Late-Onset Systemic Lupus Erythematosus With Lupus Nephritis in a 74-Year-Old Male: A Brief Case and Review

Rationale: Late-onset systemic lupus erythematosus (SLE) represents a specific subgroup of SLE, and although there is no strict age cut-off, 50 years is commonly used as the minimum age for disease onset. In this report, we present a case of a 74-year-old male with late-onset SLE and biopsy-proven lupus nephritis (LN). Presenting concerns of the patient: A 74-year-old male was referred to the nephrology clinic with a rapidly rising creatinine from a baseline of 60 µmol/L to 176 µmol/L. His labs showed pancytopenia, a positive antinuclear antibodies (ANA), and hypocomplementemia. Diagnoses: Renal biopsy showed focal proliferative glomerulonephritis that was immune-mediated and immunofluorescence showed C3, IgM, IgA, IgG, lambda, and C1q diffuse mesangial and glomerular basement membrane staining. Together these findings were in keeping with a diagnosis of stage III LN. Interventions: Treatment included hemodialysis and induction with pulse methylprednisone and cyclophosphamide. He was then placed on the Euro-Lupus Protocol. Outcomes: One year after the diagnosis, he was off dialysis, had no signs of fluid retention or uremia, and his creatinine had stabilized at ~ 330 µmol/L. Lessons learned: To the best of our knowledge, this case represents the oldest known biopsy-confirmed case of late-onset SLE and LN. Late-onset SLE is uncommon and often overlooked as classical symptoms such as malar rash or photosensitivity may not be present. The American College of Rheumatology (ACR) guidelines for treatment of LN can be applied to these patients but physicians should be cognizant of the fact that these patients may not tolerate immunosuppressive therapy as well as younger patients

Pokeweed Antiviral Protein Increases HIV-1 Particle Infectivity by Activating the Cellular Mitogen Activated Protein Kinase Pathway

Pokeweed antiviral protein (PAP) is a plant-derived N-glycosidase that exhibits antiviral activity against several viruses. The enzyme removes purine bases from the messenger RNAs of the retroviruses Human immunodeficiency virus-1 and Human T-cell leukemia virus-1. This depurination reduces viral protein synthesis by stalling elongating ribosomes at nucleotides with a missing base. Here, we transiently expressed PAP in cells with a proviral clone of HIV-1 to examine the effect of the protein on virus production and quality. PAP reduced virus production by approximately 450-fold, as measured by p24 ELISA of media containing virions, which correlated with a substantial decline in virus protein synthesis in cells. However, particles released from PAP-expressing cells were approximately 7-fold more infectious, as determined by single-cycle infection of 1G5 cells and productive infection of MT2 cells. This increase in infectivity was not likely due to changes in the processing of HIV-1 polyproteins, RNA packaging efficiency or maturation of virus. Rather, expression of PAP activated the ERK1/2 MAPK pathway to a limited extent, resulting in increased phosphorylation of viral p17 matrix protein. The increase in infectivity of HIV-1 particles produced from PAP-expressing cells was compensated by the reduction in virus number; that is, virus production decreased upon de novo infection of cells over time. However, our findings emphasize the importance of investigating the influence of heterologous protein expression upon host cells when assessing their potential for antivira

Infectivity of HIV-1 particles decreases upon treatment with MEK inhibitor.

<p>293 T cells were transfected with 5 µg pNL4-3 and 0.5 µg 3x-Flag-PAP or pcDNA3 plasmids. Cells transfected with 3x-Flag-PAP plasmid or untransfected were also treated with PD98059 (20 µM). Virus particles were purified from the media of cells collected 40 hours following transfection and equivalent amounts of virus particles (3 ng) were used to infect 1×10⁵ 1G5 cells. Cells were washed and re-fed with fresh medium containing AZT (10 µM) 8 hours following infection and incubated for an additional 40 hours. Cells were harvested, lysed and a luciferase assay was performed to determine relative virus infectivity. Values are means ± S.E. from triplicate samples of three different experiments.</p

Decreased synthesis of HIV-1 particles by PAP reduces productive infection.

<p>Equivalent volume of medium (200 µL) containing virus from 293 T cells transfected with the pNL4-3 proviral clone (5 µg) and 0.5 µg 3x-Flag-PAP or pcDNA3 vectors was used to infect 1×10⁵ MT-2 cells in a 24-well plate. Following 4 hours of incubation, the cells were washed with fresh medium and incubated for another 21 days. Aliquots of media were collected every three days and virus production was determined by p24 CA ELISA. Medium from cells expressing PAP or vector control is indicated by circles or squares, respectively. Values are means ± S.E. from triplicate samples of three different experiments.</p

PAP activates ERK1/2 MAPK in cells.

<p>(A) 293 T cells were transfected with 3x-Flag-PAP (0.5, 1.0, or 2.5 µg), 3x-Flag-PAPx (2.5 µg), pcDNA3 (2.5 µg) or MEK_AC (1.0 µg) vectors. 24 hours prior to transfection, cells were refed with DMEM without FBS until they were harvested. A portion of cells transfected with 3x-Flag-PAP (2.5 µg) was treated with the MEK inhibitor PD98059 (20 µM). Total cellular protein (100 µg) was resolved through 12% SDS-PAGE, followed by sequential immunoblot analysis using antibodies specific for phospho-MEK (1∶2,000), MEK (1∶2,000), phospho-ERK1/2 (1∶2,000), ERK1/2 (1∶2,000) and β-actin (1∶5,000). (B) Total ERK1/2 was immunoprecipitated from lysate of cells (300 µg total protein), followed by a kinase assay using MBP as substrate. Cells transfected with 3x-Flag-PAP (1.0 µg) or MEK_AC (1.0 µg) were also treated with 20 µM PD98059 MEK inhibitor. Phosphorylated proteins were visualized with a phosphorimager. A fraction of immunoprecipitated proteins was analyzed by immunoblotting using an ERK1/2 specific antibody (1∶5,000). Blots are representative of four independent experiments.</p

PAP does not alter HIV-1 virus particle composition.

<p>Virus particles were collected from media of 293 T cells transfected with the pNL4-3 proviral clone (5 µg) and 0.5 µg 3x-Flag-PAP, 3x-Flag-PAPx or pcDNA3 vectors. (A) Particles were purified by ultracentrifugation through a 20% sucrose cushion and equivalent amounts of purified virus (100 ng) were resolved on 10% SDS-PAGE. Viral proteins were then transferred to nitrocellulose and probed with antibodies specific for RT (1∶2,500), gp120 (1∶5,000) and p24 CA (1∶5,000) proteins. (B) RNA was isolated from equivalent amounts of purified virus particles (100 ng) and analyzed by RNase protection assay using an antisense riboprobe complementary to the gag ORF (nt 600–820). A fraction of the purified viruses was used for immunoblot analysis of p24 CA to control for equal virus pelleting from each sample (lower panel). Blots are representative of five independent experiments performed in triplicate.</p

PAP reduces HIV-1 production from cells.

<p>(A) Immunoblot analysis of PAP expression in 293 T cells transfected with 3x-Flag-PAP (0.5, 1.0 or 2.5 µg), 3x-Flag-PAPx (0.5 µg) or pcDNA3 (2.5 µg) plasmids. Total cellular protein (100 µg) was resolved on a 12% SDS-PAGE, transferred to nitrocellulose and probed with Flag monoclonal antibody (1∶1,000) and β-actin monoclonal antibody (1∶5,000). (B) 293 T cells were transfected with pMenv(-) proviral clone (5 µg) and 3x-Flag-PAP (0.12, 0.25, 0.5 or 1.0 µg), 3x-Flag-PAPx (1.0 µg) or pcDNA3 (1.0 µg). Media of cells were collected 40 hours following transfection and virus production was estimated using a p24 CA ELISA. Values are plotted on log scale and are means ± S.E. from triplicate samples of three different experiments. (C) Equal volume of media (1 mL) was centrifuged and pelleted virus particles were separated through 12% SDS-PAGE followed by immunoblotting using a p24 CA-specific monoclonal antibody (1∶5,000). The blot is representative of three separate experiments.</p

PAP decreases expression of HIV-1 proteins without toxicity to cells.

<p>293 T cells were transfected with the pNL4-3 proviral clone (5 µg) and 2 µg 3x-Flag-PAP, 3x-Flag-PAPx or pcDNA3 vectors. Cells were harvested 40 hours following transfection. (A) Viability was tested by an MTT conversion assay. Values are percentages relative to pcDNA3, as means ± S.E. for three independent experiments. (B) Total cellular protein (150 µg) was separated through 10% SDS-PAGE, followed by immunoblot analysis using p24 CA-specific monoclonal antibody (1∶5,000), or polyclonal antibodies specific for Nef (1∶2,000), RT (1∶2,500) or gp120 (1∶5,000). Samples were probed with β-actin monoclonal antibody (1∶5,000) to control for equal protein loading in each lane. The blots are representative of three separate experiments.</p

PAP increases the phosphorylation of p17 matrix protein.

<p>293 T cells were transfected with the pMenv(-) proviral clone (5 µg) and 0.5 µg 3x-Flag-PAP, 3x-Flag-PAPx, pcDNA3 or MEK_AC vectors. Cells co-transfected with PAP and HIV-1 and cells transfected with HIV-1 alone were additionally treated with 20 µM of the MEK inhibitor PD98059. Particles were purified by ultracentrifugation through a 20% sucrose cushion and equivalent amounts of purified virus (30 ng) were pelleted, lysed and subjected to an endogenous kinase assay using [γ33P-ATP] as a phosphate donor. Samples were resolved on 15% SDS-PAGE and phosphorylated p17 was visualized using a phosphorimager. Fractions of pelleted virus were analyzed via immunoblot using a p17-MA-specific antibody (1∶1,000). Blots are representative of four separate experiments.</p