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Obesity is characterized by chronic low-grade inflammation. Caveolin-1 (CAV1), a structural and functional protein found in adipose tissues (AT), is augmented in obese individuals. We aimed to define the inflammatory mediators that influence CAV1 gene regulation and associated mechanism in obesity. Using subcutaneous AT from 27 (7 lean/20 obese) normoglycemic individuals, in vitro human adipocyte models, and in vivo mice models, we found elevated CAV1 expression in obese AT and a positive correlation between the gene expression of CAV1, tumor necrosis factor alpha (TNF-α), and the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB). CAV1 gene expression was associated with that of proinflammatory cytokines/chemokines, and their cognate receptor (r ≥ 0.447, p ≤ 0.030) but not with anti-inflammatory markers. CAV1 expression was correlated with CD163, indicating a prospective role for CAV1 in adipose inflammatory microenvironment. Unlike wild-type animals, mice lacking TNF-α exhibited reduced levels of CAV1 mRNA/proteins, which were elevated by administering exogenous TNF-α. Mechanistically, TNF-α induces CAV1 gene transcription by mediating NF-kB binding to its two regulatory elements located in the CAV1 proximal regulatory region. The interplay between CAV1 and TNF-α signaling pathway is interesting and has potential as a target for therapeutic interventions in obesity and metabolic syndromes.

Background End-stage renal disease (ESRD) is a growing cause of morbidity worldwide. Protein malnutrition is common among patients with ESRD. Peritoneal dialysis (PD) offers greater lifestyle flexibility and independence compared to the widely used treatments for ESRD. In this study, we aimed to evaluate the nutritional status and the quality of life (QOL) along with the factors influencing these two outcomes among Palestinian patients undergoing PD. Methods We performed a cross-sectional study on patients receiving PD at Najah Hospital University, Palestine. Malnutrition was assessed by the malnutrition-inflammation scale (MIS) and the QOL score was evaluated by using the Dutch WHOQOL-OLD module. Results A total of 74 patients on PD were included with the mean age of participants was 50.5 ± 16.38, more than half of them were females. We observed a significant association between the MIS and the WHOQOL-OLD scores (p p = 0.01). Patients with pitting edema and diabetes had higher risk of a lower QOL (p

The imipramine ONC201 has antiproliferative effects in several cancer cell types and activates integrated stress response pathway associated with the induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We investigated the signaling pathways through which ONC201/CHOP crosstalk is regulated in ONC201-treated nonmetastatic and metastatic cancer cell lines (Dukes' type B colorectal adenocarcinoma nonmetastatic SW480 and metastatic LS-174T cells, respectively). Cell proliferation and apoptosis were evaluated by MTT assays and flow cytometry, gene expression was assessed by Affymetrix microarray, signaling pathway perturbations were assessed in silico, and key regulatory proteins were validated by Western blotting. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment; Gene Ontology analysis of differentially expressed genes showed that cellular responsiveness to ONC201 treatment also differed substantially. In both ONC201-treated cell lines, CHOP expression was upregulated; however, its upstream regulatory mechanisms were perturbed. Although, PERK, ATF6 and IRE1 ER-stress pathways upregulated CHOP in both cell types, the Bak/Bax pathway regulated CHOP only LS-174T cells. Additionally, CHOP RNA splicing profiles varied between cell lines; these were further modified by ONC201 treatment. In conclusion, we delineated the signaling mechanisms by which CHOP expression is regulated in ONC201-treated non-metastatic and metastatic colorectal cell lines. The observed differences could be related to cellular plasticity and metabolic reprogramming, nevertheless, detailed mechanistic studies are required for further validations.

Studies have implicated CAV1 in the pathophysiology of diabetes and obesity. Previously, we demonstrated a potential association between the CAV1 rs1997623 C/A variant and metabolic syndrome (MetS) in Kuwaiti children. In the present study, we substantiate the association of CAV1 with MetS in adult Arab individuals. Method: The CAV1 rs1997623 was genotyped in three cohorts of Arabs (n=479) , South Asians (n=660) and South East Asians (n=362) . MetS status of the individuals was diagnosed using the IDF criteria (i.e., presence of central obesity and of at least two abnormalities of high TG, low HDL, hypertension, or type 2 diabetes) . The quantitative measure of MetS was calculated as siMS=2*WC/Height + FBG/5.6 + TG/1.7 + SBP/130 - HDL/1.02 for males or HDL/1.28 for females. Allelic associations with quantitative and dichotomous traits were assessed using linear and logistic regression, respectively, with and adjustment for age and sex. CAV1 transcripts were quantified in adipose tissues from a cohort of 50 Arab individuals differing in siMS score. Results: The CAV1 variant was significantly associated with MetS status (OR=1.8[1.25-2.61]; P-value=0.0015; Pemp=0.0013) and with siMS (Effect size = 0.206; P-value=0.0035; Pemp=0.0028) in the cohort of Arab individuals. The variant was weakly and insignificantly associated in the cohorts of South Asian and South East Asian individuals (OR= 1.and 1.11; P-values= 0.25 and 0.67, respectively) . siMS scores correlated positively with mRNA levels of CAV1 in adipose tissue. Conclusion: The association of CAV1 rs1997623 C/A with MetS in Arab pediatric is now demonstrated in adults as well. Higher expression levels were seen in individuals poses increasing MetS rates further validates the implication of CAV1 rs1997623 C/A variant in MetS phenotype. Only a weak association signal was seen in the South Asian and South East Asian populations leading us to propose that the CAV1 rs1997623 association with MetS is probably specific to Arab ethnicity. Disclosure A.Al madhoun: None. P.Hebbar: None. R.Nizam: None. D.Haddad: None. M.H.Dashti: None. M.Abu-farha: None. R.Ahmad: None. A.T.Thangavel: None. F.Almulla: None. Funding Kuwait Foundation for the Advancement of Sciences (KFAS) and Dasman Diabetes Institute, Project RA CB-2021-007

We Have previously established that caveolin-1 (CAV1) rs1997623 C/A variant is associated with metabolic syndrome in obese children (PMID: 30622557) and adults (ADA 82) . Here, we evaluated the functional role of the identified variant in association with obesity. Sequence analysis revealed that the variant is located at CAV1 regulatory region and creates a site for EBF Transcription Factor 1 (EBF1) . DNA fragment (240pb) flanking the variant was cloned upstream luciferase reporter gene. Wildtype, CAV1-C-Luc, and the generated mutant, CAV1-A-Luc, were individually transfected into pre-adipocytes. CAV1-A-luc significantly upregulated Luciferase activity indicating a prospective functional role of CAV1-A allele in CAV1 regulation. The reporter gene activity was differential regulated in lean versus obese pre-adipocytes. Co-transfections with EBF1 construct resulted in a significant upregulation in CAV1-A Luc activity, particularly in pre-adipocytes from obese individuals. The role of EBF1 was further evaluated in vivo using ChIP assays. PCR analysis using specific primers flanking the CAV1 variant and anti-bodies against EBF1 showed a significant elevated enrichment of chromatins from obese individuals, which was also associated with an enrichment for H3K27ac at the same locus, indicating active transcription. Moreover, CAV1 expression was differentially regulated in response to EBF1 siRNA/overexpression in pre-adipocytes harboring CAV1 A/A or A/C alleles relative to that with C/C allele. Furthermore, a significant reduction of DNA methylation at the variant locus was observed particularly in preadipocytes with A/A alleles. Finally, RNA isolated from adipose tissue of lean and obese individuals showed a significant correlation between CAV1 and cytokines transcripts in healthy obese individuals, at least in part, is due to CAV1 rs1997623 variant as verified by sequence analysis. Taken together, our study delineates the role of CAV1 rs1997623 variant in the context of obesity and MetS. Disclosure A.Al madhoun: None. D.Haddad: None. S.P.Kochumon: None. R.Nizam: None. S.Jacob: None. S.T.Sindhu: None. R.Ahmad: None. F.Almulla: None. Funding Kuwait Foundation for the Advancement of Sciences (KFAS) and Dasman Diabetes Institute, Project RA CB-2021-007

The imipramine ONC201 exerts a novel anti-proliferative activity over a wide spectrum of cancer cell types. ONC201 activates integrated stress response pathway that is associated with induction of Damage Inducible Transcript 3 (DDIT3, also known as C/EBP homologous protein or CHOP). We questioned whether the ONC201/CHOP crosstalk is regulated by diverse signaling pathways in non-metastatic versus metastatic cancer cell lines. Therefore, the Dukes' type B colorectal adenocarcinoma non-metastatic (SW480) and metastatic (LS-174T) cell lines were treated with ONC201. Cell proliferation and apoptosis were evaluated by MTT assay, flow cytometry analysis, gene expression was assessed by Affymetrix microarray, and key regulatory proteins were validated by Western blot assays. Unlike LS-174T cells, SW480 cells were resistant to ONC201 treatment. Gene ontology pathway enrichment analysis of differentially expressed genes revealed substantial differences between LS-174T and SW480 responsiveness to ONC201 treatment. In both cell lines, CHOP expression was upregulated in response to ONC201 treatment, however, its upstream regulatory mechanisms were not identical. Although, PERK, ATF6 and IRE1 ER-stress pathways were found to upregulated CHOP in both cell types, the BAK/BAX pathway was a notable regulator of CHOP in the metastatic LS-174T cells alone. In addition, CHOP RNA splicing profiles were varied between the two cell lines, which was further modified in response to ONC201 treatments. In conclusion, we delineated the signaling mechanisms regulating the expression of CHOP in non-metastatic versus metastatic colorectal cells in response to ONC201 treatment. The observed differences were related to cellular plasticity and metabolic reprogramming.

COVID-19 is challenging healthcare preparedness, world economies, and livelihoods. The infection and death rates associated with this pandemic are strikingly variable in different countries. To elucidate this discrepancy, we analyzed 2431 early spread SARS-CoV-2 sequences from GISAID. We estimated continental-wise admixture proportions, assessed haplotype block estimation, and tested for the presence or absence of strains recombination. Herein, we identified 1010 unique missense mutations and seven different SARS-CoV-2 clusters. In samples from Asia, a small haplotype block was identified; whereas, samples from Europe and North America harbored large and different haplotype blocks with nonsynonymous variants. Variant frequency and linkage disequilibrium varied among continents, especially in North America. Recombination between different strains was only observed in North American and European sequences. Additionally, we structurally modeled the two most common mutations D614G and P314L which suggested that these linked mutations may enhance viral entry and stability. Overall, we propose that COVID-19 virulence may be more severe in Europe and North America due to coinfection with different SARS-CoV-2 strains leading to genomic recombination which might be challenging for current treatment regimens and vaccine development. Furthermore, our study provides a possible explanation for the more severe second wave of COVID-19 that many countries are currently experiencing presented as higher rates of infection and death.

COVID-19 is challenging healthcare preparedness, world economies, and livelihoods. The infection and death rates associated with this pandemic are strikingly variable in different countries. To elucidate this discrepancy, we analyzed 2431 early spread SARS-CoV-2 sequences from GISAID. We estimated continental-wise admixture proportions, assessed haplotype block estimation, and tested for the presence or absence of strains’ recombination. Herein, we identified 1010 unique missense mutations and seven different SARS-CoV-2 clusters. In samples from Asia, a small haplotype block was identified, whereas samples from Europe and North America harbored large and different haplotype blocks with nonsynonymous variants. Variant frequency and linkage disequilibrium varied among continents, especially in North America. Recombination between different strains was only observed in North American and European sequences. In addition, we structurally modelled the two most common mutations, Spike_D614G and Nsp12_P314L, which suggested that these linked mutations may enhance viral entry and replication, respectively. Overall, we propose that genomic recombination between different strains may contribute to SARS-CoV-2 virulence and COVID-19 severity and may present additional challenges for current treatment regimens and countermeasures. Furthermore, our study provides a possible explanation for the substantial second wave of COVID-19 presented with higher infection and death rates in many countries.

Wharton&rsquo, s jelly-derived mesenchymal stem cells (WJ-MSCs) are a valuable tool in stem cell research due to their high proliferation rate, multi-lineage differentiation potential, and immunotolerance properties. However, fibroblast impurity during WJ-MSCs isolation is unavoidable because of morphological similarities and shared surface markers. Here, a proteomic approach was employed to identify specific proteins differentially expressed by WJ-MSCs in comparison to those by neonatal foreskin and adult skin fibroblasts (NFFs and ASFs, respectively). Mass spectrometry analysis identified 454 proteins with a transmembrane domain. These proteins were then compared across the different cell-lines and categorized based on their cellular localizations, biological processes, and molecular functions. The expression patterns of a selected set of proteins were further confirmed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blotting, and immunofluorescence assays. As anticipated, most of the studied proteins had common expression patterns. However, EphA2, SLC25A4, and SOD2 were predominantly expressed by WJ-MSCs, while CDH2 and Talin2 were specific to NFFs and ASFs, respectively. Here, EphA2 was established as a potential surface-specific marker to distinguish WJ-MSCs from fibroblasts and for prospective use to prepare pure primary cultures of WJ-MSCs. Additionally, CDH2 could be used for a negative-selection isolation/depletion method to remove neonatal fibroblasts contaminating preparations of WJ-MSCs.

Adenylate cyclases (ADCYs) catalyze the conversion of ATP to cAMP, an important co-factor in energy homeostasis. Giving ADCYs role in obesity, diabetes and inflammation, we questioned whether calcium-stimulated ADCY isoforms may be variably detectable in human plasma. We report the results of a cross-sectional study assessing circulating levels of functional ADCY1, &minus, 3 and &minus, 8 in patients with T2D vs. non-diabetic (ND) controls in association with obesity. ADCY1 levels exhibited no significant change between ND and T2D groups. ADCY3 levels were lower in obese individuals, albeit not statistically significantly. In contrast, ADCY8 plasma levels were significantly higher in obese and T2D patients compared to controls (p = 0.001) and patients with T2D only (p = 0.039). ADCY8 levels correlated positively with body mass index and Hb1Ac levels. Parallel to the increased ADCY8 levels, significantly higher cAMP levels were observed in patients with T2D compared with ND controls, and further elevated in obese individuals, irrespective of T2D status. Additionally, cAMP levels positively correlated with fasting plasma glucose levels. In conclusion, the current cross-sectional study demonstrated elevated levels of circulating plasma ADCY8 and cAMP in obesity and T2D.

The severity of the new COVID-19 pandemic caused by the SARS-CoV-2 virus is strikingly variable in different global populations. SARS-CoV-2 usesACE2as a cell receptor,TMPRSS2protease, andFURINpeptidase to invade human cells. Here, we investigated 1,378 whole-exome sequences of individuals from the Middle Eastern populations (Kuwait, Qatar, and Iran) to explore natural variations in theACE2,TMPRSS2,andFURINgenes. We identified two activating variants (K26R and N720D) in theACE2gene that are more common in Europeans than in the Middle Eastern, East Asian, and African populations. We postulate that K26R can activateACE2and facilitate binding to S-protein RBD while N720D enhancesTMPRSS2cutting and, ultimately, viral entry. We also detected deleterious variants inFURINthat are frequent in the Middle Eastern but not in the European populations. This study highlights specific genetic variations in theACE2andFURINgenes that may explain SARS-CoV-2 clinical disparity. We showed structural evidence of the functionality of these activating variants that increase the SARS-CoV-2 aggressiveness. Finally, our data illustrate a significant correlation betweenACE2variants identified in people from Middle Eastern origins that can be further explored to explain the variation in COVID-19 infection and mortality rates globally.

It is estimated that in 2017 there were 451 million people with diabetes worldwide. These figures are expected to increase to 693 million by 2045; thus, innovative preventative programs and treatments are a necessity to fight this escalating pandemic disorder. Caveolin-1 (CAV1), an integral membrane protein, is the principal component of caveolae in membranes and is involved in multiple cellular functions such as endocytosis, cholesterol homeostasis, signal transduction, and mechanoprotection. Previous studies demonstrated that CAV1 is critical for insulin receptor-mediated signaling, insulin secretion, and potentially the development of insulin resistance. Here, we summarize the recent progress on the role of CAV1 in diabetes and diabetic complications.

During an adaptive immune response, B cells can change their surface immunoglobulins from IgM to IgG, IgE or IgA through a process called class switch recombination (CSR). Switching is preceded by inducible non-coding germline transcription (GLT) of the selected constant gene(s), which is largely controlled by a super-enhancer called the 3′ regulatory region (3′RR). Despite intense efforts, the precise mechanisms that regulate GLT are still elusive. In order to gain additional insights into these mechanisms, we analyzed GLT and CSR in mutant B cells carrying a duplication of the promoter of the α constant gene (Iα) downstream of 3′RR. Duplication of the Iα promoter affected differently GLT and CSR. While for most isotypes a drop in GLT was accompanied by a decrease in CSR, that was not the case for switching to IgA, which diminished despite unchanged GLT. Unexpectedly, there was no obvious effect on GLT and CSR to IgG3. Remarkably, specific stimuli that normally induce switching to IgG2b had contrasting effects in mutant B cells; Iγ2b was now preferentially responsive to the stimulus that induced Iα promoter. We propose that one mechanism underlying the induced 3′RR-mediated activation of GL promoters involves, at least in part, specific transcription factories.

International audience; Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to switch from IgM expression to the expression of downstream isotypes. CSR is preceded by inducible germline (GL) transcription of the constant genes and is controlled by the 3′ regulatory region (3′RR) in a stimulus-dependent manner. Why the 3′RR-mediated up-regulation of GL transcription is delayed to the mature B-cell stage is presently unknown. Here we show that mice devoid of an inducible CTCF binding element, located in the α constant gene, display a marked isotype-specific increase of GL transcription in developing and resting splenic B cells and altered CSR in activated B cells. Moreover, insertion of a GL promoter downstream of the CTCF insulator led to premature activation of the ectopic promoter. This study provides functional evidence that the 3′RR has a developmentally controlled potential to constitutively activate GL promoters but that this activity is delayed, at least in part, by the CTCF insulator, which borders a transcriptionally active domain established by the 3′RR in developing B cells.

Post-translational modification of the p53 signaling pathway plays an important role in cell cycle progression and stress-induced apoptosis. Indeed, a large body of work has shown that dysregulation of p53 and its E3 ligase MDM2 by the ubiquitin-proteasome system (UPS) promotes carcinogenesis and malignant transformation. Thus, drug discovery efforts have focused on the restoration of wild-type p53 activity or inactivation of oncogenic mutant p53 by targeted inhibition of UPS components, particularly key deubiquitinases (DUBs) of the ubiquitin-specific protease (USP) class. However, development of selective small-molecule USP inhibitors has been challenging, partly due to the highly conserved structural features of the catalytic sites across the class. To tackle this problem, we devised a protein engineering strategy for rational design of inhibitors for DUBs and other UPS proteins. We employed a phage-displayed ubiquitin variant (UbV) library to develop inhibitors targeting the DUBs USP7 and USP10, which are involved in regulating levels of p53 and MDM2. We were able to identify UbVs that bound USP7 or USP10 with high affinity and inhibited deubiquitination activity. We solved the crystal structure of UbV.7.2 and rationalized the molecular basis for enhanced affinity and specificity for USP7. Finally, cell death was increased significantly by UbV.7.2 expression in a colon cancer cell line that was treated with the chemotherapy drug cisplatin, demonstrating the therapeutic potential of inhibiting USP7 by this approach.

Class switch recombination (CSR) in B cells requires the timely repair of DNA double-stranded breaks (DSBs) that result from lesions produced by activation-induced cytidine deaminase (AID). Through a genome-wide RNAi screen, we identified Kin17 as a gene potentially involved in the maintenance of CSR in murine B cells. In this study, we confirm a critical role for Kin17 in CSR independent of AID activity. Furthermore, we make evident that DSBs generated by AID or ionizing radiation require Kin17 for efficient repair and resolution. Our report shows that reduced Kin17 results in an elevated deletion frequency following AID mutational activity in the switch region. In addition, deficiency in Kin17 affects the functionality of multiple DSB repair pathways, namely homologous recombination, non-homologous end-joining, and alternative end-joining. This report demonstrates the importance of Kin17 as a critical factor that acts prior to the repair phase of DSB repair and is of bona fide importance for CSR.

Class switch recombination (CSR) occurs between highly repetitive sequences called switch (S) regions and is initiated by activation-induced cytidine deaminase (AID). CSR is preceded by a bidirectional transcription of S regions but the relative importance of sense and antisense transcription for CSR in vivo is unknown. We generated three mouse lines in which we attempted a premature termination of transcriptional elongation by inserting bidirectional transcription terminators upstream of Sμ, upstream of Sγ3 or downstream of Sγ3 sequences. The data show, at least for Sγ3, that sense transcriptional elongation across S region is absolutely required for CSR whereas its antisense counterpart is largely dispensable, strongly suggesting that sense transcription is sufficient for AID targeting to both DNA strands.

Class switch recombination (CSR) is mediated by G-rich tandem repeated sequences termed switch regions. Transcription of switch regions generates single-stranded R loops that provide substrates for activation-induced cytidine deaminase. Mice deficient in MSH2 have a mild defect in CSR and analysis of their switch junctions has led to a model in which MSH2 is more critical for switch recombination events outside than within the tandem repeats. It is also known that deletion of the whole Sμ region severely impairs but does not abrogate CSR despite the lack of detectable R loops. Here, we demonstrate that deficiency of both MSH2 and the Sμ region completely abolishes CSR and that the abrogation occurs at the genomic level. This finding further supports the crucial role of MSH2 outside the tandem repeats. It also indicates that during CSR, MSH2 has access to activation-induced cytidine deaminase targets in R-loop-deficient Iμ-Cμ sequences rarely used in CSR, suggesting an MSH2-dependent DNA processing activity at the Iμ exon that may decrease with transcription elongation across the Sμ region.

The NeoR gene has often been used to unravel the mechanisms underlying long-range interactions between promoters and enhancers during V(D)J assembly and class switch recombination (CSR) in the immunoglobulin heavy chain (IgH) locus. This approach led to the notion that CSR is regulated through competition of germ-line (GL) promoters for activities displayed by the 3' regulatory region (3'RR). This polarized long-range effect of the 3'RR is disturbed upon insertion of NeoR gene in the IgH constant (C(H)) region, where only GL transcription derived from upstream GL promoters is impaired. In the context of V(D)J recombination, replacement of Emu enhancer or Emu core enhancer (cEmu) by NeoR gene fully blocked V(D)J recombination and mu0 GL transcription which originates 5' of DQ52 and severely diminished Imu GL transcription derived from Emu/Imu promoter, suggesting a critical role for cEmu in the regulation of V(D)J recombination and of mu0 and Imu expression. Here we focus on the effect of NeoR gene on mu0 and Imu GL transcription in a mouse line in which the Imu-Cmu intron was replaced by a NeoR gene in the sense-orientation. B cell development was characterized by a marked but incomplete block at the pro-B cell stage. However, V(D)J recombination was unaffected in sorted pro-B and pre-B cells excluding an interference with the accessibility control function of Emu. mu0 GL transcription initiation was relatively normal but the maturation step seemed to be affected most likely through premature termination at NeoR polyadenylation sites. In contrast, Imu transcription initiation was impaired suggesting an interference of NeoR gene with the IgH enhancers that control Imu expression. Surprisingly, in stark contrast with the NeoR effect in the C(H) region, LPS-induced NeoR expression restored Imu transcript levels to normal. The data suggest that Emu enhancer may be the master control element that counteracts the down-regulatory "Neo effect" on Imu expression upon LPS stimulation. More importantly, they reveal a complex and developmentally regulated interplay between IgH enhancers in the control of Imu expression.

Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG(-/-) cells, uracil levels increase by 11- to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG(-/-)splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels.

Upon activation with T-dependent Ag, B cells enter germinal centers (GC) and upregulate activation-induced deaminase (AID). AID+ GC B cells then undergo class-switch recombination and somatic hypermutation. Follicular dendritic cells (FDC) are stromal cells that underpin GC and require constitutive signaling through the lymphotoxin (LT) β receptor to be maintained in a fully mature, differentiated state. Although it was shown that FDC can be dispensable for the generation of affinity-matured Ab, in the absence of FDC it is unclear where AID expression occurs. In a mouse model that lacks mature FDC, as well as other LT-sensitive cells, we show that clusters of AID+PNA+GL7+ Ag-specific GC B cells form within the B cell follicles of draining lymph nodes, suggesting that FDC are not strictly required for GC formation. However, later in the primary response, FDC-less GC dissipated prematurely, correlating with impaired affinity maturation. We examined whether GC dissipation was due to a lack of FDC or other LTβ receptor–dependent accessory cells and found that, in response to nonreplicating protein Ag, FDC proved to be more critical for long-term GC maintenance. Our study provides a spatial-temporal analysis of Ag-specific B cell activation and AID expression in the context of a peripheral lymph node that lacks FDC-M1+ CD35+ FDC and other LT-sensitive cell types, and reveals that FDC are not strictly required for the induction of AID within an organized GC-like environment.

In B lymphocytes, class switch recombination (CSR) machinery targets highly repetitive sequences, called switch (S) sequences, in the constant domain of the immunoglobulin heavy chain (IgH) locus. Cotranscriptional generation of R loops at S sequences provides the substrate for the mutagenic enzyme AID (Activation-Induced cytidine Deaminase), which initiates the DNA breaks at the transcribed sequences. Both sense and antisense transcripts across the S regions have been reported. Our recent work shows that, unlike its sense counterpart, antisense transcription of S sequences is dispensable for CSR in vivo.