Influence of preservation solution on the isolation and culture of human hepatocytes from liver grafts

A major problem for the isolation and transplantation of hepatocytes is the lack of resources for obtaining viable hepatocytes. Improving this situation would enhance hepatic cell transplantation programs. Our objective was to evaluate the influence of the preservation solutions used during organ retrieval on the quality of hepatocytes isolated from liver tissue. We compared the results of the collagenase perfusion technique for isolation of hepatocytes in human livers flushed with University of Wisconsin (UW) and Celsior preservation solutions. Yield (number of viable cells per gram of tissue), cellular viability, efficiency of cells to attach to culture plates and form a monolayer, and drug metabolizing competence of the hepatocytes were measured. Successful isolation was achieved in 63% of the procedures using the UW solution and 100% of the procedures using the Celsior solution. In the UW group, significantly lower cell viability (38 +/- 41% vs. 79 +/- 14%, p < 0.05), yield of cells (4.0 +/- 5.2 x 10(6) vs. 8.2 +/- 5.6 x 10(6) cells/g, p < 0.05), and protein content at 24 h of culture (0.6 +/- 0.6 vs. 1.2 +/- 0.3 mg protein per plate, p < 0.05) than in Celsior solution were found. However, similar values of P450 activities were found in both groups. The more successful isolation, better yield, and higher cell viability obtained from human liver grafts preserved in Celsior solution, in comparison to UW solution, suggest Celsior solution as the most appropriate for preserving cadaveric hepatic tissue to be used for hepatocyte harvesting.     

AT-1 receptor antagonism modifies the mediation of endothelin-1, thromboxane A2, and catecholamines in the renal constrictor response to angiotensin II

OBJECTIVE: The present study investigated the consequences of partial AT(1) receptor blockade on the participation of catecholamines, thromboxane A(2) (TXA(2)), and endothelin-1 (ET-1) in the renal vasoconstriction induced by angiotensin II (Ang II). METHODS: For this purpose, the increase in renal perfusion pressure (RPP) produced by Ang II was studied in isolated kidneys from untreated or irbesartan-treated Wistar Kyoto and spontaneously hypertensive rats (SHR), in absence or presence of the alfa-1 receptor antagonist, prazosin, the TXA(2) receptor antagonist, ifetroban, or the ET(A)/ET(B) receptor antagonist, PD145065. RESULTS: Systolic arterial pressure (SAP) was higher (P < 0.05) in SHR than in WKY. Increases in RPP produced by Ang II were comparable in kidneys from both untreated groups. Treatment with irbesartan reduced SAP and RPP in both strains in a comparable extent. Presence of prazosin, ifetroban, or PD145065 in perfusion media reduced (P < 0.05) Ang II maximal response in all groups. Maximal inhibition of Ang II-induced vasoconstriction produced by the 3 antagonists was comparable in untreated WKY, but that of ifetroban and PD145065 was lower (P < 0.05) than that of prazosin in untreated SHR. Maximal inhibition of Ang II-induced vasoconstriction produced by the 3 antagonists was comparable in WKY treated with irbesartan, and not different to that observed in untreated WKY. Maximal inhibitory effect of the 3 antagonists was higher (P < 0.05) in treated than in untreated SHR. CONCLUSION: The study further supports the importance of catecholamines, TXA(2), and ET-1 as mediators of the renal vasoconstriction induced by Ang II in both normotensive and hypertensive rats. Hypertensive conditions appeared to reduce the participation of TXA(2) and ET-1 in Ang II-induced vasoconstriction when compared with normotensive conditions. Chronic partial blockade of AT(1) receptors did not affect the participation of catecholamines, TXA(2), and ET-1 in normotensive rats, but increased the participation of the 3 mediators in SHR. This suggests that when AT(1) receptors are partially blocked, other vasoconstrictor factors could exert part of the renal vasoconstrictor effects of Ang II.     

Sodium retention in cirrhotic rats is associated with increased renal abundance of sodium transporter proteins

BACKGROUND: Liver cirrhosis with ascites is associated with a decrease in renal sodium excretion and therefore sodium retention. METHODS: In this paper, we utilize transporter-specific antibodies to address the hypothesis that dysregulation of one or more sodium transporters or channels is associated with sodium chloride (NaCl) retention in a rat model of cirrhosis induced by repeated exposure to carbon tetrachloride. Age-matched controls and cirrhotic rats were pair fed to ensure identical NaCl and water intake for 4 days prior to euthanasia for quantitative immunoblotting studies. RESULTS AND CONCLUSION: The rats manifested marked extracellular fluid volume expansion with massive ascites. Plasma aldosterone levels were markedly elevated. Analysis of immunoblots revealed marked increases in the abundances of both of the major aldosterone-sensitive apical transport proteins of the renal tubule, namely the thiazide-sensitive NaCl cotransporter NCC and the epithelial sodium channel alpha subunit (alpha-ENaC). These results are consistent with an important role for hyperaldosteronism in the pathogenesis of sodium retention and ascites formation in cirrhosis. In addition, we observed a large decrease in cortical NHE3 abundance (proximal tubule) and a large increase in NKCC2 abundance (thick ascending limb), potentially shifting premacula densa sodium absorption from proximal tubule to loop of Henle (which powers urinary concentration and dilution).     

The invisible trauma patient: emergency department discharges

BACKGROUND: As the malpractice and financial environment has changed, injured patients evaluated by the trauma team and discharged from the emergency department (ED) are now commonplace. The evaluation, care, and disposition of this population has become a significant workload component but is not reported to accrediting organizations and is relatively invisible to hospital administrators. Our objective was to quantify and begin to qualify the evolving picture of the trauma ED discharge population as a work component of trauma service function in an urban, Level I trauma center with an aeromedical program. METHODS: Trauma registry (contacts, mechanism, transport, injuries, and disposition) and hospital databases (ED closure, occupancy rates) were queried for a 5-year period (1999-2003). Trend analysis provided statistical comparisons for questions of interest. RESULTS: During the 5-year study period, the total number of trauma contacts rose by 18.1% (2,220 in 1999 vs. 2,622 in 2003; trend p < 0.05). This increase in total contacts was not a manifestation of an increase in admissions (1,672 in 1999 vs. 1,544 in 2003) but rather a reflection of a marked increase in patients seen primarily by the trauma team and discharged from the ED (473 in 1999 vs. 1,000 in 2003; trend p < 0.05). These ED discharge patients were increasingly transported by helicopter (12.3% in 1999 vs. 29.2% in 2003; trend p < 0.05) and less frequently from urban areas (57.1% in 1999 vs. 48.1% in 2003; trend p < 0.05) over the course of the study period. Average injury severity of this group increased over the study period (Injury Severity Score of 2.7 +/- 0.1 in 1999 vs. 3.3 +/- 0.1 in 2003; trend p < 0.05). ED length of stay for this group increased 19.8% over the study period (trend p < 0.05), averaging nearly 5 hours in 2003. CONCLUSION: The total number, relative percentage, and injury severity of patients evaluated by the trauma team and discharged from the ED has significantly increased over the last 5 years, representing nearly 5,000 patient care hours in 2003. Systems to care for these patients in a cost- and resource-efficient fashion should be put in place. The impact of this growing population of patients on the workload of the trauma center should be recognized by accrediting agencies, hospital administration, and Emergency Medical Services.     

Pseudogranulomatous Spitz nevus: a variant of Spitz nevus with heavy inflammatory infiltrate mimicking a granulomatous dermatitis

Spitz nevus is a benign melanocytic proliferation that shows relatively characteristic clinicopathologic features. Despite this, Spitz nevus is clinically confused with many other lesions, and histopathologically it is sometimes difficult to distinguish it from melanoma. However, Spitz nevus rarely causes differential diagnostic problems with granulomatous dermatitis. This article describes an 8‐year‐old girl who presented with a nodule on her right arm, a clinical appearance of a pyogenic granuloma. Histopathologically, there was a dermal lesion composed of aggregates of large epithelioid cells surrounded by a heavy inflammatory infiltrate, mimicking a sarcoid‐like granulomatous dermatitis. Immunohistochemistry showed epithelioid cells with strong nuclear and cytoplasmic staining with S‐100 protein, thus establishing the diagnosis of a melanocytic tumor. The heavy T‐cell lymphocytic infiltrate that accompanies the large epithelioid cells caused its granulomatous appearance. Molecular assessment showed H27H mutation in the HRAS gene. We suggest the term ‘pseudogranulomatous’ for this variant of Spitz nevus because it indicates that the lesion is not authentically granulomatous and simply mimics a granulomatous dermatitis.     

The role of repeat transurethral resection in stage A1 carcinoma of the prostate

Between 1983 and 1987 repeat transurethral resection (TUR) was performed in 20 patients with a diagnosis of stage A1 prostatic carcinoma. The average weight of the resected tissue in the repeat TUR was 8.81 g (range 3-20 g). In 5 of 20 cases (25%) residual prostatic cancer was identified. By adding the number of foci with cancer obtained at the initial operation with the repeat TUR, 16 cases (80%) remained as A1 and 4 cases (20%) were reclassified (upstaged) to A2. The usefulness of this technique in the restaging of patients with incidental carcinoma of the prostate is analyzed.     

Adjunctive azithromycin in the treatment of aggressive periodontitis: microbiological findings of a 12-month randomized clinical trial

Objectives

To compare the subgingival microbiological outcomes of azithromycin or placebo as adjuncts to scaling and root planing (SRP) in the treatment of aggressive periodontitis (AgP), and to secondarily evaluate the microbiological effect of supragingival scaling in AgP patients.

Methods

Twenty-four AgP subjects 13–26 years of age received a 15-day programme of supragingival scaling (SC) and were then randomly assigned to SRP with systemic azithromycin or placebo. Subgingival samples were taken with sterile paper points at baseline, 15 days after SC, and at 3, 6 and 12 months following SRP. Microbiological analysis was performed by the checkerboard DNA–DNA hybridization.

Results

Changes in bacterial levels from baseline to 15 days after SC were similar in the 2 groups. When subjects were analysed as a single group, significant reductions after SC were observed for Actinomyces gerencseriae, Capnocytophaga ochracea, and Treponema denticola. During the 12-month follow-up, levels of most of the bacteria decreased in both groups in a similar pattern. For instance, Actinomyces israelli, Veillonella parvula, Streptococcus gordonii, C. ochracea, Eikenella corrodens, Eubacterium nodatum, Fusobacterium periodonticum and Fusobacterium nucleatum ssp. polymorphum decreased significantly within the groups.

Conclusions

Azithromycin was ineffective in lowering the subgingival levels of important putative periodontal pathogens in young AgP subjects compared to placebo.

Clinical significance

Scaling and root planing with adjunctive systemic azithromycin provides little additional benefit compared to placebo in reductions of major subgingival periodontal pathogens.     

Immunohistochemistry and polymerase chain reaction on paraffin-embedded material improve the diagnosis of cutaneous leishmaniasis in the Amazon region

Background  Recently, there has been an increase in the incidence of cutaneous leishmaniasis (CL), which represents an important health problem. This increase may be related to the epidemiologic expansion of the infective agent and the increase in tourism in tropical areas. The difficulty in clinical diagnosis, mainly in areas in which CL is not the first consideration of local physicians, has intensified efforts to describe diagnostic tests, which should be specific, sensitive, and practical. Amongst the new tests described are those including nucleic acid amplification (polymerase chain reaction, PCR) and immunohistochemistry (IHC).
Methods  In this study, we evaluated the sensitivity of a PCR based on small subunit (SSU) ribosomal DNA, in comparison with IHC using Leishmania spp. antibodies, in biopsies embedded in paraffin.
Result  The results indicated a total sensitivity of 96% (90.9% with PCR and 68.8% with IHC), showing the possibility of using paraffin-embedded biopsies to diagnose CL.
Conclusion  We propose the use of the two tests together as a routine protocol for diagnosis. This would require the provision of local medical services to perform molecular biology techniques and adequate Leishmania antibodies.     

The influence of posterior capsular opacification removal on anterior segment birefringence parameters as measured by scanning laser polarimetry

PURPOSE: To determine the effect of posterior capsular opacification (PCO) removal on anterior segment birefringence (ASB) and its influence on peripapillary retinal nerve fibre layer (RNFL) retardation measurements obtained by means of scanning laser polarimetry. METHODS: In this prospective interventional study, scanning laser polarimetry was performed using GDx variable corneal compensation on 26 eyes of 26 patients who developed clinically significant PCO (after uncomplicated cataract surgery and with no other ocular pathology) both before and between 1 and 4 weeks after Nd:YAG capsulotomy. Best-corrected visual acuity (BCVA), intraocular pressure, corneal polarization axis (CPA), corneal polarization magnitude (CPM) were compared using the Student t-test and Wilcoxon signed ranks test. Spearman correlations between changes (differences between values after and before capsulotomy) in the CPA, CPM, BCVA and RNFL data were also performed. RESULTS: PCO removal is associated with a shift in CPA (from 10.86 to 15.03 degrees, P = 0.004) and CPM (from 28.54 to 37.92 nm, P = 0.004). Significant correlations were found between changes in the parameters of ASB and BCVA. Furthermore, RNFL measurements (nerve fibre indicator, temporal-superior-nasal-inferior-temporal average and superior average) were also well related to the CPA and CPM shifts. CONCLUSIONS: PCO induces an inaccurate compensation of ASB which affects RNFL assessment. Thus, it is necessary to recompensate ASB after posterior capsulotomy.     

Topical anesthesia: possible risk factor for endophthalmitis after cataract extraction

PURPOSE: To assess the relationship between the risk for acute endophthalmitis after cataract extraction and whether certain factors, such as surgeon qualification, numerical order, duration of surgery, operating theater, and type of anesthesia (topical or retrobulbar), could be modified to decrease the risk. SETTING: Single-center academic practice. METHODS: Two epidemiological studies were performed: a case-control study and a retrospective cohort study. The surgical records of all patients with clinically diagnosed endophthalmitis within 30 days after cataract surgery performed between February 2002 and September 2003 were reviewed. The endophthalmitis cases were compared with 108 randomly selected controls (4 controls per case). The global incidence of endophthalmitis and the incidence according to type of anesthesia were calculated. RESULTS: Of 5011 cataract extractions performed, 27 cases of endophthalmitis occurred. The incidence was 5.39 per 1000 procedures. An independent statistically significant relationship was found between endophthalmitis and the use of topical anesthesia (odds ratio [OR], 11.8; 95% confidence interval [CI], 2.4-58.7) and surgery longer than 45 minutes (OR, 7.2; 95% CI, 1.7-29.7) but not between the other variables. The incidence of endophthalmitis was 1.8 per 1000 cataract extractions with retrobulbar anesthesia and 6.76 per 1000 with topical anesthesia (relative risk [RR], 3.76; 95% CI, 0.89-15.85). After the start of the study period was extended to May 2001, the incidence of endophthalmitis was 1.3 per 1000 cataract extractions with retrobulbar anesthesia and 8.7 per 1000 with topical anesthesia (RR, 6.72; 95% CI, 1.63-27.63). CONCLUSION: Results suggest that there may be an association between topical anesthesia and endophthalmitis after cataract extraction.