Disruption of the genes for ClpXP protease in Salmonella enterica serovar Typhimurium results in persistent infection in mice, and development of persistence requires endogenous gamma interferon and tumor necrosis factor alpha.

The enteric pathogen Salmonella enterica serovar Typhimurium, similar to other facultative intracellular pathogens, has been shown to respond to the hostile conditions inside macrophages of the host organism by producing a set of stress proteins that are also induced by various environmental stresses. The stress-induced ClpXP protease is a member of the ATP-dependent proteases, which are known to be responsible for more than 90% of all proteolysis in Escherichia coli. To investigate the contribution of the ClpXP protease to the virulence of serovar Typhimurium we initially cloned the clpP and clpX operon from the pathogenic strain serovar Typhimurium chi3306 and then created insertional mutations in the clpP and/or clpX gene. The Delta clpP and Delta clpX mutants were used to inoculate BALB/c mice by either the intraperitoneal or the oral route and found to be limited in their ability to colonize organs of the lymphatic system and to cause systemic disease in the host. A variety of experiments were performed to determine the possible reasons for the loss of virulence. An oxygen-dependent killing assay using hydrogen peroxide and paraquat (a superoxide anion generator) and a serum killing assay using murine serum demonstrated that all of the serovar Typhimurium Delta clpP and Delta clpX mutants were as resistant to these killing mechanisms as the wild-type strain. On the other hand, the macrophage survival assay revealed that all these mutants were more sensitive to the intracellular environment than the wild-type strain and were unable to grow or survive within peritoneal macrophages of BALB/c mice. In addition, it was revealed that the serovar Typhimurium ClpXP-depleted mutant was not completely cleared but found to persist at low levels within spleens and livers of mice. Interferon gamma-deficient mice and tumor necrosis factor alpha-deficient mice failed to survive the attenuated serovar Typhimurium infections, suggesting that both endogenous cytokines are essential for regulation of persistent infection with serovar Typhimurium.

Plasma endotoxin and serum cytokine levels in patients with alcoholic hepatitis: relation to severity of liver disturbance.

BACKGROUND: Endotoxin plays an important role in the initiation and aggravation of alcoholic liver disease. In this study, we evaluated plasma endotoxin levels and serum concentrations of cytokines and lipopolysaccharide binding protein (LBP) during the acute and recovery phase of patients with alcoholic hepatitis; we also explored the prognostic factors associated with a fatal outcome. METHODS: Fourteen patients, consisting of eight patients with alcoholic hepatitis (AH), five cirrhotics with superimposed AH (LC+AH), and one patient with severe alcoholic hepatitis (SAH), were studied. Among these, two with LC+AH died of hepatic failure. RESULTS: Plasma endotoxin levels in the acute phase were higher in patients with AH (184.4 +/- 159.4 pg/ml) and LC+AH (206.9 +/- 174.9 pg/ml) than in healthy subjects (10.4 +/- 5.5 pg/ml, p < 0.001). In particular, in one patient with SAH and one of two nonsurvivors, plasma endotoxin levels were markedly high relative to the other cases. In most survivors, plasma endotoxin levels decreased in the recovery phase, whereas they further increased at the terminal stage in one of two nonsurvivors. Serum interleukin (IL)-6 and IL-8 levels in the acute phase were significantly higher in patients with AH and LC+AH as compared with healthy subjects. These levels were especially high in nonsurvivors and in one patient with SAH. IL-10 increased in two nonsurvivors, one patient with SAH, and one with LC+AH. In the recovery phase, these cytokine levels in survivors tended to decrease, but in nonsurvivors, IL-6 remained high, and IL-8 and IL-10 further increased. Tumor necrosis factor-alpha levels were below the detection limit throughout the course in all patients. Serum lipopolysaccharide binding protein (LBP) generally was elevated in the acute phase and decreased in the recovery phase in all survivors, but in one of the nonsurvivors, LBP was elevated markedly at the terminal stage. In the acute phase, plasma endotoxin levels were correlated positively with white blood cell counts, neutrophil counts, and serum IL-8. IL-8 was correlated positively with neutrophil counts and negatively with serum cholinesterase, hepaplastin test, and serum albumin levels. IL-6 was correlated positively with white blood cell and neutrophil counts, C-reactive protein, and serum total bilirubin and negatively with hepaplastin test and serum total protein levels. Serum LBP was correlated positively with white blood cell and neutrophil counts. CONCLUSIONS: Endotoxemia and related elevation of IL-8 may play an important role in the activation and migration of neutrophils in patients with alcoholic hepatitis. Marked elevation of inflammatory cytokines, IL-6 and IL-8, are related to severity and poor prognosis of alcoholic hepatitis. Serum LBP may serve as an index of inflammatory reaction in alcoholics.

Mixed endothelin receptor antagonist, SB209670, decreases portal pressure in biliary cirrhotic rats in vivo by reducing portal venous system resistance.

BACKGROUND/AIMS: This study aimed to evaluate the hemodynamic effects of endothelin-1 or mixed endothelin receptor antagonist, SB209670 in cirrhotic rats, and to elucidate the role of endothelin in cirrhotic portal hypertension. METHODS: Secondary biliary cirrhosis was induced by bile duct ligation. Hemodynamics were studied using the radioactive microsphere technique. RESULTS: Plasma and hepatic endothelin levels in cirrhotic rats were significantly higher than those in normal rats (plasma, 9.0+/-1.3 vs. 2.6+/-0.5 pg/ml, p<0.001; liver, 74.8+/-13.3 vs. 12.6+/-2.5 pg/g wet tissue, p<0.001). Intraportal administration of endothelin-1 (3 nmol/kg) progressively raised portal pressure without an initial transient reduction, which was observed in systemic arterial pressure, in both cirrhotic and normal rats. SB209670 (5.4 micromol/kg) reduced portal pressure in cirrhotic rats (-19+/-5%, p<0.01) without modifying systemic arterial pressure and renal blood flow, but not in normal rats. This reduction was associated with reduced portal venous system resistance (vehicle, 2.5+/-0.2 vs. SB209670, 1.7+/-0.1 mmHg x min x 100 g bw/ml, p<0.01), but not with change in portal venous inflow and collateral blood flow. CONCLUSIONS: Mixed endothelin antagonist, SB209670, decreased portal pressure by reducing portal venous system resistance without modifying systemic arterial pressure and renal blood flow in cirrhotic rats. This result, together with the findings that plasma and hepatic endothelin levels were elevated in cirrhotic rats and that exogenous endothelin-1 increased portal pressure, provides further support for a role of endothelin in portal hypertension and suggests a potential use of mixed endothelin antagonist in the pharmacological treatment of portal hypertension.

Prognostic factors in severe alcoholic liver injury. Nara Liver Study Group.

Severe alcoholic liver injury has been relatively rare, but is gradually increasing in Japan. The clinical features and prognostic factors in severe alcoholic liver injury were retrospectively investigated in 105 patients, consisting of 3 with severe alcoholic hepatitis (SAH), 43 with cirrhosis with superimposed alcoholic hepatitis [liver cirrhosis (LC)+alcoholic hepatitis (AH)], 38 with AH, and 21 with alcoholic cirrhosis. Seven of the 105 patients (6.7%, 2 with SAH and 5 with LC+AH) died of hepatic failure. Patients with SAH showed severe hyperbilirubinemia, reduced hepatic biosynthetic capacity, and marked acute inflammatory reactions, and developed multiple organ failure, such as disseminated intravascular coagulation (DIC), renal failure, acute pancreatitis, or pneumonia. Two SAH patients died within 1 month, whereas five with LC+AH died within 77 days during the second episode of AH. In these nonsurvivors, the serum total bilirubin (T.Bil) level was not normalized, and the hepaplastin test (HPT), serum albumin, cholesterol, and platelet count were not markedly improved after the first episode of AH. In the survivors, elevation of AST lasted longer, and the improvement of T.Bil, hepatic biosynthetic capacity, and the platelet count were much less in patients with LC+AH than in those with AH. Multivariate analysis using the Cox proportional hazards model showed serum C-reactive protein (CRP) and DIC as significant independent prognostic factors among SAH, LC+AH, and AH groups. When factors related to multiple organ failure, such as DIC and renal failure, were excluded, T.Bil and CRP were selected as independent prognostic factors. In patients with LC+AH and AH, CRP, and HPT were shown to be significant independent prognostic factors. These results suggest that SAH with multiple organ failure, and another episode of AH in advanced LC with hyperbilirubinemia and reduced hepatic biosynthetic capacity, are indicative of an extremely poor prognosis in chronic alcoholics.

Effects of "body compression" on parameters related to ascites formation: therapeutic trial in cirrhotic patients.

Decreased effective circulating blood volume is an important factor in ascites formation in liver cirrhosis. We designed a "body compression" apparatus as a means to restore effective blood volume and investigated its effectiveness in reducing ascites formation in cirrhotics in terms of its effect on parameters of ascites formation noted below. The subjects, eight cirrhotics with ascites and eight cirrhotics without ascites were given spironolactone (50-75 mg/day) and furosemide (40-80 mg/day) while they received a diet containing 85 mEq of sodium per day. All four limbs and the lower abdomen were compressed with constant pressure [height (cm) divided by 13.6 mmHg] once, for 3h, using stroke rehabilitation splints, while patients lay supine. In cirrhotics both with and without ascites, urine volume, urinary sodium excretion, and creatinine clearance during the body compression were greater than values during control (non-compression) periods (urine volume, means 285 vs 169 ml/3h; P < 0.001, urinary sodium excretion 15.8 vs 9.5 mEq/3h; p < 0.001, creatinine clearance 74 vs 59 ml/min, P < 0.001, respectively). The increased basal plasma renin activity, angiotensin II, aldosterone, and norepinephrine levels in all cirrhotics were significantly decreased by the body compression. In another group of six cirrhotics who received no diuretics or albumin, repeat body compression alleviated ascites in three with well preserved renal function, but was ineffective in three with markedly impaired renal function. These results suggest that the improvement in renal function brought about by the body compression is attributable to an increase in effective circulating blood volume. This maneuver may be a useful complementary therapy in patients with cirrhotic ascites with well preserved renal function.

Increased plasma levels of substance P and disturbed water excretion in patients with liver cirrhosis.

BACKGROUND: The pathogenesis of impaired water excretion in liver cirrhosis has not been fully elucidated. METHODS: We induced an intravenous water overload of 20 ml/kg body weight in 10 cirrhotics without ascites (CLC), 11 cirrhotics with ascites (DLC), and 10 normal subjects (N) and investigated the relationship of plasma levels of substance P (SP), norepinephrine (NE), and antidiuretic hormone (ADH) to impaired water excretion. RESULTS: Free water clearance (CH2O) was lower in DLC (mean, 2.7 ml/min) than in N (8.3 ml/min; P < 0.001) and CLC (6.9 ml/min; P < 0.001). In DLC the creatinine clearance (CCr), maximal urine flow rate/CCr, (CH2O + CNa)/CCr, and mean arterial pressure (MAP) were significantly lower than in N and CLC. There was a progressive increase in basal SP, from lowest in N to CLC, to highest in DLC. Basal NE increased in CLC and DLC. Basal ADH did not differ among N, CLC, and DLC. In cirrhotics CH2O was correlated positively with serum albumin and cholinesterase and negatively with the retention rate of indocyanine green at 15 min. Basal SP was negatively correlated with CH2O (r= -0.71: P < 0.001) and MAP (r= -0.56; P < 0.005). Basal NE was correlated positively with basal SP (r= 0.67, P < 0.01 ). CONCLUSIONS: Decreased CH2O is closely related to the severity of the liver disturbance. Decreased CCr and reduced delivery of filtrate to the ascending limb of the loop of Henle secondary to an increased sodium reabsorption in the proximal tubule may play an important role in the impairment of water excretion. The increase in SP, which has a potent vasodilatory action, and the associated enhanced activity of the sympathetic nervous system may be responsible for the mild or moderate impairment of water excretion in the absence of nonosmotic hypersecretion of ADH in cirrhotics with ascites.

Significance of increased plasma adrenomedullin concentration in patients with cirrhosis.

BACKGROUND/AIMS: Adrenomedullin recently discovered in human pheochromocytoma is a potent vasodilatory peptide mainly derived from vascular endothelial and smooth muscle cells. Hyperdynamic circulation, ultimately leading to ascites formation, has been attributed to peripheral vasodilatation in liver cirrhosis. However, little is known about the role of adrenomedullin in this condition. METHODS: Plasma adrenomedullin concentrations were measured by radioimmunoassay after extraction and purification in 28 cirrhotic patients without ascites, 12 cirrhotic patients with ascites and 10 healthy subjects. RESULTS: Plasma adrenomedullin concentrations in cirrhotic patients with ascites (12.7+/-4.5 fmol/ml) were significantly higher than those in cirrhotic patients without ascites (8.2+/-2.3 fmol/ml, p<0.005) and healthy subjects (5.8+/-0.8 fmol/ml, p<0.005). Interestingly, plasma adrenomedullin concentrations were highest in patients with refractory ascites (n=5, 15.8+/-3.0 fmol/ml) and were positively correlated with the Child-Pugh score (r=0.44, p<0.01). Moreover, plasma adrenomedullin concentrations were positively correlated with plasma renin activity (r=0.63, p<0.0001), plasma aldosterone (r=0.60, p<0.0001) and plasma norepinephrine concentrations (r=0.60, p<0.0001), and negatively correlated with creatinine clearance (r=-0.61, p<0.0005) and urinary sodium excretion (r=-0.44, p<0.02). Stepwise multiple regression analysis using certain independent variables, including Pugh's score, vasoactive substances, renal function and hemodynamic parameters, showed that the adjusted R square was highest when plasma renin activity and creatinine clearance (standard coefficient=0.53, -0.49, respectively) were considered (adjusted R square=0.61, p<0.0001). CONCLUSIONS: Plasma adrenomedullin concentrations increased with the progression of liver cirrhosis and were highest in cirrhotic patients with refractory ascites. In addition, elevated adrenomedullin was associated with activation of the renin-angiotensin-aldosterone and sympathetic nervous systems, and with functional renal impairment in cirrhosis. Considering the potent vasodilatory action of adrenomedullin, increased adrenomedullin may participate in the hyperdynamic circulation, ultimately leading to ascites formation, in patients with liver cirrhosis.

Role of albumin and high-density lipoprotein as endotoxin-binding proteins in rats with acute and chronic alcohol loading.

In the present study, the role of albumin and high-density lipoprotein (HDL) as endotoxin (Et)-binding proteins in chronically alcohol-fed rats was studied. In acute ethanol-loaded rats, the Et clearance in the blood was slightly prolonged, and the amount of albumin and HDL- bound Et in the blood was markedly increased. In chronic ethanol-loaded rats, the Et clearance was significantly faster than that in the control, and HDL-bound Et was increased. In the chronic ethanol-fed rats with an additional 5 g/kg body weight of ethanol load, the Et clearance was much prolonged, and blood tumor necrosis factor and ALT was elevated, when HDL-bound Et was not further increased. Et-binding capacity of total proteins, albumin, and HDL in the hepatocyte culture medium were increased when the Kupffer cells were preincubated in the medium containing ethanol, and the resultant culture supernatant was added to the hepatocyte culture system. In the culture experiment in the chronic ethanol-loaded rats, such increases were not observed. These results suggest that the increase in Et-binding capacity of HDL and albumin may serve as a protective mechanism against Et in chronic ethanol-loaded rats. An addition of high-dose ethanol to these rats may lead to impaired Et binding and inactivation, which may finally result in increased endotoxicity.

[Involvement of substance P in gastric mucosal lesions of patients with rheumatoid arthritis].

We investigated the relation between gastric mucosal lesions and substance P (SP) in 64 patients with rheumatoid arthritis (RA) taking nonsteroidal antiinflammatory drugs (NSAIDs). In these patients, the incidence of gastric mucosal lesions was as high as 53.1%. Serum SP levels were significantly higher in patients with gastric mucosal lesions than in those without gastric lesions. Erythrocyte sedimentation rate, serum C-reactive protein and rheumatoid factor (RF) levels were also higher in patients with gastric mucosal lesions. A positive correlation between serum SP and RF levels was found in patients with RA. Experimental gastric mucosal lesions induced by an oral administration of indomethacin in rats were significantly enhanced by an additional intraperitoneal injection of SP. From these observations, it is suggested that, in addition to the effect of NSAIDs, SP elevation in blood has a role in the development of gastric mucosal lesion in patients with RA.

Stepped care medical treatment for cirrhotic ascites: analysis of factors influencing the response to treatment.

Thirty-two patients with liver cirrhosis and ascites were treated by stepped care diuretic treatment as follows: step 1, placed on a 35 mEq sodium diet; step 2, given 400 mg/day of potassium canrenoate in addition to step 1 treatment; step 3, given 40-80 mg/day of furosemide in addition to step 2 treatment. Eleven out of 32 patients (34.4%, group 1) and 12 of 21 patients (57.1%, group 2) lost their ascites at step 1 and step 2, respectively. The remaining nine patients (group 3) required step 3 treatment. Basal urinary sodium excretion and creatinine clearance were significantly lower and beta 2-microglobulin was significantly higher in group 3 than those in groups 1 and 2. Elevation of basal plasma renin activity and norepinephrine was evident only in group 3. In group 1, urinary sodium excretion decreased after the treatment. In group 2, plasma alpha-atrial natriuretic polypeptide was lowered and plasma renin activity and norepinephrine were elevated after the treatment. These results suggest that basal renal function and plasma renin activity and norepinephrine levels are useful indices to predict the effect of ascites treatment and that responders to sodium restriction or potassium canrenoate may be in the state of vascular overflow, while non-responders to potassium canrenoate may be in the state of vascular underfilling. In summary, this stepped care treatment is safe without any side effects, although the diuretics themselves may lead to relative vascular underfilling.

Plasma endotoxin concentration and endotoxin binding capacity of plasma acute phase proteins in cirrhotics with variceal bleeding: an analysis by new methods.

Plasma endotoxin levels in 12 cirrhotics with bleeding from oesophageal varices and 50 cirrhotics without bleeding were measured by the chromogenic assay after the pretreatment of sample by perchloric acid (HClO4) and triethylamine. Endotoxin in cirrhotics with bleeding from varices was significantly higher than those without bleeding. In patients with bleeding, endotoxin increased for 3 days after the bleeding, first in the supernatant fraction and then in the precipitate fraction by HClO4 treatment. Peak plasma alpha 1-acid glycoprotein and haptoglobin were observed 3 days after the bleeding. Alpha 1-antitrypsin gradually increased for 14 days. Transferrin did not markedly change. The endotoxin-binding capacity of transferrin and alpha 1-acid glycoprotein increased immediately after bleeding and thereafter decreased, but that of alpha 1-antitrypsin tended to increase in the recovery period. In summary, the plasma endotoxin concentration and endotoxin-binding capacity of alpha 1-acid glycoprotein and transferrin were shown to have increased after bleeding from varices by this new method. There may be a close relationship between endotoxaemia and acute phase reaction in this situation.

Hepatitis C virus infection in patients with chronic liver diseases.

Antibodies against hepatitis C (HCV) in 512 patients were measured by an enzyme immunoassay (Ortho-HCV ELISA). The frequency of anti-HCV was 80%, 86%, 85% in nonB (NB) chronic hepatitis (CH), cirrhosis (LC), hepatocellular carcinoma (HCC), respectively; 70%, 90% in alcoholic (AL) LC, HCC; 15%, 33%, 58% in hepatitis B (HB) CH, LC, HCC, respectively. Anti-HCV positive cirrhotics had a shorter survival time and earlier development of HCC than anti-HCV negative cirrhotics. The findings suggest that HCV is a major cause of NB chronic liver diseases and may play a pathogenetic role in AL and HB liver diseases.

Urinary prostaglandins and renal function in obstructive jaundice.

Changes in urinary prostaglandin E2 (PGE2), 6-keto PGF1 alpha, and thromboxane (TXB2) excretion in 12 patients with obstructive jaundice were observed in relation to renal function and the renin-angiotensin (R-A) system. In obstructive jaundice before percutaneous biliary drainage the creatinine clearance (CCr) was significantly lower (p less than 0.001) and the PGE2 and plasma angiotensin II (AII) concentrations were significantly higher (p less than 0.005 and p less than 0.005, respectively) than those in normal subjects. Both 6-keto PGF1 alpha and TXB2 were widely distributed. When CCr returned to normal after drainage, PGE2 and plasma AII also returned to normal, but when CCr decreased after drainage, PGE2 and plasma AII increased. Before drainage, PGE2 correlated negatively with CCr (r = -0.72, p less than 0.01) and positively with plasma AII(r = 0.69, p less than 0.02). 6-Keto PGF1 alpha correlated positively with serum total bilirubin (r = 0.66, p less than 0.02). The percentage change in PGE2 after drainage correlated negatively with that in CCr (r = -0.95, p less than 0.005). The percentage chang in plasma AII correlated positively with that in urine PGE2 (r = 0.94, p less than 0.005) and negatively with that in CCr (r = -0.85, p less than 0.02). These results suggest that PGE2 is closely related to the R-A system and might assist in the maintenance of renal circulation in obstructive jaundice.

Transcatheter arterial embolization using iodized oil (lipiodol) mixed with an anticancer drug for the treatment of hepatocellular carcinoma.

The therapeutic results of Lp-TAE (transcatheter arterial embolization with Gelfoam particles preceded by the infusion of a mixture of lipiodol and an anticancer drug via the proper hepatic artery) were evaluated in hepatocellular carcinomas (523 non-resected and 24 resected cases). Excellent therapeutic effects were confirmed not only for the main tumor but also for the daughter nodules by a histological examination of the liver tissues resected after Lp-TAE. The cumulative 1-year, 2-year and 3-year survival rates in the 523 non-resected cases were 60.4%, 42.9% and 28.0% respectively. These survival rates were all higher than those achieved by Gelfoam TAE. The above results suggest the usefulness of Lp-TAE in the treatment of hepatocellular carcinoma.

Hb Hope, beta 136(H14)Gly----Asp, in a diabetic Japanese female and its functional characterization.

A beta-variant hemoglobin, first misjudged as a marked elevation of Hb A1, was found in a 68-year-old Japanese female with diabetes mellitus. This hemoglobin was isolated by Bio-Rex 70 chromatography combined with chromatofocusing, and was found to be Hb Hope, beta 136(H14)Gly----Asp, by classical and high performance liquid chromatographic peptide mapping techniques. Intrinsic oxygen affinity of this hemoglobin was approximately one-third as compared with that of Hb A0. This property was still observed in the constituent beta subunits isolated. Effects of such allosteric effectors as H+ (at a fixed concentration of Cl-), anion (Cl-), 2,3-diphosphoglycerate and carbon dioxide were more or less depressed. Among others, a marked reduction in the carbamate effect should be noted in a structural interpretation of the functional modifications. Subunit cooperativity, on the contrary, was not different from that in Hb A0 (n = 2.8-2.9). Explanation of these altered functions were attempted on the basis of the altered structure. The reduced stability of Hb Hope is also described.

Urinary prostaglandins and renal function in chronic liver diseases.

Changes in urinary PGE2 and PGF2 alpha excretion in chronic liver diseases were observed in relation to renal hemodynamics and sodium balance. After equilibration on a 110-170-meq sodium diet, daily urine collections were analyzed for PGE2 and PGF2 alpha by a new extraction and radioimmunoassay method. PGE2 was significantly greater in cirrhotics than in healthy subjects and in chronic hepatitis. The value was greater in cirrhotics with ascites than in those without ascites (p less than 0.05). PGF2 alpha did not differ among the groups. In cirrhotics PGE2 was correlated negatively with creatinine clearance (Ccr)(r = -0.76, p less than 0.001). After administration of 200 mg indomethacin, a significant fall in Ccr was seen only in cirrhotics with ascites. The percentage fall in PGE2 after indomethacin correlated with that in Ccr (r = 0.89, p less than 0.05) and with that in urinary sodium excretion (r = 0.68, p less than 0.02). These results suggest that PGE2 is essential in the maintenance of renal function in liver cirrhosis with ascites.