Component resolved diagnosis by recombinant allergens in patients with allergies to inhalants.

Molecular characterization of IgE reactivity of specific individual components of allergenic extracts is now possible due to the technology of recombinant allergens derived from studies of molecular biology of allergic pathology. The identification of the immunoreactivity to single allergenic components in allergic subjects allows to specifically define her/his allergic profile and obtain the so-termed Component Resolved Diagnosis (CRD). Molecular allergens can be classified into those that induce the respiratory allergic reactivity and those that identify the food-related allergic pathology. It is also essential to identify those molecular allergens whose immunoreactivity is able to connect the two clinical conditions: respiratory symptoms and food allergy symptoms. The present study was conducted on 50 patients with a clinical history of hypersensitivity to pollen and/or allergy and positivity to Skin Prick Test. The sera were analyzed in our laboratories and the panel of recombinant allergens was applied in the case of positivity of the specific IgE. Of the 50 patients enrolled, 31 were selected as positive to 4 main pan-allergen Bet v1, Par j2, Art v1 and Phl p1; among these, 14 subjects showed one allergen-specific IgE towards natural extracts of tested foods even in absence of clinical history. CRD allows for an increased accuracy in allergy diagnosis and prognosis and plays an important role in: a) resolving genuine vs cross-reactive sensitization in poly-sensitized patients, b) assessing, in selected cases, the risk of severe, systemic vs mild, local reactions in food allergy, and c) identifying patients and triggering allergens for specific immunotherapy (ITS). In light of our results, we believe that the transition from a diagnostic based on the use of allergenic extracts to another one based on the use of single allergenic molecules that is able to define the specific allergenic profile of each patient, seems to be able to revolutionize the allergy diagnosis.

Seminal ribonuclease inhibits tumor growth and reduces the metastatic potential of Lewis lung carcinoma.

The role of some RNases as antitumoral agents has been recently emphasized. We have previously demonstrated a striking inhibitory effect of bovine seminal RNase on the in vitro growth of tumor cells of metastatic origin. This has prompted us to test the effects of this protein in vivo on the induction of metastatic foci in mice lungs after i.m. injection of a highly metastatic Lewis lung carcinoma cell line. The results presented here, while confirming and expanding upon those previously reported on the antitumor effects of bovine seminal RNase in vivo on primary thyroid epithelial tumors, indicate for the first time that bovine seminal RNase can also be regarded as a potent antimetastatic agent on in vivo spontaneous metastases.

The cobas p 630 instrument: a dedicated pre-analytic solution to optimize COBAS® AmpliPrep/COBAS® TaqMan® system workflow and turn-around-time.

The cobas p 630, a fully automated pre-analytical instrument for primary tube handling recently introduced to complete the Cobas(®) TaqMan systems portfolio, was evaluated in conjunction with: the COBAS(®) AmpliPrep/COBAS(®) TaqMan HBV Test, v2.0, COBAS(®) AmpliPrep/COBAS(®) TaqMan HCV Test, v1.0 and COBAS(®) AmpliPrep/COBAS(®) TaqMan HIV Test, v2.0. The instrument performance in transferring samples from primary to secondary tubes, its impact in improving COBAS(®) AmpliPrep/COBAS(®) TaqMan workflow and hands-on reduction and the risk of possible cross-contamination were assessed. Samples from 42 HBsAg positive, 42 HCV and 42 HIV antibody (Ab) positive patients as well as 21 healthy blood donors were processed with or without automated primary tubes. HIV, HCV and HBsAg positive samples showed a correlation index of 0.999, 0.987 and of 0.994, respectively. To assess for cross-contamination, high titer HBV DNA positive samples, HCV RNA and HIV RNA positive samples were distributed in the cobas p 630 in alternate tube positions, adjacent to negative control samples within the same rack. None of the healthy donor samples showed any reactivity. Based on these results, the cobas p 630 can improve workflow and sample tracing in laboratories performing molecular tests, and reduce turnaround time, errors, and risks.

Inhibition of MAPK activity, cell proliferation, and anchorage-independent growth by N-Ras antisense in an N-ras-transformed human cell line.

Mammalian ras genes encode a family of plasma membrane-bound proteins that function as intermediates in signal transduction pathways involved in cell growth and differentiation. Ras oncogene is frequently involved in neoplastic transformation of different cellular histotypes. In this study, we tested the ability of antisense oligodeoxyribonucleotides (AS-ODN) that have mixed phosphodiester/phosphorothioate backbone, targeted against human N-Ras, to inhibit N-ras gene expression and to specifically interfere with the Ras-dependent activity of mitogen-activated protein kinase (MAPK) in two human cell lines carrying an endogenous N-ras mutated allele at codon 61. Three AS-ODN that inhibit basal MAPK activity have been identified. Moreover, AS-ODN treatment resulted in potent antiproliferative effects in cell culture and great inhibition of N-ras mRNA levels in one of two cell lines. These studies suggest that antisense molecules, targeted against N-Ras, could be of considerable value as a tool to study the N-Ras-specific transduction pathway.

Highly selective toxic and proapoptotic effects of two dimeric ribonucleases on thyroid cancer cells compared to the effects of doxorubicin.

The lack of selectivity of conventional antitumour drugs against cancer cells is responsible for their high toxicity. The development of new tumour-specific drugs is therefore highly needed. We tested the cytotoxic effects and the nature of cell death induced by a naturally dimeric bovine RNase and a newly engineered dimeric human RNase upon three genetically well-defined normal and malignant thyroid cell systems. RNases effects were compared with those of doxorubicin, a conventional antineoplastic drug. Our results show significant and selective proapoptotic effects exerted on tumour cells by both RNases, the strength of their cytotoxic and apoptotic activity being directly related to the degree of cell malignancy. No toxic effects were observed upon normal cells. Doxorubicin showed, instead, cytotoxic and apoptotic effects also against normal cells. The in vitro results were corroborated by the antitumour action of both dimeric RNases towards a malignant human thyroid tumour grown in nude mice. These results indicate a selective action of dimeric RNases against cancer cells and suggest the potential application of these molecules or their derivatives to the treatment of aggressive subtypes of thyroid cancer.

A dimeric mutant of human pancreatic ribonuclease with selective cytotoxicity toward malignant cells.

Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.