What is the best immunosuppression in living donor liver transplantation?

INTRODUCTION: As we have learned, there are no golden rules of immunosuppression in solid organ transplantation, and every transplant program is using its own regimen to prevent or treat rejection. We have retrospectively analyzed the incidence and severity of acute rejection in a consecutive series of living donor liver transplants. The major objective during the whole study period was to ultimately avoid any steroids from the beginning. METHODS: Twenty one adult patients and five children received 23 right, one left, and two left lateral lobe grafts from genetically or emotionally related living donors, including four ABO-incompatible pairs. The majority of patients had triple initial immunosuppression, based on tacrolimus, mycophenolate mofetil or sirolimus, and basiliximab or daclizumab. Except methylprednisolone administered before reperfusion in 13 patients, only seven had prednisolone after transplantation, and 12/26 had a completely steroid-free regimen. RESULTS: The overall incidence of biopsy-proven acute rejection was 4/21 in adults (19%) and 4/5 in children (80%). Rejections were mild in five and moderate in three cases, respectively, and easily reversed with steroids in all patients. Different combinations of immunosuppressive drugs or ABO incompatibility did not seem to have an influence on the risk of rejection. CONCLUSION: Despite the small number of patients in this series, completely steroid-free triple-drug immunosuppression with tacrolimus, mycophenolate mofetil, and basiliximab is safe and efficient to prevent acute rejection in adult recipients of living donor liver transplants. At least short-term administration of prednisolone should be considered in pediatric patients.

Safety and efficacy of living donor liver preservation with HTK solution.

BACKGROUND: In living donor liver transplantation (LDLTx) organ procurement is usually well controlled, and allows to assess liver preservation and graft function under standardized conditions. Because publications on histidine-tryptophan-ketoglutarate (HTK) solution are limited, we prospectively studied its safety and efficacy in a consecutive series of LDLTx. METHODS: Twenty-four patients received 22 right, 1 left, and 1 left lateral lobe graft. Liver preservation was done by gravity perfusion with HTK through portal vein, and hepatic artery, and flushing of bile ducts. Total ischemia time was 191 +/- 68 minutes. RESULTS: There was no primary nonfunction, and all partial liver grafts showed good recovery: peak aspartate aminotransferase 577 U/L, total bilirubin 15.15 mg/dL, and partial thromboplastin time 49.37 seconds. One graft was lost from parenchymal fracture secondary to portal hyperperfusion after 6 days, and the patient was salvaged with retransplantation. Thirty-day mortality, including sudden cardiac death, pancreatitis, and hepatic artery rupture, was not related to graft dysfunction. Eight of 24 recipients developed early biliary leakage. There was no late ischemic type biliary lesion. CONCLUSION: These results confirm that HTK solution is safe and effective when used in LDLTx. Potential advantages of HTK in comparison to other preservation solutions are low potassium concentration, low viscosity, no particles, in situ perfusion, no need to flush before reperfusion, improved biliary protection, better recovery of microcirculatory changes, ready to use, and lower costs. Because the risk-benefit ratio is of particular importance in LDLTx the use of HTK solution should be encouraged.

The triple classification of dry eye for practical clinical use.

PURPOSE: "Dry Eye is a condition produced by the inadequate interrelation between lacrimal film and ocular surface epithelium, and is caused by quantitative and qualitative deficits in one or both of them. It can be produced by one or combined etiologic causes, affecting one or several of the secretions of the glands serving the ocular surface, and producing secondary manifestations of different grades of severity". Clinicians need a practical classification to face diagnosis, prognosis and treatment. Dry eyes have many etiologies and pathogenesis, different affectation of the various dacryoglands and ocular surface epithelium, and diverse grades of severity. The specialists in xero-dacryology must know these three parameters to evaluate any case of dry eye, and to establish an adequate treatment. METHODS: To facilitate this, an open session in the 8th congress of the International Society of Dacryology and Dry Eye (Madrid, April, 2005) proposed modifying the Triple Classification of dry eye approved in the XIV congress of the European Society of Ophthalmology (Madrid, June, 2003). There was consensus of all conclusions. CONCLUSIONS: The following classification has been established: First, a classification of the etio-pathogenesis, distributed in ten groups: age-related, hormonal, pharmacologic, immunopathic, hyponutritional, dysgenic, infectious/inflammatory, traumatic, neurologic and tantalic. Second, a classification of the affected glands and tissues, which under the acronym of ALMEN includes the Aqueo-serousdeficient, Lipodeficient, Mucindeficient and Epitheliopatic dry eyes, and the Non dacryological affected exocrine glands (saliva, nasal secretion, tracheo-pharyngeal secretion, etc). And thirdly, a classification of severity, in three grades: Grade 1 or mild (symptoms without slitlamp signs), grade 2 or moderate (symptoms with reversible signs), and grade 3 or severe (symptoms with permanent signs).

Retroviral gene transfer into the intestinal epithelium.

The epithelial cells of the gastrointestinal tract may be attractive targets for somatic gene therapy. In these studies, we have used rats and mice to explore the feasibility of gene transfer into the small intestinal epithelium using retroviral vectors. The first series of experiments was conducted in mature Sprague-Dawley rats using an ecotropic retroviral vector that has bacterial beta-galactosidase (beta-Gal) as the reporter gene. The vector was introduced into the lumen of ligated segments of terminal ileum. After a 4-hr exposure period, the ligatures were removed. Sham-operated animals were subjected to the same ligation procedure but received only tissue culture medium in the ligated segment. All animals were sacrificed 6 days later, and tissue from both the experimental segment and an upstream control segment was assessed for cytoplasmic beta-Gal activity using X-Gal histochemistry. Expression of the reporter gene was observed in the crypt epithelium of tissue exposed to the vector. In the villus epithelium, high background staining precluded accurate assessment of reporter gene expression. To obviate the latter problem, we sought an alternative reporter gene for which there would be no background staining in control animals. We repeated the experiments with beta-glucuronidase as the reporter gene in MPS VII mutant mice, which are devoid of this enzyme. In these studies, ileal segments exposed to the vector demonstrated expression of the reporter gene in both the crypt and villus epithelium 4 days after exposure. These results indicate that genes can be transferred into the intestinal epithelium using retroviral vectors introduced luminally.(ABSTRACT TRUNCATED AT 250 WORDS)

A critique of oral therapy of dehydration due to diarrheal syndromes.

PIP: Under the circumstances of limited health resources and immediate need for preventing the dehydration associated with diarrhea in infants, breastfeeding should be encouraged throughout the diarrheal episode. When this is not possible because of cessation or failure of lactation, an oral electrolyte solution should be administered. It should be sterile and provide a quantity of electrolytes not greatly in excess of 30 mEq/liter of sodium and potassium. There should be little possibility of an error in the dilution of the mixture if it is to be supplied in powdered form. Milk should be reintroduced after 24 hours and the electrolyte mix rapidly discontinued so as to minimize nutritional deficits. If no such electrolyte mixture is available, it is reasonable to alternate feedings of commercial soft drinks or bland teas with milk feedings. There should be specific instructions that the infant should be brought to the hydration center if more than 3 sequential feedings are lost by vomiting, if fever is present, or it the stools exceed the volume of 3 feedings. In general, dehydration of less than 5% of body weight can be managed by this program in the house, dehydration greater than 5% but less than 10% requires supervision by health authorities, and dehydration greater than 10% requires intravenous therapy in a hydration center. In those countries with cholera and during epidemics of shigellosis or enterotoxigenic Escherichia coli, solutions containing 90 mEq/liter of sodium should be given under ambulatory supervision. This solution should be discontinued when fecal losses moderate (less than 60 ml/kg/day) and the lower electrolyte solution (30 mEq/liter) substituted.^ieng

The use of DiI-marked hepatocytes to demonstrate orthotopic, intrahepatic engraftment following hepatocellular transplantation.

A novel method is described for marking primary hepatocytes with the fluorescent dye DiI prior to hepatocellular transplantation and identifying these cells within the hepatic parenchyma of recipient animals by fluorescence microscopy and flow cytometry. Optimal conditions are described for marking cells with DiI in suspension or in monolayer cultures prior to transplantation. DiI is shown to be nontoxic to hepatocytes and not to be exchanged between adjacent cells in vitro. Histological analysis of transplanted tissues shows DiI staining of engrafted hepatocytes and phagocytotic cells (Kupffer cells). This analysis shows that hepatocytes engraft within the hepatic parenchyma and exhibit a histological appearance indistinguishable from normal by conventional hematoxylin and eosin staining. Many previous reports of hepatocellular transplantation have been limited by their inability to unequivocally identify transplanted cells within the liver. These data illustrate the importance of having specific markers for transplanted cells that engraft in an orthotopic location and assume a normal morphological appearance.

DiI as a marker for cellular transplantation into solid organs.

Research in cellular transplantation is frequently compromised by an inability to identify transplanted cells engrafting into orthotopic sites if they exhibit normal morphology and no unique antigenic markers. A method is described for using the fluorescent dye DiI as a marker for cell transplantation studies. This dye is not metabolized or exchanged between cells in vitro or in vivo and enables identification of engrafted cells by fluorescence microscopy, flow cytometry or fluorescence-activated cell sorting. Applications are described in autologous hepatocellular and thyroid follicular cell transplantation.

Bismuth subsalicylate in the treatment of acute diarrhea in children: a clinical study.

Bismuth subsalicylate (BSS) and placebo were evaluated in a double-blind, placebo-controlled study as adjunct to rehydration therapy in 123 children, aged 4 to 28 months, hospitalized with acute diarrhea. The dosing regimen was 20 mg/kg five times daily for 5 days. Significant benefits were noted in the BSS group compared with placebo as manifested by decreases in stool frequency and stool weights and an improvement in stool consistency, significant improvement in clinical well-being, and shortening of the disease duration. Patients treated with BSS had a significant reduction in duration of hospital stay (6.9 days) compared with placebo-treated patients (8.5 days). Also, intravenous fluid requirements decreased significantly more rapidly and to a greater degree in the BSS-treated group. Bismuth subsalicylate was associated with clearance of pathogenic Escherichia coli from the stools in 100% of cases but was not different from placebo in rotavirus elimination. Bismuth subsalicylate was well tolerated with no reported adverse effects. Blood bismuth and serum salicylate levels were well below levels considered toxic. In this study, BSS provided effective adjunctive therapy for acute diarrhea, allowing children to get well sooner with less demand on the nursing and hospital staff.

A "real time" PCR assay to detect transplanted human liver cells in RAG-1-/- mice.

Xenotransplantation of human liver cells is an expanding field in need of new and precise quantitative techniques. "Real time" PCR is a sensitive and accurate method of quantifying picogram quantities of DNA. We used "real time" PCR with primers complementary to the human alpha-1-antitrypsin gene to assess the efficiency of engraftment of human liver cells transplanted into immunotolerant RAG-1-/- mice. Standard curves were created by mixing known proportions of human and mouse cells. There was a linear relationship between the PCR cycle at which DNA was amplified [threshold cycle (C(T)] and the percent human cells (linear regression, p < 0.00009). Results were reliable, with a maximum 1.27-fold variation in the slopes of repeated standard curves. Linearity was maintained from 100% to as low as 0.01%. Therefore, 1 in 10,000 mouse cells could be detected in a 100 ng DNA sample. We measured the percent engraftment of human liver cells transplanted into the spleen of RAG-1-/- mice. By "real time" PCR assay, 0.23% human cells could be detected at 1 day after human cell transplantation. These results show that "real time" PCR assay is highly sensitive, reproducible, and accurate for detecting human cells in xenotransplanted mice.

Apoptosis occurs in isolated and banked primary mouse hepatocytes.

Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.

DNA binding by C/EBP proteins correlates with hepatocyte proliferation.

The leucine zipper transcription factors C/EBP alpha and C/EBP beta exhibit growth-related variations of expression and DNA binding during liver regeneration. We examined the expression of C/EBP proteins in relation to hepatocyte proliferation by studying their DNA-binding activity in primary mouse hepatocytes in vitro. Mouse hepatocytes were dissociated by collagenase perfusion and cultured in a serum-free, defined medium containing a variety of growth factors and hormones. Cell protein extracts were collected every 24 h for up to 10 d and examined for DNA-binding activity by gel retardation analysis using a C/EBP consensus sequence oligomer (bZIP). C/EBP alpha is the major bZIP-binding protein present in the dissociated cells prior to plating. With the culture conditions we employed, little or no binding of C/EBP proteins was observed in the first 24 to 48 h of cultivation. After 48 h, C/EBP beta binding activity was elevated relative to the level seen in freshly dissociated cells. In contrast, C/EBP alpha binding continued to be greatly reduced and no C/EBP delta binding was observed. C/EBP beta binding remained elevated for the duration of the experiment. Additional growth factor treatment (EGF, FGF, TGF alpha, and HGF) of the hepatocytes did not appreciably alter the pattern of C/EBP binding. However, TGF beta treatment, known to decrease hepatocyte proliferation, increased C/EBP beta binding activity earlier and more actively than in control cells. This study confirms a negative correlation between DNA binding by the C/EBP transactivator proteins and the proliferation of primary mouse hepatocytes in vitro.

Induction of three-dimensional assembly of human liver cells by simulated microgravity.

The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

Limy bile syndrome.

Limy bile syndrome (LBS) is a rare condition in which a radiopaque gallbladder and/or bile ducts are noted on plain roentgenograms. LBS is caused by calcium carbonate precipitation in the bile and is usually associated with distal biliary tract obstruction. The etiology of limy bile syndrome is unclear; however, it may be a long-term complication of total parenteral nutrition.

Cultures of human liver cells in simulated microgravity environment.

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

Hemodynamic indexes in newborns using the arteriovenous oxygen content difference.

OBJECTIVE: To determine in peripheral blood samples of newborns (NB) cardiac output (Q), cardiac index (CI), systemic vascular resistance index (SVRI), and effective oxygen transport (EO2T), through arteriovenous oxygen content difference ([C(a-v) O2]). DESIGN: Comparative survey. SETTING: Healthy NBs and NBs in intermediate care in third level medical attention units. MATERIAL AND METHODS: Forty-seven NB (17 pre-term) were prospectively studied in August and September/1995. A blood sample of 0.4 mL was taken from the umbilical or femoral vein and from the umbilical, radial or femoral artery. The inferencial statistics were done with a t test and Pearson's correlation coefficient. Significance was considered if p < 0.05. RESULTS: Cardiac output ranged from 0.3 to 1.4, mean = 0.6 L/min +/- 0.24 (+/- SD); CI ranged from 1.8 to 6.4 L/min/m2 body surface area (mean = 3.3 +/- 1.2); SVRI ranged from 533 to 2,391 dyne/sec/cm-5/m2 BSA (mean = 1,317 +/- 494); EO2T ranged from 307 to 1,017 mL/min/m2 BSA (mean = 549 +/- 186); the [C(a-v) O2] ranged from 3.1 to 10.7% in volume (mean = 6.8 +/- 2.1). No significant differences were found in Q between pre-term and full-term NB nor was there any correlation between Q and gestational age. CONCLUSIONS: The [C (a-v)O2] is a good alternative to obtain indexes in peripheral blood of NB without cardiopathy, whenever other less invasive and more sophisticated methods are unavailable. In order to calculate the indexes in critically-ill patients, it is necessary to measure O2 consumption prior to applying this method.