Current histological measurement techniques for interstitial collagen, the basis of interstitial fibrosis, are semi-quantitative at best and only provide a ratio of collagen levels within tissues. The Genesis200 imaging system and supplemental image analysis software, FibroIndex from HistoIndex, is a novel, automated platform that uses secondharmonic generation (SHG) for imaging and characterization of interstitial collagen deposition and additional characteristics, in the absence of any staining. However, its ability to quantify renal fibrosis requires investigation. This study compared SHG imaging of renal fibrosis in mice with unilateral ureteric obstruction (UUO), to that of Masson's trichrome staining (MTS) and immunohistochemistry (IHC) of collagen I. Additionally, the platform generated data on collagen morphology and distribution patterns. While all three methods determined that UUO-injured mice underwent significantly increased renal fibrosis after 7 days, the HistoIndex platform additionally determined that UUO-injured mice had a significantly increased collagen-to-tissue cross reticulation ratio (all P < .001 vs sham group). Furthermore, in UUO-injured mice treated with the relaxin family peptide receptor-1 agonists, relaxin (0.5 mg/kg/ day) or B7-33 (0.25 mg/kg/day), or angiotensin converting enzyme-inhibitor, perindopril (1 mg/kg/day) over the 7-day period, only the HistoIndex platform determined that the drug-induced prevention of renal fibrosis correlated with significantly reduced collagen fiber thickness and collagen-to-tissue cross reticulation ratio, but increased collagen fiber counts. Relaxin or B7-33 treatment also increased renal matrix metalloproteinase-2 and reduced tissue inhibitor of metalloproteinase-1 levels (all P < .01 vs UUO alone). This study demonstrated the diagnostic value of the HistoIndex platform over currently used staining techniques.

The highly pathogenic avian influenza (HPAI) H5N1 virus remains to be one of the world’s largest pandemic threats due to the emergence of new variants. The rapid evolution of new sub-lineages is currently the greatest challenge in vaccine development. In this study, we developed an epitope modified non-pathogenic H5N3 (A/duck/Singapore/97) vaccine for broad protection against influenza H5 subtype. H5N3 hemagglutinin (HA) mutant reassortant viruses with A/Puerto Rico/8/34 (PR8) backbone were generated by mutating amino acids at the 140th loop and 190th α-helix of hemagglutinin. The cross-neutralizing efficacy of reverse genetics-derived H5N3HA (RG-H5N3HA) mutants was confirmed by testing reactivity with reference chicken anti-H5N1 clade 2 virus sera. Furthermore, RG-H5N3HA mutant immunized mice induced cross-neutralizing antibodies and cross-protection against distinct H5N1 viral infection. Our findings suggest that the use of non-pathogenic H5 viruses antigenically related to HPAI-H5N1 allows for the development of broadly protective vaccines and reduces the need for biosafety level 3 (BSL3) containment facilities.

New strategies are needed to combat pathological organ fibrosis that leads to end stage organ failure. We have identified the enzyme insulin regulated aminopeptidase (IRAP) as one such promising new target, with IRAP KO mice completely protected, and pharmacological inhibition of IRAP reversing, age-induced cardiac fibrosis. However, the effect of IRAP inhibition in renal fibrosis is unknown. Therefore the objective of this study was to: (1) compare anti-fibrotic efficacy of 2 chemically distinct IRAP inhibitors, HFI-419 and SJM4-164 to the ACE inhibitor, perindopril (PER) in a murine model of unilateral ureteral obstructive (UUO)-induced renal fibrosis; and (2) investigate role of IRAP substrates in mediating ant-fibrotic effect of IRAP inhibitors. Male C57Bl/6J mice (8 weeks old) were assigned to one of the following 7 day protocols (n=6/gp): sham, UUO+Veh, UUO+HFI (0.72mg/kg/d), UUO+SJM (0.72mg/kg/d), or UUO+PER (1mg/kg/d). IRAP substrate involvement was determined using specific receptor blockers for the oxytocin receptor (OTR; Atosiban; 0.6mg/kg/d) or the AT2 receptor (AT2R; PD123319; 10mg/kg/d). Interstitial fibrosis was significantly increased in UUO+Veh-injured kidneys compared to sham (% positive staining: UUO=5.9±0.7 vs Sham=1.2±0.3; p<0.05). HFI and SJM treatment were equally effective in preventing UUO-induced increase in interstitial renal fibrosis (% positive staining: HFI=2.5±0.3, SJM=2.8±0.3, all p<0.05 vs UUO). PER had no effect on UUO-induced increase in interstitial renal fibrosis (PER=6.6±0.7), however it was as effective as the IRAP inhibitors in reducing markers of inflammation, including NFκB activation and macrophage infiltration. The anti-fibrotic effects produced with IRAP inhibition were prevented by OTR or AT2R blockade (% positive staining: OTR=5.0±0.55, AT2R=6±0.8). Overall, this study demonstrated greater reno-protection with IRAP inhibition compared to that achieved using an ACE inhibitor, with IRAP inhibitors exhibiting potent anti-fibrotic and anti-inflammatory effects. Moreover, for the first time we show that the anti-fibrotic effects of IRAP inhibition involve activation of receptors associated with endogenous IRAP substrates.