Cytotoxic effect of adriamycin and agarose-coupled adriamycin on glomerular epithelial cells: Role of free radicals

Summary It has been suggested that the generation of toxic radicals plays an important role in toxicity by Adriamycin (ADR) on cancer cell lines and in vivo. We have examined the role of free radicals in determining toxicity and resistance to ADR of rat glomerular epithelial cells in culture; this method provides a good model for analyzing the mechanisms responsible for ADR experimental nephrosis in rats. Three points were established: a) the intra- or extracellular site of ADR toxicity; b) the role of the superoxide anion and of the hydroxyl radical in determining intra- and-extracellular cytotoxicity; and c) the implication of oxido-reduction cycling as a potential route for ADR semiquinone transformation. Free ADR was found to induce the same inhibition of [³H]thymidine incorporation into DNA as ADR bound to an agarose macroporous bed which prevents the intracellular incorporation of the drug. Specific scavenging of free radical activity by the enzymes catalase and superoxide dismutase, the hydroxyl radical inhibitors dimethyl sulfoxide and dimethylthiourea (DMTU) and by chelation of intracellular free iron with deferoxamine produced only a partial restoration of [³H]thymidine incorporation into DNA, which was maximal for DMTU (30% of normal incorporation). DMTU treatment was unsuccessful in preventing the extracellular cytostatic effect of ADR. Finally, glomerular epithelial cell killing (⁵¹Cr-release method) by 5-iminodaunorubicin, an ADR analogue with a modified quinone function that prohibits oxido-reduction cycling, was higher than unmodified ADR. These results indicate that ADR may exert its cytotoxic effects on glomerular epithelial cells by interaction at the cell surface, whereas the intracellular compartment, principally DNA, does not seem to be the target of ADR effects. They also suggest that the free radicals are in part responsible for ADR intracellular cytotoxicity, but other mechanisms should also be hypothesized. Finally, the participation of the ADR semiquinone radical in oxido-reduction cycling seems not important for the induction of the cellular damage.     

Structural organization of the menisci of the human knee: scanning electron microscopy study]

The structure of the meniscus of the knee joint was studied in 10 human subjects after meniscectomy for acute trauma. The specimens were studied by scanning electron microscopy after cryofracture. Collagen fibres are arranged in two layers: a thin superficial zone with radially oriented fibres and a deep zone with circumferentially oriented ones. Deep fibres are surrounded by oblique fascicules. The cells are rare and oval-shaped. The observations enhance the hypothesis that the meniscus of the knee joint is not a real fibrocartilage.     

Murine red blood cells as efficient carriers of three bacterial antigens for the production of specific and neutralizing antibodies.

Three bacterial toxoids, CRM 197 (mutagenized diphtheria toxin), tetanus toxoid (formaldehyde-treated tetanus toxin), and PT-9K/129G (double mutant of pertussin toxin) were encapsulated within red blood cells (RBCs) of B6D2F1 and Balb/C mice according to a mild procedure based on hypotonic dialysis-isotonic resealing that yielded undamaged RBCs. The toxoid-loaded RBCs were injected intravenously in order to immunize animals and their effects were compared to those of identical amounts (30-95 micrograms per mouse subdivided into multiple injections) of the corresponding free toxoids injected intravenously in saline. Sera from treated mice were collected and tested for titers of specific antibodies against each of the three antigens and also for titers of neutralizing antibodies, i.e., affording protection from toxic effects induced by the corresponding native toxins. In all experiments, significant seroconversion was observed with both immunization systems. Titers of both specific and neutralizing antibodies against CRM 197 and tetanus toxoid were several-fold higher upon immunization with the RBC-encapsulated toxoids, than with the free toxoids. These differences were not due to qualitatively different recognition patterns of antigenic determinants by the two types of sera. Conversely, intravenous immunization with pertussis toxoid either as RBC-encapsulated or as free antigen elicited a comparably high production of specific and of neutralizing antibodies. These data demonstrate that properly engineered RBCs behave as natural carriers and possibly adjuvants for antigens of vaccinal interest.     

High conservation of sequences involved in cystic fibrosis mutations in five mammalian species

Several mutations have been identified in the first nucleocide binding fold (NBF) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We have analyzed the DNA sequences of exons 10 and 11 in five different mammalian species, marmoset, mouse, cow, pig, and sheep; the amino acid conservation studied for nine disease mutations; and two “benign” mutations. For exon 10,87% homology at the DNA level and 93.5% at the amino acid level were found for these species. For exon 11, the lowest homology (70%), as found in mouse and the highest in marmoset (93%), whereas the amino acid sequence conservation ranged from 82.5 to 100%. All codons involved in CF mutations are highly conserved throughout evolution.     

The search for south European cystic fibrosis mutations: identification of two new mutations, four variants, and intronic sequences

The major mutation in the cystic fibrosis (CF) gene is a 3-bp deletion (delta F508) in exon 10. About 50% of the CF chromosomes in Southern Europe carry this mutation, while other previously described mutations account for less than 4%. To identify other common mutations in CF patients from the Mediterranean area, we have sequenced, exon by exon, 16 chromosomes that did not show the delta F508 deletion from a selected panel of eight unrelated CF patients. We describe here one missense and one nonsense mutation, and four sequence polymorphisms. We have also found two previously reported mutations in three chromosomes. Overall, these mutations may account for about 20% of CF alleles in the Italian and Spanish populations. No other mutations were detected in 10 out of 16 CF chromosomes after analyzing about 90% of the coding region of the CF gene, and 39 out of 54 intron/exon boundaries. Therefore, about 26% of CF mutations remain to be identified. In addition we provide the intron/exon boundary sequences for exons 4 to 9. These results together with previously reported linkage data suggest that in the Mediterranean populations further mutations may lie in the promoter region, or in intron sequences not yet analyzed.     

High molecular weight RNA containing histone messenger in the sea urchin Paracentrotus lividus

Sea urchin RNA extracted from early and mesenchyme blastula embryos and oocytes and fractionated on denaturing sucrose density gradients, was hybridized with histone DNA recombinants of Psammechinus miliaris (clone λh22) and of Paracentrotus lividus (clone pPH70). Histone sequences are found in the 9 S and larger than 9 S regions of the formamide/sucrose density gradients. The melting of the RNA-DNA duplexes obtained by hybridization of polysomal and high molecular weight RNA of embryos of P. lividus at the stage of early blastula, suggests a degree of heterogeneity in the high M_r RNA. The high M_r RNA contains at least four of the five histone gene sequences covalently linked.     

Chromosome constitution of in vitro segregated haploid and diploid cells of the mouse

The composition in segregated haploid sets of paternal and maternal chromosomes has been studied in order to verify whether their composition is uniparental of mixed, fixed or variable. Primary cultures where prepared using kidneys from hybrids of strains of Mus musculus in which the parental chromosomes are distinguishable; the maternal set consists of 20 teleocentric chromosomes, the paternal set of 9 metacentric chromosomes, derived by Robertsonian fusion and 2 telocentrics. Applying Seabright's banding technique, an analysis of segregated haploid and diploid cells, which have originated spontaneously through polyploidisation-segregation processes was carried out. It was concluded that the haploid sets have a variable composition of paternal and maternal chromosomes.     

Different Placebos,Different Mechanisms,Different Outcomes: Lessons for Clinical Trials

Clinical trials use placebos with the assumption that they are inert, thus all placebos are considered to be equal. Here we show that this assumption is wrong and that different placebo procedures are associated to different therapeutic rituals which, in turn, trigger different mechanisms and produce different therapeutic outcomes. We studied high altitude, or hypobaric hypoxia, headache, in which two different placebos were administered. The first was placebo oxygen inhaled through a mask, whereas the second was placebo aspirin swallowed with a pill. Both placebos were given after a conditioning procedure, whereby either real oxygen or real aspirin was administered for three consecutive sessions to reduce headache pain. We found that after real oxygen conditioning, placebo oxygen induced pain relief along with a reduction in ventilation, blood alkalosis and salivary prostaglandin (PG)E2, yet without any increase in blood oxygen saturation (SO2). By contrast, after real aspirin conditioning, placebo aspirin induced pain relief through the inhibition of all the products of cyclooxygenase, that is, PGD2, PGE2, PGF2, PGI2, thromboxane (TX)A2, without affecting ventilation and blood alkalosis. Therefore, two different placebos, associated to two different therapeutic rituals, used two different pathways to reduce headache pain. The analgesic effect following placebo oxygen was superior to placebo aspirin. These findings show that different placebos may use different mechanisms to reduce high altitude headache, depending on the therapeutic ritual and the route of administration. In clinical trials, placebos and outcome measures should be selected very carefully in order not to incur in wrong interpretations.     

An extracellular vesicle epitope profile is associated with acute myocardial infarction

The current standard biomarker for myocardial infarction (MI) is high‐sensitive troponin. Although powerful in clinical setting, search for new markers is warranted as early diagnosis of MI is associated with improved outcomes. Extracellular vesicles (EVs) attracted considerable interest as new blood biomarkers. A training cohort used for diagnostic modelling included 30 patients with STEMI, 38 with stable angina (SA) and 30 matched‐controls. Extracellular vesicle concentration was assessed by nanoparticle tracking analysis. Extracellular vesicle surface‐epitopes were measured by flow cytometry. Diagnostic models were developed using machine learning algorithms and validated on an independent cohort of 80 patients. Serum EV concentration from STEMI patients was increased as compared to controls and SA. EV levels of CD62P, CD42a, CD41b, CD31 and CD40 increased in STEMI, and to a lesser extent in SA patients. An aggregate marker including EV concentration and CD62P/CD42a levels achieved non‐inferiority to troponin, discriminating STEMI from controls (AUC = 0.969). A random forest model based on EV biomarkers discriminated the two groups with 100% accuracy. EV markers and RF model confirmed high diagnostic performance at validation. In conclusion, patients with acute MI or SA exhibit characteristic EV biomarker profiles. EV biomarkers hold great potential as early markers for the management of patients with MI.