NF-κB Inducing Kinase Attenuates Colorectal Cancer by Regulating Noncanonical NF-κB Mediated Colonic Epithelial Cell Regeneration.

BACKGROUND & AIMS: Dysregulated colonic epithelial cell (CEC) proliferation is a critical feature in the development of colorectal cancer. We show that NF-κB-inducing kinase (NIK) attenuates colorectal cancer through coordinating CEC regeneration/differentiation via noncanonical NF-κB signaling that is unique from canonical NF-κB signaling. METHODS: Initial studies evaluated crypt morphology/functionality, organoid generation, transcriptome profiles, and the microbiome. Inflammation and inflammation-induced tumorigenesis was initiated in whole-body NIK knockout mice (Nik-/-)and conditional-knockout mice following administration of azoxymethane and dextran sulfate sodium (AOM/DSS). RESULTS: Human transcriptomic data revealed dysregulated noncanonical NF-κB signaling. In vitro studies evaluating Nik-/- crypts and organoids derived from mature, nondividing CECs and colonic stem cells (CSCs) exhibited increased accumulation and stunted growth, respectively. Transcriptomic analysis of Nik-/- cells revealed gene expression signatures associated with altered differentiation-regeneration. When assessed in vivo, Nik-/- mice exhibited more severe colitis with DSS administration and an altered microbiome characterized by increased colitogenic microbiota. In the inflammation-induced tumorigenesis model, we observed both increased tumor burdens and inflammation in mice where NIK is knocked out in CECs (NikΔCEC). Interestingly, this was not recapitulated when NIK was conditionally knocked out in myeloid cells (NikΔMYE). Surprisingly, conditional knockout of the canonical pathway in myeloid cells (RelAΔMYE) revealed decreased tumor burden and inflammation and no significant changes when conditionally knocked out in CECs (RelAΔCEC). CONCLUSIONS: Dysregulated noncanonical NF-κB signaling is associated with the development of colorectal cancer in a tissue dependent manner and defines a critical role for NIK in regulating gastrointestinal inflammation and regeneration associated with colorectal cancer.

Gram-scale synthesis of a covalent nanocage that preserves the redox properties of encapsulated fullerenes.

Discrete nanocages provide a way to solubilize, separate, and tune the properties of fullerenes, but these 3D receptors cannot usually be synthesized easily from inexpensive starting materials, limiting their utility. Herein, we describe the first fullerene-binding nanocage (Cage4+) that can be made efficiently on a gram scale. Cage4+ was prepared in up to 57% yield by the formation of pyridinium linkages between complemantary porphyrin components that are themselves readily accessible. Cage4+ binds C60 and C70 with large association constants (>108 M-1), thereby solubilizing these fullerenes in polar solvents. Fullerene association and redox-properties were subsequently investigated across multiple charge states of the host-guest complexes. Remarkably, neutral and singly reduced fullerenes bind with similar strengths, leaving their 0/1- redox couples minimally perturbed and fully reversible, whereas other hosts substantially alter the redox properties of fullerenes. Thus, C60@Cage4+ and C70@Cage4+ may be useful as solubilized fullerene derivatives that preserve the inherent electron-accepting and electron-transfer capabilities of the fullerenes. Fulleride dianions were also found to bind strongly in Cage4+, while further reduction is centered on the host, leading to lowered association of the fulleride guest in the case of C60 2-.

Uptake, Trapping, and Release of Organometallic Cations by Redox-Active Cationic Hosts.

The host-guest chemistry of metal-organic nanocages is typically driven by thermodynamically favorable interactions with their guests such that uptake and release of guests can be controlled by switching this affinity on or off. Herein, we achieve this effect by reducing porphyrin-walled cationic nanoprisms 1a12+ and 1b12+ to zwitterionic states that rapidly uptake organometallic cations Cp*2Co+ and Cp2Co+, respectively. Cp*2Co+ binds strongly (Ka = 1.3 × 103 M-1) in the neutral state 1a0 of host 1a12+, which has its three porphyrin walls doubly reduced and its six (bipy)Pt2+ linkers singly reduced (bipy = 2,2'-bipyridine). The less-reduced states of the host 1a3+ and 1a9+ also bind Cp*2Co+, though with lower affinities. The smaller Cp2Co+ cation binds strongly (Ka = 1.7 × 103 M-1) in the 3e- reduced state 1b9+ of the (tmeda)Pt2+-linked host 1b12+ (tmeda = N,N,N',N'-tetramethylethylenediamine). Upon reoxidation of the hosts with Ag+, the guests become trapped to provide unprecedented metastable cation-in-cation complexes Cp*2Co+@1a12+ and Cp2Co+@1b12+ that persist for >1 month. Thus, dramatic kinetic effects reveal a way to confine the guests in thermodynamically unfavorable environments. Experimental and DFT studies indicate that PF6- anions kinetically stabilize Cp*2Co+@1a12+ through electrostatic interactions and by influencing conformational changes of the host that open and close its apertures. However, when Cp*2Co+@1a12+ was prepared using ferrocenium (Fc+) instead of Ag+ to reoxidize the host, dissociation was accelerated >200× even though neither Fc+ nor Fc have any observable affinity for 1a12+. This finding shows that metastable host-guest complexes can respond to subtler stimuli than those required to induce guest release from thermodynamically favorable complexes.

In pursuit of a better broiler: carcass traits and muscle myopathies in conventional and slower-growing strains of broiler chickens.

Selection for accelerated growth rate and high breast yield in broiler chickens have been associated with an increase in myopathies, including wooden breast (WB) and white striping (WS). To investigate effects of growth rate on carcass traits and incidence of myopathies, 14 strains were evaluated, encompassing 2 conventional (CONV; strains B and C: ADG0-48 > 60 g/d) and 12 slower-growing (SL) strains. The latter were categorized based on growth rate: FAST (strains F, G, I and M; ADG0-62=53-55 g/d), MOD (strains E, H, O and S; ADG0-62=50-51 g/d), and SLOW (strains D, J, K and N; ADG0-62<50 g/d). In a randomized incomplete block design, 7,216 mixed-sex birds were equally allocated into 164 pens (44 birds/pen; 30 kg/m2), with each strain represented in 8 to 12 pens over 2 to 3 production cycles. From each pen, 4 males and 4 females were processed at 2 Target Weights (TWs) based on their expected time to reach 2.1 kg BW (TW 1: 34 d for CONV; 48 d for SL strains) and 3.2 kg BW (TW 2: 48 d for CONV; 62 d for SL strains). Weights and yields for the carcass, breast, drumsticks, thighs, and wings were obtained; breast fillets were assessed to determine the presence and severity of WB and WS. At both TWs, breast yield was higher as growth rate increased (P < 0.001), with CONV having greater breast yield than other categories. Strain F had the greatest breast yield at both TWs (P < 0.001) within the FAST category. At TW 2, CONV had the greatest incidence of WB and WS (P < 0.001). However, within FAST, strain F had the greatest incidence of myopathies (P < 0.001) at both TWs, exhibiting values as high or as greater than CONV birds. The incidence of WB and WS in strains with differing growth rates but high breast meat yield suggests that the latter may play a major role in the occurrence of these myopathies.

The influence of polarity items on inferential judgments.

Polarity items are linguistic expressions such as any, at all, some, which are acceptable in some linguistic environments but not others. Crucially, whether a polarity item is acceptable in a given environment is argued to depend on the inferences (in the reasoning sense) that this environment allows. We show that the inferential judgments reported for a given environment are modified in the presence of polarity items. Hence, there is a two-way influence between linguistic and reasoning abilities: the linguistic acceptability of polarity items is dependent on reasoning facts and, conversely, reasoning judgments can be altered by the mere addition of seemingly innocuous polarity items.

In pursuit of a better broiler: growth, efficiency, and mortality of 16 strains of broiler chickens.

To meet the growing consumer demand for chicken meat, the poultry industry has selected broiler chickens for increasing efficiency and breast yield. While this high productivity means affordable and consistent product, it has come at a cost to broiler welfare. There has been increasing advocacy and consumer pressure on primary breeders, producers, processors, and retailers to improve the welfare of the billions of chickens processed annually. Several small-scale studies have reported better welfare outcomes for slower-growing strains compared to fast-growing, conventional strains. However, these studies often housed birds with range access or used strains with vastly different growth rates. Additionally, there may be traits other than growth, such as body conformation, that influence welfare. As the global poultry industries consider the implications of using slower growing strains, there was a need for a comprehensive, multidisciplinary examination of broiler chickens with a wide range of genotypes differing in growth rate and other phenotypic traits. To meet this need, our team designed a study to benchmark data on conventional and slower-growing strains of broiler chickens reared in standardized laboratory conditions. Over a 2-year period, we studied 7,528 broilers from 16 different genetic strains. In this paper, we compare the growth, efficiency, and mortality of broilers to one of two target weights (TW): 2.1 kg (TW1) and 3.2 kg (TW2). We categorized strains by their growth rate to TW2 as conventional (CONV), fastest-slow strains (FAST), moderate-slow strains (MOD), and slowest-slow strains (SLOW). When incubated, hatched, housed, managed, and fed the same, the categories of strains differed in body weights, growth rates, feed intake, and feed efficiency. At 48 d of age, strains in the CONV category were 835 to 1,264 g heavier than strains in the other categories. By TW2, differences in body weights and feed intake resulted in a 22 to 43-point difference in feed conversion ratios. Categories of strains did not differ in their overall mortality rates.

The Influence of Redox-Active Linkers on the Stability and Physical Properties of a Highly Electroactive Porphyrin Nanoprism.

Redox-active metal-organic nanocages are of interest for many applications, but the development of cages with extensive redox activity is often hindered by their limited stability and solubility across multiple charge states. This report reveals that these properties can be tuned for cages with redox-active walls by incorporating additional redox activity into the linkers. In particular, new +12 charged triangular nanoprisms 1a,b were formed from three electroactive tetrakis(3-pyridyl)porphyrin walls linked by six [(TMEDA)Pt]2+ (for 1a) or [(2,2'-bipy)Pt]2+ (for 1b) vertices, the latter of which are also electroactive. Thus, 1b exhibits extensive redox activity, consisting of two porphyrin-centered (x3) and two 2,2'-bipy-centered (x6) reductions that provide reversible access to +12, +9, +3, 0, and -6 charge states, whereas 1a undergoes only two, porphyrin-centered (x3) reversible reductions. Comparisons of 1a and 1b (and monomeric control compounds) by cyclic voltammetry and UV-vis-NIR spectroelectrochemistry show that the redox-activity of the linkers in 1b lowers the second reduction potential of the porphyrins by 100 mV and improves the stability and solubility of this structure under highly reducing conditions (e.g., -2.25 V vs Fc+/0 in MeCN). These findings reveal new principles for controlling the properties of highly electroactive molecular nanostructures. Anion exchange rates (≫103 s-1) were also probed, showing that the narrow apertures (≤3 Å van der Waals width) of 1a,b do not impede the loss/gain of PF6- anions during redox processes.

Modeling the structure and infrared spectra of omega-3 fatty acid esters.

Omega-3 dietary supplements provide a rich source of the active moieties eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which exist in the form of triacylglycerols or ethyl esters. Infrared (IR) spectroscopy provides a rapid and quantitative tool to assess the quality of these products as specific normal modes, in particular the ester carbonyl stretch modes, exhibit characteristic spectral features for the two ester forms of omega-3 fatty acids. To uncover the origin of the observed spectra, in this work, we perform molecular dynamics simulations of EPA and DHA ethyl esters and triacylglycerols to characterize their conformation, packing, and dynamics in the liquid phase and use a mixed quantum/classical approach to calculate their IR absorption spectra in the ester carbonyl stretch region. We show that the ester liquids exhibit slow dynamics in spectral diffusion and translational and rotational motion, consistent with the diffusion ordered NMR spectroscopy measurements. We further demonstrate that the predicted IR spectra are in good agreement with experiments and reveal how a competition between intermolecular and intramolecular interactions gives rise to distinct absorption peaks for the fatty acid esters.

Ultrasound-accelerated thrombolysis (USAT) versus standard catheter-directed thrombolysis (CDT) for treatment of pulmonary embolism: A retrospective analysis.

Ultrasound-accelerated thrombolysis (USAT) is advocated in pulmonary embolism (PE) based on the hypothesis that adjunctive ultrasound provides superior clinical efficacy compared to standard catheter-directed thrombolysis (CDT). This retrospective study was designed to compare outcomes between the two modalities. We analyzed patients with computed tomography-diagnosed PE at our institution treated with either USAT or standard CDT. Efficacy parameters assessed included invasive pulmonary artery systolic pressure (PASP; pre- and 24 hours post-treatment), non-invasive right-to-left ventricle (RV/LV) ratio (pre- and post-treatment), and general clinical outcomes (length-of-stay, significant bleeding, and mortality). We analyzed 98 cases (62 USAT and 36 CDT), in whom massive PE was diagnosed in 7%, intermediate/high risk in 81%, and intermediate/low risk in 12%. Overall, 92% had bilateral clot and 40% saddle embolus. At 24 hours, PASP decreased similarly in both groups (CDT Δ14.7 mmHg, USAT Δ10.8 mmHg; p = 0.14). Post-treatment, CDT showed similar improvement in the RV/LV ratio (CDT Δ0.58 vs USAT Δ0.45; p = 0.07), despite the baseline ratio being greater in the CDT group, indicating more severe RV strain (1.56 ± 0.36 vs 1.40 ± 0.29; p = 0.01). Intensive care unit and hospital length-of-stays were similar in both groups. A trend toward lesser significant bleeding rates in the CDT group (8.3% vs 12.9%, p = 0.74) as well as improved survival-to-discharge (97.2% vs 91.9%, p = 0.66) was observed. Compared to USAT, standard CDT achieves similar beneficial effects on hemodynamics, RV/LV ratios, and clinical outcomes. These observations suggest that salutary clinical results may be achieved without the need for very expensive devices.

Low-dose systemic thrombolytic therapy for treatment of submassive pulmonary embolism: Clinical efficacy but attendant hemorrhagic risks.

OBJECTIVES: The purpose of the present study is to evaluate the safety and efficacy of "low-dose" systemic thrombolytic therapy (TT) for treatment of patients with intermediate-high risk submassive pulmonary embolism (PE). BACKGROUND: TT is increasingly utilized in acute submassive PE. Strategies for TT include catheter-directed administration as well as traditional IV systemic therapy. Regardless of the route, most studies document the attendant significant bleeding complication rates expected from induction of a systemic lytic state. To mitigate bleeding, "low-dose" systemic TT (Alteplase 50 mg) has been advocated, based on recent studies which demonstrated clinical efficacy with elimination of any significant bleeding complications. METHODS: Over a 24-month period, our institutional PE Response Team treated 45 acute submassive PE patients with "Low Dose" IV Alteplase 50 mg. Clinical outcomes and bleeding complications were assessed. RESULTS: Overall clinical outcome was excellent, with 97.8% of patients surviving to discharge and a 30-day, all-cause mortality of 4.4%. Despite no patients having a HAS-BLED score > 2 (average score = 0.8 +/-), ISTH major and GUSTO moderate bleeding was observed in 11% (n = 5) of cases. CONCLUSIONS: The present observations document that low-dose systemic TT is associated with excellent clinical outcome for intermediate-high risk submassive PE, but with attendant risk for bleeding. These findings are consistent with the concept that induction of a therapeutic lytic state carries inextricable bleeding risk.

Modulating inflammation through the negative regulation of NF-κB signaling.

Immune system activation is essential to thwart the invasion of pathogens and respond appropriately to tissue damage. However, uncontrolled inflammation can result in extensive collateral damage underlying a diverse range of auto-inflammatory, hyper-inflammatory, and neoplastic diseases. The NF-κB signaling pathway lies at the heart of the immune system and functions as a master regulator of gene transcription. Thus, this signaling cascade is heavily targeted by mechanisms designed to attenuate overzealous inflammation and promote resolution. Mechanisms associated with the negative regulation of NF-κB signaling are currently under intense investigation and have yet to be fully elucidated. Here, we provide an overview of mechanisms that negatively regulate NF-κB signaling through either attenuation of signal transduction, inhibition of posttranscriptional signaling, or interference with posttranslational modifications of key pathway components. While the regulators discussed for each group are far from comprehensive, they exemplify common mechanistic approaches that inhibit this critical biochemical signaling cascade. Despite their diversity, a commonality among these regulators is their selection of specific targets at key inflection points in the pathway, such as TNF-receptor-associated factor family members or essential kinases. A better understanding of these negative regulatory mechanisms will be essential to gain greater insight related to the maintenance of immune system homeostasis and inflammation resolution. These processes are vital elements of disease pathology and have important implications for targeted therapeutic strategies.

Noncanonical NF-κB signaling and the essential kinase NIK modulate crucial features associated with eosinophilic esophagitis pathogenesis.

Eosinophilic esophagitis (EoE) is an allergic disease of the esophagus driven by T cell and eosinophil responses to dietary allergens, resulting in chronic mucosal inflammation. Few spontaneous animal models of esophageal eosinophilia exist, with most studies relying on artificial sensitization procedures. NF-κB-inducing kinase (NIK; MAP3K14) is a key signaling molecule of the noncanonical NF-κB (NFKB1) pathway, an alternative signaling cascade producing chemokines involved in lymphoid stroma development and leukocyte trafficking. Nik-/- mice have been shown to develop a hypereosinophilic syndrome in peripheral blood and major filtering organs; however, the gastrointestinal mucosa of these mice has not been well characterized. We show that Nik-/- mice develop significant, localized eosinophilic esophagitis that mimics human EoE, including features such as severe eosinophil accumulation, degranulation, mucosal thickening, fibrosis and basal cell hyperplasia. The remainder of the GI tract, including the caudal stomach, small intestine and colon, in mice with active EoE are unaffected, also similar to human patients. Gene expression patterns in esophageal tissue of Nik-/- mice mimics human EoE, with thymic stromal lymphopoetin (TSLP) in particular also elevated at the protein level. In gene expression data sets from human biopsy specimens, we further show that many genes associated with noncanonical NF-κB signaling are significantly dysregulated in EoE patients, most notably a paradoxical upregulation of NIK itself with concurrent upregulation of powerful protein-level destabilizers of NIK. These findings suggest that Nik-/- mice could be useful as a spontaneous model of specific features of EoE and highlight a novel role for noncanonical NF-κB signaling in human patients.

ERpS294 is a biomarker of ligand or mutational ERα activation and a breast cancer target for CDK2 inhibition.

ERα phosphorylation at hinge site S294 (pS294) was recently shown to be essential for ER-dependent gene transcription and mediated by an unknown cyclin-dependent kinase (CDK). This study was undertaken to identify the exact CDK pathway mediating pS294 formation, and to determine if this phosphorylation event occurs with, and can be targeted to treat, the ligand-independent growth of breast cancers expressing endocrine-refractory ESR1 mutations. Using a newly developed anti-pS294 monoclonal antibody, a combination of CDK specific siRNA knockdown studies and a broad panel of CDK selective inhibitors against ligand (E2)-stimulated MCF7 cells, we first identified CDK2 as the primary mediator of pS294 formation and showed that CDK2-selective inhibitors like Dinaciclib, but not CDK4/6 inhibitors like Palbociclib, can selectively prevent pS294 formation and repress ER-dependent gene expression. We then expressed the ER-activating mutations ERmut(Y537S) and ERmut(D538G) in MCF7 cells, and demonstrated their ability to induce ligand-independent and tamoxifen-resistant growth, associated with constitutive and CDK2-dependent pS294 expression. Following robust growth of E2-independent and TAM-resistant MCF7mutER(Y537S) tumors in vivo, nude mice were also treated with either Dinaciclib or Palbociclib at doses and injection schedules unable to retard tumor growth as single agents; the TAM plus Palbociclib combination arrested further tumor growth without affecting pS294 formation, while the TAM plus Dinaciclib combination produced tumor regression associated with loss of pS294 expression. These findings, and our proposed mechanistic model, provide new rationale for the clinical evaluation of CDK2 inhibitors given in combination with endocrine agents as a new treatment strategy against ESR1 mutation expressing breast cancers.

Pacemaker-induced tricuspid regurgitation is uncommon immediately post-implantation.

BACKGROUND: Prior studies report permanent pacemaker (PPM)-induced tricuspid regurgitation (TR) in up to one third of cases late post-implantation. We sought to assess the extent of immediate PPM-induced TR. METHODS: Forty patients undergoing PPM implant were prospectively enrolled. Patients with pre-existing moderate or severe TR or an RVSP >50 mmHg were excluded. Pre- and immediate post-implantation transthoracic echocardiography (TTE) analyzed TR grade according to established methods. 3D TTE was utilized to determine lead position in relation to tricuspid leaflets as well as lead mobility across the TV. RESULTS: Of 40 patients, four were excluded due to baseline moderate TR (n = 3) or RVSP >50 mmHg (n = 1). In the remaining cohort (n = 36), immediate post-implantation TTE showed no increase in TR grade in 30 patients (83%), whereas a one-grade increase from no/trace to mild occurred in six (17%) others. In no patient did immediate moderate or severe TR develop. Exclusive RV pacing was present in 47% of the patients; however, only two of the six patients with increased TR were paced. 3D TTE identified lead position in 92% of the cases-more than 50% of the cases showed RV lead distribution in the middle or post eroseptal commissure of the TV. Lead immobility was seen in only three of the six patients with increased TR. CONCLUSIONS: These findings show that significant PPM-induced TR is uncommon immediately post-implantation and, when it occurs, causes no greater than mild TR. RV pacing and lead mobility do not correlate with worsening of TR. 3D TTE is highly reliable at identifying lead position.

TIPS pentacene loaded PEO-PDLLA core-shell nanoparticles have similar cellular uptake dynamics in M1 and M2 macrophages and in corresponding in vivo microenvironments.

Nanoparticle based drug delivery platforms have the potential to transform disease treatment paradigms and therapeutic strategies, especially in the context of pulmonary medicine. Once administered, nanoparticles disperse throughout the lung and many are phagocytosed by macrophages. However, there is a paucity of knowledge regarding cellular up-take dynamics of nanoparticles due largely to macrophage heterogeneity. To address this issue, we sought to better define nanoparticle up-take using polarized M1 and M2 macrophages and novel TIPS-pentacene loaded PEO-PDLLA nanoparticles. Our data reveal that primary macrophages polarized to either M1 or M2 phenotypes have similar levels of nanoparticle phagocytosis. Similarly, M1 and M2 polarized macrophages isolated from the lungs of mice following either acute (Th1) or allergic (Th2) airway inflammation also demonstrated equivalent levels of nanoparticle up-take. Together, these studies provide critical benchmark information pertaining to cellular up-take dynamics and biodistribution of nanoparticles in the context of clinically relevant inflammatory microenvironments.

A case of reverse takotsubo cardiomyopathy caused by an eating disorder.

Takotsubo cardiomyopathy is an acquired transient cardiomyopathy that causes severe systolic dysfunction in the absence of coronary disease. Here we present a case of reverse takotsubo cardiomyopathy. A 67-year-old female was admitted for weakness and falls due to severe malnutrition. During her stay she was diagnosed with an eating disorder. She also developed chest pain during her hospitalization and was taken urgently for catheterization which showed normal coronary arteries with akinetic basal and inferior walls and hyperkinesis of the apex. Her ejection fraction decreased to 25% from 60% five days previously. She was diagnosed with reverse takotsubo. She was placed on an appropriate heart failure regimen and eventually transferred to inpatient rehabilitation. In its typical form, takotsubo causes apical ballooning due to hyperkinetic basal and inferior wall motion and apical hypokinesis. Reverse takotsubo causes a depressed basal segment with apical hyperkinesis usually brought on by severe emotional or physical stress. Our case is the first documented report of an eating disorder causing the reverse type of takotsubo cardiomyopathy. .

Enhanced Mucosal Defense and Reduced Tumor Burden in Mice with the Compromised Negative Regulator IRAK-M.

Aberrant inflammation is a hallmark of inflammatory bowel disease (IBD) and colorectal cancer. IRAK-M is a critical negative regulator of TLR signaling and overzealous inflammation. Here we utilize data from human studies and Irak-m-/- mice to elucidate the role of IRAK-M in the modulation of gastrointestinal immune system homeostasis. In human patients, IRAK-M expression is up-regulated during IBD and colorectal cancer. Further functional studies in mice revealed that Irak-m-/- animals are protected against colitis and colitis associated tumorigenesis. Mechanistically, our data revealed that the gastrointestinal immune system of Irak-m-/- mice is highly efficient at eliminating microbial translocation following epithelial barrier damage. This attenuation of pathogenesis is associated with expanded areas of gastrointestinal associated lymphoid tissue (GALT), increased neutrophil migration, and enhanced T-cell recruitment. Further evaluation of Irak-m-/- mice revealed a splice variant that robustly activates NF-κB signaling. Together, these data identify IRAK-M as a potential target for future therapeutic intervention.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.