Shpigel Lab

Recent Publications

Xenotransplantation of human intestine into mouse abdomen or subcutaneous tissue: Novel platforms for the study of the human enteric nervous system

N. Nagy, N. Marsiano, R. S. Bruckner, M. Scharl, M. J. Gutnick, S. Yagel, E. Arciero, A. M. Goldstein, and N. Y. Shpigel. 2018. “Xenotransplantation of human intestine into mouse abdomen or subcutaneous tissue: Novel platforms for the study of the human enteric nervous system.” Neurogastroenterol MotilNeurogastroenterol Motil, 30, Mar.Abstract
BACKGROUND: Current efforts to develop stem cell therapy as a novel treatment for neurointestinal diseases are limited by the unavailability of a model system to study cell transplantation in the human intestine. We propose that xenograft models support enteric nervous system (ENS) development in the fetal human intestine when transplanted into mice subcutaneously or intra-abdominally. METHODS: Fetal human small and large intestine were grafted onto the small intestinal mesentery and into the subcutaneous tissue of immunodeficient mice for up to 4 months. Intestinal cytoarchitecture and ENS development were studied using immunohistochemistry. KEY RESULTS: In both abdominal and subcutaneous grafts, the intestine developed normally with formation of mature epithelial and mesenchymal layers. The ENS was patterned in two ganglionated plexuses containing enteric neurons and glia, including cholinergic and nitrergic neuronal subtypes. c-Kit-immunoreactive interstitial cells of Cajal were present in the gut wall. CONCLUSIONS & INFERENCES: Abdominal xenografts represent a novel model that supports the growth and development of fetal human intestine. This in vivo approach will be a useful method to study maturation of the ENS, the pathophysiology of neurointestinal diseases, and the long-term survival and functional differentiation of neuronal stem cells for the treatment of enteric neuropathies.

Microbial communities and inflammatory response in the endometrium differ between normal and metritic dairy cows at 5-10 days post-partum

R. Sicsic, T. Goshen, R. Dutta, N. Kedem-Vaanunu, V. Kaplan-Shabtai, Z. Pasternak, Y. Gottlieb, N. Y. Shpigel, and T. Raz. 2018. “Microbial communities and inflammatory response in the endometrium differ between normal and metritic dairy cows at 5-10 days post-partum.” Vet ResVet Res, 49: 77, Aug 2.Abstract
Post-partum metritis is among the most prevalent disease in dairy cows affecting animal welfare and inflicting considerable economic loses. While post-partum contamination of the uterus is rife in dairy cows, only a fraction of these animals will develop metritis. Our main objective was to compare the bacterial communities and the inflammatory response in the endometrium of healthy and metritic dairy cows. Holstein-Friesian cows (n = 35) were sampled immediately following clinical classification as healthy (n = 21), suffering from metritis (n = 13) or septic metritis (n = 1), based on veterinary examination at 5-10 days post-partum. Polymorphonuclear cells (PMN) percentage in endometrial cytology was significantly higher in cows with metritis. Full-thickness uterine biopsy analysis revealed that the luminal epithelium in inter-caruncle areas was preserved in healthy cows, but in metritis it was compromised, with marked PMN infiltration particularly in the apical endometrium. Gram staining revealed that bacterial load and spatial distribution was associated with disease severity. 16S-rDNA bacterial community analysis revealed unique endometrial bacterial community composition in metritic cows, as compared to more diverse communities among healthy cows. The most abundant phyla in healthy cows were Proteobacteria (31.8 +/- 9.3%), Firmicutes (27.9 +/- 8.4%) and Bacteroidetes (19.7 +/- 7.2%), while Bacteroidetes (60.3 +/- 10.3%), Fusobacteria (13.4 +/- 5.9%) and Firmicutes (10.5 +/- 3.3%) were most abundant in the endometrial mucosa of metritic cows. Relative abundance of Bacteroidetes (19.7 +/- 7.2% vs. 60.3 +/- 10.3%), Fusobacteria (7.5 +/- 5.2% vs. 13.4 +/- 5.9%) and Proteobacteria (31.8 +/- 9.3% vs. 7.3 +/- 5.6%) phyla differed significantly between healthy and metritic cows. In summary, endometrial PMN abundance, spatial distribution and bacterial communities differed between healthy and metritic dairy cows at early post-partum.

T3SS-dependent microvascular thrombosis and ischemic enteritis in human gut xenografts infected with enteropathogenic Escherichia coli

E. Nissim-Eliraz, E. Nir, I. Shoval, N. Marsiano, I. Nissan, H. Shemesh, N. Nagy, A. M. Goldstein, M. Gutnick, I. Rosenshine, S. Yagel, and N. Y. Shpigel. 2017. “T3SS-dependent microvascular thrombosis and ischemic enteritis in human gut xenografts infected with enteropathogenic Escherichia coli.” Infect ImmunInfect Immun, Aug 07.Abstract
Enteropathogenic Escherichia coli (EPEC) is a leading cause of severe intestinal disease and infant mortality in developing countries. Virulence is mediated by a type three secretion system (T3SS) causing the hallmark lesions of attaching and effacing (AE) and actin-rich pedestal formation beneath the infecting bacteria on the apical surface of enterocytes. EPEC is a human-specific pathogen whose pathogenesis cannot be studied in animal models. We therefore established an EPEC infection model in human gut xenografts in SCID mice and used it to study the role of T3SS in the pathogenesis of the disease. Following EPEC O127:H6 strain E2348/69 infection, T3SS-dependent AE lesions and pedestals were demonstrated in all infected xenografts. We report here the development of T3SS-dependent intestinal thrombotic microangiopathy (iTMA) and ischemic enteritis in approximately 50% of infected human gut xenografts. Using species-specific CD31 immunostaining, we showed that iTMA was limited to the larger human-mouse chimeric blood vessels which are located between the muscularis mucosa and circular muscular layer of the human gut. These blood vessels were massively invaded by bacteria which adhered to and formed pedestals on endothelial cells and aggregated with mouse neutrophils in the lumen. We conclude that endothelial infection, iTMA and ischemic enteritis might be central mechanisms underlying severe EPEC-mediated disease.

Characterization and identification of microbial communities in bovine necrotic vulvovaginitis

N. Y. Shpigel, L. Adler-Ashkenazy, S. Scheinin, T. Goshen, A. Arazi, Z. Pasternak, and Y. Gottlieb. 2017. “Characterization and identification of microbial communities in bovine necrotic vulvovaginitis.” The Veterinary JournalThe Veterinary Journal, 219: 34-39, 1//.Abstract
Bovine necrotic vulvovaginitis (BNVV) is a severe and potentially fatal disease of post-partum cows that emerged in Israel after large dairy herds were merged. While post-partum cows are commonly affected by mild vulvovaginitis (BVV), in BNVV these benign mucosal abrasions develop into progressive deep necrotic lesions leading to sepsis and death if untreated. The etiology of BNVV is still unknown and a single pathogenic agent has not been found. We hypothesized that BNVV is a polymicrobial disease where the normally benign vaginal microbiome is remodeled and affects the local immune response. To this end, we compared the histopathological changes and the microbial communities using 16S rDNA metagenetic technique in biopsies taken from vaginal lesions in post-partum cows affected by BVV and BNVV. The hallmark of BNVV was the formation of complex polymicrobial communities in the submucosal fascia and abrogation of neutrophil recruitment in these lesions. Additionally, there was a marked difference in the composition of bacterial communities in the BNVV lesions in comparison to the benign BVV lesions. This difference was characterized by the abundance of Bacteroidetes and lower total community membership in BNVV. Indicator taxa for BNVV were Parvimonas, Porphyromonas, unclassified Veillonellaceae, Mycoplasma and Bacteroidetes, whereas unclassified Clostridiales was an indicator for BVV. The results support a polymicrobial etiology for BNVV.

OP028 Gut specific regulatory T cells – a new frontier for Crohn's disease therapy

R. Goldberg, C. Scotta, D. Cooper, E. Eliraz, E. Nir, P. Irving, J. Sanderson, N. Shpigel, F. Marelli-Berg, G. Lombardi, and G. Lord. 2017. “OP028 Gut specific regulatory T cells – a new frontier for Crohn's disease therapy.” Journal of Crohn's and ColitisJournal of Crohn's and Colitis, 11: S17-S17.Abstract
Background: We have recently shown that Tregs isolated from the peripheral blood of patients with Crohn's Disease (CD) play a critical role in controlling both phenotype and expansion of auto-reactive T cells (1). This is a critical step to developing cell based therapy for Crohn's disease, where where primary and secondary non response rates to biologics remain unacceptably high. The immune system used retinoic acid (ATRA) to induce a4b7 and thus prime Tregs to home to the gut. We sought use ATRA in-vitro in order to engineer gut specific Tregs for our Phase 1 trial in Crohn's Disease. We then validated our findings in-vitro and in-vivo.Methods: Tregs were isolated from peripheral blood of CD patients. ATRA supplementation was tested in standard culture conditions. The expression of a4b7 was assessed by flow cytometry. Suppression assays were performed using autologous effector T cells (Teff). An ibidi flow chamber system coated with recombinant human MAdCAM-1 was used for in-vitro trafficking experiments. SCID mice xenografted with foetal intestinal small bowel were used for in-vivo experiments. Parametric and non-parametric data were calculated as the mean ± s.d. and median (IQR). For comparison of parametric and non-parametric data, t-test, or ANOVA were used.Results:Ex-vivo expansion of Tregs in the presence of ATRA significantly induced the expression of a4b7 compared to Rapamycin alone (5.57%±3.12 vs 82.8%±9.5, p=0.0057) Cells treated with Rapa+ATRA maintained their superior suppressive ability compared to Rapamycin treated Tregs (95.8%±3.5% vs 91.15%±10.1% p=ns; at Treg:Teff 1:1 ratio). RAPA+ATRA Tregs did not produce IFNy or IL17 under pro-inflammatory cytokine challenge. When flowed through a MAdCAm-1 coated chamber, significantly higher numbers of Rapa+ATRA treated cells were observed to roll (Rapa 0.83±0.40 vs Rapa+ATRA 10.17±2.54 p=0.005), crawl (Rapa 0 vs Rapa+ATRA 4±0.89 p=0.001) and firmly adhere (Rapa 0.33±0.21 vs Rapa+ATRA 36.8±1.78, p<0.001) than those treated with Rapa alone. When Tregs were transferred into mice, a higher proportion of Tregs were found in xenografts of animals treated with Rapa+ATRA Tregs compared Rapa Tregs (12.10 (7.54–22.83) vs 4.97 (1.72–7.63), p=0.0056). Importantly there was a higher proportion of Tregs in inflamed xenografts of animals treated with Rapa+ATRA Tregs compared to those treated with Rapa Tregs (18.35 (12.95–28.63) vs 6.78 (2.65–9.61), p=0.0095).Conclusions: The addition of ATRA to Treg culture in-vitro confers a gut homing phenotype. This is functionally relevant in-vitro and in-vivo. The treatment maintains the highly suppressive and phenotypically stable phenotype of these cells. These gut specific Tregs will be implemented in our first in man trial Treg therapy for Crohn's disease.