乳酸脱氢酶实验检测细胞新陈代谢的实验研究

目的 探索LDH实验检测细胞活力的可行性。方法 原代培养骨髓细胞和软骨细胞，用LDH实验测定上述两组细胞的活力，并与镜下活体观察到细胞的生长状况相比较。与目前比较成熟的测定细胞活力的MTS实验的测得的值相比较。结果 LDH实验对上述两组细胞的活力的测定结果与镜下活体观察到的结果相符合。与MTS实验的测得的结果经统计学处理无显著差异。结论 LDH实验可用于细胞活力的直接测定，而对活细胞的生存、繁殖无影响。     

Two novel mutations at exon 8 of the Sequestosome 1 (SQSTM1) gene in an Italian series of patients affected by Paget's disease of bone (PDB).

PDB is genetically heterogeneous. Mutations of the sequestosome1 gene have been reported in sporadic and familial forms of Paget's in patients of French Canadian and British descent. Mutational analyses in different ethnic groups are needed to accurately investigate hereditary diseases. We describe two novel mutations of sequestosome1 in 62 Italian sporadic patients, confirming the role of the encoded protein in this disorder. INTRODUCTION: Paget's disease of bone (PDB) is a relatively common disease of bone metabolism reported to affect up to 3% of whites over 55 years of age. The disorder is genetically heterogeneous, and at present, there is scientific evidence that at least eight different human chromosomal loci are correlated with its pathogenesis. Mutations of the sequestosome1 (SQSTM1) gene were identified as responsible for most of the sporadic and familial forms of Paget in patients of French Canadian and British descent. Such mutations were located at exon 7 and 8 levels, encoding for the ubiquitin protein-binding domain (UBA) and representing a mutational hot spot area. MATERIALS AND METHODS: To verify the involvement of this gene in Italian subjects affected by PDB, we performed mutational analysis in 62 sporadic PDB cases. RESULTS: We described three different mutations at exon 8 level: P392L, already described in the French Canadian population and families predominantly of British descendent, and two novel mutations consisting of the amino acid substitutions M404V and G425R. No significant differences in the clinical history of PDB have been observed in patients with SQSTM1 mutations in respect to those without. CONCLUSIONS: Even though our findings suggest a minor involvement of the SQSTM1 gene in the pathogenesis of sporadic Italian Paget's cases, the identification of different significant mutations within the SQSTM1 gene in unrelated, but clinically similar individuals, offers extremely convincing evidence for a causal relationship between this gene and PDB. Longitudinal studies are needed to assess the penetrance of genotype/phenotype correlations. Our findings confirm the evidence of a clustered mutation area at this level in this disorder.     

Mutations in the Ca(2+)-sensing receptor gene cause autosomal dominant and sporadic hypoparathyroidism

Parathyroid hormone secretion is negatively regulated by a 7- transmembrane domain, G-protein coupled Ca(2+)-sensing receptor. We hypothesized that activating mutations in this receptor might cause autosomal dominant hypoparathyroidism (ADHP). Consistent with this hypothesis, we identified, in two families with ADHP, heterozygous missense mutations in the Ca(2+)-sensing receptor gene that cosegregated with the disorder. None of 50 normal controls had either mutation. We also identified a de novo, missense Ca(2+)-sensing receptor mutation in a child with severe sporadic hypoparathyroidism. The amino acid substitution in one ADHP family affected the N-terminal, extracellular domain of the receptor. The other mutations involved the transmembrane region. Unlike patients with acquired hypoparathyroidism, patients with these mutations had hypercalciuria even at low serum calcium concentrations. Their greater hypercalciuria presumably reflected activation of Ca(2+)-sensing receptors in kidney cells, where the receptor negatively regulates calcium reabsorption. This augmented hypercalciuria increases the risk of renal complications and thus has implications for the choice of therapy.      

Constitutive activation of the PI3K‐AKT pathway and cardiovascular abnormalities in an individual with Kosaki overgrowth syndrome

Kosaki overgrowth syndrome is a recently described syndrome characterized by distinctive facial features, brain white matter lesions, and developmental delay. Germline activating heterozygous PDGFRB mutations have been reported in this condition. Systemic connective tissue‐type findings have been described in some individuals. We describe a 19‐year‐old Caucasian female with a history of hydrocephalus, Dandy–Walker malformation, cervical spine arachnoid cyst, progressive scoliosis, and overgrowth. Her physical exam included distinctive craniofacial dysmorphism, as well as soft and hyperextensible skin. Cardiovascular imaging during adolescence revealed saccular aneurysms in both coronary artery systems and subtle tortuosity of the cervical vertebral arteries. Exome sequencing trio analysis identified a de novo previously reported pathogenic variant in PDGFRB, c.1696T>C (p.[Trp566Arg]). Further functional studies included platelet‐derived growth factor cellular metabolic pathway activity that confirmed the variant causes a constitutive activation of the PI3K‐AKT pathway. This is the first report to characterize the activating nature of this PDGFRB variant. We also highlight the connective tissue findings seen in Kosaki overgrowth syndrome and recommend baseline echocardiographic evaluation in all individuals with this condition with particular emphasis on coronary arteries.     

Efficacy and Safety of Atezolizumab Plus Bevacizumab Following Disease Progression on Atezolizumab or Sunitinib Monotherapy in Patients with Metastatic Renal Cell Carcinoma in IMmotion150: A Randomized Phase 2 Clinical Trial

BackgroundThe use of immune checkpoint inhibitors combined with vascular endothelial growth factor (VEGF)-targeted therapy as second-line treatment for metastatic clear cell renal cancer (mRCC) has not been evaluated prospectively.ObjectiveTo evaluate the efficacy and safety of atezolizumab + bevacizumab following disease progression on atezolizumab or sunitinib monotherapy in patients with mRCC.Design, setting, and participantsIMmotion150 was a multicenter, randomized, open-label, phase 2 study of patients with untreated mRCC. Patients randomized to the atezolizumab or sunitinib arm who had investigator-assessed progression as per RECIST 1.1 could be treated with second-line atezolizumab + bevacizumab.InterventionPatients received atezolizumab 1200 mg intravenously (IV) plus bevacizumab 15 mg/kg IV every 3 wk following disease progression on either atezolizumab or sunitinib monotherapy.Outcome measurements and statistical analysisThe secondary endpoints analyzed during the second-line part of IMmotion150 included objective response rate (ORR), progression-free survival (PFS), and safety. PFS was examined using Kaplan-Meier methods.Results and limitationsFifty-nine patients in the atezolizumab arm and 78 in the sunitinib arm were eligible, and 103 initiated second-line atezolizumab + bevacizumab (atezolizumab arm, n = 44; sunitinib arm, n = 59). ORR (95% confidence interval [CI]) was 27% (19–37%). The median PFS (95% CI) from the start of second line was 8.7 (5.6–13.7) mo. The median event follow-up duration was 19.4 (12.9–21.9) mo among the 25 patients without a PFS event. Eighty-six (83%) patients had treatment-related adverse events; 31 of 103 (30%) had grade 3/4 events. Limitations were the small sample size and selection for progressors.ConclusionsThe atezolizumab + bevacizumab combination had activity and was tolerable in patients with progression on atezolizumab or sunitinib. Further studies are needed to investigate sequencing strategies in mRCC.Patient summaryPatients with advanced kidney cancer whose disease had worsened during treatment with atezolizumab or sunitinib began second-line treatment with atezolizumab + bevacizumab. Tumors shrank in more than one-quarter of patients treated with this combination, and side effects were manageable.     

Dibenzo[a,l]pyrene-induced DNA adduction, tumorigenicity, and Ki-ras oncogene mutations in strain A/J mouse lung

Dibenzo[a,l]pyrene (DB[a,l]P), an environmental polycyclic aromatic hydrocarbon, is the most potent carcinogen ever tested in mouse skin and rat mammary gland. In this study, DB[a,l]P was examined for DNA adduction, tumorigenicity, and induction of Ki-ras oncogene mutations in tumor DNA in strain A/J mouse lung. Groups of mice received a single i.p. injection of 0.3, 1.5, 3.0, or 6.0 mg/kg DB[a,l]P in tricaprylin. Following treatment, DNA adducts were measured at times between 1 and 28 days, while tumors were counted at 250 days and analyzed for the occurrence of point mutations in codons 12 and 61 of the Ki-ras oncogene. DB[a,l]P in strain A/J mouse lung induced six major and four minor DNA adducts. Maximal levels of adduction occurred between 5 and 10 days after injection followed by a gradual decrease. DB[a,l]P-DNA adducts in lung tissue were derived from both anti- and syn-11,12- dihydroxy-13,14-epoxy- 11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) and both deoxyadenosine (dAdo) and deoxyguanosine (dGuo) residues in DNA as revealed by cochromatography. The major adduct was identified as a product of the reaction of an anti-DB[a,l]PDE with dAdo in DNA. DB[a,l]P induced significant numbers of lung adenomas in a dose- dependent manner, with the highest dose (6.0 mg/kg) yielding 16.1 adenomas/mouse. In tricaprylin-treated control animals, there were 0.67 adenomas/mouse. Based on the administered dose, DB[a,l]P was more active than other environmental carcinogens including benzo[a]pyrene. As a function of time-integrated DNA adduct levels, DB[a,l]P induced lung adenomas with about the same potency as other PAHs, suggesting that the adducts formed by DB[a,l]P are similar in carcinogenic potency to other PAHs in the strain A/J mouse lung model. Analysis of the Ki- ras mutation spectrum in DB[a,l]P-induced lung tumors revealed the predominant mutations to be G-->T transversions in the first base of codon 12, A-->G transitions in the second base of codon 12, and A-->T transversions in the second or third base of codon 61, concordant with the DNA adduct profile.      

Prevenzione delle infezioni in odontoiatria: disinfezione e sterilizzazione

Objectives. The aim of this paper is to provide an easy-to-consult vademecum on methods for protecting patients and members of the dentistry staff from infections. Materials and methods. We conducted a review of the main scientific data evidence related to the prevention of infections in dental patients. Results and conclusions. Dental procedures represent a potential risk of infection for patients and staff members alike. It is difficult, however, to correlate the development of specific infections with specific types of treatment. Furthermore, epidemiologic data are not available on the actual number of infections contracted in the dentistry environment. Nevertheless, it is clear that, alongside technical and operational skills, there is an essential need for knowledge relating to the most basic standards of hygiene, sterilization and disinfection processes, and procedures used to verify the latter.