Stomatal conductance, not biochemistry, drives low temperature acclimation of photosynthesis in Populus balsamifera, regardless of nitrogen availability.

Low-temperature thermal acclimation may require adjustments to N and water use to sustain photosynthesis because of slow enzyme functioning and high water viscosity. However, understanding of photosynthetic acclimation to temperatures below 11 °C is limited. We acclimated Populus balsamifera to 6 °C and 10 °C (6A and 10A, respectively) and provided the trees with either high or low N fertilizer. We measured net CO2 assimilation (Anet ), stomatal conductance (gs ), maximum rates of Rubisco carboxylation (Vcmax ), electron transport (Jmax ) and dark respiration (Rd ) at leaf temperatures of 2, 6, 10, 14 and 18 °C, along with leaf N concentrations. The 10A trees had higher Anet than the 6A trees at warmer leaf temperatures, which was correlated with higher gs in the 10A trees. The instantaneous temperature responses of Vcmax , Jmax and Rd were similar for trees from both acclimation temperatures. While soil N availability increased leaf N concentrations, this had no effect on acclimation of photosynthesis or respiration. Our results indicate that acclimation below 11 °C occurred primarily through changes in stomatal conductance, not photosynthetic biochemistry, and was unaffected by short-term N supply. Thermal acclimation of stomatal conductance should therefore be a priority for future carbon cycle model development.

New tree-ring data from Canadian boreal and hemi-boreal forests provide insight for improving the climate sensitivity of terrestrial biosphere models.

Understanding boreal/hemi-boreal forest growth sensitivity to seasonal variations in temperature and water availability provides important basis for projecting the potential impacts of climate change on the productivity of these ecosystems. Our best available information currently comes from a limited number of field experiments and terrestrial biosphere model (TBM) simulations of varying predictive accuracy. Here, we assessed the sensitivity of annual boreal/hemi-boreal forest growth in Canada to yearly fluctuations in seasonal climate variables using a large tree-ring dataset and compared this to the climate sensitivity of annual net primary productivity (NPP) estimates obtained from fourteen TBMs. We found that boreal/hemi-boreal forest growth sensitivity to fluctuations in seasonal temperature and precipitation variables changed along a southwestern to northeastern gradient, with growth limited almost entirely by temperature in the northeast and west and by water availability in the southwest. We also found a lag in growth climate sensitivity, with growth largely determined by the climate during the summer prior to ring formation. Analyses of NPP sensitivity to the same climate variables produced a similar southwest to northeast gradient in growth climate sensitivity for NPP estimates from all but three TBMs. However, analyses of growth from tree-ring data and analyses of NPP from TBMs produced contrasting evidence concerning the key climate variables limiting growth. While analyses of NPP primarily indicated a positive relationship between growth and seasonal temperature, tree-ring analyses indicated negative growth relationships to temperature. Also, the positive effect of precipitation on NPP derived from most TBMs was weaker than the positive effect of precipitation on tree-ring based growth: temperature had a more important limiting effect on NPP than tree-ring data indicated. These mismatches regarding the key climate variables limiting growth suggested that characterization of tree growth in TBMs might need revision, particularly regarding the effects of stomatal conductance and carbohydrate reserve dynamics.

Genetic variants of antioxidant enzymes and environmental exposures as molecular biomarkers associated with the risk and aggressiveness of bladder cancer.

Bladder cancer (BC) is one of the top 10 most common tumours worldwide; however, no molecular markers are currently available for tumour management and follow-up. BC could benefit from molecular biomarkers in environmental disease, which provide mechanistic understanding of individual susceptibility to exposure-related cancers and allow characterizing genetic alterations in the molecular pathway for malignancy. This case-control study performed a molecular analysis in 99 BC and 125 controls. Buccal swabs were collected to assess SNPs in eleven genes coding for xenobiotic detoxification enzymes, cellular antioxidant defences, and hormone synthesis and signalling (NAT2 (rs1801280), GPX1 (rs1050450 and rs17650792), TXNRD1 (rs7310505), PRDX3 (rs3740562), PON1 (rs662), SOD1 (rs10432782), SOD2 (rs4880), CAT (rs1001179), CYP17A1 (rs743572) and ESR1 (rs746432)). A structured questionnaire was administered to study participants to assess environmental and dietary chemical exposures. Several miRNAs associated with BC and detoxification/antioxidant pathways were analysed in a subsample of the study population, including miR-93-5p, miR-221-3p, miR-126, miR-27a-3p, miR-193b, and miR-193a-5p. Levels of selected environmental pollutants (polycyclic aromatic hydrocarbons and endocrine disrupting chemicals) were determined in urine from a subsample of BC cases and controls. We found that CYP17A1, CAT, SOD1, ESR1, PON1, and GPX1 (rs17650792) were associated with BC risk. Furthermore, exposure to smoke and/or dust, and alcohol intake were identified as risk factors for BC. Increased urinary levels of benzo[a]pyrene and bisphenol A were observed in BC patients relative to controls, along with an increased expression of miR-193b, miR-27a and miR-93-5p in BC. Nevertheless, further studies with a larger sample size are warranted to confirm these exploratory results. This study also shows that the combination of genetic markers (PON1 and CYP17A1) and miRNA (miR-221-3p and miR-93-5p) open a new scenario in the use of non-invasive biomarkers in the stratification of BC to guide personalized medicine, which is extremely urged in the current clinical setting.

Panenteric capsule endoscopy identifies proximal small bowel disease guiding upstaging and treatment intensification in Crohn's disease: A European multicentre observational cohort study.

BACKGROUND: Endoscopically defined mucosal healing in Crohn's disease is associated with improved outcomes. Panenteric capsule endoscopy enables a single non-invasive assessment of small and large bowel mucosal inflammation. AIMS AND METHODS: This multicentre observational study of patients with suspected and established Crohn's disease examined the feasibility, safety and impact on patient outcomes of panenteric capsule endoscopy in routine clinical practice. The potential role in assessment of disease severity and extent by a comparison with existing clinical and biochemical markers is examined. RESULTS: Panenteric capsule endoscopy was performed on 93 patients (71 with established and 22 with suspected Crohn's disease). A complete examination occurred in 85% (79/93). Two cases (2.8%) of capsule retention occurred in patients with established Crohn's disease. Panenteric capsule resulted in management change in 38.7% (36/93) patients, including 64.6% (32/48) of those with an established diagnosis whose disease was active, and all three patients with newly diagnosed Crohn's disease. Montreal classification was upstaged in 33.8% of patients with established Crohn's disease and mucosal healing was demonstrated in 15.5%. Proximal small bowel disease upstaged disease in 12.7% and predicted escalation of therapy (odds ratio 40.3, 95% confidence interval 3.6-450.2). Raised C-reactive protein and faecal calprotectin were poorly sensitive in detecting active disease (0.48 and 0.59 respectively). CONCLUSIONS: Panenteric capsule endoscopy was feasible in routine practice and the ability to detect proximal small bowel disease may allow better estimation of prognosis and guide treatment intensification. Panenteric capsule endoscopy may be a suitable non-invasive endoscopic investigation in determining disease activity and supporting management decisions.

Small-calibre percutaneous nephrolithotomy (SC-PCNL). Therapeutic decision algorithm. / Nefrolitectomía percutánea de calibre reducido (NLP-CR). Algoritmo de decisión terapéutica.

INTRODUCTION: The progressive reduction in the calibre of the tract in percutaneous kidney surgery to the point of miniaturisation has expanded its use to smaller stones that until now have been treated with extracorporeal shock wave lithotripsy (ESWL) and retrograde intrarenal surgery (RIRS). OBJECTIVE: To provide an update on the various techniques of small-calibre nephrolithotomy (SC-PCNL) analyse their efficacy, safety and indications and determine their degree of implantation at this time. MATERIAL AND METHODS: We performed a review in PubMed of Spanish and English medical literature on the various techniques of SC-PCNL. RESULTS: The use of SC-PCNL has reduced the morbidity associated with standard PCNL, particularly bleeding, and has enabled tubeless nephrolithotomy with greater safety. There are various techniques with blurred terminology (Miniperc, Microperc, Mini-microperc, Ultraminiperc), which differ in terms of gauge employed and in certain technical aspects that require their indications be specified. Currently, SC-PCNL competes with techniques that are less invasive than standard PCNL such as ESWL and the RIRS in treating small stones, but the role of SC-PCNL is still not sufficiently understood and continues to be the subject of debate. CONCLUSIONS: The indications for PCNL are expanding to small stone sizes due to the miniaturisation of the technique. PCNL competes in this field with ESWL and RIRS. Larder studies are needed to establish the specific indications for PCNL in treating nephrolithiasis.

Psychometric validation of the Spanish version of the USS-PROM questionnaire for patients who undergo anterior urethral surgery. / Validación psicométrica de la versión en castellano del cuestionario USS-PROM para pacientes sometidos a cirugía de uretra anterior.

OBJECTIVE: To translate into Spanish and validate the Urethral Stricture Surgery Patient-Reported Outcome Measure (USS-PROM) questionnaire, assessing its psychometric properties and determining its suitability for clinical use in our community. We also assessed the potential changes in ejaculatory function using the Male Sexual Health Questionnaire-Ejaculatory Dysfunction (MSHQ-EjD). MATERIAL AND METHODS: A systematic translation of the British version was performed. Patients scheduled for anterior urethral stricture surgery between September 2014 and September 2015 were prospectively included in the study. All patients completed the questionnaire before and after the surgery. We conducted an in-depth psychometric study of the questionnaire. RESULTS: We assessed the responses of a total of 40 patients. The questionnaire showed its validity, presenting an excellent negative correlation between the voiding symptom scores and the maximum flow (r=-0.6, P<.001), and also showed significant improvement in the EQ5D-VAS (visual analogue scale) and the time trade-off. For internal consistency, the Cronbach's alpha was 0.701. For the test-retest reliability, the overall intraclass correlation coefficient (ICC) was 0.974, and the ICC for each item separately ranged from 0.799 to 0.980. We observed significant improvement in all items regarding urinary symptoms and health-related quality of life (P<.001), thereby demonstrating the response capacity to changing the questionnaire. There were no significant changes in the MSHQ-EjD. CONCLUSIONS: The Spanish version of the USS-PROM questionnaire is a valid instrument for quantifying changes in voiding symptoms and the health-related quality of life of patients undergoing anterior urethral surgery.

Regional blood flow effects of dopexamine versus enalaprilat during propofol anaesthesia in rabbits with experimental chronic heart failure.

OBJECTIVE: Chronic congestive heart failure (CHF) was induced in rabbits with doxorubicin in order to evaluate: (1) haemodynamic and regional blood flow responses to propofol anaesthesia; (2) modification of these cardiovascular responses with background intravenous infusions of enalaprilat or dopexamine. METHODS: Rabbits received either doxorubicin, 2 mg.kg-1 weekly intravenously for seven weeks (CHF, n = 6), or saline (controls, n = 6). Doppler flow probes were implanted on the ascending aorta, left renal artery, and lower abdominal aorta. In three separate studies propofol was infused for 40 min periods at 0.6 and then 1.2 mg.kg-1.min-1 after background infusions of either saline, enalaprilat (0.2 mg.kg-1 + 0.003 mg.kg-1.min-1), or dopexamine (0.008 mg.kg-1.min-1). RESULTS: In normal rabbits propofol (1.2 mg.kg-1.min-1) reduced mean arterial pressure from awake control by 33(SEM 3)%, cardiac output by 24(4)%, and hindlimb blood flow (HBF) by 10(2)%, but did not change renal blood flow. In rabbits with CHF, although resting mean blood pressure was lower, propofol did not alter blood pressure or hindlimb blood flow, but renal blood flow was reduced by 37(6)%. CONCLUSIONS: Both enalaprilat and dopexamine increased renal blood flow in the control and CHF groups. Enalaprilat caused marked hypotension during anaesthesia in the CHF group. Dopexamine increased mean arterial pressure, heart rate, and hindlimb blood flow during anaesthesia in controls, but not in CHF.

The restricted cellular host range of human herpesvirus 8.

DESIGN: A selection of primary and transformed cell types were evaluated for their susceptibility to infection with human herpesvirus 8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus. METHODS: Sources of HHV-8 included Kaposi's sarcoma lesion punch biopsies that were either cocultured directly with target cells or that were first cocultured with human lymphocytes to derive HHV-8-containing fluids that were inoculated onto target cells. HHV-8 was also obtained from primary effusion lymphoma-derived cell lines. Techniques to detect infection included the PCR, immunofluorescence assays and in situ hybridization. RESULTS: Susceptible cells included human umbilical cord blood mononuclear cells (UCMC), adult CD19 B cells, macrophages and certain endothelial cells of human and animal origin, including some that are transformed with human papilloma virus type 16 E6 and E7 genes. The infection of lymphocytes did not yield established lymphoblastoid cell lines (LCL) and virus infection persisted for only 4-7 days. However, long-term HHV-8 infection of UCMC could be achieved by coinfection with Epstein-Barr virus. HHV-8 could also infect UCMC LCL recently derived by Epstein-Barr virus transformation, but long-established LCL could not be infected with HHV-8. CONCLUSIONS: These data provide further biological evidence in cell culture for the limited cellular host range of HHV-8 to CD19 B cells, macrophages, and certain endothelial cells.

Characterization of cytosolic estrogen sulfotransferase from RL95-2 endometrial cancer cells.

Estrogen sulfoconjugation previously was reported in normal endometrium and in RL95-2 cells, a cell line derived from a human endometrial cancer maintained in continuous in vitro culture. In the present study the estrogen sulfurylation activity in the cytosolic fraction of RL95-2 cells was characterized using [3H]estrone as substrate. Estrone sulfate was separated from unreacted estrone by thin layer chromatography. Activity was proportional to cytosol concentration, with a pH optimum at pH 8. There was marked temperature dependence between 24 and 40 C. The apparent Km for estrone conjugation was 3.6 nM, with a maximum velocity of 135 fmol/micrograms DNA . h. No complex kinetic behavior was found at estrone concentrations up to 1 microM. The apparent Km for the cosubstrate 3'-phosphoadenosine 5'-phosphosulfate was 0.6 microM. Inhibition experiments demonstrated that the sulfurylating activity studied under these conditions was specific for estrogens. Only estradiol and estriol, in addition to estrone itself, inhibited conjugation to any significant degree. Dehydroepiandrosterone had only 1% the inhibitory activity of estrone. Other androgens, corticoids, progestins, phenols, nonsteroidal estrogens and antiestrogens, and bile acids had no significant effects on the sulfurylation of estrone. An estrogen-sulfoconjugating activity with the characteristics of estrogen sulfotransferase (EST) was demonstrated in RL95-2 cells. The Km of EST for estrone in the RL95-2 cells closely approximated the value reported for the enzyme in normal endometrium. The affinity of EST for estrogens is within the range of the Kd of estrogen receptor and of the physiological concentrations of estrogens reported in the endometrium, suggesting that EST could serve as a regulator of intracellular estrogen levels.

Correlation of DNA content and histology in prognosis of astrocytomas.

Thirty-two cases of astrocytoma were analyzed for DNA content and cell-cycle proliferation features by flow cytometry using paraffin-embedded tissue. The findings were correlated with histologic grading and survival. Abnormal DNA (aneuploidy or elevated G2-M fraction greater than or equal to 7%) was present in 18 cases (56%). Glioblastoma multiforme (GBM) had 11 of 16 (69%), anaplastic astrocytomas (ANA) 7 of 11 (64%), and low-grade (LG) neoplasms 0 of 5 cases with abnormal DNA content. Short-term survival (less than or equal to 26 months) occurred in all 16 patients with GBM (100%), 7 of 11 patients with ANA (64%), and 1 of 5 patients with LG neoplasms (20%). Seventeen of 18 patients (94%) with abnormal DNA content were short-term survivors (P less than 0.0002). Abnormal DNA content was found in 17 of 24 short-term survivors (71%), whereas histologic grading identified 16 of 24 such cases (67%). A combination of grading and abnormal DNA content identified 22 of 24 (92%) of the poor survival cases. DNA content was most useful in the anaplastic group. Six of seven cases (86%) with abnormal DNA content had short survival (P less than 0.055), and three of four (75%) with normal DNA content had long survival. DNA analysis combined with histologic grading improves prognosis designation.

Suppression of noradrenaline spillover by the dopamine prodrug gamma-L-glutamyl-L-dopa: a central effect?

The DA prodrug gamma-L-glutamyl-L-dopa (gludopa) has a high degree of renal selectivity with 2-step conversion to DA in the kidney. The effects of gludopa, with and without DA-2 receptor blockade, on renal and total noradrenaline (NA) spillover, were studied in two groups of rabbits. Eight rabbits received gludopa infusion (25 and 100 micrograms/kg/min and 8 received an infusion of gludopa and DA-2 receptor antagonist, YM-09151 (50 micrograms/kg i.v.). Renal and total NA spillover rates were measured by 3H-NA tracer method before and after gludopa infusion. Brain NA, DA, gludopa and L-dopa content were measured after gludopa infusion in 5 rabbits; control values for tissue catecholamine and drug levels were obtained in 5 untreated rabbits. Gludopa infusion markedly increased kidney DA content (300-fold) and DA excretion (6000-fold) but had little effect on plasma DA. It produced a dose-related fall in mean (+/- SEM) renal NA spillover (21.6 +/- 3.7 to 10.6 +/- 2.7, 7.2 +/- 2.7 ng/min, p < 0.01). Even greater falls were observed in total NA spillover after gludopa (43.1 +/- 10.2 to 19.7 +/- 3.4, 9.4 +/- 1.8 ng/min, p < 0.01). DA-2 receptor antagonism had no influence on the effects of gludopa on either renal or total NA spillover. Significant amounts of gludopa were detected in the brain after drug infusion (0.28 +/- 13 nmol/g brain tissue). Gludopa, a putative renal selective dopamine prodrug with effects mediated via DA-1 receptors also significantly inhibits both renal and extra-renal NA spillover. This effect is not a DA-2 effect but may be mediated centrally.

Absence of monoclonal antibody detectable Kaposi sarcoma-specific antigens on lesion-derived cultured cells.

To define the histogenesis and cell origin of Kaposi sarcoma (KS), we cultured KS cells without retrovirally conditioned media from three HIV seropositive AIDS patients and then attempted to raise mouse hybrid monoclonal antibodies (Mabs) specific to these AIDS-KS cells. After both in vivo and in vitro immunization trials, all putative Mabs reacted positively to KS cells but also non-specifically with other human (CH5 and OM) and non-human (RSE-1) control endothelial cell lines. To overcome this crossreactivity, we further "absorbed" previously cloned hybrids and pre-hybrid splenocytes by incubating them with the control endothelial cell lines to eliminate splenocytes and/or hybridomas reactive to normal endothelium. Whereas absorption successfully eliminated immunoreactivity to control endothelium, it also excluded reactivity to KS cells. These findings (lack of specific antigenicity and immunoresponsiveness of KS similar to non-KS control endothelium) suggest that AIDS-KS cells are neither antigenically transformed nor neoplastic, but instead represent dedifferentiated or transdifferentiated endothelium which retains immunogenicity of its original endothelial cell prototype.

Results of a national survey on ethics education in general surgery residency programs.

BACKGROUND: Medical ethics is a required part of the curriculum in all medical schools in the United States, and an essential component of the educational guidelines for most postgraduate residency programs. Currently, general surgery does not specify ethics education in its essential curriculum for surgical training. This study was designed to determine the existing educational activities in ethics for residents in general surgery, as well as to characterize the attitudes of surgical educators about the role of ethics teaching in residency training. METHODS: An 80-item questionnaire was mailed to the program directors of all accredited general surgery residencies in the United States. They were requested to provide information about their teaching activities in ethics, their resources for ethics instruction, and their attitudes about the importance of education in clinical ethics for surgical residents. RESULTS: The survey had a 71% response rate with a representative distribution of programs based on size, geographic location, and community versus university affiliation. Fifty-six programs (28%) offered no formal ethics education, 94 (48%) held one teaching event in ethics, and 48 (24%) conducted two or more activities. The format for instruction in ethics included grand rounds (50%), resident conferences (41%), and ethics rounds (9%). Residencies with a faculty surgeon having expertise or special interest in ethics had a greater number of ethics teaching activities (P <0.05), whereas programs with a hospital ethicist were more likely to provide ethics rounds (P <0.01). A standardized curriculum in ethics was favored by 85% of respondents with critical content in end-of-life decisions, managing ethical conflict, and informed consent. The majority of program directors were opposed to (50%) or undecided (20%) about inclusion of ethics questions on the American Board of Surgery Inservice Training Examination (ABSITE) and Qualifying Examination in General Surgery. CONCLUSIONS: The majority of program directors of general surgery residencies support the teaching of clinical ethics and favor a standardized curriculum. However, most residencies in general surgery do not include ethics instruction as part of their on-going, regular educational schedule.

Glutathione-S-Transferase as a selective inhibitor of oncogenic ras-p21-induced mitogenic signaling through blockade of activation of jun by jun-N-terminal kinase.

We have identified the intracellular detoxification enzyme, glutathione-S-transferase (GST), as a potent inhibitor of the activation of jun by its kinase, jun-N-terminal kinase (JNK), in vitro. All three major isozymes (alpha, mu, and pi) bind to JNK-jun complexes and inhibit activation of jun by JNK. We now find that GST inhibits JNK-induced oocyte maturation in vivo and strongly inhibits oocyte maturation induced by oncogenic ras-p21 protein, but not by insulin-activated normal cellular p21 protein. These results correlate with the finding that oncogenic, but not insulin-activated normal, p21 induces high levels of activated JNK. GST also strongly blocks induction of oocyte maturation by protein kinase C (PKC) which is a critical downstream target of oncogenic but not normal ras-p21. Thus, we have established a new function for GST as a potent physiological inhibitor of the ras-JNK-jun pathway.

Effective stabilization of ethanol levels in multiple-well tissue culture plates.

Underestimation of ethanol (EtOH) volatility in vitro is a potential source of experimental error. EtOH (0-5% in culture medium) was added to 24- or 96-well tissue culture plates with standard low evaporation lids and incubated at 37 degrees C in humidified 7.5% CO2 and 92.5% air. After 72 hours, approximately 70% of the initial EtOH had disappeared from the aqueous phase of the plate (EtOH volatilization). EtOH concentrations gradually decreased in high-concentration wells (1-5%) and increased in low-concentration wells (0-0.1%) over time. This temporal redistribution of EtOH (EtOH diffusion) was detected after only 1 hour of incubation. Parafilm, Blenderm surgical tape, and ELISA plate-sealing tape barriers inconsistently or inadequately prevented EtOH volatilization and diffusion, but a newly designed plate-sealing clamp (PSC) apparatus inhibited this phenomenon. Rat hepatic sinusoidal endothelial cells cultured with the PSC apparatus maintained intact cell membranes for 72 hours and stable levels of monolayer permeability for at least 48 hours. By stabilizing in vitro EtOH concentrations, the PSC apparatus eliminates a potential source of major experimental error.

AIDS, alcohol, endothelium, and immunity.

Analogies are drawn between important unknowns in AIDS and alcohol research, related to underlying common pathogenetic mechanisms, immunodysregulation, cofactors, and prominent vascular manifestations. The central role of the blood and lymphatic vasculatures and specifically their endothelial lining in many facets of the immune response is reviewed. Evidence is presented that both alcohol and HIV (as well as other coinfecting viruses in AIDS) target and alter endothelial cells and the angiogenic process. These concepts are further illustrated by a serendipitous viral epidemic among rats on continuous long-term alcoholic and control nonalcoholic diets, where synergism between alcohol and virus appeared to underlie multiple vascular proliferative lesions in the liver. In AIDS and alcoholism/alcoholic liver disease (ALD), the prominent features of dysregulated angiogenesis point to the endothelium as a key player in pathogenesis of these seemingly disparate disorders and potentially in immunomodulation.

Alcohol, hepatic sinusoidal microcirculation, and chronic liver disease.

According to the "intact cell hypothesis," ethanol (EtOH) primarily targets nonparenchymal hepatic sinusoidal and perisinusoidal cells, thereby promoting sinusoidal capillarization, which impairs microcirculatory exchange of nutrients and wastes, promotes tissue fibrosis, and only indirectly damages hepatic parenchyma. To test this hypothesis, sinusoidal ultrastructure and hepatic lymph flow and protein composition were examined in rats up to 16 weeks after intragastric EtOH (36% calories)-high fat infusion (Tsukamoto-French model) (TF). The findings were compared to dietary controls and interpreted in light of restricted transsinusoidal protein movement observed in patients with alcoholic cirrhosis. In vitro, alterations in rat hepatic sinusoidal endothelial cell (RSE) morphology, proliferative index, and transendothelial macromolecular permeability (Evans blue-albumin uptake into microcarrier beads) were determined after acute and more chronic exposure to 0.1%-5 vol% EtOH. TF displayed 75% increased liver size, perisinusoidal collagenosis, and basal lamina deposition, ascitic fluid, and doubling of hepatic lymph liquid and protein flux. In vitro, 1% EtOH retracted RSE cell margins, enhanced transendothelial Evans blue-albumin flux and suppressed proliferative index. Thus, high EtOH concentration, clinically attainable in the portal blood during an alcoholic binge, both in vivo and in vitro, promotes early structural and functional alterations in sinusoidal endothelium, which over time may be responsible for progressive restriction of free intrahepatic exchange of liquid, macromolecules, and migrating immune cells.

Expression of multiple matrix metalloproteinases and urokinase type plasminogen activator in cultured Kaposi sarcoma cells.

Kaposi's sarcoma (KS) cells are considered to be of endothelial origin. KS lesions are characterized by hyperproliferation and an invasive phenotype. We have determined that KS cell cultures constitutively secrete multiple forms of several matrix metalloproteinases (MMPs) and an altered form of urokinase plasminogen activator (uPA) by zymogram and Western analysis of the culture media. MMPs are a family of secreted endoproteinases which degrade components of the extracellular matrix. Their enhanced expression and activity are strongly correlated with cellular processes involving tissue remodeling and invasion. The KS cells secrete increased levels of gelatinase A and B and a high molecular weight uPA in vitro when compared with non-KS endothelial or epithelial cells. Multiple forms of gelatinases A and B were observed on gelatin zymograms. Caseinolytic bands observed were confirmed by Western blot analysis to be due to stromelysin activity, whereas matrilysin was not detected by casein zymography. Western blot analysis also detected secretion of interstitial collagenase and high molecular weight uPA. Gelatinolytic activity with the mobility of gelatinase B was detected on gelatin zymograms, but not by Western analysis. This unusual constitutive expression pattern of MMPs and uPA by KS cells in vitro is characterized by elevated levels of gelatinase A, gelatinase B, interstitial collagenase, stromelysin and a high molecular weight form of uPA, and the lack of expression of matrilysin. These secreted MMPs, taken together, are capable of digesting a broad range of components of the extracellular matrix. This unusual pattern is likely to contribute to the characteristic hyperproliferative and invasive phenotype of KS lesions.

AIDS, Kaposi sarcoma, and the lymphatic system: update and reflections.

Based on ongoing basic and clinical investigations, further evidence is presented that the AIDS-Kaposi sarcoma (AIDS-KS) complex involves a progressive disturbance of the blood-lymph circulatory loop of fluid, macromolecules, and migrating cells. We first surveyed the spectrum of vascular abnormalities including Kaposi sarcoma (KS) found in the AIDS/ARC population, then non-invasively imaged lymphatic system abnormalities in AIDS-KS by whole body lymphangioscintigraphy, and finally examined the biologic behavior of AIDS-KS cells in long-term tissue culture. These observations are viewed in terms of the lymphatic and blood vascular route of entry and transport of free and cell-associated virus and other opportunistic pathogens as well as poorly understood host endothelial-immune system interactions.

Refinement of a rodent model of peripheral lymphedema.

A reliable, inexpensive experimental counterpart of peripheral lymphedema has been notoriously difficult to reproduce thereby stifling basic and clinical research into this frustrating clinical condition. Accordingly, in 45 adult Wistar-Fuzzy rats, we attempted to produce sustained hindlimb lymphedema by either groin nodal/lymphatic microsurgical ablation (S) (guided by visual blue dye lymphography) or limited field-groin irradiation (R) alone (4500 rads) or combined S followed by R or R followed by S with an additional non-manipulated group serving as controls. Observations were made for 30-100 days thereafter. Hindlimb volumes were determined serially using the truncated cone formula based on multiple circumferential measurements at standardized intervals along the affected hindlimb and the findings compared with similar measurements in the contralateral non-manipulated hindlimb. In randomly selected rats from each group, lymphatic drainage was assessed by lymphangioscintigraphy (LAS), soft tissue swelling by magnetic resonance imaging (MRI), and edema fluid total protein content by refractometry. Whereas S or R alone produced only transient or mild hindlimb edema without associated morbidity or mortality, S-R or R-S induced moderate to severe sustained protein-rich hindlimb lymphedema associated with 9-13% early mortality and notable late local limb morbidity. Lymphatic obstruction was documented by sustained maintenance of increased hindlimb volume, subcutaneous fluid accumulation (MRI), and impaired lymphatic drainage (LAS). This reproducible rodent model of secondary lymphedema reliably simulates a stable clinical condition for a window of up to 100 days and should thereby facilitate standardized testing of therapeutic/preventive protocols and basic research into lymphatic dynamics in secondary lymphedema.