Creation of an Isogenic H/N/KRAS-Less Mouse Embryonic Fibroblast Cell Line Panel Derived from a Size-Sorted Diploid Clone.

Cell line panels have proven to be an invaluable tool for investigators researching a range of topics from drug mechanism or drug sensitivity studies to disease-specific etiology. The cell lines used in these panels may range from heterogeneous tumor populations grown from primary tumor isolations to genetically engineered clonal cell lines which express specific gene isoforms. Mouse embryonic fibroblast (MEF) cells are a commonly used cell line for biological research due to their accessibility and ease of genetic manipulation. This chapter will describe the process of creating a size-sorted diploid (SSDC) clonal cell panel expressing specific RAS isoforms from a previously engineered RAS-less MEF cell line pool.

[Comparative immunohistochemistry study of different antibodies clones for detection of breast cancer markers (estrogen receptor, progesterone receptor, HER2/neu, Ki-67)]. / Sravnitel'noe issledovanie antitel razlichnykh klonov dlya vyyavleniya markerov raka molochnoi zhelezy (retseptorov estrogenov, progesterona, HER2/neu, Ki-67) immunogistokhimicheskim metodom.

OBJECTIVE: A comparative study of detection of breast cancer markers (estrogen receptors, progesterone receptors, HER2/neu, Ki-67) by immunohistochemical method with antibodies produced by PrimeBioMed (Russia) and antibodies produced by Roche Ventana (USA). MATERIAL AND METHODS: Surgical specimens and biopsies from 37 patients with invasive breast cancer were used. Sections were stained with antibodies of clones ER SP1 and GM030, PR 1E2 and PBM-5B8, HER2/neu 4B5 and PBM-46A6, Ki-67 30-9 and GM010. RESULTS: There was a high positive and significant correlation between the immunohistochemistry results and antibodies of the clones ER-SP1 and GM030, PR1E2 and PBM-5B8, HER2/neu4B5 and PBM-46A6, Ki-67 30-9 and GM010. CONCLUSION: The study showed the possibility of using antibodies of clones GM030, HER2/neu 4B5, PBM-46A6, GM010 (PrimeBioMed) on the Ventana Bench Marck Ultra automatic immunostainer using the detection system UltraView Universal DAB Detection Kit.

Pursuing dynamics of minimal residual leukemic subclones in relapsed and refractory acute myeloid leukemia during conventional therapy.

BACKGROUND: Acute myeloid leukemia (AML) is characterized by clonal heterogeneity, leading to frequent relapses and drug resistance despite intensive clinical therapy. Although AML's clonal architecture has been addressed in many studies, practical monitoring of dynamic changes in those subclones during relapse and treatment is still understudied. METHOD: Fifteen longitudinal bone marrow (BM) samples were collected from three relapsed and refractory (R/R) AML patients. Using droplet digital polymerase chain reaction (ddPCR), the frequencies of patient's leukemic variants were assessed in seven cell populations that were isolated from each BM sample based on cellular phenotypes. By quantifying mutant clones at the diagnosis, remission, and relapse stages, the distribution of AML subclones was sequentially monitored. RESULTS: Minimal residual (MR) leukemic subclones exhibit heterogeneous distribution among BM cell populations, including mature leukocyte populations. During AML progression, these subclones undergo active phenotypic transitions and repopulate into distinct cell population regardless of normal hematopoiesis hierarchic order. Of these, MR subclones in progenitor populations of patient BM predominantly carry MR leukemic properties, leading to more robust expansion and stubborn persistence than those in mature populations. Moreover, a minor subset of MR leukemic subclones could be sustained at an extremely low frequency without clonal expansion during relapse. CONCLUSIONS: In this study, we observed treatment persistent MR leukemic subclones and their phenotypic changes during the treatment process of R/R AML patients. This underscores the importance of preemptive inhibition of clonal promiscuity in R/R AML, proposing a practical method for monitoring AML MR subclones.

Comprehensive Molecular Profiling of NPM1-Mutated Acute Myeloid Leukemia Using RNAseq Approach.

Acute myeloid leukemia (AML) is a complex hematologic malignancy with high morbidity and mortality. Nucleophosmin 1 (NPM1) mutations occur in approximately 30% of AML cases, and NPM1-mutated AML is classified as a distinct entity. NPM1-mutated AML patients without additional genetic abnormalities have a favorable prognosis. Despite this, 30-50% of them experience relapse. This study aimed to investigate the potential of total RNAseq in improving the characterization of NPM1-mutated AML patients. We explored genetic variations independently of myeloid stratification, revealing a complex molecular scenario. We showed that total RNAseq enables the uncovering of different genetic alterations and clonal subtypes, allowing for a comprehensive evaluation of the real expression of exome transcripts in leukemic clones and the identification of aberrant fusion transcripts. This characterization may enhance understanding and guide improved treatment strategies for NPM1mut AML patients, contributing to better outcomes. Our findings underscore the complexity of NPM1-mutated AML, supporting the incorporation of advanced technologies for precise risk stratification and personalized therapeutic strategies. The study provides a foundation for future investigations into the clinical implications of identified genetic variations and highlights the importance of evolving diagnostic approaches in leukemia management.

High-risk Escherichia coli clones that cause neonatal meningitis and association with recrudescent infection.

Neonatal meningitis is a devastating disease associated with high mortality and neurological sequelae. Escherichia coli is the second most common cause of neonatal meningitis in full-term infants (herein NMEC) and the most common cause of meningitis in preterm neonates. Here, we investigated the genomic relatedness of a collection of 58 NMEC isolates spanning 1974-2020 and isolated from seven different geographic regions. We show NMEC are comprised of diverse sequence types (STs), with ST95 (34.5%) and ST1193 (15.5%) the most common. No single virulence gene profile was conserved in all isolates; however, genes encoding fimbrial adhesins, iron acquisition systems, the K1 capsule, and O antigen types O18, O75, and O2 were most prevalent. Antibiotic resistance genes occurred infrequently in our collection. We also monitored the infection dynamics in three patients that suffered recrudescent invasive infection caused by the original infecting isolate despite appropriate antibiotic treatment based on antibiogram profile and resistance genotype. These patients exhibited severe gut dysbiosis. In one patient, the causative NMEC isolate was also detected in the fecal flora at the time of the second infection episode and after treatment. Thus, although antibiotics are the standard of care for NMEC treatment, our data suggest that failure to eliminate the causative NMEC that resides intestinally can lead to the existence of a refractory reservoir that may seed recrudescent infection.

Convergence, plasticity, and tissue residence of regulatory T cell response via TCR repertoire prism.

Suppressive function of regulatory T cells (Treg) is dependent on signaling of their antigen receptors triggered by cognate self, dietary, or microbial peptides presented on MHC II. However, it remains largely unknown whether distinct or shared repertoires of Treg TCRs are mobilized in response to different challenges in the same tissue or the same challenge in different tissues. Here we use a fixed TCRß chain FoxP3-GFP mouse model to analyze conventional (eCD4) and regulatory (eTreg) effector TCRα repertoires in response to six distinct antigenic challenges to the lung and skin. This model shows highly 'digital' repertoire behavior with easy-to-track challenge-specific TCRα CDR3 clusters. For both eCD4 and eTreg subsets, we observe challenge-specific clonal expansions yielding homologous TCRα clusters within and across animals and exposure sites, which are also reflected in the draining lymph nodes but not systemically. Some CDR3 clusters are shared across cancer challenges, suggesting a response to common tumor-associated antigens. For most challenges, eCD4 and eTreg clonal response does not overlap. Such overlap is exclusively observed at the sites of certain tumor challenges, and not systematically, suggesting transient and local tumor-induced eCD4=>eTreg plasticity. This transition includes a dominant tumor-responding eCD4 CDR3 motif, as well as characteristic iNKT TCRα CDR3. In addition, we examine the homeostatic tissue residency of clonal eTreg populations by excluding the site of challenge from our analysis. We demonstrate that distinct CDR3 motifs are characteristic of eTreg cells residing in particular lymphatic tissues, regardless of the challenge. This observation reveals the tissue-resident, antigen-specific clonal Treg populations.

Single-cell characterisation of tissue homing CD4 + and CD8 + T cell clones in immune-mediated refractory arthritis.

BACKGROUND: Immune-mediated arthritis is a group of autoinflammatory diseases, where the patient's own immune system attacks and destroys synovial joints. Sustained remission is not always achieved with available immunosuppressive treatments, warranting more detailed studies of T cell responses that perpetuate synovial inflammation in treatment-refractory patients. METHODS: In this study, we investigated CD4 + and CD8 + T lymphocytes from the synovial tissue and peripheral blood of patients with treatment-resistant immune-mediated arthritis using paired single-cell RNA and TCR-sequencing. To gain insights into the trafficking of clonal families, we compared the phenotypes of clones with the exact same TCRß amino acid sequence between the two tissues. RESULTS: Our results show that both CD4 + and CD8 + T cells display a more activated and inflamed phenotype in the synovial tissue compared to peripheral blood both at the population level and within individual T cell families. Furthermore, we found that both cell subtypes exhibited clonal expansion in the synovial tissue. CONCLUSIONS: Our findings suggest that the local environment in the synovium drives the proliferation of activated cytotoxic T cells, and both CD4 + and CD8 + T cells may contribute to tissue destruction and disease pathogenesis.

Detecting T-cell clonal expansions and quantifying clone survival using deep profiling of immune repertoires.

An individual's T-cell repertoire constantly changes under the influence of external and internal factors. Cells that do not receive a stimulatory signal die, while those that encounter and recognize a pathogen or receive a co-stimulatory signal divide, resulting in clonal expansions. T-cell clones can be traced by monitoring the presence of their unique T-cell receptor (TCR) sequence, which is assembled de novo through a process known as V(D)J rearrangement. Tracking T cells can provide valuable insights into the survival of cells after hematopoietic stem cell transplantation (HSCT) or cancer treatment response and can indicate the induction of protective immunity by vaccination. In this study, we report a bioinformatic method for quantifying the T-cell repertoire dynamics from TCR sequencing data. We demonstrate its utility by measuring the T-cell repertoire stability in healthy donors, by quantifying the effect of donor lymphocyte infusion (DLI), and by tracking the fate of the different T-cell subsets in HSCT patients and the expansion of pathogen-specific clones in vaccinated individuals.

High clonal diversity and spatial genetic admixture in early prostate cancer and surrounding normal tissue.

Somatic copy number alterations (SCNAs) are pervasive in advanced human cancers, but their prevalence and spatial distribution in early-stage, localized tumors and their surrounding normal tissues are poorly characterized. Here, we perform multi-region, single-cell DNA sequencing to characterize the SCNA landscape across tumor-rich and normal tissue in two male patients with localized prostate cancer. We identify two distinct karyotypes: 'pseudo-diploid' cells harboring few SCNAs and highly aneuploid cells. Pseudo-diploid cells form numerous small-sized subclones ranging from highly spatially localized to broadly spread subclones. In contrast, aneuploid cells do not form subclones and are detected throughout the prostate, including normal tissue regions. Highly localized pseudo-diploid subclones are confined within tumor-rich regions and carry deletions in multiple tumor-suppressor genes. Our study reveals that SCNAs are widespread in normal and tumor regions across the prostate in localized prostate cancer patients and suggests that a subset of pseudo-diploid cells drive tumorigenesis in the aging prostate.

A Case of Chronic Eosinophilic Leukemia with CSF3R-Mutant Clone and Transformed to Secondary Acute Myeloid Leukemia.

BACKGROUND: Chronic eosinophilic leukemia (CEL) is a rare invasive disease characterized by non-specific cytogenetic abnormalities or elevated mother cells, poor prognosis, and a high risk of conversion to acute leukemia. METHODS: We described the data of a patient with CEL-NOS. RESULTS: This case is a CEL-NOS with four mutations in CSF3R-T618I, DNMT3A Q816, ASXL1, and IDH2. CONCLUSIONS: The patient rapidly evolves into secondary acute myeloid leukemia (AML).

HIV-1-infected T cell clones are shared across cerebrospinal fluid and blood during ART.

The central nervous system HIV reservoir is incompletely understood and is a major barrier to HIV cure. We profiled people with HIV (PWH) and uninfected controls through single-cell transcriptomic and T cell receptor (TCR) sequencing to understand the dynamics of HIV persistence in the CNS. In PWH on ART, we found that most participants had single cells containing HIV-1 RNA, which was found predominantly in CD4 central memory T cells, in both cerebrospinal fluid (CSF) and blood. HIV-1 RNA-containing cells were found more frequently in CSF than blood, indicating a higher burden of reservoir cells in the CNS than blood for some PWH. Most CD4 T cell clones containing infected cells were compartment specific, while some (22%) - including rare clones with members of the clone containing detectable HIV RNA in both blood and CSF - were found in both CSF and blood. These results suggest that infected T cells trafficked between tissue compartments and that maintenance and expansion of infected T cell clones contributed to the CNS reservoir in PWH on ART.

[Guideline of the diagnosis and treatment of eosinophilic disorders (2024)].

The eosinophilias encompass a broad range of nonhematologic (secondary or reactive) and hematologic (primary or clonal) disorders with potential for end-organ damage. Based on new clinical data and increased understanding of disease molecular genetics, the World Health Organization (WHO) and the international consensus classification (ICC) has provided updated criteria and classifications for eosinophilic disorders in 2022. This guideline represents an update of Chinese expert consensus on the diagnosis and treatment of eosinophilia published in 2017 and aim to provide Chinese hematologist with clear guidance on management for eosinophilic disorders.

The effect of cooperator recognition on competition among clones in spatially structured microbial communities.

In spatially structured microbial communities, clonal growth of stationary cells passively generates clusters of related individuals. This can lead to stable cooperation without the need for recognition mechanisms. However, recent research suggests that some biofilm-forming microbes may have mechanisms of kin recognition. To explore this unexpected observation, we studied the effects of different types of cooperation in a microbial colony using spatially explicit, agent-based simulations of two interacting strains. We found scenarios that favor a form of kin recognition in spatially structured microbial communities. In the presence of a "cheater" strain, a strain with greenbeard cooperation was able to increase in frequency more than a strain with obligate cooperation. This effect was most noticeable in high density colonies and when the cooperators were not as abundant as the cheaters. We also studied whether a polychromatic greenbeard, in which cells only cooperate with their own type, could provide a numerical benefit beyond a simple, binary greenbeard. We found the greatest benefit to a polychromatic greenbeard when cooperation is highly effective. These results suggest that in some ecological scenarios, recognition mechanisms may be beneficial even in spatially structured communities.

Characterization of Sf9 cell clones with differential susceptibilities to Sf-rhabdovirus X+3.7 and Sf-rhabdovirus X- replication.

Our laboratory previously discovered a novel rhabdovirus in the Spodoptera frugiperda Sf9 insect cell line that was designated as Sf-rhabdovirus. Using limiting dilution, this cell line was found to be a mixed population of cells infected by Sf-rhabdovirus variants containing either the full length X accessory gene with a 3.7 kb internal duplication (designated as Sf-rhabdovirus X+3.7) or lacking the duplication and part of the X gene (designated as Sf-rhabdovirus X-), and cells that were negative for Sf-rhabdovirus. In this paper, we found that the Sf-rhabdovirus negative cell clones had sub-populations with different susceptibilities to the replication of Sf-rhabdovirus X+3.7 and X- variants: cell clone Sf9-13F12 was more sensitive to replication by both virus variants compared to Sf9-3003; moreover, Sf9-3003 showed more resistance to X+3.7 replication than to X- replication. RNA-Seq analysis indicated significant differentially expressed genes in the Sf9-13F12 and Sf9-3003 cell clones further supporting that distinct sub-populations of virus-negative cells co-exist in the parent Sf9 cell line.

Specific interaction between Group B Streptococcus CC17 hypervirulent clone and phagocytes.

Streptococcus agalactiae also named Group B Streptococcus (GBS) is the most significant pathogen causing invasive infections, such as bacteremia and meningitis, in neonates. Worldwide epidemiological studies have shown that a particular clonal complex (CC) of capsular serotype III, the CC17, is strongly associated with meningitis in neonates and is therefore, designated as the hypervirulent clone. Macrophages are a permissive niche for intracellular bacteria of all GBS clones. In this study, we deciphered the specific interaction of GBS CC17 strains with macrophages. Our study revealed that CC17 strains are phagocytosed at a higher rate than GBS non-CC17 strains by human monocytes and macrophages both in cellular models and in primary cells. CC17-enhanced phagocytosis is due to an initial enhanced-attachment step to macrophages mediated by the CC17-specific surface protein HvgA and the PI-2b pilus (Spb1). We showed that two different inhibitors of scavenger receptors (fucoidan and poly(I)) specifically inhibited CC17 adhesion and phagocytosis while not affecting those of non-CC17 strains. Once phagocytosed, both CC17 and non-CC17 strains remained in a LAMP-1 positive vacuole that ultimately fuses with lysosomes where they can survive at similar rates. Finally, both strains displayed a basal egress which occurs independently from actin and microtubule networks. Our findings provide new insights into the interplay between the hypervirulent GBS CC17 and major players of the host's innate immune response. This enhanced adhesion, leading to increased phagocytosis, could reflect a peculiar capacity of the CC17 lineage to subvert the host immune defenses, establish a niche for persistence or disseminate.

Evolutionary trajectories of small cell lung cancer under therapy.

The evolutionary processes that underlie the marked sensitivity of small cell lung cancer (SCLC) to chemotherapy and rapid relapse are unknown1-3. Here we determined tumour phylogenies at diagnosis and throughout chemotherapy and immunotherapy by multiregion sequencing of 160 tumours from 65 patients. Treatment-naive SCLC exhibited clonal homogeneity at distinct tumour sites, whereas first-line platinum-based chemotherapy led to a burst in genomic intratumour heterogeneity and spatial clonal diversity. We observed branched evolution and a shift to ancestral clones underlying tumour relapse. Effective radio- or immunotherapy induced a re-expansion of founder clones with acquired genomic damage from first-line chemotherapy. Whereas TP53 and RB1 alterations were exclusively part of the common ancestor, MYC family amplifications were frequently not constituents of the founder clone. At relapse, emerging subclonal mutations affected key genes associated with SCLC biology, and tumours harbouring clonal CREBBP/EP300 alterations underwent genome duplications. Gene-damaging TP53 alterations and co-alterations of TP53 missense mutations with TP73, CREBBP/EP300 or FMN2 were significantly associated with shorter disease relapse following chemotherapy. In summary, we uncover key processes of the genomic evolution of SCLC under therapy, identify the common ancestor as the source of clonal diversity at relapse and show central genomic patterns associated with sensitivity and resistance to chemotherapy.

Construction of an infectious clone for enterovirus A89 and mutagenesis analysis of viral infection and cell binding.

Enterovirus A89 (EV-A89) is an unconventional strain belonging to the Enterovirus A species. Limited research has been conducted on EV-A89, leaving its biological and pathogenic properties unclear. Developing reverse genetic tools for EV-A89 would help to unravel its infection mechanisms and aid in the development of vaccines and anti-viral drugs. In this study, an infectious clone for EV-A89 was successfully constructed and recombinant enterovirus A89 (rEV-A89) was generated. The rEV-A89 exhibited similar characteristics such as growth curve, plaque morphology, and dsRNA expression with parental strain. Four amino acid substitutions were identified in the EV-A89 capsid, which were found to enhance viral infection. Mechanistic studies revealed that these substitutions increased the virus's cell-binding ability. Establishing reverse genetic tools for EV-A89 will significantly contribute to understanding viral infection and developing anti-viral strategies.IMPORTANCEEnterovirus A species contain many human pathogens and have been classified into conventional cluster and unconventional cluster. Most of the research focuses on various conventional members, while understanding of the life cycle and infection characteristics of unconventional viruses is still very limited. In our study, we constructed the infectious cDNA clone and single-round infectious particles for the unconventional EV-A89, allowing us to investigate the biological properties of recombinant viruses. Moreover, we identified key amino acids residues that facilitate EV-A89 infection and elucidate their roles in enhancing viral binding to host cells. The establishment of the reverse genetics system will greatly facilitate future study on the life cycle of EV-A89 and contribute to the development of prophylactic vaccines and anti-viral drugs.

Role of seasonal importation and genetic drift on selection for drug-resistant genotypes of Plasmodium falciparum in high-transmission settings.

Historically Plasmodium falciparum has followed a pattern of drug resistance first appearing in low-transmission settings before spreading to high-transmission settings. Several features of low-transmission regions are hypothesized as explanations: higher chance of symptoms and treatment seeking, better treatment access, less within-host competition among clones and lower rates of recombination. Here, we test whether importation of drug-resistant parasites is more likely to lead to successful emergence and establishment in low-transmission or high-transmission periods of the same epidemiological setting, using a spatial, individual-based stochastic model of malaria and drug-resistance evolution calibrated for Burkina Faso. Upon controlling for the timing of importation of drug-resistant genotypes and examination of key model variables, we found that drug-resistant genotypes imported during the low-transmission season were (i) more susceptible to stochastic extinction due to the action of genetic drift, and (ii) more likely to lead to establishment of drug resistance when parasites are able to survive early stochastic loss due to drift. This implies that rare importation events are more likely to lead to establishment if they occur during a high-transmission season, but that constant importation (e.g. neighbouring countries with high levels of resistance) may produce a greater risk during low-transmission periods.

KPC-2 and VIM-1 producing Klebsiella pneumoniae ST39 high-risk clone isolated from a clinical sample in Volos, Greece.

Klebsiella pneumoniae is a major human pathogen, because it causes both community- and hospital-acquired infections. Several multidrug-resistant high-risk clones of K. pneumoniae have been reported worldwide, and these are responsible for high numbers of difficult-to-treat infections. In Greece, a K. pneumoniae ST39 high-risk clone was detected in 2019 in a survey of carbapenem- and/or colistin-resistant Enterobacteriacae. The present study included nine carbapenem-resistant K. pneumoniae (CRKP) isolates collected during a retrospective analysis from October 2020 to December 2020. They were isolated from nine different patients hospitalized in the intensive care unit (ICU) of a hospital in Volos, Greece, and they were selected for analysis due to their phenotypic profile. In this study, we analyzed A165 strain K. pneumoniae ST39 isolated from a blood culture in November 2020. Whole-genome sequencing (WGS) was performed using Ion Torrent Platform, and resistance genes, virulence determinants, capsular types, insertion sequences, phage regions, and clustered regularly interspaced palindromic repeats (CRISPR) regions were detected by bioinformatic analysis. The molecular characterization revealed antimicrobial resistance genes, including sul2 for sulfamethoxazole; dfrA1 for trimethoprim; blaVIM-1 and blaKPC-2 for carbapenems; aac(6')-II for aminoglycosides; fosA for fosfomycin and aad1 for streptomycin, blaSHV-40, blaSHV-85, blaSHV-79, blaSHV-56, and blaSHV-89 for beta-lactams. Point mutations were identified in ompK36, and ompK37 and in acrR, gyrA, parC. Several replicons were found, including CoIRNA, IncC, IncFIB(K), IncFIB(pQiL), and IncFII(K). The capsular typing revealed that the strain was KL23, O2afg. The genome sequence of A165 was submitted to NCBI under PRJNA1074377 and have been assigned to Genbank accession number JAZIBV000000000.

Anti-inflammatory and antioxidative actions of tacrolimus (FK506) on human microglial HMC3 cell line.

Microglial cells are indispensable for the normal development and functioning of neurons in the central nervous system, where they play a crucial role in maintaining brain homeostasis by surveilling the microenvironment for signs of injury or stress and responding accordingly. However, in neurodegenerative diseases, the density and phenotypes of microglial cells undergo changes, leading to chronic activation and inflammation. Shifting the focus from neurons to microglia in drug discovery for neurodegenerative diseases has become an important therapeutic target. This study was aimed to investigate the potential of Tacrolimus (FK506) an FDA-approved calcineurin inhibitor, to modulate the pathology of neurodegenerative diseases through anti-inflammatory and antioxidative effects on microglial activation. The human microglia clone 3 (HMC3) cells were exposed to 1 µg/mL LPS in the presence and absence of doses of FK506. Survival rates of cells were determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) method. Morphological evaluation of cells showed that FK506 restored the normal morphology of activated microglia. Furthermore, FK506 treatment increases the total antioxidant capacity and reduces the total oxidative capacity, indicating its potential antioxidant effects. Data from ELISA and RT-PCR analyses showed that LPS abolished its promoting effects on the release of proinflammatory IL-1ß and IL-6 cytokines in HMC3 cells, reflecting the anti-inflammatory effect of FK506. These findings support the idea that FK506 could be a promising therapeutic agent for neurodegenerative diseases by modulating microglial activation and reducing inflammation and oxidative stress.