Nonclinical pharmacokinetics and biodistribution of VSV-GP using methods to decouple input drug disposition and viral replication.

Viral replication places oncolytic viruses (OVs) in a unique niche in the field of drug pharmacokinetics (PK) as their self-amplification obscures exposure-response relationships. Moreover, standard bioanalytical techniques are unable to distinguish the input from replicated drug products. Here, we combine two novel approaches to characterize PK and biodistribution (BD) after systemic administration of vesicular stomatitis virus pseudotyped with lymphocytic choriomeningitis virus glycoprotein (VSV-GP) in healthy mice. First: to decouple input drug PK/BD versus replication PK/BD, we developed and fully characterized a replication-incompetent tool virus that retained all other critical attributes of the drug. We used this approach to quantify replication in blood and tissues and to determine its impact on PK and BD. Second: to discriminate the genomic and antigenomic viral RNA strands contributing to replication dynamics in tissues, we developed an in situ hybridization method using strand-specific probes and assessed their spatiotemporal distribution in tissues. This latter approach demonstrated that distribution, transcription, and replication localized to tissue-resident macrophages, indicating their role in PK and BD. Ultimately, our study results in a refined PK/BD profile for a replicating OV, new proposed PK parameters, and deeper understanding of OV PK/BD using unique approaches that could be applied to other replicating vectors.

Mast cell function in prostate inflammation, fibrosis, and smooth muscle cell dysfunction.

Intraurethral inoculation of mice with uropathogenic Escherichia coli (CP1) results in prostate inflammation, fibrosis, and urinary dysfunction, recapitulating some but not all of the pathognomonic clinical features associated with benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS). In both patients with LUTS and CP1-infected mice, we observed increased numbers and activation of mast cells and elevated levels of prostate fibrosis. Therapeutic inhibition of mast cells using a combination of a mast cell stabilizer, cromolyn sodium, and the histamine 1 receptor antagonist cetirizine di-hydrochloride in the mouse model resulted in reduced mast cell activation in the prostate and significant alleviation of urinary dysfunction. Treated mice showed reduced prostate fibrosis, less infiltration of immune cells, and decreased inflammation. In addition, as opposed to symptomatic CP1-infected mice, treated mice showed reduced myosin light chain-2 phosphorylation, a marker of prostate smooth muscle contraction. These results show that mast cells play a critical role in the pathophysiology of urinary dysfunction and may be an important therapeutic target for men with BPH/LUTS.NEW & NOTEWORTHY LUTS-associated benign prostatic hyperplasia is derived from a combination of immune activation, extracellular matrix remodeling, hyperplasia, and smooth muscle cell contraction in prostates of men. Using a mouse model, we describe the importance of mast cells in regulating these multiple facets involved in the pathophysiology of LUTS. Mast cell inhibition alleviates both pathology and urinary dysfunction in this model, suggesting the potential for mast cell inhibition as a therapeutic that prevents and reverses pathology and associated symptomology.

Uropathogenic Escherichia coli-induced fibrosis, leading to lower urinary tract symptoms, is associated with type 2 cytokine signaling.

Chronic inflammation and prostate fibrosis have been identified as contributors to lower urinary tract symptoms (LUTS) pathophysiology in humans. It has been shown that transurethral infection of an Escherichia coli strain named CP1, which was isolated from a patient with chronic prostatitis, can lead to the develop of differential chronic inflammation and pain in certain mouse strains. Therefore, we hypothesized that differential inflammation would influence fibrotic response in the prostate. This study showed that while prostatic infection by CP1 causes the development of chronic tactile allodynia in NOD/ShiltJ (NOD) but not C57BL/6 (B6) mice, both mice developed evidence of prostate inflammation, prostate fibrosis, and urinary dysfunction. Fibrosis was confirmed by the upregulation of fibrosis-associated messenger RNAs (mRNAs), α-smooth muscle actin immunohistochemistry, and collagen staining with picrosirius red. These findings were mainly focused on the dorsolateral lobes of the prostate. Both mouse strains also developed smaller, more frequent voiding patterns postinfection, examined via cystometry. B6 mice responded to CP1 infection with type 2 cytokines (IL-4 and IL-13), while NOD mice did not, which may explain the differing tactile allodynia responses and level of collagen deposition. When mice lacking signal transducer and activator of transcription 6 (STAT6), a transcription factor known to be important for the production and signaling of IL-4 and IL-13, were infected with CP1, fibrosis was attenuated. This study provides a potential model for studying the development of infection-induced prostatic fibrosis and LUTS. This study also demonstrates that CP1-induced prostate fibrosis has a STAT6-dependent mechanism in B6 mice.

IL17 Mediates Pelvic Pain in Experimental Autoimmune Prostatitis (EAP).

Chronic pelvic pain syndrome (CPPS) is the most common form of prostatitis, accounting for 90-95% of all diagnoses. It is a complex multi-symptom syndrome with unknown etiology and limited effective treatments. Previous investigations highlight roles for inflammatory mediators in disease progression by correlating levels of cytokines and chemokines with patient reported symptom scores. It is hypothesized that alteration of adaptive immune mechanisms results in autoimmunity and subsequent development of pain. Mouse models of CPPS have been developed to delineate these immune mechanisms driving pain in humans. Using the experimental autoimmune prostatitis (EAP) in C57BL/6 mice model of CPPS we examined the role of CD4+T-cell subsets in the development and maintenance of prostate pain, by tactile allodynia behavioral testing and flow cytometry. In tandem with increased CD4+IL17A+ T-cells upon EAP induction, prophylactic treatment with an anti-IL17 antibody one-day prior to EAP induction prevented the onset of pelvic pain. Therapeutic blockade of IL17 did not reverse pain symptoms indicating that IL17 is essential for development but not maintenance of chronic pain in EAP. Furthermore we identified a cytokine, IL7, to be associated with increased symptom severity in CPPS patients and is increased in patient prostatic secretions and the prostates of EAP mice. IL7 is fundamental to development of IL17 producing cells and plays a role in maturation of auto-reactive T-cells, it is also associated with autoimmune disorders including multiple sclerosis and type-1 diabetes. More recently a growing body of research has pointed to IL17's role in development of neuropathic and chronic pain. This report presents novel data on the role of CD4+IL17+ T-cells in development and maintenance of pain in EAP and CPPS.

Introducing Amylo-Glo, a novel fluorescent amyloid specific histochemical tracer especially suited for multiple labeling and large scale quantification studies.

One of the hallmark pathologies associated with Alzheimer's disease (AD) is the conspicuous deposition of extracellular amyloid plaques within the forebrain. These plaques are primarily composed of fibrular aggregates of the A-beta peptide. Traditional methods for the histological localization of these plaques typically rely on the use of the tracers Congo Red or Thioflavin S. This study describes the characterization of a novel fluorescent histochemical probe, Amylo-Glo, for the high resolution and contrast localization of amyloid plaques in brain tissue sections. Potential advantages over conventional amyloid plaque stains such as Congo Red or Thioflavin S can be attributed to its unique chemical and spectral properties. Specifically, it results in a very bright blue UV excitable stain under physiological conditions that will not bleed through when illuminated with other filters. Its brightness makes it ideal for low magnification quantification studies, while its unique excitation/emission profile and mild staining conditions makes it ideal for combination with multiple immunofluorescent labeling studies.