Evaluation of stability of allergen extracts for sublingual immunotherapy during transport under unfavourable temperature conditions with an innovative thermal insulating packaging.

Many pharmaceutical and biotechnological products are temperature-sensitive and should normally be kept at a controlled temperature, particularly during transport, in order to prevent the loss of their stability and activity. Therefore, stability studies should be performed for temperature-sensitive products, considering product characteristics, typical environmental conditions, and anticipating environmental extremes that may occur during product transport in a specific country. Staloral products for sublingual immunotherapy are temperature sensitive and are labelled for maintenance under refrigerated conditions (2-8°C). Given the peculiar climatic context of Italy and the great temperature fluctuations that may occur during transport, this study was aimed at evaluating the impact of a new engineered thermal insulating packaging for Staloral. In particular, the purpose was to assess whether the new packaging could create a container condition able to preserve the stability and immunological activity of the product during the transport phase throughout Italy. The results showed that the range of temperatures that can affect the product, in the area surrounding the product packaging, may reach a peak of 63°C during transport under the most unfavourable climatic conditions, i.e. in a non-refrigerated van during the summer season, from the site of production in France to the patient's house in Catania, the city with the highest temperatures in Italy. However, the highest temperature reached inside the vaccine did not exceed 45°C over a period of about 2 h. The ELISA inhibition test on samples subjected to the extreme temperature conditions previously defined (45°C) showed an immunological activity higher than 75% of that initially measured and was comparable to those obtained with samples stored at controlled temperature (5°C). This means that, even in the worst case scenario, the structure of the allergen extracts is not influenced and the vaccine potency is preserved.

A monoclonal antibody (AB/3) reactive with human breast cancer.

Monoclonal antibody AB/3 was produced from a fusion of spleen cells of a human breast cancer cell-primed BALB/c mouse with the murine myeloma cell line P3-NS1-Ag4-1. The antibody reacted strongly with the plasma membrane of human breast cancer cells. Tissue sections of both malignant and benign human mammary carcinomas and tumors of non-breast origin as well as apparently normal tissues were tested with immunoperoxidase. Ninety-six of 124 (77%) primary human breast cancers, 12 of 14 (86%) metastatic breast lesions, and 12 of 44 (27%) benign breast lesions reacted positively. Little or no appreciable reactivity was observed with apparently normal human tissues and carcinomas of non-breast origin, with the exception of colon carcinoma. Antibody AB/3 did not immunoprecipitate any identifiable protein from radiolabeled extracts of the immunizing cell line.

Glucocorticoid receptor determinations in leukemia patients using cytosol and whole-cell assays.

Parallel determinations of glucocorticoid receptors in the cells of patients with various forms of leukemia were made by two assay methods, one using cell-free cytosolic extracts and the other using whole-cell preparations. Both assays revealed saturable binding of triamcinolone acetonide in all cases. The mean equilibrium dissociation constant for the interaction of triamcinolone acetonide with the cytoplasmic receptor at 2 degrees was 9.45 +/- 6.33 (S.D.) nM while that for the whole-cell binding at 37 degrees was 6.13 +/- 3.25 nM, suggesting an increase in receptor affinity at physiological temperatures. Competition experiments with various unlabeled steroids revealed a higher degree of glucocorticoid specificity at 37 degrees in whole-cell suspensions than at 2 degrees in cytosol. In a comparative analysis of 41 leukemic cell specimens, it was found that determinations carried out by whole-cell assay, calculated as number of sites per cell correlated well with those performed by cytosol assay, calculated as fmol/mg protein, independent of the type of leukemia. However, for cells with low receptor content, the two assay methods were more difficult to compare. In agreement with previous reports, the cytosol assay consistently underestimated the number of receptors with respect to the whole-cell assay, particularly in cases of lymphatic leukemia. Furthermore, the underestimation decreased for increasing levels of total cellular receptor. These results suggest that, in addition to possible defects in the cytoplasm-to-nucleus translocation process, the acceptor-binding capacity of the nucleus may also represent one of the factors which determines the levels of assayable cytoplasmic receptors. Moreover, they indicate that the two assay methods furnish nonequivalent information.

An action spectrum for photoinduction of prolonged cutaneous photosensitivity by topical 8-MOP.

Suberythemogenic exposure of human skin treated with aqueous 8-MOP to radiation greater than 380 nm prolongs photosensitization to subsequent UVA from 6 to 24-72 h. One hypothesis for prolonged photosensitization is that greater than 380 nm irradiation forms 8-MOP-DNA monoadducts, which are removed more slowly than free 8-MOP and serve as a substrate for crosslinking by further UVA exposure. Sufficient DNA crosslinking results in erythema. We have examined this hypothesis by measuring the action spectrum for induction of prolonged photosensitization. Skin of normal volunteers was treated with aqueous 8-MOP (0.003%) and immediately received suberythemogenic monochromatic exposures between 334 and 430 nm. twenty-four hours later, the presence of prolonged sensitization was tested by small exposures of UVA. Erythema was evaluated 3 and 5 d later, and an action spectrum for prolonged sensitization was determined. The minimum phototoxic dose (MPD) was also determined at each wavelength. The action spectrum for prolonged photosensitization declined gradually between 334 and 425 nm. The action spectrum for delayed erythema induced by a single exposure of 8-MOP-treated skin declined rapidly from 334-390 nm. These findings are consistent with prolonged binding of 8-MOP in the skin by an initial exposure, possibly as 8-MOP-DNA monoadducts, allowing the second exposure to induce an erythemogenic event, possibly crosslinking of DNA.

Steroid-induced proteins in human endometrium.

The synthesis of soluble proteins in human endometria at various phases of menstrual cycle was evaluated by polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Densitometric analysis of the gels revealed alterations in the rate of synthesis of single protein bands throughout the cycle. Administration of conjugated estrogens (Premarin) to women undergoing hysterectomy, or exposure, in vitro, of the endometrial tissue to 17 beta-estradiol produced an increased incorporation of [35S]methionine into a specific protein which migrated on SDS-polyacrylamide gels at a molecular weight of about 55 000. Induction of this protein was observed only in those endometria showing a secretory histological appearance. The protein was resolved into at least 2 different spots in two-dimensional gel electrophoresis. An increase in the rate of synthesis of another endometrial protein with an apparent molecular weight of 51 000 was observed in tissues exposed in vitro to medroxyprogesterone acetate. These steroid-induced proteins may be a useful marker for studying hormone action in both normal and neoplastic endometria.

Estrogen responsive creatine kinase in human breast cancer cells.

The brain type (BB) isoenzyme of creatine kinase (CK) is a very sensitive marker of estrogen action in rat uterus and in experimental mammary tumors. In order to detect useful markers for estrogen dependent human breast cancer cell proliferation we measured CK levels and isoenzyme composition in the CG5 cells, an estrogen supersensitive variant of the MCF-7 human breast cancer cell line. Under basal conditions, CK-BB accounted for 10%-15% of total enzymatic activity, while the MM isoenzyme represented the overwhelming component. In cells cultured in the presence of 5% charcoal-treated fetal calf serum, physiological concentrations (0.1-1 nM) of estradiol increased CK-BB levels in a dose- and time-related fashion. The effect was specific for estrogens and was prevented by antiestrogens. Our results show that CK-BB is estrogen regulated in the CG5 cells and suggest a possible role for this enzyme as an additional marker of the hormonal responsiveness of breast cancer.

[Evaluation of two rapid tests for the determination of microalbuminuria and the urinary albumin/creatinine ratio]. / Evaluación de dos métodos rápidos para la determinación de microalbuminuria y de la relación albúmina/creatinina en orina.

OBJECTIVE: Microalbuminuria screening is justified on the grounds of its cost-benefit ratio in patients at risk of kidney damage while the process is still reversible. The aim of the present study was to evaluate the DCA 2000 analyser and the Clinitek 50 system (Bayer), which simultaneously measure urinary albumin and creatinine levels to adopt them as rapid methods for microalbuminuria detection. METHODS: One hundred twenty-seven urine samples from pediatric patients with various disorders were assessed. Albumin, creatinine, and the albumin/creatinine ratio were determined using the DCA 2000 analyzer and the Clinitek 50 system, which were compared against the usual reference laboratory methods. RESULTS: The correlation coefficient of nephelometric values vs the DCA 2000 analyzer was 0.914 for albumin, 0.970 for creatinine and 0.839 for the albumin/creatinine ratio. At an albumin cut-off concentration of 30 mg/l, the sensitivity, specificity, positive predictive value and negative predictive value were 100 %, 93 %, 84 % and 100 % for the DCA 2000 analyzer and 91.7 %, 86 %, 55 % and 98 % for the Clinitek 50 system. ROC curve analysis showed that the DCA 2000 system was more effective than the Clinitek 50 in microalbuminuria screening. CONCLUSIONS: The data obtained with the DCA 2000 system showed close agreement with those obtained with reference laboratory methods. The immediate availability of results is a great advantage in clinical practice. The Clinitek-Microalbumin dipstick system is a semiquantitative method that is easy to use, low in cost, simple and useful for screening, but it is less reliable as a follow-up method.

Utilidad de la tomografía computarizada multidetector en la evaluación postoperatoria de cirugías de derivación urinaria / Usefulness of multidetector computed tomography in the postoperative evaluation of urinary diversion surgery

Las cirugías de derivación urinaria son procedimientos que cada vez son más frecuentes, ya que sus indicaciones no son sólo neoplásicas, siendo también útiles en el manejo de otras patologías. Debido a este incremento, no es infrecuente observar complicaciones secundarias, ya sean en el postoperatorio temprano (menos de 30 días después de la cirugía) o tardío (más de 30 días). Dentro de éstas tenemos alteraciones de la motilidad intestinal (íleo paralítico, obstrucción), fugas anastomóticas, colecciones líquidas (linfocele, urinoma, absceso), fístulas, herniación paraestomal, estenosis ureterales, litiasis y recurrencia tumoral. Dada la gran cantidad de técnicas quirúrgicas usadas en estos procedimientos, es importante conocer los cambios anatómicos resultantes, ocasionalmente de difícil valoración. La tomografía computarizada multidetector (TCMD) tiene gran utilidad en el estudio de estos pacientes, especialmente mediante las técnicas de reconstrucción multiplanar, representando adecuadamente las estructuras urinarias y extraurinarias afectadas, y sus relaciones con estructuras adyacentes, permitiendo identificarlas acertada y rápidamente.

Urinary diversion surgeries are procedures that are becoming more frequent, as their indications are not only neoplastic, being useful also in managing other diseases. Due to this increase, it is not uncommon to observe secondary complications, whether in the early postoperative period (less than 30 days after surgery) or later (more than 30 days). Within these are alterations in intestinal motility (paralytic ileus, blockage), anastomotic leaks, fluid collections (lymphocele, urinoma, abscess), fistulas, parastomal herniation, ureteral obstruction, urolithiasis and tumor recurrence. Given the large number of surgical techniques used in these procedures, it is important to know the resulting anatomical changes, occasionally difficult to evaluate. Multidetector computed tomography (MDCT) is of great use in the study of these patients, especially with multiplanar reconstruction techniques, adequately representing the affected urinary and extra-urinary structures, and their relationship to adjacent structures, enabling their accurate and quick identification.

Effect of natural beta-interferon on cell proliferation and steroid receptor level in human breast cancer cells.

The effect of natural beta-interferon (beta-IFN) on cell proliferation and steroid receptor level was investigated in CG-5 human breast cancer cell line. beta-interferon determines an appreciable diminution of cell growth, at concentrations ranging from 100 to 1000 IU/ml, which is enhanced when serum content of the culture medium is lowered. Low concentrations of beta-IFN (10-100 IU/ml) produce, after a 5-day treatment, an increase in estrogen receptors (ER) and progesterone receptors (PR). No variation of ER and PR levels is observed when beta-IFN is added directly to the cell homogenate before the assay. Our data suggest that beta-IFN could affect hormone sensitivity through a modification of ER and PR in neoplastic mammary cells.

Two new estrogen-supersensitive variants of the MCF-7 human breast cancer cell line.

Two new estrogen-sensitive variants of MCF-7 human breast cancer cells, CG-4 and CG-5, are described in this report. These cells were derived from a casual contamination by MCF-7 cells of primary cultures from a human adenocarcinoma of the breast and a pleural effusion of a patient with advanced breast cancer, respectively. Careful characterization of these variants revealed chromosomal properties highly similar to and alloenzyme phenotypes identical to those of MCF-7 cells which were simultaneously cultured in the laboratory. MCF-7, CG-4 and CG-5 cells were tested for estrogen responsiveness under identical growth conditions, that is in the presence of culture medium supplemented with 5% charcoal-treated serum. While the number of MCF-7 cells increases by about 40% over the controls in the presence of physiological concentrations of estradiol, the number of CG-4 cells doubles and the number of CG-5 cells triples. All these cell lines have approximately the same number of estrogen, androgen, glucocorticoid, and progesterone receptor sites/cell. Since several difficulties arise in demonstrating the estrogen responsive growth stimulation of currently available human breast cancer cells, these two new variants, characterized by a high and reproducible estrogen responsiveness, afford a new model for studying the mechanisms by which estrogen regulates cell proliferation. The problems related to the careful characterization of every established cell line are discussed.

Presence and steroid inducibility of glutamine synthetase in human leukemic cells.

Glutamine synthetase (EC 6.3.1.2; GS) is present in lymphoblasts from patients with acute lymphoblastic leukemia (ALL) as well as in normal peripheral blood lymphocytes. In 16 out of 20 ALL patients studied exposure of the cells to physiological concentrations of dexamethasone in vitro increased enzyme activity above the control levels. The increase was specific for glucocorticoid receptor ligands. A direct correlation was found between the magnitude of glucocorticoid-mediated increase of GS activity and the cellular levels of specific glucocorticoid receptors assayed in the same cell specimen. Moreover, the basal levels of the enzyme measured in cells prior to exposure to dexamethasone correlated negatively with receptor density. It is suggested that the presence of steroid-inducible GS in ALL cells may prove to be a marker for functional receptor sites.

Estrogen stimulates cell proliferation and the increase of a 52,000 dalton glycoprotein in human breast cancer cells.

In an estrogen supersensitive variant of the MCF-7 cell line, CG-5, estrogen was found to stimulate the labelling of a glycoprotein released into the culture medium which has the same electrophoretic migration pattern as that previously reported in MCF-7 cells (Biochem. Biophys. Res. Commun., 90: 410-416, 1979). To test the possibility that the 52 K is a marker of estrogen-dependent breast cancer cell proliferation, we have correlated the effect of estrogen and antiestrogen on protein labelling and cell proliferation under different experimental conditions. In cells cultured in the presence of 5% charcoal-treated fetal calf serum, physiological concentrations (0.1-1 nM) of estradiol stimulated in a dose- and time-related fashion both 52 K labelling and cell proliferation. However at high concentrations (10-100 nM) estrogen decreased 52 K labelling while it still stimulated cell proliferation. Concentrations of the tamoxifen derivative, 4-hydroxytamoxifen, which effectively prevented estrogen-stimulated cell proliferation also blocked estrogen-stimulated increase of 52 K labelling. Time-course experiments suggest that the estrogen-stimulated increase of 52 K labelling (detectable after 22 h of hormone exposure) precedes the effect of cell proliferation (detectable after 3 days of hormone exposure). In cells cultured under serum-free conditions there was no effect of estradiol at any of the concentrations and times used on either 52 K labelling or cell proliferation.