Modeling delay to diagnosis for Amyotrophic lateral sclerosis: under reporting and incidence estimates

Background

This paper provides a strategy to obtain a reliable estimate of the incidence rate for Amyotrophic lateral sclerosis based on data from the National Registry of Rare Diseases (NRRD). In fact, unobserved cases may be due to the fact that “a long time” may intercour between the suspect of having the disease (onset) and the date the disease is diagnosed. Potential factors that may influence the probability of experiencing the event (diagnosis) conditionally on the onset (suspected) are investigated. Since we are treating rare diseases, the role of social and economic factors is not that obvious; latent as well as observed factors may influence the delay to diagnosis.

Methods

We use a semiparametric estimator based on the distribution of delay to diagnosis to account for potential underreporting. In particular, we propose to adopt an Horvitz-Thompson based estimator to correct the incidence figure that can be derived for the period 2007-2009 from the NRRD, Italy.

Results

The incidence estimates obtained by adopting the proposed approach are about 1 case per 100000 inhabitants and despite they let recovering a good part of underreporting, they are still far from ALS incidence international ranges between 1.5 and 2.5. However, by looking only at northern Italy, the incidence estimates we can derive are coherent with those known internationally.

Conclusions

These results confirm the existence of substantial differences in reporting accuracy, and point out where the system of data collection must be improved. In particular, when reliable individual characteristics will be available, they could be employed to refine the proposed estimator.

     

Chemoselective Modification of Viral Surfaces via Bioorthogonal Click Chemistry

The modification of virus particles has received a significant amount of attention for its tremendous potential for impacting gene therapy, oncolytic applications and vaccine development.^1,2,3 Current approaches to modifying viral surfaces, which are mostly genetics-based, often suffer from attenuation of virus production, infectivity and cellular transduction.^4,5 Using chemoselective click chemistry, we have developed a straightforward alternative approach which sidesteps these issues while remaining both highly flexible and accessible.^1,2The goal of this protocol is to demonstrate the effectiveness of using bioorthogonal click chemistry to modify the surface of adenovirus type 5 particles. This two-step process can be used both therapeutically¹ or analytically,^2,6 as it allows for chemoselective ligation of targeting molecules, dyes or other molecules of interest onto proteins pre-labeled with azide tags. The three major advantages of this method are that (1) metabolic labeling demonstrates little to no impact on viral fitness,^1,7 (2) a wide array of effector ligands can be utilized, and (3) it is remarkably fast, reliable and easy to access.^1,2,7In the first step of this procedure, adenovirus particles are produced bearing either azidohomoalanine (Aha, a methionine surrogate) or the unnatural sugar O-linked N-azidoacetylglucosamine (O-GlcNAz), both of which contain the azide (-N₃) functional group. After purification of the azide-modified virus particles, an alkyne probe containing the fluorescent TAMRA moiety is ligated in a chemoselective manner to the pre-labeled proteins or glycoproteins. Finally, an SDS-PAGE analysis is performed to demonstrate the successful ligation of the probe onto the viral capsid proteins. Aha incorporation is shown to label all viral capsid proteins (Hexon, Penton and Fiber), while O-GlcNAz incorporation results in labeling of Fiber only.In this evolving field, multiple methods for azide-alkyne ligation have been successfully developed; however only the two we have found to be most convenient are demonstrated herein – strain-promoted azide-alkyne cycloaddition (SPAAC) and copper-catalyzed azide-alkyne cycloaddition (CuAAC) under deoxygenated atmosphere.     

Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol

Background aimsWe have recently shown that thymoglobulin (TG) efficiently expands cytokine-induced killer (CIK) cells in combination with interferon (IFN)-γ and interleukin (IL)-2 (ITG2 protocol). It is presently unknown whether the infusion of autologous immune effector cells generated by TG, IFN-γ and IL-2 is feasible and safeMethodsFive patients with advanced and/or refractory solid tumors were enrolled in the present phase I/II study. Peripheral blood mononuclear cells (PBMC) collected by leukapheresis were stimulated under good manufacturing practice (GMP)-conditions with IFN-γ, followed by TG and IL-2. After 2–3 weeks in culture, a median of 4.65 × 10⁶ immune effector cells per kilogram of recipient's body weight was obtained and infused intravenously. The median time from enrollment into the study to infusion of the expanded CIK cells was 30 daysResultsITG2 efficiently expanded immune effector cells that comprised both conventional natural killer (NK) cells and CD3⁺ CD16⁺ CD56⁺ CIK cells. One patient with advanced melanoma died because of disease progression before the infusion of CIK cells. The target dose of at least 2.5 × 10⁶ CIK cells/kg of recipient's body weight was reached in four out of five evaluable patients. CIK cells were administered intravenously without any measurable toxicity. In vitro, CIK cells exerted lytic activity against cervical cancer cells. The median survival was 4.5 months (range 1–13) from the first infusion of CIK cells.ConclusionsThis study has highlighted the feasibility and safety of the administration of CIK cells generated with the ITG2 protocol. Whether CIK cells can help control disease burden in patients with advanced malignancies will be determined in future clinical trials.     

Evaluation of microvascular density in tumors: pro and contra

Microvascular density (MVD) counting protocols have become the morphological gold standard to assess the neovasculature in human tumors. This method requires the use of specific markers to vascular endothelium and of immunohistochemical procedures to visualize microvessels. MVD determined in primary tumors is significantly associated with metastasis and prognosis in several tumors and is most predictive in those tumors that induce significant angiogenesis, namely carcinomas of breast and prostate, and haematological malignancies. There is such a wide range of antibodies and suppliers, antigen retrieval methods, designation of high and low vessel count groups, patient study groups and data interpretation, that it is exceedingly difficult to compare results.     

Patterns of molecular evolution associated with two selective sweeps in the Tb1-Dwarf8 region in maize

We focused on a region encompassing a major maize domestication locus, Tb1, and a locus involved in the flowering time variation, Dwarf8 (D8), to investigate the consequences of two closely linked selective sweeps on nucleotide variation and gain some insights into maize geographical diffusion, through climate adaptation. First, we physically mapped D8 at approximately 300 kb 3' of Tb1. Second, we analyzed patterns of nucleotide variation at Tb1, D8, and seven short regions (400-700 bp) located in the Tb1-D8 region sequenced on a 40 maize inbred lines panel encompassing early-flowering temperate and late-flowering tropical lines. The pattern of polymorphism along the region is characterized by two valleys of depleted polymorphism while the region in between exhibits an appreciable amount of diversity. Our results reveal that a region approximately 100 kb upstream of the D8 gene exhibits hallmarks of divergent selection between temperate and tropical lines and is likely closer than the D8 gene to the target of selection for climate adaptation. Selection in the tropical lines appears more recent than in the temperate lines, suggesting an initial domestication of early-flowering maize. Simulation results indicate that the polymorphism pattern is consistent with two interfering selective sweeps at Tb1 and D8.     

Characterization and quantification of bacterial pathogens and indicator organisms inhousehold kitchens with and without the use of a disinfectant cleaner

This two year study evaluated the prevalence of indicator bacteria and specific pathogens in10 'normal' kitchens in the United States. In Phase I, none of the kitchens wascleaned with an antimicrobial cleaner or disinfectant. Eight locations within the kitchens weremonitored for: total heterotrophs, staphylococci, Pseudomonas , total coliforms andfaecal coliforms. Almost all locations at all households exhibited contamination, with the sink andsponge samples exhibiting large bacterial concentrations. The faecal coliform concentrations insink and sponge samples were very high, with 63 and 67% of all samples being positive,respectively. Escherichia coli was detected in 16·7% of all sink surfaces and33·3% of all sponges. Salmonella was detected once and Campylobacter , on two occasions. In a second phase, households were provided with an antimicrobialdisinfectant cleaner which families were encouraged to use but not forced to do so; in some cases,the product was used infrequently or not at all. This regimen did not demonstrate any consistentreduction in the incidence of bacterial contamination. By contrast, in the final phase of the studywhere disinfectant use was targeted for surfaces soon after contamination with foods or hands,the incidence of contamination decreased dramatically. These data show that normal kitchens caneasily be contaminated with a variety of bacterial contaminants including faecal coliforms, E.coli, Salmonella and Campylobacter . Irregular use, or not using antimicrobialagents, is unlikely to reduce the risk of these infectious agents. By contrast, targeted use is likelyto reduce the incidence of bacterial contaminants.     

Conventional and molecular approaches to isolates of Salmonella hadar from sporadic and epidemic cases

In September 1994 an outbreak of gastroenteritis occurred in 437 people who had consumed lunch in the canteen of a factory in Central Italy. Salmonella sp. was isolated from stools of 99 patients and in 73 of them Salmonella hadar was identified. This is the first outbreak caused by this serotype described in Italy. In order to examine the genotypic basis of the epidemic strains, molecular typing was applied to sporadic strains isolated before and after the outbreak episode. For this purpose phage type, resistance to antibiotics, DNA plasmid profile and sites of insertion of the mobile element of IS200 were determined. The epidemic strains were genetically distinct from the non-epidemic isolates; they were shown to be phage type 26, harbouring four small plasmids, were resistant to nalidixic acid and showed a unique characteristic IS200 fingerprint. The typing methods used in this study allowed the identification and discrimination of the outbreak strains from related isolates. They can thus be considered as a tool for epidemiological purposes. In addition we should point out the emerging resistance to nalidixic acid, largely used in veterinary medicine, in Salm . hadar .     

Measurement of surface contamination from nucleoside analogue antineoplastic drugs by high-performance liquid chromatography in occupational hygiene studies of oncologic hospital departments

Within the frame of a continuing interest in occupational hygiene of hospitals as workplaces, we describe an automated analytical method by reversed-phase high-performance liquid chromatography for the measurement of contamination from the three most important nucleoside analogue antineoplastic drugs (5-fluorouracil, 5FU; cytarabin, CYA; gemcytabin, GCA) on such surfaces as those of preparation hoods and work-benches in departmental pharmacies of oncologic departments. Our method is characterized by a short analysis time (7 min) under isocratic conditions, by the use of a mobile phase devoid of organic solvent and by high sensitivity (LOD≥40 μg/l for all compounds), adequate to detect surface contamination above a threshold of 4 μg/m² for wide surfaces and of 30 μg/m² for small irregular objects. We present some results from a preliminary survey study recently performed in seven oncologic departments of two large general hospitals in Milan. To exemplify the contamination levels on various surfaces (such as on handles, floor surfaces and window glass panes, even far from the preparation hood), analyte concentrations in the order of 0.03–0.06 μg/ml, corresponding to 0.8–1.5 μg of 5FU were measured on telephones, of 0.02–0.6 μg/ml (0.85–28 μg/m²) of CYA were measured on table boards, of 0.05–10.6 μg/ml (1.2–1150 μg/m²) of GCA on furniture and floors. Spillage fractions up to 1% of the employed ANDs (employed daily 5FU 7–13 g; CYA 0.1–7.1 g; GCA 0.2–5 g) are measured on the polyethylene-backed paper disposable cover sheet of the preparation hood.