A comparison of alternative assays to measure DNA damage in stallion spermatozoa: TUNEL test versus ‘Nicoletti assay’

The aberrations of sperm DNA may cause various problems and have negative consequences on fertility. These influence embryonic development or might lead to early embryo loss. Sperm Chromatin Structure Assay (SCSA) is the flow cytometric method most often used for the detection of DNA lesions; however, some studies using that method reached confusing conclusions. The aim of this pilot study was to adjust and compare two alternative tests, namely the TUNEL test and the Nicoletti assay. The above-mentioned two flow cytometric methods capable of detecting the fragmented DNA of sperm were tested on 12 frozen-thawed stallion semen samples. The TUNEL test demonstrated much higher DNA fragmentation ratio than the Nicoletti assay (mean ± SD: 30.77 ± 13.03% vs. 1.93 ± 0.89%, respectively). A fluorescent microscopic check of the samples showed that TUNEL labelled the plasma membrane and the mitochondria in a nonspecific way, rather than detecting only the fragmented DNA, thus eventually resulting in a false positive sign. The Nicoletti assay is simpler, quicker and does not detect nonspecific binding; however, further analyses are required to determine its diagnostic value

A bispecific T cell engager recruits both type 1 NKT and Vy9V52-T cells for the treatment of CD1d-expressing hematological malignancies

Bispecific T cell engagers (bsTCEs) hold great promise for cancer treatment but face challenges due to the induction of cytokine release syndrome (CRS), on-target off-tumor toxicity, and the engagement of immuno-suppressive regulatory T cells that limit efficacy. The development of Vy9V52-T cell engagers may overcome these challenges by combining high therapeutic efficacy with limited toxicity. By linking a CD1d-specific single-domain antibody (VHH) to a V52-TCR-specific VHH, we create a bsTCE with trispecific properties, which engages not only Vy9V52-T cells but also type 1 NKT cells to CD1d+ tumors and triggers robust proin-flammatory cytokine production, effector cell expansion, and target cell lysis in vitro. We show that CD1d is expressed by the majority of patient MM, (myelo)monocytic AML, and CLL cells and that the bsTCE triggers type 1 NKT and Vy9V52-T cell-mediated antitumor activity against these patient tumor cells and improves survival in in vivo AML, MM, and T-ALL mouse models. Evaluation of a surrogate CD1d-y5 bsTCE in NHPs shows Vy9V52-T cell engagement and excellent tolerability. Based on these results, CD1d-V52 bsTCE (LAVA-051) is now evaluated in a phase 1/2a study in patients with therapy refractory CLL, MM, or AML.Transplantation and autoimmunit

IFN-Stimulated Gene 15 Is an Alarmin that Boosts the CTL Response via an Innate, NK Cell-Dependent Route

Type I IFN is produced upon infection and tissue damage and induces the expression of many IFN-stimulated genes (ISGs) that encode host-protective proteins. ISG15 is a ubiquitin-like molecule that can be conjugated to proteins but is also released from cells in a free form. Free, extracellular ISG15 is suggested to have an immune-regulatory role, based on disease phenotypes of ISG15-deficient humans and mice. However, the underlying mechanisms by which free ISG15 would act as a "cytokine" are unclear and much debated. We, in this study, demonstrate in a clinically relevant mouse model of therapeutic vaccination that free ISG15 is an alarmin that induces tissue alert, characterized by extracellular matrix remodeling, myeloid cell infiltration, and inflammation. Moreover, free ISG15 is a potent adjuvant for the CTL response. ISG15 produced at the vaccination site promoted the vaccine-specific CTL response by enhancing expansion, short-lived effector and effector/memory differentiation of CD8(+) T cells. The function of free ISG15 as an extracellular ligand was demonstrated, because the equivalents in murine ISG15 of 2 aa recently implicated in binding of human ISG15 to LFA-1 in vitro were required for its adjuvant effect in vivo. Moreover, in further agreement with the in vitro findings on human cells, free ISG15 boosted the CTL response in vivo via NK cells in the absence of CD4(+) T cell help. Thus, free ISG15 is part of a newly recognized innate route to promote the CTL response.Tumorimmunolog

Radiotherapy and Cisplatin Increase Immunotherapy Efficacy by Enabling Local and Systemic Intratumoral T-cell Activity

Contains fulltext : 202601.pdf (publisher's version ) (Closed access

Barley starch

This thesis examined barley amylopectin structure and looked for correlations between the structure and physical properties of starch. The structure of amylopectin and gelatinisation and retrogradation of starch were studied in 10 different barley cultivars/breeding lines with differing genetic background. Amylopectin is built up of thousands of chains of glucose monomers, organised into clusters. The detailed fine structure of amylopectin was studied by isolating clusters of amylopectin and their building blocks, which are the tightly branched units building up the clusters. Barley cultivars/breeding lines possessing the amo1 mutation had fewer long chains of DP≥38 in amylopectin and more large building blocks. The structure of building blocks was rather conserved between the different barley cultivars/breeding lines studied and was categorized into different size groups. These different building blocks were shown to be randomly distributed in the amylopectin molecule. The C-chains in amylopectin can be of any length and are a category of chains different from the B-chains. The backbone in amylopectin consists of a special type of B-chains which, when cleaved by α-amylase, become chains of a similar type to C-chains. Gelatinisation and retrogradation (recrystallisation of gelatinised starch) of barley starch was studied by differential scanning calorimetry. The amo1 mutation resulted in a broader gelatinisation temperature range and a higher enthalpy of retrogradation. Other structural features were also found to influence the physical properties of starch. Small clusters and denser structure of the building blocks resulted in higher gelatinisation temperature. Fast retrogradation was observed in barley which had amylopectin with shorter chains and many large building blocks consisting of many chains. Amylopectin structure was also studied in developing barley kernels. Three barley cultivars/breeding lines were grown in a phytotron and kernels were harvested at 9, 12 and 24 days after flowering. The results showed that amylopectin synthesized at later stages of development had a more tightly branched structure. Expression of the enzymes involved in starch biosynthesis is also known to change during endosperm development