Copper vapor laser fragmentation of gallstones: in vitro measurements of wall heat transmission

Laser fragmentation is a promising new modality in management of retained CBD stones. Recent reports demonstrate the feasibility of lasers for this, but few studies have evaluated their safety (e.g., thermal injury may occur at greater than 43 degrees C). This study was conducted to measure heat transmission from lased bilirubinate and mixed stones to a simulated CBD wall. Four welded thermocouples were passed to the inside wall of 6-mm polyvinyl tubing 90 degrees apart to surround the lumen stone. The thermocouples were interfaced to a computer and temperatures were recorded every 270 msec. The tubing was submerged in a 37 degrees C water bath for all lasing work. A copper vapor laser (wavelength, 510 nm; 5.6 W; 5 kHz; pulse length, 30 ns) was attached to a 650-micron quartz fiber. A stone was "impacted" in the tubing and the laser fiber was pushed against the stone while making multiple passes to fragment it. Thirty mixed gallstones (mean size, 6.9 X 5.1 mm) and 20 bilirubinate gallstones (mean size, 7.1 X 5.2 mm) were fragmented during the study. Maximum temperature (Tmax), duration of Tmax (TmaxD), interval to stone piercing (TiP), and interval to fragmentation (TiF) were measured and comparisons were carried out with the SPSS statistical package using the t test procedure. The Tmax generated during fragmentation of bilirubinate stones (43.4 +/- 1.7 degrees C) was significantly less (P less than 0.002) than the Tmax for mixed stones (54.0 +/- 2.7 degrees C) but both Tmax values represented potentially injurious temperature levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.     

Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen.

Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a negative population (n = 160) from assay cutoffs. With a selected population (n = 307), inclusion of reductant eliminated apparent anti-HBc activity in 54 of 81 samples in the 30 to 70% inhibition range. Reductant-stable anti-HBc samples were strongly associated with the presence of antibody to hepatitis B surface antigen (21 of 27). The association persisted below the detection limits of current assays to 0.3 to 0.4 Paul Ehrlich Institute units per ml. Only 1 of 54 reduction-sensitive borderline samples was confirmed to be positive for antibody to hepatitis B surface antigen. The modified procedures had unchanged or slightly improved sensitivity for immunoglobulin G (IgG)-associated anti-HBc activity. Although IgM anti-HBc detection was reduced from four- to eightfold in the presence of reductants, sensitivities remained at least twofold greater than tha of an enzyme immunoassay (Corzyme M; Abbott) designed to detect acute-phase levels of IgM anti-HBc. The use of reducing agents should significantly improve the reliability of anti-HBc testing, especially near assay cutoffs.     

Mechanisms of nitric oxide interplay with Rho GTPase family members in modulation of actin membrane dynamics in pericytes and fibroblasts

Migration of pericytes such as hepatic stellate cells is fundamentally important for diverse biological and pathological processes including tumor invasion and fibrosis. In prototypical migratory cells such as fibroblasts, the small GTPases Rac1 and RhoA govern the assembly of lamellipodia and stress fibers, respectively, cytoskeletal structures that are integral to the cell migration process. The gaseous signaling molecule nitric oxide (NO) influences growth factor chemotactic responses, although this occurs primarily in cell-type-specific ways and through cell biological effects that are poorly characterized. In this study, we use complementary molecular and cell biological approaches to delineate important roles for Rac1, RhoA, and NO in migration of the human hepatic stellate cell line LX2 and primary rat hepatic stellate cells. Both platelet-derived growth factor (PDGF) and Rac1 overexpression drove migration through formation of actin-positive filopodia spikes in LX2 as compared to the formation of lamellipodia in fibroblasts. NO inhibited PDGF- and Rac1-driven migration in LX2 by abrogating filopodia formation and inhibited migration of fibroblasts by attenuating lamellipodial protrusions. Additionally, RhoA conferred resistance to NO inhibition of migration and restored chemotactic responses to PDGF in the absence of functional Rac1 in LX2. In conclusion, these studies identify novel crosstalk between small GTPases, cytoskeletal structures, and NO in pericyte-specific pathways, providing counterbalances in the chemotactic responses to growth factors.     

Cytogenetic and molecular studies of a familial renal cell carcinoma.

In a previously studied family with inherited renal cell carcinoma (RCC), RCC was shown to segregate with a constitutional balanced t(3;8)(p14.2;q24.1). In addition, we recently showed that in a RCC tumor from this family the constitutional translocation became unbalanced, suggesting a genetic mechanism that may be associated with the primary genetic events of tumorigenesis. We now report that the RCC tumor cells from this case showed additional cytogenetic alterations, possibly related to tumor progression, which include an additional tumor-specific translocation involving band 14 of chromosome 13. Because this band contains the retinoblastoma (RB) gene, we examined the tumor for aberrations in the RB gene using DNA sequence polymorphism analysis and pulsed-field gel electrophoresis (PFGE), but did not detect alterations in the RB gene.     

Effects of placental tissue on immunological responses

Using in vivo assays for alloreactivity, the responsiveness of maternal lymph node cells was shown to be increased when cells were harvested from both syngeneically and allogeneically pregnant mice. In addition, foetal liver and thymus cells induced a state of delayed type hypersensitivity to alloantigens whereas trophoblastic tissue, in the form of 12 to 15 day metrial glands, did not.

The influence of mouse placenta on in vitro immune responses was tested using 7-day ectoplacental cones as sources of trophoblastic tissue. Ectoplacental cones increased a T cell dependent but not a T cell independent antibody response, whereas T cell dependent cytotoxicity was reduced. The findings reported in this paper suggest that murine ectoplacental cones are a particularly useful tissue with which to study both specific and non-specific immunological activities of the trophoblast in syngeneic and allogeneic pregnancies.

     

Effects of methylprednisolone on electrolyte transport by in vitro rat ileum

Administration of the glucocorticoid methylprednisolone (MP) (30 mg/kg body wt for 3 days) to rats increased intestinal mucosal guanylate cyclase and Na-K-ATPase activities, short-circuit current (Isc), electrical potential difference (PD), net Na absorption, and net Cl secretion and reversed HCO3 transport from secretion to absorption. In the MP-treated animals, removal of HCO3 from both the mucosal and serosal bathing solutions increased Cl secretion but did not alter the Isc, PD, and net Na flux. Removal of Cl abolished the MP-induced increase in Isc but did not affect the MP-induced changes in net Na and HCO3 fluxes. At 6 h, after a single dose of MP, stimulation of guanylate cyclase activity was already maximal, whereas Na-K-ATPase activity was not detectably altered. The changes in intestinal transport properties present 6 h after MP treatment and associated with the increased guanylate cyclase activity were an increase in Isc and PD and a reversal of net Cl absorption to net secretion. These results suggest that an initial response to MP administration is a persistent increase in intestinal guanylate cyclase activity that mediates an electrogenic Cl secretory process, then is followed by a superimposed effect of increased Na-K-ATPase activity that mediates an increase in net Na absorption.     

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Serodiagnosis of viral hepatitis A by a modified competitive binding radioimmunoassay for immunoglobulin M anti-hepatitis A virus.

A competitive binding radioimmunoassay (CBA) for antibody to hepatitis A virus (HAV) was evaluated and compared with a standard solid-phase radioimmunoassay for anti-HAV, CBA was found to be sensitive and specific for the detection of anti-HAV, as demonstrated by the 98% concordance of CBA and solid-phase radioimmunoassay test results. The standard CBA test was modified for the differential detection of acute (immunoglobulin M) and convalescent (immunoglobulin G) anti-HAV by incorporation of a step in which immunoglobulin G anti-HAV was preferentially absorbed with S. aureus cells (protein A). The modified CBA test was shown to be capable of differentiating between acute- and convalescent-phase sera. The modified CBAM test was able to detect immunoglobulin M anti-HAV up to approximately 4 weeks after the onset of illness.