B Lymphocytes in Multiple Sclerosis : Bregs and BTLA/CD272 Expressing-CD19+ Lymphocytes Modulate Disease Severity

B lymphocytes contribute to the pathogenesis of Multiple Sclerosis (MS) by secreting antibodies and producing cytokines. This latter function was analyzed in myelin olygodendrocyte protein (MOG)-stimulated CD19+B lymphocytes of 71 MS patients with different disease phenotypes and 40 age- and sex-matched healthy controls (HC). Results showed that: 1) CD19+/TNF alpha+, CD19+/IL-12+ and CD19+/IFN gamma+ lymphocytes are significantly increased in primary progressive (PP) compared to secondary progressive (SP), relapsing-remitting (RR), benign (BE) MS and HC; 2) CD19+/IL-6+ lymphocytes are significantly increased in PP, SP and RR compared to BEMS and HC; and 3) CD19+/IL-13+, CD19+/IL-10+, and CD19+/IL-10+/TGF beta+ (Bregs) B lymphocytes are reduced overall in MS patients compared to HC. B cells expressing BTLA, a receptor whose binding to HVEM inhibits TcR-initiated cytokine production, as well as CD19+/ BTLA+/IL-10+ cells were also significantly overall reduced in MS patients compared to HC. Analyses performed in RRMS showed that fingolimod-induced disease remission is associated with a significant increase in Bregs, CD19+/BTLA+, and CD19+/BTLA+/IL-10+ B lymphocytes. B lymphocytes participate to the pathogenesis of MS via the secretion of functionally-diverse cytokines that might play a role in determining disease phenotypes. The impairment of Bregs and CD19+/BTLA+ cells, in particular, could play an important pathogenic role in MS

The NLRP3 and NLRP1 Inflammasomes are Activated in Alzheimer’s Disease

Background Interleukin-1 beta (IL-1\u3b2) and its key regulator, the inflammasome, are suspected to play a role in the neuroinflammation observed in Alzheimer\u2019s disease (AD); no conclusive data are nevertheless available in AD patients. Results mRNA for inflammasome components (NLRP1, NLRP3, PYCARD, caspase 1, 5 and 8) and downstream effectors (IL-1\u3b2, IL-18) was up-regulated in severe and MILD AD. Monocytes co-expressing NLRP3 with caspase 1 or caspase 8 were significantly increased in severe AD alone, whereas those co-expressing NLRP1 and NLRP3 with PYCARD were augmented in both severe and MILD AD. Activation of the NLRP1 and NLRP3 inflammasomes in AD was confirmed by confocal microscopy proteins co-localization and by the significantly higher amounts of the pro-inflammatory cytokines IL-1\u3b2 and IL-18 being produced by monocytes. In MCI, the expression of NLRP3, but not the one of PYCARD or caspase 1 was increased, indicating that functional inflammasomes are not assembled in these individuals: this was confirmed by lack of co-localization and of proinflammatory cytokines production. Conclusions The activation of at least two different inflammasome complexes explains AD-associated neuroinflammation. Strategies targeting inflammasome activation could be useful in the therapy of AD

Tectono-stratigraphic response of the Sandino Forearc Basin (N-Costa Rica and W-Nicaragua) to episodes of rough crust and oblique subduction

The southern Central American active margin is a world-class site where past and present subduction processes have been extensively studied. Tectonic erosion/accretion and oblique/orthogonal subduction are thought to alternate in space and time along the Middle American Trench. These processes may cause various responses in the upper plate, such as uplift/subsidence, deformation, and volcanic arc migration/ shut-off. We present an updated stratigraphic framework of the Late Cretaceous– Cenozoic Sandino Forearc Basin (SFB) which provides evidence of sedimentary response to tectonic events. Since its inception, the basin was predominantly filled with deep-water volcaniclastic deposits. In contrast, shallow-water deposits appeared episodically in the basin record and are considered as tectonic event markers. The SFB stretches for about 300 km and varies in thickness from 5 km (southern part) to about 16 km (northern part). The drastic, along-basin, thickness variation appears to be the result of (1) differential tectonic evolutions and (2) differential rates of sediment supply. (1) The northern SFB did not experience major tectonic events. In contrast, the reduced thickness of the southern SFB (5 km) is the result of at least four uplift phases related to the collision/accretion of bathymetric reliefs on the incoming plate: (i) the accretion of a buoyant oceanic plateau (Nicoya Complex) during the middle Campanian; (ii) the collision of an oceanic plateau (?) during the late Danian–Selandian; (iii) the collision/accretion of seamounts during the late Eocene–early Oligocene; (iv) the collision of seamounts and ridges during the Pliocene–Holocene. (2) The northwestward thickening of the SFB may have been enhanced by high sediment supply in the Fonseca Gulf area which reflects sourcing from wide, high relief drainage basins. In contrast, sedimentary input has possibly been lower along the southern SFB, due to the proximity of the narrow, lowland isthmus of southern Central America. Moreover, two phases of strongly oblique subduction affected the margin, producing strike-slip faulting in the forearc basin: (1) prior to the Farallon Plate breakup, an Oligocene transpressional phase caused deformation and uplift of the basin depocenter, triggering shallowing-upward of the Nicaraguan Isthmus in the central and northern SFB; (2) a Pleistocene–Holocene transtensional phase drives the NW-directed motion of a forearc sliver and reactivation of the graben-bounding faults of the late Neogene Nicaraguan Depression. We discuss arguments in favour of a Pliocene development of the Nicaraguan Depression and propose that the Nicaraguan Isthmus, which is the apparent rift shoulder of the depression, represents a structure inherited from the Oligocene transpressional phase

Identificazione del liquido seminale: metodiche a confronto

Nei casi di violenza sessuale è di fondamentale importanza, oltre ad un accurato esame obiettivo della vittima, la raccolta di tutti gli elementi che possono costituire la prova dell’abuso subito. Tra gli elementi di prova assume particolare rilievo la ricerca del liquido seminale che, come altri fluidi corporei, può essere evidenziato su reperti diversi quali indumenti, lenzuola, fazzoletti, ecc. Tra le metodiche attualmente impiegate nei laboratori medico-legali vanno segnalate quelle basate su metodi immunocromatografici finalizzati all’identificazione di sostanze presenti in elevata concentrazione nel liquido seminale quali l’Antigene Prostatico Specifico (PSA) e la Semenogelina (Sg). Nel presente lavoro illustriamo i risultati da noi ottenuti utilizzando in parallelo due diversi kit commerciali per l’identificazione del PSA e della Sg. Al fine di verificare la sensibilità dei kit le indagini sono state condotte su reperti di interesse forense conservati nel nostro laboratorio per un periodo di tempo variabile da 3 mesi a 10 anni. Sebbene le metodiche immunocromatografiche, per la rapidità e l’efficacia dei test stessi, rappresentino un valido mezzo per l’identificazione del liquido seminale, è comunque necessario, per i campioni risultati negativi e/o con risultati discordanti nei test, eseguire le analisi genetiche al fine di giungere ad una corretta definizione del caso

GENETIC ANALYSES ON BONE REMAINS: THE UNIVERSITY OF ROME “SAPIENZA” LABORATORY OF FORENSIC GENETICS’ EXPERIENCE

Genetic analyses on bone remains in the field of human identification represent one of the most stimulating and complex challenges for forensic geneticists. Unlike the analysis of biological traces such as blood, semen, saliva or urine, that usually do not present any particular technical and operational difficulty so that personal identification can be achieved, as appropriate, more or less easily through comparing the genetic profile obtained from the sample with the one available as reference, the identification of bone remains forces the analysts to face multiple and complex variable factors (e.g. the degradation of genetic material and the environmental contamination of the samples) that can affect the success of the analysis in the sense of obtaining a complete and interpretable STR profile. In such cases an accurate evaluation of the characteristics of the sample and the environmental conditions to which this finding has been exposed is extremely important. This presentation describes the methods used in the Laboratory of Forensic Genetics of the Department S.A.I.M.L.A.L of the University of Rome "Sapienza" for the analysis of bone remains, to the purpose of either personal identification or the assessment of a parental relationship. The authors will present a selection of 20 cases came under their observation during the years 2007-2011, for which the genetic analyses were performed on different bone samples (femur, tibia, humerus, mandible) using different extraction, amplification and STR typing methods. The results obtained will be compared in order to assess for each case the specific role of 3 important variable factors: the age of the remains, the environmental conditions of storage/finding and the cause of death. 6 out of the 20 cases showed interpretation problems related to the Low Copy Number (LCN, or Low Template -LT) DNA condition due to DNA degradation (i.e. the effects of high temperatures in case of charred remains; the acceleration of autolytic processes in case of hexumation of a corpse) and/or the presence of DNA inhibitors (e.g. Calcium Phosphate, Humic Acid) that likely were co-extracted with the DNA from the evidence sample. The results show that the possibility of obtaining a complete and interpretable genetic profile depends largely on the 3 variable factors mentioned above, particularly with regard to the environmental conditions of storage/finding of the remains, thus confirming the need to optimize the analytical methods in order to minimize the effects of environmental inhibitors. After attending this presentation, attendees will understand some principles of genetic analysis on bone remains, the challenges related to this kind of investigation especially for what concerns criminal cases and the importance of the honesty of the forensic scientist when a certain and unequivocal interpretation of the DNA profile obtained cannot be provided. This presentation will impact the forensic community by highlighting the importance of bone remains as an evidentiary sample in forensic caseworks and the difficulties related to the genetic analysis of such samples due to degradation and/or inhibition factors: in these cases it is fundamental for the scientist to consider that asserting that a complete and interpretable genetic profile is not obtained from a sample (thus the sample cannot be considered useful for a comparison) does not mean a failure but, on the contrary, reveals scientific honesty and should stimulate the necessary progress in this field

Indagini genetiche su reperti ossei: esperienza del laboratorio di genetica forense dell'Università di Roma "Sapienza"

L’analisi di reperti ossei in ambito identificativo rappresenta ad oggi una delle sfide più stimolanti e complesse per i genetisti forensi. A differenza dell’analisi di tracce biologiche quali sangue, sperma, saliva o urina, le quali non presentano particolari difficoltà dal punto di vista tecnico-operativo per cui l’identificazione personale può essere raggiunta, a seconda dei casi, più o meno facilmente tramite il confronto del profilo genetico ottenuto con quello di riferimento disponibile caso per caso, nell’identificazione di resti ossei ci si trova a dover affrontare molteplici e complesse variabili (degradazione del materiale genetico nonché contaminazione ambientale dei reperti) in grado di condizionare il buon esito dell’analisi nel senso dell’ottenimento di un profilo STR completo ed interpretabile. In tali casi risulta infatti spesso tanto difficile quanto auspicabile riconoscere e valutare a priori la specifica tipologia del reperto in esame e le condizioni ambientali a cui tale reperto è stato esposto. Nel presente lavoro vengono illustrate le metodiche impiegate nel Laboratorio di Genetica Forense del Dipartimento SAIMLAL dell'Università di Roma “Sapienza” nell’analisi di resti ossei, ai fini sia identificativi che di accertamento del rapporto parentale. Nei casi esaminati, le analisi genetiche sono state eseguite su campioni ossei diversi (femore, tibia, omero, mandibola) utilizzando la medesima metodica di estrazione del DNA abbinata a kit commerciali di amplificazione e tipizzazione degli STR differenti ed i risultati ottenuti sono stati confrontati al fine di evidenziare il ruolo specifico, per ogni singolo caso in esame, dei seguenti fattori variabili: età dei reperti, condizioni ambientali di conservazione/ritrovamento e modalità del decesso. I risultati ottenuti mostrano che la possibilità di ottenere un profilo genetico utile dipende strettamente dalle variabili precedentemente indicate, con particolare riguardo alle condizioni ambientali di conservazione/ritrovamento dei resti ossei, confermando la necessità di approfondire le conoscenze sulla natura e gli effetti degli inibitori dell’amplificazione del DNA al fine di ottimizzare le metodiche analitiche riducendo al minimo gli effetti inibenti di tali sostanze

DNA quantification by real time PCR and short tandem repeats (STRs) amplification results.

Determining the DNA amount in a forensic sample is fundamental for PCR-based analyses because if on one hand an excessive amount of template may cause the appearance of additional or out-of-scale peaks, by the other a low quantity can determine the appearance of stochastic phenomena affecting the PCR reaction and the subsequent interpretation of typing results. In the common practice of forensic genetics laboratories, the quantification results provided by Real Time PCR (qPCR) assume the role of “boundary line” between the possibility for a given DNA sample to be subjected or not to the subsequent analytical steps, on the basis of an optimal amount of DNA in the range indicated by the manufacturer of the specific commercial kit. However, some studies have shown the possibility to obtain STR typing results even with an extremely low DNA concentration or, paradoxically, equal to zero (1). Regardless of the amount of DNA used for the quantification of the testing sample, specific software are able to use the standard curve to calculate concentration values far below the manufacturer’s reported optimal detection limit (0.023 ng/μL). Consequently, laboratories have to face the critical decision to interrupt the analyses giving up the possibility to obtain a genetic profile -although partial- or to try the amplification of the extract with the awareness of the interpretation issues that this implies. The authors will present the quantification results obtained by qPCR performed on numerous samples collected from items of forensic interest, subjected to DNA extraction using magnetic beads. Following the quantification step, the extracts were subjected to DNA amplification and STR typing using last generation commercial kits. Samples that showed quantification values below the limit of detection for the method were included in the analysis in order to check the existence of a correlation between the DNA quantification results by qPCR and the possibility of obtaining a genetic profile useful for identification purposes. Our study, performed on 558 samples from forensic casework items, has shown a correlation between the DNA amount resulted from qPCR analysis and the possibility of obtaining a genetic profile useful for identification purposes. In spite of the increasing sensitivity of last generation commercial kits for STR analysis, as demonstrated by the ability to detect allelic peaks from extremely low DNA quantities (with concentrations far below the limit of detection for the specific quantification kit, even corresponding to 0 or “Undetermined”), the results obtained show a correlation between qPCR quantification values and STR typing results. Thus the qPCR method confirms being today a useful and valid instrument for both qualitative and quantitative evaluation of genetic samples for human identification purposes

An Investigation of the Effect of DNA Degradation and Inhibition on PCR Amplification of Single Source and Mixed Forensic Samples

The goal of this proposal was to examine the mechanisms for PCR inhibition and degradation and their effects on forensic DNA typing. The effects of these problems are well known; poor amplification and allele dropout. However, there are very few studies in the forensic literature that explore the issue of how inhibitors produce poor PCR results and even less is known about the mechanisms for degradation commonly present in typical forensic samples. A better understanding of these inhibition mechanisms could lead to the development of more sensitive, more robust analytical protocols. In this proposal we performed controlled studies to clarify the mechanisms of environmental and chemical degradation and PCR inhibition on single source samples and mixtures. To do this we utilized real time PCR and HPLC/EC to evalutate the mechanisms of DNA degradation, oxidative damage and PCR inhibition on the recovery of STR profiles. Both degraded and pristine DNA were examined. In particular we performed the following experiments: 1) An analysis of the effects of various inhibitors on PCR amplification using real time PCR with high resolution DNA melt curves. 2) an analysis of the effect of natural and enzymatic degradation on PCR profiles. 3) An analysis of the effect of chemical oxidation on DNA profiles and 4) a correlation between PCR inhibition and DNA amplification. Our overall conclusions are that 1) Environmental damage to DNA in tissue samples occurs rapidly to the point that DNA becomes nearly unrecoverable. The template in such samples breaks down to very small pieces in as little as 3 weeks. 2) The effects of oxidative damage on such samples was minimal. We utilized HPLC with electrochemical detection to monitor base damage to heavily degraded tissue samples. No oxidation of DNA bases was found for environmentally degraded DNA, although it was present in saliva samples. 3) . The combination of real time PCR and DNA melt curves is an effective tool for the detection of PCR inhibition and permits classification of various inhibitors based on their behavior. Our experiments on the effect of DNA template sequence, DNA template length and inhibitor concentration reveal that PCR inhibitors may affect STR results in several different fashions. Real time PCR results reveal that PCR inhibitors can affect Taq polymerase reactions reducing the total amount of DNA produced and/or can bind DNA, resulting in a loss of available template. 4) The effects of DNA binding also appear to be sequence and/or length specific. PCR inhibitors that mainly affect taq tend to inhibit DNA by affecting the largest alleles first, while inhibitors that bind DNA may affect smaller alleles as well as larger ones. 5) It has been widely reported that MiniSTRs improve resistance to PCR inihibition. Based on our results, a caveat should be that such improvements may depend on the type of inhibition. Sequence specific inhibition may still cause problems even with reduced sized amplicons