Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells

The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.     

Online determination of sulfide using an optical immersion probe combined with headspace liquid-phase microextraction

A new design for headspace liquid phase microextraction in combination with an optical immersion probe (HS-LPME-OIP) was proposed and successfully tested for the determination of sulfide in wine and water samples. The developed method is based on the release of hydrogen sulfide from the aqueous phase after the addition of orthophosphoric acid and its extraction with an aqueous solution of 5,5′-dithiobis-(2-nitrobenzoic) acid (DTNB). The analytical signal was recorded using an optical probe immersed in a vial containing 200 μL of 0.1 mM DTNB solution. Using the optical immersion probe in combination with HS-LPME allowed to register the analytical signal online and significantly improve the reproducibility of sulfide determination compared to known microextraction approaches. In the proposed approach, the problems with drop stability, limitations in mixing rate or extraction time, too small volume of the acceptor phase and stability of the holding the acceptor phase in the hole of the optical probe were also satisfactorily solved. The calibration graph was linear in the range of 16–256 μg L⁻¹ with a correlation coefficient of 0.9992. The limit of detection was 6 μg L⁻¹.

A new design for headspace liquid phase microextraction combined with an optical probe.     

A Feasibility Study of High-Entropy Alloy Coating Deposition by Detonation Spraying Combined with Laser Melting

In this work, a new two-stage approach to the deposition of high-entropy alloy coatings is proposed. At the first stage, a composite precursor coating is formed by detonation spraying of the metal powder mixtures. At the second stage, the precursor coating is re-melted by a laser, and the formation of multi-component solid solution phases can be expected upon solidification. The feasibility of the proposed approach was validated using three different mixtures of Fe, Ni, Cu, Co and Al powders. It was shown that detonation spraying allows forming composite coatings with a uniform distribution of the lamellae of different metals. The results of the structural analysis of the laser-treated coatings suggest that complete alloying occurred in the melt and face-centered cubic solid solutions formed in the coatings upon cooling.     

Impaired expression of perforin and granulysin in CD8+ T cells at the site of infection in human chronic pulmonary tuberculosis

Protective immunity in tuberculosis is dependent on the coordinated release of cytolytic effector molecules from effector T cells and the subsequent granule-associated killing of infected target cells. In this study, we investigated the expression of cytolytic (perforin and granzyme A) and antimicrobial (granulysin) molecules at the single-cell level in cryopreserved lung tissue from patients with chronic, progressive tuberculosis disease. Quantification of protein-expressing cells was performed by in situ imaging, while mRNA levels in the infected tissue were analyzed by real-time PCR. Persistent inflammation, including excessive expression of inducible nitric oxide synthase in CD68+ macrophages and significant infiltration of CD3+, CD8+ and CD4+ T cells, was evident in tuberculosis lesions in all patients. However, despite the accumulation of CD3+ T cells, perforin- and granulysin-expressing CD3+ T cells were detected at two- to threefold-lower ratios in the tuberculosis lesions than in distal lung parenchyma and uninfected control lungs, respectively. This was evident at both the protein and mRNA levels. Moreover, perforin- and granulysin-expressing CD8+ T cells were scarce in individual granulomas within the tuberculosis lesions. In contrast, significant up-regulation of granzyme A-expressing CD3+ T cells was evident in the lesions from all patients. Confocal microscopy revealed coexpression of perforin and granulysin, primarily in CD8+ T cells; however, this expression was lower in the tuberculosis lesions. These findings suggest that symptomatic, chronic tuberculosis disease is associated with insufficient up-regulation of perforin and granulysin coexpression in CD8+ T cells at the local site of infection.     

Development of overt hemolytic anemia after splenectomy for thrombocytopenia in Evans syndrome with negative Coombs test

A 69-year-old man was diagnosed as having idiopathic thrombocytopenic purpura (ITP) in April 2000, and treated with prednisolone (PSL) without effect. Splenectomy performed in June 2000 had only a transient and marginal influence on his platelet count. Two months later, he developed autoimmune hemolytic anemia (AIHA) without Coombs test positivity, and his diagnosis was changed to Coombs-negative Evans syndrome. Treatment with PSL led to recovery of his hemoglobin level, but not his platelet count. Although the mechanism responsible for development of AIHA after splenectomy in this patient with ITP remains unknown, close observation is required for any association with other autoimmune diseases such as SLE.     

Delaying haematopoietic stem cell transplantation in children with viral respiratory infections reduces transplant-related mortality

Viral respiratory infections (VRIs) contribute to the morbidity and transplant-related mortality (TRM) after allogeneic haematopoietic stem cell transplantation (HSCT) and strategies to prevent and treat VRIs are warranted. We monitored VRIs before and after transplant in children undergoing allogeneic HSCT with nasopharyngeal aspirates (NPA) and assessed the impact on clinical outcome. Between 2007 and 2017, 585 children underwent 620 allogeneic HSCT procedures. Out of 75 patients with a positive NPA screen (12%), transplant was delayed in 25 cases (33%), while 53 children started conditioning with a VRI. Patients undergoing HSCT with a positive NPA screen had a significantly lower overall survival (54% vs. 79%) and increased TRM (26% vs. 7%) compared to patients with a negative NPA. Patients with a positive NPA who delayed transplant and cleared the virus before conditioning had improved overall survival (90%) and lower TRM (5%). Pre-HSCT positive NPA was the only significant risk factor for progression to a lower respiratory tract infection and was a major risk factor for TRM. Transplant delay, whenever feasible, in case of a positive NPA screen for VRIs can positively impact on survival of children undergoing HSCT.     

Interaction of Interoceptive Perception and Emotional Intelligence: A Functional Neuroimaging Study

Neuroscience and Behavioral Physiology - Impairments to nociception are currently regarded as the pathogenetic mechanism of psychosomatic disorders and as a potential therapeutic target. An aim of...     

Evaluation of the ERIC-PCR as a probable method to differentiate Avibacterium paragallinarum serovars

Infectious coryza, an upper respiratory tract disease in chickens, caused by Avibacterium paragallinarum, leads to huge economic losses. The disease is controlled through vaccination; but vaccination efficacy is dependent on correct identification of the infecting serovar, as limited cross-protection is reported amongst some serovars. Current identification methods include the heamagglutination inhibition test, which is demanding and could be subjective. To overcome this, molecular typing methods proposed are the Multiplex polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism-PCR, but low reproducibility is reported. Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR has been suggested for molecular groupings of various bacterial species. This study focuses on evaluating the ERIC-PCR as a probable method to differentiate between different Av. paragallinarum serovars by grouping with reference isolates, based on clonal relations. The ERIC-PCR was performed on 12 reference isolates and 41 field isolates originating from South Africa and South America. The data indicate that the ERIC-PCR is not ideal for the differentiation or for molecular typing of Av. paragallinarum serovars, as no correlation is drawn upon comparison of banding patterns of field isolates and reference strains. However, the results do indicate isolates from the same origin sharing unique banding patterns, indicating potential clonal relationship; but when compared to the reference isolates dominant in the specific area, no correlation could be drawn. Furthermore, although the ERIC-PCR serves a purpose in epidemiological studies, it has proved to have little application in differentiating amongst serovars of Av. paragallinarum and to group untyped field strains with known reference strains.