Quantitative assessment of mitophagy in irradiated cancer cells.

Mitophagy is a finely regulated mechanism through which eukaryotic cells selectively dispose of supernumerary, permeabilized or otherwise damaged mitochondria through lysosomal degradation. Dysfunctional mitochondria are prone to release potentially cytotoxic factors including reactive oxygen species (ROS) and caspase activators, such as cytochrome c, somatic (CYCS). Thus, proficient mitophagic responses mediate prominent cytoprotective functions. Moreover, the rapid degradation of permeabilized mitochondria limits the release of mitochondrial components that may drive inflammatory reactions, such as mitochondrial DNA (mtDNA) and transcription factor A, mitochondrial (TFAM), implying that mitophagy also mediates potent anti-inflammatory effects. Here, we detail a simple, flow cytometry-assisted protocol for the specific measurement of mitophagic responses as driven by radiation therapy (RT) in mouse hormone receptor (HR)+ mammary carcinoma TS/A cells. With some variations, this method - which relies on the mitochondria-restricted expression of a fluorescent reporter that is sensitive to pH and hence changes excitation wavelength within lysosomes (mt-mKeima) - can be adapted to a variety of human and mouse cancer cell lines and/or straightforwardly implemented on fluorescence microscopy platforms.

Cytofluorometric assessment of cell cycle progression in irradiated cells.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects upon the accumulation of unrepaired damage to macromolecules, notably DNA. The ability of ionizing radiation to prevent malignant cells from replicating and to cause their demise is indeed an integral component of the anticancer activity of RT. Neoplastic cells are generally more sensitive to the cytostatic and cytotoxic effects of RT than their healthy counterparts as they exhibit increased proliferative rate and limited capacity for DNA repair. This provides a rather comfortable therapeutic window for clinical RT usage, especially with the development of novel, technologically superior RT modalities that minimize the exposure of normal tissues. Thus, while accumulating evidence indicates that cancer control by RT also involves the activation of tumor-targeting immune responses, assessing cell cycle progression in irradiated cells remains a central approach for investigating radiosensitivity in preclinical tumor models. Here, we detail a simple, flow cytometry-assisted method to simultaneously assess cell cycle distribution and active DNA replication in cultured estrogen receptor (ER)+ breast cancer MCF7 cells. With minimal variations, the same technique can be straightforwardly implemented to a large panel of human and mouse cancer cell lines.

Cytofluorometric assessment of acute cell death responses driven by radiation therapy.

Radiation therapy (RT) is well known for its capacity to mediate cytostatic and cytotoxic effects on malignant cells, largely reflecting the ability of ionizing radiation to cause direct and indirect damage to macromolecules including DNA and lipids. While low-dose RT generally causes limited cytotoxicity in an acute manner (as it imposes insufficient cellular damage to compromise homeostasis, or instead induces the delayed demise of cells that fail to complete mitosis successfully), high RT doses can mediate an acute wave of cell death that begins to manifest shortly (24-72h) after irradiation. Here, we provide two straightforward techniques to assess the acute cytotoxic effects of RT by the flow cytometry-assisted quantification of plasma membrane permeabilization (PMP, a late-stage manifestation of cell death) and either mitochondrial outer membrane permeabilization (MOMP) or phosphatidylserine (PS) externalization (two early-stage signs of cell death) in mouse mammary adenocarcinoma TS/A cells. With minor variations, the same protocols can be straightforwardly adapted to measure acute cell death responses as elicited by RT in a large panel of human and mouse cancer cells lines of different histological derivation.

Cytofluorometric assessment of dendritic cell-mediated uptake of cancer cell apoptotic bodies.

Dendritic cells (DCs) are specialized antigen presenting cells (APCs) able to intake and crosspresent antigens (Ags) on major histocompatibility complex (MHC) class I and II molecules to T cells thus initiating primary and memory immune responses. DC-mediated Ag uptake and crosspresentation represent crucial steps toward cancer recognition and eventually elimination. Cytofluorometry is a standardized procedure to study phagocytosis. By fast and reproducible single cell measurements, flow cytometry allows for simultaneous biochemical and functional analyses of Ag intake. In this chapter, we discuss a two-color flow cytometric analysis of DC-mediated uptake of apoptotic bodies. We also show data on the adjuvanticity of Type-I-interferons (Type-I-IFNs) during Ag retention as we offer a guideline and a range of advice on sample preparation and acquisition.

Assessment of IFN-γ and granzyme-B production by in "sitro" technology.

Tumor neantigens (TNAs) and tumor-associated antigens (TAAs) are crucial triggers of anticancer immune responses. Through major histocompatibility complex, such antigens activate T cells, which, by releasing interferon gamma (IFN-γ) and granzyme B (GRZB), act as crucial effectors against tumor onset and progression. However, in response to immune pressure, cancer cells use different strategies to favor the establishment of an immunosuppressive tumor microenvironment (TME). Elucidating the dynamics of tumor-host co-evolution provides novel opportunities for personalized cancer immunotherapies. The in sitro (in vitro+in situ) technology is an experimental approach involving the preparation of heterocellular cell suspensions from fresh tumors and their use in vitro. The in sitro experimental setup offers the possibility to (1) analyze immune-related parameters (e.g., quantification of cytokines released in the TME), (2) reveal the mechanism of action of drugs, and (3) unveil crucial cell-intrinsic and cell-extrinsic processes boosting anticancer immune responses. Nonetheless, the in sitro technology does not fully recapitulate the complexity of the tissue "in situ" nor models the patterns of infiltrating immune cell localization, and hence parallel experimentation should be scheduled. In this chapter we discuss in sitro technology to analyze and quantify IFN-γ and GRZB production by T cells either co-cultured with cancer cells in the presence of exogenous adjuvant stimuli (i.e., an antibody targeting the immune checkpoint programmed cell death protein 1, and recombinant calreticulin) and boosting with TAAs (i.e., the model SIINFEKL ovalbumin antigen). Specifically, we describe IFN-γ and GRZB quantification by flow cytometry, ELISA and ELISpot technologies.

[Covid-19 and clinical-epidemiological research in Italy: proposal of a research agenda on priority topics by the Italian association of epidemiology]. / Covid-19 e ricerca clinico-epidemiologica in Italia: proposta di un'agenda di ricerca su temi prioritari da parte dell'Associazione italiana di epidemiologia.

BACKGROUND: the Covid-19 pandemic has provoked a huge of clinical and epidemiological research initiatives, especially in the most involved countries. However, this very large effort was characterized by several methodological weaknesses, both in the field of discovering effective treatments (with too many small and uncontrolled trials) and in the field of identifying preventable risks and prognostic factors (with too few large, representative and well-designed cohorts or case-control studies). OBJECTIVES: in response to the fragmented and uncoordinated research production on Covid-19, the   italian Association of Epidemiology (AIE) stimulated the formation of a working group (WG) with the aims of identifying the most important gaps in knowledge and to propose a structured research agenda of clinical and epidemiological studies considered at high priority on Covid-19, including recommendations on the preferable methodology. METHODS: the WG was composed by 25 subjects, mainly epidemiologists, statisticians, and other experts in specific fields, who have voluntarily agreed to the proposal. The agreement on a list of main research questions and on the structure of the specific documents to be produced were defined through few meetings and cycles of document exchanges. RESULTS: twelve main research questions on Covid-19 were identified, covering aetiology, prognosis, interventions, follow-up and impact on general and specific populations (children, pregnant women). For each of them, a two-page form was developed, structured in: background, main topics, methods (with recommendations on preferred study design and warnings for bias prevention) and an essential bibliography. CONCLUSIONS: this research agenda represents an initial contribution to direct clinical and epidemiological research efforts on high priority topics with a focus on methodological aspects. Further development and refinements of this agenda by Public Health Authorities are encouraged.

The socioeconomic determinants of cancer.

This paper provides a synthesis on socioeconomic inequalities in cancer incidence, mortality and survival across countries and within countries, with particular focus on the Italian context; the paper also describes the underlying mechanisms documented for cancer incidence, and reports some remarks on policies to tackle inequalities.From a worldwide perspective, the burden of cancer appears to be particularly increasing in developing countries, where many cancers with a poor prognosis (liver, stomach and oesophagus) are much more common than in richer countries. As in the case of incidence and mortality, also in cancer survival we observe a great variability across countries. Different studies have suggested a possible impact of health care on the social gradients in cancer survival, even in countries with a National Health System providing equitable access to care.In developed countries, there is increasing awareness of social inequalities as an important public health issue; as a consequence, there is a variety of strategies and policies being implemented throughout Europe. However, recent reviews emphasize that present knowledge on effectiveness of policies and interventions on health inequalities is not sufficient to offer a robust and evidence-based guide to the choice and design of interventions, and that more evaluation studies are needed.The large disparities in health that we can measure within and between countries represent a challenge to the world; social health inequalities are avoidable, and their reduction therefore represents an achievable goal and an ethical imperative.

[Objectives, tools and methods for an epidemiological use of electronic health archives in various areas of Italy]. / Obiettivi, strumenti e metodi per un utilizzo epidemiologico di archivi sanitari elettronici correnti in diverse aree italiane.

The availability of Electronic Health Archives (EHA) has increased remarkably over the last twenty years. As part of a joint project of the Italian Association of Epidemiology (AIE) and the Italian Association of Medical Statistics and Clinical Epidemiology (SISMEC), a workgroup of experts was set up in 2005 with the aim of comparing various experiences and of standardizing the procedures by which electronic sources can be integrated. In particular, the workgroup's aim was to estimate the frequency of certain major diseases using standard algorithms applied to EHA. This volume is published with the purpose of making available in a common publication the methods and the results obtained. The results from a multicentre study using a standard approach to probabilistic record-linkage procedures are also included in a specific chapter. Eleven Italian centres from five Italian regions with an overall population of 11,932,026 collected and treated more than 21,374,426 records (year 2003) from five electronic information sources: death certificates, hospital discharge records (including outpatient discharges), drug prescriptions, tax- exemptions, and pathology records in order to estimate the frequency of the following diseases: diabetes, ischemic heart diseases, acute myocardial infarction, stroke, asthma, chronic obstructive pulmonary disease, obstructive lung diseases. For each pathology a specific algorithm was developed and used by all centres for the identification of the prevalent/incident cases of the selected diseases. Standardized methods were used to estimate the rates. The results confirm the need for a common standard approach to produce estimates based on EHA, considering the variability of the quality and of the completeness of the archives, and the difficulties of standardizing record-linkage operations in the various centres. The main achievement of this work was the elimination of the variability due to the use of different algorithms to identify cases using EHA.

[Chronic obstructive pulmonary disease prevalence estimated using a standard algorithm based on electronic health data in various areas of Italy]. / Stima della prevalenza di broncopneumopatia cronico-ostruttiva basata su dati sanitari correnti, mediante l'uso di un algoritmo comune, in differenti aree italiane.

AIM: to estimate the prevalence of chronic obstructive pulmonary disease (COPD) by integrating various administrative health information systems. METHODS: prevalent COPD cases were defined as those reported in the hospital discharge registry (HDR) and cause of mortality registry (CMR) with codes 490*, 491*, 492*, 494* and 496* of the International diseases classification 9th revision. Annual prevalence was estimated in 35+ year-old residents in six Italian areas ofb different sizes, in the period 2002-2004. We included cases observed in the previous four years who were alive at the beginning of each year. RESULTS: in 2003, age-standardized prevalence rates varied from 1.6% in Venice to 5% in Taranto. Prevalence was higher in males and increased with age. The highest rates were observed in central (Rome) and southern (Taranto) cities, especially in the 35-64 age group. HDR contributed 91% of cases. Health-tax exemption registry would increase the prevalence estimate by 0.2% if used as a third data source. CONCLUSIONS: with respect to the National Health Status survey, COPD prevalence is underestimated by 1%-3%; this can partly be due to the selection of severe and exacerbated COPD by the algorithm used. However, age, gender and geographical characteristics of prevalent cases were comparable to national estimates. Including cases observed in previous years (longitudinal estimates) increased the point estimate (yearly) of prevalence two or three times in each area.

[Obstructive lung disease prevalence estimated using a standard algorithm based on electronic health data in various areas of Italy]. / Stima della prevalenza di malattie polmonari cronico-ostruttive basata su dati sanitari correnti mediante un algoritmo comune in differenti aree italiane.

OBJECTIVE: development of an algorithm to estimate the prevalence of obstructive lung diseases (OLD) through record linkage of administrative health data sources in three Italian areas. SETTING: AULSS 12 Veneziana, city of Torino, ASL10 Firenze. PARTICIPANTS: all residents in the three areas in the period 2002-2004 (N = 1,944,471 on 30th June 2003). MAIN OUTCOME: crude prevalence, standardized prevalence with 95% confidence intervals. METHODS: the following data sources were used to identify OLD cases: hospital discharges (HD), health-tax exemptions (HTE), death causes (DC) and drug prescriptions (DP). All patients diagnosed with (from HD) or dead because of chronic bronchitis, emphysema and asthma have been included in the analysis. We defined as a prevalent case a subject found in each year in at least one of the four data sources. We reported the absolute and relative contribution of each information system by area, age, gender and year of interest. We performed a sensitivity analysis using more restrictive criteria to identify prevalent cases (two or more DPs per year). RESULTS: DP was the most relevantsource in identifying cases (from 86 to 88%). The relative contribution ofHD ranged from 3 to 5%. In 2003, standardized prevalence of OLD ranged from 5.35% in Firenze to 6.02% in Venezia. Venezia showed a higher prevalence in children aged 0-14years and a lower prevalence in older age groups (> 64 years) compared to other centers. Overall, the prevalence was higher among males. The use of more restrictive criteria in case identification substantially reduces the estimated prevalence, particularly in younger age-groups and to a lesser extent, in older age-groups. CONCLUSIONS: the algorithm provides estimates with differences between centres. The validity of this algorithm (in terms of sensitivity and positive predictive value) needs to be evaluated through further ad hoc studies.

[Respiratory and allergic disorders in children: differences in socio-economic status]. / Differenze sociali nella frequenza di disturbi respiratori e allergici nell'infanzia.

The association between socio-economic status (SES) and respiratory and allergic disorders is controversial, and conflicting results are reported in literature. We examined this association using as indicators of SES parental education and occupation. Persistent cough resulted inversely associated with SES, while current wheezing and atopic dermatitis were directly associated with SES. The role of SES on frequency of respiratory and allergic conditions in Italy needs to be further investigated, although our results confirm a higher frequency of allergic dermatitis and a lower frequency of persistent cough in more advantaged families.

[Access to health care for asthma in Italian children and adolescents]. / Ricorso alle cure per asma in bambini e adolescenti italiani.

Bronchial asthma represents the most frequent chronic illness in the paediatric age. Although a number of guidelines for the diagnosis, treatment and prevention of the disease exist, some studies have shown that their application on a large scale is still lacking, in this way leading to the inadequate treatment of symptoms and the frequent use of emergency visits and hospitalization. Within the scope of the SIDRIA-2 project, this article describes the main aspects of diagnosis, management and treatment of asthma in children (6/7 years old) and adolescents (13/14 years old). The information was obtained from the SIDRIA questionnaire completed by the parents. The analysis of data demonstrated some relevant differences between the actual management of asthma in the paediatric age in Italy and the suggestions available from the guidelines and the literature. Moreover, some choices in the management of asthma were associated to the socio-economic level of families of children and adolescents, such as a lower use of spirometry and a higher frequency of hospitalization in disadvantaged subjects.