Evaluation of Tissue Stem Cell-Derived Human Intestinal Organoids, a Physiologically Relevant Model to Evaluate Cytochrome P450 Induction in Gut.

Induction of cytochrome P450 can cause drug-drug interactions and efficacy failure. Induction risk in liver and gut is typically inferred from experiments with plated hepatocytes. Organoids are physiologically relevant, multicellular structures originating from stem cells. Intestinal stem cell-derived organoids retain traits of normal gut physiology, such as an epithelial barrier and cellular diversity. Matched human enteroid and colonoid lines, generated from ileal and colon biopsies from two donors, were cultured in extracellular matrix for 3 days, followed by a single 48-hour treatment with rifampin, omeprazole, CITCO, and phenytoin at concentrations that induce target genes in hepatocytes. After treatment, mRNA was analyzed for induction of target genes. Rifampin induced CYP3A4; estimated EC50 and maximal fold induction were 3.75 µM and 8.96-fold, respectively, for ileal organoids and 1.40 µM and 11.3-fold, respectively, for colon organoids. Ileal, but not colon, organoids exhibited nifedipine oxidase activity, which was induced by rifampin up to 14-fold. The test compounds did not increase mRNA expression of CYP1A2, CYP2B6, multidrug resistance transporter 1 (P-glycoprotein), breast cancer resistance protein, and UDP-glucuronosyltransferase 1A1 in ileal organoids. Whereas omeprazole induced CYP3A4 (up to 5.3-fold, geometric mean, n = 4 experiments), constitutive androstane receptor activators phenytoin and CITCO did not. Omeprazole failed to induce CYP1A2 mRNA but did induce CYP1A1 mRNA (up to 7.7-fold and 15-fold in ileal and colon organoids, respectively, n = 4 experiments). Despite relatively high intra- and interexperimental variability, data suggest that the model yields induction responses that are distinct from hepatocytes and holds promise to enable evaluation of CYP1A1 and CYP3A4 induction in gut. SIGNIFICANCE STATEMENT: An adult intestinal stem cell-derived organoid model to test P450 induction in gut was evaluated. Testing several prototypical inducers for mRNA induction of P450 isoforms, UDP-glucuronosyltransferase 1A1, P-glycoprotein, and breast cancer resistance protein with both human colon and ileal organoids resulted in a range of responses, often distinct from those found in hepatocytes, indicating the potential for further development of this model as a physiologically relevant gut induction test system.

Optimized Culture Conditions for Improved Growth and Functional Differentiation of Mouse and Human Colon Organoids.

Background & Aims: Diligent side-by-side comparisons of how different methodologies affect growth efficiency and quality of intestinal colonoids have not been performed leaving a gap in our current knowledge. Here, we summarize our efforts to optimize culture conditions for improved growth and functional differentiation of mouse and human colon organoids. Methods: Mouse and human colon organoids were grown in four different media. Media-dependent long-term growth was measured by quantifying surviving organoids via imaging and a cell viability readout over five passages. The impact of diverse media on differentiation was assessed by quantifying the number of epithelial cell types using markers for enterocytes, stem cells, Goblet cells, and enteroendocrine cells by qPCR and histology upon removal of growth factors. Results: In contrast to Wnt3a-conditioned media, media supplemented with recombinant Wnt3a alone did not support long-term survival of human or mouse colon organoids. Mechanistically, this observation can be attributed to the fact that recombinant Wnt3a did not support stem cell survival or proliferation as demonstrated by decreased LGR5 and Ki67 expression. When monitoring expression of markers for epithelial cell types, the highest level of organoid differentiation was observed after combined removal of Wnt3a, Noggin, and R-spondin from Wnta3a-conditioned media cultures. Conclusion: Our study defined Wnt3a-containing conditioned media as optimal for growth and survival of human and mouse organoids. Furthermore, we established that the combined removal of Wnt3a, Noggin, and R-spondin results in optimal differentiation. This study provides a step forward in optimizing conditions for intestinal organoid growth to improve standardization and reproducibility of this model platform.

Structure and N-acetylglucosamine binding of the distal domain of mouse adenovirus 2 fibre.

Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple ß-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut.

Alpha-defensin-dependent enhancement of enteric viral infection.

The small intestinal epithelium produces numerous antimicrobial peptides and proteins, including abundant enteric α-defensins. Although they most commonly function as potent antivirals in cell culture, enteric α-defensins have also been shown to enhance some viral infections in vitro. Efforts to determine the physiologic relevance of enhanced infection have been limited by the absence of a suitable cell culture system. To address this issue, here we use primary stem cell-derived small intestinal enteroids to examine the impact of naturally secreted α-defensins on infection by the enteric mouse pathogen, mouse adenovirus 2 (MAdV-2). MAdV-2 infection was increased when enteroids were inoculated across an α-defensin gradient in a manner that mimics oral infection but not when α-defensin levels were absent or bypassed through other routes of inoculation. This increased infection was a result of receptor-independent binding of virus to the cell surface. The enteroid experiments accurately predicted increased MAdV-2 shedding in the feces of wild type mice compared to mice lacking functional α-defensins. Thus, our studies have shown that viral infection enhanced by enteric α-defensins may reflect the evolution of some viruses to utilize these host proteins to promote their own infection.

Defensins Potentiate a Neutralizing Antibody Response to Enteric Viral Infection.

α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture.

Defensins at the Mucosal Surface: Latest Insights into Defensin-Virus Interactions.

Defensins are innate immune effector peptides expressed at mucosal surfaces throughout the human body and are potently antiviral in vitro The role of defensins in viral pathogenesis in vivo is poorly understood; however, recent studies have revealed that defensin-virus interactions in vivo are complicated and distinct from their proposed antiviral mechanisms in vitro These findings highlight the need for additional research that connects defensin neutralization of viruses in cell culture to in vivo antiviral mechanisms.

Antiviral mechanisms of human defensins.

Defensins are an effector component of the innate immune system with broad antimicrobial activity. Humans express two types of defensins, α- and ß-defensins, which have antiviral activity against both enveloped and non-enveloped viruses. The diversity of defensin-sensitive viral species reflects a multitude of antiviral mechanisms. These include direct defensin targeting of viral envelopes, glycoproteins, and capsids in addition to inhibition of viral fusion and post-entry neutralization. Binding and modulation of host cell surface receptors and disruption of intracellular signaling by defensins can also inhibit viral replication. In addition, defensins can function as chemokines to augment and alter adaptive immune responses, revealing an indirect antiviral mechanism. Nonetheless, many questions regarding the antiviral activities of defensins remain. Although significant mechanistic data are known for α-defensins, molecular details for ß-defensin inhibition are mostly lacking. Importantly, the role of defensin antiviral activity in vivo has not been addressed due to the lack of a complete defensin knockout model. Overall, the antiviral activity of defensins is well established as are the variety of mechanisms by which defensins achieve this inhibition; however, additional research is needed to fully understand the role of defensins in viral pathogenesis.

Critical determinants of human α-defensin 5 activity against non-enveloped viruses.

Human α-defensins, such as human α-defensin 5 (HD5), block infection of non-enveloped viruses, including human adenoviruses (AdV), papillomaviruses (HPV), and polyomaviruses. Through mutational analysis of HD5, we have identified arginine residues that contribute to antiviral activity against AdV and HPV. Of two arginine residues paired on one face of HD5, Arg-28 is critical for both viruses, while Arg-9 is only important for AdV. Two arginine residues on the opposite face of the molecule (Arg-13 and Arg-32) and unpaired Arg-25 are less important for both. In addition, hydrophobicity at residue 29 is a major determinant of anti-adenoviral activity, and a chemical modification that prevents HD5 self-association was strongly attenuating. Although HD5 binds to the capsid of AdV, the molecular basis for this interaction is undefined. Capsid binding by HD5 is not purely charge-dependent, as substitution of lysine for Arg-9 and Arg-28 was deleterious. Analysis of HD5 analogs that retained varying levels of potency demonstrated that anti-adenoviral activity is directly correlated with HD5 binding to the virus, confirming that the viral capsid rather than the cell is the relevant target. Also, AdV aggregation induced by HD5 binding is not sufficient for neutralization. Rather, these studies confirm that the major mechanism of HD5-mediated neutralization of AdV depends upon specific binding to the viral capsid through interactions mediated in part by critical arginine residues, hydrophobicity at residue 29, and multimerization of HD5, which increases initial binding of virus to the cell but prevents subsequent viral uncoating and genome delivery to the nucleus.

Bridging the gap between preclinical and clinical microbicide trials: blind evaluation of candidate gels in murine models of efficacy and safety.

BACKGROUND: Despite significant protection in preclinical studies, cellulose sulfate (CS) failed to protect women against HIV-1/2 and was associated with a trend toward increased HIV-1 acquisition in one of the clinical trials. These results highlight the need for preclinical tests more predictive of clinical outcomes. The objective of this study was to test coded vaginal gels, including CS, in murine models of safety and efficacy to determine the models' utility for evaluating future products. METHODS: Four coded formulations, including 6% CS, 2% PRO 2000 and two placebo gels, were administered intravaginally to medroxyprogesterone-treated mice and their ability to prevent genital herpes (efficacy) or to alter the susceptibility to low dose HSV challenge (safety) was determined. Nonoyxnol-9 served as a positive toxicity control. RESULTS: CS and PRO 2000 significantly protected mice from genital herpes following infection with a laboratory or clinical isolate of HSV-2 introduced in buffer (p<0.001). However, protection was reduced when virus was introduced in seminal plasma. Moreover, mice were significantly more susceptible to infection with low doses of HSV-2 when challenged 12 h after the 7th daily dose of CS or nonoxynol-9 (p<0.05). The increased susceptibility was associated with alterations in epithelial architecture. CONCLUSIONS: CS prevented genital herpes when present at the time of viral challenge, but increased the rate of infection when gel was applied daily for 7 days with a vaginal wash prior to viral inoculation. The findings presumably reflect altered epithelial architecture, which may have contributed to the trend towards increased HIV observed clinically.

Interlaboratory reproducibility of female genital tract cytokine measurements by Luminex: implications for microbicide safety studies.

The interlaboratory reproducibility of cytokine measurements from cervicovaginal samples by Luminex has not been reported. Using cervicovaginal lavage specimens collected on three study days from 12 women participating in a Phase I microbicide study, we measured a panel of eight cytokines in three independent laboratories. Four (IFN-γ, IL-10, IL-17, and TNF) were below the limit of detection in the majority (85%) of samples in either two or all three laboratories, an observation that may guide analyte selection for future studies. Good interlaboratory agreement (intraclass correlation coefficient, r>0.7) in absolute levels was observed for IL-1ß, IL-6, and IL-8, while poor agreement was seen for IFN-α2 (r=0.47). When considering within-subject change from baseline (pre-product, at study-day 0) to either post-product visit (study-days 7 and 14), IL-1ß and IL-6 exhibited good interlaboratory agreement (r>0.7), while IFN-α2 and IL-8 did not. Future studies addressing the clinical utility of specific biomarkers of inflammation for microbicide trials should consider reproducibility in the context of defining biologically meaningful thresholds of change for candidate biomarkers, ensuring that such change can be reliably distinguished from background variability.

Susceptibility to genital herpes as a biomarker predictive of increased HIV risk: expansion of a murine model of microbicide safety.

BACKGROUND: A crucial gap in the development of microbicides for HIV prevention is the absence of models predictive of safety. Previous studies have demonstrated an increased susceptibility to genital herpes in mice following repeated applications of nonoxynol-9 (N-9). This study was designed to explore the underlying mechanisms, focusing on the effects that N-9 has on genital tract epithelium and to apply this expanded model to evaluate the safety of microbicides that have been advanced to clinical trials. METHODS: Mice were treated intravaginally with formulated 3.5% N-9, 1% tenofovir, 0.5% or 2% PRO 2000, hydroxyethylcellulose (HEC) placebo or no treatment and the effect on herpes simplex virus 2 (HSV-2) susceptibility, epithelial cell architecture, junctional proteins and inflammation were assessed. RESULTS: Mice treated with seven daily doses of N-9, but not tenofovir, PRO 2000 or HEC, were significantly more susceptible to challenge with low doses of HSV-2; confocal microscopy demonstrated increased numbers of viral particles deep within the genital tract. N-9 disrupted the epithelium with loss of tight and adherens junctional proteins. By contrast, the epithelium was relatively preserved following tenofovir, PRO 2000 and HEC exposure. Additionally, N-9, but not the other microbicides, triggered a significant inflammatory response relative to untreated mice. CONCLUSIONS: These findings indicate that disruption of the epithelium contributes to increased HSV-2 susceptibility and might provide a biomarker predictive of increased risk for HIV acquisition. The results are consistent with the safety outcomes of the recently completed Phase IIb clinical trial with 0.5% PRO 2000 gel, and predict that tenofovir gel will not adversely affect the genital tract.

Disruption of tight junctions by cellulose sulfate facilitates HIV infection: model of microbicide safety.

BACKGROUND: The lack of biomarkers that are predictive of safety is a critical gap in the development of microbicides. The present experiments were designed to evaluate the predictive value of in vitro models of microbicide safety. METHODS: Changes in the epithelial barrier were evaluated by measuring transepithelial electrical resistance (TER) after exposure of human epithelial cells to candidate microbicides in a dual-chamber system. The significance of observed changes was addressed by challenging cultures with human immunodeficiency virus (HIV) and measuring the ability of virus to cross the epithelium and infect target T cells cultured in the lower chamber. RESULTS: Exposure to nonoxynol-9 (N-9) or cellulose sulfate (CS), but not 9-[2-(phosphonomethoxy)propyl]adenine (also referred to as tenofovir) or PRO2000, resulted in a rapid and sustained reduction in TER and a marked increase in HIV infection of T cells cultured in the lower chamber. Moreover, CS triggered nuclear factor kappaB activation in peripheral blood mononuclear cells and increased HIV replication in chronically infected U1 cells. CONCLUSIONS: Epithelial barrier disruption and enhanced viral replication may have contributed to the increased risk of HIV acquisition observed in phase 3 trials of N-9 and CS. Expansion of in vitro safety testing to include these models would provide a more stringent preclinical assessment of microbicide safety and may prove to be more predictive of clinical outcomes.

Novel approaches in fighting herpes simplex virus infections.

The development of novel strategies to eradicate herpes simplex virus (HSV) is a global public health priority. While acyclovir and related nucleoside analogues provide successful modalities for treatment and suppression, HSV remains highly prevalent worldwide and is a major cofactor fueling the HIV epidemic. HSV is the predominant cause of genital ulcerative disease, and neonatal and sporadic infectious encephalitis. Asymptomatic shedding, which occurs more frequently than previously appreciated, contributes to viral transmission. Acyclovir resistance may be problematic for immunocompromised patients and highlights the need for new safe and effective agents. Ideally, vaccines to prevent infection, drugs to inhibit the establishment of or reactivation from latency, or vaginal microbicides to prevent sexual and perinatal transmission are needed to control the epidemic. This review summarizes current therapeutic options and strategies in development.

Herpes simplex virus downregulates secretory leukocyte protease inhibitor: a novel immune evasion mechanism.

Secretory leukocyte protease inhibitor (SLPI), an anti-inflammatory mediator of mucosal immunity, inhibits human immunodeficiency virus (HIV) and herpes simplex virus (HSV) in cell culture. Epidemiological studies demonstrate that higher concentrations of SLPI in mucosal secretions are associated with a reduced risk of HIV transmission. The current studies were designed to test the hypothesis that HSV triggers a loss of SLPI to evade innate immunity and that this response may contribute to the increased risk of HIV infection in the setting of HSV infection. Exposure of human cervical epithelial cells to HSV-1 or HSV-2, but not HIV or vesicular stomatitis virus, triggered a significant and sustained reduction in SLPI levels. The reduction persisted when cells were infected in the presence of acyclovir but not following infection with UV-inactivated virus, indicating that viral gene expression, but not replication, is required. Reverse transcriptase PCR studies demonstrated that the loss of SLPI is mediated by downregulation of gene expression. SLPI downregulation was associated with activation of NF-kappaB signaling pathways and upregulation of proinflammatory cytokines, consistent with the known inhibitor effects of SLPI on NF-kappaB pathways. The downregulation mapped to viral early-gene expression, as variants impaired in expression of the ICP4 or ICP0 immediate-early gene failed to downregulate SLPI or activate NF-kappaB. Together, these results identify a novel role for HSV immediate-early-gene expression in regulating mucosal immune responses.

Candidate microbicide PPCM blocks human immunodeficiency virus type 1 infection in cell and tissue cultures and prevents genital herpes in a murine model.

A structurally novel candidate microbicide, PPCM, which is formed from the reaction of D,L-mandelic acid with sulfuric acid, provides activity against human immunodeficiency virus (HIV) and herpes simplex virus (HSV) and is not cytotoxic. The objectives of the current studies were to comprehensively evaluate the activity of PPCM in cell and explant cultures, explore the possibility of combining PPCM with HIV-specific reverse transcriptase inhibitors, and evaluate the efficacy of a formulated gel against genital herpes in a murine model. PPCM inhibited infection by laboratory and clinical R5 and X4 clade B and clade C HIV strains in cell culture. Ectocervical and endocervical tissue explants exposed to HIV-1(BaL) in the presence of PPCM were protected (50% inhibitory concentrations [IC(50)] of 3.9 microg/ml for ectocervix and 3.1 microg/ml for endocervix), and transfer of virus to target T cells via migratory cells was significantly impaired (IC(50) of 35.7 microg/ml for ectocervix and 54.6 microg/ml for endocervix). The drug also blocked infection by cell-associated virus. Combinations of PPCM with UC-781 or PMPA in vitro exhibited additive anti-HIV activity. PPCM was incorporated into stable, low-pH gel formulations at concentrations of 0.4% and 4%. Both gels prevented genital herpesvirus infection in mice, even when virus was introduced in human seminal plasma. The abilities of PPCM to inhibit primary HIV isolates, reduce infection by cell-associated virus, and transfer of HIV from migratory to T cells, combined with the complete protection provided by formulated gel against genital herpes, indicate that this drug is an excellent candidate for inclusion in a combination microbicide and would provide protection against both HIV and HSV.

A comprehensive murine model to evaluate topical vaginal microbicides: mucosal inflammation and susceptibility to genital herpes as surrogate markers of safety.

A critical gap in microbicide development is the absence of surrogate safety markers. The objective of the present study was to develop a murine model to examine the mucosal response to microbicides and to assess the functional implication of observed changes. Mice received 14 daily intravaginal doses of nonoxynol-9, PRO 2000, or placebo gel. Nonoxynol-9 induced an inflammatory response characterized by increases in levels of cytokines and chemokines, recruitment of neutrophils and monocytes into the genital tract, and activation of the transcription factors NF- kappa B and activator protein-1. Minimal inflammation was observed in response to 2% PRO 2000. Nonoxynol-9-treated mice were significantly more susceptible to challenge with a low dose of herpes simplex virus type 2; the response of PRO 2000-treated mice was similar to the response to placebo. These findings suggest that PRO 2000 has little deleterious effect on mucosal immunity and, if validated by clinical experiences, support the inclusion of this model in the preclinical evaluation of future candidate microbicides.