Variable Region Sequences Research Articles

Role of gut microbiome in developing necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is one of the most devastating intestinal diseases observed in preterm in the first days of life. Researchers have recently focused on potential predictive biomarkers for early and concomitant diagnoses. Thus, we inquired about the linkage of intestinal dysbiosis, one of the most important factors in NEC development to the gut microbiota. In this study, the systematic differences in the bacterial composition between neonates affected by NEC and healthy newborns were highlighted by metagenomic analysis. The next-generation sequencing of the V3-V4 variable region of the 16S rRNA gene and gene-specific qPCR analyzed the untargeted gut microbiota. Total bacteria, total and fecal coliform loads in stool samples with NEC were higher than control. OTU-level relative abundances of NEC infant was characterized by Firmicutes and Bacteroidetes at phylum levels. At the genus level, NEC stool was identified by the lack of Klebsiella and the presence of Roseburia, Blautia, and Parasutterella. Finally, Clostridium fessum was the predominant species of Clostridium genus in disease and healthy specimens at the species level, whereas Clostridium jeddahitimonense was at NEC diagnosis. Despite a strong relationship between pathophysiology and characterization of gut microbiota at a clinical diagnosis of NEC, our results emphasize the broad difficulty in identifying potential biomarkers.

Hydrocarbon-Oxidizing Bacteria of the Bottom Ecotopes of the Barents and Pechora Seas

Microorganisms capable of degrading hydrocarbons are regular components of natural microbial communities and play an important role in self-purification of marine environments from oil contamination. High-throughput sequencing of the 16S rRNA gene V4 variable region was used to analyze microbial communities of the Barents and Pechora seas and of the microcosms with a spectrum of hydrocarbon substrates: oil, n-nonane, n-undecane, and phenanthrene. The Barents Sea communities of hydrocarbon-oxidizing microorganisms were characterized by predominance of the genera Pseudoalteromonas, Pseudomonas, Porticoccus, and Oleispira, while those of the Pechora Sea contained members of the genera Rhodococcus, Dietzia, Sphingorhabdus, and Hyphomonas. Pure cultures of these microorganisms were shown to utilize the major oil hydrocarbons: n-alkanes, cycloalkanes, and aromatic compounds.

Full-length single-cell BCR sequencing paired with RNA sequencing reveals convergent responses to pneumococcal vaccination

Single-cell RNA sequencing (scRNA-seq) can resolve transcriptional features from individual cells, but scRNA-seq techniques capable of resolving the variable regions of B cell receptors (BCRs) remain limited, especially from widely-used 3′-barcoded libraries. Here, we report a method that can recover paired, full-length variable region sequences of BCRs from 3′-barcoded scRNA-seq libraries. We first verify this method (B3E-seq) can produce accurate, full-length BCR sequences. We then apply this method to profile B cell responses elicited against the capsular polysaccharide of Streptococcus pneumoniae serotype 3 (ST3) by glycoconjugate vaccines in five infant rhesus macaques. We identify BCR features associated with specificity for the ST3 antigen which are present in multiple vaccinated monkeys, indicating a convergent response to vaccination. These results demonstrate the utility of our method to resolve key features of the B cell repertoire and profile antigen-specific responses elicited by vaccination.

Construction of on-site DNA pre-treatment device and rapid electrochemical biosensor set for Escherichia coli detection in milk

Restaurants and cafeterias are constantly exposed to bacterial contamination. In such environments, it is highly needed the rapid bacteria detection system with simple pre-treatment device. To solve this problem, this study presented the pre-treatment device and Rapid electrochemical (REC) biosensor set for detecting Escherichia coli (E. coli) gene in milk sample. The pre-treatment device is consisted of tetraethyl orthosilicate (TEOS)-coated bead that operated with sonication method, and it can effectively remove the E. coli residues and extract E. coli DNA well. By analyzing the sequences of various foodborne pathogens, we selected the highly variable region sequences of E. coli DNA to use as the bioprobe for REC biosensor. The selected thiol-modified bioprobe was immobilized onto the fabricated biosensor electrode. For detecting the extracted E. coli gene fragment rapidly, the alternating current electrothermal flow (ACEF) technique was introduced to REC-biosensor. ACEF technique minimize the target-bioprobe binding time to 10 mins. Using the extracted E. coli DNA fragments with the pre-treatment device, the proposed REC-biosensor shows the limit of detection (LOD) in milk samples was 9.235 × 10-5 ng/μL, and the target detection accuracy was 92.5 %, with a maximal error rate up to 6.730 %. Moreover, the biosensor demonstrated high selectivity in electrochemical performance evaluation (Matrix effect = 7.425 %). The developed pre-treatment device provides simple and quick preprocessing time (within 20 min) of E. coli, and REC biosensor provides the rapid detection of E. coli (within 10 min). In conclusion, the proposed pre-treatment device and biosensor set can be easily applied to other types of bacteria, virus or germs in the near future.

Correlation between Serum Levels of Factor I, CD59, Interferon-gamma, and Interleukin-6 with the Response to Rituximab in Iraqi Patients with Rheumatoid Arthritis

Background: Rituximab is a chimeric IgG1 kappa immunoglobulin that has been genetically modified to incorporate human constant region sequences together with murine light- and heavy-chain variable region sequences. People use it to treat rheumatoid arthritis and certain malignancies. Objective: The study aimed to assess the potential association between the serum levels of Factor I, CD59, interleukins (IL)-6, and interferon-gamma (IFN)-γ and the response to Rituximab treatment in Iraqi rheumatoid arthritis patients. Methods: A cross-sectional study was conducted at the rheumatology center at Baghdad Teaching Hospital. Ninety adult patients who have been diagnosed with rheumatoid arthritis and are receiving Rituximab intravenous infusions were included. The enrolled patients were divided into a responder group (45 patients) and a non-responder group (45 patients). The response to Rituximab was assessed according to the 28-joint Disease Activity Score (DAS28). Results: The serum levels of Factor I and CD59 were significantly higher in the non-responders group in comparison to the responders group. In addition, the serum IL-6 and IFN-γ levels were significantly elevated in the non-responders group in comparison to the responders group. The estimated marker serum levels showed a strong, significant correlation with the 6-month change in DAS28. Conclusions: In Rituximab nonresponder RA patients, serum levels of Factor I, CD59, Factor H, IL-6, and IFN-γ are higher, and they have good potential to be used in the assessment of the response to Rituximab therapy.

Fecal Microbiota of Transbaikal Camels (Camelus bactrianus) under Different Systems of Grazing Management

Microbial diversity in the fecal samples of Bactrian camels in Transbaikalia under various grazing management (free grazing (group I), mixed (group II) and stall housing (group III)) was revealed using high-throughput sequencing of the 16S rRNA gene variable regions. The microbial community of the fecal microbiota was found to be diverse and to depend on the camel grazing management. The most common phyla of the camel fecal microbiota were Bacillota and Bacteroidota. The phylum Verrucomicrobiota was a codominant in the fecal microbiota of groups I and II of animals, and Actinomycetota, in the feces of camels of group III. Changes in the fecal microbiota structure and taxonomic diversity occurred as camel grazing management and feeding conditions changed. Free grazing resulted in high diversity of the prokaryotic community in the fecal microbiota. In addition, differences in taxonomic composition depending on sex were found, which were in the abundance of taxa rather than in their presence or absence. The results contribute to the current understanding of the fecal microbiota of camels under different management conditions and provide evidence of the influence of nutrition on the fecal microbiota under different management conditions. Our results may be useful for addressing the issues of reproduction and conservation of the Transbaikal camel (Camelus bactrianus).

At Shores of a Vanishing Sea: Microbial Communities of Aral and Southern Aral Sea Region

Since the early 60s of the 20th century, as a result of agricultural development in the irrigated areas of Uzbekistan, the area of the Aral Sea has decreased by 90%, while the water salinity has increased from 1% to 20%. The aim of our work was to investigate the diversity of microbial communities of water and sediments of the Western Aral Sea, as well as of the adjacent soils and reservoirs using high-throughput sequencing of the 16S rRNA genes variable V4 region. It was found that the Aral Sea water with a salinity of 22% was dominated by uncultured Archaea of the family Haloferacaceae (22‒43%), as well as by bacteria of the genera Spiribacter and Psychroflexus. In the Aral Sea sediments the share of archaea was much lower (2‒17%), and among them the uncultured Woesearchaeales predominated. Among bacteria, dominating in Aral sediments, there were sulfate reducers of the phylum ‘Desulfobacterota’, as well as representatives of the genera Fusibacter, Halanaerobium, Guyparkeria, Marinobacter, Idiomarina and Thiomicrospira. In soil samples of the former Aral Sea bed with salinity of 8.2%, a variety of archaea of the phylum Halobacterota were present, as well as uncultured bacteria of the family Nitrosococcaceae. However, in the rhizosphere of Ewresmann’s teresken plant (Kraschennininikovia ewresmanniana) growing there, archaea accounted for only 4% and mainly represented the family Nitrososphearaceae. 33% of all prokaryotes in the rhizosphere microbiome were the uncultured representatives of the phylum Actinomycetota. The microbial community of the teresken rhizosphere turned out to be similar to the microbial communities of the soil of the Ustyurt plateau, located in 3 km from the Aral Sea shore. The fresh water flowing along the former Aral Sea bed from an artificially drilled well also causes significant changes in the microbial communities: cyanobacterial mats and associated organotrophic bacteria develop along the stream bed with the increasing salinity (0.5‒2%). Finally, the greatest diversity of prokaryotes was found in the microbial community of Sudochie Lake sediment with salinity of 1%, which is probably a modern analogue of the Aral Sea microbiome before its shallowing.

The chloroplast genomes of two medicinal species (Veronica anagallis-aquatica L. and Veronica undulata Wall.) and its comparative analysis with related Veronica species

Veronica anagallis-aquatica L. and Veronica undulata Wall. are widely used ethnomedicinal plants in China. The two species have different clinical efficacies, while their extremely similar morphology and unclear interspecific relationship make it difficult to accurately identify them, leading to increased instances of mixed usage. This article reports on the complete chloroplast genomes sequence of these two species and their related Veronica species to conduct a comparative genomics analysis and phylogenetic construction. The results showed that the chloroplast (cp) genomes of Veronica exhibited typical circular quadripartite structures, with total lengths of 149,386 to 152,319 base pairs (bp), and GC content of 37.9 to 38.1%, and the number of genes was between 129–134. The total number of simple sequence repeats (SSRs) in V. anagallis-aquatica and V. undulata is 37 and 36, while V. arvensis had the highest total number of 56, predominantly characterized by A/T single bases. The vast majority of long repeat sequence types are forward repeats and palindromic repeats. Selective Ka/Ks values showed that three genes were under positive selection. Sequence differences often occur in the non-coding regions of the large single-copy region (LSC) and small single-copy region (SSC), with the lowest sequence variation in the inverted repeat regions (IR). Seven highly variable regions (trnT-GGU-psbD, rps8-rpl16, trnQ-UUG, trnN-GUU-ndhF, petL, ycf3, and ycf1) were detected, which may be potential molecular markers for identifying V. anagallis-aquatica and V. undulata. The phylogenetic tree indicates that there is a close genetic relationship between the genera Veronica and Neopicrorhiza, and V. anagallis-aquatica and V. undulata are sister groups. The molecular clock analysis results indicate that the divergence time of Veronica may occur at ∼ 9.09 Ma, and the divergence time of these two species occurs at ∼ 0.48 Ma. It is speculated that climate change may be the cause of Veronica species diversity and promote the radiation of the genus. The chloroplast genome data of nine Veronica specie provides important insights into the characteristics and evolution of the chloroplast genome of this genus, as well as the phylogenetic relationships of the genus Veronica.

A proposed update of African swine fever virus (genotype II) subgenotyping based on the central variable region (CVR) of Russian isolates.

African swine fever virus (ASFV) isolates are grouped and tracked through analysis of their central variable region (CVR) sequences. In this study, sequences of 70 ASFV isolates collected from different regions of Russia between 2018 and 2022 were analyzed. The analysis based on the CVR sequences indicated that the isolates belonged to three distinct groups. Group 1 shared 100% sequence identity to the isolate Georgia 2007/1. Group 5 had a C > A single-nucleotide polymorphism (SNP) at position 601, while group 13 is new and unique to the Far East of Russia, with five isolates from the Amur, Khabarovsk, and Primorsky regions. These findings demonstrate a new approach to phylogenomics and cladistics of ASFV isolates within genotype II on the basis of the CVR.

Construction of CD138-targeted chimeric antigen receptor- modified T cells and their effect in multiple myeloma therapy

Objective: To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells. Methods: The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects. Results: ① We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. ② The purified CD138 (5G2) mAb can especially recognize CD138(+) cells with a binding affinity constant (K(D)) of 6.011×10(-9) mol/L and showed no significant binding activity with CD138(-) cells. ③The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.④ The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. ⑤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138(+) myeloma cell line H929, whereas CD138(-) cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (E∶T=1∶2; P<0.001). ⑥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P<0.001]. ⑦After co-culturing with CD138(+) cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P<0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P<0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P<0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138(-) cells. Conclusion: This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.

Beyond 16S sequencing for microbiome research: Nanopore sequencing resource at The University of Toledo Microbiome Consortium

Objective: The recognition that humans are holobionts has opened a new research area to examine the contributions of the microbiome to health and disease. This requires a full understanding of the identities of microbiota at the taxonomical level of the species. Introduction: The University of Toledo Microbiome Consortium currently offers the second-generation Illumina sequencing platform for 16S sequencing to determine microbiota composition. 16S sequencing of variable regions of the 16S operon, such as V3, V4, and the combination of V3 to V4, has long been the main stay for analyzing the composition of microbiota. The advantage of this approach is the ability to screen large numbers of samples at a lower cost per sample. However, the major drawback of this approach is a lower resolution whereby taxonomical identities of microbiota are limited to the genus level. To overcome this disadvantage, we introduced the third-generation sequencing technology from Nanopore into our pipeline. The known advantages of this approach are longer reads and the ability to sequence DNA in the native form without PCR-induced bias. Here we sought to conduct a comparative study of the relative effciencies of the 16S versus the nanopore technology for metagenomics. Methods: Genomic DNA was isolated from control and experimental rat fecal samples provided by an end-user. Libraries were prepared using Ligation sequencing DNA V14 (SqK-LSK114) on a Flow cell R10.4.1and Lambda DNA was used as a sequencing control. The native Libraries were run for 72hr, and high accuracy base calling was performed. The Fastq files (passed) generated by GridION were run on cloud-based CZ-ID pipeline with host and human read filtering, the results were normalized to one million bases sequenced from reads of each sample. Results and Conclusions: The 16S sequencing data highlighted the relative abundance of different taxa up to genus level such as Lactobacillus and Erysipelatoclostridium in the experimental group where as Stepcococcus and prevotellaceaeGa6A1group in the control group. However, Nanopore sequencing further identified twenty different species of Lactobacillus and , four different species of Erysipelatoclostridium. Similarly, nanopore sequencing was able to profile diverse species belonging to genera prevotellaceaeGa6A1group and Streptococcus. This species level resolution will greatly help in focusing on specific species and the pathways that a researcher intends to pursue. Therefore, the nanopore sequencing presents an opportunity to explore sequencing with a small footprint in terms of instrumentation and associated costs that greatly helps even a smaller laboratory to adopt and reap the benefits of species level resolution of metagenomic data. Summary: This study correlated well with 16S sequencing results at the genus level, while offering the added benefit of species level Identification and classification. Authors acknowledge the National Institutes of Health for funding support to Bina Joe (R01HL1430820). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Analysis of the Diversity and Functional Potential of Bacterial Communities in Tuberculomas.

The dynamics of lung microbiota in tuberculosis remains poorly understood. Sequencing of variable regions of the 16S rRNA gene from surgically excised tuberculosis foci and biopsy specimens of normal lung tissue allowed characterization of the diversity and predictive potential of bacterial communities. Taxonomic diversity indices attested to differences in the structure of microbial communities between "healthy" lungs and tuberculomas. The microbial composition of "healthy" lungs varied in taxonomic diversity and was presented by both gram-positive and gram-negative bacteria with sufficiently similar metabolic potential. The microbiota of the examined tuberculomas consisted of Mycobacterium tuberculosis in 99.9% of cases. A significant part of the metabolic pathways predicted by PICRUSt2 included cholesterol catabolism, sulfate assimilation, and various pathways for the biosynthesis of cell wall components.

Effect of composting and storage on the microbiome and resistome of cattle manure from a commercial dairy farm in Poland.

Manure from food-producing animals, rich in antibiotic-resistant bacteria and antibiotic resistance genes (ARGs), poses significant environmental and healthcare risks. Despite global efforts, most manure is not adequately processed before use on fields, escalating the spread of antimicrobial resistance. This study examined how different cattle manure treatments, including composting and storage, affect its microbiome and resistome. The changes occurring in the microbiome and resistome of the treated manure samples were compared with those of raw samples by high-throughput qPCR for ARGs tracking and sequencing of the V3-V4 variable region of the 16S rRNA gene to indicate bacterial community composition. We identified 203 ARGs and mobile genetic elements (MGEs) in raw manure. Post-treatment reduced these to 76 in composted and 51 in stored samples. Notably, beta-lactam, cross-resistance to macrolides, lincosamides and streptogramin B (MLSB), and vancomycin resistance genes decreased, while genes linked to MGEs, integrons, and sulfonamide resistance increased after composting. Overall, total resistance gene abundance significantly dropped with both treatments. During composting, the relative abundance of genes was lower midway than at the end. Moreover, higher biodiversity was observed in samples after composting than storage. Our current research shows that both composting and storage effectively reduce ARGs in cattle manure. However, it is challenging to determine which method is superior, as different groups of resistance genes react differently to each treatment, even though a notable overall reduction in ARGs is observed.

The Transmission of Campylobacter Strains in Dairy Herds in Different Housing Systems.

Cattle are frequent carriers of Campylobacter spp.; therefore, these bacteria may be transmitted to humans through meat or milk. Campylobacter spp. in raw milk derives most commonly from secondary fecal contamination during the milking process; however, the udder excretion of Campylobacter may be a cause of milk-borne infection. Studies were carried out on a Campylobacter-positive farm with two different housing systems (with free-stall and tie-stall systems). The sampling process comprised several stages, including samples being taken from animals, such as from raw milk and feces, and from the environment, such as the from floor in the milking parlor and from teat cups. None of the individual raw milk samples or swabs from the floor in the parlor before the milking process were positive for Campylobacter spp. Simultaneously, Campylobacter spp. was isolated from all swabs from the floor after the milking process and in the bulk tank milk samples from the two farms. The incidence of Campylobacter isolated from fecal and teat swab samples ranged from 15.4% to 26.7% and from 8.9% to 25%, respectively. Altogether, 59 recovered Campylobacter isolates were classified, based on sequencing of the flaA short variable region, showing 15 different allele types, and the majority of them were distributed among one farm. Analysis of the virulence and antimicrobial properties showed that genes related to adherence, invasion and cytotoxicity were widely distributed among the Campylobacter recovered strains. In relation to AMR, multidrug resistance was noted in 16.1% of strains.

Prediction of polyspecificity from antibody sequence data by machine learning.

Antibodies are generated with great diversity in nature resulting in a set of molecules, each optimized to bind a specific target. Taking advantage of their diversity and specificity, antibodies make up for a large part of recently developed biologic drugs. For therapeutic use antibodies need to fulfill several criteria to be safe and efficient. Polyspecific antibodies can bind structurally unrelated molecules in addition to their main target, which can lead to side effects and decreased efficacy in a therapeutic setting, for example via reduction of effective drug levels. Therefore, we created a neural-network-based model to predict polyspecificity of antibodies using the heavy chain variable region sequence as input. We devised a strategy for enriching antibodies from an immunization campaign either for antigen-specific or polyspecific binding properties, followed by generation of a large sequencing data set for training and cross-validation of the model. We identified important physico-chemical features influencing polyspecificity by investigating the behaviour of this model. This work is a machine-learning-based approach to polyspecificity prediction and, besides increasing our understanding of polyspecificity, it might contribute to therapeutic antibody development.

Generation of human antibodies targeting human platelet antigen (HPA)-1a.

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a condition during pregnancy, which can lead to thrombocytopenia and a bleeding tendency with intracranial hemorrhage (ICH) being the most concerning complication in the fetus or neonate. An incompatibility between human platelet antigen (HPA)-1a accounts for the majority of FNAIT cases. Binding of HPA-1a-specific alloantibodies to their target on fetal platelets and endothelial cells can induce apoptosis of megakaryocytes, disrupt platelet function, and impair angiogenesis. Currently, there is no screening program to identify pregnancies at risk for severe disease. A better understanding of HPA-1a-specific antibody heterogeneity in FNAIT could aid in identifying pathogenic antibody properties linked to severe disease. This study aimed to isolate HPA-1a-specific B-cells from an HPA-1a-alloimmunized pregnant woman. Using fluorescently labeled HPA-1a-positive platelets, single B-cells were sorted and cultured for 10 days to stimulate antibody production. Subsequently, supernatants were tested for the presence of antibodies by enzyme-linked immunosorbent assay and their reactivity towards HPA-1a-positive platelets. Amplification and sequencing of variable regions allowed the generation of monoclonal antibodies using a HEK-Freestyle-based expression system. Three platelet-specific B-cells were obtained and cloned of which two were specific for HPA-1a, named D- and M-204, while the third was specific for HLA class I, which was named L-204. This study outlined an effective method for the isolation of HPA-1a-specific B-cells and the generation of monoclonal antibodies. Further characterization of these antibodies holds promise for better understanding the pathogenic nature of alloantibodies in FNAIT.

Chicken γδ T cells proliferate upon IL-2 and IL-12 treatment and show a restricted receptor repertoire in cell culture.

In chickens, γδ T cells represent a large fraction of peripheral T cells; however, their function remains largely unknown. Here, we describe the selective in vitro expansion of γδ T cells from total splenocytes by stimulation with the cytokines IL-2 and IL-12. Under these conditions, γδ T cells proliferated preferentially and reached frequencies of >95% within three weeks. Although IL-2 alone also triggered proliferation, an increased proliferation rate was observed in combination with IL-12. Most of the expanded cells were γδ TCR and CD8 double-positive. Splenocytes sorted into TCR1+CD8+, TCR1highCD8-, and TCR1lowCD8- subsets proliferated well upon dual stimulation with IL-2/IL-12, indicating that none of the three γδ T cell subsets require bystander activation for proliferation. TCR1+CD8+ cells maintained CD8 surface expression during stimulation, whereas CD8- subpopulations showed varied levels of CD8 upregulation, with the highest upregulation observed in the TCR1high subset. Changes in the γδ T-cell receptor repertoire during cell culture from day 0 to day 21 were analyzed by next-generation sequencing of the γδ variable regions. Overall, long-term culture led to a restricted γ and δ chain repertoire, characterized by a reduced number of unique variable region clonotypes, and specific V genes were enriched at day 21. On day 0, the δ chain repertoire was highly diverse, and the predominant clonotypes differed between animals, while the most frequent γ-chain clonotypes were shared between animals. However, on day 21, the most frequent clonotypes in both the γ and δ chain repertoires were different between animals, indicating that selective expansion of dominant clonotypes during stimulation seems to be an individual outcome. In conclusion, IL-2 and IL-12 were sufficient to stimulate the in vitro outgrowth of γδ T cells. Analyses of the TCR repertoire indicate that the culture leads to an expansion of individual T cell clones, which may reflect previous in vivo activation. This system will be instrumental in studying γδ T cell function.

Genetic Characteristics of Non B Cell-Derived Immunoglobulin Genes.

It is widely acknowledged that immunoglobulins (Igs) are produced solely by B-lineage cells. The Ig gene is created by the rearrangement of a group of gene segments [variable (V), diversity (D), and joining (J) segments rearrangement, or V(D)J recombination], which results in the vast diversity of B cell-derived Ig responsible for recognising various antigens. Ig subsequently undergoes somatic hypermutation (SHM) and class switch recombination (CSR) after exposure to antigens, thus converting the low-affinity IgM to IgG, IgA, or IgE antibodies. IgM and IgD are primarily expressed in naïve B cells that have not been exposed to antigens, they do not undergo somatic hypermutation; hence, their variable region sequences remain the same as those in the germline. In contrast, IgG, IgA, and IgE are expressed in antigen-stimulated memory B cells or plasma cells, and thus, they often possess high-frequency mutations in their variable region sequences. Since the discovery that Ig can be produced by non-B cells, Qiu's group has investigated and compared the genetic characteristics of B cell-derived Ig and non-B cell-derived Ig. These findings demonstrated that non-B cell-derived Ig shares certain similarities with B cell-derived Ig in that the sequence of its constant region is identical to that of B cell-derived Ig, and its variable region is also strictly dependent on the rearrangement of V, D, and J gene segments. Moreover, akin to B cell-derived Ig, the V regions of IgM and IgD are rarely mutated, while IgG, IgA, and IgE produced by cancer cells are frequently mutated. However, the non-B cell-derived Ig V region sequence displays unique characteristics. (1) Unlike the vast diversity of B cell-derived Igs, non-B cell-derived Igs exhibit restricted diversity; cells from the same lineage always select the same V(D)J recombination patterns; (2) Both mRNA and proteins of RAG1/RAG2 recombinase have been detected in Ig positive cancer cell lines and normal tissues. But Ig recombination could also be found in RAG1-/- and RAG2-/- mice, suggesting that they are not necessary for the rearrangement of non-B cell-derived Igs. These features of non-B cell-derived Igs suggest a potentially undiscovered mechanism of V(D)J recombination, ligation, and SHM in non-B cells, which necessitates further investigation with advanced technology in molecular biology.

Functions and Clinical Significance of Myocardial Cell-Derived Immunoglobulins.

Immunoglobulins (Igs) have been widely accepted to be exclusively expressedby B cells. Nonetheless, this theory is challenged by mounting evidence which suggests thatIgs can also be generated by nonB cells (nonB-Ig), including cardiomyocytes (CM). NonB-Ig exhibits unique physical and chemical characteristics, unique variable region sequences and functions, which diverge from those of B-Ig. For instance, nonB-Ig demonstrates hydrophobicity, limited diversity in the variable region, and extracellular matrix protein activity. Likewise, cardiomyocytes can express different classes of Igs, including IgM, IgG, and free Igκ light chains (cardiomyocyte derived-Igs, CM-Igs). In particular, CM-Igs can be secreted into the extracellular space in various cardiovascular diseases, such as myocardial ischaemia and myocardial fibrosiswhere they might be involved in complement activationand direct damage to cardiomyocytes. Nevertheless, the precise pathological activity of CM-Igs remains unclear. Recently, Zhu et al. focused on studying the sequence characteristics and functions of CM-Igκ; they discovered that the CM-Igκ exhibits a unique VJ recombination pattern, high hydrophobicity, and isprincipally located on the intercalated discs and cross striations of the cardiomyocytes. Interestingly, loss of Igκ in cardiomyocytes results in structural disorders in intercalated discs and dysfunction in myocardial contraction and conduction. Mechanically, Igκ promotes the stabilisation of plectin, a cytoskeleton cross-linker protein that connects desmin to desomsome, to maintain the normal structure of the intercalated disc. This finding indicatesthatCM-Igκ plays an integral role in maintaining cytoskeleton structure. Consequently, it is imperative to reveal the physiological functions and mechanisms of pathological injury associated with CM-Igs.

Cancer-Derived Immunoglobulin G and Pancreatic Cancer.

Immunoglobulin (Ig) is traditionally believed to be produced solely by B cells. Nonetheless, mounting evidence has demonstrated that various types of Igs are extensively expressed in many cell types. Among them, IgG is found to be highly expressed in cancer cells and is thus labeled as cancer-derived IgG. Cancer-derived IgG shares identical fundamental structures with B cell-derived IgG, but displays several unique characteristics, including restricted variable region sequences and unique glycosylation modifications for those expressed by epithelial cancers. Cancer-derived IgG plays multiple crucial roles in carcinogenesis, including facilitating cancer invasion and metastasis, enhancing cancer stemness, contributing to chemoresistance, and remodeling the tumour microenvironment. Recent studies have discovered that cancer-derived sialylated IgG (SIA-IgG) is extensively expressed in pancreatic cancer cells and is predominantly located in the cytoplasm and on the cell membrane. Cancer-derived IgG expressed by pancreatic cancer presents a restrictive variable region sequence and contains a unique sialylation site of the Fab region. Functionally, cancer-derived IgG participates in pancreatic cancer progression via different mechanisms, such as promoting proliferation, facilitating migration and invasion, resisting apoptosis, inducing inflammation, and modulating the tumour microenvironment. SIA-IgG has shown potential as a clinical biomarker. The expression of SIA-IgG is associated with poor tumour differentiation, metastasis, and chemoresistance in pancreatic cancer. High expression of SIA-IgG can serve as an independent prognostic factor for pancreatic cancer. Additionally, SIA-IgG expression elevated with malignant progression for the precursor lesions of pancreatic cancer. These findings present a prospect of applying cancer-derived IgG as a novel diagnostic and therapeutic target in the management of pancreatic cancer, and aiding in overcoming the challenge in the treatment of this stubborn malignancy.