Faculty Bibliography

Background/Purpose: Tubulointerstitial damage in lupus nephritis (LN) is a strong predictor of progression to chronic kidney disease (CKD) and end stage renal disease (ESRD). While prior studies showed complement activation mediates glomerular injury, the role of complement in renal tubular damage has not been evaluated. The objective of this study is to investigate the association between complement activation as measured by tubular complement 9 (C9) deposition with interstitial fibrosis and tubular atrophy (IFTA) in LN.
Method(s): LN biopsies from July 2014 to July 2016 were evaluated. Chromogenic immunohistochemistry was performed on formalin-fixed, paraffin-embedded, 4-mum human renal biopsy sections using unconjugated, murine anti-human Complement C9 (Hycult Biotech, clone X197) as a marker of the membrane attack complex (MAC) activation. C3 glomerulopathy was used as a positive control and normal kidney served as a negative control. Tubular basement membrane C9 staining intensity was analyzed as absent (0) versus present (1 to 3) scored on a semi-quantitative scale by a renal pathologist. IFTA was categorized as 0-10%, 11-20%, and >20%. Clinical parameters were assessed at the time of biopsy and 6 months post biopsy.
Result(s): Renal biopsies from 30 LN patients were studied, of which 23 (77%) were proliferative LN. There were 24 (80%) women, mean age 33 (standard deviation) (12) years. Positive tubular C9 staining (C9+) was observed in 7 (23%) biopsies. At the time of renal biopsy, C9+ patients had significantly higher proteinuria, compared to C9- patients: median (interquartile range) 6.2g (3.3-13.1) vs. 2.4g (1.3-4.6), p< 0.01. The differences persisted at 6 months after induction therapy: 1.08g (1.0-8.3) in C9+ vs. 0.68g (0.2-2.1) in C9- patients, p=0.06. Tubular C9 deposition was associated with the finding of interstitial fibrosis on biopsy: 3 out of 7 (42.9%) had >20% interstitial fibrosis as compared to none in the C9- group, p=< 0.01. Higher proportion of C9+ patients had moderate NIH Chronicity index: 3 out of 7 (42.9%) vs 2 out of 23 (8.7%) in the C9- group, p=0.07. There was no significant difference in eGFR at renal biopsy between the two groups.
Conclusion(s): Tubular C9 deposition is associated with proteinuria, interstitial fibrosis and increased chronicity which are predictors of progression to ESRD. Complement activation may play an important role in tubulointerstitial damage in LN

Adeno-associated virus (AAV) vector serotypes vary in their ability to transduce hepatocytes from different species. Chimeric mouse models harboring human hepatocytes have shown translational promise for liver-directed gene therapies. However, many variables that influence human hepatocyte transduction and transgene expression in such models remain poorly defined. Here, we aimed to test whether three experimental conditions influence AAV transgene expression in immunodeficient, fumaryl-acetoactetate-hydrolase-deficient (Fah^-/-) chimeric mice repopulated with primary human hepatocytes. We examined the effects of the murine liver injury cycle, human donor variability, and vector doses on hepatocyte transduction with various AAV serotypes expressing a green fluorescent protein (GFP). We determined that the timing of AAV vector challenge in the liver injury cycle resulted in up to 7-fold differences in the percentage of GFP expressing human hepatocytes. The GFP+ hepatocyte frequency varied 7-fold between human donors without, however, changing the relative transduction efficiency between serotypes for an individual donor. There was also a clear relationship between AAV vector doses and human hepatocyte transduction and transgene expression. We conclude that several experimental variables substantially affect human hepatocyte transduction in the Fah^-/- chimera model, attention to which may improve reproducibility between findings from different laboratories.

In diagnostic breast pathology, there is no reliable applicable immunostain to help discern atypical and in situ apocrine lesions from benign apocrine tissue. At present, the diagnosis of non-invasive apocrine lesions remains challenging with current diagnoses rendered based on discrete morphologic characteristics on conventional hematoxylin and eosin staining. Interobserver variability is significant even among subspecialists partly due to lack of adjuvant diagnostic immunohistochemical stains. Herein, we set to elucidate the potential utility of EZH2 and Ki-67 immunostains as tangible tools in non-invasive apocrine proliferations. A cohort of apocrine breast lesions [Benign apocrine hyperplasia (BAH), n = 10; Atypical apocrine hyperplasia (AAH), n = 16; Apocrine ductal carcinoma in situ (ADCIS), n = 12] were subjected to EZH2 immunostaining and analyzed via H-scoring of nuclear expression. Mean H-scores for EZH2 progressively increased from BAH (23.5), to AAH (47.4) and ADCIS (196.4), and showed a significant difference utilizing the Kruskal-Wallis test (p < 0.0001). Further interrogation of Ki-67 demonstrated incremental expression from BAH to AAH and ADCIS at 1.6 %, 4.7 % and 24.7 %, respectively (p < 0.0001, Kruskal-Wallis test), suggesting an association with increased proliferation. Our results demonstrate that a combination of EZH2 and Ki-67 immunostaining may be employed in differentiating among challenging apocrine breast lesions and suggest a putative diagnostic utility for EZH2 and Ki-67 in non-invasive apocrine breast lesions.

Background: Fibroconnective tissues of the body are traditionally conceived as layers of densely compacted collagen. Recent in vivo microscopy, however, demonstrates that at least some, including visceral submucosae, dermis, fascia, adventitia and perineurium, are actually a reticular network of fluid-filled sinuses supported by a complex scaffold of thick collagen bundles (Benias et al Sci Rep 2018: 8). The interstitial fluid is rich in hyaluronic acid (HA). Whether these large scale interstitial spaces are continuous between tissues/organs or separate is unclear. Continuity was investigated by two methods: 1. movement of non-biological pigment (tattoo pigment, colloidal silver) in colon and skin specimens; 2. localization of HA by IHC.
Design(s): H&E-stained sections of FFPE tissues from resected colons following endoscopic submucosal tattoo for malignant polyps (n=5) and from skin biopsies with either cosmetic tattoos (n=3) or colloidal silver (n=2) were examined. Location of particles was assessed. The slides were then scanned, decolorized, and stained by multiplex chromogenic IHC assay (Discovery Ultra, Ventana) for HA-binding protein (brown), vimentin (magenta) and CD34 (teal) to label interstitial lining cells.
Result(s): Tattoo pigment and colloidal silver within the interstitial spaces was identified in the dermis (Fig. 1) and colonic submucosa and in the dependent mesenteric and subcutaneous fascias. In all colon specimens HA IHC highlighted the spatial continuity of all layers of the colon from lamina propria through muscularis mucosae to submucosa (Fig. 2A), then through the muscularis propria into mesenteric fascia (Fig. 2B, C). Continuity between these spaces and perivascular stroma/adventitia and perineurium in the bowel wall was also evident. Continuity of HA-filled spaces is also demonstrated from papillary to reticular dermis and then to subcutaneous fascia interface. (Figure presented)
Conclusion(s): Interstitial spaces are neither virtual nor a result of processing artifact, but are filled with physiologically relevant fluid rich in HA. Their continuity across tissue compartments is demonstrated by movement of non-biological pigments and by spatial continuity of HA. The implications of such multisystem continuity are protean, but may particularly explain commonly observed modes of discontinuous cancer spread through tissue planes, such as mesenteric tumor deposits in colon cancer and subcutaneous in-transit melanoma metastasis, and spread of infection (e.g. necrotizing fasciitis)

Primary human hepatocytes (PHHs) are an essential tool for modeling drug metabolism and liver disease. However, variable plating efficiencies, short lifespan in culture, and resistance to genetic manipulation have limited their use. Here, we show that the pyrrolizidine alkaloid retrorsine improves PHH repopulation of chimeric mice on average 10-fold and rescues the ability of even poorly plateable donor hepatocytes to provide cells for subsequent ex vivo cultures. These mouse-passaged (mp) PHH cultures overcome the marked donor-to-donor variability of cryopreserved PHH and remain functional for months as demonstrated by metabolic assays and infection with hepatitis B virus and Plasmodium falciparum mpPHH can be efficiently genetically modified in culture, mobilized, and then recultured as spheroids or retransplanted to create highly humanized mice that carry a genetically altered hepatocyte graft. Together, these advances provide flexible tools for the study of human liver disease and evaluation of hepatocyte-targeted gene therapy approaches.

Background/UNASSIGNED:Glioma is a family of primary brain malignancies with limited treatment options and in need of novel therapies. We previously demonstrated that the adhesion G protein-coupled receptor GPR133 (ADGRD1) is necessary for tumor growth in adult glioblastoma, the most advanced malignancy within the glioma family. However, the expression pattern of GPR133 in other types of adult glioma is unknown. Methods/UNASSIGNED:We used immunohistochemistry in tumor specimens and non-neoplastic cadaveric brain tissue to profile GPR133 expression in adult gliomas. Results/UNASSIGNED:We show that GPR133 expression increases as a function of WHO grade and peaks in glioblastoma, where all tumors ubiquitously express it. Importantly, GPR133 is expressed within the tumor bulk, as well as in the brain-infiltrating tumor margin. Furthermore, GPR133 is expressed in both isocitrate dehydrogenase (IDH) wild-type and mutant gliomas, albeit at higher levels in IDH wild-type tumors. Conclusion/UNASSIGNED:The fact that GPR133 is absent from non-neoplastic brain tissue but de novo expressed in glioma suggests that it may be exploited therapeutically.

Microvascular rarefaction, or the decrease in vascular density, has been described in the cerebrovasculature of aging humans, rats, and, more recently, mice in the presence and absence of age-dependent diseases. Given the wide use of mice in modeling age-dependent human diseases of the cerebrovasculature, visualization, and quantification of the global murine cerebrovasculature is necessary for establishing the baseline changes that occur with aging. To provide in vivo whole-brain imaging of the cerebrovasculature in aging C57BL/6 mice longitudinally, contrast-enhanced magnetic resonance angiography (CE-MRA) was employed using a house-made gadolinium-bearing micellar blood pool agent. Enhancement in the vascular space permitted quantification of the detectable, or apparent, cerebral blood volume (aCBV), which was analyzed over 2 years of aging and compared to histological analysis of the cerebrovascular density. A significant loss in the aCBV was detected by CE-MRA over the aging period. Histological analysis via vessel-probing immunohistochemistry confirmed a significant loss in the cerebrovascular density over the same 2-year aging period, validating the CE-MRA findings. While these techniques use widely different methods of assessment and spatial resolutions, their comparable findings in detected vascular loss corroborate the growing body of literature describing vascular rarefaction aging. These findings suggest that such age-dependent changes can contribute to cerebrovascular and neurodegenerative diseases, which are modeled using wild-type and transgenic laboratory rodents.