Validation Checklist for Massive Vaccination Centers.

Abstract: Starting from the minimum requirements indicated by Lombardy Region, a validation checklist has been developed by experts in design, healthcare layout planning, hygiene and public health, planning and compliance, in order to provide managers of COVID-19 massive vaccination centers with a useful and easy-to-use tool to ensure quality, safety and efficiency of the different activities performed.

Donepezil plasma concentrations, CYP2D6 and CYP3A4 phenotypes, and cognitive outcome in Alzheimer's disease.

PURPOSE: The purpose of the study is to evaluate whether donepezil (D) plasma concentrations and activity of CYP2D6 and CYP3A4 are associated with the therapeutic response of patients with mild to moderate Alzheimer's disease (AD). METHODS: This study comprised 54 patients affected by probable AD in therapy with D 10 mg/daily for at least 3 months. Plasma concentrations of D and its three main metabolites (6DD, 5DD, DNox) were assayed with a novel high performance liquid chromatography (HPLC) technique. Cognitive progression was assessed at baseline and at 9 months of follow-up with the mini mental state examination (MMSE). The activities of the two cytochromes involved in D metabolism-CYP2D6 and CYP3A4-were evaluated according to their metabolic ratios in plasma or urine, after test doses of probe drugs (dextromethorphan and omeprazole). RESULTS: A significant correlation was found between plasma levels of D and variations in MMSE scores after 9 months of therapy (r (2) = 0.14; p = 0.006). Neither the concentrations of D metabolites nor the metabolic ratios of CYP2D6 and CYP3A4 showed any correlations with cognitive variations. Low CYP2D6 activity and advanced age were associated with high D concentrations. Patients who were treated with CYP2D6 and P-glycoprotein (P-gp) inhibitors also had higher D plasma levels (mean difference = 19.6 ng/mL; p = 0.01) than those who were not. CONCLUSIONS: D plasma concentrations, but not cytochrome phenotyping, are associated with cognitive outcomes in AD patients.

QUANTITATIVE ANALYSIS OF PERIODONTAL PATHOGENS IN PERIODONTITIS AND GINGIVITIS.

Periodontal tissues surround the teeth and provide their attachment. Periodontal diseases include a mild and reversible form named gingivitis and periodontitis that is the main cause of tooth loss in adults. Gingivitis, that affects gums and coronal junctional epithelium, as well as periodontitis, that is characterized by loss of connective tissue attachment, are caused by a persistent inflammatory response promoted by alteration of periodontal biofilm. The aim of the study was to test whether the prevalence or relative amount of each species was associated with a particular clinical condition. Periodontal evaluation of 539 unrelated patients was performed by the Periodontal Screening and Recording (PSR) system. Subgingival samples were obtained from the site with the worst PSR score. A selection of eleven bacterial species was evaluated by quantitative real time PCR. Some bacterial species were found to be associated with all phases of periodontal disease, such as Tannerella forsythia, Treponema denticola, and Treponema lecithinolyticum, while other species were more specifically associated with periodontitis, such as Porphyromonas endodontalis and Porphyromonas gingivalis, or with gingivitis, such as Capnocytophaga ochracea and Campylobacter rectus. Quantitative and qualitative analyses helps to better understand the microbial changes associated with different stages of periodontal disease.

INTERLEUKIN-6 GENE POLYMORPHISM MODULATES THE RISK OF PERIODONTAL DISEASES.

Gingivitis and periodontitis are the two main periodontal diseases. Both are characterized by inflammation of the tissues surrounding the teeth but while tissue damages observed in gingivitis are mild and reversible, destruction caused by periodontitis is deeper and irreversible. Periodontal diseases and levels of degeneration of tissues surrounding teeth depend on several interacting endogenous and exogenous factors. Polymorphisms of genes encoding molecules that modulate the immune response and tissue homeostasis are the main causes of individual susceptibility to periodontal diseases. The aim of this study was to investigate IL6, IL10 and VDR gene polymorphisms in a large number of subjects affected by either gingivitis or chronic periodontitis. The sample included 750 Italian patients. We found that the rs1800795 SNP located in the IL6 gene promoter was strongly associated with the occurrence of both gingivitis and periodontitis. Indeed, homozygous individuals with variant allele appeared less-susceptible to both gingivitis OR=0.47 (95% C.I. 0.27-0.82) and periodontitis OR=0.36 (95% C.I. 0.21-0.64). No evidence of association between periodontal diseases and IL10 or VDR polymorphisms was obtained. This data confirmed the role of IL6 in susceptibility to periodontitis among the Italian population. The evidence that IL6 polymorphisms are also involved in gingivitis has implications in periodontal disease pathogenesis and reduces the appeal of IL6 as a periodontitis biomarker.

E-CADHERIN CODING GENE (CDH1) AND NONSYNDROMIC CLEFT LIP WITH OR WITHOUT CLEFT PALATE: IS THERE ANY ASSOCIATION?

Epithelial to Mesenchymal Transition (EMT) is an important process involved in cancer, embryogenesis and organ development. Its role in nonsyndromic cleft lip with or without cleft palate (NSCL/P) has been extensively investigated and successfully linked to the disease. In this study, we focused on a gene, CDH1, encoding for E-cadherin, a key protein in EMT. We carried out an association study on an Italian sample group, genotyping four single nucleotide variations within the CDH1 gene, in order to verify the potential role of this gene in NSCL/P etiology. Neither the haplotype nor the family-based association test revealed any association between the genotyped SNPs and the pathology. Our results demonstrate that, in our Italian sample study, the analyzed single nucleotide polymorphisms are not associated to NSCL/P.

Bio-Revitalization: Effects of NASHA on Genes Involving Tissue Remodeling.

BACKGROUND: The "non-animal stabilized hyaluronic acid" (NASHA) is a widely used product in bio-revitalization injective procedures in esthetic medicine. The present research aimed to quantitatively evaluate the therapeutic effect of one of the more used bio-revitalization products on cultured dermal fibroblasts. RT-PCR was used for gene expression profiling of some proteins known to be relevant in skin homeostasis. METHODS: Human dermal fibroblasts were seeded on a culture medium enriched with a product for dermal bio-revitalization, consisting of stabilized hyaluronic acid gel 20 mg/ml. After 24, 48, and 72 h of exposure, the cDNA was amplified by real-time PCR. Gene expression was quantified with the delta/delta calculation method. RESULTS: In this study, the gene of metalloproteinase (MMP)-13 is strongly expressed after NASHA incubation. The MMP-2 encoding gene instead is less expressed, but both evidence the same temporal trend, being progressively up-regulated after 24 and 48 h, thereafter the expression decreases, whereas MMP-3 maintains the same up-regulation at 72 h. Hyaluronan synthase 1 and desmoplakin are progressively up-regulated and increase at 24, 48, and 72 h. Hyaluronidase 1 and neutrophil elastase genes are overexpressed, but at 72 h they both exhibit the same behavior as the other degradative enzymes MMP-13 and MMP-2. CONCLUSIONS: Skin bio-revitalization by injecting the tested NASHA gel produces an enhancement in the expression of some genes involved in extracellular matrix degradation and organization. In this study, a time-dependent behavior, different for genes encoding degradative compared to synthesis proteins, was demonstrated.

Psychometric properties of the pain assessment in advanced dementia scale compared to self assessment of pain in elderly patients.

AIM: The aims of the present study were to report on the psychometric properties of the Pain Assessment in Advanced Dementia (PAINAD) scale by comparing it with the gold standard method of self-reporting on a numerical rating scale (NRS), and to provide a categorical version of the PAINAD scale comparable with the verbal descriptor scale of the NRS. METHODS: Six hundred elderly patients with various degrees of cognitive impairment consecutively admitted to the acute geriatric section at Padua University were evaluated. Cognitive, functional, and health statuses were evaluated using the Mini-Mental State Examination (MMSE), activities of daily living, and the Cumulative Illness Rating Scale (CIRS), respectively. Pain measurements were obtained by administering the NRS and the PAINAD scale. RESULTS: Cognitive decline was recorded in 310 subjects (52%). The internal reliability of the PAINAD scale was adequate for all items, both in patients with dementia (α = 0.90) and in those with no cognitive impairment (α = 0.94). The psychometric evaluation demonstrated a stronger level of concurrent validity (Kendall's τ = 0.73, p < 0.0001) and inter-rater agreement (ĸ = 0.74, p < 0.0001) for the PAINAD compared with the NRS. CONCLUSION: Our findings clearly indicate that the PAINAD scale is a reliable and easily administered tool for assessing pain intensity also in elderly patients with advanced dementia.

No association between polymorphisms in cubilin, a gene of the homocysteine metabolism and the risk of non-syndromic cleft lip with or without cleft palate.

Epidemiological studies have correlated lower maternal periconceptional levels of plasma folate and cobalamin with increased risk of delivering offspring presenting congenital malformations such as cleft lip with or without cleft palate (CL/P) or neural tube defects. A number of genetic studies aimed at correlating these biochemical levels or the occurrence of malformations with specific genetic defects or polymorphisms have been successfully performed. The cubilin gene (CUBN) codes for a carrier that plays a crucial role in cobalamin cell internalization. CUBN polymorphisms were previously found to be associated with spina bifida occurrence. In this work, a family-based association study was performed to test CUBN involvement in CL/P. A sample of 391 CL/P triads was investigated with three single nucleotide polymorphisms mapping on the cubilin gene. Association tests indicated no significant association between CL/P and marker alleles or marker haplotypes. No evidence of maternal effect and imprinting were obtained. These data suggest that CUBN is not involved in CL/P onset in the investigated Italian population.

Evidence of LEF1 fetal-maternal interaction in cleft lip with or without cleft palate in a consistent Italian sample study.

Epithelial mesenchymal transformation is considered a cardinal process in orofacial development. Several molecular players appear to be involved in this delicate mechanism; the activation of LEF1 transcription factor by transforming growth factor beta 3 seems to be a key step for the correct flow of events. The failure of orofacial processes during embryonic development may provoke cleft lip and/or cleft palate malformations. The scope of the present investigation was to verify whether genetic variants at LEF1 could influence the risk of orofacial clefting. The approach was a family based association study involving a total of 512 Italian patients and their parents, 401 having cleft lip with or without cleft palate (CL/P) and 111 with cleft palate only (CPO). Haplotype association analysis provided moderate evidence of an association with clefting (p 0.01). A log-linear likelihood-based method was used to verify maternal and foetal-maternal association. An association between the maternal genotype and the occurrence of CL/P was observed at two polymorphic loci, at rs10022956 (P = 0.0049) and rs10025431 (P = 0.0065) respectively, while a foetal-maternal effect modulating the risk of clefting was found at locus rs10025431 (P = 0.0071). These data further corroborate the importance of the mother's genotype with regard to susceptibility to malformations and early-onset diseases.

Study of the 12q13 region in nonsyndromic cleft lip with or without cleft palate.

The 12q13 region has been suggested as a candidate locus for orofacial cleft by different investigators. In the present study we tested the region for linkage with non syndromic cleft lip with or without cleft palate in a collection of 39 Italian multigenerational families, using microsatellite markers. No evidence of linkage was detected between the marker map and NSCLP under different mode of inheritance nor with a nonparametric method. Formal level of linkage exclusion, were obtained for each point of the map. Genetic heterogeneity and the different impact of the candidate locus among populations could explain conflicting results obtained in different studies.

Study of ABCB1 multidrug resistance protein in a common orofacial malformation.

The onset of embryonic malformations is greatly determined by the intrauterine environment, conditioned by maternal lifestyle, diet, drugs and medication intake, in addition to both foetal and maternal genotypes. Maternal C677T MTHFR genotype has been identified as important factor in cleft lip with or without cleft palate (CL/P) etiology. In the present study we evaluated the possible interaction between maternal methylenetetrahydrofolate reductase (MTHFR) and foetal ABCB1 genotypes. ABCB1 gene codes for a drug-transport pump in charge to protect the cell by extruding a variety of harmful exogens, but with a reduced activity in a folate-restricted condition. Maternal 677T genotype is translated in a reduced folate availability for the developing embryo who consequently may becomes more exposed to external insults. A family based association analysis was performed to test the effect of ABCB1 polymorphisms in clefting, in the whole sample and in the stratified sample accordingly to maternal MTHFR genotype. No evidence of association between ABCB1 polymorphisms and CL/P was detected. This suggests that ABCB1 or ABCB1-MTHFR feto-maternal interaction could have no effect in orofacial clefting or could play a role in a limited number of cases.

Evidence of an involvement of TFAP2A gene in non-syndromic cleft lip with or without cleft palate: an Italian study.

Unraveling of factors involved in multifactorial diseases is a great challenge. Different approaches can be contemplate and applied to a variety of congenital malformations. In the present investigation TFAP2A has been considered a good candidate gene for nonsyndromic cleft lip with or without cleft palate (NSCLP) aetiology, basing on a sum of considerations. TFAP2A has been seen involved in orofacial development in mice; it is located in the NSCLP candidate region 6p24; it codes for a transcription factor which regulates expression of IRF6, a gene implied in NSCLP; finally, it is embroiled in the branchiooculofacial syndrome, that includes clefting as feature. A family based association analysis was performed with a sample study of 405 NSCLP triads. Evidence of association was obtained with both single marker and haplotype analyses, thus providing a support for TFAP2A in NSCLP aetiology.

Incidence of low risk human papillomavirus in oral cancer: a real time PCR study on 278 patients.

Squamous cell carcinoma is the most frequent malignant tumour of the oral cavity. It is widely known that tobacco and alcohol consumption are the major causes of the development of oral squamous cell carcinoma (OSCC). The human papilloma virus infection has also been postulated as a risk factor for squamous cell carcinoma, although conflicting results have been reported. The aim of this study is to evaluate the presence of high-risk and low-risk type human papillomavirus in a large sample of squamous cell carcinoma limited to the oral cavity by means of quantitative real-time polymerase chain reaction. Data were obtained from 278 squamous cell carcinoma limited to oral cavity proper. Sequencing revealed that 5 samples were positive for HPV type 16, 5 for HPV type 11, and 1 for HPV type 6. Human papillomavirus 11 was detected in 5 tumours out of the 278 examined. The prevalence rate for Human papillomavirus 11 was 1.8% (C.I. 0.7-3.9). The matched case-controls analysis indicated that the prevalence among controls did not significantly differ with respect to cases and that Human papillomavirus 11 alone did not correlate with squamous cell carcinoma.

LOH at PDCD4, CTNNB1, and CASP4 loci contributes to stage progression of oral cancer.

Squamous cell carcinoma is the most frequent malignant tumor of the oral cavity. Markers of tumor progression that could help to define diagnosis, plan treatment and implement prognosis have still to be identified. Seven candidate markers for tumor progression were investigated using a loss of heterozygosity (LOH) assay. The sample was made up of 51 squamous cell carcinoma and adjacent normal tissues from the same patients. LOH at one, or more, markers was a relatively frequent event that was observed in 53% of tumors. The number of losses detected in each tumor was significantly associated with tumor severity. Significant association between UICC stage grouping and LOH was found for 3 gene loci: programmed cell death 4 (PDCD4), catenin beta 1 (CTNNB1), and caspase 4 (CASP4). No association between allelic loss and the occurrence of lymph node metastasis was found for any of the seven investigated loci. Overall, LOH contributes to tumor progression of oral SCC. A specific role for PDCD4, CTNNB1, and CASP4 was found.

MicroRNA expression profiling of oral carcinoma identifies new markers of tumor progression.

Oral squamous cell carcinoma, the most frequently occurring malignant head and neck tumour, generally exhibits poor prognosis and metastases are the main cause of death. The discovery of reliable prognostic indicators of tumour progression could greatly improve clinical practice. MicroRNAs are involved in the regulation of basic cellular processes such as cell proliferation, differentiation, and apoptosis. Since miRNAs have been shown to be abnormally expressed in different tumours their importance as potential cancer prognostic indicators is increasing. To define the role of miRNA in OSCC tumours we investigated the expression profile of 15 OSCC (8 without metastasis and 7 with lymph node metastasis) using microarray analysis. Thirteen miRNA were significantly overexpressed (miR-489, miR-129, miR-23a, miR-214, miR-23b, miR-92, miR-25, miR-210, miR-212, miR-515, miR-146b, miR-21, miR-338) and 6 miRNA were underexpressed (miR-520h, miR-197, miR-378, miR-135b, miR-224, miR-34a) in oral tumours. Underexpression of mir-155, let-7i, mir-146a was found to characterize progression to metastastatic tumours. Further investigations will elucidate whether differentially expressed miRNAs will help to better classify OSCCs, thus improving diagnoses and patient care.

Long-term regulation of vacuolar H(+)-ATPase by angiotensin II in proximal tubule cells.

Long-term effects of angiotensin II (Ang II) on vacuolar H(+)-ATPase were studied in a SV40-transformed cell line derived from rat proximal tubules (IRPTC). Using pH(i) measurements with the fluorescent dye BCECF, the hormone increased Na(+)-independent pH recovery rate from an NH(4)Cl pulse from 0.066 +/- 0.014 pH U/min (n = 7) to 0.14 +/- 0.021 pH U/min (n = 13; p < 0.05) in 10 h Ang II (10(-9) M)-treated cells. The increased activity of H(+)-ATPase did not involve changes in mRNA or protein abundance of the B2 subunit but increased cell surface expression of the V-ATPase. Inhibition of tyrosine kinase by genistein blocked Ang II-dependent stimulation of H(+)-ATPase. Inhibition of phosphatidylinositol-3-kinase (PI3K) by wortmannin and of p38 mitogen-activated protein kinase (MAPK) by SB 203580 also blocked this effect. Thus, long-term exposure of IRPTC cells to Ang II causes upregulation of H(+)-ATPase activity due, at least in part, to increased B2 cell surface expression. This regulatory pathway is dependent on mechanisms involving tyrosine kinase, p38 MAPK, and PI3K activation.

Pattern of drug use among preterm neonates: results from an Italian neonatal intensive care unit.

BACKGROUND: Drug use in preterm neonates admitted to Neonatal Intensive Care Unit (NICU) has been investigated, so far, in terms of unauthorized or off-label use; very little is known on the use of combinations of different active substances, which is frequently required in this population (prophylaxis of infections, treatment of concomitant diseases). The aim of this study was to describe the most common patterns of drug use in an Italian NICU, focusing on those with nephrotoxic potential. METHODS: Medical records of preterm neonates (<37 weeks of gestational age) weighing less than 1,500 g at birth and admitted to an Italian NICU were scrutinized in a 3-year retrospective investigation. Analysis included drug exposure, duration of therapies, co-administration of drugs with potential renal side effects; also daily protein supplement was calculated from parenteral nutrition. RESULTS: A cohort of 159 preterm neonates was selected; 68 were born weighing less than 1,000 g (extremely low birth weight infants, ELBW, Group A), 91 weighed between 1,000 and 1,500 g at birth (Group B). Compared to Group B, neonates of Group A were more likely to receive pharmacological treatments: the most used drugs were antibiotics (especially ampicillin and amikacin, p = .07 and p < .001, respectively), antifungals (especially fluconazole, p < .001), and diuretics (especially furosemide, p < .001). Analysis of co-administration of drugs with potential nephrotoxicity showed ampicillin and amikacin as the most reported combination (94.1% of Group A and 31.9% of Group B), the combination of furosemide with antibacterials (ampicillin or amikacin) was also frequently reported, with average period of combination shorter than 2 days. CONCLUSIONS: ELBW infants were exposed to a higher number of drugs compared to other neonates and were more likely to receive associations of drugs with nephrotoxic potential (e.g. furosemide and amikacin), though only for short cycles. Further studies should evaluate the safety profile (especially potential renal side effects) related to most commonly used combinations.

Immunologic studies with mouse mammary tumor viruses: comparative studies with spontaneous mammary tumor cell vaccines from five inbred mouse strains.

Vaccines were prepared from cells of primary spontaneous mammary tumors (MT) of RIII/Imr, GR/Imr, C3H/Imr, A/Imr, and C3HfC57BL/Imr mice. Each vaccine was used to immunize "murine mammary tumor virus (MuMTV)-free" BALB/c/Imr and C57BL/Imr mice before challenge with infectious RIII-MuMTV, GR-MuMTV, C3H-MuMTV, and A-MuMTV. RIII-MT and C3H-MT cells protected mice against tumorigenesis by all four MuMTV's; GR-MT cells protected against all but the RIII-MuMTV challenge; A-MT cells were ineffective against challenge by all four MuMTV's and significantly enhanced development of A-MuMTV-induced tumors. The activity of the C3H-MT cells was not seen when C3HfC57BL cells, free of the standard C3H-MuMTV, were used. The same cell vaccines were inoculated into the five donor strains of mice in attempts to prevent spontaneous MT in these high-incidence strains, which are naturally infected with MuMTV at birth. None of the vaccines prevented tumorigenesis in these mouse strains although delays in the appearance of tumors were observed in RIII and GR mice inoculated with either isologous MT cells or C3Hf-MT cells. The role of viral antigens acting alone or in concert with cellular antigens is discussed.

Serologic responses to murine mammary tumor virus (MuMTV) in MuMTV-exposed laboratory personnel.

A retrospective study was conducted to determine whether exposure to murine mammary tumor virus (MuMTV) induced MuMTV-specific serologic responses among intramural laboratory personnel. Results obtained with a panel of five purified structural proteins of the RIII mouse strain milk-derived MuMTV (gp55, gp34, p28, p18, and p12), by means of the enzyme-linked immunosorbent assay to assess antibody binding, established that MuMTV exposure resulted in highly significant increases in serologic responses to these test antigens as compared to age- and gender-matched controls without overt contact with MuMTV. Furthermore, immunoreactions to gp55 and gp34 were found not to be directed to the carbohydrate moieties of these glycoproteins. Similar results were obtained by assays of human immunoglobulin binding to Western blots of MuMTV proteins. These increased MuMTV-specific immunoreactivities, in general, were found to be related to degree and length of exposure to this virus. These results with MuMTV suggest the possibility of important human immune response differences between exposure to type B (MuMTV) and animal type C (leukemia-sarcoma) RNA tumor viruses, perhaps reflective of sensitivity to antibody dependence of complement-induced virolysis.

Comparative studies of purified subviral component vaccines and formalin-treated mammary tumor viruses from four inbred strains of mice.

Formalin-treated virus vaccines were prepared from purified murine mammary tumor viruses (MuMTV) from 4 inbred strains of mice: RIII/Imr, GR/Imr, C3H/Imr, and A/Imr. In addition, subviral components were isolated from these 4 strains and purified to homogeneity. The inactivated viruses, their major envelope glycoproteins (gp50-gp55), and their major internal core protein (p28) were emulsified in complete Freund's adjuvant and used as vaccines for prevention of mammary tumors in mice. All 4 Formalin-treated virus vaccines reduced significantly the incidence of mammary tumors in "virus-free" C57BL and BALB/c mice when inoculated prior to challenge with live MuMTV. The RIII-, GR-, and A-MuMTV strains showed extensive heterologous cross-protection, whereas the C3H-MuMTV strain showed significant protection only against C3H- and A-MuMTV challenge. The major viral glycoproteins gp50-gp55 reduced significantly the tumor incidence when mice were challenged with isologous infectious virus after immunization, although these glycoproteins showed different degrees of cross-protection than did the same virus strains used as "intact" but Formalin-treated preparations. RIII-gp55 and GR-gp55 cross-protected against each other but not against challenge with C3H- and A-MuMTV strains; the A-gp50 protected against challenge with A- and RIII-MuMTV strains; C3H-gp55 demonstrated limited activity against C3H-MuMTV challenge only. The internal viral core proteins (p28) were ineffective in all systems studied. The same vaccines were tested in MuMTV-positive, high-tumor-incidence strains from which they were derived. At best, the appearance of spontaneous tumors was delayed in a few experimental sets; eventually, all mice developed mammary tumors. The foster-nursed C3HfC57BL strain of mice, which is not exposed to exogenous MuMTV during suckling and which develops mammary tumors after activation of the endogenous virus genome later in life, was responsive only when the heterologous GR-MuMTV Formalin-treated vaccine was used. The association between the ability of virus vaccines to protect a mouse strain and the degree of natural virus expression in that strain is discussed.