Mutually Repulsive EphA7-EfnA5 Organize Region-to-Region Corticopontine Projection by Inhibiting Collateral Extension.

Coordination of skilled movements and motor planning relies on the formation of regionally restricted brain circuits that connect cortex with subcortical areas during embryonic development. Layer 5 neurons that are distributed across most cortical areas innervate the pontine nuclei (basilar pons) by protrusion and extension of collateral branches interstitially along their corticospinal extending axons. Pons-derived chemotropic cues are known to attract extending axons, but molecules that regulate collateral extension to create regionally segregated targeting patterns have not been identified. Here, we discovered that EphA7 and EfnA5 are expressed in the cortex and the basilar pons in a region-specific and mutually exclusive manner, and that their repulsive activities are essential for segregating collateral extensions from corticospinal axonal tracts in mice. Specifically, EphA7 and EfnA5 forward and reverse inhibitory signals direct collateral extension such that EphA7-positive frontal and occipital cortical areas extend their axon collaterals into the EfnA5-negative rostral part of the basilar pons, whereas EfnA5-positive parietal cortical areas extend their collaterals into the EphA7-negative caudal part of the basilar pons. Together, our results provide a molecular basis that explains how the corticopontine projection connects multimodal cortical outputs to their subcortical targets.SIGNIFICANCE STATEMENT Our findings put forward a model in which region-to-region connections between cortex and subcortical areas are shaped by mutually exclusive molecules to ensure the fidelity of regionally restricted circuitry. This model is distinct from earlier work showing that neuronal circuits within individual cortical modalities form in a topographical manner controlled by a gradient of axon guidance molecules. The principle that a shared molecular program of mutually repulsive signaling instructs regional organization-both within each brain region and between connected brain regions-may well be applicable to other contexts in which information is sorted by converging and diverging neuronal circuits.

DBZ regulates cortical cell positioning and neurite development by sustaining the anterograde transport of Lis1 and DISC1 through control of Ndel1 dual-phosphorylation.

Cell positioning and neuronal network formation are crucial for proper brain function. Disrupted-in-Schizophrenia 1 (DISC1) is anterogradely transported to the neurite tips, together with Lis1, and functions in neurite extension via suppression of GSK3ß activity. Then, transported Lis1 is retrogradely transported and functions in cell migration. Here, we show that DISC1-binding zinc finger protein (DBZ), together with DISC1, regulates mouse cortical cell positioning and neurite development in vivo. DBZ hindered Ndel1 phosphorylation at threonine 219 and serine 251. DBZ depletion or expression of a double-phosphorylated mimetic form of Ndel1 impaired the transport of Lis1 and DISC1 to the neurite tips and hampered microtubule elongation. Moreover, application of DISC1 or a GSK3ß inhibitor rescued the impairments caused by DBZ insufficiency or double-phosphorylated Ndel1 expression. We concluded that DBZ controls cell positioning and neurite development by interfering with Ndel1 from disproportionate phosphorylation, which is critical for appropriate anterograde transport of the DISC1-complex.

WAVE2-Abi2 complex controls growth cone activity and regulates the multipolar-bipolar transition as well as the initiation of glia-guided migration.

Glia-guided migration (glia-guided locomotion) during radial migration is a characteristic yet unique mode of migration. In this process, the directionality of migration is predetermined by glial processes and not by growth cones. Prior to the initiation of glia-guided migration, migrating neurons transform from multipolar to bipolar, but the molecular mechanisms underlying this multipolar-bipolar transition and the commencement of glia-guided migration are not fully understood. Here, we demonstrate that the multipolar-bipolar transition is not solely a cell autonomous event; instead, the interaction of growth cones with glial processes plays an essential role. Time-lapse imaging with lattice assays reveals the importance of vigorously active growth cones in searching for appropriate glial scaffolds, completing the transition, and initiating glia-guided migration. These growth cone activities are regulated by Abl kinase and Cdk5 via WAVE2-Abi2 through the phosphorylation of tyrosine 150 and serine 137 of WAVE2. Neurons that do not display such growth cone activities are mispositioned in a more superficial location in the neocortex, suggesting the significance of growth cones for the final location of the neurons. This process occurs in spite of the "inside-out" principle in which later-born neurons are situated more superficially.

Measurement of Mitochondrial Respiration in Cryopreserved Human Peripheral Blood Mononuclear Cells (PBMCs).

Inflammatory diseases caused by infectious agents such as the SARS-CoV-2 virus can lead to impaired reductive-oxidative (REDOX) balance and disrupted mitochondrial function. Peripheral blood mononuclear cells (PBMCs) provide a useful model for studying the effects of inflammatory diseases on mitochondrial function but can be limited by the need to store these cells by cryopreservation prior to assay. Here, we describe a method for improving and determining PBMC viability with normalization of values to number of living cells. The approach can be applied not only to PBMC samples derived from patients with diseases marked by an altered inflammatory response such as viral infections.

Phldb2 is essential for regulating hippocampal dendritic spine morphology through drebrin in an adult-type isoform-specific manner.

Morphologically dynamic dendritic spines are the major sites of neuronal plasticity in the brain; however, the molecular mechanisms underlying their morphological dynamics have not been fully elucidated. Phldb2 is a protein that contains two predicted coiled-coil domains and the pleckstrin homology domain, whose binding is highly sensitive to PIP3. We have previously demonstrated that Phldb2 regulates synaptic plasticity, glutamate receptor trafficking, and PSD-95 turnover. Drebrin is one of the most abundant neuron-specific F-actin-binding proteins that are pivotal for synaptic morphology and plasticity. We observed that Phldb2 bound to drebrin A (adult-type drebrin), but not to drebrin E (embryonic-type drebrin). In the absence of Phldb2, the subcellular localization of drebrin A in the hippocampal spines and its distribution in the hippocampus were altered. Immature spines, such as the filopodium type, increased relatively in the CA1 regions of the hippocampus, whereas mushroom spines, a typical mature type, decreased in Phldb2-/- mice. Phldb2 suppressed the formation of an abnormal filopodium structure induced by drebrin A overexpression. Taken together, these findings demonstrate that Phldb2 is pivotal for dendritic spine morphology and possibly for synaptic plasticity in mature animals by regulating drebrin A localization.

LL5beta directs the translocation of filamin A and SHIP2 to sites of phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P3) accumulation, and PtdIns(3,4,5)P3 localization is mutually modified by co-recruited SHIP2.

Phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P(3)) accumulates at the leading edge of migrating cells and works, at least partially, as both a compass to indicate directionality and a hub for subsequent intracellular events. However, how PtdIns(3,4,5)P(3) regulates the migratory machinery has not been fully elucidated. Here, we demonstrate a novel mechanism for efficient lamellipodium formation that depends on PtdIns(3,4,5)P(3) and the reciprocal regulation of PtdIns(3,4,5)P(3) itself. LL5beta, whose subcellular localization is directed by membrane PtdIns(3,4,5)P(3), recruits the actin-cross-linking protein Filamin A to the plasma membrane, where PtdIns(3,4,5)P(3) accumulates, with the Filamin A-binding Src homology 2 domain-containing inositol polyphosphate 5-phosphatase 2 (SHIP2). A large and dynamic lamellipodium was formed in the presence of Filamin A and LL5beta by the application of epidermal growth factor. Conversely, depletion of either Filamin A or LL5beta or the overexpression of either an F-actin-cross-linking mutant of Filamin A or a mutant of LL5beta without its PtdIns(3,4,5)P(3)-interacting region inhibited such events in COS-7 cells. Because F-actin initially polymerizes near the plasma membrane, it is likely that membrane-recruited Filamin A efficiently cross-links newly polymerized F-actin, leading to enhanced lamellipodium formation at the site of PtdIns(3,4,5)P(3) accumulation. Moreover, we demonstrate that co-recruited SHIP2 dephosphorylates PtdIns(3,4,5)P(3) at the same location.

Autistic-Like Behavior and Impairment of Serotonin Transporter and AMPA Receptor Trafficking in N-Ethylmaleimide Sensitive Factor Gene-Deficient Mice.

Autism spectrum disorder (ASD), characterized by profound impairment in social interactions and communication skills, is the most common neurodevelopmental disorder. Many studies on the mechanisms underlying the development of ASD have focused on the serotonergic system; however, these studies have failed to completely elucidate the mechanisms. We previously identified N-ethylmaleimide-sensitive factor (NSF) as a new serotonin transporter (SERT)-binding protein and described its importance in SERT membrane trafficking and uptake in vitro. In the present study, we generated Nsf +/- mice and investigated their behavioral, neurotransmitter, and neurophysiological phenotypes in vivo. Nsf +/- mice exhibited abnormalities in sociability, communication, repetitiveness, and anxiety. Additionally, Nsf loss led to a decrease in membrane SERT expression in the raphe and accumulation of glutamate alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors at the synaptic membrane surface in the hippocampal CA1 region. We found that postsynaptic density and long-term depression were impaired in the hippocampal CA1 region of Nsf +/- mice. Taken together, these findings demonstrate that NSF plays a role in synaptic plasticity and glutamatergic and serotonergic systems, suggesting a possible mechanism by which the gene is linked to the pathophysiology of autistic behaviors.

PIP3-Phldb2 is crucial for LTP regulating synaptic NMDA and AMPA receptor density and PSD95 turnover.

The essential involvement of phosphoinositides in synaptic plasticity is well-established, but incomplete knowledge of the downstream molecular entities prevents us from understanding their signalling cascades completely. Here, we determined that Phldb2, of which pleckstrin-homology domain is highly sensitive to PIP3, functions as a phosphoinositide-signalling mediator for synaptic plasticity. BDNF application caused Phldb2 recruitment toward postsynaptic membrane in dendritic spines, whereas PI3K inhibition resulted in its reduced accumulation. Phldb2 bound to postsynaptic scaffolding molecule PSD-95 and was crucial for localization and turnover of PSD-95 in the spine. Phldb2 also bound to GluA1 and GluA2. Phldb2 was indispensable for the interaction between NMDA receptors and CaMKII, and the synaptic density of AMPA receptors. Therefore, PIP3-responsive Phldb2 is pivotal for induction and maintenance of LTP. Memory formation was impaired in our Phldb2-/- mice.

A novel sensitive immunoassay method based on the Invader technique.

A novel and sensitive immunoassay method has been developed in which the conventional sandwich immunoassay and the highly sensitive DNA detection method, the Invader method, are combined. The signal amplification function of the latter method has been successfully used to enhance the sensitivity of the sandwich immunoassay. The new assay method may be called the Immuno-Invader assay. The assay format involves three important steps: (1) a target antigen is captured and flagged with a biotin-conjugated detection antibody by the sandwich method, (2) streptavidin and a biotin-conjugated oligonucleotide are added to form a complex with the detection antibody, and (3) the oligonucleotide in the complex is detected using the Invader method. The method was applied to the assay of human tumor necrosis factor-alpha (hTNF-alpha). Detection limits obtained were 0.1 pg/ml hTNF-alpha when a luminescent europium chelate was used with a time-resolved measurement mode, and 0.8 pg/ml when fluorescein was used with a normal prompt fluorescence measurement mode. On the other hand, the detection limit of a commercially available hTNF-alpha enzyme-linked immunosorbent assay that uses horseradish peroxidase was 3.5 pg/ml. These results demonstrate the feasibility and potential of the new assay method for highly sensitive immunoassay.

Subcellular distribution of non-muscle myosin IIb is controlled by FILIP through Hsc70.

The neuronal spine is a small, actin-rich dendritic or somatic protrusion that serves as the postsynaptic compartment of the excitatory synapse. The morphology of the spine reflects the activity of the synapse and is regulated by the dynamics of the actin cytoskeleton inside, which is controlled by actin binding proteins such as non-muscle myosin. Previously, we demonstrated that the subcellular localization and function of myosin IIb are regulated by its binding partner, filamin-A interacting protein (FILIP). However, how the subcellular distribution of myosin IIb is controlled by FILIP is not yet known. The objective of this study was to identify potential binding partners of FILIP that contribute to its regulation of non-muscle myosin IIb. Pull-down assays detected a 70-kDa protein that was identified by mass spectrometry to be the chaperone protein Hsc70. The binding of Hsc70 to FILIP was controlled by the adenosine triphosphatase (ATPase) activity of Hsc70. Further, FILIP bound to Hsc70 via a domain that was not required for binding non-muscle myosin IIb. Inhibition of ATPase activity of Hsc70 impaired the effect of FILIP on the subcellular distribution of non-muscle myosin IIb. Further, in primary cultured neurons, an inhibitor of Hsc70 impeded the morphological change in spines induced by FILIP. Collectively, these results demonstrate that Hsc70 interacts with FILIP to mediate its effects on non-muscle myosin IIb and to regulate spine morphology.

Filamin A interacting protein plays a role in proper positioning of callosal projection neurons in the cortex.

The callosal connections between the two hemispheres of the neocortex are altered in certain psychiatric disorders including schizophrenia. However, how and why the callosal connection is impaired in patients suffering from psychiatric diseases remain unclear. Filamin A interacting protein (FILIP), whose alteration through mutation relates to schizophrenic pathogenesis, binds to actin-binding proteins and controls neurotransmission. Because cortical excitatory neurons, including callosal projection neurons, migrate to the cortical plate during development, with the actin-binding proteins playing crucial roles during migration, we evaluated whether FILIP is involved in the development of the callosal projection neurons by histological analysis of Filip-knockout mice. The positioning of the callosal projection neurons, especially those expressing Plxnd1, in the superficial layer of the cortex is disturbed in these mice, which suggests that FILIP is a key molecule that links callosal projections to the pathogenesis of brain disorders.

Superoxide dismutase is induced during rat pancreatic acinar cell injury.

INTRODUCTION: Free radicals and their scavengers are supposed to be involved in pancreatitis. AIMS: To investigate the expression of superoxide dismutase (SOD) in rat pancreatic acinar cell injury. METHODOLOGY AND RESULTS: As an in vivo model, male WBN/Kob rats were used. Chronic pancreatitis developed spontaneously at 12 weeks in this model and progressed thereafter, but acinar regeneration was recognized at 20 weeks. By semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), manganese SOD (MnSOD) mRNA expression peaked at 8 and 20 weeks, whereas copper/zinc SOD (CuZnSOD) mRNA expression peaked at 12 and 20 weeks. Immunohistochemistry confirmed the localization of SOD in acinar cells. Acinar cell apoptosis peaked at 12 and 20 weeks. In an in vitro study, MnSOD mRNA expression peaked at 2 hours after the addition of arginine to culture medium, whereas apoptosis was increased at 24 hours. CONCLUSION: Thus, the induction of SOD around the onset and at the late stage of chronic pancreatitis in the WBN/Kob rats implies pancreatic ischemia and acinar remodeling, respectively. From the in vitro results, MnSOD expression might reflect a defensive mechanism of acinar cells against oxidative stress or pro-apoptotic stimuli.

Induction of chemokines in rat pancreatic acinar cell injury.

INTRODUCTION: The pathogenesis of chronic pancreatitis (CP), especially of acinar cell injury, is still unclear. Interleukin (IL)-8 is a chemokine that is involved in various inflammatory diseases. AIM: To examine whether IL-8 and other chemokines are expressed in experimental acinar cell injury. METHODOLOGY: IL-8 expression was analyzed in spontaneous CP in the WBN/Kob rat and in rat pancreatic acinar AR4-2J cells treated with various stimuli using reverse transcription-polymerase chain reaction (semiquantitative) and immunohistochemistry. RESULTS: Chronic pancreatitis developed at 12 weeks in the WBN/Kob rats. IL-8, macrophage chemoattractant protein-1, and macrophage inflammatory protein-2 mRNA was expressed from 4 weeks and peaked at 12 weeks. Immunohistochemistry showed a strong expression of IL-8 in acinar cells, proliferating ductular cells, and interstitial infiltrating cells. In contrast, normal pancreatic tissues lacked IL-8 expression. Further, IL-8 mRNA and protein were detectable in AR4-2J cells treated with the various stimuli, such as menadione, tumor necrosis factor-alpha and transforming growth factor beta1. CONCLUSION: These results suggest that IL-8 is expressed in the pancreatic parenchyma and infiltrates in CP and that it plays a role in the initial pathogenesis of CP together with other chemokines and cytokines.

Expression of interleukin-8 in human obstructive pancreatitis.

CONTEXT: Obstructive pancreatitis is a specific form of pancreatitis, which is caused by the obstruction of the main pancreatic duct due to tumors or some other causes. Interleukin-8 is induced in acute pancreatitis, but its expression in obstructive pancreatitis has not been clarified. OBJECTIVE: We attempted to provide some insight into the significance of interleukin -8 in the pathogenesis of pancreatic fibrosis. PATIENTS: Fifteen cases of pancreatic cancer, 7 cases of mucinous cystadenoma, 3 cases of Vater's papilla cancer and 9 normal pancreases were included in this study. MAIN OUTCOME MEASURES: The obstructive pancreatitis portions of the above pathologies were evaluated for interleukin-8 expression by means of immunohistochemistry and in situ hybridization. RESULTS: Interleukin-8 was positive in 72% of cases of obstructive pancreatitis. The positive rate was not significantly related to the etiology of the obstruction (P=0.972). Interleukin-8 was expressed in infiltrating cells, proliferating ductular cells and acinar cells. In contrast, normal pancreases and tumor cells lacked interleukin-8 expression (P<0.001 vs. obstructive pancreatitis). Both immunohistochemistry and in situ hybridization demonstrated that interleukin-8 was expressed mostly in acinar cells in mild pancreatic fibrosis, whereas it was expressed in stromal and ductular cells in moderate and severe pancreatic fibrosis. CONCLUSIONS: These results suggest that interleukin-8 expression is related to the fibrotic process in obstructive pancreatitis.

Tumor protein p53-induced nuclear protein 1 (TP53INP1) in spontaneous chronic pancreatitis in the WBN/Kob rat: drug effects on its expression in the pancreas.

CONTEXT: The tumor protein p53-induced nuclear protein 1 (TP53INP1) gene was found using DNA microarray technology as an overexpressed gene in acute pancreatitis. However, expression of TP53INP1 in chronic pancreatitis has not been previously reported. OBJECTIVE: This study investigated TP53INP1 gene expression and its relationship with p53 and apoptosis in spontaneous chronic pancreatitis in the Wistar-Bonn/Kobori rat. METHODS: Ninety four-week-old male Wistar-Bonn/Kobori rats were fed a special breeding diet until sacrifice. Camostat mesilate (n=30) or a herbal medicine (Saiko-keishi-to; n=30) were mixed with the diet, while the other 30 rats were untreated. The rats were sacrificed every 4 weeks for 20 weeks, and the pancreas was examined. In addition, 6 four-week-old male Wistar-Bonn/Kobori rats were sacrificed and studied as starting reference. Finally, Wistar rats (n=36) were studied as controls. MAIN OUTCOME MEASURE: TP53INP1 mRNA expression was determined by reverse transcription-polymerase chain reaction using semi-quantitative analysis, direct sequencing and in situ hybridization. RESULTS: TP53INP1 mRNA was strongly expressed at 12 weeks when chronic pancreatitis developed, with a second peak at 20 weeks. The expression kinetics of TP53INP1 mRNA paralleled acinar cell apoptosis assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling. The p53 mRNA expression showed a single peak at 12 weeks. In situ hybridization revealed that TP53INP1 mRNA was expressed mainly in acinar cells. Therapeutic drugs such as camostat mesilate and a herbal medicine Saiko-keishi-to suppressed the TP53INP1 mRNA expression. TP53INP1 mRNA induction in acinar cells was confirmed with in vitro experiments using an arginine-induced rat pancreatic acinar AR4-2J cell injury model. CONCLUSIONS: TP53INP1 expression may reflect the acute-phase response and apoptosis of acinar cells in the course of chronic pancreatitis.

Filamin A-interacting protein (FILIP) is a region-specific modulator of myosin 2b and controls spine morphology and NMDA receptor accumulation.

Learning and memory depend on morphological and functional changes to neural spines. Non-muscle myosin 2b regulates actin dynamics downstream of long-term potentiation induction. However, the mechanism by which myosin 2b is regulated in the spine has not been fully elucidated. Here, we show that filamin A-interacting protein (FILIP) is involved in the control of neural spine morphology and is limitedly expressed in the brain. FILIP bound near the ATPase domain of non-muscle myosin heavy chain IIb, an essential component of myosin 2b, and modified the function of myosin 2b by interfering with its actin-binding activity. In addition, FILIP altered the subcellular distribution of myosin 2b in spines. Moreover, subunits of the NMDA receptor were differently distributed in FILIP-expressing neurons, and excitation propagation was altered in FILIP-knockout mice. These results indicate that FILIP is a novel, region-specific modulator of myosin 2b.

Anxiety- and depression-like behavior in mice lacking the CD157/BST1 gene, a risk factor for Parkinson's disease.

CD157, known as bone marrow stromal cell antigen-1, is a glycosylphosphatidylinositol-anchored ADP-ribosyl cyclase that supports the survival and function of B-lymphocytes and hematopoietic or intestinal stem cells. Although CD157/Bst1 is a risk locus in Parkinson's disease (PD), little is known about the function of CD157 in the nervous system and contribution to PD progression. Here, we show that no apparent motor dysfunction was observed in young knockout (CD157 (-/-)) male mice under less aging-related effects on behaviors. CD157 (-/-) mice exhibited anxiety-related and depression-like behaviors compared with wild-type mice. These behaviors were rescued through treatment with anti-psychiatric drugs and oxytocin. CD157 was weakly expressed in the amygdala and c-Fos immunoreactivity in the amygdala was less evident in CD157 (-/-) mice than in wild-type mice. These results demonstrate for the first time that CD157 plays a role as a neuro-regulator and suggest a potential role in pre-motor symptoms in PD.

Suppressive effect of herbal medicine saikokeishito on acinar cell apoptosis in rat spontaneous chronic pancreatitis.

BACKGROUND/AIMS: Chronic pancreatitis is characterized by acinar destruction and fibrosis. We previously reported that apoptosis is involved in acinar destruction in chronic pancreatitis in the WBN/Kob rat. This study aimed to elucidate the antiapoptotic effect of Saikokeishito (TJ-10). METHODS: Four-week-old male WBN/Kob rats were fed a special pellet diet (MB-3) with or without TJ-10 (80 mg/100 g body weight) for 20 weeks. Pancreas was histopathologically examined every 4 weeks, and the expression of apoptosis-related factors such as Fas and Fas ligand (FasL) mRNA and protein was analyzed with RT-PCR, in situ hybridization and immunohistochemistry. Apoptosis was detected with a TUNEL method. RESULTS: In untreated WBN/Kob rats, chronic pancreatitis developed at 12 weeks and progressed with marked acinar cell destruction at 16 weeks. The expression of Fas and FasL peaked at 12 and 20 weeks. An apoptotic index in acinar cells correlated to the expression of Fas and FasL mRNA. However, in the TJ-10-treated rats, the rate of pancreatic acinar cell destruction, the apoptotic index at 12-20 weeks, and the expression of Fas and FasL at 12 and 20 weeks decreased significantly compared to those in untreated rats. CONCLUSION: These results suggest that TJ-10 has a therapeutic effect on chronic pancreatitis by the suppression of acinar cell apoptosis via the Fas/FasL system.

Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development.

Pancreatic cancer is a disease with an extremely poor prognosis. Tumor protein 53-induced nuclear protein 1 (TP53INP1) is a proapoptotic stress-induced p53 target gene. In this article, we show by immunohistochemical analysis that TP53INP1 expression is dramatically reduced in pancreatic ductal adenocarcinoma (PDAC) and this decrease occurs early during pancreatic cancer development. TP53INP1 reexpression in the pancreatic cancer-derived cell line MiaPaCa2 strongly reduced its capacity to form s.c., i.p., and intrapancreatic tumors in nude mice. This anti-tumoral capacity is, at least in part, due to the induction of caspase 3-mediated apoptosis. In addition, TP53INP1(-/-) mouse embryonic fibroblasts (MEFs) transformed with a retrovirus expressing E1A/ras(V12) oncoproteins developed bigger tumors than TP53INP1(+/+) transformed MEFs or TP53INP1(-/-) transformed MEFs with restored TP53INP1 expression. Finally, TP53INP1 expression is repressed by the oncogenic micro RNA miR-155, which is overexpressed in PDAC cells. TP53INP1 is a previously unknown miR-155 target presenting anti-tumoral activity.

Effect of carboxamide derivative IS-741 on rat spontaneous chronic pancreatitis.

This study aimed to analyze the therapeutic effects of the carboxamide derivative IS-741 on spontaneous chronic pancreatitis (CP) in the WBN/Kob rat. Four-week-old male WBN/Kob rats were fed with MB-3, and IS-741 (0.012% in MB-3) was orally administered for 12 weeks. Cytokine-induced neutrophil chemoattractant (CINC), interleukin (IL) -6, pancreatitis-associated protein (PAP), and p8 mRNAs in the pancreas were detected with semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Histological examination of the pancreas showed that hemorrhage, interstitial edema, and inflammatory cell infiltration were markedly less severe at 12 weeks, while fibrosis and acinar degeneration were also milder at 16 weeks, in the IS-741-treated rats than in untreated rats. The above-mentioned genes were significantly suppressed in IS-741-treated rats at 12 weeks. Our results show that IS-741 exerts a potentially therapeutic effect on experimental CP by suppressing the expressions of the proinflammatory cytokines (IL-6 and CINC) and pancreatic acute phase proteins (PAP and p8).