Mycophenolic Acid Research Articles

Identification of IMP Dehydrogenase as a Potential Target for Anti-Mpox Virus Agents.

Mpox virus (formerly monkeypox virus [MPXV]) is a neglected zoonotic pathogen that caused a worldwide outbreak in May 2022. Given the lack of an established therapy, the development of an anti-MPXV strategy is of vital importance. To identify drug targets for the development of anti-MPXV agents, we screened a chemical library using an MPXV infection cell assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV propagation. These compounds showed broad-spectrum anti-orthopoxvirus activities and presented lower 90% inhibitory concentrations (0.026 to 0.89 μM) than brincidofovir, an approved anti-smallpox agent. These three compounds have been suggested to target the postentry step to reduce the intracellular production of virions. Knockdown of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanosine biosynthesis and a target of MPA, dramatically reduced MPXV DNA production. Moreover, supplementation with guanosine recovered the anti-MPXV effect of MPA, suggesting that IMPDH and its guanosine biosynthetic pathway regulate MPXV replication. By targeting IMPDH, we identified a series of compounds with stronger anti-MPXV activity than MPA. This evidence shows that IMPDH is a potential target for the development of anti-MPXV agents. IMPORTANCE Mpox is a zoonotic disease caused by infection with the mpox virus, and a worldwide outbreak occurred in May 2022. The smallpox vaccine has recently been approved for clinical use against mpox in the United States. Although brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration, their efficacy against mpox has not been established. Moreover, these drugs may present negative side effects. Therefore, new anti-mpox virus agents are needed. This study revealed that gemcitabine, trifluridine, and mycophenolic acid inhibited mpox virus propagation and exhibited broad-spectrum anti-orthopoxvirus activities. We also suggested IMP dehydrogenase as a potential target for the development of anti-mpox virus agents. By targeting this molecule, we identified a series of compounds with stronger anti-mpox virus activity than mycophenolic acid.

Management of immunosuppressive therapy in kidney transplant recipients with COVID-19. A multicentre national study derived from the Spanish Society of Nephrology COVID registry

IntroductionSARS CoV2 infection has had a major impact on renal transplant patients with a high mortality in the first months of the pandemic. Intentional reduction of immunosuppressive therapy has been postulated as one of the cornerstone in the management of the infection in the absence of targeted antiviral treatment. This has been modified according to the patient`s clinical situation and its effect on renal function or anti-HLA antibodies in the medium term has not been evaluated. ObjectivesEvaluate the management of immunosuppressive therapy made during SARS-CoV2 infection, as well as renal function and anti-HLA antibodies in kidney transplant patients 6 months after COVID19 diagnosis. Material and methodsRetrospective, national multicentre, retrospective study (30 centres) of kidney transplant recipients with COVID19 from 01/02/20 to 31/12/20. Clinical variables were collected from medical records and included in an anonymised database. SPSS statistical software was used for data analysis. Resultsrenal transplant recipients with COVID19 were included (62.6% male), with a mean age of 57.5 years.The predominant immunosuppressive treatment prior to COVID19 was triple therapy with prednisone, tacrolimus and mycophenolic acid (54.6%) followed by m-TOR inhibitor regimens (18.6%). After diagnosis of infection, mycophenolic acid was discontinued in 73.8% of patients, m-TOR inhibitor in 41.4%, tacrolimus in 10.5% and cyclosporin A in 10%. In turn, 26.9% received dexamethasone and 50.9% were started on or had their baseline prednisone dose increased.Mean creatinine before diagnosis of COVID19, at diagnosis and at 6 months was: 1.7 ± 0.8, 2.1 ± 1.2 and 1.8 ± 1 mg/dl respectively (p < 0.001).56.9% of the patients (N = 350) were monitored for anti-HLA antibodies. 94% (N = 329) had no anti-HLA changes, while 6% (N = 21) had positive anti-HLA antibodies. Among the patients with donor-specific antibodies post-COVID19 (N = 9), 7 patients (3.1%) had one immunosuppressant discontinued (5 patients had mycophenolic acid and 2 had tacrolimus), 1 patient had both immunosuppressants discontinued (3.4%) and 1 patient had no change in immunosuppression (1.1%), these differences were not significant. ConclusionsThe management of immunosuppressive therapy after diagnosis of COVID19 was primarily based on discontinuation of mycophenolic acid with very discrete reductions or discontinuations of calcineurin inhibitors. This immunosuppression management did not influence renal function or changes in anti-HLA antibodies 6 months after diagnosis.

Early conversion to belatacept-based immunosuppression regimen promotes improved long-term renal graft function in kidney transplant recipients

BackgroundBelatacept has been demonstrated as an effective alternative immunosuppressant in kidney transplant recipients. This study focuses on outcomes of early and late conversion to Belatacept-based immunosuppression after kidney transplant. Materials and methodsThis retrospective analysis of a prospectively collected database included all adult kidney transplants patients at SUNY Upstate Medical Hospital from 1 January 2014 to 30 December 2022. Early conversion was defined as all conversions done at <6 months after kidney transplantation, and late conversion to belatacept was defined as conversion at >6 months after kidney transplantation. ResultsOut of 61 patients included in this study, 33 patients (54%) were in the early conversion group, and 28 patients (46%) were in the late conversion group. The mean eGFR in the early conversion group was 26.73 ± 16.26 ml/min/1.73 m2 before conversion to belatacept, which improved to 45.3 ± 21.01 ml/min/1.73 m2 at one-year post-conversion (p = 0.0006). Furthermore, eGFR changes in the late conversion group were insignificant, with 46.30 ± 15.65 ml/min/1.73 m2 before conversion to belatacept, and 44.76 ± 22.91 ml/min/1.73 m2 after one year of follow-up (p = 0.72). All four biopsy-proven allograft rejections in the early conversion group were acute T-cell-mediated rejections (ATMR). In the late conversion group, out of three biopsy-proven rejections, one was chronic antibody-mediated rejection (CAMR), one was ATMR, and one was mixed ATMR/CAMR. All four patients with ATMR rejection received mycophenolic acid (MPA) as part of their immunosuppressive regimen, and none received tacrolimus. The one-year post-conversion allograft survival rate in early and late conversion groups was 100%. However, the one-year post-conversion patient survival rate was 90.9% in the early conversion group and 100% in the late conversion group (P = 0.11). ConclusionsEarly post-transplant conversion to belatacept can improve the eGFR more meaningful when compared to late conversion. Patients who receive belatacept and MPA rather than tacrolimus may have increased rates of T-cell-mediated rejection.

High-throughput detection of mycophenolic acid in human plasma based on sensitive and rapid fluorescence nitrogen-doped carbon dots sensing platform

In this experiment, a water-soluble, nitrogen-doped yellow-green fluorescent N-doped carbon dots (N-CDs) were synthesized by one-step hydrothermal method using β-cyclodextrin as carbon source and L-phenylalanine as nitrogen source. The fluorescence quantum yield of the obtained N-CDs was as high as 9.96%, and the N-CDs exhibited photostability at different pH, ionic strength and temperature. The morphology of the N-CDs was approximately spherical with an average particle size of about 9.4 nm. Based on the fluorescence enhancement effect of mycophenolic acid (MPA) on N-CDs, a quantitative detection method of MPA was established. This method had good selectivity and high sensitivity for MPA. The fluorescence sensing system was applied to the detection of MPA in human plasma. The linear range of MPA were 0.06–3 μg·mL−1 and 3–27 μg·mL−1 with a detection limit of 0.016 μg·mL−1, and the recoveries were 97.03∼100.64 % with the RSDs of 0.13∼2.90 %. The interference experiment results showed that the interference of other coexisting substances, including Fe3+, can be ignored in the actual detection. Comparing the results measured by the established method with the EMIT method, it was found that the results obtained by the two methods were similar, and the relative error was within ± 5 %. This study provided a simple, rapid, sensitive, selective and effective method for the quantitative analysis of MPA, and was expected to be applied to clinical MPA blood concentration monitoring.

Specific inhibitory effects of guanosine on breast cancer cell proliferation

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer-related death. Drug therapy for breast cancer is currently selected based on the subtype classification; however, many anticancer drugs are highly cytotoxic. Since intracellular levels of GTP are elevated in many cancer cells that undergo a specific cell proliferation cycle, GTP has potential as a target for cancer therapy. The present study focused on nucleosides and nucleotides and examined intracellular GTP-dependent changes in cell proliferation rates in normal (MCF-12A) and cancer (MCF-7) breast cell lines. Decreased cell proliferation due to a reduction in intracellular GTP levels by mycophenolic acid (MPA), an inosine monophosphate dehydrogenase inhibitor, was observed in both cell lines. The inhibitory effects of MPA on cell proliferation were suppressed when it was applied in combination with Guanosine (Guo), a substrate for GTP salvage synthesis, while the single exposure to Guo suppressed the proliferation of MCF-7 cells only. Although the underlying mechanisms remain unclear, since the inhibitory effects of Guo on cell proliferation did not correlate with GTP or ATP intracellular levels or the GTP/ATP ratio, there may be another cause besides GTP metabolism. Guo inhibited the proliferation of MCF-7, a human breast cancer cell line, but not MCF-12A, a human normal breast cell line. Further studies are needed to investigate the potential of applying Guo as a target for the development of a novel cancer treatment system.

Cardiovascular Outcomes in De Novo Kidney Transplant Recipients Receiving Everolimus and Reduced Calcineurin Inhibitor or Standard Triple Therapy: 24-month Post Hoc Analysis From TRANSFORM Study.

The comparative impact of everolimus (EVR)-based regimens versus standard of care (mycophenolic acid+standard calcineurin inhibitor [MPA+sCNI]) on cardiovascular outcomes in de novo kidney transplant recipients (KTRs) is poorly understood. The incidence of major adverse cardiac events (MACEs) in KTRs receiving EVR+reduced CNI (rCNI) or MPA+sCNI from the TRANSplant eFficacy and safety Outcomes with an eveRolimus-based regiMen study was evaluated. The incidence of MACE was determined for all randomized patients receiving at least 1 dose of the study drug. Factors associated with MACEs were determined by logistic regression. Risk of MACE out to 3 y post-study was calculated using the Patient Outcome in Renal Transplantation equation. MACE occurred in 81 of 1014 (8.0%; EVR+rCNI) versus 89 of 1012 (8.8%; MPA+sCNI) KTRs (risk ratio, 0.91 [95% confidence interval [CI], 0.68-1.21]). The incidence of cardiac death, myocardial infarction, revascularization, or angina was similar between the arms. Incidence of MACE was similar between EVR+rCNI and MPA+sCNI arms with a higher incidence in prespecified risk groups: older age, pretransplant diabetes (15.1% versus 15.9%), statin use (8.5% versus 10.8%), and low estimated glomerular filtration rate (Month 2 estimated glomerular filtration rate <30 versus >60 mL/min/1.73 m2; odds ratio, 2.23 [95% CI, 1.02-4.86]; P = 0.044), respectively. Predicted risk of MACE within 3 y of follow-up did not differ between the treatment arms. Cardiovascular morbidity and mortality were similar between de novo KTRs receiving EVR+rCNI and MPA+sCNI. EVR+rCNI is a viable alternative to the current standard of care in KTRs.

Booster effect of the third dose of SARS-CoV-2 mRNA vaccine in Japanese kidney transplant recipients

The humoral response of kidney transplant recipients (KTR) to the mRNA vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is generally poor. We evaluated the booster effect of the third dose (D3) of two SARS-CoV-2 mRNA vaccines 6 months after the second dose (D2) in Japanese KTR. The anti-spike (anti-S) antibody titer 1 and 3 months after the D3 was evaluated in 82 Japanese KTR. The primary endpoint was the seropositivity rate, and factors associated with the lack of a response were evaluated in a logistic regression model. Overall, the anti-S antibody seropositivity rate 1 and 3 months after the D3 was 74.7% and 76.0%. The anti-S antibody titers after the first and second doses were higher in patients vaccinated with the mRNA-1273 than with the BNT162b2 vaccine. Among the 38 KTR who were seronegative 5 months after the D2, 18 (47.4%) became seropositive following the D3. Factors associated with a non-response were mycophenolic acid dose, post-transplant duration, hemoglobin, and lymphocyte count. A humoral response 1 and 3 months after the D3 was obtained in ~ 75% of KTR, but 20% were non-responders. Additional studies are needed to clarify the factors hindering a vaccine response.

Gut Microbiota-Mediated Pharmacokinetic Drug-Drug Interactions between Mycophenolic Acid and Trimethoprim-Sulfamethoxazole in Humans.

Mycophenolic acid (MPA) and trimethoprim-sulfamethoxazole (TMP-SMX) are commonly prescribed together in certain groups of patients, including solid organ transplant recipients. However, little is known about the pharmacokinetic drug-drug interactions (DDIs) between these two medications. Therefore, the present study aimed to determine the effects of TMP-SMX on MPA pharmacokinetics in humans and to find out the relationship between MPA pharmacokinetics and gut microbiota alteration. This study enrolled 16 healthy volunteers to take a single oral dose of 1000 mg mycophenolate mofetil (MMF), a prodrug of MPA, administered without and with concurrent use of TMP-SMX (320/1600 mg/day) for five days. The pharmacokinetic parameters of MPA and its glucuronide (MPAG) were measured using high-performance liquid chromatography. The composition of gut microbiota in stool samples was profiled using a 16S rRNA metagenomic sequencing technique during pre- and post-TMP-SMX treatment. Relative abundance, bacterial co-occurrence networks, and correlations between bacterial abundance and pharmacokinetic parameters were investigated. The results showed a significant decrease in systemic MPA exposure when TMP-SMX was coadministered with MMF. Analysis of the gut microbiome revealed altered relative abundance of two enriched genera, namely the genus Bacteroides and Faecalibacterium, following TMP-SMX treatment. The relative abundance of the genera Bacteroides, [Eubacterium] coprostanoligenes group, [Eubacterium] eligens group, and Ruminococcus appeared to be significantly correlated with systemic MPA exposure. Coadministration of TMP-SMX with MMF resulted in a reduction in systemic MPA exposure. The pharmacokinetic DDIs between these two drugs were attributed to the effect of TMP-SMX, a broad-spectrum antibiotic, on gut microbiota-mediated MPA metabolism.

#6517 CLINICOHISTOLOGICAL CHARACTERISTICS, THERAPEUTIC APPROACH AND RENAL OUTCOME IN PATIENTS WITH C3 GLOMERULOPATHY: A SINGLE-CENTER EXPERIENCE

Abstract Background and Aims C3 glomerulopathy has been identified as a distinct disease in the last decade but it has already been associated with a high burden of end stage renal disease (ESRD). Due to the rarity and heterogeneity of the disease, data regarding the prognosis and optimal treatment are still lacking. We aimed to describe the clinical and histological phenotype of patients with C3 glomerulopathy followed at our center, the treatment regimens applied and their long-term renal outcome. Method We conducted a retrospective medical chart review of 19 patients with C3 glomerulopathy followed at our center and we reported clinical, histological and therapeutic parameters as well as their long-term renal outcome. Results Median age of patients at the time of diagnosis was 23 years (IQR 28), median proteinuria 2.6 g/d (IQR 2.8) and median eGFR 63ml/min (IQR 63). 26% of patients (5/19) presented with an eGFR &amp;lt;30ml/min. Low serum C3 levels were reported in 11/19 patients (58%). Α monoclonal paraprotein was detected in three patients and pathogenous mutations of CFH/CFI genes in 4 out of 10 patients who underwent genetic testing. 41% of patients (7/17) were treated with RAAS inhibitors only, while 59% (10/17) received immunosuppressive treatment. Immunosuppressive treatment consisted of different combinations of steroids with mycophenolic acid, cyclophosphamide, cyclosporine or rituximab and steroid monotherapy in one patient. Treatment data were not available for two patients. There was a significant difference in baseline proteinuria between patients treated with RAAS inhibitors only (median 1.5g/d, IQR 1.14) and those who were treated with immunosuppressants (median 3.95g/d, IQR 4.8) (p = 0.003). Remission (complete or partial) was achieved by 43% of patients at 6 months and by 50% at 12 months. Median follow up time was 45 months (IQR 66). Eight patients (42%) reached ESRD in a median time of 58.5 months (IQR 62.5) while other three patients (16%) ended up with a GFR decline &amp;gt;30%. Among patients with adverse renal outcome (ESRD, GFR decline &amp;gt;30%), 54.5% (6/11) had baseline proteinuria &amp;gt;3g/d, 73% (8/11) had low serum C3 and 54.5% (6/11) had interstitial fibrosis/tubular atrophy &amp;gt;25%, while the median percentage of glomerulosclerosis was 33% (IQR 58). On the contrary, in the group of patients with favorable renal outcome, baseline proteinuria &amp;gt;3g/d was reported in only 25% (2/8), low serum C3 in 37.5% (3/8), interstitial fibrosis/tubular atrophy &amp;gt;25% in 25% (2/8), while median glomerulosclerosis was only 8% (IQR 15). Attainment of complete remission was associated with favorable renal outcome (p = 0.001). Five of eight patients who reached ESRD underwent kidney transplantation from a living related donor. All of them received basiliximab, mycophenolic acid, tacrolimus and steroids. Recurrence of C3 glomerulopathy was reported in four of these patients and eculizumab was given in two of them. Graft loss due to disease recurrence was observed in one patient. Conclusion The present case series of patients with C3 glomerulopathy highlights the great heterogeneity in the clinical phenotype, therapeutic approach and renal prognosis of the disease. It also underscores the importance of attainment of complete remission regarding the long-term renal survival. Large studies are needed in order to better define clinical and histological predictors of adverse renal outcome as well as the optimal treatment regimens.

#6915 IMMUNOLOGIC EFFECTS OF THE DECREASE OF IMMUNOSUPPRESSION AFTER SARS-COV-2 INFECTION IN KIDNEY TRASPLANT RECIPIENTS

Abstract Background and Aims The decrease of immunosuppression (IS) in kidney transplant (KT) recipients with SARS-CoV-2 infection was proposed during the first years of the pandemic due to the lack of knowledge of the course of the infection and the absence of vaccines and specific treatment. The effects of this decrease are being assessed in a medium-long term. Method Unicentric retrospective study that included 19 patients with a kidney biopsy after a SARS-CoV-2 infection (120 days). We measured acute kidney injury (AKI) after de infection, decrease in the IS during the infection, rejection episodes and renal function evolution during 24 months after the infection. Results The studied group was constituted by 19 patients from which 57.9% (11/19) men, age 56 (51, 70), being the first KT in 78.9% (15/19) of them. The IS induction was in a 57.9% (11/19) Basilximab and in a 42.1% (8/19) thymoglobulin, and the usual IS of all of them was done with prednisone, tacrolimus and mycophenolic acid. There was a graft loss in 26.3% (5/19), being all of immunologic cause, and 10.5% (2/19) of patients died. The severity of the SARS-CoV-2 infection was different between patients (21.1% (4/11) asymptomatic, 47.1% (9/19) mild respiratory symptoms, 5.3% (1/19) severe pneumonia not requiring critical care unit and 26.3% (5/19) severe pneumonia requiring critical care unit, with a decrease in IS in a 42% (8/19) of patients (steroids in monotherapy in a 31.6% (6/19) and bitherapy with steroids and tacrolimus in a 10,5% (2/19), to achieve 5-7ng/mL blood levels). AKI was found after the first month after the infection in a 57.9% (11/19) of patients, being in a 42% (8/19) AKI KDIGO1 and in a 16% (3/19) AKI KDIGO3. Renal function kept worsening until the sixth month after the infection [CKDEPI before SARS-CoV-2 37mL/min (32, 45) vs CKDEPI 6months after 34mL/min (22, 48.3); p = 0.3]. There was an improvement in renal function in 12 months after the infection (38mL/min (25, 48)). Acute graft rejection was detected in renal biopsies after the infection in 31.6% (6/19) being cellular in 15.8% (3/19), humoral in 10.5% (2/19) and mixed in 5.3% (1/19), de novo DSA were found in 15.8% (3/19). Conclusion The decrease in IS after SARS-CoV-2 in KT recipient has an elevated immunologic price. Glomerular filtrate rate decreases after 6 months from infection with a recovery at 1 year.

#5492 TRANSCRIPTOME ANALYSIS IN LUPUS NEPHRITIS: DIFFERENCES IN GENE EXPRESSION BETWEEN DIAGNOSIS AND RENAL REMISSION IN KIDNEY TISSUE

Abstract Background and Aims Transcriptome analysis in kidney tissue from lupus nephritis patients (LN) provide useful information about gene expression. We designed a pilot study in paraffin-embedded kidney tissue (FFPE) from LN patients to evaluate differences between gene expression at diagnosis (kidney biopsy diagnosis) and 2 years after achieve a complete renal response (protocol kidney biopsy). We evaluated if transcriptome analysis results could be verified through immunofluorescence staining (IF) in kidney biopsies (diagnosis biopsy vs protocol biopsy). Method We included diagnosis and protocol biopsies from 16 LN patients with class III and/or IV. All patients received prednisone (0.5 mg/kg) plus mycophenolic acid. Complete renal remission was defined as normal renal function, uPCOR &amp;lt; 300 mg/gr and inactive urine sediment. RNA Sequencing (RNA-Seq) was performed in FFPE, Human MSigDB Collections were used for gene enrichment analysis and Gene sets derived from the Biological Process Gene Ontology (GO) V7.2 is the library used in the study. Results 16 LN patients were included, Class III (43.8%), Class IV (43.8%) and Mixed Class (12.5%). 100% patients were women and Caucasian with mean age 40.9±7.1 years. RNA-Seq showed 2 overexpressed groups of genes (GO) in diagnosis biopsies, expression of this GO was not detected in protocol biopsies: GO-Complement_activation (NES 2.26, p = 4.2E -0.5): Properdin, Ficolin, C3, Factor B, C3AR1, C4 y CDC59. GO- Humoral_Inmune_Response_Mediated_Circulating_Inmunoglobulin (NES 2.10, p = 7,6E -0.6): IgG1, IgG2, IgA, chemokine receptors 2 y 7 and receptor 13 TNF. Results were verified using IF staining in kidney biopsies, in protocol biopsies were observed a significant reduction in C1q and C3 complement proteins staining: C1q (78% vs 22.2%, p = 0,006), C3 (73.3% vs 27.8%, p = 0.005); results in immunoglobulins staining also agree with transcriptome analysis results, so that we observed significant reduction in IgA and IgG: IgG (69% vs 36.4%, p = 0.009) and IgA (83.3% vs 16.7%, p = 0.02) while IgM staining not showed differences between diagnosis biopsy and protocol biopsy (43.8% vs 45.5%). Conclusion Transcriptome analysis in kidney tissue from LN patients, identified complement proteins and humoral mediators overexpressed at LN diagnosis and infraexpressed at complete renal remission; the results could help nephrologist to investigate the role of these protein as LN biomarkers.

#4487 AN UNEXPECTED CASE OF 2,8-DIHYDROXYADENINE NEPHROPATHY AFTER KIDNEY TRANSPLANTATION RELATED TO NEW VARIANTS OF ADENINE PHOSPHORIBOSILTRANSFERASE GENE

Abstract Background and Aims Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of the purine metabolism which results in the conversion of adenine into 2,8 dihydroxyadenine (DHA) due to the activity of the xanthine oxidoreductase (XOR). Patients affected by APRT deficiency if not treated with inhibitors of XOR may develop 2,8-DHA nephropathy that might progress to end-stage kidney disease (ESKD) with the need of kidney transplant. The high rate of misdiagnosis of 2,8-DHA nephropathy in native kidneys could lead to the failure of kidney graft in transplanted patients affected by APRT deficiency. Method Here, we report the case of a female, 63-years old patient with ESKD of unknown cause on regular hemodialysis treatment from 2018 after a period of three years on peritoneal dialysis, who underwent kidney transplantation in our Center in 2022. Her medical history showed a metabolic syndrome. She did not experience episodes of renal colic whereas family history reported a brother affected by frequent renal colic of unknown cause. In March 2022, she underwent kidney transplantation from a deceased death brain donor. Induction therapy includes basiliximab, tacrolimus, mycophenolic acid and steroids. Due to the persistence of delay graft function, ten days after kidney transplantation an allograft biopsy has been performed. The histological examination revealed tubular damage surrounded by inflammation cells and intratubular crystals in the renal cortex. The crystals were reddish brown tinged in hematoxylin and eosin stain and were birefringent under polarized light Fig. 1 A, B, so they were strongly related to the hypothesis of DHA crystals. Consistently, the urinalysis showed yellow-brown crystals of DHA. Thus, a genetic analysis of APRT gene has been performed showing two novel heterozygous variants c.388_397p.(Leu130ValfsTer4) and exon 3 deletion, expected pathogenic. The patient was treated with bolus of methylprednisolone (4mg/kg alternate to 50 mg) and a therapy with febuxostat 80 mg/die was started to reduce the amount of plasma DHA. In addition, based on our previous experience of recurrence DHA nephropathy after transplantation, we treated the patient with six consecutive hemodiafiltration (HDF) sessions without ultrafiltration, to promptly remove the serum DHA avoiding their precipitation in the graft while waiting for the lowering effect of febuxostat. At discharge the patient showed an increase of the urine output not associated with a complete recovery of kidney function (sCr 3.88 mg/dl, uric acid 1.6 mg/dl), so two other hemodialysis treatments were performed in the next two weeks. Results At present, almost one year after kidney transplant, the patient is doing well and the graft function is stable with a sCr of 1.6 mg/dl without significant presence of DHA crystals in the urine. Conclusion In conclusion, we find out an unexpected recurrence of 2,8-DHA nephropathy due to novel expected pathogenetic variants of APRT gene in patient without medical history of kidney stones successfully treated with steroid, febuxostat and hemodiafiltration.

#6071 RITUXIMAB FOR MAINTENANCE OF REMISSION IN ADULT MINIMAL CHANGE DISEASE

Abstract Background and Aims Maintaining remission in a minority of adult patients with minimal change disease remains challenging. Prolonged corticosteroid use is associated with severe adverse effects. A limited number of observational studies suggest that rituximab may be an alternative therapeutic option for remission maintenance, allowing steroid withdrawal. Method We retrospectively analysed the data of all adult patients with minimal change disease, who received rituximab in our centre between 2017 and 2022. Results A total of 14 adults with minimal change disease were treated with rituximab, six of whom had childhood-onset nephrotic syndrome. Six patients were steroid dependent. The median number of relapses was 6.5 (IQR 4.5-15.2) prior to rituximab. Thirteen patients had received at least ≥ 2 different immunosuppressive agents during their disease. Corticosteroid-related adverse events, including osteopenia, hypertension, cataract and diabetes mellitus were observed in 10 subjects. Rituximab was administered in a median time of 10.6 years (IQR 4.5-16.3) after diagnosis. All patients received 2 doses of 1g rituximab 2 weeks apart. The median age at rituximab administration was 31 years (IQR 18.7-49.0); all patients were in remission. Besides corticosteroids, concomitant immunosuppressive therapy included calcineurin inhibitors (n = 6) and mycophenolate acid analogues (n = 1). The median follow-up time after the first dose of rituximab was 44 months (IQR 9.5-67.2). Relapses occurred in 6/14 patients in a median time of 17.5 months (IQR 10.2-26.5). The number of relapses per year decreased to 0.27 from 0.87 (p &amp;lt; 0.001) after rituximab. Six patients received a repeat course of rituximab. One patient developed Pneumocystis Jirovecii pneumonia, that was successfully treated with cotrimoxazole. At the last follow-up visit, corticosteroids were discontinued in 8 out of 14 patients and calcineurin inhibitors in 3 out of 7 patients. Conclusion Rituximab is a reasonable therapeutic approach for maintaining remission and avoiding steroid toxicity in adult minimal change disease. Randomized controlled studies are needed to further evaluate the safety and efficacy of rituximab in these patients.

#4488 OLD BIOPSY IS ALWAYS GOLD, REVISING MEMBRANOPROLIFERATIVE GN DIAGNOSIS IN A RENAL TRANSPLANT PATIENT

Abstract Background and Aims MPGN resulting from hepatitis C virus (HCV) infection typically shows granular deposition of immunoglobulin M (IgM), C3, and both kappa and lambda light chains. Immunoglobulin G (IgG) may or may not be present, and C1q is typically negative, the same pathology can be found in other viral infections. The recurrence rate post-transplantation is high, with 33% of immune-complex MPGN. Graft loss is even higher 65%, especially among patients with idiopathic MPGN. Treatment of a patient with significant proteinuria can be tricky, as no proven treatment is described yet, but plasmapheresis and induction with cyclophosphamide were described for heavy proteinuria &amp;gt;3.5 g/day. Method 79 year old female patient, known case of live unrelated renal transplantation in December 2013, primary renal disease is unknown. She had a history of equivocal hepatitis C virus antibodies, but Hepatitis C virus RNA was not detected. She is also known to have Type II Diabetes Mellitus and hypertension. The patient had baseline creatinine of 100-120 micromol/L. 24 hr urine protein was 0.8 g/Day but started rising quickly to reach a peak of 9.2 g/g seven months post-transplantation to nephrotic range proteinuria but her serum albumin level was always above 35 g/L. Serum protein electrophoresis showed no M band, a kappa/ Lambda ratio 1.21 within a normal range (0.260-1.65). Anti- nuclear antibody (ANA), P-ANCA, C-ANCA, and Double-stranded DNA all were negative. Complements were within normal range C3 1.39 g/L (0.79 -1.52), C4 0.44 g/L(0.17-0.57)m rheumatoid factor and Anti- citrullinated peptide antibodies were negative. Hepatitis b Virus surface antigen and core antibodies were not reactive, HIV screening was negative. Urine analysis showed RBCs but no RBC casts. Renal biopsy showed mild mesangial expansion and mild l increase in mesangial cells and matrix. Some glomeruli show increased lobulation with increase in mesangial cells and matrix with segmental endocapillary proliferation There is double contour of the glomerular basement membrane with scattered spikes and holes within the basement membrane seen best with silver stain. There is mild increase in the thickness of glomerular basement membrane. There is focal mild lymphocytic infiltration. Tubulitis is not seen in the examined material. One Focal area of mild interstitial fibrosis. Sampled blood vessels are unremarkable. The stain for C4d is negative. Immunofluorescence showed granular capillary wall immunoreactivity to IgM (+2), C4 (+1), C1q (+1), and Lambda light chain (+2). The glomeruli show no immunoreactivity to IgG, IgA, C3, Kappa and fibrin. The glomeruli show (+) staining for albumin. Tubular protein reabsorption droplets showed immunoreactivity for albumin, IgG, IgM, C3, kappa and lambda light chains. Electron microscopy showed many subendothelial, and mesangial electron-dense deposits. The patient was on maintenance immunosuppression medications inform of mycophenolic acid 720 mg, cyclosporine; 250mg BID, and Prednisolone 5 mg daily.Revisiting the new classification of membranoproliferative glomerulonephritis is challenging in this patient, since there is the dominance of IgM deposition by immunofluorescence and C4 but not C3 makes it challenging, the negative monoclonal band in serum electrophoresis and negative autoimmune workup excludes immune complex mediate MPGN, leave idiopathic MPGN a possibility, keeping in mind that hepatitis C virus infection was never confirmed. On the other hand, IF showed IgM with C4 subendothelial and mesangial dense deposits that excludes away the C4 glomerulopathy. Results Whether this is a recurrence of glomerular disease early on post-transplantation or denovo membranoproliferative disease, she responded well to the addition of losartan 100 mg daily, with a reduction of her urinary protein to less than 1 g/g. the patient never received treatment for the hepatitis C virus. Conclusion In our patient, it is difficult to conclude if the cause of MPGN is recurrence or denovo GN, especially with severe proteinuria that responded well to ACEi treatment. The pathological findings can be tricky.

Outcomes With Belatacept Exposure During Pregnancy in Kidney Transplant Recipients: A Case Series.

Posttransplant fertility returns quickly, and female recipients of child-bearing age may conceive while on immunosuppression. However, pregnancy after transplantation confers risks to the recipient, transplant, and fetus, including gestational hypertension, preeclampsia, gestational diabetes, transplant dysfunction, preterm labor, and low birthweight infants. Additionally, mycophenolic acid (MPA) products are teratogenic. Literature evidence regarding belatacept, a selective T-cell costimulation blocker, during pregnancy and while breastfeeding is extremely limited. When female transplant recipients on a belatacept-based regimen are desirous of pregnancy or at the time of conception, transplant providers manage the immunosuppression regimen in 1 of 2 ways: (1) switch both belatacept and MPA to a calcineurin inhibitor-based regimen with or without azathioprine, which is the more common practice but requires several modifications, having potential negative outcomes; or (2) only switch MPA to azathioprine while continuing belatacept. This case series includes 16 pregnancies in 12 recipients with exposure to belatacept throughout pregnancy and while breastfeeding. Patient information was obtained from several sources, including Transplant Pregnancy Registry International, providers at Emory University, and Columbia University, as well as literature review. Pregnancy outcomes included 13 live births and 3 miscarriages. No birth defects or fetal deaths were reported in any of the live births. Seven infants were breastfed while their mothers continued belatacept. Outcomes appear comparable to those documented with the administration of calcineurin inhibitors. This case series provides data supporting the continued administration of belatacept during pregnancy. Additional research will assist in developing better guidelines to counsel female transplant recipients on belatacept desiring to pursue pregnancy.

Machine learning-guided the fabrication of nanozyme based on highly-stable violet phosphorene decorated with phosphorus-doped hierarchically porous carbon microsphere for portable intelligent sensing of mycophenolic acid in silage

Violet phosphorene (VP) have been proved to be more stable than black phosphorene, but few reports for its application in electrochemical sensors. In this study, a highly-stable VP decorated with phosphorus-doped hierarchically porous carbon microsphere (PCM) with multiple enzyme-like activities as a nanozyme sensing platform for portable intelligent analysis of mycophenolic acid (MPA) in silage with machine learning (ML) assistance is successfully fabricated. The pore size distribution on the PCM surface is discussed using N2 adsorption tests, and morphological characterization indicates that the PCM is embedded in the layers of lamellar VP. The affinity of the VP-PCM nanozyme obtained under the guidance of the ML model reaches Km = 12.4 μmol/L for MPA. The VP-PCM/SPCE for the efficient detection of MPA exhibits high sensitivity, a wide detection range of 2.49 μmol/L - 71.14 μmol/L with a low limit of detection of 18.7 nmol/L. The proposed ML model with high prediction accuracy (R2 = 0.9999, MAPEP = 0.0081) assists the nanozyme sensor for intelligent and rapid quantification of MPA residues in corn silage and wheat silage with satisfactory recoveries of 93.33%–102.33%. The excellent biomimetic sensing properties of the VP-PCM nanozyme are driving the development of a novel MPA analysis strategy assisted by ML in the context of production requirements of livestock safety.

Identification of In Vitro Inhibitors of Monkeypox Replication.

Monkeypox virus (MPXV) infections in humans have historically been restricted to regions of endemicity in Africa. However, in 2022, an alarming number of MPXV cases were reported globally, with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. The supply of MPXV vaccines is limited, and only two antivirals, tecovirimat and brincidofovir, approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (mScarlet or green fluorescent protein [GFP]) and luciferase (Nluc) reporter genes to identify compounds with antiorthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed inhibitory activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating their inhibitory activity in vitro against two orthopoxviruses. IMPORTANCE Despite the eradication of smallpox, some orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, access to those vaccines is limited. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV infection and other potentially zoonotic orthopoxvirus infections. Here, we show that 13 compounds, derived from two different libraries, previously found to inhibit several RNA viruses, also inhibit VACV. Notably, 11 compounds also displayed inhibitory activity against MPXV.

Development of a novel UPLC-MS/MS method for the simultaneous quantification of mycophenolic mofetil, mycophenolic acid, and its major metabolites: Application to pharmacokinetic and tissue distribution study in rats

Mycophenolate mofetil (MMF) is a prodrug of mycophenolic acid (MPA) used to prevent rejection in organ transplant patients. The purpose of this study is to develop a sensitive LC-MS/MS method to simultaneously quantify MMF, MPA, and two major metabolites, mycophenolic acid-glucuronide (MPAG) and Acyl-mycophenolic acid-glucuronide (AcMPAG) and applied this method in a pharmacokinetic (PK) and tissue distribution study. A Shimadzu UHPLC system coupled to an AB Sciex QTrap 4000 mass spectrometer was used for the analysis. Protein precipitation with a mixture of methanol: acetonitrile (2:1, v:v) was used to process the plasma samples and tissue samples. Separation was achieved using an Ultra Biphenyl 5 µm column (100 × 2.1 mm) with 0.1% formic acid in water (A) and acetonitrile (B) as the mobile phases. Quantification analysis was performed under positive ionization mode using the multiple reaction monitoring (MRM) approach. The method was linear in the range of 1.22 – 1250.00 nM for all four analytes with correlation coefficient values > 0.99. The method was reproducible, with intra- and inter-day accuracy ranging from 85.0 ± 11.2–108.3 ± 6.50 for all analytes in both plasma, liver and intestine homogenates. The extraction recovery and matrix effect of plasma sample using a mixture methanol/acetonitrile (2:1, V:V) can achieve an acceptable range (<20%), but extraction recovery and matrix effect of AcMPAG decreased to 64.10 ± 15.42 in the liver and intestine homogenates. The analytes in plasma were found to be stable under bench-top, freeze-thaw, and storage conditions. The validated method was successfully applied to quantify MMF, MPA, MPAG, and AcMPAG in a rat PK study. The PK results showed MPA was the major form exposed in the plasma in rats after oral administration of MMF, but the major metabolites in the rat’s tissue disposition were MPAG.

Resistant dermatomyositis as a manifestation of post COVID-19 infection: A case report

Many autoimmune diseases were reported as a sequel of SARS CoV-2 infection. We report here a 51-year-old female who presented with myalgia, progressive quadriparesis, and skin rash 2 weeks after COVID-19 infection. She improved with high-dose methylprednisolone. She was given oral methotrexate and mycophenolic acid during follow up. However, her condition deteriorated until she passed away half a year after the initial illness. This case showed that dermatomyositis can be associated with COVID-19 infection.

Biomimetic liposomes hybrid with erythrocyte membrane modulate dendritic cells to ameliorate systemic lupus erythematosus.

Dendritic cells (DCs)-based immunotherapy has shown immense promise in systemic lupus erythematosus (SLE) treatment. However, existing carrier strategies such as polymers, liposomes, and polypeptides, are difficult to achieve active targeting to DCs due to their intricate interaction with biological systems. Since DCs represent a class of phagocytes responsible for the removal of senescent or damaged erythrocytes, we hypothesize that hybrid vesicles containing erythrocytes membrane components could be presented to be potent drug carriers to target DCs specifically. Herein, inspired by the cell membrane fusion technique, we develop hybrid biomimetic liposomes (R-Lipo) by fusing natural erythrocyte membrane vesicles and artificial liposomes for DCs-targeted SLE therapy. The resultant R-Lipo exhibited excellent biocompatibility and was shown to be effectively internalized by DCs both in vitro and in vivo. Using an immunosuppressant, mycophenolic acid (MPA), as the model drug, MPA-loaded R-Lipo powerfully suppressed DCs maturation and efficiently controlled the duration of lupus nephritis without apparent side effects. Our findings provide a safe, effective, and easy-to-prepare biomimetic vesicle platform for the treatment of SLE and other DC-associated diseases.