A deeply invasive Phoma species infection in a renal transplant recipient.

Phoma sp, a fungus routinely isolated from the soil and a known plant pathogen, was found to be the cause of an aggressive, deep compartment hand infection in a renal transplant recipient. Previous reports have described minimally invasive Phoma sp infections with isolates recovered from the skin or subcutaneous tissue. This case, however, is the first reported in which Phoma sp was found to be both aggressive and deeply invasive. Histologic sections obtained from the synovium of the fourth and fifth dorsal hand compartments revealed invasive hyphal elements. Detailed examination with Grocott-Gomori methenamine-silver staining revealed branching filaments and pycnidia. A Phoma sp was isolated from culture after 2 weeks of incubation. Antifungal agent sensitivity testing found the organism to be sensitive to amphotericin B but resistant to both fluconazole and 5-flucytosine. Treatment required surgical debridement and the use of prolonged systemic amphotericin B therapy in order to effect cure. This is a unique case of a deeply invasive Phoma sp infection, indicating that such processes are not strictly indolent as previously reported.

Liver and circulating NK1.1(+)CD3(-) cells are increased in infection with attenuated Salmonella typhimurium and are associated with reduced tumor in murine liver cancer.

An attenuated (DeltacyA, Deltacrp) strain of Salmonella typhimurium (chi4550) containing a gene for human IL-2 (chi4550pIL2) reduces hepatic tumor burden when orally inoculated into mice with liver cancer; however, wild-type S. typhimurium is also associated with cancer regression. Therefore, experiments were designed to clarify the invasiveness and the anti-tumor properties of three strains of S. typhimurium. S. typhimurium chi4550pIL2, chi4550, or wild type (WT) was incubated with mature Caco-2 and HT-29 enterocytes, and S. typhimurium internalization was assessed. For infectivity experiments, mice were orally inoculated with saline or 10(9)S. typhimurium chi4550pIL2, chi4550, or WT; 48 h later mice were sacrificed for analysis of cecal bacteria and S. typhimurium translocation to mesenteric lymph nodes. For experiments involving tumor implantation, four groups were studied: saline control, tumor alone, chi4550pIL2+tumor, and chi4550+tumor. Mice were orally inoculated with saline or S. typhimurium and underwent laparotomy 24 h later with 5 x 10(4) MCA38 murine adenocarcinoma cells injected into the spleen. On day 14, liver tumors were counted and peripheral blood and hepatic lymphocyte populations were analyzed by FACScan. Attenuated S. typhimurium exhibited decreased internalization by cultured enterocytes and decreased infectivity after oral inoculation. Mice treated with chi4550pIL2 or chi4550 had fewer liver tumors and increased populations of hepatic and circulating NK1.1(+)CD3(-) lymphocytes compared to mice treated with saline (P < 0.01). These data suggest that attenuated S. typhimurium may have an application as an anti-tumor agent.

Recurrence of hydatid disease.

Surgical management of hepatic hydatid disease has been associated with an overall local recurrence rate of approximately 10%. Local recurrence is rarely seen following complete resection of an intact cyst and is usually the result of spillage of live parasites or leaving a residual cyst wall containing germinal epithelium, daughter cysts, or protoscolices during surgery. Recurrence is frequently asymptomatic, so the diagnosis depends on dedicated follow-up of treated patients with serology and either ultrasonography or computed tomography. The management of locally recurrent disease should include administration of albendazole followed by the appropriate application of interventional radiotherapy or operation. As with the treatment of primary disease, the preservation of liver function and minimizing the risk to the patient remain the guiding principles of therapy of local recurrence.

Split liver transplantation for two adult recipients: an initial experience.

The shortage of cadaver donor livers has been most severe for adult patients. Split liver transplantation is one method to expand the donor pool, but to have a significant impact on the waiting list, it needs to be applied for 2 adult recipients. We split livers from 6 cadaver donors, and transplanted 12 adult recipients. All splits were performed in situ with transection through the midplane of the liver, resulting in a right lobe and a left lobe graft. Mean donor age was 19.7 years; mean donor weight was 79.1 kg. Mean recipient age was 41.5 years. Mean weight of right lobe recipients was 89 kg; left lobe recipients, 60 kg. All donors were hemodynamically stable and had normal liver function tests. Mean operative time for the procurement was 7.4 h. Average blood loss during the transection of the liver was 490 mL. Mean GW/ RW ratio for all recipients was 0.87%; right lobe recipients, 0.86%; and left lobe recipients, 0.88%. With mean follow-up of 9.3 months, patient and graft survival rates were both 83.3%. There were 2 deaths: 1 after hepatic artery thrombosis (HAT) and subsequent multiorgan failure; the other after HAT, a liver retransplant, and subsequent gram-negative sepsis. The remaining 10 recipients are doing well. We observed no cases of primary nonfunction. Other complications included bile leak and/or stenosis (n = 3), bleeding from the Roux loop (n = 1), bleeding after percutaneous biopsy (n = 1), and incisional hernia (n = 1). In conclusion, split liver transplantation, using 1 cadaver liver for 2 adult recipients, can be performed successfully. Crucial to success is proper donor and recipient selection.

Hepatic resection of noncolorectal nonneuroendocrine metastases.

Because hepatic resection is generally a safe procedure, the indications for resection of noncolorectal nonneuroendocrine (NCNNE) hepatic metastases have broadened. The prognostic features of NCNNE metastases treated surgically were reviewed to define better the value of resection. A retrospective review of patients undergoing liver resection for NCNNE metastases between 1978 and 1998 was undertaken. Thirty-seven patients were identified. Mean age was 56 years, with a median follow-up of 22 months. Primary tumor sites were grouped into gastrointestinal (GI) adenocarcinoma (small bowel, n = 4; pancreas, n = 2; esophagus, n = 1) and other (renal cell, n = 7; sarcoma, n = 7; melanoma, n = 5; adrenal, n = 3; unknown adenocarcinoma, n = 3; thyroid, n = 2; testicular, n = 1; ovarian, n = 1; breast, n = 1). All patients underwent surgery for cure. Metastases were synchronous in 14 patients. There was no surgical mortality. Overall 5-year survival rate was 45%. Five-year survival rates were better for patients with non-GI-origin metastases (60% v 0%; P =.01). Long-term survival was seen only in patients with non-GI-origin metastases. The extent of resection, presence of synchronous metastases, or disease-free interval from time of original disease to presentation with liver metastases were not predictive of outcome. We conclude that patients with NCNNE hepatic metastases can undergo liver resection with an expectation of prolonged survival. However, patients with liver metastases from GI primary tumors other than the colorectum are unlikely to show extended survival.

Extracorporeal tissue engineered liver-assist devices.

The treatment of acute liver failure has evolved to the current concept of hybrid bioartificial liver (BAL) support, because wholly artificial systems have not proved efficacious. BAL devices are still in their infancy. The properties that these devices must possess are unclear because of our lack of understanding of the pathophysiology of liver failure. The considerations that attend the development of BAL devices are herein reviewed. These considerations include choice of cellular component, choice of membrane component, and choice of BAL system configuration. Mass transfer efficiency plays a role in the design of BAL devices, but the complexity of the systems renders detailed mass transfer analysis difficult. BAL devices based on hollow-fiber bioreactors currently show the most promise, and available results are reviewed herein. BAL treatment is designed to support patients with acute liver failure until an organ becomes available for transplantation. The results obtained to date, in this relatively young field, point to a bright future. The risks of using xenogeneic treatments have yet to be defined. Finally, the experience gained from the past and current BAL systems can be used as a basis for improvement of future BAL technology.

Characterization of the three-compartment gel-entrapment porcine hepatocyte bioartificial liver.

A hybrid bioartificial liver device supporting a large mass of cells expressing differentiated hepatocyte metabolic capabilities is necessary for the successful treatment of fulminant hepatic failure. The three-compartment gel-entrapment porcine hepatocyte bioartificial liver was designed to provide "bridge" support to transplantation or until native liver recovery is achieved for patients with acute liver failure. The device is an automated mammalian cell culture system supporting 6-7 x 10(9) porcine hepatocytes entrapped in a collagen matrix and inoculated into the capillary lumen spaces of two 100 kDa molecular mass cut-off hollow fiber bioreactors. Gel contraction recreates a small lumen space within the hollow fiber which allows for the delivery of a nutrient medium. This configuration supported hepatocyte viability and differentiated phenotype as measured by albumin synthesis, ureagenesis, oxygen consumption, and vital dye staining during both cell culture and ex vivo application. The hollow fiber membrane was also shown to isolate the cells from xenogenic immunoglobulin attack. The gel-entrapment bioartificial liver maintained a large mass of functional hepatocytes by providing a three-dimensional cell culture matrix, by delivering basal nutrients through lumen media perfusion, and by preventing rejection of the xenocytes. These features make this device a favorable candidate for the treatment of clinical fulminant hepatic failure.

Development of a bioartificial liver employing xenogeneic hepatocytes.

Liver failure is a major cause of mortality. A bioartificial liver (BAL) employing isolated hepatocytes can potentially provide temporary support for liver failure patients. We have developed a bioartificial liver by entrapping hepatocytes in collagen loaded in the luminal side of a hollow fiber bioreactor. In the first phase of development, liver-specific metabolic activities of biosynthesis, biotransformation and conjugation were demonstrated. Subsequently anhepatic rabbits were used to show that rat hepatocytes continued to function after the BAL was linked to the test animal. For scale-up studies, a canine liver failure model was developed using D-galactosamine overdose. In order to secure a sufficient number of hepatocytes for large animal treatment, a collagenase perfusion protocol was established for harvesting porcine hepatocytes at high yield and viability. An instrumented bioreactor system, which included dissolved oxygen measurement, pH control, flow rate control, an oxygenator and two hollow fiber bioreactors in series, was used for these studies. An improved survival of dogs treated with the BAL was shown over the controls. In anticipated clinical applications, it is desirable to have the liver-specific activities in the BAL as high as possible. To that end, the possibility of employing hepatocyte spheroids was explored. These self-assembled spheroids formed from monolayer culture exhibited higher liver-specific functions and remained viable longer than hepatocytes in a monolayer. To ease the surface requirement for large-scale preparation of hepatocyte spheroids, we succeeded in inducing spheroid formation in stirred tank bioreactors for both rat and porcine hepatocytes. These spheroids formed in stirred tanks were shown to be morphologically and functionally indistinguishable from those formed from a monolayer. Collagen entrapment of these spheroids resulted in sustaining their liver-specific functions at higher levels even longer than those of spheroids maintained in suspension. For use in the BAL, a mixture of spheroids and dispersed hepatocytes was used to ensure a proper degree of collagen gel contraction. This mixture of spheroids and dispersed cells entrapped in the BAL was shown to sustain the high level of liver-specific functions. The possibility of employing such a BAL for improved clinical performance warrants further investigations.

Mycoplasma hominis infections occurring in cardiovascular surgical patients.

BACKGROUND: Postoperative Mycoplasma hominis sternal would or mediastinal infections are uncommon and difficult to diagnose. Atypical growth characteristics in routine bacterial culture, and the inability to demonstrate the organism on Gram stain, lead to delayed diagnosis of M hominis infections and increased morbidity. METHODS: Postoperative purulent would drainage or acute mediastinitis caused by M hominis developed in 3 cardiovascular surgery patients. These patients were considered along with 9 patients previously reported in the literature. RESULTS: Operative findings included moderately thick, gray purulent fluid with the degree of tissue necrosis related to duration of infection. Intraoperative Gram stain of wound or mediastinal drainage demonstrated no microorganisms, and initial bacterial cultures did not reveal microbial growth. After an average of 4.5 days of culture, minute translucent colonies of M hominis were identified. The institution of appropriate antimycoplasma therapy (doxycycline and clindamycin) was associated with clinical or microbiological cure in all patients. Sternal wound complications developed in 3 patients, and a chronic infection developed in 1 patient. CONCLUSIONS: Empiric therapy for M hominis infection should be considered in patients with mediastinitis or a sternal wound infection in which organisms are not observed on Gram stain and are not readily cultured.

Review of support systems used in the management of fulminant hepatic failure.

Fulminant hepatic failure has an exceedingly high mortality. Liver transplantation is the treatment option of choice. Unfortunately, one-third of patients with fulminant hepatic failure die awaiting a donor liver. For over 35 years attempts to remove or dilute putative toxins in the blood have been unsuccessful in improving survival rates. The use of biocompatible interfaces with blood or plasma and current hepatocyte culture techniques have led to the development of new support systems. This generation of bioartificial livers will hopefully provide the necessary hepatic functions and prevent many of the complications associated with fulminant hepatic failure. This paper will review the support systems tried and currently under investigation, with an emphasis on bioartificial livers.

Effects of hepatocyte growth factor on viability and biotransformation functions of hepatocytes in gel entrapped and monolayer culture.

OBJECTIVES: An extracorporeal bioartificial liver device must maintain viability and differentiated function of hepatocytes cultivated at high cell density. Growth factors, such as hepatocyte growth factor, found in high concentrations in the plasma of patients with fulminant hepatic failure, have the potential to promote hepatocyte dedifferentiation and thus, decrease function. We tested the hypothesis that hepatocyte growth factor would improve viable cell density and decrease biotransformation functions of liver cells in monolayer culture and in hepatocytes entrapped in collagen cylindrical gel "noodles" as found in the extracorporeal bioartificial liver. DESIGN: In vitro, controlled study. SETTING: University research laboratory. SUBJECTS: Adult Sprague Dawley Rats. INTERVENTIONS: Hepatocytes were harvested by a two-step collagenase technique. Harvested hepatocytes were plated onto type 1 collagen coated plates or entrapped in type 1 collagen cylindrical gels and cultured in different concentrations of hepatocyte growth factor. Interval measurements of 3H-thymidine incorporation, albumin synthesis, biotransformation functions, and viability were made. MEASUREMENTS AND MAIN RESULTS: In monolayer culture, the addition of hepatocyte growth factor caused a dramatic increase in 3H-thymidine incorporation. This increase was accompanied by a decrease in the appearance of the lidocaine metabolite, monoethyglycinexylidide. Albumin production was unchanged. In cylindrical gel entrapment cultures, hepatocyte growth factor caused a significant increase in 2-day viability but had no effect on the metabolite appearance of lidocaine or 4-methyl umbelliferone or albumin production. CONCLUSIONS: Hepatocyte growth factor induces dedifferentiation of hepatocytes in monolayer culture. Collagen matrix entrapment appears to abrogate this effect and improve liver cell viability. There may be reciprocal regulation of hepatocyte reproductive and differentiated functions, such as biotransformation, which can be influenced by the entrapment of hepatocytes in an extracellular type 1 collagen matrix.

Gel-entrapment bioartificial liver therapy in galactosamine hepatitis.

A need exists for an effective, safe bioartificial liver to support patients in fulminant hepatic failure (FHF). The purpose of this study was to determine the treatment efficacy of the novel gel-entrapment porcine hepatocyte bioartificial liver (BAL) in a fatal model of canine hepatic failure. FHF was produced in 27- to 30-kg halothane-anesthetized dogs by bolus infusion of the hepatotoxin D-galactosamine (D-Gal). Three groups were studied during the 48-hr experiment: Group D-Gal (n = 5) received galactosamine, 1.0 g/kg, iv at Time O, Group HepBAL (n = 5) received D-Gal followed by continuous hemoperfusion with the BAL device loaded with approximately 6 billion viable pig hepatocytes starting at Time 24 hr, and three dogs served as healthy controls (Group Control) and received no galactosamine. The primary endpoints were survival and coma development. Group D-Gal demonstrated 100% mortality from liver failure by 42 hr, characterized by a progressive rise in liver enzymes, total bilirubin, ammonia, and lactate and associated with coagulopathy, hypoglycemia, coma, and brain death. BAL therapy significantly delayed the onset of coma and improved survival (median 47 hr vs D-Gal median 36 hr). A significant delay in the rise of lactate and ammonia was also noted. BAL therapy prolonged survival and improved both laboratory and clinical markers of fatal liver failure. These data indicate that this BAL may have clinical utility in supporting human liver failure.

A technique for porcine hepatocyte harvest and description of differentiated metabolic functions in static culture.

Current bioartificial liver devices are based on the use of a large mass of hepatocytes exhibiting differentiated metabolic function. The pig has become a source of interest for the acquisition of such cells-however, harvesting a large mass of highly viable cells has met with difficulty. This study describes a technique for harvesting large quantities of hepatocytes at viabilities greater than 90% and also describes several features documenting differentiated function. Pigs, 6 to 10 kg body weight, underwent in situ two-step whole liver perfusion (ethylene glycol tetraacetic acid and collagenase) and ex vivo cell harvest. Harvests yielded an average of 19.5 billion cells with an average viability of 94.6%. Hepatocytes were then entrapped in type I collagen (3 x 10(5) cells/well) and cultured in serum-free media for 5 days. Pig hepatocytes produced stable amounts of albumin and maintained cytochrome P-450 and glucuronidation activity over 5 days, as shown by the metabolism of lidocaine and 4-methylumbelliferone. These data indicate that pig hepatocytes can be harvested with high yields and can retain viability and differentiated function over at least 5 days of culture, and therefore should prove to be an excellent source of hepatocytes for bioartificial liver devices.

An anesthetized model of lethal canine galactosamine fulminant hepatic failure.

A reproducible large animal model of fulminant hepatic failure was developed in the anesthetized dog by the administration of the amino sugar D-galactosamine. Galactosamine in 5% dextrose in water (D5W), was given as an intravenous bolus to 10 young male dogs weighing 27 to 30 kg. Three dogs that received an equal volume of D5W alone served as controls. Galactosamine at 0.5 g/kg (n = 5) produced significant biochemical evidence of liver injury with 100% survival at 48 hours. Galactosamine 1.0 g/kg (n = 5) yielded in 100% 48-hour mortality resulting from fulminant liver failure characterized by a progressive increase in liver enzymes, total bilirubin, ammonia, and lactate and associated coagulopathy, hypoglycemia, coma, and increased intracranial pressure. Necropsy showed liver pallor, ascites, and brain swelling. Liver histology showed significant hepatocellular necrosis. This clinically relevant large animal model will enable the quantitative evaluation of new technologies, such as the bioartificial liver, for the support of hepatic failure in humans.

Extracorporeal application of a gel-entrapment, bioartificial liver: demonstration of drug metabolism and other biochemical functions.

Metabolic activity of a gel-entrapment, hollow fiber, bioartificial liver was evaluated in vitro and during extracorporeal hemoperfusion in an anhepatic rabbit model. The bioartificial liver contained either 100 million rat hepatocytes (n = 12), fibroblasts (n = 3), or no cells (n = 7) during hemoperfusion of anhepatic rabbits. Eight other anhepatic rabbits were studied without hemoperfusion as anhepatic controls, and three sham rabbits served as normal controls. Albumin production rates (mean +/- SEM) were similar during in vitro (17.0 +/- 2.8 micrograms/h) and extracorporeal (18.0 +/- 4.0 micrograms/h) application of the hepatocyte bioartificial liver. Exogenous glucose requirements were reduced (p < 0.01) and euglycemia was prolonged (p < 0.001) in anhepatic rabbits treated with the hepatocyte bioartificial liver. The maximum rate of glucose production by the hepatocyte bioartificial liver ranged from 50-80 micrograms/h. Plasma concentrations of aromatic amino acids, proline, alanine, and ammonia were normalized in anhepatic rabbits during hepatocyte hemoperfusion. Gel-entrapped hepatocytes in the bioartifical liver performed sulfation and glucuronidation of 4-methylumbelliferone. P450 activity was demonstrated during both in vitro and extracorporeal application of the BAL device by the formation of 3-hydroxy-lidocaine, the major metabolite of lidocaine biotransformation by gel-entrapped rat hepatocytes. In summary, a gel-entrapment, bioartificial liver performed multiple hepatocyte-specific functions without adverse side effects during extracorporeal application in an anhepatic, small animal model. With its potential for short term support of acute liver failure, scale-up of the current bioartificial liver device is indicated for further investigations in large animal, preclinical trials.

Ibuprofen prevents deterioration in static transpulmonary compliance and transalveolar protein flux in septic porcine acute lung injury.

UNLABELLED: The effects of intravenous ibuprofen on measurements of pulmonary function and alveolar capillary membrane permeability to protein in sepsis-induced porcine acute lung injury (ALI) were studied. Young swine (15-25 kg) were anesthetized, cannulated, and ventilated (5 cm H2O PEEP, 0.5 FIO2, and 15 cc/kg tidal volume). Three groups were studied: septic animals (Ps, n = 10) received Pseudomonas aeruginosa for 1 hr IV, controls (C, n = 9) received 0.9% NaCl, and ibuprofen-treated septic animals (Ps + Ibu, n = 7) received ibuprofen 12.5 mg/kg at 0 and 120 min post Ps. Systemic (SAP) and pulmonary (PAP) arterial pressures, PaO2, cardiac index (CI), static lung compliance (CL), EVLW (thermal cardiogreen), and peripheral white blood cell counts (WBC) were measured. Bronchoalveolar lavage (BAL) was performed for protein and % neutrophil (%PMN) content. RESULTS: Ps produced significant (p less than 0.05) decreases in CL, PaO2, SAP, CI, and peripheral WBC and increases in PAP, EVLW, BAL protein, and %PMN's vs. controls. Ibu prevented the early increase in PAP and attenuated the late increase in PAP and EVLW. Ibu also maintained PaO2, CL, BAL protein, and %PMN's in BAL at control levels, but exhibited no significant effect on peripheral leukopenia. These data strongly suggest that ibuprofen administered before and at 120 min after onset of Pseudomonas infusion improves lung compliance and affects neutrophil function sufficiently to significantly ameliorate many of the physiologic derangements in acute sepsis.

Platelet-activating factor in porcine Pseudomonas acute lung injury.

We investigated the role of platelet-activating factor (PAF) in acute septic lung injury by examining the effects of the selective PAF antagonist SRI 63-675 and by measuring PAF in lung tissue in the porcine model. Four groups of pigs (15-25 kg) were studied: saline control (C, n = 5); Pseudomonas (Ps, n = 9), given 5 x 10(8) CFU/ml at 0.3 ml/20 kg/min intravenously over 1 hr; SRI (n = 3), given SRI 63-675 in a 40 mg/kg bolus; and SRI + Ps (n = 5). Ps infusion produced a fulminant lung injury characterized by a threefold increase in pulmonary arterial pressure at 30 min and persistent pulmonary hypertension (P less than 0.05 vs C), a significant (P less than 0.05 vs C) decrease in arterial oxygen tension (PaO2) from 60 min, a significant (P less than 0.05 vs C) increase in extravascular lung water (EVLW) from 120 min, and a significant (P less than 0.05 vs C) increase in albumin flux determined scintigraphically as slope index at 150-180 min. Systemic arterial pressure and cardiac index (CI) decreased significantly (P less than 0.05) in the Ps group vs C at 60 and 180 min, respectively. Bolus injection of SRI 63-675 at the time of Ps infusion blocked the early pulmonary hypertension, attenuated the early and late fall in PaO2, ameliorated the increase in EVLW, and prevented the late (150-180 min) increase in albumin flux. SRI 63-675 had minimal effects on Ps-induced hypotension or alterations in CI.(ABSTRACT TRUNCATED AT 250 WORDS)

Pulmonary compliance: early assessment of evolving lung injury after onset of sepsis.

We compared the sensitivity of dynamic (Cdyn) and static lung compliance (CL) with indicators of permeability injury in a model of septic porcine adult respiratory distress syndrome. Two groups of anesthetized ventilated swine (15-25 kg) were studied. Septic animals (Ps, n = 13) received Pseudomonas aeruginosa intravenously for 1 h, which resulted in severe adult respiratory distress syndrome. Controls (C, n = 13) received 0.9% NaCl. Cdyn, CL, bronchoalveolar lavage for protein estimation, and thermal cardiogreen extravascular lung water (EVLW) measurements were performed in seven C and eight Ps animals. Six C and five Ps animals underwent gamma camera measurement of lung-to-heart ratio (slope index) of 99Tc-labeled human serum albumin. Both Cdyn and CL decreased significantly (P less than 0.01) at 30 min and thereafter in Ps vs. C. EVLW, slope index, and bronchoalveolar protein content increased significantly (P less than 0.05) in Ps vs. C at 120, 150, and 300 min, respectively. Cdyn and CL decreased well before onset of permeability injury. These early changes may be due to release of vasoactive mediators and sequestration of neutrophils in the pulmonary capillaries and later to increases in EVLW. Measurement of Cdyn and CL represents an early means of assessing evolving lung injury in this acute septic porcine model.