Arkadiusz Miazek

Neurodegenerative disorders manifest with markedly different phenotypes, with motor- and memory impairments dominating both clinical and preclinical phenotyping efforts. However, neurodegeneration in unlikely to be confined to these two functional domains alone, and its broader behavioral consequences, including social alterations remain underexplored. Here, we present a non-invasive, high-content home-cage phenotyping framework to investigate social and exploratory dysfunction in SPTAN1 R1098Q mice, a model of early infantile epileptic encephalopathy (EIEE5). Our pipeline integrates three open-source AI engines: SLEAP.ai for markerless multi-animal pose estimation, DeepOF for automated classification of autonomous and social behaviors, and SAM2 for segmentation and quantification of nest-building material usage. Long-term monitoring revealed that SPTAN1 R1098Q mutants exhibit impaired spatial habituation in mutant-mutant (MUT - MUT) pairs. Mutants also displayed persistent, unreciprocated trailing (following) of cage mates and a pronounced reduction of nest-building behavior alongside. Notably, co-housing with a wild-type littermate (MUT - WT) attenuated mutant hyperactivity and restored more typical rest pattern, suggesting that social context can partially buffer these behavioral abnormalities. By revealing social and exploratory alterations that expand conventional phenotyping, this computational ethology pipeline advances 3R principles while revealing that intrinsic executive state-switching failures in neurodegenerative models can be functionally buffered by social dynamics.

Background/Objectives: Canine Diffuse Large B-cell Lymphoma (cDLBCL) is characterized by a high prevalence of MHC II DR (DLA-DR) antigen overexpression. Murine anti-pan-DLA-DR monoclonal antibodies (mAbs) B5 and E11 have been previously observed to promote death of cDLBCL cells in vitro and in vivo. Consequently, DLA-DR antigens are considered a prospective target for passive immunotherapy aside from CD20. While infusion of anti-pan MHC II mAbs has demonstrated tumor suppression in cDLBCL xenografted immunodeficient mice, the relative contributions of direct cellular versus immune-mediated mechanisms to this therapeutic effect remain undefined. This study aimed to dissect these potential mechanisms of mAb E11. Methods: Canine lymphoma and leukemia cell lines CLBL1 and CLB70 were incubated with full E11 antibody or its F(ab′)2 and Fab fragments and cell viability was assessed with sub-G1 assay then, NOD-SCID mice were xenotransplanted with 1.5 × 10⁷ canine CLBL1 cells expressing nanoluciferase and were infused either with mAb E11 or its fragments, each at 1 mg/kg body mass, twice weekly for three consecutive weeks. Tumor burden was monitored by assessing body weight, nanoluciferase activity in blood, and by flow cytometric analyses of bone marrow tumor cell content. Time to tumor progression (TTP) was calculated based on weight loss and luminescence measurements. Results: We observed cytotoxic activity of monovalent E11-Fab fragments in vitro and in vivo. The mean TTP for mice treated with irrelevant mouse IgG antibodies was 9.8 ± 4.65 days. In contrast, treatment with E11 Fab fragments resulted in a TTP of 19.1 ± 2.67 days, which was similar to that achieved with the full E11 mAb (19.5 ± 1.73 days) and E11 F(ab′)2 fragments (18.1 ± 2.9 days). Conclusions: Our findings demonstrate a potent antibody cytotoxicity mechanism that operates in vivo and is independent of cell surface MHC II crosslinking or Fc engagement. These data support the promising potential of E11-Fab fragments for further clinical development as a therapeutic agent in canine lymphoma.

Background/Objectives: Canine Diffuse Large B-cell Lymphoma (cDLBCL) is characterized by a high prevalence of MHC II DR (DLA-DR) antigen overexpression. Murine anti-pan-DLA-DR monoclonal antibodies (mAbs) B5 and E11 have been previously shown to induce cDLBCL cell death in vitro and in vivo. Consequently, DLA-DR antigens are considered a promising alternative target to CD20 for passive immunotherapy. While infusion of anti-pan MHC II mAbs has demonstrated tumor suppression in cDLBCL xenografted immunodeficient mice, the relative contributions of direct cellular versus immune-mediated mechanisms to this therapeutic effect remain undefined. This study aimed to dissect these potential mechanisms of mAb E11.Methods: Canine lymphoma and leukemia cell lines CLBL1 and CLB70 were incubated with full E11 antibody or its F(ab’)2 and Fab fragments and cell viability was assessed with sub-G1 assay then, NOD-SCID mice were xenotransplanted with 15×106 canine CLBL1 cells expressing nanoluciferase and were infused either with mAb E11 or its fragments, each at 1 mg/kg body mass, twice weekly for three consecutive weeks. Tumor burden was monitored by assessing body weight, nanoluciferase activity in blood, and by flow cytometric analyses of bone marrow tumor cell content. Time to tumor progression (TTP) was calculated based on weight loss and luminescence measurements. Results: We observed cytotoxic activity of monovalent E11-Fab fragments in vitro and in vivo. The mean TTP for mice treated with irrelevant mouse IgG antibodies was 9.8±4.65 days. In contrast, treatment with E11 Fab fragments resulted in a TTP of 19.1±2.67 days, which was similar to that achieved with the full E11 mAb (19.5±1.73 days) and E11 F(ab')2 fragments (18.1±2.9 days). Conclusions: Our findings demonstrate a potent antibody cytotoxicity mechanism that operates in vivo and is independent of cell surface MHC II crosslinking or Fc engagement. These data support the promising potential of E11-Fab fragments for further clinical development as a therapeutic agent in canine lymphoma.

The subtle sequence diversity and mutually exclusive expression patterns of T cell receptor beta chain constant genes, TRBC1 and TRBC2 , in mature human T cells, provide the basis for immune‐targeting strategies designed to eliminate clonally expanded malignant T cells while sparing a subset of normal T cells capable of maintaining immunocompetence. The evolutionarily conserved gene arrangement and regulation of TRBC loci in mammals make these genes attractive targets for translational immune‐targeting strategies in companion species, including dogs. However, available TRBC sequence data relevant to common dog breeds remains limited. In this study, we investigated the sequence diversity and mRNA expression profiles of canine TRBC1 and TRBC2 genes in peripheral blood mononuclear cell (PBMC) samples representing 14 different dog breeds, and in six established canine haematopoietic cell lines of both T‐cell and non‐T‐cell origin (i.e., B and NK cell lines). Our analysis uncovered a previously unreported variation in the TRBC1 sequence encoding the transmembrane region but found no sequence diversity in the extracellular domain of TRBC1 and TRBC2. A nearly equal mRNA expression of TRBC1 and TRBC2 was consistently observed in bulk samples of canine PBMCs across all breeds, in contrast to canine cell lines, which exhibited a more skewed expression profile. Unexpectedly, germline mRNA expression of TRBC was present in some (i.e., CLB70, GL1) but not other (i.e., CLBL1) canine cell lines of B cell origin. In conclusion, our findings indicate that the fully conserved amino acid sequence in the extracellular domain of canine TCR beta chain variants presents a challenge for the development of differential therapeutic antibodies. Additionally, the presence of germline TRBC transcripts in certain canine B‐cell neoplasms, but not others, may provide additional insights into the developmental stages from which these neoplasms originate.

This paper describes the effects of murine norovirus (MNV) infection on oxidative stress and histopathological changes in mice. This study uses histopathological assays, enzymatic and non-enzymatic antioxidant markers, and total oxidative status and capacity (TOS, TAC). The results suggest that MNV infection can lead to significant changes with respect to the above-mentioned parameters in various organs. Specifically, reduced superoxide dismutase (SOD), Mn superoxide dismutase (MnSOD), catalase (CAT), and glutathione reductase (GR) activities were observed in liver tissues, while higher MnSOD activity was observed in kidney tissues of MNV-infected mice when compared to the control. GR activity was lower in all tissues of MNV-infected mice tested, with the exception of lung tissue. This study also showed that norovirus infection led to increased TOS levels in the brain and liver and TAC levels in the brain, while TOS levels were significantly reduced in the kidneys. These changes may be due to the production of reactive oxygen species (ROS) caused by the viral infection. ROS can damage cells and contribute to oxidative stress. These studies help us to understand the pathogenesis of MNV infection and its potential effects on oxidative stress and histopathological changes in mice, and pave the way for further studies of the long-term effects of MNV infection.

Background/Aim: c-MYC promoter binding protein (MBP-1) is a product of alternatively translated mRNA encoding alpha-enolase (ENO1). In contrast to ENO1, MBP-1 possesses no enzymatic activity but acts as a transcriptional repressor of c-MYC. Ectopic over-expression of MBP-1 in tumor cells was shown to reduce cell proliferation and tumorigenicity, thus making it an attractive target for anticancer strategies. This study aimed to assess the effects of MBP-1 over-expression on human cutaneous melanoma cell lines. Materials and Methods: We overexpressed the full-length MBP-1 or its C-terminal truncated variant (MBP-1ΔC), in two human melanoma cell lines (A375, WM9) and assessed their subcellular localization. qPCR was then used to quantitate c-MYC transcription. Further, 5-ethynyl-2′-deoxyuridine incorporation assay was used to measure cell proliferation and a lactate assay was performed to measure the glycolysis rate of cells in normoxia and hypoxia. Finally, an in vitro wound-healing assay was performed to evaluate cell migration. Results: The overexpressed MBP-1 variants predominantly localized in the cytoplasm and barely decreased c-MYC expression. Unexpectedly, the proliferation rate of MBP-1- transduced cells increased in comparison to controls, as did the rate of glucose metabolism in hypoxia. Furthermore, over-expression of MBP-1, but not MBP-1ΔC, led to a substantial decrease in the cell migration capacity of metastatic WM9 cells but not A375 cells from the primary tumor lesion. Conclusion: Misslocalization of over-expressed MBP-1 in the cytoplasm of two melanoma cell lines resulted in an unexpected tumor promoting activity by increasing cell proliferation and glycolysis rates in hypoxia.

SPTAN1 spectrinopathies refer to a group of rare, inherited diseases associated with damage to non-erythrocytic α-II spectrin (α-II). They are linked to a range of mild to severe neuropathologies of the central and peripheral nervous systems, such as early infantile epileptic encephalopathy type 5, cerebellar ataxia, inherited peripheral neuropathy, and spastic paraplegia. Modeling human SPTAN1 encephalopathies in laboratory animals has been challenging partially because no haploinsufficiency-related phenotypes unfold in heterozygous Spna2 deficient mice nor stable transgenic lines of mice mimicking missense human SPTAN1 mutations have been created to date. Here, we assess the motor and memory performance of a dominant-negative murine Spna2 (SPTAN1) variant carrying a spontaneous point mutation replacing an arginine 1098 in the repeat 10th of α-II with the glutamine (R1098Q). By comparing groups of heterozygous R1098Q mice at different ages, we find evidence for progressive ataxia, and age-related deterioration of motor performance and muscle strength. We also document stress-induced, long-lasting seizure episodes of R1098Q mice and their poor performance in novel object recognition memory tests. Overall, we propose that the complexity of neuropathology-related phenotypes presented by the R1098Q mice recapitulates a number of symptoms observed in human patients carrying SPTAN1 mutations affecting α-II scaffold stability. This makes the R1098Q mice a valuable animal model for preclinical research.

Alpha-enolase (ENO1) is a glycolytic metalloenzyme, and its overexpression occurs in numerous cancers, contributing to cancer cell survival, proliferation, and maintenance of the Warburg effect. Patients with an overexpression of ENO1 have a poor prognosis. The aim of the present study was to investigate the prognostic significance of ENO1 in surgical resections from 112 melanoma patients and to assess its expression and enzymatic activity in normoxia and hypoxia in several melanoma cell lines. Overexpression of ENO1 in tumor cells from patients was correlated with unfavorable prognosticators such as Breslow thickness, Clark level, mitotic activity, and the presence of ulceration. The expression of ENO1 also positively correlated with a greater thickness of the neoplastic infiltrate and a worse long-term prognosis for patients with cutaneous melanoma. We report significantly higher expression of ENO1 in melanoma cell lines in comparison to normal melanocytes. To conclude, our in vitro and clinical models showed that overexpression of ENO1 promotes invasiveness of melanoma cells and correlates with aggressive clinical behavior. These observations open the way to further search of a potential prognostic and therapeutic target in cutaneous melanoma.

Intracellular signaling through the T cell receptor (TCR) is essential for T cell development and function. Proper TCR signaling requires the sequential activities of Lck and ZAP-70 kinases, which result in the phosphorylation of tyrosine residues located in the CD3 ITAMs and the LAT adaptor, respectively. LAT, linker for the activation of T cells, is a transmembrane adaptor protein that acts as a scaffold coupling the early signals coming from the TCR with downstream signaling pathways leading to cellular responses. The leukemic T cell line Jurkat and its derivative mutants J.CaM1.6 (Lck deficient) and J.CaM2 (LAT deficient) have been widely used to study the first signaling events upon TCR triggering. In this work, we describe the loss of LAT adaptor expression found in a subline of J.CaM1.6 cells and analyze cis-elements responsible for the LAT expression defect. This new cell subline, which we have called J.CaM1.7, can re-express LAT adaptor after Protein Kinase C (PKC) activation, which suggests that activation-induced LAT expression is not affected in this new cell subline. Contrary to J.CaM1.6 cells, re-expression of Lck in J.CaM1.7 cells was not sufficient to recover TCR-associated signals, and both LAT and Lck had to be introduced to recover activatory intracellular signals triggered after CD3 crosslinking. Overall, our work shows that the new LAT negative J.CaM1.7 cell subline could represent a new model to study the functions of the tyrosine kinase Lck and the LAT adaptor in TCR signaling, and their mutual interaction, which seems to constitute an essential early signaling event associated with the TCR/CD3 complex.