Search Results (61)

Background: In the adaptive immune response of the dromedary (Camelus dromedarius, Camdro), the T cell receptor (TR) repertoire of the gamma–delta (γδ) T cells is unusually diversified both by somatic hypermutation in rearranged TR gamma (TRG) and delta (TRD) genes and by the diversity in sequence and length of the third complementarity-determining region (CDR3) of the TRD chain. Methods: The purpose was to investigate, in the absence of 3D structures, the role of Camdro γδ T cells, focusing on the binding interactions at the interface between the V-gamma and V-delta domains, and in complex with the CD1D, a major histocompatibily class I (MH1)-like glycoprotein presenting lipid antigen in association with B2M. A combination of hypermutated TRG dromedary cDNA clones was paired with TRD clones bearing very long, long, or short CDR3s, all isolated from the spleen of a single animal. Results: The 3D models of the Camdro TRG_TRD/CD1D_B2M complexes were inferred using the Homo sapiens 3D structure and the ImMunoGeneTics (IMGT) numbering for V, C, and G domains, and investigated for binding interactions at the interface of the paired V-gamma_V-delta and at the interface with CD1D. Our results suggest that transcripts with long CDR3s may derive from a population of CD1D-restricted γδ T cells. Both the CD1D G-alpha1-like and G-alpha-2 like domain helices were contacted by both the V-gamma and V-delta CDR-IMGT loops. Conclusions: Our findings further emphasize the similarity between the γδ T cells population we analyzed in Camelus dromedarius and the CD1D-restricted γδ NKT cells in Homo sapiens. Full article

The poor prognosis for high-grade serous ovarian cancer (HGSOC), the dominant subtype of ovarian cancer, reflects its aggressive nature, late diagnosis, and the highest mortality rate among all gynaecologic cancers. Apart from late diagnosis, the main reason for the poor prognosis and its unsuccessful treatment is primarily the emergence of chemoresistance to carboplatin. Although there is a good response to primary treatment, the disease recurs in 80% of cases, at which point it is largely resistant to carboplatin. The introduction of novel targeted therapies in the second decade of the 21st century has begun to transform the treatment of HGSOC, although their impact on overall survival remains unsatisfactory. Targeting the specific pathways known to be abnormally activated in HGSOC is especially difficult due to the molecular diversity of its subtypes. Moreover, a range of molecular changes are associated with acquired chemoresistance, e.g., reversion of BRCA1 and BRCA2 germline alleles. In this review, we examine the advantages and disadvantages of approved targeted therapies, including bevacizumab, PARP inhibitors (PARPis), and treatments targeting cells with neurotrophic tyrosine receptor kinase (NTRK), B-rapidly accelerated fibrosarcoma (BRAF), and rearranged during transfection (RET) gene alterations, as well as antibody–drug conjugates. Additionally, we explore promising new targets under investigation in ongoing clinical trials, such as immune checkpoint inhibitors, anti-angiogenic agents, phosphatidylinositol-3-kinase (PI3K) inhibitors, Wee1 kinase inhibitors, and ataxia telangiectasia and Rad3-related protein (ATR) inhibitors for platinum-resistant disease. Despite the development of new targeted therapies, carboplatin remains the fundamental medicine in HGSOC therapy. The correct choice of treatment strategy for better survival of patients with advanced HGSOC should therefore include a prediction of patients’ risks of developing chemoresistance to platinum-based chemotherapy. Moreover, effective targeted therapy requires the selection of patients who are likely to derive clinical benefit while minimizing potential adverse effects, underscoring the essence of precision medicine. Full article

Recent discoveries revealed mechanistic insights into the control of adipogenesis by the Constitutive Photomorphogenesis 9 Signalosome (CSN) and its variants, CSN^CSN7A and CSN^CSN7B, which differ in the paralog subunits, CSN7A and CSN7B. CSN^CSN7A and CSN^CSN7B variants form permanent complexes with cullin-RING-ubiquitin ligases 3 and 4A (CRL3 and CRL4A), respectively. These complexes can be found in most eukaryotic cells and represent a critical reservoir for cellular functions. In an early stage of adipogenesis, mitotic clonal expansion (MCE), CSN-CRL1, and CSN^CSN7B-CRL4A are blocked to ubiquitinate the cell cycle inhibitor p27^KIP, leading to cell cycle arrest. In addition, in MCE CSN-CRL complexes rearrange the cytoskeleton for adipogenic differentiation and CRL3^KEAP1 ubiquitylates the inhibitor of adipogenesis C/EBP homologous protein (CHOP) for degradation by the 26S proteasome, an adipogenesis-specific proteolysis. During terminal adipocyte differentiation, the CSN^CSN7A-CRL3 complex is recruited to a lipid droplet (LD) membrane by RAB18. Currently, the configuration of the substrate receptors of CSN^CSN7A-CRL3 on LDs is unclear. CSN^CSN7A-CRL3 is activated by neddylation on the LD membrane, an essential adipogenic step. Damage to CSN/CUL3/CUL4A genes is associated with diverse diseases, including obesity. Due to the tremendous impact of CSN-CRLs on adipogenesis, we need strategies for adequate treatment in the event of malfunctions. Full article

The carotid body (CB), the main peripheral arterial chemoreceptor, exhibits considerable structural and neurochemical plasticity in response to pathological conditions such as high blood pressure. Previous studies have shown that morphological alterations in the hypertensive CB are characterized by enlarged parenchyma due to cellular hypertrophy and hyperplasia, and vasodilation. To test whether hypertension can also induce neoangiogenesis and modulate its chemosensory function, we examined the immunohistochemical expression of two angiogenic factors, vascular endothelial growth factor (VEGF) and endothelin-1 (ET), and their corresponding receptors in the CB of adult spontaneously hypertensive rats (SHRs), and compared their expression patterns to that of age-matched normotensive Wistar rats (NWR). We found an increased VEGF-A and B, and VEGFR-2 expression in glomus and endothelial cells in the enlarged CB glomeruli of SHRs compared with that in NWR. Conversely, weaker immunoreactivity to VEGFR-1 was detected in cell clusters of the hypertensive CB. The expression of endothelin-converting enzyme 1 and its receptor ETA was higher in a subset of glomus cells in the normotensive CB, while the immunoreactivity to the ETB receptor was enhanced in endothelial cells of CB blood vessels in SHRs. The elevated endothelial expression of VEGF and ET-1 suggests their role as local vascular remodeling factors in the adaptation to hypertension, though their involvement in the cellular rearrangement and modulation of chemosensory function could also be implied. Full article

Amyloid-β peptide (Aβ) is a critical cause of Alzheimer’s disease (AD). It is generated from amyloid precursor protein (APP) through cleavages by β-secretase and γ-secretase. γ-Secretase, which includes presenilin, is regulated by several stimuli. Tau protein has also been identified as a significant factor in AD. In particular, Tau phosphorylation is crucial for neuronal impairment, as phosphorylated Tau detaches from microtubules, leading to the formation of neurofibrillary tangles and the destabilization of the microtubule structure. This instability in microtubules damages axons and dendrites, resulting in neuronal impairment. Notably, Aβ is linked to Tau phosphorylation. Another crucial factor in AD is neuroinflammation, primarily occurring in the microglia. Microglia possess several receptors that bind with Aβ, triggering the expression and release of an inflammatory factor, although their main physiological function is to phagocytose debris and pathogens in the brain. NF-κB activation plays a major role in neuroinflammation. Additionally, the production of reactive oxygen species (ROS) in the microglia contributes to this neuroinflammation. In microglia, superoxide is produced through NADPH oxidase, specifically NOX2. Rho GTPases play an essential role in regulating various cellular processes, including cytoskeletal rearrangement, morphology changes, migration, and transcription. The typical function of Rho GTPases involves regulating actin filament formation. Neurons, with their complex processes and synapse connections, rely on cytoskeletal dynamics for structural support. Other brain cells, such as astrocytes, microglia, and oligodendrocytes, also depend on specific cytoskeletal structures to maintain their unique cellular architectures. Thus, the aberrant regulation of Rho GTPases activity can disrupt actin filaments, leading to altered cell morphology, including changes in neuronal processes and synapses, and potentially contributing to brain diseases such as AD. Full article

Flow cytometric (FC) immunophenotyping and T-cell receptor (TCR) gene rearrangement studies are essential ancillary methods for the characterisation of T-cell lymphomas. Traditional manual gating and polymerase chain reaction (PCR)-based analyses can be labour-intensive, operator-dependent, and have limitations in terms of sensitivity and specificity. The objective of our study was to investigate the efficacy of the Phenograph and t-SNE algorithms together with an antibody specific for the TCR β-chain constant region 1 (TRBC1) to identify monoclonal T-cell populations. FC- and PCR-based clonality analyses were performed on 275 samples of T-cell lymphomas, B-cell lymphomas, and reactive lymphocytic proliferations. Monotypic T-cell populations were identified in 65.1% of samples by manual gating and 72.4% by algorithm-driven analysis, while PCR-based analysis detected clonal T cells in 68.0%. Of the 262 monotypic populations identified, 46.6% were classified as T-cell lymphomas and 53.4% as T-cell populations of uncertain significance (T-CUS). Algorithm-driven gating identified monotypic populations that were overlooked by manual gating or PCR-based methods. The study highlights the difficulty in distinguishing monotypic populations as T-cell lymphoma or T-CUS. Further research is needed to establish criteria for distinguishing between these populations and to improve FC diagnostic accuracy. Full article

The immune repertoire (IR) is a term that defines the combined unique genetic rearrangements of antigen receptors expressed by B and T lymphocytes. The IR determines the ability of the immune system to identify and respond to foreign antigens while preserving tolerance to host antigens. When immune tolerance is disrupted, development of autoimmune diseases can occur due to the attack of self-antigens. Recent technical advances in immune profiling allowed identification of common patterns and shared antigen-binding sequences unique to diverse array of diseases. However, there is no current literature to date evaluates IR findings in autoimmune and skin inflammatory conditions. In this review, we provide an overview of the past and current research findings of IR in various autoimmune and dermatologic conditions. Enriching our understanding of IRs in these conditions is critical for understanding the pathophysiology behind autoimmune skin disease onset and progression. Furthermore, understanding B-cell and T-cell IR will help devise therapeutic treatments in the hopes of restoring immune tolerance and preventing disease onset and progression. Full article

Toceranib phosphate (toceranib) is approved for canine mast cell tumor treatment. However, no long-term response to toceranib in canine HSTCL has been reported. Here, we describe a case of a 10-year-old castrated mixed-breed dog that presented with a 3-month history of weight loss, polydipsia, and polyuria. The clinicopathological and imaging abnormalities included icterus, biliary obstruction, and splenomegaly with multiple diffuse splenic hypoechoic nodules. On day 21, a cholecystectomy was performed to remove the obstruction, followed by a liver biopsy and splenectomy. Cytology of the spleen and liver showed many small lymphocytes with intracytoplasmic granules (sGLs). Splenic and hepatic infiltration of neoplastic CD3/granzyme B-positive small cells and lymphocytic cholecystitis with granzyme B-negative small cells were noted. T-cell receptor gene clonal rearrangements were observed in the liver tissues. The dog was diagnosed with a hepatosplenic T-cell lymphoma (HSTCL) of sGLs concurrent with lymphocytic cholecystitis. The icterus resolved after surgery, but there was progressive elevation of liver enzyme levels. Toceranib was administered from day 39, resulting in decreased liver enzyme levels, and the dog remained in good condition. The dog stayed in remission after toceranib administration and survived for 460 days. Toceranib should be considered an effective treatment option for canine HSTCL. Full article

Feline lymphoma, a prevalent cancer in cats, exhibits varied prognoses influenced by anatomical site and cellular characteristics. In this study, we investigated the utility of flow cytometry and clonality analysis via PCR for antigen receptor rearrangement (PARR) with respect to characterizing the disease and predicting prognosis. For this purpose, we received fine needle aspirates and/or blood from 438 feline patients, which were subjected to flow cytometry analysis and PARR. We used a subset of the results from patients with confirmed B- or T-cell lymphomas for comparison to cytological or histological evaluation (n = 53). Using them as a training set, we identified the optimal set of flow cytometry parameters, namely forward scatter thresholds, for cell size categorization by correlating with cytology-defined sizes. Concordance with cytological sizing among this training set was 82%. Furthermore, 90% concordance was observed when the proposed cell sizing was tested on an independent test set (n = 24), underscoring the reliability of the proposed approach. Additionally, lymphoma subtypes defined by flow cytometry and PARR demonstrated significant survival differences, validating the prognostic utility of these methods. The proposed methodology achieves high concordance with cytological evaluations and provides an additional tool for the characterization and management of feline lymphoproliferative diseases. Full article

Colorectal cancer (CRC) is one of the most diagnosed cancers and a leading contributor to cancer-related deaths in the United States. Clinically, standard treatment regimens include surgery, radiation, and chemotherapy; however, there has been increasing development and clinical use of targeted therapies for CRC. Unfortunately, many patients develop resistance to these treatments. Cetuximab, the first targeted therapy approved to treat advanced CRC, is a monoclonal antibody that targets the epidermal growth factor receptor and inhibits downstream pathway activation to restrict tumor cell growth and proliferation. CRC resistance to cetuximab has been well studied, and common resistance mechanisms include constitutive signal transduction through downstream protein mutations and promotion of the epithelial-to-mesenchymal transition. While the most common resistance mechanisms are known, a proportion of patients develop resistance through unknown mechanisms. One protein predicted to contribute to therapy resistance is RAC1, a small GTPase that is involved in cytoskeleton rearrangement, cell migration, motility, and proliferation. RAC1 has also been shown to be overexpressed in CRC. Despite evidence that RAC1 and its alternative splice isoform RAC1B play important roles in CRC and the pathways known to contribute to cetuximab resistance, there is a need to directly study the relationship between RAC1 and RAC1B and cetuximab resistance. This review highlights the recent studies investigating RAC1 and RAC1B in the context of CRC and suggests that these proteins could play a role in resistance to cetuximab. Full article

Recombinase-activating gene (RAG)-deficient SCID patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. The two RAG genes act as a required dimer to initiate gene recombination. Gene therapy is a valid treatment alternative for RAG-SCID patients who lack a suitable bone marrow donor, but developing such therapy for RAG1/2 has proven challenging. Using a clinically approved lentiviral vector with a codon-optimized RAG1 gene, we report here preclinical studies using CD34+ cells from four RAG1-SCID patients. We used in vitro T cell developmental assays and in vivo assays in xenografted NSG mice. The RAG1-SCID patient CD34⁺ cells transduced with the RAG1 vector and transplanted into NSG mice led to restored human B and T cell development. Together with favorable safety data on integration sites, these results substantiate an ongoing phase I/II clinical trial for RAG1-SCID. Full article

Mucosa-associated lymphoid tissue (MALT) lymphomas are slow-growing B-cell lymphomas mainly diagnosed in the stomach and termed gastric MALT lymphoma (G-MALT). Despite histological evaluation, immunostaining, and additional B-cell clonality analysis by fragment analysis, a clear-cut diagnosis is not feasible in all cases, especially for clinical follow-up of patients after treatment. We examined clonally rearranged immunoglobulin heavy- and light-chain gene sequences of 36 genomic DNA samples from six different patients obtained at different time points over the course of several years using the Oncomine^TM B-cell receptor pan-clonality next-generation sequencing (NGS) assay. Each case consisted of samples diagnosed with G-MALT and samples without evidence of lymphoma, based on histological examinations. We show a robust correlation (100%) of the results between the applied NGS method and histology-diagnosed G-MALT-positive patients. We also detected malignant clonotypes in samples where histology assessment failed to provide clear evidence of G-MALT (15 out of 19 samples). Furthermore, this method revealed malignant clonotypes much earlier in the disease course, with NGS of the immunoglobulin light chain being crucial in complementing immunoglobulin heavy-chain analysis. Hence, the value of NGS in routine lymphoma diagnostics is greatly significant and can be explored in order to provide better diagnoses and proffer the early detection of lymphoma relapse. Full article

The human immunodeficiency virus (HIV-1) matrix protein p17 (p17) is released from infected cells as a protein capable of deregulating the biological activity of different cells. P17 variants (vp17s), more frequently detected in the plasma of HIV-1⁺ patients with rather than without lymphoma and characterized by amino acids insertions in their C-terminal region, were found to trigger B cell growth and clonogenicity. Vp17s endowed with B-cell-growth-promoting activity are drastically destabilized, whereas, in a properly folded state, reference p17 (refp17) does not exert any biological activity on B cell growth and clonogenicity. However, misfolding of refp17 is necessary to expose a masked functional epitope, interacting with the protease-activated receptor 1 (PAR-1), endowed with B cell clonogenicity. Indeed, it is worth noting that changes in the secondary structure can strongly impact the function of a protein. Here, we performed computational studies to show that the gain of function of vp17s is linked to dramatic conformational changes due to structural modification in the secondary-structure elements and in the rearrangement of the hydrogen bond (H-bond) network. In particular, all clonogenic vp17s showed the disengagement of two critical residues, namely Trp16 and Tyr29, from their hydrophobic core. Biological data showed that the mutation of Trp16 and Tyr29 to Ala in the refp17 backbone, alone or in combination, resulted in a protein endowed with B cell clonogenic activity. These data show the pivotal role of the hydrophobic component in maintaining refp17 stability and identify a novel potential therapeutic target to counteract vp17-driven lymphomagenesis in HIV-1⁺ patients. Full article

Sequencing of the T-cell repertoire is an innovative method to assess the cellular responses after immunization. The purpose of this study was to compare T-cell repertoires after COVID-19 immunization with homologous (HOB) and heterologous (HEB) boosting. The study included 20 participants with a median age of 27.5 (IQR:23) years, who were vaccinated with one dose of the Ad26.COV2.S vaccine and were boosted with either Ad26.COV2.S (n = 10) or BNT162b2 (n = 10) vaccine. Analysis of the T-cell receptor beta locus (TCRβ) sequencing one month after the booster dose identified that the HEB compared to the HOB group exhibited a higher number of both total and COVID-19-related functional T-cell rearrangements [mean of total productive rearrangements (TPRs): 63151.8 (SD ± 18441.5) vs. 34915.4 (SD ± 11121.6), p = 0.001 and COVID-19–TPRs: 522.5 (SD ± 178.0) vs. 298.3 (SD ± 101.1), p = 0.003]. A comparison between the HOB and HEB groups detected no statistically significant differences regarding T-cell Simpson clonality [0.021 (IQR:0.014) vs. 0.019 (IQR:0.007)], richness [8734.5 (IQR:973.3) vs. 8724 (IQR:383.7)] and T-cell fraction [0.19 (IQR:0.08) vs. 0.18 (IQR:0.08)]. HEB also exhibited a substantially elevated humoral immune response one month after the booster dose compared to HOB [median antibody titer (IQR): 10115.0 U/mL (6993.0) vs. 1781.0 U/mL (1314.0), p = 0.001]. T-cell repertoire sequencing indicated that HEB had increased SARS-CoV-2-related T-cell rearrangements, which was in accordance with higher humoral responses and possibly conferring longer protection. Data from the present study indicate that the administration of different COVID-19 vaccines as a booster may provide better protection. Full article

B-cell neoplasms possess clonal B-cell receptor rearrangements (BCR clonotype lineages) that can be identified by sequencing the B-cell repertoire for use in diagnostics, risk stratification, and high-sensitivity monitoring. BCR somatic hypermutation (SHM) can result in clonality detection failure from point mutations in PCR primer binding regions, often necessitating splitting samples into multiple reactions which increases test costs, turnaround times, and sample requirements. We evaluated the Oncomine BCR Pan-Clonality Assay, a novel single-tube PCR reaction that simultaneously amplifies all BCR loci for next-generation DNA sequencing, using neoplastic B-cell lines and clinical research samples from multiple myeloma (MM) patients, a plasma cell neoplasm associated with high SHM levels. The assay showed a linear detection range down to 1 ng of clonal DNA input, sensitivity to 10⁻⁶ in a polyclonal background, and high reproducibility. Clonotype lineages were identified in 42/45 (93%) MM samples. Ion Reporter software packaged with the assay permitted straightforward identification of MM subgroups. As expected, SHM was identified in 94% of MM cases, but several unexpected subgroups were identified including biased IGHV3-11 or IGHV4-34 usage in 20% of MM samples, and two cases with very low levels of SHM. Evidence of intraclonal diversity/ongoing SHM was identified in 18% of samples, suggesting a possible germinal center origin for some MM cases. The single-tube Oncomine BCR Pan-Clonality assay efficiently detects BCR clonotype lineages at rates comparable to existing multiple reaction assays and permits their characterization for cell of origin studies and lymphoma classification. Full article