Differential splenic migration of dendritic cells after immunologic unresponsiveness in rat hepatic allografts induced by pretransplant donor-specific transfusion.

BACKGROUND: Donor dendritic cells migrate into the recipient spleen after hepatic transplantation. We previously reported that immunologic unresponsiveness to rat hepatic allografts can be induced by prior donor-specific blood transfusion (DST). We investigated the phenotype and splenic distribution of donor dendritic cells after allografting and DST. METHODS: Donor dendritic cells were identified with anti-rat dendritic cell (OX-62) and anti-donor class II MHC (RT1B(a)) (OX-76) antibodies. The phenotype of dendritic cells was determined with antibodies to CD45RC, CD62L, and the maturation markers CD80 (B7-1) and CD86 (B7-2). The cytokine profile of sorted CD45RC(+) OX-62(+) and CD45RC(-) OX-62(+) dendritic cells was analyzed by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Pretransplant DST significantly prolonged rat hepatic allograft survival. Immunostaining revealed OX76(+)/OX-62(+) cells in the splenic red pulp of animals receiving pretransplant DST and in the white pulp of untreated animals after transplantation. The ratio of splenic CD45RC(-) OX-62(+) cells to CD45RC(+) OX-62(+) cells was significantly higher in DST recipients than in untreated animals. CD62L, CD80, and CD86 were lower on CD45RC(-) OX-62(+) than CD45RC(+) OX-62(+) cells. RT-PCR revealed that sorted CD45RC(-) OX-62(+) cells expressed interleukin (IL)-4 and IL-10. In contrast, sorted CD45RC(+) OX-62(+) cells expressed only IL-2 and interferon gamma (IFN-gamma). CONCLUSION: Differential splenic migration of CD45RC(-) dendritic cells is associated with immunologic unresponsiveness to rat hepatic allografts.

Oxidation-active flavin models: oxidation of alpha-hydroxy acids by benzo-dipteridine bearing metal-binding site in the presence of divalent metal ion and base in organic solvents.

The oxidizing ability of benzo-dipteridine bearing a bipyridin-6-ylmethyl moiety (4) was found to be increased with Zn(2+) by approximately 10(3)-fold for sulfite addition in MeOH and approximately 10(2)-fold for oxidation of an NADH model in MeCN. It was found for the first time that 4 is able to oxidize alpha-hydroxy acids to alpha-keto acids in the presence of a divalent metal ion such as Zn(2+), Co(2+), and Ni(2+) and an amine base in MeCN or t-BuOH, whereas benzo-dipteridine having a bipyridin-5-ylmethyl moiety (3) is unable to oxidize them under the same conditions. The oxidation reaction was kinetically investigated including the kinetic isotope effect for deuterated mandelic acids (k(H)/k(D) = 2.1-3.7) and the Hammett plots for substituted mandelic acids (V-shaped plots). In the reaction of alpha-substituted alpha-hydroxy acids such as alpha-methyl mandelic and benzylic acids with 4, novel oxidative decarboxylation was found to take place, giving acetophenone and benzophenone, respectively. The oxidation mechanism for mandelic acid was proposed to proceed via a ternary complex of 4.Zn(2+).PhCH(OH)CO(2)(-), in which alpha-oxyanion of mandelate attacks C(4a)-position of 4 to form an adduct followed by 1,2-elimination to afford benzoyl formate and 2e-reduced 4. The roles of the metal ion were proposed as follows; (i) activation of 4, (ii) substrate-binding site, and (iii) activation of the bound alpha-hydroxy acid by lowering pK(a)'s of alpha-OH and alpha-CH. This is a first example that a flavin model oxidizes alpha-hydroxy acids in the presence of a metal ion.

CD45RC gammadelta T-cell infiltration is associated with immunologic unresponsiveness induced by prior donor-specific blood transfusion in rat hepatic allografts.

Little is known regarding the role of gammadelta(+) T cells in organ transplantation. We previously reported that immunologic unresponsiveness is induced by prior donor-specific blood transfusion (DST) in rat hepatic allografts. We investigated the phenotype and distribution of gammadelta(+) T cells in the hepatic allograft, spleen, and peripheral blood of recipient rats with immunologic unresponsiveness induced by DST. gammadelta(+) T cells were enumerated in allograft livers and spleens by immunostaining and in blood by flow cytometric analysis. The phenotype of gammadelta(+) T cells was determined using CD45RC isoforms derived from alternative mRNA splicing. The cytokine profile of CD45RC(+) and CD45RC(-) gammadelta(+) T cells was analyzed by reverse transcription polymerase chain reaction. The number of gammadelta(+) T cells in hepatic infiltrates in recipient rats pretreated with DST was significantly greater than in untreated animals. This correlated with significantly higher levels of gammadelta T cell receptor (TCR) mRNA in hepatic allografts of DST-treated rats as compared with untreated animals. The gammadelta(+) T cell/alphabeta(+) T-cell ratio increased in hepatic infiltrates in DST-treated recipient rats but not in untreated animals. CD45RC(-)gammadelta(+) T cells were predominantly increased in DST-treated hepatic allografts compared with untreated allografts. Most of the intestinal intraepithelial T cells were CD45RC(-)gammadelta(+). Interleukin (IL)-10 and IL-4 mRNA were detected more in CD45RC(-)gammadelta(+) T cells than CD45RC(+)gammadelta(+) T cells. CD45RC(-)gammadelta(+) T cells infiltrating liver allografts produce Th2-type cytokines and are associated with immunologic unresponsiveness induced by DST.

A serine protease inhibitor, N-alpha-tosyl-l-lysine chloromethyl ketone, prolongs rat hepatic allograft survival.

BACKGROUND: Serine protease inhibitors have profound suppressive effects on cellular and humoral immune responses. We investigated the effect of a serine protease inhibitor, N-alpha-tosyl-l-lysine chloromethyl ketone (TLCK), on hepatic allograft survival in rats. Methods. Orthotopic hepatic transplantation was performed in an ACI (RT1(a))-to-LEW (RT1(1)) rat combination. TLCK was administered continuously at a dose of 4.4 mg/kg/day using an osmotic subcutaneous infusion minipump. RESULTS: TLCK prolonged hepatic allograft survival. Histologic staging of acute rejection based on Banff criteria in TLCK-treated hepatic allografts was significantly lower than in untreated allografts. TLCK significantly reduced serum concentrations of interferon (IFN)-gamma and tumor necrosis factor (TNF) alpha in allograft recipients. TNF-alpha mRNA levels in TLCK-treated allografts were significantly lower than in untreated allografts. TLCK also decreased perforin mRNA levels in hepatic allografts. Hepatic infiltrates eluted from TLCK-treated allografts showed significantly lower cell-mediated lympholytic activity against donor Con A blast cervical lymph node cells than those from untreated allografts. In vitro, TLCK suppressed interleukin-2 production and [(3)H]thymidine incorporation into an allogeneic mixed lymphocyte reaction. CONCLUSION: TLCK suppressed acute allograft rejection, suggesting a novel immunosuppressive strategy for therapy of acute organ rejection.

[Study on the effects of walking on functional fitness in aged patients with the hand-arm vibration syndrome].

PURPOSE: This article reports the effectiveness of mild walking exercise for maintaining functional fitness in aged patients with the hand-arm vibration syndrome (HAVS). SUBJECTS AND METHODS: Fifty-two patients suffering from vibration syndrome, for which they received annual compulsory examination from December 1998 to March 1999 at the San-in Rosai Hospital, were examined. They all were male, with a mean age (standard deviation) of 69.1 (7.3) years, and were randomly allocated to an intervention group (N = 26) and a control group (N = 26). The goal of the intervention was to achieve and maintain at least 30 minutes of walking a day. Functional fitness was assessed by a sitting and standing test, a zigzag walking test, a hand working test with a pegboard for dexterity, and a self-care working test proposed by the Physical Fitness Research Institute, Meiji Life Foundation of Health and Welfare. RESULTS: There were no significant differences between the two groups regarding baseline characteristics. The proportions of subjects with regular exercise habits after the intervention were 84.6% (22/26) and 53.8% (14/26) in the intervention and control groups, respectively, the difference being significant. The total scores for functional fitness were improved in the intervention, while decline was noted in the control group. Sitting and standing and self-care working ability were also improved in the intervention group as against the deterioration with age in the control group. CONCLUSIONS: Our findings show to some extent that intervention using mild exercise, walking for 30 min, is effective for aged patients with HAVS to maintain and improve functional fitness.

Urinary trypsin inhibitor reduces C-X-C chemokine production in rat liver ischemia/reperfusion.

BACKGROUND AND AIM: Protease inhibitors attenuate ischemia/reperfusion injury. However, the underlying mechanisms by which protease inhibitors prevent reperfusion injury remain obscure. Neutrophils play an important role in reperfusion injury. We studied the effects of urinary trypsin inhibitor (UTI) on production of the C-X-C chemokine, cytokine-induced neutrophil chemoattractant (CINC), by Kupffer cells during ischemia/reperfusion of the liver. METHODS: Liver ischemia was induced in rats by occlusion of the portal vein for 30 min. UTI (50,000 U/kg) was injected intravenously 5 min before vascular clamping. Serum CINC concentrations were measured by enzyme-linked immunosorbent assay. Levels of CINC mRNA in the liver were determined by Northern blot analysis. We also examined the inhibitory effects of UTI on in vitro CINC production by peritoneal macrophages in response to neutrophil elastase (NE). RESULTS: Serum CINC concentrations increased and peaked 6 h after reperfusion. However, pretreatment of animals with UTI blunted this increase in CINC and significantly reduced CINC mRNA levels in the liver after ischemia/reperfusion. UTI also decreased neutrophil accumulation in the liver 24 h after reperfusion. In vitro CINC production by Kupffer cells from rats pretreated with UTI 3 h after ischemia/reperfusion was significantly decreased compared to those from untreated animals. UTI reduced NE activity in vitro in a dose-dependent manner, and UTI significantly reduced in vitro CINC production by peritoneal macrophages stimulated with NE. CONCLUSION: UTI reduces the production of CINC by Kupffer cells stimulated with NE, attenuating ischemia/reperfusion injury of the liver.

Atomic force microscopy to study direct neurite-mast cell (RBL) communication in vitro.

Communication between nerves and mast cells is a prototypic demonstration of neuroimmune interaction. We used an in vitro co-culture approach comprising cultured murine superior cervical ganglia (SCG) and rat basophilic leukemia (RBL-2H3) cells. Atomic force microscopy (AFM) showed how neurites attached to a pseudopodium or a cell body of an RBL cell. After stimulation of SCG neurites with bradykinin or scorpion venom, RBL cells attached to neurites spread and flattened, and several discharged granules (0. 5-1.0 microm in diameter) were found on the surface of the RBL cells. A neurokinin (NK)-1 receptor (i.e. substance P receptor) antagonist prevented the RBL degranulation. The results showed that activation of the SCG neurites with bradykinin or scorpion venom was able to elicit degranulation in RBL cells which were attached to neurites.

Posttransplant infusion of donor-specific blood induces immunological unresponsiveness in rat hepatic allografts.

BACKGROUND: We previously reported that pretransplant donor-specific blood transfusion (DST) induces CD45RC-CD4+ T cells, Th2-like effector cells, and prolongs rat hepatic allograft survival. Our study investigated the effects of posttransplant DST on rat hepatic allograft survival. METHODS: Three days after transplantation, LEW (RT1(1)) recipient rats with ACI (RT1a) livers were injected i.v. with freshly heparinized donor-specific blood. The time kinetics of CD45RC-CD4+ and CD45RC+CD4+ T cell subsets in hepatic infiltrates were examined. RESULTS: Posttransplant DST significantly prolonged rat hepatic allograft survival. Interferon (IFN)-gamma, interleukin (IL)-12, and IL-18 mRNA levels in hepatic allografts of untreated recipients were significantly greater than in recipients treated with posttransplant DST. However, hepatic allografts of recipients treated with posttransplant DST showed significantly higher IL-4, IL-10, and transforming growth factor (TGF)-beta mRNA levels than untreated recipients. The ratio of CD45RC-CD4+ T cells to CD45RC+CD4+ T cells was significantly higher in hepatic allografts treated with posttransplant DST than in untreated animals. Immunostaining with anti-rat dendritic cell (OX-62) monoclonal antibody revealed that OX-62+ cells were distributed to the splenic red pulp of animals treated with posttransplant DST and to the splenic white pulp in untreated animals. Most OX62+ cells isolated from the spleen of recipients treated with posttransplant DST expressed donor RT1Ba class II major histocompatibility complex antigens, suggesting that OX-62+ cells were of donor origin. CONCLUSION: Posttransplant DST was associated with persistent infiltration of CD45RC-CD4+ T cells, Th2-like effector cells, in rat hepatic allografts, causing immunologic unresponsiveness and establishment of microchimerism in the spleen.

Increasing incidence of distal radius fractures in Japanese children and adolescents.

To investigate recent trends in the incidence of fractures in children and adolescents, we conducted a survey of distal radius fractures in 1992 and 1995 in Tottori Prefecture, Japan. Seven-hundred and forty-five patients under age 20 years were registered (562 males and 183 females). The age and sex-specific incidences of fractures in patients under 20 years of age were higher in males than in females, showing peaks at age 12-13 years for males and at age 10-11 years for females. The incidences had an obvious monthly variation, with peaks in spring and autumn. The age and sex-specific incidences were compared with those in the period 1986-1988, which we reported previously. The incidence in each year showed a significant increase with time for males but not for females. We conclude that the incidence of distal radius fractures in children and adolescents has a prominent peak matched by the age of the growth spurt, and the incidence of the fracture has increased in males during the past decade in Tottori Prefecture.

Thrombin and factor Xa enhance neutrophil chemoattractant production after ischemia/reperfusion in the rat liver.

BACKGROUND: Clotting proteases may affect leukocyte effector function. Activation of the coagulation cascade after ischemia/reperfusion stimulates cytokine production by activated macrophages. Cytokine-induced neutrophil chemoattractant (CINC) may also be important in the pathophysiology of liver ischemia/reperfusion injury. We investigated the effects of a selective factor Xa inhibitor, DX-9065a, on CINC expression after ischemia/reperfusion in the rat liver. METHODS: Liver ischemia was induced in rats by occluding the portal vein for 30 min. DX-9065a (9 mg/kg) was injected intravenously 5 min before vascular clamping. Serum CINC concentrations were measured by enzyme-linked immunosorbent assay. Levels of CINC mRNA in the liver were determined by Northern blot analysis. We also examined in vitro CINC production by peritoneal macrophages in response to alpha-thrombin or factor Xa. RESULTS: Serum CINC concentrations increased and peaked 6 h after reperfusion. However, pretreatment of animals with DX-9065a resulted in significantly smaller increases in CINC after reperfusion. Pretreatment with DX-9065a also significantly reduced CINC mRNA levels in the liver after ischemia/reperfusion. In vitro CINC production by peritoneal macrophages was enhanced by alpha-thrombin, as well as factor Xa. CONCLUSIONS: Thrombin and factor Xa stimulate CINC production by macrophages. A selective inhibitor of factor Xa, DX-9065a, attenuates neutrophil chemoattractant production after ischemia/reperfusion injury of the rat liver.

Manganese deposition in the globus pallidus in patients with biliary atresia.

BACKGROUND: Chronic liver diseases may alter trace element contents in the brain. Among these trace elements, manganese is a ubiquitous transition metal excreted by the liver into the bile. Blood concentrations of manganese are elevated in patients with biliary atresia who have undergone hepatic portoenterostomy. The present study investigated the effects of liver transplantation on manganese deposition in the brain in such patients. METHODS: The signal intensity of the globus pallidus was calculated as an index defined as the percentile ratio of signal intensity in the globus pallidus to the subcortical frontal white-matter in sagittal T1-weighted magnetic resonance imaging planes. RESULTS: Brain magnetic resonance imaging revealed hyperintense signals in the globus pallidus due to manganese deposition in biliary atresia patients. Few neurologic symptoms related to manganese intoxication were observed. However, one 23-year-old female with biliary atresia had depressive symptoms and dyskinesia; she improved after oral administration of the dopamine precursor, L-DOPA. Manganese deposition disappeared in two patients after living-related reduced-size hepatic transplantation. CONCLUSIONS: Manganese accumulates in the brain during cholestasis associated with biliary atresia and disappears after hepatic transplantation. Manganese deposition is likely to be subclinical and reversible but may be associated with some age-related neurologic symptoms.

Calcium-channel blocker attenuates Kupffer cell production of cytokine-induced neutrophil chemoattractant following ischemia-reperfusion in rat liver.

We investigated the effects of the calcium-channel blocker verapamil hydrochloride on the production of cytokine-induced neutrophil chemoattractant (CINC) following reperfusion injury in rat liver. Ischemia was induced for 30 min by portal vein occlusion. Animals were pretreated with intravenous injection of verapamil hydrochloride (2.5 mg/kg) 5 min before vascular clamp. Verapamil hydrochloride limited increases in the chemoattractant compared with nonpretreated rats. Most cells immunostained for chemoattractant were ED2-positive macrophages in sinusoids. In vitro chemoattractant production by Kupffer cells isolated from animals pretreated with verapamil hydrochloride was significantly lower than by Kupffer cells from nonpretreated animals. Expression of transcripts in liver for chemoattractant peaked 3 hr after reperfusion in nonpretreated animals, while pretreatment with verapamil hydrochloride significantly decreased chemoattractant mRNA levels. In vitro chemoattractant production could be induced in naive Kupffer cells after stimulation with oxygen radicals generated by hypoxanthine and xanthine oxidase, but verapamil hydrochloride prevented these increases. We concluded that the calcium-channel blocker verapamil hydrochloride significantly attenuates chemoattractant release by Kupffer cells after ischemia-reperfusion in the rat liver.

Manganese deposits in patients with biliary atresia after hepatic porto-enterostomy.

PURPOSE: The aim of this study was to determine if there is latent manganese toxicity in patients with biliary atresia. METHODS: Fifteen children with biliary atresia were examined postoperatively with regard to whole-blood manganese levels using brain magnetic resonance imaging (MRI) and I-123 iodoamphetamine (IMP) per rectal portal scintigraphy. RESULTS: Nine (60%) of the 15 had high whole-blood manganese levels (mean, 4.1 microg/dL; range, 1.2 to 9.6; normal, 0.5 to 2.5), and these 9 had hyperintense globus pallidus on T1-weighted images, with no corresponding signal change in T2 sequences. I-123 IMP per rectal portal scintigraphy was done for 13 patients to evaluate portosystemic shunt flow. 12 (92%) of these patients had an increased flow. Mean shunt ratio was estimated to be 41% (range, 0.6 to 98; normal, <5%). Encephalopathy was evident in only 1 patient. CONCLUSIONS: Some patients with biliary atresia in the postoperative period have manganese deposits in globus pallidus on T1-weighted images and high whole-blood manganese levels, possibly caused by increased portsystemic shunt, and a latent or subclinical encephalopathy is also present.

Reduced interleukin-12, interleukin-18, and interferon-gamma production with prolonged rat hepatic allograft survival after donor-specific blood transfusion.

Interferon-gamma is a key immunoregulatory cytokine involved in acute graft rejection. Immunologic unresponsiveness to organ allografts has been induced by pretransplantation donor-specific blood transfusion, both experimentally and clinically. We investigated interferon-gamma production and intragraft gene expression of type-1 T-helper cytokines such as interleukin-12 and -18 and type-2 T-helper cytokines such as interleukin-10 and transforming growth factor-beta in rats receiving hepatic allografts after such transfusions. The animals were divided into four groups: group I received isografts; group II received allografts; group III received allografts after donor-specific transfusion; and group IV received allografts and was treated with FK 506. Donor blood given seven days prior to transplantation significantly prolonged allograft survival. The serum interferon-gamma concentrations in group II increased, peaking on day 5 and then decreasing. Serum interferon-gamma concentrations in groups I, III, and IV were significantly lower than those observed in group II, as were levels of interleukin-12 and interleukin-18 mRNA in the graft. Transforming growth factor-beta and interleukin-10 mRNA levels in grafts in transfused animals were significantly greater than those in the untreated allograft group. Interleukin-12 and -18 mRNA transcripts in an allogeneic mixed lymphocyte reaction were inhibited by interleukin-10 and transforming growth factor-beta. These results suggest that interleukin-12 and -18 expression in hepatic allografts is inhibited in the immunologically unresponsive state induced by donor-specific transfusion.

Neutrophil elastase and oxygen radicals enhance monocyte chemoattractant protein- expression after ischemia/reperfusion in rat liver.

BACKGROUND: The monocyte chemoattractant protein-1 (MCP-1) is produced during reperfusion injury and induces tissue factor that is the initiator of the clotting cascade. Neutrophil elastase is a crucial mediator of inflammatory tissue damage. Activation of the coagulation system stimulates cytokine production by activated leukocytes. We investigated the effects of neutrophil elastase and oxygen radicals generated by hypoxia associated with microthrombus formation on MCP-1 expression after ischemia/reperfusion in rat liver. METHODS: In vitro MCP-1 production by macrophages after stimulation with human neutrophil elastase (HNE) or oxygen radicals generated by hypoxanthine and xanthine oxidase was examined. Liver ischemia was induced in rats by occluding the portal vein for 30 min. An inhibitor of human neutrophil elastase (ONO-5046*Na, 10 mg/kg) and antithrombin III (AT-III, 250 U/kg) were injected i.v. 5 min before vascular clamping. Serum concentrations of MCP-1 were measured by enzyme-linked immunosorbent assay. RESULTS: Human neutrophil elastase or oxygen radicals significantly enhanced in vitro MCP-1 production by macrophage. Serum MCP-1 concentrations reached a peak at 6 hr after reperfusion and then gradually decreased. However, pretreatment of animals with AT-III or ONO-5046*Na alone resulted in significantly smaller increases in serum concentrations of MCP-1 after reperfusion. Pretreatment with both ONO-5046*Na and AT-III produced additive effects. The combined treatment with ONO-5046*Na and AT-III significantly reduced MCP-1 mRNA in liver after ischemia/reperfusion. CONCLUSIONS: MCP-1 production by macrophages is stimulated by neutrophil elastase and oxygen radicals generated by hypoxia, probably due to microthrombus formation after ischemia/reperfusion of the rat liver.

Surgical indications for patients with hyperammonemia.

BACKGROUND/PURPOSE: The authors surgically treated seven of eight patients with congenital portosystemic shunt and hyperammonemia. This entity is uncommon in children. METHODS: The patients included five boys and three girls with a mean age of 8 years (range, 7 months to 24 years). Preoperative symptoms included hyperammonemia. Hepatic encephalopathy was evident in five patients. Diagnosis and assessment were made by ultrasound scan, magnetic resonance imaging (MRI), angiography, and 123 I-iodoamphetamine per-rectal portal scintigraphy. Surgical banding was done for five patients and transvenous coil embolization for two. One patient was not a surgical candidate because there were no intrahepatic portal veins. RESULTS: In four of the five patients treated using surgical banding, and in both patients who underwent coil embolizations, hyperammonemia and clinical symptoms improved soon after surgery. However, in the remaining patient, hyperammonemia worsened after surgery, and reoperation was needed because of a severe portal hypertension, possibly caused by malconformation of hepatic veins. CONCLUSIONS: For patients with congenital portosystemic shunt, early diagnosis and surgery are needed to prevent damage to central nerves caused by persistent hyperammonemia. Maldevelopment of the intrahepatic portal veins is a surgical option, if the patient has a normal liver architecture, but malconformation of hepatic veins or severe anomalies such as cardiac defects would rule out surgical intervention.

Reversibility of hyperintense globus pallidus on T 1-weighted MRI follow- ing surgery for a portosystemic shunt in an 8-year-old girl.

An 8-year-old Japanese girl with a portosystemic shunt had shown hyperammonaemia since she was 3 years of age. MRI of her brain showed bilateral hyperintense globus pallidus. A portosystemic shunt was evident on US and angiography. She underwent surgical banding of the shunt, after which the lesion and clinical symptoms disappeared.

Platelet-activating factor antagonist (TCV-309) attenuates the priming effects of bronchoalveolar macrophages in cerulein-induced pancreatitis rats.

We investigated the role of platelet-activating factor (PAF) as a priming signal for cytokine-induced neutrophil chemoattractant (CINC) expression by bronchoalveolar macrophages in acute pancreatitis. Pancreatitis was induced by four intramuscular injections of cerulein (50 micrograms/kg at 1-h intervals) in Wistar rats. The animals were injected intraperitoneally with 10 micrograms/kg of lipopolysaccharide (LPS) as a septic challenge. Pancreatitis rats were treated with a bolus intravenous injection of TCV-309 (3 or 30 micrograms/kg) 30 min before the septic challenge. Intense mononuclear cell infiltration and lung hemorrhage occurred in pancreatitis rats complicated with sepsis but were not seen in pancreatitis rats receiving a bolus TCV-309. Pancreatitis rats treated with TCV-309 had lower serum concentrations of CINC after septic challenge and lower levels of CINC messenger RNA (mRNA) in the lung, as well as fewer pulmonary infiltrates immunoreactive for CINC or Mac-1 (CD11b/CD18). In vitro CINC production in response to LPS by bronchoalveolar macrophages obtained from pancreatitis rats 6 h after the first cerulein injection, immediately before septic challenge, was enhanced but was significantly reduced in a TCV-309-sensitive manner. LPS-stimulated in vitro CINC production by naive bronchoalveolar macrophages was significantly enhanced by pretreatment with PAF. TMB-8 (an inhibitor of calcium release from endoplasmic reticulum) or W7 (calmodulin antagonist) completely abrogated the chemoattractant production by bronchoalveolar macrophages pretreated with PAF after LPS stimulation. Altered intracellular calcium, due to Ca2+ efflux from intracellular stores, may be involved in the "priming" of bronchoalveolar macrophages to release CINC after triggering with LPS during acute cerulein-induced pancreatitis. The PAF antagonist TCV-309 effectively prevented hyperactivity of bronchoalveolar macrophages and pancreatitis-associated lung injury after the septic challenge.