Ahmad Zaheen

AID constrains germinal center size by rendering B cells susceptible to apoptosis

The germinal center (GC) is a transient lymphoid tissue microenvironment that fosters T cell-dependent humoral immunity. Within the GC, the B cell-specific enzyme, activation-induced cytidine deaminase (AID), mutates the immunoglobulin locus, thereby altering binding affinity for antigen. In the absence of AID, larger GC structures are observed in both humans and mice, but the reason for this phenomenon is unclear. Because significant apoptosis occurs within the GC niche to cull cells that have acquired nonproductive mutations, we have examined whether a defect in apoptosis could account for the larger GC structures in the absence of AID. In this report, we reveal significantly reduced death of B cells in AID(-/-) mice as well as in B cells derived from AID(-/-) bone marrow in mixed bone marrow chimeric mice. Furthermore, AID-expressing B cells show decreased proliferation and survival compared with AID(-/-) B cells, indicating an AID-mediated effect on cellular viability. The GC is an etiologic site for B-cell autoimmunity and lymphomagenesis, both of which have been linked to aberrant AID activity. We report a link between AID-induced DNA damage and B-cell apoptosis that has implications for the development of B-cell disorders.

The RNF8/RNF168 ubiquitin ligase cascade facilitates class switch recombination

An effective immune response requires B cells to produce several classes of antibodies through the process of class switch recombination (CSR). Activation-induced cytidine deaminase initiates CSR by deaminating deoxycytidines at switch regions within the Ig locus. This activity leads to double-stranded DNA break formation at the donor and recipient switch regions that are subsequently synapsed and ligated in a 53BP1-dependent process that remains poorly understood. The DNA damage response E3 ubiquitin ligases RNF8 and RNF168 were recently shown to facilitate recruitment of 53BP1 to sites of DNA damage. Here we show that the ubiquitination pathway mediated by RNF8 and RNF168 plays an integral part in CSR. Using the CH12F3-2 mouse B cell line that undergoes CSR to IgA at high rates, we demonstrate that knockdown of RNF8, RNF168, and 53BP1 leads to a significant decrease in CSR. We also show that 53BP1-deficient CH12F3-2 cells are protected from apoptosis mediated by the MDM2 inhibitor Nutlin-3. In contrast, deficiency in either E3 ubiquitin ligase does not protect cells from Nutlin-3–mediated apoptosis, indicating that RNF8 and RNF168 do not regulate all functions of 53BP1.

Negative Supercoiling Creates Single-Stranded Patches of DNA That Are Substrates for AID–Mediated Mutagenesis

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID.

Activation-Induced Cytidine Deaminase and Aberrant Germinal Center Selection in the Development of Humoral Autoimmunities

Humoral immunity, which is the branch of the immune system governed by B cells, protects the body from extracellular pathogens through the secretion of immunoglobulins. Given the unpredictability of exogenous antigens, B cells must be accommodating to numerous genetic alterations to mold immunoglobulin specificity to recognize offending pathogens. Abnormalities in this process leave the host susceptible to permanent pathological modifications and in particular humoral autoimmunities in which secreted immunoglobulins mistake host proteins as pathogenic targets. Underlying the development of self-reactive immunoglobulins is activation-induced cytidine deaminase (AID), a mutagenic enzyme responsible for modifying the specificity of B cells by producing point mutations at the immunoglobulin gene locus. Ideally, these mutations result in an increased affinity for exogenous antigens. However, in pathological scenarios, these mutations produce or enhance a B cells ability to target the host. AID-induced mutations occur in the germinal center microenvironment of peripheral lymphoid tissue, where pathogenic B-cell clones must evade overwhelming selection pressures to be released systemically. Recent research has revealed numerous genes and pathways responsible for eliminating self-reactive clones within the germinal center. On the basis of these studies, this review aims to clarify the link between AID and the generation of pathogenic immunoglobulins. Furthermore, it describes the selective pressures that pathogenic B cells must bypass within the germinal center to secrete immunoglobulins that ultimately result in disease.

Improved Survival in Patients with Viral Hepatitis-Induced Hepatocellular Carcinoma Undergoing Recommended Abdominal Ultrasound Surveillance in Ontario: A Population-Based Retrospective Cohort Study.

The optimal schedule for ultrasonographic surveillance of patients with viral hepatitis for the detection of hepatocellular carcinoma (HCC) remains unclear owing to a lack of reliable studies. We examined the timing of ultrasonography in patients with viral hepatitis-induced HCC and its impact on survival and mortality risk while determining predictors of receiving surveillance before HCC diagnosis. A population-based retrospective cohort analysis of patients with viral hepatitis-induced HCC in Ontario between 2000 and 2010 was performed using data from the Ontario Cancer Registry linked health administrative data. HCC surveillance for 2 years preceding diagnosis was assigned as: i) ≥2 abdominal ultrasound screens annually; ii) 1 screen annually; iii) inconsistent screening; and iv) no screening. Survival rates were estimated using the Kaplan-Meier method and parametric models to correct for lead-time bias. Associations between HCC surveillance and the risk of mortality after diagnosis were examined using proportional-hazards regression adjusting for confounding factors. Overall, 1,483 patients with viral hepatitis-induced HCC were identified during the study period; 20.2% received ≥1 ultrasound screen annually (routine surveillance) for the 2 years preceding diagnosis. The 5-year survival of those receiving routine surveillance was 31.93% (95% CI: 25.77–38.24%) and 31.84% (95% CI: 25.69–38.14%) when corrected for lead-time bias (HCC sojourn time 70 days and 140 days, respectively). This is contrasted with 20.67% (95% CI: 16.86–24.74%) 5-year survival in those who did not undergo screening. In the fully adjusted model, compared to unscreened patients, routine surveillance was associated with a lower mortality risk and a hazard ratio of 0.76 (95% CI: 0.64–0.91) and 0.81 (95% CI: 0.68–0.97), corrected for the respective lead-time bias. Our findings suggest that routine ultrasonography in patients with viral hepatitis is associated with improved survival and reduced mortality risk in a population-based setting. The data emphasizes the importance of surveillance for timely intervention in HCC-diagnosed patients.

The Effect of HIV-Hepatitis C Co-Infection on Bone Mineral Density and Fracture: A Meta-Analysis

Objective There is a variable body of evidence on adverse bone outcomes in HIV patients co-infected with hepatitis C virus (HCV). We examined the association of HIV/HCV co-infection on osteoporosis or osteopenia (reduced bone mineral density; BMD) and fracture. Design Systematic review and random effects meta-analyses. Methods A systematic literature search was conducted for articles published in English up to 1 April 2013. All studies reporting either BMD (g/cm2, or as a T-score) or incident fractures in HIV/HCV co-infected patients compared to either HIV mono-infected or HIV/HCV uninfected/seronegative controls were included. Random effects meta-analyses estimated the pooled odds ratio (OR) and the relative risk (RR) and associated 95% confidence intervals (CI). Results Thirteen eligible publications (BMD N = 6; Fracture = 7) of 2,064 identified were included with a total of 427,352 subjects. No publications reported data on HCV mono-infected controls. Meta-analysis of cross-sectional studies confirmed that low bone mineral density was increasingly prevalent among co-infected patients compared to HIV mono-infected controls (pooled OR 1.98, 95% CI 1.18, 3.31) but not those uninfected (pooled OR 1.47, 95% CI 0.78, 2.78). Significant association between co-infection and fracture was found compared to HIV mono-infected from cohort and case-control studies (pooled RR 1.57, 95% CI 1.33, 1.86) and compared to HIV/HCV uninfected from cohort (pooled RR 2.46, 95% CI 1.03, 3.88) and cross-sectional studies (pooled OR 2.30, 95% CI 2.09, 2.23). Conclusions The associations of co-infection with prevalent low BMD and risk of fracture are confirmed in this meta-analysis. Although the mechanisms of HIV/HCV co-infection’s effect on BMD and fracture are not well understood, there is evidence to suggest that adverse outcomes among HIV/HCV co-infected patients are substantial.

Trends in relative survival in patients with a diagnosis of hepatocellular carcinoma in Ontario: a population-based retrospective cohort study.

BACKGROUND The incidence of hepatocellular carcinoma (HCC) is increasing and survival rates are poor. Our objectives were to estimate the relative survival over time in patients with HCC in Ontario and to examine potential factors associated with excess mortality risk. METHODS We performed a population-based retrospective cohort analysis involving patients with a diagnosis of HCC in Ontario between 1990 and 2009 using data extracted from the Ontario Cancer Registry. Relative survival was estimated by controlling for background mortality using expected mortality from Ontario life tables. A generalized linear model was used to estimate the excess mortality risk for important factors. RESULTS A total of 5645 patients had HCC diagnosed during the study period; 4412 (78.2%) of these patients were male. Improvements in 1-year relative survival were observed across all age groups over time: the highest was among those patients less than 60 years of age who had a diagnosis of HCC during 2005-2009, with 1-year survival exceeding 50% for both sexes. However, the overall 5-year relative survival did not exceed 28%. The excess mortality risk decreased with increased years of follow-up, recent diagnosis, and curative or noncurative treatments for HCC, whereas excess mortality risk increased with age. INTERPRETATION Although improving, the prognosis for HCC remains poor. Our findings highlight the importance of effective prevention and treatment for HCC to reduce the burden of disease and improve health care systems.

AID and Caspase 8 Shape the Germinal Center Response through Apoptosis

Germinal centers (GCs) are clusters of activated B cells that form in secondary lymphoid organs during a T-dependent immune response. B cells enter GCs and become rapidly proliferating centroblasts that express the enzyme activation-induced deaminase (AID) to undergo somatic hypermutation and class-switch recombination. Centroblasts then mature into centrocytes to undergo clonal selection. Within the GC, the highest affinity B cell clones are selected to mature into memory or plasma cells while lower affinity clones undergo apoptosis. We reported previously that murine Aicda−/− GC B cells have enhanced viability and accumulate in GCs. We now show that murine Aicda−/− GC B cells accumulate as centrocytes and inefficiently generate plasma cells. The reduced rate of plasma cell formation was not due to an absence of AID-induced DNA lesions. In addition, we show that the deletion of caspase 8 specifically in murine GC-B cells results in larger GCs and a delay in affinity maturation, demonstrating the importance of apoptosis in GC homeostasis and clonal selection.

Lack of MSH2 involvement differentiates V(D)J recombination from other non-homologous end joining events

V(D)J recombination and class switch recombination are the two DNA rearrangement events used to diversify the mouse and human antibody repertoires. While their double strand breaks (DSBs) are initiated by different mechanisms, both processes use non-homologous end joining (NHEJ) in the repair phase. DNA mismatch repair elements (MSH2/MSH6) have been implicated in the repair of class switch junctions as well as other DNA DSBs that proceed through NHEJ. MSH2 has also been implicated in the regulation of factors such as ATM and the MRN (Mre11, Rad50, Nbs1) complex, which are involved in V(D)J recombination. These findings led us to examine the role of MSH2 in V(D)J repair. Using MSH2−/− and MSH2+/+ mice and cell lines, we show here that all pathways involving MSH2 are dispensable for the generation of an intact pre-immune repertoire by V(D)J recombination. In contrast to switch junctions and other DSBs, the usage of terminal homology in V(D)J junctions is not influenced by MSH2. Thus, whether the repair complex for V(D)J recombination is of a canonical NHEJ type or a separate microhomology-mediated-end joining (MMEJ) type, it does not involve MSH2. This highlights a distinction between the repair of V(D)J recombination and other NHEJ reactions.

Cost-effectiveness analysis of treatment with non-curative or palliative intent for hepatocellular carcinoma in the real-world setting

Hepatocellular carcinoma (HCC) presentation is heterogeneous necessitating a variety of therapeutic interventions with varying efficacies and associated prognoses. Poor prognostic patients often undergo non-curative palliative interventions including transarterial chemoembolization (TACE), sorafenib, chemotherapy, or purely supportive care. The decision to pursue one of many palliative interventions for HCC is complex and an economic evaluation comparing these interventions has not been done. This study evaluates the cost-effectiveness of non-curative palliative treatment strategies such as TACE alone or TACE+sorafenib, sorafenib alone, and non-sorafenib chemotherapy compared with no treatment or best supportive care (BSC) among patients diagnosed with HCC between 2007 and 2010 in a Canadian setting. Using person-level data, we estimated effectiveness in life years and quality-adjusted life years (QALYs) along with total health care costs (2013 US dollars) from the health care payer’s perspective (3% annual discount). A net benefit regression approach accounting for baseline covariates with propensity score adjustment was used to calculate incremental net benefit to generate incremental cost-effectiveness ratio (ICER) and uncertainty measures. Among 1,172 identified patients diagnosed with HCC, 4.5%, 7.9%, and 5.6%, received TACE alone or TACE+sorafenib, sorafenib, and non-sorafenib chemotherapy clone, respectively. Compared with no treatment or BSC (81.9%), ICER estimates for TACE alone or TACE+sorafenib was