The present study assessed whether peritoneal macrophages isolated from cirrhotic patients produce nitric oxide (NO) and express NO synthase type II (NOS II) mRNA and protein. Patients with cirrhosis and ascites without peritonitis or with unresolved or resolved spontaneous bacterial peritonitis (SBP) were studied. Following paracentesis, ascites NO 2 − + NO 3 − content (NOx) was measured. Peritoneal macrophages from ascites were seeded on well plates, and NO 2 − in the medium was determined. NOx was higher in patients with unresolved or resolved SBP than in cirrhotic patients without peritonitis. Macrophages of patients with SBP or resolved SBP produced NO 2 − after 30 hours in culture, but those obtained from patients without peritonitis did not. Reverse-transcription polymerase chain reaction (RT-PCR) and immunocytochemical analysis revealed the presence of a clear signal for NOS II mRNA and protein in macrophages of SBP patients, regardless of whether or not the infection subsided. Therefore, peritoneal macrophages isolated from cirrhotic patients with unresolved or resolved SBP produce NO and express the NOS II mRNA and protein, suggesting that NOS II may contribute to the control of SBP, or to its associated pathology, in human cirrhosis.
Rhodococcus equi is a Gram-positive, aerobic, nonmotile, non-spore-forming bacillus related to Mycobacteriaceae, Nocardiaceae, and Corynebacteriaceae. It is a well-established pathogen in veterinary medicine, usually causing pneumonia and sepsis to cattle. R equi was first isolated by Magnusson in 1923 from suppurating lung lesions in foals (1). Its natural reservoir is the soil with herbivore manure. Farm animals acquire the infection through the respiratory tract, when contaminated dust is inhaled (2). Human infections due to R equi are uncommon. However, this bacterium is becoming an increasingly important opportunistic pathogen in immunosuppressed patients. The first human infection was reported in 1967 in a patient with plasma cell hepatitis under steroid and 6-mercaptopurine treatment (3). Since then, several cases have been described in patients with immunosuppression due to either human immunodeficiency (HIV)* infection (4, 5), therapy for neoplastic disorders (6) or kidney (7-9) and heart transplantation (10). We report herein the first case of R equi infection in a liver transplant recipient. A 58-year-old man with end-stage primary biliary cirrhosis underwent liver transplantation in July 1992, with an uncomplicated course during hospital admission. The immunosuppression regimen consisted of cyclosporine (CsA), steroids, and azathioprine (the last was administered only during the first month). Complications after hospital discharge included an ophthalmic varicella zoster virus infection resolved without sequelae; osteoporosis with several compression fractures of the spine; arterial hypertension requiring antihypertensive drugs (nifedipine, prazosin), and moderate renal dysfunction, with serum creatinine levels ranging between 1.5 and 2.4 mg/dl, attributed to CsA nephrotoxicity. Ten months after transplantation the patient had an episode of acute cellular rejection that was treated with high-dose steroid therapy (methylprednisolone, 1 g/day for three days) and subsequently with OKT3 (5 mg/day for 10 days). After OKT3 administration, liver function tests progressively improved. In the next months, doses of immunosuppressants were slowly tapered in an attempt to reduce their toxic side effects. Two years after transplantation the patient had a second episode of acute cellular rejection and was treated with high-dose steroid therapy (methylprednisolone, 1 g/day for three days) with only a partial response. Since then, moderate liver dysfunction remained, and triple immunosuppressive therapy with CsA (400 mg/day), prednisone (20 mg/day) and azathioprine (50 mg/day) was administered. On November 1994 (three months after the second episode of rejection) the patient came to the emergency room complaining of two painful nodules on the left arm and fever up to 39°C. Physical examination revealed moderate jaundice, temperature of 38.5°C, blood pressure 160/90 mmHg, and a heart rate of 88 beats/min. Two nodules were observed in the left shoulder and elbow 8 cm and 3 cm in diameter, respectively. Both were erythematous and painful, and fluctuation was present. Percutaneous needle aspiration of the nodules obtained purulent fluid. No skin wound was apparent near the nodules. No peripheral lymph node enlargement was found. Cardiopulmonary exploration was normal. A roentgenogram of the left arm was also normal. A chest X-ray film revealed a small round lesion on the base of the left lung (Fig. 1). Fundoscopy was found to be normal. The white blood cell count was 22.880 cells/mm3 with 71% neutrophils, 16% band forms, 4% lymphocytes, 4% monocytes, 4% myelocytes, and 1% metamyelocytes. The hemoglobin level was 9.2 g/dl and the platelet count 261,000/mm3. The serum bilirubin level was 5.1 mg/dl; alkaline phosphatase 3415 U/L; gamma-glutamyl transpeptidase 3024 U/L; aspartate aminotransferase 78 U/L; alanine aminotransferase 166 U/L, and prothrombin activity 100%. The serum creatinine was 2.8 mg/dl and BUN 79 mg/dl. Incision and drainage of the nodules were performed under local anesthesia, and samples of the pus were sent for standard bacterial and fungal cultures. Samples of blood, urine, and sputum were also obtained for culture. The patient was admitted to the hospital, and intravenous antibiotic therapy was started with vancomycin (500 mg/12 hr), ceftriaxone (1 g/24 hr), and metronidazole (500 mg/8 hr). At 48 hr after admission, the culture of the pus specimens obtained from the arm and forearm abscesses showed growth of Gram-positive coccobacillary structures that were subsequently identified as R equi. The identification of R equi was based on the morphology of the colonies (large, mucoid, pale salmon-pink colonies), the catalase activity, and the API Coryne test (bioMerieux, Marcy-l'Etoile, France). The resistance to penicillin showed by the germ isolated also helped in the identification of R equi, since other diphtheroids are usually susceptible. Blood, sputum, and urine cultures were negative. Although the patient denied specifically the presence of cough, dyspnea, or chest pain, the presence of R equi in the abscess purulent fluid and the existence of a round condensed area in the lower lobe of the left lung found in the chest radiograph led us to suspect the presence of a possible pneumonia. A thoracic computerized tomography (CT) scan showed a thick-walled cavitated mass at the left lung base, with minimal pleural effusion. In addition, several small areas of consolidation and cavitation not seen in the chest radiograph taken at admission were found in the right lung (Fig. 2). These findings suggested the presence of necrotizing bilateral pneumonia. Antibiotic therapy was changed, and intravenous erythromycin (1 g/6 hr), rifampicin (600 mg/24 hr), and ciprofloxacin (200 mg/12 hr) administration was initiated. After that, the patient remained afebrile and asymptomatic. After three weeks of intravenous antibiotic therapy, and once the resolution of the left arm abscesses was achieved, the patient was discharged from the hospital and the triple antibiotic therapy was continued orally. A liver biopsy obtained during hospital admission showed marked centrilobular cholestasis and interlobular bile duct cell dysplasia, both findings considered to be residual after the previous episode of rejection. Since no signs of cellular rejection were apparent, the doses of the immunosuppressive drugs were slightly reduced. A chest radiograph and CT scan obtained 2 months after the initiation of the triple antibiotic therapy only showed a small residual lesion in the left lower lobe and complete resolution of the right lung lesions (Fig. 3). Nevertheless, double antibiotic therapy including erythromycin and ciprofloxacin was maintained in order to avoid potential reactivation of residual septic foci. R equi, formerly known as Corynebacterium equi, is a well known pathogen for farm animals, particularly foals, and is an increasingly important opportunistic respiratory pathogen in immunocompromised hosts. These patients are at high risk of developing serious infections caused by microorganisms rarely pathogenic to man. HIV-infected patients constitute the largest group of humans in which R equi infections have been reported. Immunosuppressive therapy used in organ transplant recipients can also be an important risk factor for R equi infection. However, only 6 cases in kidney transplant recipients and one case in a heart transplant recipient have been reported (7-10). R equi resides in the soil, and humans acquire the infection through the respiratory tract. In the lung R equi tends to produce a cavitating pneumonia, with symptoms such as cough, fever, or pleuritic chest pain, as well as a wasting syndrome. In some instances the initial clinical picture may be insidious. Subcutaneous abscesses are uncommon and probably represent hematogenous dissemination from the pulmonary lesion to the soft tissues. Three remarkable characteristics can be pointed out in this case. First, the route of infection-our patient had been working occassionally in his garden, which he used to fertilize with calf manure, thus suggesting a respiratory tract acquisition of the infection. Second, the intense immunosuppression state-the patient had been treated for a late-onset rejection episode few weeks before the infection, and was being maintained on a triple-immunosuppressive-drug regimen. Finally, the patient was first seen a clinical picture consisting of fever and soft tissue abscesses in the left arm, but without any symptom suggesting respiratory tract involvement. Only at follow-up during admission was the radiographic lesion in the left lung recognized to be cavitated pneumonia; subsequently the left arm soft tissue abscesses could be interpreted as septic embolisms. The CT scan also revealed other foci of pneumonia in the right lung that were not apparent in the chest roentgenogram performed at admission. This finding, and the peripheral character of the main pneumonic focus at the left lung, could lead to the hypothesis that the primary infection was located at the subcutaneous tissue of the left arm. Therefore, the lung lesions could be septic embolisms. However, no skin wound was apparent besides the subcutaneous abscesses to support this latter pathogenetic hypothesis. Since R equi is an intracellular pathogen surviving in macrophage phagosomes, the use of two lipophylic bactericidal antibiotics for long periods is generally accepted. Erythromycin, rifampicin, vancomycin, ciprofloxacin, and imipenem have been used with different clinical results (10, 11). In our case a combination of erythromycin, ciprofloxacin, and rifampicin was started when R equi was isolated. The case reported herein constitutes the first description of R equi infection in a liver transplant recipient.Figure 1: Chest radiograph at admission: antero-posterior and lateral films. Note the round-shape lesion located at the posterobasal area of the left lung, partly overlapping the cardiac apex silhouette.Figure 2: CT scan of thorax obtained 1 week after admission. The round-shape lesion observed in the chest X-ray film corresponds to a thick-wall cavitated mass at the left lung base. In addition, at the right lung base a small consolidated area is apparent.Figure 3: CT scan of thorax obtained 2 months after the initiation of triple antibiotic therapy. Only a small residual lesion is apparent at the left lung base.
Background and Aims: News strategies for the accurate assessment of the state of immunosuppression (IS) in liver transplant recipients are needed to prevent rejection and minimize drug-related side effects. miRNAs can potentially be used as diagnostic or prognostic biomarkers in transplant patients. This study evaluated the capacity of a plasmatic miRNA panel (miR-155-5p, miR-122-5p, miR-181a-5p, and miR148-3p) as an early non-invasive prognostic and diagnostic biomarker for T cell-mediated acute rejection (TCMAR) and subclinical rejection (SCR) in adult liver recipients. Methods: A total of 145 liver recipients were included. All patients received a calcineurin inhibitor with or without mycophenolate mofetil and methylprednisolone. Plasmatic miRNA expression was assessed by qPCR before and at different time-points after liver transplantation. Results: Seventeen patients experienced TCMAR, and eight were diagnosed with SCR during the protocol biopsy at the 3rd month post-transplantation. Pre-transplantation, miR-155-5p expression was significantly higher in TCMAR patients and in SCR patients than in non-rejectors, and miR-181a-5p expression was also significantly higher in SCR patients than in non-rejectors. Post-transplantation, before transaminase-level modification, significantly increased miR-181a-5p, miR-155-5p, and miR-122-5p expression was observed in TCMAR and SCR patients. Binary logistic regression analyses showed, post-transplantation, that TCMAR risk was better predicted by individual expression of miR-181a-5p (LOGIT = -6.35 + 3.87*miR-181a-5p), and SCR risk was better predicted by the combination of miR-181a-5p and miR-155-5p expression (LOGIT = -5.18 + 2.27*miR-181a-5p+1.74*miR-155-5p). Conclusions: Pre-transplantation plasmatic miR-155-5p expression may be useful for stratifying low-immunologic-risk patients, and post-transplantation miR-181a-5p and miR-155-5p may be candidates for inclusion in early, non-invasive prognostic biomarker panels to prevent TCMAR or SCR and better identify patient candidates for IS minimization. Large prospective randomized multicenter trials are needed to refine the cut-off values and algorithms and validate the clinical usefulness of these biomarkers.
Massive ascites after liver transplantation, although uncommon, usually represents a serious adverse event. The pathogenesis of this complication has not been adequately investigated. To determine the incidence, characteristics, and pathogenic factors of massive ascites after liver transplantation (ascitic fluid > 500 mL/d for > 10 days), the charts of 378 liver transplant recipients were reviewed. Massive ascites occurred in 25 patients (7%). Mean ascitic fluid production was 960 mL/d (range, 625 to 2,350 mL/d), and the duration of ascites was 77 days (range, 15 to 223 days). The ascitic fluid had a high protein content (36 ± 7 g/L; range, 25 to 50 g/L). When patients who did and did not develop massive ascites were compared, significant differences were found in receptor sex (men, 88% v 60%, respectively; P < .01) and surgical technique (inferior vena cava preservation with piggyback technique, 72% v 41%; P < .01). Significantly increased wedged and free hepatic venous pressures and gradients between hepatic vein and right atrial pressures were found in patients who developed ascites, suggesting a difficulty in graft blood outflow. Massive ascites was associated with renal impairment, increased incidence of abdominal infection, prolonged hospitalization, and a tendency toward reduced survival. In conclusion, massive ascites after liver transplantation is relatively uncommon but associated with increased morbidity and mortality and is predominantly related to difficulties of hepatic venous drainage. Measurement of hepatic vein and atrial pressures to detect a significant gradient and correct possible alterations in hepatic vein outflow should be the first approach in the management of these patients.
Akt is expected to be an effective target for the treatment of ischemia-reperfusion injury (I/R) due to its anti-apoptotic properties and its ability to activate the endothelial nitric oxide synthase (eNOS) enzyme. Therefore, this study was aimed to determine the efficacy of an active mutant of Akt (myr-Akt) to decrease I/R injury in a model of orthotopic liver transplantation in pigs. In addition, we analyzed the contribution of nitric oxide in the Akt-mediated effects by using an eNOS mutant (S1179DeNOS) that mimics the phosphorylation promoted by Akt in the eNOS sequence. Donors were treated with adenoviruses codifying for myr-Akt, S1179DeNOS or beta-galactosidase 24 h before liver harvesting. Then, liver grafts were orthotopically transplanted into their corresponding recipients. Levels of transaminases and lactate dehydrogenase (LDH) increased in all recipients after 24 h of transplant. However, transaminases and LDH levels were significantly lower in the myr-Akt group compared with vehicle. The percentage of apoptotic cells and the amount of activated-caspase 3 protein were also markedly reduced in myr-Akt-treated grafts after 4 days of liver transplant compared with vehicle and S1179DeNOS groups. In conclusion, myr-Akt gene therapy effectively exerts cytoprotection against hepatic I/R injury regardless of the Akt-dependent eNOS activation.
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