International Harmonization of Nomenclature and Diagnostic Criteria (INHAND): Progress to Date and Future Plans.

The International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice proposal (INHAND) has been operational since 2005. A Global Editorial Steering Committee manages the overall objectives of the project, and the development of harmonized terminology for each organ system is the responsibility of the Organ Working Groups, drawing upon experts from North America, Europe, and Japan. Great progress has been made with 9 systems published to date--respiratory, hepatobiliary, urinary, central/peripheral nervous systems, male reproductive and mammary, zymbals, clitoral, and preputial glands in Toxicologic Pathology and the integument and soft tissue and female reproductive in the Journal of Toxicologic Pathology as supplements and on a Web site--www.goReni.org. INHAND nomenclature guides offer diagnostic criteria and guidelines for recording lesions observed in rodent toxicity and carcinogenicity studies. The guides provide representative photomicrographs of morphologic changes, information regarding pathogenesis, and key references. The purpose of this brief communication is to provide an update on the progress of INHAND.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition.

OBJECTIVE: Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN: The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl's reagent. RESULTS: Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS: Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.

Inhibiting endocannabinoid biosynthesis: a novel approach to the treatment of constipation.

BACKGROUND AND PURPOSE: Endocannabinoids are a family of lipid mediators involved in the regulation of gastrointestinal (GI) motility. The expression, localization and function of their biosynthetic enzymes in the GI tract are not well understood. Here, we examined the expression, localization and function of the enzyme diacylglycerol lipase-α (DAGLα), which is involved in biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). EXPERIMENTAL APPROACH: Cannabinoid CB1 receptor-deficient, wild-type control and C3H/HeJ mice, a genetically constipated strain, were used. The distribution of DAGLα in the enteric nervous system was examined by immunohistochemistry. Effects of the DAGL inhibitors, orlistat and OMDM-188 on pharmacologically induced GI hypomotility were assessed by measuring intestinal contractility in vitro and whole gut transit or faecal output in vivo. Endocannabinoid levels were measured by mass spectrometry. KEY RESULTS: DAGLα was expressed throughout the GI tract. In the intestine, unlike DAGLß, DAGLα immunoreactivity was prominently expressed in the enteric nervous system. In the myenteric plexus, it was colocalized with the vesicular acetylcholine transporter in cholinergic nerves. In normal mice, inhibiting DAGL reversed both pharmacologically reduced intestinal contractility and pharmacologically prolonged whole gut transit. Moreover, inhibiting DAGL normalized faecal output in constipated C3H/HeJ mice. In colons incubated with scopolamine, 2-AG was elevated while inhibiting DAGL normalized 2-AG levels. CONCLUSIONS AND IMPLICATIONS: DAGLα was expressed in the enteric nervous system of mice and its inhibition reversed slowed GI motility, intestinal contractility and constipation through 2-AG and CB1 receptor-mediated mechanisms. Our data suggest that DAGLα inhibitors may be promising candidates for the treatment of constipation.

AM841, a covalent cannabinoid ligand, powerfully slows gastrointestinal motility in normal and stressed mice in a peripherally restricted manner.

BACKGROUND AND PURPOSE: Cannabinoid (CB) ligands have been demonstrated to have utility as novel therapeutic agents for the treatment of pain, metabolic conditions and gastrointestinal (GI) disorders. However, many of these ligands are centrally active, which limits their usefulness. Here, we examine a unique novel covalent CB receptor ligand, AM841, to assess its potential for use in physiological and pathophysiological in vivo studies. EXPERIMENTAL APPROACH: The covalent nature of AM841 was determined in vitro using electrophysiological and receptor internalization studies on isolated cultured hippocampal neurons. Mouse models were used for behavioural analysis of analgesia, hypothermia and hypolocomotion. The motility of the small and large intestine was assessed in vivo under normal conditions and after acute stress. The brain penetration of AM841 was also determined. KEY RESULTS: AM841 behaved as an irreversible CB1 receptor agonist in vitro. AM841 potently reduced GI motility through an action on CB1 receptors in the small and large intestine under physiological conditions. AM841 was even more potent under conditions of acute stress and was shown to normalize accelerated GI motility under these conditions. This compound behaved as a peripherally restricted ligand, showing very little brain penetration and no characteristic centrally mediated CB1 receptor-mediated effects (analgesia, hypothermia or hypolocomotion). CONCLUSIONS AND IMPLICATIONS: AM841, a novel peripherally restricted covalent CB1 receptor ligand that was shown to be remarkably potent, represents a new class of potential therapeutic agents for the treatment of functional GI disorders.

Protection of rat gastric mucosa by sucralfate. Effects of luminal stasis and of inhibition of prostaglandins synthesis.

Studies using a gastric chamber model demonstrated that sucralfate protected the rat gastric mucosa against hemorrhagic erosions induced by 40 percent ethanol and by acidified 80 mM sodium taurocholate. Protection required continuous contact of sucralfate with the gastric mucosa but it occurred without the production, by sucralfate alone, of significant damage to the luminal epithelium. Ultrastructural examination indicated that sucralfate stimulated mucus secretion by surface epithelial cells. Furthermore, sucralfate was "cytoprotective" in that, in addition to its anti-ulcer effects, it significantly reduced the damaging effects of luminal ethanol on the surface epithelium. Luminal stasis also significantly reduced the extent of hemorrhagic erosions produced by both ethanol and sodium taurocholate, but the most effective reduction in erosions occurred when sucralfate and luminal stasis were combined. Pretreatment with indomethacin abolished the protection provided by luminal stasis, but this protection was restored by sucralfate. Thus, these studies suggest that protection of the gastric mucosa by sucralfate results in part from effects on the unstirred layer. Sucralfate or its products also interact with the epithelial cells and stimulate mucus release and synthesis or release of inflammatory mediators.

Biological consequences of thrombin receptor deficiency in mice.

The thrombin receptor (ThrR) is a membrane-bound, G-protein-coupled receptor for the serine protease thrombin. This receptor is expressed in a wide variety of cells and tissues, and elicits a range of physiological responses associated with tissue injury, inflammation, and wound repair. To achieve a better understanding of the physiological role of the ThrR, we have employed homologous recombination to create mice with a disrupted ThrR gene. Following heterozygous (+/-) intercrosses, a total of 351 surviving offspring were genotyped. Only 7% of these offspring were identified as homozygous (-/-) for the disrupted allele, indicating a profound effect on embryonic development. Paradoxically, adult ThrR-/- mice appeared to be normal by anatomical and histological analysis, including their platelet number and function. Similarly, ThrR deficiency had no detectable effect in adult ThrR-/- mice on basal heart rate, arterial blood pressure, vasomotor responses to angiotensin II and acetycholine, and coagulation parameters, even though the ThrR is expressed in many cardiovascular tissue types. In addition, the loss of ThrR function in the peripheral vasculature of adult ThrR-/- mice was confirmed by the absence of various standard hemodynamic effects of the ThrR-activating peptides SFLLRN-NH2 and TFLLRNPNDK-NH2. Our results indicate that ThrR deficiency has a strong impact on fetal development; however. ThrR-/- mice that proceed to full development display surprisingly little change in phenotype compared to the wild-type.

Immune complex decomplementation of canine sera for use in a complement-fixation test for diagnosis of visceral leishmaniasis.

Canine sera frequently become anti-complementary when heat-inactivated at 56 degrees C for 30 min, and generally cannot be used in standard complement-fixation (CF) assays. Therefore, a procedure was developed for decomplementing canine sera by absorption with particulate immune complexes consisting of sheep erythrocyte stroma optimally sensitized with anti-sheep erythrocyte antibody (hemolysin). Canine sera incubated for 20 min at 30 degrees C with sensitized stroma consistently showed less than 10% residual complement and were not anti-complementary. This decomplementation procedure was applied in a complement-fixation (CF) test for detection of serum antibodies during canine visceral leishmaniasis. Two groups of German shepherd dogs were injected intravenously with Leishmania donovani or L. donovani chagasi amastigotes, and the course of infections was followed for 12 weeks. Using freeze-thaw sonicate preparations of L. donovani parasites as antigen, reciprocal CF antibody titers above 24 were detectable in sera 7 weeks after infection and gradually increased to a maximum titer of 775 at 12 weeks. Sera from control dogs had mean titers of 24. This improved methodology enhances the potential of the CF test in the serodiagnosis of canine leishmaniasis.

The expression and role of Fas ligand in intestinal inflammation.

Fas ligand (FasL) is involved in the pathogenesis of inflammatory diseases and immune privilege. We examined the expression of FasL in the enteric nervous system (ENS) in murine colitis and guinea-pig ileitis. We studied FasL immunoreactivity, functional integrity of the ENS, severity of colitis, and distribution of neutrophils in wild type and B6/gld mice that lack functional FasL. In ileitis, the distribution of FasL, CD4+ and CD8+ T cells was examined. FasL expression was increased in the ENS of wild type mice with colitis, but decreased labelling of nerve fibres was noted in B6/gld mice. Neutrophils were more abundant and widely distributed in B6/gld mice. Colitis was more severe and persistent in B6/gld mice 7 days after induction. Functional parameters of intestinal secretion and motility in B6/gld mice were the same as controls. In ileitis, FasL expression was increased in the guinea-pig ENS and returned to control levels following the resolution of inflammation. While T cells were not present in the ENS of controls, they were observed during inflammation, but were excluded from ganglia. The number of enteric neurons was unchanged over the course of inflammation. The expression of FasL is altered in intestinal inflammation and contributes to its resolution in experimental colitis.

Reduction of the severity of experimental gastric and duodenal ulceration by interleukin-1 beta.

Interleukin-1 beta (IL-1 beta) has been reported to stimulate prostaglandin synthesis by the rat stomach in vitro and to inhibit gastric acid secretion in vivo. We have therefore tested the hypothesis that IL-1 beta might have protective actions in experimental models of gastroduodenal ulceration. IL-1 beta, given i.p., dose and time dependently reduced the severity of ethanol-induced gastric damage. A pretreatment time of 90 min was found to produce the greatest reduction of damage, while doses of 0.1 micrograms/kg or greater were found to produce significant effects. The protective actions of IL-1 beta were abolished by prior boiling or by pretreatment of the animals with indomethacin, and were not shared by the nonapeptide fragment 163-171. IL-1 beta also reduced the severity of gastric damage induced by indomethacin and the duodenal ulceration induced by cysteamine. The results indicate that IL-1 beta has protective actions in three separate experimental models of gastroduodenal ulceration. The mechanism of action of IL-1 beta is not entirely clear, but contributions of endogenous prostaglandin synthesis and inhibition of gastric acid secretion cannot be excluded.

Comparison of the effects of endothelin-1 and endothelin-3 on the rat stomach.

The effects of systemic administration of endothelin-1 and endothelin-3 on the susceptibility of the stomach to injury were compared in the anesthetized rat, as were their effects on gastric vascular tone, systemic blood pressure and hematocrit. When infused at concentrations in the 10(-7)-10(-6) M range, endothelin-1 was a far more potent hypertensive agent than endothelin-3. Endothelin-1 caused significant hemoconcentration, while endothelin-3 did not Endothelin-1 was approximately 5- to 10-times more potent as a vasoconstrictor in the stomach and a similar difference in potencies was observed when the ability of these peptides to increase the susceptibility of the stomach to ethanol-induced damage was compared. The two peptides were equipotent in producing gastric mucosal hemorrhage in the absence of any exogenous irritant. These results demonstrate that like endothelin-1, endothelin-3 has ulcerogenic and vasoconstriction actions in the stomach. While there are very large differences in the potencies of the two peptides in terms of producing systemic hypertension and hemoconcentration, the differences in the potency of the gastric ulcerogenic and vasoconstrictor effects are much less marked.

Pharmacological investigation of the role of leukotrienes in the pathogenesis of experimental NSAID gastropathy.

The role of leukotrienes in the pathogenesis of acute gastric ulceration induced by nonsteroidal antiinflammatory drugs was investigated using a rat model. One part of the study involved oral pretreatment with a leukotriene synthesis inhibitor 1 h prior to administration of indomethacin (20 mg/kg per os). Three hours after indomethacin, the extent of macroscopically visible gastric damage was determined, and gastric LTB4 synthesis was determined. The compounds tested were PF-5901, A-64077, nordihydroguaiaretic acid, and L-698,037. Each compound produced dose-related inhibition of gastric LTB4 synthesis and a parallel reduction in the severity of indomethacin-induced damage. The antioxidant properties of these compounds was assessed using an in vitro assay. There was no correlation between the antioxidant properties of the compounds and their ability to reduce the severity of indomethacin-induced gastric damage. In the second part of the study, the effects of intravenous, administration of LTD4 and LTB4 receptor antagonists on indomethacin-induced gastric epithelial damage (measured by permeability to [51Cr]EDTA) were assessed. The two LTD4 receptor antagonists (MK-571 and ICI-204,219) significantly reduced the permeability changes induced by indomethacin, while the two LTB4 antagonists (SC-41930 and LY-255,283) were without significant effect. Despite the reduction of gastric epithelial injury, blockade of LTD4 receptors did not markedly affect the extent of macroscopically visible injury. These data are consistent with the hypothesis that leukotrienes contribute to the epithelial injury and macroscopically visible damage induced by NSAIDs. However, it remains unclear to what extent leukotrienes are involved in the initiation of the injury, as opposed to its amplification.

Ear tag induced Staphylococcus infection in mice.

Mice used in a 2-year oral toxicity study developed a progressive, moist dermatitis. The initial lesions were seen around the ears in which metal identification tags had been placed and usually progressed to include the skin of the neck and shoulder. Clinically, the mice were pruritic, lost weight, had rough coats, and became moribund. The predominant finding at necropsy was pale brown kidneys with irregular granular surfaces. Histologically, there was inflammation and focal-to-diffuse necrosis in the visceral organs and affected skin. The predominant organism isolated from the skin, kidneys and heart blood was Staphylococcus aureus. This bacterium is a common inhabitant of the skin of conventionally housed mice and its isolation from the kidneys and blood suggested that the portal of entry was the wound caused by the insertion of the metal ear tag.

Endogenous cellular prion protein regulates contractility of the mouse ileum.

BACKGROUND: Cellular prion protein (PrP(C) ) is expressed in the enteric nervous system (ENS), however, its physiological role has not been identified. Studies suggest that PrP(C) can function as a metal-binding protein, as absence of the protein has been linked to altered copper metabolism and atypical synaptic activity. Because copper is known to modulate smooth muscle relaxation, we tested the hypothesis that PrP(C) deficiency would alter intestinal contractility. METHODS: We examined electrically evoked ileal contractility in Prnp(-/-) or wild type littermate mice and the effects of copper or copper chelation. PrP(C) expression was studied in whole mount ileal preparations of mice and guinea pigs by immunohistochemistry. KEY RESULTS: Relative to wild type mice, ileal tissues of Prnp(-/-) mice exhibited reduced electrical field stimulation (EFS)-evoked contractility. Furthermore, EFS-induced relaxation, as a percentage of that induced by a nitric oxide donor, was enhanced. Addition of a copper donor to the organ bath increased, whereas the addition of a copper chelator inhibited, nitric oxide donor-induced ileal relaxation in Prnp(-/-) mice. PrP(C) was expressed on nerve fibers or terminals, and some cell bodies in the myenteric and submucosal plexuses of wild type mice. PrP(C) colocalized with a neuron-specific ectonucleotidase, nucleoside triphosphate diphosphohydrolase 3 (NTPDase3), but to only a limited extent with GFAP, a marker of enteric glia. Guinea pigs expressed PrP(C) in nerve fibers or terminals and enteric glia in the myenteric and submucosal plexuses. CONCLUSIONS & INFERENCES: Our findings suggest that PrP(C) , which is abundant in the ENS, has a role in the regulation of ileal contractility.

Inhibiting fatty acid amide hydrolase normalizes endotoxin-induced enhanced gastrointestinal motility in mice.

BACKGROUND AND PURPOSE: Gastrointestinal (GI) motility is regulated in part by fatty acid ethanolamides (FAEs), including the endocannabinoid (EC) anandamide (AEA). The actions of FAEs are terminated by fatty acid amide hydrolase (FAAH). We investigated the actions of the novel FAAH inhibitor AM3506 on normal and enhanced GI motility. EXPERIMENTAL APPROACH: We examined the effect of AM3506 on electrically-evoked contractility in vitro and GI transit and colonic faecal output in vivo, in normal and FAAH-deficient mice treated with saline or LPS (100 µg·kg(-1), i.p.), in the presence and absence of cannabinoid (CB) receptor antagonists. mRNA expression was measured by quantitative real time-PCR, EC levels by liquid chromatography-MS and FAAH activity by the conversion of [(3)H]-AEA to [(3)H]-ethanolamine in intestinal extracts. FAAH expression was examined by immunohistochemistry. KEY RESULTS: FAAH was dominantly expressed in the enteric nervous system; its mRNA levels were higher in the ileum than the colon. LPS enhanced ileal contractility in the absence of overt inflammation. AM3506 reversed the enhanced electrically-evoked contractions of the ileum through CB(1) and CB(2) receptors. LPS increased the rate of upper GI transit and faecal output. AM3506 normalized the enhanced GI transit through CB(1) and CB(2) receptors and faecal output through CB(1) receptors. LPS did not increase GI transit in FAAH-deficient mice. CONCLUSIONS AND IMPLICATIONS: Inhibiting FAAH normalizes various parameters of GI dysmotility in intestinal pathophysiology. Inhibition of FAAH represents a new approach to the treatment of disordered intestinal motility.

Differential effects of CB(1) neutral antagonists and inverse agonists on gastrointestinal motility in mice.

BACKGROUND: Cannabinoid type 1 (CB(1)) receptors are involved in the regulation of gastrointestinal (GI) motility and secretion. Our aim was to characterize the roles of the CB(1) receptor on GI motility and secretion in vitro and in vivo by using different classes of CB(1) receptor antagonists. METHODS: Immunohistochemistry was used to examine the localization of CB(1) receptor in the mouse ileum and colon. Organ bath experiments on mouse ileum and in vivo motility testing comprising upper GI transit, colonic expulsion, and whole gut transit were performed to characterize the effects of the inverse agonist/antagonist AM251 and the neutral antagonist AM4113. As a marker of secretory function we measured short circuit current in vitro using Ussing chambers and stool fluid content in vivo in mouse colon. We also assessed colonic epithelial permeability in vitro using FITC-labeled inulin. KEY RESULTS: In vivo, the inverse agonist AM251 increased upper GI transit and whole gut transit, but it had no effect on colonic expulsion. By contrast, the neutral antagonist AM4113 increased upper GI transit, but unexpectedly reduced both colonic expulsion and whole gut transit at high, but not lower doses. CONCLUSIONS & INFERENCES: Cannabinoid type 1 receptors regulate small intestinal and colonic motility, but not GI secretion under physiological conditions. Cannabinoid type 1 inverse agonists and CB(1) neutral antagonists have different effects on intestinal motility. The ability of the neutral antagonist not to affect whole gut transit may be important for the future development of CB(1) receptor antagonists as therapeutic agents.

The identification of peroxisome proliferator-activated receptor alpha-independent effects of oleoylethanolamide on intestinal transit in mice.

Oleoylethanolamide (OEA) is an endogenous lipid produced in the intestine that mediates satiety by activation of peroxisome proliferator-activated receptor alpha (PPARalpha). OEA inhibits gastric emptying and intestinal motility, but the mechanism of action remains to be determined. We investigated whether OEA inhibits intestinal motility by activation of PPARalpha. PPARalpha immunoreactivity was examined in whole mount preparations of mouse gastrointestinal (GI) tract. The effect of OEA on motility was assessed in wildtype, PPARalpha, cannabinoid CB(1) receptor and CB(2) receptor gene-deficient mice and in a model of accelerated GI transit. In addition, the effect of OEA on motility was assessed in mice injected with the PPARalpha antagonist GW6471, transient receptor potential vanilloid 1 antagonist SB366791 or the glucagon-like peptide 1 antagonist exendin-3(9-39) amide. PPARalpha immunoreactivity was present in neurons in the myenteric and submucosal plexuses throughout the GI tract. OEA inhibited upper GI transit in a dose-dependent manner, but was devoid of an effect on whole gut transit or colonic propulsion. OEA-induced inhibition of motility was still present in PPARalpha, CB(1) and CB(2) receptor gene-deficient mice and in the presence of GW6471, SB366791 and exendin-3(9-39) amide, suggesting neither PPARalpha nor the cannabinoids and other likely receptors are involved in mediating the effects of OEA. OEA blocked stress-induced accelerated upper GI transit at a dose that had no effect on physiological transit. We show that PPARalpha is found in the enteric nervous system, but our results suggest that PPARalpha is not involved in the suppression of motility by OEA.

Leukotriene B4 potentiates colonic ulceration in the rat.

The ability of various leukotrienes, platelet-activating factor and N-formyl-methyl-leucyl-phenylalanine to augment colonic damage induced by 30% ethanol was investigated in the rat. Each of the mediators was tested at a dose of 2 nmol, administered intracolonically in the ethanol vehicle. When colonic damage was assessed 72 hr later, only leukotriene B4 significantly augmented damage compared to the controls. The incidence of ulcers increased from 35% in the control group to 90% in the group receiving leukotriene B4. Leukotriene B4 administration also resulted in significant increases in colonic myeloperoxidase activity and colonic leukotriene B4 synthesis. To assess the possible contribution of infiltrating neutrophils to the increase in colonic leukotriene B4 synthesis that accompanies colonic inflammation, colitis was induced in normal and neutropenic rats by intracolonic administration of trinitrobenzene sulfonic acid. Neutropenia was achieved by treatment with an antineutrophil serum. In the neutropenic animals killed 4 hr after induction of colitis significant changes in leukotriene B4 synthesis were not observed, whereas a fourfold increase was observed in the controls. From these studies we conclude the following: (1) leukotriene B4, at a dose of 2 nmol, can significantly potentiate the colonic ulceration induced by 30% ethanol; (2) this action of leukotriene B4 is not shared by the same dose of the other inflammatory mediators tested; and (3) infiltrating neutrophils are the major source of colonic leukotriene B4 synthesis in a rat model of colitis.

Eicosanoid interactions in the canine proximal colon.

Effects of eicosanoids on the canine proximal colonic mucosa were examined. Both arachidonic acid (AA) and prostaglandin E2 (PGE2) increased short-circuit currents. Tetrodotoxin did not affect these responses, suggesting that functioning nerves are not required. Indomethacin abolished responses to AA, indicating that the cyclooxygenase pathway is the primary metabolic pathway. Indomethacin significantly potentiated responses to PGE2, suggesting that in the presence of cyclooxygenase inhibition either 1) a normally inhibitory cyclooxygenase product is not present or 2) a potentiating lipoxygenase product is being produced in greater amounts. PGE2 is produced in significant quantities, whereas leukotriene B4 (LTB4) is produced in smaller amounts. Cyclooxygenase inhibitors significantly decreased PGE2 production but had no effect on LTB4. This suggests that an inhibitory PG may be opposing the response to PGE2. Therefore, we tested the effects of several cyclooxygenase products on PGE2 responsiveness. PGD2 alone significantly reduced responses to PGE2. In the canine proximal colon the response to AA is apparently the algebraic sum of the opposing responses of PGE2 and PGD2.