Beatriz Santamaría

Prevalence of IgG Anti-α-Fodrin Antibodies in Sjögren's Syndrome

Abstract: The objective of this study was to determine the prevalence of antibodies against alpha‐fodrin (α‐fodrin) of the immunoglobulin G (IgG) isotype in Sjögrens syndrome (SS), as defined by European Community Study Group (ESG) and ESG‐modified criteria. We arrived at the prevalence and mean concentrations of IgG anti‐α‐fodrin antibodies using enzyme‐linked immunosorbent assay (ELISA) in 507 patients with SS, primary SS (pSS), and secondary SS (sSS), classified according to either the ESG or the ESG‐modified criteria. IgG anti‐α‐fodrin antibodies were detected in 6/507 (1.2%) and 4/228 (1.7%) of the SS group, according to the ESG or ESG‐modified criteria, respectively. Similar prevalence was found for patients with pSS or sSS. Anti‐Ro/SSA antibodies were present in 151/409 (36.9%) vs. 149/213 (70.0%) of the SS group, 85/195 (43.6%) vs. 83/101 (82.2%) of the pSS group, and 66/214 (30.8%) vs. 66/112 (58.9%) of the sSS group. Anti‐La/SSB antibodies were detected in 77/403 (19.1%) vs. 73/212 (34.4%) of the SS group, 47/194 (24.2%) vs. 45/101 (44.5%) of the pSS group, and 30/209 (14.3%) vs. 28/111 (25.2%) of the sSS group. No clinical associations were found. Only two IgG anti‐α‐fodrin‐positive sera were anti‐Ro/SSA‐negative. We conclude that IgG antibodies against α‐fodrin are present in a small percentage of people with SS, pSS, and sSS. The lower prevalence in patients classified according to the ESG criteria reflects the lower specificity of these criteria. IgG anti‐α‐fodrin antibodies can be detected in some SS patients whose sera do not contain anti/Ro/SSA antibodies.

Prognostic value of peripheral blood mononuclear cell-associated HIV-1 DNA for virological outcome in asymptomatic HIV-1 chronic infection

BACKGROUND Studies in primary HIV-1 infection and advanced HIV-1 disease have demonstrated that HIV-1 DNA associated with peripheral blood mononuclear cells (PBMC HIV-1 DNA) has predictive value for disease progression. OBJECTIVES To analyse in asymptomatic HIV-1 chronic infection the predictive value of PBMC HIV-1 DNA for virological failure. STUDY DESIGN In 115 individuals who had previously participated in study STIR-2102, we retrospectively analysed the PBMC HIV-1 DNA by quantitative real-time PCR. Antiretroviral naïve patients (baseline pre-ART) received 6 weeks of ART prior to randomisation (baseline post-ART). The predictive value of PBMC HIV-1 DNA, HIV-1 RNA in plasma and CD4+ T cells, at baselines pre-ART and post-ART, was determined by Kaplan-Meier and Proportional Hazards Regression analyses. RESULTS At baseline post-ART, 82% of patients showed suppression of HIV-1 RNA, however they maintained significant amounts of HIV-1 DNA (geometric mean: 690 copies/10(6) PBMC). Pre-ART and post-ART levels of HIV-1 DNA and pre-ART levels of HIV-1 RNA showed predictive value (Log-Rank test: p<0.001, p<0.001, p=0.003, respectively). In a multivariate model post-ART PBMC HIV-1 DNA was the stronger predictive variable (adjusted HR, 2.51 [95% CI, 1.33-4.73, p=0.004]) independently of HIV-1 RNA (HR 1.74 [95% CI, 1.16-2.61, p=0.007]). CONCLUSIONS PBMC HIV-1 DNA is an effective prognostic marker for virological outcome in individuals with asymptomatic HIV-1 chronic infection.

Prevalencia del polimorfismo C77G en el exón 4 del gen CD45 en la población española

Fundamento y objetivo: El cambio C77G en el exon 4 del gen CD45 produce un splicing anormal frecuente en poblaciones sanas europeas y relacionado con la infeccion por el virus de la inmunodeficiencia humana. El objetivo de este trabajo es analizar la frecuencia de C77G en la poblacion espanola. Pacientes y metodo: Se incluyeron 517 muestras de sangre anticoagulada con acido etilendiaminotetraacetico procedentes de donantes sanos, en las que se determino la expresion de CD45RA y CD45RO sobre linfocitos T circulantes mediante citometria de flujo. Se realizo asimismo la secuenciacion del exon 4 de CD45 en las muestras con coexpresion anormal de ambas isoformas de CD45. Resultados: En 6 de 517 individuos se detecto persistencia de la expresion de CD45RA en los linfocitos T memoria; todos ellos eran heterocigotos para C77G. La frecuencia alelica es del 0,58% (intervalo de confianza del 95%, 0,23-1,32). Conclusiones: La mutacion C77G esta presente en la poblacion sana espanola. Establecer su significado clinico requiere estudios con grupos de pacientes

Development towards Compact Nitrocellulose-Based Interferometric Biochips for Dry Eye MMP9 Label-Free In-Situ Diagnosis

A novel compact optical biochip based on a thin layer-sensing surface of nitrocellulose is used for in-situ label-free detection of metalloproteinase (MMP9) related to dry eye disease. In this article, a new integrated chip with different interferometric transducers layout with an optimal sensing surface is reported for the first time. We demonstrate that specific antibodies can be immobilized onto these transducers with a very low volume of sample and with good orientation. Many sensing transducers constitute the presented biochip in order to yield statistical data and stability in the acquired measurements. As a result, we report the recognition curve for pure recombinant MMP9, tests of model tears with MMP9, and real tear performance from patients, with a promising limit of detection.

A Proof-of-Concept of Label-Free Biosensing System for Food Allergy Diagnostics in Biophotonic Sensing Cells: Performance Comparison with ImmunoCAP

Food allergy is a common disease worldwide with over 6% of the population (200–250 million people) suffering from any food allergy nowadays. The most dramatic increase seems to be happening in children and young people. Therefore, improvements in the diagnosis efficiency of these diseases are needed. Immunoglobulin type E (IgE) biomarker determination in human serum is a typical in vitro test for allergy identification. In this work, we used a novel biosensor based on label-free photonic transducers called BICELLs (Biophotonic Sensing Cells) for IgE detection. These BICELLs have a thin film of nitrocellulose over the sensing surface, they can be vertical optically interrogated, and are suitable for being integrated on a chip. The BICELLs sensing surface sizes used were 100 and 800 µm in diameter. We obtained calibration curves with IgE standards by immobilizating anti-IgE antibodies and identified with standard IgE calibrators in minute sample amounts (3 µL). The results, in similar assay format, were compared with commercially available ImmunoCAP®. The versatility of the interferometric nitrocellulose-based sensing surface was demonstrated since the limit of detections for BICELLs and ImmunoCAP® were 0.7 and 0.35 kU/L, respectively.

On the Determination of Uncertainty and Limit of Detection in Label-Free Biosensors

A significant amount of noteworthy articles reviewing different label-free biosensors are being published in the last years. Most of the times, the comparison among the different biosensors is limited by the procedure used of calculating the limit of detection and the measurement uncertainty. This article clarifies and establishes a simple procedure to determine the calibration function and the uncertainty of the concentration measured at any point of the measuring interval of a generic label-free biosensor. The value of the limit of detection arises naturally from this model as the limit at which uncertainty tends when the concentration tends to zero. The need to provide additional information, such as the measurement interval and its linearity, among others, on the analytical systems and biosensor in addition to the detection limit is pointed out. Finally, the model is applied to curves that are typically obtained in immunoassays and a discussion is made on the application validity of the model and its limitations.

Resonant nanopillars as label-free optical biosensors

In recent works it has been demonstrated the suitability of using resonant nanopillars (R-NPs) as biochemical. In this work it has been shown the capability of the R-NPs to behave as label-free multiplexed biological sensors. Each R-NP is formed by silicon oxide (SiO2) and silicon nitride (Si3N4) Bragg reflectors and a central cavity of SiO2, and they are grouped into eight arrays called BICELLs, which are distributed on a single chip of quartz substrate for multiplexing measurements. For the biological sensing assessment it was developed an immunoassay on the eight single BICELLs. The biofunctionalization process was performed by a silanization protocol based on 3-aminopropyltrymethoxysilane (APTMS) and glutaradheyde (GA) as a linker between APTMS and the IgG which acted as biorreceptor for the anti-IgG recognition. In this work, there were compared two forms of immobilization: on one hand by incubating the R-NPs under static drop of 50 μg/mL and on the second hand by introducing the sensing chip in a flow cell with a continuous flow of the same concentration of IgG. The eight arrays of R-NPs or BICELLs were independently optically interrogated by a bundle of fiber connected to a spectrometer. The multiplexing analysis showed reproducibility among the BICELLs, suggesting the potentially of using R-NPs for multiplexed biosensors. Performance in the immobilization process apparently does not have a signification effect. However the election of one method or another should be a commitment between time and resources.

Development towards compact nitrocellulose interferometric biochips for dry eye diagnosis based on MMP9, S100A6 and CST4 biomarkers using a Point-of-Care device

A novel compact optical biochip based on a thin layer-sensing BICELL surface of nitrocellulose is used for in-situ labelfree detection of dry eye disease (DED). In this work the development of a compact biosensor that allows obtaining quantitative diagnosis with a limited volume of sample is reported. The designed sensors can be analyzed with an optical integrated Point-of-Care read-out system based on the “Increase Relative Optical Power” principle which enhances the performance and Limit of Detection. Several proteins involved with dry eye dysfunction have been validated as biomarkers. Presented biochip analyzes three of those biomarkers: MMP9, S100A6 and CST4. BICELLs based on nitrocellulose permit to immobilize antibodies for each biomarker recognition. The optical response obtained from the biosensor through the readout platform is capable to recognize specifically the desired proteins in the concentrations range for control eye (CE) and dry eye syndrome (DES). Preliminary results obtained will allow the development of a dry eye detection device useful in the area of ophthalmology and applicable to other possible diseases related to the eye dysfunction.

Performance evaluation for different sensing surface of BICELLs bio-transducers for dry eye biomarkers

Biophotonic Sensing Cells (BICELLs) are demonstrated to be an efficient technology for label-free biosensing and in concrete for evaluating dry eye diseases. The main advantage of BICELLs is its capability to be used by dropping directly a tear into the sensing surface without the need of complex microfluidics systems. Among this advantage, compact Point of Care read-out device is employed with the capability of evaluating different types of BICELLs packaged on Biochip-Kits that can be fabricated by using different sensing surfaces material. In this paper, we evaluate the performance of the combination of three sensing surface materials: (3-Glycidyloxypropyl) trimethoxysilane (GPTMS), SU-8 resist and Nitrocellulose (NC) for two different biomarkers relevant for dry eye diseases: PRDX-5 and ANXA-11.