Heat treatment of hepatocellular carcinoma cells: increased levels of heat shock proteins 70 and 90 correlate with cellular necrosis.

Immunotherapy, i.e. stimulation of the body's immune response against tumor cells, is a promising approach in cancer treatment. In this context, heat shock proteins (HSP) have been shown to function in tumor antigen chaperoning. HSP are evolutionarily conserved and show increased expression in response to chemical and physical stress. Two members of the HSP family, HSP 70 and 90, seem to further act as immunostimulating agents because of their possible involvement in tumor antigen presentation. We cultured the human hepatocellular carcinoma cell line HepG2 and investigated its HSP content under normal and hyperthermic conditions. Flow cytometry showed increased levels of HSP 70 and 90 after heat shock at 41.8 degrees C for 60 minutes, measured after a subsequent incubation time of five hours, as compared to untreated cells in vitro. We further observed a clear correlation between the HSP 70 and 90 levels and the necrotic cell subpopulation in heat shocked tumor cells. We conclude that HSP expression in HepG2 cells can be enhanced by heat shock treatment in vitro. We suggest that this mechanism can be exploited in increasing tumor immunogenicity.

Tumor antigen pulsed dendritic cells enhance the cytolytic activity of tumor infiltrating lymphocytes in human hepatocellular cancer.

OBJECTIVE: Tumor infiltrating lymphocytes (TILs) stimulated with interleukin-2 (IL-2) ex vivo have been successfully used therapeutically in some cancer patients, but their potency in eliciting an effective anti-tumor response is variable. We have tried to augment killing activity of tumor infiltrating lymphocytes derived from hepato-cellular carcinoma (HCC) using autologous monocytes derived dendritic cells. METHODS: Tumor infiltrating lymphocytes (TILs) from 6 patient with hepatocellular carcinoma were isolated and the phenotype were further characterized. From the same patients, autologous dendritic cells were generated from CD14+ monocytes that were cultured for 6 days in the presence of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Those professional antigen presenting cells were pulsed with whole autologous hepatoma tumor lysates (pDC). TILs were cocultured with pDC or unpulsed DC. To assess the cytotoxic potency of TILs, the ability to lyse the tumor cell targets K652, Daudi and an allogeneic HCC celline was determined in a standard cytotoxic assay. RESULTS: Tumor cells targets in vitro are poorly lysed by tumor infiltrating lymphocytes indicating T-cell hyporesponsiveness. In contrast, the killing activity of HCC derived TILs against Daudi (9.15% +/- 7.5) and allogeneic HCC tumor target (18.2% +/- 9.2) could be significantly augmented when stimulated with pDC (Daudi: 38% +/- 6.8 and allogeneic HCC: 55% +/- 10). The killing activity of TILs against K562 was unaffected by pDC. CONCLUSION: The low cytotoxic activity profile of HCC derived TILs in vitro can be increased by tumor lysate pulsed dendritic cells and may therefore be more effective in vivo when used for adoptive immunotherapy.

Cost analysis of an Italian ICU.

BACKGROUND: Cost reduction is an important issue in medicine today, especially when considering ICUs, since they account for a large percentage of all hospital expenditure. Through a retrospective analysis of the data regarding the expenses incurred during the years 1996-97, we have been able to evaluate the total costs of our ICU and the influence that each component had on the final costs, thus gathering the necessary information for the improvement of the unit itself. METHODS: Retrospective analysis of a 5-bedded multidisciplinary ICU activity over a two-year period (1996-1997). Cost-related data have been supplied by the Hospital Administration as to wages, infrastructures, equipment buying and maintenance; by Hospital Pharmacy as to drugs and devices supplies; and by Laboratory and Radiology as to diagnostic investigations. RESULTS: According to our experience, physicians and non-medical staff account for more than 50% of the total expenditure--the latter slightly prevailing. Furthermore, we have assessed that the cost distribution is hardly comparable to that reported by other authors. CONCLUSIONS: It is useful to analyse the total distribution and to evaluate their nature only to gain the necessary data that will lead to a more effective management of the unit. Nevertheless, this methodology is valid within the cost analysis of our ICUs but the ICUs of other countries show great differences in the way they are structured and some of the use more reliable activity-based costing methodology.

Physiopathology of the renin-angiotensin system in the ovary.

The present review confirms the existence of the so-called "ovarian-derived prorenin-angiotensin cascade". It also describes the physiopathology of the system and, consequently, its role in the genesis of phenomena concerning reproductive function such as ovulation, steroid synthesis and folliculogenesis. Moreover, the "ovarian-derived prorenin-angiotensin cascade" appears to play an important role in the aetiopathogenesis of diseases such as ovarian tumours, ovarian hyperstimulation syndrome and ectopic pregnancy.

Physiopathology of the renin-angiotensin system in the ovary.

The present review confirms the existence of the so-called "ovarian-derived prorenin-angiotensin cascade". It also describes the physiopathology of the system and, consequently, its role in the genesis of phenomena concerning reproductive function such as ovulation, steroid synthesis and folliculogenesis. Moreover, the "ovarian-derived prorenin-angiotensin cascade" appears to play an important role in the aetiopathogenesis of diseases such as ovarian tumours, ovarian hyperstimulation syndrome and ectopic pregnancy.

[Gastrin and gastrointestinal neoplasias]. / Gastrina e neoplasie gastrointestinali.

In this paper the authors focus attention on the role of gastrin as a carcinogenic factor. The aim was to bring together the numerous controversial studies on this subject, adding the authors' own personal clinical experience. Gastrin (G) is responsible for the development of carcinoids, as has been experimentally shown in Mastomys rats, and more recently in man. This hormone is regarded as a mitogen for cells in the gastroenteric tract; it acts through specific reactors and messengers, including AMPc and protein kinase A (PKA). Its role in the development of other neoplasias of the gastroenteric tract appears to be linked, but not always subordinate, to the presence of growth factors such as: EGF and TGF-alpha, and also to the possible stimulation of oncogens induced by hypergastrinemia.

Effects of Panax Ginseng C.A. Meyer saponins on male fertility.

Sixty-six patients have been treated with Panax Ginseng C.A. Meyer extract, of whom 30 oligoastenospermic sine causa (group A), 16 oligoastenospermic with idiopathic varicocele (group B). Twenty age-matched volunteers were used as controls (group C). Use of Panax Ginseng extract showed an increase in spermatozoa number/ml and progressive oscillating motility, an increase in plasma total and free testosterone, DHT, FSH and LH levels, but a decrease in mean PRL. It is suggested that ginsenosides may have an effect at different levels of the hypothalamus-pituitary-testis axis.

Relationship between autoimmune thyroid disease Rand Alzheimer's disease.

Alzheimer's Disease (AD) is a particular form of degenerative dementia probably due to deposit in the brain cortex of a non soluble protein called beta-A4 amyloid in senile plaque form. beta A4 is an aberrant mutant proteolytic product of Amyloid Protein Precursor (APP) codified on chromosome 21. Trisomy 21 is responsible for Down's Syndrome (DS). Down's patients have been shown to develop a form of Alzheimer's after 50 years of age, and high blood levels of antithyroid antibodies are also present in a significant percentage of these cases. In the present investigation, antithyroid antibody titres have been studied by means of RIA in group of 34 AD patients. As compared to 30 non-demented controls, AD subjects showed a significant increase in the mean values of antithyroglobulin (TgAb) and antimicrosomial (MCSAb) autoantibodies. The physiopathological relationship regarding this association is discussed.

Sphingosine effects on the contractile behavior of skinned cardiac myocytes.

Sphingosine modulates myocyte beating behavior by acting on the sarcoplasmic reticulum calcium release channel, the ryanodine receptor. Chemically skinned myocytes isolated from adult rabbit ventricles exhibited spontaneous asynchonous contractions in response to micromolar levels of calcium. These cells do not have a functional sarcolemma but exhibit spontaneous contraction-relaxation cycles which are controlled by the sarcoplasmic reticulum. The intracellular second messenger, sphingosine, significantly reduced myocyte beat frequency in a biphasic manner with an IC50 of c. 0.5 microM. A computerized video-enhancement micrography system was used to determine the effect of sphingosine on sarcomere contractile parameters and to determine the potential source of the altered beating behavior produced by sphingosine. Contraction parameters related to sarcomere shortening were unaffected by sphingosine in the submicromolar range, suggesting that sphingosine had no effect on the contractile machinery itself. However, submicromolar sphingosine had a significant inhibitory effect on the spread of activation from sarcomere to sarcomere in these cells. Activation waves were propagated with an average velocity of 331 and 199 microns/s in control and sphingosine (0.58 microM) treated cells, respectively. Permeabilized myocyte calcium uptake was markedly increased by treatment with sphingosine, consistent with an inhibitory effect of sphingosine on sarcoplasmic reticulum calcium release. Sphingosine blocked calcium-induced calcium release from isolated cardiac sarcoplasmic reticulum membranes containing the ryanodine receptor. The results suggest that the site of sphingosine action on calcium signaling and beating behavior in the cardiac cell is the sarcoplasmic reticulum ryanodine receptor. By inhibiting channel opening sphingosine may increase the calcium threshold necessary to trigger calcium-induced calcium release, thus modulating cardiac excitation-contraction coupling.

[Long-term sedation with propofol in ICU: hemocoagulation problems]. / Sedazione di lunga durata con propofol in ICU: problemi emocoagulativi.

The Authors have studied the effects of propofol on coagulation in 15 patients admitted to ICU. Propofol was used for long-term sedation (therapeutic range 3 mg/kg/h). Variables monitored included: platelets, PTT, PT, Fibrinogen, FDP, AT III. The effects on coagulation has been investigated in three groups of patients: group I) 5 patients that received propofol for 1-3 days; group II) 5 patients that received propofol for 4-10 days; group III) 5 patients that received propofol for 11-35 days. No difference were found about blood coagulation in this groups of patients before and after administration of propofol.

[Fluconazole in the therapy of candidiasis in intensive care]. / Fluconazolo nella terapia delle candidosi in terapia intensiva.

The authors studied four patients in ICU suffering from Candida infections who were treated with a new bis-triazole antimycotic, fluconazole. Various parameters of blood chemistry and blood and urine drug levels were monitored. After treatment all microbiological tests had become negative and clinical conditions had improved considerably within 30 to 60 days. No significant side effects were observed.

Patterns of sarcomere activation, temperature dependence, and effect of ryanodine in chemically skinned cardiac fibers.

Functionally skinned and electrochemically shunted myocytes were prepared by perfusing rat hearts with collagenase in order to obtain a technically improved measurement of sarcomere dynamics and to evaluate the role of sarcoplasmic reticulum in situ with respect to contractile activation. In the presence of micromolar calcium, the myocytes exhibited phasic and propagated contraction waves beginning at one end and proceeding along the myocyte. Beating rates, the propagation velocity of the activation wave, and single sarcomere shortening and relaxation velocities were obtained by manual or automated analysis of 16-mm film recorded at 170 frames/s from a camera attached to a microscope that was equipped with a temperature-controlled stage. In parallel experiments, calcium accumulation by the sarcoplasmic reticulum of the myocytes in situ was measured by direct isotopic tracer methods. The frequency (10-38 min-1) of spontaneous contractions, the velocity (1.9-7.4 microns . s-1) of sarcomere shortening, and the velocity (1.7-6.8 microns . s-1) of sarcomere relaxation displayed identical temperature dependences (Q10 = 2.2), which are similar to that of the calcium pump of sarcoplasmic reticulum and are consistent with a rate limit imposed by enzyme-catalyzed mechanisms on all these parameters. On the other hand, the velocity (77-159 microns . s-1) of sequential sarcomere activation displayed a lower temperature dependence (Q10 = 1.5), which is consistent with a diffusion-limited and self-propagating release of calcium from one sarcomere to the other. The phasic contractile activity of the dissociated myocytes was inhibited by 10(-8)-10(6) M ryanodine (and not by myolemmal calcium blockers) under conditions in which calcium accumulation by sarcoplasmic reticulum in situ was demonstrated to proceed optimally. The effect of ryanodine is attributed to an interaction of this drug with sarcotubular structures, producing inhibition of calcium release from the sarcoplasmic reticulum. The consequent lack of sarcomere activation underlines the role of sarcoplasmic reticulum uptake and release in the phasic contractile activation of the electrochemically shunted myocytes.

The effects of chemical cross-linking agents on calcium-induced structural changes in skinned muscle fibers. Origin within thick filaments detected by optical diffraction methods.

We have reported earlier (Sabbadini, R.A., Rieser, G.D. and Paolini, P.J. (1979) Biochim. Biophys. Acta 578, 526-533) that physiological levels of calcium (pCa 6.95-5.49) can produce structural changes in thick filaments which are detectable as an intensity loss of the first-order optical diffraction lines from chemically skinned skeletal muscle fibers stretched beyond myofilament overlap. We now show that the calcium-induced intensity decrease results from structural changes within, rather than between, thick filaments. Glycerinated, detergent-treated fibers from frog semitendinosus muscle were incubated in 1-10 mM concentrations of dimethylsuberimidate (DMS), dithiobis(succinimidylpropionate) (DTSP) or dimethyl-3,3'-dithiobispropionimidate (DTBP) for 4 h. These substances are homobifunctional lysine-modifying cross-linking reagents known to restrict movement of S-1 heads and limit changes in the association of myosin rods within the core of the thick filament without affecting interfilament lattice spacing. Diffraction patterns from cross-linked cells in relaxing solution were identical to those in control cells, but Ca2+ (pCa 5.49) totally failed to produce the typical 50-70% attenuation of first-order line intensity. Cleavage of the disulfide bond in DTBP-treated cells with dithiothreitol fully restored the Ca2+ sensitivity. Lysine group modification with methylacetimidate, a monofunctional lysine modification reagent equivalent to DMS, did not block the Ca2+ sensitivity. We observed that intensity reductions can also be produced by numerous other agents and mechanisms, such as nonionic polymeric solutions of polyvinylpyrrolidone, which reduces the lattice spacing, and alkaline pH, which probably displaces the S-1 heads from a resting position close to the thick filament surface. However, the prevention of the Ca2+ effect by cross-linkers indicates that intrafilament rather than interfilament changes in structure are responsible for the light diffraction intensity decrease accompanying activation.

Calcium-induced structural changes in chemically skinned muscle fibers. Detection by optical diffractometry.

The intensity of the first order diffraction line produced by chemically skinned muscle fibers was detected by a self scanning photodiode array and minicomputer system. Line intensity was observed to decrease in fibers stretched to zero filament overlap when subjected to calcium-EGTA buffers in the physiological pCa range. Calcium dependent intensity decreases were not observed for myosin extracted fibers indicating that the thick filament proteins may be the source of the calcium effect seen in non-extracted fibers. These results can be interpreted in terms of calcium dependent effects on thick filament disordering which are not dependent upon cross bridge formation.

Sarcomere motion in isolated cardiac cells.

Computerized image-analysis techniques have been employed to examine the sarcomere dynamics of isolated mammalian cardiac myocytes. The cells were prepared by perfusion of adult rabbit hearts with hyaluronidase-collagenase solutions; they exhibited phasic contractions in the presence of 10(-6) M Ca2+. The dissociated cells were visualized by phase microscopy and a video camera interfaced in a minicomputer. Digitized cell images were processed by an algorithm utilizing signal averaging and contrast enhancement to yield data showing individual sarcomere position and shortening vs. time, so that patterns of sarcomere activation could be observed in spontaneously contracting cells. Compared to records of whole-cell shortening and of striation displacement, computerized image analysis provided a much more faithful indication of time course and sequence of sarcomere shortening. Spontaneously contracting cells showed sequential sarcomere shortening beginning at one end and propagating longitudinally with a constant velocity, typically at 100--150 micron/s for beat rates of 40 min-1. Velocities of initial sarcomere shortening appeared to increase with elevated Ca2+. These observations are consistent with a regenerative mechanism of calcium-induced calcium release.

Light diffraction studies of sarcomere dynamics in single skeletal muscle fibers.

A position-sensitive optical diffractometer has been used to examine the diffraction spectra produced by single skeletal muscle fibers during twitch and tetanic contraction. First-order diffraction lines were computer-analyzed for mean sarcomere length, line intensity, and percent dispersion in sarcomere length. Line intensity was observed to decrease rapidly by about 60 percent during a twitch, with an exponential recovery to resting intensity persisting well beyond cessation of sarcomere shortening; recovery was particularly prolonged at zero myofilament overlap. A number of single fibers at initial lengths from 2.5 to 3.5 MICRON EXHIBITED a splitting of the first-order line into two or more components during relaxation, with components merging back into a single peak by 200 ms after stimulation. This splitting reflects the asynchronous nature of myofibrillar relaxation within a single fiber. During tetanus, the dispersion decreased by more than 10 percent from onset to plateau, implying a gradual stabilization of sarcomeres.

Sarcomere length dispersion in single skeletal muscle fibers and fiber bundles.

Light diffraction patterns produced by single skeletal muscle fibers and small fiber bundles of Rana pipiens semitendinosus have been examined at rest and during tetanic contraction. The muscle diffraction patterns were recorded with a vidicon camera interfaced to a minicomputer. Digitized video output was analyzed on-line to determine mean sarcomere length, line intensity, and the distribution of sarcomere lengths. The occurrence of first-order line intensity and peak amplitude maxima at approximately 3.0 mum is interpreted in terms of simple scattering theory. Measurements made along the length of a singel fiber reveal small variations in calculated mean sarcomere length (SD about 1.2%) and its percent dispersion (2.1% +/- 0.8%). Dispersion in small multifiber preparations increases approximately linearly with fiber number (about 0.2% per fiber) to a maximum of 8-10% in large bundles. Dispersion measurements based upon diffraction line analysis are comparable to SDs calculated from length distribution histograms obtained by light micrography of the fiber. First-order line intensity decreases by about 40% during tetanus; larger multifibered bundles exhibit substantial increases in sarcomere dispersion during contraction, but single fibers show no appreciable dispersion change. These results suggest the occurrence of asynchronous static or dynamic axial disordering of thick filaments, with a persistence in long range order of sarcomere spacing during contraction in single fibers.