressed tumor growth in a mouse model. In conclusion, this new benzoporphyrin was found to be an effective photosensitizer, showing rapid uptake and clearance both in vitro and in vivo. This rapid photosensitization of tumors could be useful in therapies requiring a potent, rapidly accumulating photosensitizer, while minimizing the potential for skin photosensitivity reactions to sunlight following treatment.

Selective action of the photosensitizer QLT0074 on activated human T lymphocytes.

A new photosensitizer, presently designated QLT0074, may have the potential for the treatment of immune and nonimmune conditions with photodynamic therapy (PDT). The activity of QLT0074 was tested against human peripheral blood T cells and Jurkat T lymphoma cells. At low nanomolar concentrations of QLT0074 in combination with blue light, apoptosis was rapidly induced in Jurkat and blood T cells in vitro as indicated by the expression of the apoptosis-associated mitochondrial 7A6 marker and Annexin-V labeling. Further studies performed with Jurkat T cells showed that PDT-induced apoptosis with QLT0074 was associated with caspase-3 activation and the cleavage of the caspase substrate poly(adenosine diphosphate-ribose)polymerase. Flow cytometry studies revealed that blood T cells with high expression of the interleukin-2 receptor (CD25) took up greater amounts of QLT0074 and were eliminated to a greater extent with PDT than T cells with low levels of this activation marker. This selective action of PDT was confirmed by similar reductions in the percentage of T cells that expressed other activation-related markers, including very late activation antigen-4 (CD49d), human leukocyte antigen DR (HLA-DR), intercellular adhesion molecule-1 (CD54) and Fas (CD95). For activated T cells treated with a specific dose of QLT0074 and light 24 h earlier, CD25 expression density was significantly less, whereas CD54, CD95 and HLA-DR levels were similar to those for control cells treated with light alone. This work shows that PDT with QLT0074 exerts selective, dose-related effects on T cells in vitro.