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.

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.

Metal transport capabilities of anticancer copper chelators.

In the present study, several Cu chelators [2,2'-biquinoline, 8-hydroxiquinoline (oxine), ammonium pyrrolidinedithiocarbamate (APDTC), Dp44mT, dithizone, neocuproine] were used to study Cu uptake, depletion and localization in different cancer cell lines. To better understand the concentration dependent fluctuations in the Cu intracellular metal content and Cu-dependent in vitro antiproliferative data, the conditional stability constants of the Cu complex species of the investigated ligands were calculated. Each investigated chelator increased the intracellular Cu content on HT-29 cells causing Cu accumulation depending on the amount of the free Cu(II). Copper accumulation was 159 times higher for Dp44mT compared to the control. Investigating a number of other transition metals, intracellular accumulation of Cd was observed only for two chelators. Intracellular Zn content slightly decreased (cca. 10%) for MCF-7 cells, while a dramatic decrease was observed on MDA-MB-231 ones (cca. 50%). A similar decrease was observed for HCT-116, while Zn depletion for HT-29 corresponded to cca. 20%. The IC50 values were registered for the investigated four cell lines at increasing external Cu(II) concentration, namely, MDA-MB-231 cells had the lowest IC50 values for Dp44mT ranging between 7 and 35 nM. Thus, Zn depletion could be associated with lower IC50 values. Copper depletion was observed for all ligands being less pronounced for Dp44mT and neocuproine. Copper localization and its colocalization with Zn were determined by µ-XRF imaging. Loose correlation (0.57) was observed for the MCF-7 cells independently of the applied chelator. Similarly, a weak correlation (0.47) was observed for HT-29 cells treated with Cu(II) and oxine. Colocalization of Cu and Zn in the nucleus of HT-29 cells was observed for Dp44mT (correlation coefficient of 0.85).

Paneth-cell-disruption-induced necrotizing enterocolitis in mice requires live bacteria and occurs independently of TLR4 signaling.

Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in premature infants. Both human surgical specimens and animal models suggest a potential involvement of Paneth cells in NEC pathogenesis. Paneth cells play critical roles in epithelial homeostasis, innate immunity and host-microbial interactions. Yet, the complex interplay between Paneth cell disruption, epithelial barrier dysfunction and microbial-driven inflammation remains unclear in the immature intestine. In this study, mucosal intestinal injury consistent with human NEC was induced in postnatal day 14-16 (P14-P16) mice by disrupting Paneth cells, followed by gavage with Klebsiella pneumonia. Mucosal injury was determined by histology, serum cytokine levels and epithelial barrier dysfunction. Toll-like receptor 4 (TLR4) activation was examined using protein expression, gene expression, and TLR4-/- mice. Finally, the role of bacteria was evaluated using heat-killed bacteria, conditioned media, Bacillus cereus and cecal slurries. We found that live bacteria were required to induce injury; however, TLR4 activation was not required. NEC induced by Paneth cell disruption results in altered localization of tight junction proteins and subsequent loss of barrier function. Prior research has shown a requirement for TLR4 activation to induce NEC-like damage. However, many infants develop NEC in the absence of Gram-negative rod bacteremia, raising the possibility that alternative pathways to intestinal injury exist. In this study, we show a previously unknown mechanism for the development of intestinal injury equivalent to that seen in human NEC and that is not dependent on TLR4 pathways. These data are congruent with the new hypothesis that NEC may be the consequence of several disease processes ending in a final common inflammatory pathway.

Cadmium uptake, localization and stress-induced morphogenic response in the fern Pteris vittata.

Cadmium uptake, tissue localization and structural changes induced at cellular level are essential to understand Cd tolerance in plants. In this study we have exposed plants of Pteris vittata to different concentrations of CdCl2 (0, 30, 60, 100 µM) to evaluate the tolerance of the fern to cadmium. Cadmium content determination and its histochemical localization showed that P. vittata not only takes up, but also transports and accumulates cadmium in the aboveground tissues, delocalizing it mainly in the less bioactive tissues of the frond, the trichomes and the scales. Cadmium tolerance in P. vittata was strictly related to morphogenic response induced by the metal itself in the root system. Adaptive response regarded changes of the root apex size, the developmental pattern of root hairs, the differentiation of xylem elements and endodermal suberin lamellae. All the considered parameters suggest that, in our experimental conditions, 60 µM of Cd may represent the highest concentration that P. vittata can tolerate; indeed this Cd level even improves the absorbance features of the root and allows good transport and accumulation of the metal in the fronds. The results of this study can provide useful information for phytoremediation strategies of soils contaminated by Cd, exploiting the established ability of P. vittata to transport, delocalize in the aboveground biomass and accumulate polluting metals.

Sonic hedgehog inhibition induces mouse embryonic stem cells to differentiate toward definitive endoderm.

In the experimental group (shh inhibited group), there were significant decreases in the expression of Oct4, Nanog, Shh, GATA4, Brachyury and Goosecoid, while increases were observed for TAT and Pdx1. The expression of Sox17 did not differ between two control and experimental groups. In experimental group, the amount of GSC positive cells was somehow lower but it seems that there was no difference for Sox17. Shh inhibition induces ESCs to differentiate toward definitive endoderm by committing mesendodermal lineages.

Cryo-isolation: a novel method for enzyme-free isolation of pancreatic islets involving in situ cryopreservation of islets and selective destruction of acinar tissue.

BACKGROUND: A critical component of treating type I diabetes by transplantation is the availability of sufficient high-quality islets. Currently, islets can be obtained only by reliance on an expensive, inconsistent, and toxic enzyme digestion process. As an alternative, we hypothesize that cryobiologic techniques can be used for differential freeze destruction of the pancreas to release islets that are selectively cryopreserved in situ. METHODS: Pancreases were procured from juvenile pigs with the use of approved procedures. The concept of cryo-isolation is based on differential processing of the pancreas in 5 stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water (or saline solution) to fully distend the gland; 3) freezing the entire pancreas to -160°C, and stored in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen pancreas into small fragments; and 5) thawing, filtering and washing the frozen fragments with RPMI 1640 culture medium to remove the CPA. Finally, the filtered effluent (cryo-isolate) was stained with dithizone for identification of intact islets, and samples were taken for static glucose-stimulated insullin release assessment. RESULTS: As predicted the cryo-isolated contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact embedded islets. The degree of cleavage of the cryoprotected islets from the freeze-destroyed exocrine cells, was variable. Islets were typically larger than their counterparts isolated from juvenile pigs with conventional enzyme-digestion techniques. Functionally, the islets from replicate cryo-isolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7 (n = 3). CONCLUSIONS: An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze-destruction of acinar tissue is feasible and proposed as a novel method that avoids the problems associated with conventional collagenase digestion methods.

Effect of chelating agents and stress factors on zinc content in paneth cells and prostate gland cells.

The effects of chelating agents dithizone and sodium diethyldithiocarbamate on zinc content in Paneth cells and prostate gland cells were studied on rats preliminary exposed to stress factors (exercise and immobilization). Dithizone induced irreversible triphasic and sodium diethyldithiocarbamate reversible biphasic changes in zinc metabolism in cells. Preliminary stress potentiated the damaging effect of dithizone.

Regulation of lipid peroxidation in the retina under the effect of bright light.

Changes in LPO intensity under the effect of exposure to bright light and the possibility of their correction with antioxidants were studied on rabbits with diabetic retinopathy. It was found that enhanced LPO caused by exposure to bright light in rabbits with diabetic retinopathy can be corrected with antioxidants. Phenosan potassium salt, α-tocopherol, and oxypyridine were more effective than SOD and taurine in preventing MDA accumulation. A complex of natural and synthetic antioxidants was most efficient in correcting LPO under conditions of exposure to bright light.

Maturation of paneth cells induces the refractory state of newborn mice to Shigella infection.

The intestinal tract of adult mice is naturally resistant to infection by Shigella, the causative agent of bacillary dysentery in humans. Conversely, newborn mice are highly susceptible to intragastric Shigella infection and develop inflammatory lesions of the jejunal mucosa, very similar to those observed in the colon of dysenteric patients. However, the susceptibility period is short and one week after birth, animals have acquired a status of resistance characteristic of adult animals. To identify the developmental changes controlling the switch from disease susceptibility to resistance, we performed global gene expression analysis on noninfected and infected intestinal tissues taken from 4-day- and 7-day-old animals. Transcriptomic analysis of 4-day-old mice infected with the invasive Shigella strain showed a profile reflecting a strong inflammatory response with no evidence for retro-control, suggesting that the invasive process had occurred, whereas inflammation had been controlled after infection with the noninvasive strain. Differences in gene expression profiles between noninfected 4-day- and 7-day-old mice corresponded mainly to genes encoding anti-microbial peptides and proteases, suggesting that these molecules could be candidates for host antimicrobial resistance in the course of shigellosis. Indeed, expression of genes specific of Paneth cells was higher in 7-day- than in 4-day-old mice, and histological analysis indicated that Paneth cells were present only at day 7. Finally, using Sox9(flox/flox)-vil-cre mice, we showed that depletion of Paneth cells restored the susceptibility to Shigella of 7-day-old mice, clearly indicating that Paneth cells development is crucial for the clearance of intestinal infection.

Paneth cells and antibacterial host defense in neonatal small intestine.

Paneth cells are specialized epithelia in the small bowel that secrete antimicrobial proteins. Paneth cells are vital to the innate immunity of the small bowel in adult mammals, but their role during neonatal infection of the small bowel is not well established. Dithizone selectively damages Paneth cells, and when dithizone-treated newborn rats are infected enterally with Escherichia coli, the numbers of E. coli cells in their jejunal and ileal lavage fluid are significantly increased compared to controls. The data support that Paneth cells are necessary for neonatal antibacterial defense.

A metal chelator, diphenylthiocarbazone, induces apoptosis in acute promyelocytic leukemia (APL) cells mediated by a caspase-dependent pathway without a modulation of retinoic acid signaling pathways.

A metal chelator, diphenylthiocarbazone (dithizone), has been reported to induce differentiation and apoptosis of the human myeloid leukemia cell line HL-60, however, very little is known about the mechanism of dithizone-induced apoptosis. Here, we report for the first time that dithizone can induce inhibition of cellular growth of retinoic acid (RA)-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis but not differentiation. Treatment of NB4 cells with dithizone markedly-induced apoptosis, which was associated with the loss of mitochondrial transmembrane potentials (Delta Psi(m)) and activation of caspase-3 and -9. Further investigation of the RA-resistant UF-1 APL cells showed that dithizone-induced apoptosis to a lesser extent. However, neither dithizone alone nor in combination with all-trans RA induced the expression of myeloid differentiation antigen CD11b. Concomitantly, the degradation of PML/RARalpha fusion protein was not observed after treatment with dithizone alone, and the degradation was not enhanced by the combination of dithizone and all-trans RA. We conclude that dithizone, a metal chelator, induced apoptosis without differentiation in APL cells in association with Delta Psi(m) collapse and caspase-3 and -9 activation.

Involvement of tumor necrosis factor alpha in intestinal epithelial cell proliferation following Paneth cell destruction.

BACKGROUND: An intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induces selective killing of Paneth cells which have a large amount of zinc in their cytoplasmic granules. A transient wave of intestinal epithelial cell proliferation occurs at 12 h after the injection. Paneth cells have tumor necrosis factor (TNF)-alpha protein in their cytoplasmic granules, and TNF-alpha has a proliferative effect on intestinal epithelial cells in vitro. The aim of this study is to clarify the in vivo role of TNF-alpha in intestinal epithelial cell proliferation using a dithizone-treated rat model. METHODS: Male Wistar rats received a dithizone (100 mg/kg of body weight) injection with or without TNF-alpha inhibitor, pentoxifylline (100 mg/kg), neutralizing anti-TNF-alpha antibody (2 mg/kg), or nuclear transcription factor kappaB (NF-kappaB) inhibitors: pyrrolidine dithiocarbamate (100 mg/kg) or N-acetyl-L-cystein (100 mg/kg). The activation of NF-kappaB was examined by the electrophoretic mobility shift assay, and cellular proliferation by BrdU labeling. RESULTS: Without any inhibitors, dithizone treatment evoked NF-kappaB activation in the ileal mucosa with its peak level at 2 h after the injection. TNF-alpha inhibition reduced the NF-kappaB activation, and blocked a transient wave of epithelial cell proliferation 12 h after the injection. NF-kappaB inhibitors also reduced the NF-kappaB activation and epithelial cell proliferation. CONCLUSIONS: TNF-alpha released from degenerated Paneth cells was, in part, responsible for the intestinal cell proliferation through the activation of NF-kappaB, suggesting its proliferative effect on intestinal epithelial cells.

Enhanced expression of transforming growth factor (TGF) -alpha precursor and TGF-beta1 during Paneth cell regeneration.

An intravenous injection of diphenylthiocarbazone (dithizone), a zinc chelator, induces selective killing and rapid regeneration of Paneth cells, which have a large amount of zinc in their cytoplasmic granules. We examined the expression pattern of transforming growth factor (TGF) -alpha and TGF-beta1 in this regenerative process. Messenger RNA expression of TGF-alpha and TGF-beta1 reached their peaks at 12 and 24 hr after dithizone injection, respectively. Protein expression of TGF-alpha precursor and TGF-beta1 increased to a maximum at 24 and 72 hr, respectively. Their immunoreactivities were localized in the epithelial cells in the vicinity of Paneth cells, whereas they were prominent in the upper half of the crypts in control rats. In conclusion, destruction of Paneth cells induced TGF-alpha precursor expression, followed by an increase of TGF-beta1 especially in the crypt bases. This unique expression pattern of two growth factors may be involved in rapid regeneration of Paneth cells.

Endogenous Zn(2+) is required for the induction of long-term potentiation at rat hippocampal mossy fiber-CA3 synapses.

The functional role of the abundant Zn(2+) found in some hippocampal synapses has been an enigma. We show here, using N-[6-methoxy-8-quinolyl]-P-toluenesulfonamide (TSQ) staining, that chelatable-Zn(2+) can be removed from hippocampal synaptic boutons using dietary depletion or with Zn(2+) chelators. A chronic dietary deficiency of bouton Zn(2+) resulted in the impairment of long-term potentiation (LTP) at mossy fiber-CA3 synapses. The averaged normalized fEPSP slope 30 min after tetanus was 209 +/- 28% of baseline value in control (mean +/- SEM, n = 10), and 118 +/- 12% in Zn(2+)-deficient rats (mean +/- SEM, n = 12, P < 0.01). In the deficient rats with Zn(2+) supplements, mossy fiber LTP returned to normal levels. The acute depletion of bouton Zn(2+) in the hippocampal slice with membrane-permeable Zn(2+) chelators, dithizone, or diethyldithiocarbamic acid (DEDTC) blocked the induction of mossy fiber LTP. The mean amplitudes of EPSCs after tetanus were 194 +/- 22% of baseline value in control (n = 5), compared to 108 +/- 14% in dithizone (n = 6) and 101 +/- 12% in DEDTC (n = 5). The averaged value of LTP, at the associational commisural fiber-CA3 synapses, was 193 +/- 20% in the control (n = 6), compared to 182 +/- 21% (n = 6, P > 0.1) in the presence of dithizone. The blockade of mossy fiber LTP by dithizone was reversible after washout. In addition, normal LTP could be induced by tetanus if exogenous Zn(2+) was applied immediately following dithizone. Our results indicate that the endogenous Zn(2+) is specifically required for LTP induction at the mossy fiber input into CA3 neurons.

Enhancement by dimethyl sulfoxide of 1,25-dihydroxyvitamin D3-induced differentiation in human promyelocytic HL-60 leukemia cells requires dimethyl sulfoxide-induced G0/G1 arrest.

We investigated the induction of differentiation in human promyelocytic HL-60 leukemia cells by a relatively low concentration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). The cells were markedly induced to differentiate by combined treatment with reduced concentrations of dimethyl sulfoxide (DMSO) and 1,25(OH)2D3, neither of which alone had much of an effect on differentiation. At 48 hr post-treatment, a greater proportion of DMSO- and DMSO/1,25(OH)2D3-treated, but not 1,25(OH)2D3-treated, cells were in G0/G1 than untreated cells. Our study indicates that the synergistic effect of DMSO and 1,25(OH)2D3 on the induction of differentiation in HL-60 cells requires DMSO-induced G0/G1 arrest.

Antimitogenic effect of Larrea divaricata Cav.: participation in arachidonate metabolism.

Aqueous extracts of the leaves of Larrea divaricata Cav. exert antimitogenic effects on tumor cells (BW 5147 murine immature T-lymphoma) and normal, stimulated lymphocytes. The effective concentration was four times smaller in the case of tumor cells than in the case of normal, stimulated lymphocytes. Inhibitor studies of arachidonate pathway suggest that the proliferative effect of the extract is due to the activation of lipoxygenase metabolism, while the inhibitory action could be a direct effect.

Localization of zinc in the rat submandibular gland and the effect of its deficiency on salivary secretion.

To clarify the role of zinc in the mechanism of salivary secretion, the effects of zinc deficiency on the morphologic findings and secretory function of the salivary gland were investigated with a rat model of chronic zinc deficiency, prepared by feeding a zinc-deficient diet, and a rat model of acute zinc deficiency, prepared by administration of a zinc chelator, dithizone. In rats with chronic zinc deficiency, the granule production in the granular duct cells was decreased, but the glandular epithelial cells and myoepithelial cells showed no degenerative or other destructive morphologic changes. The degranulation of the granular duct cells and acinar cells in response to acetylcholine hydrochloride seen in control rats was strongly inhibited in rats with acute and chronic zinc deficiency. The contractile response of the actin microfilament bundles in the myoepithelial cells to acetylcholine seen in the control rats was also absent in the zinc-deficient rats. Further, electron microscopy of the submandibular gland stained by Timm's method disclosed prominent zinc localization at the membrane surface, granules, and vesicles of the glandular epithelial cells and in the pits of the myoepithelial cells. These findings suggest that zinc, together with many zinc-dependent enzymes, is closely involved in the production and degranulation of secretory granules in the glandular epithelial cells, and also in the contraction of the myoepithelial cells in the submandibular gland.

Apoptosis during iron chelator-induced differentiation in F9 embryonal carcinoma cells.

We have previously demonstrated that three potent iron chelators, hinokitiol, dithizone and deferoxamine, induce differentiation of F9 embryonal carcinoma cells, as do other well-known morphogens such as retinoic acid (RA) and sodium butyrate (NaB). In this study, we compared the patterns of cell proliferation, cell death and cell cycle arrest during the process of differentiation induced by these five agents. When F9 cells were cultured with the agents at their individual differentiation-inducing concentrations, cell proliferation was rapidly inhibited by treatment with the iron chelators and NaB. In contrast, RA did not influence the rate of increase of cell number at the concentration of 1 microm. The three chelators also caused a marked reduction in cell viability, and the treated cells exhibited internucleosomal DNA fragmentation, whereas cells treated with NaB showed no apoptotic characteristics. RA induced apoptosis weakly at 1 microm and strongly at higher concentrations. In addition, all the iron chelators hindered cell cycle progression, resulting in an arrest at the G1-S interface or S phase. The phenomena observed in chelator-treated cells were considerably different from those in RA- or NaB-treated cells. It is concluded that the three iron chelators cause both severe apoptotic cell death and cell cycle arrest of proliferating F9 cells via cellular iron deprivation, and that this apoptotic change may be independent of the process of differentiation.

Induction of differentiation and apoptosis by dithizone in human myeloid leukemia cell lines.

We investigated the effect of diphenylthiocarbazone (dithizone) and its structurally related compounds on the differentiation and apoptosis of two human myeloid leukemia cell lines. Dithizone caused a time- and concentration-dependent induction of differentiation in both the promyelocytic leukemia cell line HL-60 cells and the myeloblastic leukemia cell line ML-1 cells, as measured by nitroblue tetrazolium (NBT) reducing activity. Morphological changes and esterase activities confirmed that this differentiation took place. The induction of differentiation required the addition of dithizone to the culture medium for at least 12 h. The differentiation inducing activity was inhibited by the preincubation of dithizone with various metal ions such as Pb2+, Zn2+, Cu2+ and Mn2+ ions, but not with Fe3+ and Mg2+ ions. In addition, the DNA extracted from dithizone-treated HL-60 cells showed a typical ladder pattern characteristic of apoptosis in agarose gel electrophoresis. A quantitative analysis of DNA fragmentation revealed that this apoptosis was concentration- and time-dependent in both the HL-60 and ML-1 cells. Dithizone-induced apoptosis was also inhibited by preincubation with Mn2+ ions, but not with Mg2+ ions. These results indicate that dithizone induces both differentiation and apoptosis in HL-60 and ML-1 cells through a unique mechanism including metal chelation.