UBE2L3 regulates TLR7-induced B cell autoreactivity in Systemic Lupus Erythematosus.

Both TLR7 and NF-κB hyperactivity are known to contribute to pathogenesis in Systemic Lupus Erythematosus (SLE), driving a pro-interferon response, autoreactive B cell expansion and autoantibody production. UBE2L3 is an SLE susceptibility gene which drives plasmablast/plasma cell expansion in SLE, but its role in TLR7 signalling has not been elucidated. We aimed to investigate the role of UBE2L3 in TLR7-mediated NF-κB activation, and the effect of UBE2L3 inhibition by Dimethyl Fumarate (DMF) on SLE B cell differentiation in vitro. Our data demonstrate that UBE2L3 is critical for activation of NF-κB downstream of TLR7 stimulation, via interaction with LUBAC. DMF, which directly inhibits UBE2L3, significantly inhibited TLR7-induced NF-κB activation, differentiation of memory B cells and plasmablasts, and autoantibody secretion in SLE. DMF also downregulated interferon signature genes and plasma cell transcriptional programmes. These results demonstrate that UBE2L3 inhibition could potentially be used as a therapy in SLE through repurposing of DMF, thus preventing TLR7-driven autoreactive B cell maturation.

Persistence of antibody response to SARS-CoV-2 in a cohort of haemodialysis patients with COVID-19.

BACKGROUND: Haemodialysis patients are extremely vulnerable to COVID-19. Their immune response after infection is unclear. We have found high seroconversion rates in this population with 95% developing antibodies. It is unclear if and how long these antibodies persist. Here we investigate this with serial antibody testing. METHODS: We identified haemodialysis patients who had confirmed SARS-CoV-2 between March-May 2020 and measured monthly antibodies (IgG/IgM) in those who survived. We used a semi-quantitative cut-off index (COI) to create a qualitative result and plotted optical density (OD) over time. We used linear regression to examine the slope, as well as noting peak OD and time to peak OD. We correlated these against baseline demographics, markers of illness severity, and comorbidities. RESULTS: 122 patients were analysed. All remained antibody positive during follow-up; for a minimum of 148 days. 71% had a positive gradient indicating increasing antibody positivity over time. We found that age (p = 0.01), duration of PCR positivity (p = 0.06) and presence of symptoms (p = 0.05) were associated with a longer time to peak OD. Immunosuppression did not alter peak OD but did lead to a non-significant increase in time to peak OD and more patients had a subsequent fall in Ab levels (p = 0.02). Diabetic patients were more likely to have a positive slope (OR 2.26). CONCLUSIONS: These results indicate that haemodialysis patients have a robust and sustained antibody response after confirmed COVID-19 infection with no suggestion that immunosuppression weakens this response. Although unclear what protection these antibodies confer, this encouraging that haemodialysis patients should respond to vaccination.

Effects of targeting the transcription factors Ikaros and Aiolos on B cell activation and differentiation in systemic lupus erythematosus.

OBJECTIVE: To evaluate the effects of targeting Ikaros and Aiolos by cereblon modulator iberdomide on the activation and differentiation of B-cells from patients with systemic lupus erythematosus (SLE). METHODS: CD19+ B-cells isolated from the peripheral blood of patients with SLE (n=41) were cultured with TLR7 ligand resiquimod ±IFNα together with iberdomide or control from day 0 (n=16). Additionally, in vitro B-cell differentiation was induced by stimulation with IL-2/IL-10/IL-15/CD40L/resiquimod with iberdomide or control, given at day 0 or at day 4. At day 5, immunoglobulins were measured by ELISA and cells analysed by flow cytometry. RNA-Seq was performed on fluorescence-activated cell-sorted CD27-IgD+ naïve-B-cells and CD20lowCD27+CD38+ plasmablasts to investigate the transcriptional consequences of iberdomide. RESULTS: Iberdomide significantly inhibited the TLR7 and IFNα-mediated production of immunoglobulins from SLE B-cells and the production of antinuclear antibodies as well as significantly reducing the number of CD27+CD38+ plasmablasts (0.3±0.18, vehicle 1.01±0.56, p=0.011) and CD138+ plasma cells (0.12±0.06, vehicle 0.28±0.02, p=0.03). Additionally, treatment with iberdomide from day 0 significantly inhibited the differentiation of SLE B-cells into plasmablasts (6.4±13.5 vs vehicle 34.9±20.1, p=0.013) and antibody production. When given at later stages of differentiation, iberdomide did not affect the numbers of plasmablasts or the production of antibodies; however, it induced a significant modulation of gene expression involving IKZF1 and IKZF3 transcriptional programmes in both naïve B-cells and plasmablasts (400 and 461 differentially modulated genes, respectively, false discovery rate<0.05). CONCLUSION: These results demonstrate the relevance of Ikaros and Aiolos as therapeutic targets in SLE due to their ability to modulate B cell activation and differentiation downstream of TLR7.

Response to Vaccination Against SARS-CoV-2 in Patients With Antineutrophil Cytoplasmic Antibody-Associated Vasculitis With Renal Involvement.

BACKGROUND: Patients with anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV) present with multisystem disease including renal impairment. The treatment for AAV involves a high burden of immunosuppression. Patients with renal involvement are treated especially intensively. As a result, we identified these patients as being potentially at high risk of failure to seroconvert to COVID-19 vaccination. METHODS: We collected data on seroconversion response rates to COVID-19 vaccination in a multi-ethnic cohort of patients with AAV and renal involvement treated at a busy tertiary nephrology centre as part of a retrospective review of patient notes. Blood samples were taken following vaccination with either Pfizer or Astra-Zeneca COVID-19 vaccines and median fluorescence intensity was measured using the validated MULTICOV-Ab Magnetic LuminexⓇ Assay. We also evaluated whether seroconversion was affected by immunosuppression regimen. RESULTS: 81 patients were included. The mean age was 62, and there were 49 (60%) females. 55 patients had a blood test after the first dose; 46 after the second dose. Patients were in remission with a median BVAS of 0 (IQR 2). Seroconversion after the first dose with either vaccine was 35/55 (63.6%). After the second it was 38/46 (82.6%). Subgroup analyses revealed a trend to impaired seroconversion in non-white versus white patients (77.8 vs. 81.7% (p = 0.69) after the first dose of vaccine and in those treated with Rituximab in the last 12 months (73.3 vs. 87.1%, p = 0.41). CONCLUSIONS: These data offer real-world evidence of lower seroconversion in response to vaccination with one dose in patients with AAV and renal involvement than the general UK population. After two doses, seroconversion is in line with national data. These data provide a rationale for hospital-led identification of patients most at risk of COVID-19 and underscore the importance of local connexions between hospitals and their communities. These data provide further support for targeting booster vaccination programmes to vulnerable patient cohorts. They add to the growing evidence of reduced seroconversion in response to vaccination in patients with renal disease of any cause.

Flipped learning: Turning medical education upside down.

BACKGROUND: Flipped learning is an approach in which core teaching is delivered using online material viewed prior to face-to-face learning, applying knowledge gained from online material. Core teaching in a module for third-year undergraduate medical students was based around a 1-week course comprising 32 hours of lectures. Feedback suggested that students were poorly engaged and attendance was poor. OBJECTIVES: To develop and evaluate a programme of learning for medical students using flipped learning. METHODS: Core lectures were videoed and students were advised to watch online at home in the morning prior to a case-based interactive discussion session in the afternoon. Feedback was undertaken prior to and following change in delivery; changes in Likert scale feedback were assessed. Thematic assessment of free-text feedback was undertaken. Results of in-course assessment examinations were compared prior to and following change in delivery. RESULTS: Student feedback showed a significant improvement in satisfaction with flipped learning compared to standard lectures, both in scores and free-text feedback. Results of in-course assessments did not change between the two methods of delivery. CONCLUSIONS: Flipped learning can improve student satisfaction and engagement with teaching, but our study has not demonstrated an improvement in assessment scores.

Systematic Review and the External Validity of Randomized Controlled Trials in Lupus Nephritis.

INTRODUCTION: Randomized controlled trials (RCTs) are considered the gold standard for assessing treatment efficacy. However, sampling bias can affect the generalization of results to routine clinical practice. Here we assessed whether patients with lupus nephritis (LN) seen in routine clinical practice would have satisfied entry criteria to the major published RCTs in LN. METHODS: A systematic literature search from January 1974 to May 2015 was carried out, identifying all RCTs investigating LN induction treatment. Patients diagnosed with proliferative or membranous LN between 1995 and 2013 were identified from the Barts Lupus Centre database; baseline characteristics were compared with each RCT's entry criteria to assess hypothetical inclusion or exclusion. RESULTS: Of 363 articles, 33 RCTs met inclusion criteria. Of 137 patients newly diagnosed with LN (111 with proliferative/mixed proliferative and 26 with pure membranous LN), 32% would have been excluded from RCT entry (range 8%-73%). The main reasons for exclusion would have been too severe disease, too mild disease, or prior immunosuppressant use, which were exclusion criteria in 26, 20, and 22 RCTs, respectively. A total of 27 patients with LN (20%) were re-biopsied due to flare; 68% of these would have been ineligible to enter RCTs. CONCLUSION: Published RCTs do not truly reflect the heterogeneity of patients with LN in routine practice at our lupus center. The external validity of RCTs could be improved by including more representative patient cohorts. RCTs should be used as a guide but consideration should be given to similarities between individual patients and the characteristics of the trial cohorts before treatment decisions being made.

Leptospirosis presenting as haemolytic uraemic syndrome: a case report.

BACKGROUND: Leptospirosis is a rare infectious disease especially in Western Countries. Renal involvement is a recognised complication of leptospirosis but leptospirosis-associated haemolytic uraemic syndrome is extremely rare and to our knowledge has only been reported once, in 1985. CASE PRESENTATION: A 29-year-old male was transferred to our Renal Unit with fevers, myalgia and diarrhoeal illness. Laboratory investigations revealed an acute kidney injury, acute liver injury, significantly raised lactate dehydrogenase with marked anaemia, thrombocytopenia and schistocytes on a blood film. A diagnosis of haemolytic uraemic syndrome was made. Surprisingly, the stool culture was negative which led to a suspicion of leptospirosis as one of the differential diagnoses. This was subsequently confirmed by enzyme-linked immunosorbent assay and microscopic agglutination test. He received plasma exchange and antibiotics and made a complete recovery on discharge. CONCLUSION: Leptospirosis presenting as haemolytic uraemic syndrome is rare but should be considered in the differential diagnosis especially in the presence of significant liver injury, as current evidence suggests that the disease is re-emerging.

Utility of a repeat renal biopsy in lupus nephritis: a single centre experience.

Background: The role of repeat renal biopsy in lupus nephritis (LN) to guide treatment or predict prognosis has been controversial. We assessed glomerular and tubulointerstitial histological characteristics of serial renal biopsies, correlations with clinical variables and the impact on subsequent management. Methods: Out of a large single-centre cohort of 270 biopsy-proven LN patients, 66 (24%) had serial biopsies. LN classes based on glomerular pathology were defined according to the International Society of Nephrology/Renal Pathology Society 2003 classification, while tubulointerstitial pathologies were evaluated using the revised Austin's semi-quantitative scoring system. Results: LN class transitions from proliferative (III and IV) to non-proliferative classes (II and V) were uncommon (n = 4, 7.7%), while non-proliferatives frequently switched to proliferative classes (n = 12, 63.2%) and were more likely to receive increased immunosuppression (P = 0.040). Biochemical or serological variables could not predict these histopathological transitions. Tubulointerstitial score (mean ± standard deviation) progressed from 2.69 ± 2.03 on reference to 3.78 ± 2.03 on repeat biopsy (P = 0.001). Serum creatinine levels correlated with the degree of tubular atrophy on both reference (r = 0.33, P = 0.048) and repeat biopsy (r = 0.56, P < 0.001), and with interstitial scarring (r = 0.60, P < 0.001) on repeat biopsy. Greater interstitial inflammation on reference biopsy was associated with advanced interstitial scarring on repeat biopsies (r = 0.385, P = 0.009). Conclusions: Repeat renal biopsy is an important tool to guide management, in particular in those with initial class II or V who flare. Although class transitions cannot be predicted by clinical parameters, serum creatinine level correlates with the degree of tubulointerstitial damage.

Infliximab as long-term maintenance in steroid-resistant and recurrent sarcoidosis in a renal transplant with central nervous system involvement.

Sarcoidosis is a multisystemic, granulomatous disease of unknown aetiology, which commonly involves the lungs, skin and the eyes. Renal sarcoidosis is rare. Recurrent renal sarcoidosis leading to transplant graft failure in adults has not been reported. We report a single case of steroid-resistant sarcoid with recurrence in a renal transplant and the central nervous system that was managed with infliximab. We describe successful resolution of granulomas in the transplant kidney and stabilization of renal function with catastrophic central nervous system recurrence upon withdrawal of infliximab.

Nonresolving inflammation in gp91phox-/- mice, a model of human chronic granulomatous disease, has lower adenosine and cyclic adenosine 5'-monophosphate.

In chronic granulomatous disease (CGD), there is failure to generate reactive oxygen metabolites, resulting in recurrent infections and persistent inflammatory events. Because responses to sterile stimuli in murine models of CGD also result in nonresolving inflammation, we investigated whether defects in endogenous counterregulatory mechanisms and/or proresolution pathways contribute to the etiology of CGD. To this end, we conducted a series of experiments finding, in the first instance that adenosine and cAMP, which dampen innate immune-mediated responses, show a biphasic profile in resolving peritonitis; peaking at onset, waning as inflammation progresses, and rising again at resolution. We also found elevations in adenosine and cAMP in resolving human peritonitis. In gp91(phox-/-) mice, an experimental model of CGD, levels of adenosine and cAMP were significantly lower at onset and again at resolution. Corroborating the finding of others, we show that adenosine, signaling through its A(2A) receptor and therefore elevating cAMP, is not only anti-inflammatory, but, importantly, it does not impair proresolution pathways, properties typical of nonsteroidal anti-inflammatory drugs. Conversely, antagonizing the A(2A) receptor worsens acute inflammation and prolongs resolution. Taking this further, activating the A(2A) receptor in gp91(phox-/-) mice was dramatically anti-inflammatory regardless of the phase the inflammatory response A(2A) agonists were administered, i.e., onset or resolution, demonstrating wide and robust pharmacological flexibility that is unlikely to subvert proresolution pathways. Therefore, we describe the biphasic profile of adenosine and cAMP throughout the time course of acute inflammation that is dysregulated in CGD.

Novel biphasic role for lymphocytes revealed during resolving inflammation.

Acute inflammation is traditionally described as the influx of polymorphonuclear leukocytes (PMNs) followed by monocyte-derived macrophages, leading to resolution. This is a classic view, and despite subpopulations of lymphocytes possessing innate immune-regulatory properties, seldom is their role in acute inflammation and its resolution discussed. To redress this we show, using lymphocyte-deficient RAG1(-/-) mice, that peritoneal T/B lymphocytes control PMN trafficking by regulating cytokine synthesis. Once inflammation ensues in normal mice, lymphocytes disappear in response to DP1 receptor activation by prostaglandin D(2). However, upon resolution, lymphocytes repopulate the cavity comprising B1, natural killer (NK), gamma/delta T, CD4(+)/CD25(+), and B2 cells. Repopulating lymphocytes are dispensable for resolution, as inflammation in RAG1(-/-) and wild-type mice resolve uniformly. However, repopulating lymphocytes are critical for modulating responses to superinfection. Thus, in chronic granulomatous disease using gp91phox(-/-) mice, not only is resolution delayed compared with wild-type, but there is a failure of lymphocyte re-appearance predisposing to exaggerated immune responses upon secondary challenge that is rescued by resolution-phase lymphocytes. In conclusion, as lymphocyte repopulation is also evident in human peritonitis, we hereby describe a transition in T/B cells from acute inflammation to resolution, with a central role in modulating the severity of early onset and orchestrating responses to secondary infection.

Hematopoietic prostaglandin D2 synthase controls the onset and resolution of acute inflammation through PGD2 and 15-deoxyDelta12 14 PGJ2.

Hematopoietic prostaglandin D(2) synthase (hPGD(2)S) metabolizes cyclooxygenase (COX)-derived PGH(2) to PGD(2) and 15-deoxyDelta(12-14) PGJ(2) (15d-PGJ(2)). Unlike COX, the role of hPGD(2)S in host defense is ambiguous. PGD(2) can be either pro- or antiinflammatory depending on disease etiology, whereas the existence of 15d-PGJ(2) and its relevance to pathophysiology remain controversial. Herein, studies on hPGD(2)S KO mice reveal that 15d-PGJ(2) is synthesized in a self-resolving peritonitis, detected by using liquid chromatography-tandem MS. Together with PGD(2) working on its DP1 receptor, 15d-PGJ(2) controls the balance of pro- vs. antiinflammatory cytokines that regulate leukocyte influx and monocyte-derived macrophage efflux from the inflamed peritoneal cavity to draining lymph nodes leading to resolution. Specifically, inflammation in hPGD(2)S KOs is more severe during the onset phase arising from a substantial cytokine imbalance resulting in enhanced polymorphonuclear leukocyte and monocyte trafficking. Moreover, resolution is impaired, characterized by macrophage and surprisingly lymphocyte accumulation. Data from this work place hPGD(2)S at the center of controlling the onset and the resolution of acute inflammation where it acts as a crucial checkpoint controller of cytokine/chemokine synthesis as well as leukocyte influx and efflux. Here, we provide definitive proof that 15d-PGJ(2) is synthesized during mammalian inflammatory responses, and we highlight DP1 receptor activation as a potential antiinflammatory strategy.

Not all eicosanoids are bad.

Although considerable attention has been focused on elucidating the factors that drive inflammation, it is becoming clear that this "acceleration" state is offset by an internal "handbrake". A recent study has uncovered an essential component of this handbrake system, revealing that lipoxins trigger suppressors of cytokine signalling to dampen inflammatory responses to infection. This work bolsters the growing interest in understanding how inflammation is controlled from within and draws further attention to novel targets for drug development based on mimicking the actions of endogenous anti-inflammatory and pro-resolution signals.

COX-2 in inflammation and resolution.

Aspirin and the other NSAIDs have popularized the notion of inhibiting prostaglandins as a common anti-inflammatory strategy based on the erroneous premise that all eicosanoids are, within the context of inflammation, generally detrimental. However, our fascination with aspirin and the emergence of COX-2 has shown a more affable side to lipid mediators based on our increasing interest in the endogenous control of acute inflammation and in factors that mediate its resolution. Epilipoxins, for instance, are produced from aspirin's acetylation of COX-2 and together with Resolvins and COX-2-derived prostaglandins of the D(2) and J(2) series represent an increasingly important family of immunoregulatory lipid mediators with strong implications for disease control and drug discovery.

Essential role for hematopoietic prostaglandin D2 synthase in the control of delayed type hypersensitivity.

Hematopoietic prostaglandin D(2) synthase (hPGD(2)S) metabolizes cyclooxygenase-derived prostaglandin (PG) H(2) to PGD(2), which is dehydrated to cyclopentenone PGs, including 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)). PGD(2) acts through two receptors (DP1 and DP2/CRTH2), whereas 15d-PGJ(2) can activate peroxisome proliferator-activated receptors or inhibit a range of proinflammatory signaling pathways, including NF-kappaB. Despite eliciting asthmatic and allergic reactions through the generation of PGD(2), it is not known what role hPGD(2)S plays in T helper (Th)1-driven adaptive immunity. To investigate this question, the severity and duration of a delayed type hypersensitivity reaction was examined in hPGD(2)S knockout and transgenic mice. Compared with their respective controls, knockouts displayed a more severe inflammatory response that failed to resolve, characterized histologically as persistent acute inflammation, whereas transgenic mice had little detectable inflammation. Lymphocytes isolated from inguinal lymph nodes of hPGD(2)S(-/-) animals showed hyperproliferation and increased IL-2 synthesis effects that were rescued by 15d-PGJ(2), but not PGD(2), working through either of its receptors. Crucially, 15d-PGJ(2) exerted its suppressive effects through the inhibition of NF-kappaB activation and not through peroxisome proliferator-activated receptor signaling. In contrast, lymph node cultures from transgenics proliferated more slowly and synthesized significantly less IL-2 than controls. Therefore, contrary to its role in driving Th2-like responses, this report shows that hPGD(2)S may act as an internal braking signal essential for bringing about the resolution of Th1-driven delayed type hypersensitivity reactions. Consequently, hPGD(2)S-derived cyclopentenone PGs may protect against inflammatory diseases, where T lymphocytes play a pathogenic role, as in rheumatoid arthritis, atopic eczema, and chronic rejection.

High pre-transplant soluble CD30 levels are predictive of the grade of rejection.

In renal transplantation, serum soluble CD30 (sCD30) levels in graft recipients are associated with increased rejection and graft loss. We investigated whether pre-transplant sCD30 concentrations are predictive of the grade of rejection. Pre-transplant sera of 51 patients with tubulointerstitial rejection (TIR), 16 patients with vascular rejection (VR) and an age-matched control group of 41 patients with no rejection (NR) were analyzed for sCD30. The transplant biopsies were immunostained for C4d. The median sCD30 level was significantly elevated in the group with VR (248 Units (U)/mL, range: 92-802) when compared with TIR (103 U/mL, range: 36-309, p<0.001) and NR (179 U/mL, range: 70-343, p<0.03). Moreover, patients with TIR had significantly lower sCD30 levels compared to NR. Based on C4d staining, a TH2 driven process, the median sCD30 levels were significantly raised in C4d+ patients compared with C4d- group (177 U/mL vs. 120 U/mL, p<0.05). sCD30 levels measured at time of transplantation correlate with the grade of rejection. High pre-transplant levels are associated with antibody-mediated rejection which carries a poorer prognosis. sCD30 could be another tool to assess immunological risk prior to transplantation and enable a patient centered approach to immunosuppression.