[Laparoscopic cholecystectomy in an overnight admission]. / Internarea de o zi în colecistectomia laparoscopica.

OBJECTIVE: to determine the feasibility and desirability of laparoscopic cholecystectomy (LC) in an overnight versus a several day admission. BACKGROUND: Although LC is performed in U.S.A. as outpatient surgery this has not been generally accepted in Europe. The main objections, are the risk of complications becoming apparent at home. Considering the specific local conditions and the general attitude towards early discharge we tried to assess the degree of feasibility and acceptance of a overnight admission in our patients. METHODS: Two groups of forty patients each, with symptomatic gallstones, received LC either in an overnight or a several day admission. Preoperative and intraoperative data and postoperative recovery were prospectively assessed. RESULTS: The general and female age, the duration and frequency of the biliary pain, the duration and the degree of difficulty of the operation and the necessity of the subhepatic drainage were all significant in favour of the group with a shorter stay. No postoperative complications were recorded. CONCLUSIONS: Overnight LC is safe and effective in treating patients requiring LC. The option of the patient must also be considered.

Effects of clozapine and 2,5-dimethoxy-4-methylamphetamine [DOM] on 5-HT2A receptor expression in discrete brain areas.

Activation of 5-HT2A receptors has been shown to be an essential component of the discriminative stimulus effects of indoleamine and phenethylamine hallucinogens. The objective of the present study was to determine the neuroanatomical location of the 5HT2A receptors which may be responsible for the stimulus effects of the phenethylamine hallucinogen [-]2,5-dimethoxy-4-methylamphetamine (DOM). It was hypothesized that brain areas containing altered 5-HT2A receptor expression in the context of a similar alteration in DOM-induced stimulus control might be important in mediating the stimulus effects of DOM. Fisher 344 rats were treated with either clozapine (25 mg/kg/day) or DOM (2 mg/kg/day) for 7 days, and the consequences of these drug treatment regimens on DOM-induced stimulus control and on 5-HT2A receptor expression in several brain areas were determined. Chronic administration of clozapine was associated with a wide-spread decrease in levels of 5-HT2A/2C receptors. Conversely, treatment with DOM had varied effects including a neuroanatomically selective decrease in 5-HT2A/2C receptor levels that was restricted to the olfactory nucleus. Both chronic treatment with DOM and clozapine decreased the stimulus effects of DOM. The present findings suggest a role for the olfactory nucleus in producing the stimulus effects of DOM.

Profound apoptosis-mediated regional myocyte loss and compensatory hypertrophy in pigs with hibernating myocardium.

BACKGROUND: Myocyte apoptosis is seen in ischemic heart disease, but whether it can occur after reversible ischemia or independent of necrosis and replacement fibrosis is unknown. METHODS AND RESULTS: Pigs were instrumented with a stenosis of the left anterior descending coronary artery to chronically reduce coronary flow reserve over a period of 3 months. At this time, there was viable dysfunctional myocardium having the physiological features of hibernating myocardium. Resting subendocardial perfusion was reduced to 0.65+/-0.08 (mean+/-SEM) mL. min(-1). g(-1) in hibernating myocardium of instrumented pigs compared with 0.98+/-0.14 mL. min(-1). g(-1) in myocardium of sham-operated pigs (P<0.05). There was a critical limitation in subendocardial flow during vasodilation to 0.78+/-0.20 mL. min(-1). g(-1) in instrumented pigs versus 3. 24+/-0.50 mL. min(-1). g(-1) in sham-operated pigs (P<0.001). Histology revealed a regional reduction in myocyte nuclear density to 995+/-100 nuclei/mm(2) in hibernating myocardium from the instrumented group versus 1534+/-65 nuclei/mm(2) in myocardium from the sham-operated group (P<0.05), regional myocyte hypertrophy (myocyte volume per nucleus, 14 183+/-2594 in the instrumented group versus 9130+/-1301 microm(3) in the sham group; P<0.05), and minimal increases in connective tissue (5.8+/-0.9% in the instrumented group versus 3.0+/-0.2% in the sham group, P<0.05). Necrosis was not identified, but apoptosis was increased from 30+/-9 myocytes per 10(6) myocyte nuclei in myocardium from the sham group to 220+/-77 myocytes per 10(6) myocyte nuclei in hibernating myocardium (P<0.05). CONCLUSIONS: These findings indicate that reversible ischemia in an area of chronically reduced coronary flow reserve induces regional myocyte loss via an apoptotic mechanism. This may contribute to the progression of chronic coronary disease to heart failure and explain the lack of complete functional recovery after revascularization in hibernating myocardium.

Does cartilage down-regulate growth factor expression in tracheal epithelium?

BACKGROUND: Maintaining tracheal integrity and restoring normal physiologic function after injury is complex. Some of the critical events in this process are deposition of a provisional extracellular matrix, tissue remodeling, and angiogenesis. These events are coordinated with epithelial migration and proliferation to restore the mucosal barrier. The ability of respiratory epithelial cells (REC) to migrate and proliferate and restore denuded areas of the large conducting airway after injury is poor. OBJECTIVE: To test the hypotheses that (1) the cartilaginous framework, underlying the extracellular matrix (submucosa) and epithelium, decreases the migratory ability of REC when compared with REC on the same provisional extracellular matrix (type I collagen) alone, and (2) this phenomenon is associated with a change in expression of transforming growth factor (TGF)-alpha and TGF-beta, both of which have been demonstrated in cutaneous models to be important in epithelial migration and proliferation. DESIGN: We developed a culture system that reconstitutes the tracheal lumen in vitro, consisting of dissociated chondrocytes cultured in a manner to form cartilage, submucosa (type I collagen), and REC (termed a "composite culture"). Control cultures consisted of epithelial cells grown on type I collagen alone. Control and composite cultures were evaluated morphologically using scanning electron and light microscopy. Expression of TGF-alpha and TGF-beta was determined in day 14 cultured epithelial cells from control and composite cultures by semiquantitative polymerase chain reaction. RESULTS: Epithelial cells from composite cultures did not spread and were less squamoid in morphological appearance than epithelial cells on type I collagen alone. Expression of both growth factors was reduced in epithelial cells from composite cultures compared with those on type I collagen. CONCLUSIONS: Cartilage modulates TGF-alpha and TGF-beta expression in REC, and may contribute to regulation of REC proliferation and differentiation.

Quantitative study of germinal center area in normal and diseased tonsils using image analysis.

The aim of this study was to measure the germinal center area of secondary follicles in a consistent and unbiased manner using image analysis. Tonsil specimens were obtained from children with clinical evidence of recurrent tonsillitis and/or idiopathic tonsillar hypertrophy. Normal control biopsy specimens were obtained from children with no history of ear, nose, or throat infections. The results show a significant increase in the germinal center area in tonsils exhibiting hypertrophy when compared to normal controls (p < .002). Image analysis is an important tool for making quantitative size comparisons in lymphoid tissues.

Ciliary activity in differentiating and reactivated human respiratory epithelial cells.

OBJECTIVE: Experimental studies of mucociliary clearance, especially those involving drug effects, suffer from the difficulty of determining whether drugs act directly on ciliary motility or whether their effects are indirect, acting via changes in cell metabolism, viscous load, or alternative mechanisms. The present study provides a solution to this problem by comparing the motile characteristics of ciliated cells that have differentiated in primary cell culture with those of demembranated cilia reactivated with MgATP. METHODS: Human respiratory epithelial cells (REC) were dissociated from trachea, bronchus, nasal polyps, or turbinates and then placed in a dissociated cell culture system. Thirty-three percent of the dissociated cells contain beating cilia. Following 1 week in culture, the REC dedifferentiated, but then redifferentiated within 96 hours after they were brought to an air interface. RESULTS: The cilia on such cells beat with planar waves consisting of power and recovery strokes. Beat frequencies at 20 degrees C were 15+/-2.3 Hz. Ciliary beating often was coordinated both within and between cells with a defined antilaeoplectic pattern of coordination. Either fresh or cultured cells could be demembranated with Triton X-100 and reactivated with MgATP. The activity of these reactivated models was equivalent to those observed in living cells. CONCLUSION: The authors have demonstrated that ciliary beat frequency of demembranated human respiratory epithelial cells can be modulated by MgATP and can be adjusted to the same level of activity as measured in living cells. This allows them to selectively test whether a drug's effects on mucociliary transport are the result of direct interactions with the ciliary apparatus or are produced through other indirect mechanisms.

Respiratory syncytial virus and human bronchial epithelium.

BACKGROUND: A suitable model for respiratory syncytial virus (RSV) infection has yet to be developed. OBJECTIVE: To describe an in vitro model of human respiratory epithelium in primary cell culture linked with a computer microscope interface that allows evaluation and imaging of living RSV-infected respiratory epithelium. DESIGN: A descriptive, controlled study. Human bronchial cells were obtained from surgical samples by elastase dissociation and replated on collagen gel membranes. After 7 to 10 days, cells were brought to air interface. Baseline sampling of cell fluid for cytokine production by enzyme-linked immunosorbent assay (interleukin [IL] 1beta, IL-6, IL-8, and RANTES) and leukotriene C4 by radioimmunoassay was taken before treatment with RSV (n=30) or HEp-2 (human laryngeal carcinoma cells) control (n=25). Sampling was done at 4, 24, 72, and 120 hours thereafter. The infectious process was monitored with a microscope (Zeiss UEM, Carl Zeiss Inc, Thornwood, NY) equipped with a camera (Newvicon, Dage Corporation, Stamford, Conn). Images were either digitized using a computer (Macintosh Quadra 950, Apple Computers Inc, Cupertino, Calif) equipped with a digitizing board (Perceptics Corporation, Knoxville, Tenn) or were recorded on an SVHS videotape using a videocassette recorder (JVC, Elmwood Park, NJ). RESULTS: Respiratory syncytial virus induced profound effects on the ciliated cells: ciliostasis, clumping, and loss of cilia from live cells and sloughing of cells. Significant differences in the release of IL-6, IL-8, and RANTES (P<.03 for each cytokine) were noted in RSV-infected bronchial cultures by 24 hours with a peak at 72 hours. The IL-beta and leukotriene C4 were not altered by RSV infection in bronchial cells. CONCLUSIONS: This model closely mirrors human RSV disease and affords a unique opportunity to study interepithelial cell interactions, cytokine responses from cells of different donors, and ciliary activity of live cells undergoing RSV infection.

Visualization of kinetochores and analysis of their refractility in crane-fly spermatocytes after aldehyde fixation.

Glutaraldehyde and formaldehyde were used to fix crane-fly spermatocytes for observation with differential interference contrast (DIC) microscopy. In aldehyde-fixed cells, kinetochores exhibit contrast not normally observed in living cells. Although the mechanism underlying this result is not understood, the visualization of kinetochores as distinct refractile objects opens the way for analysis of unstained kinetochores with the light microscope. The analysis of kinetochore refractility reported in this paper is made possible by the finding that the refractility of chromosomes in formaldehyde-fixed cells decreases as the concentration of formaldehyde is increased. In 4% formaldehyde, the refractility of chromosomes is matched with that of its surround, chromosomes appear invisible, and kinetochores may be analyzed as if chromosomes were not present. Kinetochores were imaged with DIC optics, and then digital image analysis was performed. Gray-level scans through the highlight and shadow of an individual kinetochore parallel to the axis of shear resulted in a curve having a slope proportional to the DIC optical path gradient. Curves from autosomal kinetochores imaged in anaphase had slopes approximately one-half those recorded at metaphase under identical optical conditions. By contrast, kinetochore thicknesses (defined as the distance between the peak and the valley of a gray-level scan) at those two stages were not significantly different. These data suggest a loss of dry mass from autosomal kinetochores during anaphase. Neither the refractility nor thickness of lagging sex kinetochores varied as autosomes went through anaphase. The conclusion drawn from these findings is that the decreased refractility of autosomal kinetochores in anaphase is movement-related.

Effect of high-voltage pulsed current and alternating current on macromolecular leakage in hamster cheek pouch microcirculation.

BACKGROUND AND PURPOSE: Electrical stimulation (ES) is supposed to affect edema formation by inhibiting macromolecular leakage from microvessels. The purpose of the study was to determine the effects of various forms of ES on macromolecular leakage from microvessels. SUBJECTS: Fifty-three hamsters were randomly assigned to one of seven groups: a control group (histamine only); groups that received histamine with cathodal high-voltage pulsed current (HVPC) at intensities of 90%, 50%, and 10% of visible motor threshold (VMT); groups that received anodal HVPC at intensities of 90% and 50% of VMT; and a group that received alternating current (AC) at 90% of VMT. METHODS: Anesthetized animals were injected with fluorescein-labeled dextran. Macromolecular leakage was determined by computer analysis of fluorescence microscopy images for 5 minutes after treatment. RESULTS: When compared with controls, leakage was less in groups treated with cathodal HVPC at 90% and 50% of VMT and anodal HVPC at 90% of VMT. CONCLUSION AND DISCUSSION: Cathodal and anodal HVPC, but not AC, curb macromolecular leakage from the microvessels of histamine-treated hamsters. [Taylor K, Mendel FC, Fish DR, et al. Effect of high-voltage pulsed current and alternating current on macromolecular leakage in hamster cheek pouch microcirculation.

Apoptosis of late-stage erythroblasts in megaloblastic anemia: association with DNA damage and macrocyte production.

An in vitro model of folate-deficient erythropoiesis has been developed using proerythroblasts isolated from the spleens of Friend virus-infected mice fed an amino acid-based, folate-free diet. Control proerythroblasts were obtained from Friend virus-infected mice fed the same diet plus 2 mg folic acid/kg diet. Our previous studies showed that, after 20 to 32 hours of culture in folate-deficient medium with 4 U/mL of erythropoietin, the folate-deficient proerythroblasts underwent apoptosis, whereas control erythroblasts survived and differentiated into reticulocytes over a period of 48 hours. The addition of folic acid or thymidine to the folate-deficient medium prevented the apoptosis of the folate-deficient erythroblasts, thereby implicating decreased thymidylate synthesis as the main cause of apoptosis in the folate-deficient erythroblasts. In the study reported here, we examined intracellular folate levels, uracil misincorporation into DNA, p53 and p21 proteins, and reticulocyte formation in erythroblasts cultured in folate-deficient or control medium. In all experiments, the folate-deficient erythroblasts cultured in folate-deficient medium gave results that varied significantly from folate-deficient erythroblasts cultured in control medium or control erythroblasts cultured in either folate-deficient or control media. Folate-deficient erythroblasts cultured in folate-deficient medium had marked decreases in all coenzyme forms of folate that persisted throughout culture, increased uracil misincorporation into DNA, persistent accumulations of p53 and p21, and decreased reticulocyte production but increased size of individual reticulocytes. A model of folate-deficient erythropoiesis based on apoptosis of late stage erythroblasts is presented. This model provides explanations for the clinical findings in megaloblastic anemia.

The phylogenetic odyssey of the erythrocyte. IV. The amphibians.

Amphibians manifest permanently nucleated, oval, flattened, biconvex erythrocytes. These cells demonstrate a cytoskeleton which is responsible for their morphogenetic conversion from a sphere to an ellipse and imparts to their cellular mass reversibility of traumatic deformation. The class Amphibia has the largest of all erythrocytes attaining volumes greater than 10,000 femtoliters in the Amphiuma. The large dimensions reflect evolutionary processes, genomic size, ploidy and the relative size of other somatic cells. Conversely, the erythrocyte count and hemoglobin concentration of these species are low. Occasional denucleated red cells can be seen in the peripheral blood but may attain levels of 90-95% of the total circulating population in certain members of the tribe Bolitoglossini (e.g. Batrachoseps attenuatus). These erythroplastids retain the marginal band thus remaining different from mammalian erythrocytes. Embryologically, erythropoiesis initiates in the yolk sac and then progresses to the kidney, liver, and possibly spleen. The yolk sac cohort is transitory and is successively replaced by the larval and definitive populations of erythrocytes. Red cell production (along with thrombocytopoiesis) in adult urodeles is conducted intravascularly in the spleen. In anurans this organ is usually the major site although the liver also serves as a secondary locus for this activity. Medullary (bone marrow) erythropoiesis makes its phylogenetic debut in anurans and typically occurs during heightened hemopoiesis following metamorphosis or hibernation. Maturation of the erythrocyte in the circulation is commonplace (especially in urodeles) while proliferation at this site is inducible by splenectomy and/or hemolysins. Erythrocyte-related values demonstrate variable differences associated with age, weight, season, gender, and environment.

Isolation and characterization of basal cells from human upper respiratory epithelium.

Cellular pathways of normal and reparative differentiation of upper airway epithelium are not well understood. Of the three main cell types, basal and secretory cells are known to divide, while ciliated cells are considered terminally differentiated. Several investigations support the role of the basal cell as a progenitor cell type, but others suggest that the secretory cell can regenerate a complete mucocilliary epithelium. Thus, lineage relationships within renewing adult epithelia are still unclear. Understanding the pathways involved in upper airway epithelial cell differentiation is critical for studying injury and repair mechanisms and for developing clinical strategies for tracheal reconstruction. We undertook the current studies to determine the integrin profile of isolated human upper airway basal cells. Respiratory epithelial cells (REC) were isolated by elastase digestion, stained with FITC-labeled Griffonia simplicifolia isolectin B4 (GSI-B4), and sorted by flow cytometry. Approximately 80% of the lectin-positive cells were basal cells, as determined by morphology and cytokeratin staining. These cells expressed integrins alpha 1, alpha 2, alpha 3, alpha 5, alpha v beta 5, beta 1, beta 3, and alpha 6 beta 4, by immunohistochemistry. This is the first report to identify the integrin profile of isolated human upper airway basal cells. These basal cells could be maintained on type I collagen for at least 7 days, where they became partially confluent and retained expression of cytokeratins 5 and 14. Availability of pure populations of basal cells should permit investigations of their role in both normal and maladaptive repair of adult upper airway epithelium.

Morphologic and motility changes of nasal cilia in primary culture caused by Haemophilus influenzae.

We focused on the pathogenicity of otitis media with effusion (OME) with respect to the susceptibility of the upper respiratory tract mucosa to Haemophilus influenzae. Human nasal polyps in outgrowth culture were used to study H influenzae disturbance of the ciliary beat frequency (CBF) and the morphology of cilia. The CBF of control primary culture was 11.7 +/- 2.7 Hz. The CBF slowed down significantly, to 8.5 +/- 5.7 Hz, after incubation with the filtrate of 10(8) CFU/mL of H influenzae, and to 4.1 +/- 4.1 Hz with a suspension of 10(8) CFU/mL of H influenzae (p<.05). In the morphologic study, we classified the shapes of the cilia into five types: normal cilia, immotile cilia, swollen cilia, clumped cilia, and exfoliated ciliated cells. The abnormal shapes of cilia increased after incubation with the suspension or the filtrate of H influenzae. By scanning electron microscopy, we saw that aggregated bacteria tended to associate with cilia. Thus, the findings suggest that H influenzae disturbs the ciliary clearance of nasal cells and makes them more susceptible to infections.

Interdoublet sliding in bovine spermatozoa: its relationship to flagellar motility and the action of inhibitory agents.

Interdoublet sliding rates were assessed in bull sperm, utilizing a freeze-thaw procedure to allow axonemal disintegration. The sliding rate of 23 degrees C increased with increasing MgATP concentrations up to 1 mM ATP, to plateau at 8 microns/sec. The analyzed interdoublet shear in both live and demembranated (Triton X-100-extracted) bull sperm reactivated with 1 mM ATP established maximal microtubule sliding rates at 6 microns/sec during flagellar beating. Therefore, in vitro sliding rates were sufficient to account for the beat in intact flagella. The effect of inhibitors of flagellar motility on in vitro sliding rates was evaluated. While 8 microM vanadate minimally reduced the sliding rate (to approximately or equal to 4 microns/sec), only 0.5 microM vanadate was sufficient to terminate reactivated bull sperm motility. Nickel ion (0.66 mM) terminated all spontaneous motility, while only reducing microtubule sliding rates to approximately or equal to 5.0 microns/sec. Exposing intact bull sperm to theophylline (1 mM), and incubating the subsequently demembranated sperm in cAMP (3 microM), improved flagellar motility, but had little impact on microtubule sliding rates as determined by axonemal disintegration. Furthermore, deactivating live sperm with 2 mM KCN and 4 mM 2-deoxy-D-glucose renders the subsequently reactivated sperm immotile (as long as exogenous cAMP is absent). Yet, this treatment only reduced the sliding rate by 38%. Paradoxically, 4 mM MgADP reduced the sliding rates most dramatically (86%), whereas demembranated sperm models retain a strong, coordinated beating pattern in the presence of MgADP. These results demonstrate that there is no direct relationship between interdoublet sliding rates and the capacity for coordinated flagellar beating.

Immunodeficiency in RFM/(T6xRFM)F1 mouse chimaeras with lethal host-versus-graft syndrome.

Rather than central tolerance, the perinatal inoculation of related F1 hybrid spleen cells into inbred mice may result in host-versus-graft (HVG) reactions manifested as transient autoimmunity, or as a lethal immunodeficiency syndrome. RFM/(T6xRFM)F1 chimaeras with lethal disease die in 30 days with lymphosplenomegaly, immune complexes and impaired immune responses. The present studies used in vitro proliferation assays to show that the HVG reaction caused hyperplasia sufficient to account for the lymphosplenomegaly, while also causing severe impairment of splenic and nodal cell responses to concanavalin A (Con A) and to bacterial lipopolysaccharide (LPS). By 25 days, HVG mice could not distinguish between self and non-self as judged by mixed lymphocyte reactions (MLR) to RFM, (T6xRFM)F1 and third party A/J cells. There were no indications that host cells reactive to F1 donor cells had undergone clonal deletion, anergy or expansion. Flow cytometry revealed that donor T lymphocytes achieved stable engraftment, mostly in the nodes, despite the HVG reaction. Taken together with previous observations, these studies showed that HVG reactions in young parent F1/chimaeras can result in an immunodeficiency state which is characterized by an early appearing, profound and persistent impairment of both host and donor T and B cell functions. The results suggest that HVG reactions can contribute directly to immune deficits seen after clinical allogeneic bone marrow transplantation.

Cytokeratin expression in human respiratory epithelium of nasal polyps and turbinates.

The cytokertatins in respiratory epithelial cells (REC) of human nasal polyps and turbinates were analyzed by immunohistochemistry. Cytokeratin 19 (CK19) was present in all REC, CK5 and 14 were expressed primarily in basal cells, and CK7, 8, and 18 were found in suprabasal cells. Differences in cytoplasmic locations were also apparent among the individual cytokeratins. CK13 was not detected in any REC of these tissues. The results indicate the profile of cytokeratins in REC of human nasal polyps and turbinates is essentially identical to that of REC in the more distal respiratory tract.

Outer arm dynein from Newt lung respiratory cilia: purification and polypeptide composition.

Dyneins are multimeric ATPases that comprise the inner and outer arms of cilia and flagella. It previously has been shown that salt extraction of newt lung axonemes selectively removes > 95% of the outer arm dynein (OAD), and that the beat frequency of OAD-depleted axonemes cannot be activated as compared to controls [Hard et al., 1992: Cell Motil. Cytoskeleton 21:199-209]. Therefore, expression of the activated state appears to require the presence of outer dynein arms. The present study was undertaken to ascertain basic information on the structure and molecular composition of newt OAD. Populations of demembranated axonemes were extracted with 0.375 M salt. Each lung released approximately 1.4 x 10(7) axonemes during isolation, yielding approximately 120 ng of salt extractable OAD. Electron microscopy of negatively stained samples revealed that newt OAD consisted of two globular heads joined together by a Y-shaped stem, similar to sea urchin and trout sperm OAD. Each head appeared to be roughly spherical in shape, measuring approximately 17 nm in diameter. Electrophoretic analysis of whole axonemes revealed more than six dynein heavy chains when resolved in silver stained 0-8 M urea, 3-5% acrylamide gradients. Extracted OAD, either crude in high salt or purified by alloaffinity, was composed of two heavy chains. UV-induced (366 nm) photolytic cleavage at the V1 site, performed in the presence of Mg2+, vanadate, and ATP, produced four new polypeptides (M(r) 234, 232, 197, and 189 kD). Photolysis was supported by Mg2+ and Ca2+, but did not occur in the presence of Mn2+. The apparent M(r) of the dynein heavy chains was determined to lie between 430-420 kD. Eight discrete polypeptides (putative intermediate chains, IC1-IC8, M(r), 175-56 kD) copurified with the alpha- and beta-heavy chains by microtubule-alloaffinity. Based on its extraction characteristics, polypeptide composition in purified and crude samples, and structure, we conclude that this two-headed particle represents the entire newt respiratory outer arm dynein.