Paneth cells protect intestinal stem cell niche to alleviate deoxynivalenol-induced intestinal injury.

Deoxynivalenol (DON) is a common toxin in grains and feeds, and DON exposure triggers severe small intestinal injury and inflammation, which harms the health of humans and livestock. DON treatment leads to a decrease in Paneth cells, whereas the role of Paneth cells in DON-induced intestinal injury is poorly understood. We utilized dithizone (40 mg/kg) to keep murine Paneth cell number at a low level. The results showed that dithizone-mediated long-term disruption of Paneth cells aggravated intestinal injury, intestinal stem cell (ISC) loss, and microbiota disorder in DON (2 mg/kg)-treated mice. Unexpectedly, the number of goblet cells and proliferative cells was boosted in mice treated with dithizone and DON. After dithizone and DON treatments, the Firmicutes/Bacteroidetes (F/B) ratio was reduced, and the increased abundance of Dubosiella and the decreased abundance of Lactobacillus were observed in mice. The functional recovery of Paneth cells by lysozyme (200 U/day) supplementation improved intestinal injury and ISC loss in mice after DON challenge. In addition, lysozyme also promoted the growth and ISC activity of intestinal organoids. Taken together, these results demonstrate the protective role of Paneth cells in DON-induced intestinal injury. Our study raises a novel target, Paneth cell, for the treatment of DON exposure.

Ultra-trace determination of cadmium in water and food samples by a thin-film microextraction using a supported liquid membrane combined with smartphone-based colorimetric detection.

A mixture of n-octanol and dithizone was introduced as an effective and novel extraction agent in a thin-film microextraction technique for the pre-concentration of cadmium ions. The extraction agent was immobilized on small pieces of porous polypropylene flat membrane as a supported liquid membrane. The analyte extraction was performed by immersing the modified film in the sample solution, and via a complex formation between the immobilized dithizone on the film and cadmium ions. After the thin-film microextraction process, the colored cadmium-dithizone complex was directly measured by a smartphone colorimetric analysis. Under optimized conditions, the linear dynamic range, the limit of detection, and the limit of quantification were 0.5-300.0, 0.1, and 0.4 µg L-1, respectively. The developed technique was successfully employed to quantify cadmium ions in water and food samples. The high relative recovery values (95.0-103.0%) along with relative standard deviations of less than 2.5% were obtained for the spiked samples.

Transplantation of insulin-producing cells derived from human MSCs to treat diabetes in a non-human primate model.

BACKGROUND: Islet cell transplantation is an emerging therapy in the treatment of diabetes mellitus. Differentiation of islet cells from mesenchymal stem cells (MSCs) is a potential solution to the challenge of insufficient donor sources. This study investigated whether human umbilical cord-derived MSCs could effectively differentiate into insulin-producing cells (IPCs) and evaluated the therapeutic efficacy of IPCs in treating diabetes. METHODS: IPCs were induced from MSCs by a two-step protocol. IPC expression products were evaluated by western blot and real-time PCR. IPC insulin secretion was evaluated by ELISA. The viability of IPCs was measured by FDA/PI and dithizone staining. The non-human primate tree shrew was used as a diabetes model. After a single STZ induction into a diabetes model, a single intraportal transplantation of IPCs, MSCs, or normal saline was performed (n = 6 per group). Blood glucose was monitored for 3 weeks, then the animals were euthanized and the distribution of IPCs in the liver was examined pathologically. RESULTS: After about 3 weeks of in vitro induction, IPCs formed microspheres of 100-200 µm, with >95% viable cells that were dithizone stain positive. IPCs expressed islet-related genes and proteins and secreted high levels of insulin whether stimulated by low or high levels of glucose. After transplantation of IPCs into diabetic tree shrews, blood glucose levels decreased rapidly to near normal and were significantly lower than the MSC or saline groups for 3 weeks thereafter. CONCLUSION: We present the novel discovery that IPCs derived from human umbilical cord MSCs exert a therapeutic effect in a non-human primate model of diabetes. This study provides a preliminary experimental basis for the use of autologous MSC-derived IPCs in the treatment of human diabetes.

Enhancement of olfaction by femtomolar concentrations of zinc ions.

Zinc is recognized as an important element for olfaction. Zinc nanoparticles enhance olfaction in response to odors; however, the mechanisms underlying this action remain unknown. Herein, the effect of zinc on olfactory receptors was deduced using electro-olfactogram (EOG) responses recorded from the isolated olfactory mucosae of bullfrogs (Rana catesbeiana) following the administration or chelation of zinc ions. Menthone and n-amyl acetate were used as odorants, whereas forskolin (an adenylate cyclase activator) and cholera toxin (a Gαolf activator) were used as intracellular signal transduction activators. The EOG responses provoked by the odorants and cholera toxin were suppressed by dithizone-mediated zinc ion chelation, and the EOG responses were recovered by administering non-chelated zinc. However, the EOG response to forskolin was not suppressed by dithizone. In contrast, the addition of femtomolar concentrations of zinc ions enhanced the EOG responses. The above-mentioned effects on EOG responses were examined by changing the concentration of zinc ions but not zinc nanoparticles. The results of this study suggest that Gαolf alone or both olfactory receptors and Gαolf likely require zinc ions for their activation.

In vitro differentiation of human pancreatic duct-derived PANC-1 cells into ß-cell phenotype using Tinospora cordifolia.

Type 1 diabetes mellitus is an autoimmune disorder leading to loss of beta cells. There is a dire need to inhibit apoptosis and induce regeneration of new beta cells. There are plants in the Indian medicine system having the potential for rejuvenation. In the present study, we have attempted to evaluate the capacity of aqueous extract of Tinospora cordifolia to regenerate beta cells from PANC-1 ductal cells. After differentiation, the characterization of ß-cell phenotype was carried out using dithizone and Gomori's staining and further confirmed by mRNA expression study of insulin, Pdx-1, and carbonic anhydrase-9. Insulin production was estimated with ELISA. Aqueous extract of Tinospora cordifolia at 15 µg/ml concentration can effectively induce differentiation of PANC-1 cells into beta cells. The morphological observations showed brownish-colored dithizone and purple-colored Gomori's staining. The ß-cells demonstrated significant mRNA expression of insulin and Pdx-1 and downregulation of carbonic anhydrase-9. The functionality of beta cells was demonstrated by 1.5-fold increase in insulin secretion in response to high glucose. Tinospora cordifolia has potential to differentiate PANC-1 ductal cells into functional beta cells and can be a lead towards non-invasive treatment of type 1 diabetes mellitus.

Environmental monitoring of trace metal pollutants using cellulosic-paper incorporating color change of azo-chromophore.

An essential requirement for colorimetric paper-sensor is to allow the target analytes (heavy metal ions) to access the chromophore while maintaining strong chromophore immobilization on the porous substrate surface. This work evaluates the selection of sensitive chromophores (dithizone, 1-(2-pyridylazo) 2-naphthol and 4-(2-pyridylazo)-resorcinol) and their immobilization strategies on paper sensors. Dithizone (DTz) are capable of producing a significant color transition at unadjusted pH, observed by UV-Vis absorption spectroscopy and visible recognition. After immobilizing DTz on a paper substrate (cellulose acetate/chitosan substrate), the DTz-paper sensor showed a distinctive color change from blue-green to peach-pink upon reaction with Pb2+ ions, and the color intensity was proportional to the metal concentration. Quantitative analysis using RGB (R:Red; G:Green; B:Blue) plots showed that increasing DTz concentration on the CA/CS paper sensor increases the difference in total color intensity (∆IT) and the difference in red code intensity (∆IR). This is due to the formation of more DTz-Pb2+ complexes on the CA/CS paper substrate. The CA/CS paper strips immobilized with 100 ppm DTz showed practical potential for rapid detection of heavy metal ions. The DTz-CA/CS paper sensor showed significant color change when detecting spiked heavy metals ions (0.1 ppm Pb2+, 2.0 ppm Zn2+, and 0.2 ppm Cu2+) in river water samples that prepared at the maximum permissible limit for industrial effluent in Malaysia.

Dithizone-functionalized C18 online solid-phase extraction-HPLC-ICP-MS for speciation of ultra-trace organic and inorganic mercury in cereals and environmental samples.

A simple and efficient dithizone-functionalized solid-phase extraction (SPE) procedure, online coupled with high-performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry, was developed for the first time for enrichment and determination of ultra-trace mercury (Hg) species (inorganic divalent Hg (Hg(II)), methylmercury (CH3Hg(II)) and ethylmercury (C2H5Hg(II)) in cereals and environmental samples. In the proposed method, functionalization of the commercial C18 column with dithizone, enrichment, and elution of the above Hg species can be completed online with the developed SPE device. A simple solution of 2-mercaptoethanol (1% (V/V)) could be used as an eluent for both the SPE and HPLC separation of Hg species, significantly simplifying the method and instrumentation. The online SPE method was optimized by varying dithizone dose, 2-mercaptoethanol concentration, and sample volume. In addition, the effect of pH, coexisting interfering ions, and salt effect on the enrichment was also discussed. Under the optimized conditions, the detection limits of Hg species for 5 mL water sample were 0.15 ng/L for Hg(II), 0.07 ng/L for CH3Hg(II), and 0.04 ng/L for C2H5Hg(II) with recoveries in the range of 85%-100%. The developed dithizone-functionalized C18 SPE column can be reused after a single functionalization, which significantly simplifies the enrichment step. Moreover, the stability of Hg species enriched on the SPE column demonstrated its suitability for field sampling of Hg species for later laboratory analysis. This environment-friendly method offers a robust tool to detect ultra-trace Hg species in cereals and environmental samples.

Direct Reprogramming of Mice Skin Fibroblasts into Insulin-Producing Cells In Vitro.

Transdifferentiation means mature cell conversion into other mature cells. Ethical issues, epigenetic failure, or teratoma development are found in cellular reprogramming strategies. Thus, new methods are needed. This study aimed to develop a new novel formula of chemical molecules and growth factors that differentiate skin fibroblasts into insulin-producing cells (IPCs). Newborn mice fibroblasts differentiated using four induction methods into IPCs to search for the best method. Fibroblasts, stem cells, and pancreatic markers were identified using an immunocytochemistry (ICC) assay. Insulin was measured using ELISA and dithizone (DTZ) assays. The skin fibroblasts were induced successfully into IPCs. The best method to obtain IPCs was indicated by measuring insulin concentration in differentiated cell supernatant from all induced cells by the four methods. The protein expression of the pancreatic markers of induced cells increased with time, as indicated by the ICC assay. OCT3/4 increased on day 9, after which the expression tended to decrease. DTZ-positive clusters were observed on day 16. Secreted insulin of differentiated cells was injected in streptozotocin-induced diabetic mice, which decreased blood glucose levels after injection. This study indicated an efficient new chemical method for transdifferentiating skin fibroblasts into functional IPCs, which is a promising method for diabetes mellitus therapy.

The Fragility of Cryopreserved Insulin-producing Cells Differentiated from Adipose-tissue-derived Stem Cells.

The aim of our study is to determine whether insulin-producing cells (IPCs) differentiated from adipose-tissue-derived stem cells (ADSCs) can be cryopreserved. Human ADSCs were differentiated into IPCs using our two-step protocol encompassing a three-dimensional culture and xenoantigen-free method. Thereafter, IPCs were frozen using three different methods. First, IPCs were immediately frozen at -80°C (-80°C group). Second, IPCs were initially placed into a Bicell freezing container before freezing at -80°C (BICELL group). Third, a vitrification method for oocytes and embryos was used (CRYOTOP group). Cell counting kit-8 (CCK-8) assay showed that cell viability was decreased in all groups after cryopreservation (P < 0.01). Corroboratively, the amount of adenosine triphosphate was markedly decreased after cryopreservation in all groups (P < 0.01). Immunofluorescence staining showed a reduced positive staining area for insulin in all cryopreservation groups. Furthermore, 4',6-diamidino-2-phenylindole and merged immunofluorescence images showed that cryopreserved cells appeared to be randomly reduced in the -80°C group and CRYOTOP group, while only the central region was visibly reduced in the BICELL group. Using immunohistochemical staining, IPCs after cryopreservation were shown to be positive for cleaved caspase-3 antibody in all groups. Finally, insulin secretion following glucose stimulation was significantly reduced in IPCs from all groups after cryopreservation (P < 0.01). In conclusion, IPCs may be too fragile for cryopreservation with accomplished methods and further investigations for a suitable preservation method are required.

Differentiation of rat pancreatic duct stem cells into insulin-secreting islet-like cell clusters through BMP7 inducement.

To cure the epidemic of diabetes, in vitro produced ß-like cells are lauded for cell therapy of diabetic patients. In this regard, we investigated the effects of different concentrations of bone morphogenetic protein 7 (BMP7) on the differentiation of rat pancreatic ductal epithelial-like stem cells (PDESCs) into ß-like cells. For inducement of the differentiation, PDESCs were cultured in the basal media (H-DMEM + 10 % FBS + 1% penicillin-streptomycin) supplemented with 5 ng/mL, 10 ng/mL, 15 ng/mL, and 20 ng/mL of BMP7 for 28 days. To corroborate the identity of induced cells, they were examined through cell morphology, dithizone (DTZ) staining, immunofluorescence staining, real-time polymerase chain reaction (qPCR), and glucose-stimulated insulin secretion assay (GSIS). The enrichment of induced cells was high among 5 ng/mL, 10 ng/mL, 15 ng/mL, and 20 ng/mL of BMP7 supplemented groups as compared to the control group. Further, the induced cells were positive, while, the control group cells were negative to DTZ staining and the differentiated cells also have shown the upregulated expression of ß cell-specific marker genes (Ins1 and Pdx1). The GSIS assay of inducement groups for insulin and C-peptide secretion has shown significantly higher values as compared to the control group (P < 0.01). Hence, the addition of BMP7 to basal medium has effectually induced differentiation of adult rat PDESCs into islet like-cell clusters containing insulin-secreting ß-like cells.

A sensitive fluorescent probe based on dithizone-capped ZnS quantum dots for quercetin determination in biological samples.

A simple turn on/off fluorescence approach based on dithizone-capped ZnS quantum dots (ZnS@DZ QDs) with the help of lead ions as a fluorescent probe for the quantitative determination of quercetin is reported. The interaction of lead ions with dithizone led to the formation of a rigid structure on the surface of ZnS@DZ QDs and turned on the fluorescence intensity of the QDs. After addition of quercetin to this probe and interaction with lead ions, the fluorescence emission turned off. Concerning the quenching fluorescence intensity of ZnS@DZ QDs/Pb2+ QDs probe induced by the target, under the optimum conditions, the probe enabled detection of quercetin in the concentration range from 0.54 µM to 21.7 µM with a correlation coefficient of 0.993 and detection limit of 0.25 µM. The present probe was applied successfully to the determine quercetin as a nutritional biomarker in human serum and 24-h urine samples.

Cysteine Prevents the Development of Experimental Diabetes Induced by Zinc-Binding Substances.

In experimental rabbits, cysteine injected intravenously in a dose of 1000 mg/kg temporarily bound zinc in ß cells and prevented the formation of chelate zinc complexes in response to subsequent injection of diabetogenic zinc-binding substances that induce cell destruction. Injection of cysteine to animals was associated with a sharply negative reaction to zinc in ß cells, which attests to blockade of zinc ions. Injection of cysteine few minutes after dithizone and formation of zinc-dithizone complex was followed by displacement of dithizone from the complex and prevented the development of diabetes in most animals. The most plausible mechanism of preventive effect of cysteine is the formation of 2:1 zinc-cysteine complex in ß cells with possible fixation of Zn atom between sulfur atoms from SH groups of two cysteine molecules.

Automatic multicommuted flow-batch setup for photometric determination of mercury in drinking water at ppb level.

Herein, a multicommuted flow-batch setup and a photometric procedure for the determination of mercury at the ppb level in aqueous samples are described. The setup was designed to implement a versatile solvent extraction and pre-concentration strategy by combining flow-batch and multicommuted flow analysis approaches. The photometric method was based on Hg(II) reaction with dithizone in a chloroform medium, which was also used as the extracting organic solvent. The flow analysis system was composed of a homemade syringe pump module, a set of solenoid valves, two Aquarius mini-pumps, and a flow-batch chamber. The homemade photometer was comprised of a light emitting diode (LED), photodiode, and homemade flow cell (50 mm length). The flow system and photometer were controlled using an Arduino Due board, running custom-written software. After optimizing the operational conditions, the effectiveness of the developed system was evaluated for the determination of the mercury concentration in drinking water. For accuracy assessment, samples were analyzed using a spiking methodology and an independent method, yielding a recovery ranging from 92% to 108%. Other important characteristics of the proposed method were found as follows: linear response range, 0.5-10.0 µg L-1 (r = 0.9984); limit of detection 0.38 µg L-1 Hg(II); consumption of dithizone and chloroform, 1.85 µg L-1 and 0.8 mL per analysis, respectively; coefficient of variation, 2% (n = 10); sampling throughput, 20 determinations per h.

A change in the zinc ion concentration reflects the maturation of insulin-producing cells generated from adipose-derived mesenchymal stem cells.

The generation of insulin-producing cells (IPCs) from pluripotent stem cells could be a breakthrough treatment for type 1 diabetes. However, development of new techniques is needed to exclude immature cells for clinical application. Dithizone staining is used to evaluate IPCs by detecting zinc. We hypothesised that zinc ion (Zn2+) dynamics reflect the IPC maturation status. Human adipose-derived stem cells were differentiated into IPCs by our two-step protocol using two-dimensional (2D) or 3D culture. The stimulation indexes of 2D -and 3D-cultured IPCs on day 21 were 1.21 and 3.64 (P < 0.05), respectively. The 3D-cultured IPCs were stained with dithizone during culture, and its intensity calculated by ImageJ reached the peak on day 17 (P < 0.05). Blood glucose levels of streptozotocin-induced diabetic nude mice were normalised (4/4,100%) after transplantation of 96 3D-cultured IPCs. Zn2+ concentration changes in the medium of 3D cultures had a negative value in the early period and a large positive value in the latter period. This study suggests that Zn2+ dynamics based on our observations and staining of zinc transporters have critical roles in the differentiation of IPCs, and that their measurement might be useful to evaluate IPC maturation as a non-destructive method.

Paneth Cell Ablation Aggravates Pancreatic and Intestinal Injuries in a Rat Model of Acute Necrotizing Pancreatitis after Normal and High-Fat Diet.

We previously reported that acute necrotizing pancreatitis (ANP) after normal or high-fat diet is associated with a decreased number of Paneth cells in ileal crypts. Here, we ablated Paneth cells in a rat model of ANP after normal and high-fat diet to investigate the effects on disease symptoms. Adult male Sprague-Dawley rats received standard rat chow or a high-fat diet for 2 weeks, after which they were treated with dithizone to deplete Paneth cells. Six hours later, ANP was established by retrograde injection of sodium taurocholate into the biliopancreatic duct. Rats were sacrificed at 6, 12, and 24 h for assessment. We found dithizone aggravated ANP-associated pathological injuries to the pancreas and ileum in rats on high-fat or standard diets. Lysozyme expression in ileal crypts was decreased, while serum inflammatory cytokines (TNFα, IL-1ß, and IL-17A) and intestinal permeability (serum DAO activity and D-lactate) were increased. Expression of tight junction proteins (claudin-1, zo-1, and occludin) was decreased. Using high-throughput 16S rRNA sequencing, we found dithizone reduced microbiota diversity and altered microbiota composition in rats on high-fat or standard diets. Dithizone decreased fecal short-chain fatty acids (SCFAs) in rats on high-fat or standard diets. Changes in intestinal microbiota correlated significantly with SCFAs, lysozyme, DAO activity, D-lactate, inflammatory cytokines, and pathological injury to the pancreas and ileum in rats on high-fat or standard diets. In conclusion, ablation of Paneth cells exacerbates pancreatic and intestinal injuries in ANP after normal and high-fat diet. These symptoms may be related to changes in the intestinal microbiota.

Pancreatic human islets and insulin-producing cells derived from embryonic stem cells are rapidly identified by a newly developed Dithizone.

We developed an optimized Dipheylthiocarbazone or Dithizone (DTZ) with improved physical and chemical properties to characterize human islets and insulin-producing cells differentiated from embryonic stem cells. Application of the newly formulated iDTZ (i stands for islet) over a range of temperatures, time intervals and cell and tissue types found it to be robust for identifying these cells. Through high transition zinc binding, the iDTZ compound concentrated in insulin-producing cells and proved effective at delineating zinc levels in vitro.

Surfactant-Assisted Emulsification and Surfactant-Based Dispersive Liquid-Liquid Microextraction Method for Determination of Cu(II) in Food and Water Samples by Flame Atomic Absorption Spectrometry.

Background: Copper (Cu) is an essential metal for humans at certain concentrations. However, it can be toxic at higher concentrations. Therefore, determination of Cu content of foodstuff is important. Objective: The aim of the study was to develop a simple, economical, and environmentally friendly surfactant-mediated extraction method for the determination of Cu using surfactants and flame atomic absorption spectrometry (FAAS). Methods: A nonionic surfactant-assisted emulsification and surfactant-based dispersive liquid-liquid microextraction method was developed for the separation, preconcentration, and determination of Cu by FAAS. Triton X-15 nonionic surfactant, which is insoluble in water, was used as an extractive agent. Triton X-114 (TX-114) nonionic water-soluble surfactant was used as a disperser solvent. Dithizone was used as a complexing agent for complexation of Cu(II) at pH 4. Results: The detection and quantitation limits of the method were determined as 1.61 and 3.82 µg/L, respectively. The preconcentration factor was obtained as 50. Relative SD based on 10 replicates was obtained as 3.7%. Accuracy of the developed method was proved using certified standard reference materials. Cu(II) content of edible mushroom samples was determined between 12 and 19 µg/g. Recoveries were obtained between 96 and 101%. Conclusions: The developed surfactant-assisted emulsification and surfactant-based dispersive liquid-liquid microextraction method has represented the wide linear ranges, low detection limit, and high preconcentration factor for Cu ions. Highlights: TX-114 surfactant was used as both sticking agent and disperser solvent. The method does not require expert personnel and high operational costs. The method is environmentally friendly because mainly surfactants and low-toxicity organic solvents are used in the recommended procedure.

Dithizone-induced Paneth cell disruption significantly decreases intestinal perfusion in the murine small intestine.

PURPOSE: Necrotizing enterocolitis is associated with decreased intestinal perfusion and ischemia. Paneth cells, specialized epithelial cells, have been shown to regulate the intestinal vasculature and disruption of these cells has been associated with NEC. We hypothesized that Paneth cell disruption in immature mice intestine would decrease the perfusion of the intestinal microvasculature. METHODS: Paneth cells were disrupted in P14-16 mice using chemical (dithizone) and transgenic (diphtheria toxin) methodology. Six hours after Paneth cell disruption, Dylight 488 was injected directly into the left ventricle and allowed to perfuse for 5 minutes prior to intestinal harvesting. Tissue samples were evaluated with confocal fluorescence microscopy to quantify intestinal perfusion and samples were quantified by real time RT-PCR for gene expression. RESULTS: Dithizone treatment significantly decreased intestinal perfusion compared to controls (p < 0.01). However, diphtheria toxin treatment demonstrated no significant difference in perfusion (p > 0.21). Intestines from all treatment groups had similar PECAM staining, but intestines treated with dithizone had significantly decreased nNOS and iNOS gene expression compared to controls (p < 0.007). CONCLUSIONS: Paneth cell disruption significantly decreases the perfusion of the small intestinal microvasculature in a dithizone-specific manner. Dithizone has no effect on the amount of microvasculature, but does impact genes critical to nitric oxide signaling likely contributing to mesenteric vasoconstriction.

Zinc-Chelating Small Molecules Preferentially Accumulate and Function within Pancreatic ß Cells.

Diabetes is a hyperglycemic condition characterized by pancreatic ß-cell dysfunction and depletion. Whereas methods for monitoring ß-cell function in vivo exist, methods to deliver therapeutics to ß cells are lacking. We leveraged the rare ability of ß cells to concentrate zinc to preferentially trap zinc-binding molecules within ß cells, resulting in ß-cell-targeted compound delivery. We determined that zinc-rich ß cells and islets preferentially accumulated TSQ (6-methoxy-8-p-toluenesulfonamido-quinoline) in a zinc-dependent manner compared with exocrine pancreas. Next, we asked whether appending a zinc-chelating moiety onto a ß-cell replication-inducing compound was sufficient to confer preferential ß-cell accumulation and activity. Indeed, the hybrid compound preferentially accumulated within rodent and human islets in a zinc-dependent manner and increased the selectivity of replication-promoting activity toward ß cells. These data resolve the fundamental question of whether intracellular accumulation of zinc-chelating compounds is influenced by zinc content. Furthermore, application of this principle yielded a proof-of-concept method for ß-cell-targeted drug delivery and bioactivity.

Loss of murine Paneth cell function alters the immature intestinal microbiome and mimics changes seen in neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) remains the leading cause of gastrointestinal morbidity and mortality in premature infants. Human and animal studies suggest a role for Paneth cells in NEC pathogenesis. Paneth cells play critical roles in host-microbial interactions and epithelial homeostasis. The ramifications of eliminating Paneth cell function on the immature host-microbial axis remains incomplete. Paneth cell function was depleted in the immature murine intestine using chemical and genetic models, which resulted in intestinal injury consistent with NEC. Paneth cell depletion was confirmed using histology, electron microscopy, flow cytometry, and real time RT-PCR. Cecal samples were analyzed at various time points to determine the effects of Paneth cell depletion with and without Klebsiella gavage on the microbiome. Deficient Paneth cell function induced significant compositional changes in the cecal microbiome with a significant increase in Enterobacteriacae species. Further, the bloom of Enterobacteriaceae species that occurs is phenotypically similar to what is seen in human NEC. This further strengthens our understanding of the importance of Paneth cells to intestinal homeostasis in the immature intestine.