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Background: Knowledge of antigen-specific CD4(+) T cells frequencies is pivotal to the choice of the antigen to be used in anti-viral and anti-tumor vaccination procedures and for monitoring of immune responses. Methods that employ small cell numbers from patient samples, are easy to perform and do not require complex techniques/instrumentations and therefore standardization are desirable., Methodology/principal Findings: Purified blood CD4(+) T cells from healthy donors were cultured with autologous antigen presenting cells in several replicate wells in equal numbers in the absence (un-stimulated wells) or in the presence of synthetic peptides corresponding to viral antigens promiscuous HLA-DR epitopes (antigen-stimulated wells). At day 7 of culture low dose IL-2 was added and at day 14 IFN-γ and IL-5 release in the supernatant was measured. A statistical analysis approach, based on Poisson distribution, was then implemented to calculate the frequency of viral-specific CD4(+) T cells. We first determined a patient-specific exceptionality threshold of cytokine release in the un-stimulated wells and then, based on this threshold, we counted the inactive/active wells within the antigen-stimulated wells. This number, along with the number of cells per well, allowed the point and interval estimates of frequencies. A ready-to-use Excel worksheet template with automatic calculations for frequencies estimate was developed and is provided as a supplemental file (Table S9)., Conclusions/significance: We report a simple experimental procedure combining short term in vitro cell culture with statistical analysis to calculate the frequency of antigen-specific CD4(+) T cells. The detailed experimental procedure along with the Excel applicative are a valuable tool for monitoring immune responses in the clinical practice.

Aims: To achieve a reduction of time to reperfusion through the organization of an interhospital network and the involvement of the Regional Health Authority., Methods: Four major endpoints were identified: institutional governance action, clinical management of acute ST-elevation myocardial infarction (STEMI), priority actions for cardiac arrest and early defibrillation, actions to avoid the delay related to decision-making, and logistic factors. Since 2001 in the urban area of Milan a network has been operating among 23 coronary care units, the 118 Dispatch Center (national free number for medical emergencies) and the Health Country Government Agency named Group for Prehospital Cardiac Emergency. In order to monitor the network activity and time to treatment and clinical outcomes a periodic monthly survey, called MOMI (One Month Monitoring Myocardial Infarction), was undertaken and repeated twice yearly. Data were evaluated according to hospital admission modality., Results: Global times are: symptom onset to first medical contact 116 min (interquartile range [IQR] 189), time to first ECG 7 min (IQR 12), door-to-balloon time 77 min (IQR 81.7). Non-parametric test showed that the modality of hospital admittance was the most critical determinant of door-to-balloon time. The shortest one (49.5 min) was that of patients transported by means of advanced rescue units with 12-lead ECG teletransmission and activation of a fast track directly to the cath lab., Conclusions: Our data show how in a complex urban area the organization of an interhospital network and the availability of ECG teletransmission are effective in reducing time to reperfusion, in the treatment of major arrhythmias and in pre-alert of coronary care units and cath labs in case of confirmed STEMI. This experience also stimulated an improvement in technological equipment of rescue units with extension of 12-lead teletransmission to basic life support units. Through the Health Country Government Agency and the Scientific Societies we carry on with our job to create a regional network for cardiac emergency involving all the hospitals.