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).

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.

Funduplicatura de Nissen por laparoscopia como técnica de elección para el tratamiento de la enfermedad por reflujo gastroesofágico / Laparoscopic nissen fundoplication as the technique of choice in the treatment of gastroesophageal reflux disease

Introducción y objetivos. Pretendemos corroborar, mediante la aportación de nuestros resultados y la comparación de los datos manométricos y pHmétricos, pre y postoperatorios, que la funduplicatura de Nissen por vía laparoscópica es la técnica de elección para el tratamiento de la enfermedad por reflujo gastroesofágico (ERGE), obteniéndose resultados similares a los de la cirugía convencional. Material y métodos. Un total de 72 pacientes, intervenidos consecutivamente, afectados de ERGE, 54 varones y 18 mujeres, con una edad media de 42 años. Todos presentaban clínica de ERGE, con una media de evolución de 5 años y habían recibido tratamiento médico correcto durante una media de 24 meses. El estudio preoperatorio incluyó: esofagogastroduodenoscopia, TEGD, pHmetría de 24 h y manometría esofágica. La técnica quirúrgica practicada fue una funduplicatura de Nissen por vía laparoscópica. En el control postoperatorio se realizaron: esofagogastroduodenoscopia, pHmetría de 24 h y manometría esofágica, valorándose el grado de satisfacción mediante la escala de Visick. Los datos obtenidos se compararon estadísticamente mediante el test de la t de Student apareada y el test de Wilcoxon. Resultados. La pHmetría preoperatoria reveló una media de porcentaje de tiempo de pH < 4 del 9,85 por ciento y un índice de DeMeester de 37,4. La manometría dio unas presiones basales medias del EEI de 11,6 mmHg y una longitud media del mismo de 3,13 cm. El tiempo medio de la intervención fue de 75,4 min y las complicaciones intraoperatorias se produjeron en 11 pacientes, siendo el índice de reconversiones a cirugía abierta del 1,3 por ciento. La pHmetría postoperatoria obtuvo un índice de DeMeester medio de 6,06 y un porcentaje de tiempo de pH < 4 medio de 1,1 por ciento. La longitud media del EEI, en el estudio manométrico postoperatorio, fue de 3,5 cm y la presión basal media del mismo, de 20,9 mmHg. Al ser comparados los resultados preoperatorios con los postoperatorios, todos ellos resultaron ser estadísticamente significativos (p < 0,05).Conclusiones. Tras una experiencia de cuatro años y medio en funduplicatura de Nissen por laparoscopia, y a la vista de los resultados obtenidos, pensamos que es la técnica quirúrgica de elección para el tratamiento de la ERGE (AU)

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.

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.

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.

Risks of endoscopy in hospitalized pediatric patients with collagen vascular diseases.

BACKGROUND: The gastrointestinal manifestations of the collagen vascular diseases have been well described in the pediatric population. These patients frequently have symptoms that constitute indications for endoscopy. However, the risks and benefits of endoscopy in this population have not been examined. METHODS: A retrospective review of all patients with collagen vascular diseases hospitalized during a 7-year period was undertaken, and those patients who underwent endoscopy were identified. RESULTS: Nine patients (5%) underwent endoscopic procedures (eight upper and three lower endoscopy). Complications and outcomes were analyzed. Indications for endoscopy included abdominal pain, gastrointestinal (GI) bleeding, and/or vomiting and diarrhea. Two patients had complications that required surgery within 1 day of the endoscopic procedure. One of these patients subsequently died with GI bleeding. Five of the nine patients had changes in their management after endoscopy. Helicobacter pylori infection was identified and treated in two patients. Three patients had esophagitis or gastritis and acid suppression treatment was started or optimized. Vasculopathy was present in the patients who had complications. CONCLUSIONS: This series suggests that endoscopy can provide useful information for the management of the pediatric patient with GI symptoms and collagen vascular diseases. However, because serious and potentially life-threatening complications can occur, great care is needed in evaluating the risk/benefit ratio of endoscopy in these patients.

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.

Immunosuppressive therapy with microemulsion cyclosporine A shortens the hospitalization of pediatric liver transplant recipients.

Intravenous administration of cyclosporine (Sandimmune) to rapidly and effectively achieve therapeutic serum levels in transplant recipients has been the treatment standard in many transplantation centers. With the development of microemulsion cyclosporine (Neoral), that standard is changing. Neoral has greater bioavailability than the oral form of Sandimmune and, consequently, can be more efficacious and cost-effective. To test this hypothesis, we undertook a retrospective study of Sandimmune and Neoral in the treatment of 66 children who underwent uncomplicated orthotopic liver transplantation in the Texas Medical Center between April 1991 and December 1997. Both forms of cyclosporine were evaluated in terms of in-patient treatment cost, recuperative time in the intensive care unit and duration of hospitalization. Twenty-two patients were treated orally with Neoral, and 44 patients were treated intravenously with Sandimmune for a mean time of 14 d. Once the blood concentration of Sandimmune reached a steady state, as confirmed by daily measurements of the trough level, the patients in the Sandimmune group were converted to oral cyclosporine. None of the 22 patients treated with Neoral required intravenous treatment. The mean time spent in the intensive care unit was 4 d for the Neoral group and 5.5 d for the Sandimmune group. The mean duration of hospitalization from the date of transplantation to discharge was 12 d for the Neoral group and 20 d for the Sandimmune group (p < 0.001). Based on these results, we determined that the overall cost per patient in the Neoral group was $3598 less than that per patient in the Sandimmune group.

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.

Lack of C/EBP alpha gene expression results in increased DNA synthesis and an increased frequency of immortalization of freshly isolated mice [correction of rat] hepatocytes.

The CCAAT/enhancer binding protein alpha (C/EBP alpha) binds to specific promoter sequences and directs transcription of many genes expressed in the liver. Overexpression of C/EBP alpha in established cell lines inhibits cell proliferation. Primary hepatocytes from newborn C/EBP alpha(-/-) mice and normal littermates were used to determine whether the absence of C/EBP alpha increased proliferation and/or transformation of these cells in vitro. DNA synthesis, as measured by bromodeoxyuridine (BrdU) incorporation 24 hours postharvest, was fourfold higher in cells from C/EBP alpha(-/-) pups. Established cell lines were derived from 7 of 8 hepatocyte cultures initiated from null mutants, 4 of 23 cultures from heterozygotes, and 0 of 12 cultures from wild-type animals. C/EBP alpha(-/-) cultures had epithelial morphology, showed bile canaliculi, and expressed albumin messenger RNA (mRNA). When cultured on Matrigel, which promotes differentiation, cell lines derived from C/EBP alpha(-/-) mice formed cords and increased albumin mRNA expression by 1.7- to 3.8-fold. C/EBP alpha(-/-) cell lines exhibited rapid growth and rapid accumulation of chromosomal abnormalities, and were capable of forming nodules when inoculated into the abdominal subcutaneous tissue of nude mice. Our data show that C/EBP alpha is an important regulator of hepatocyte proliferation and participates in the maintenance of the nontransformed hepatic phenotype in vitro.

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.

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)