What is the meaning of ANCA positivity in IgG4-related disease?

Objectives To evaluate the prevalence and meaning of antineutrophil cytoplasmic antibodies (ANCA) positivity in a cohort of IgG4-related disease (IgG4-RD). Methods We identified patients with ANCA determination from a retrospective cohort of 69 patients with IgG4-RD. ANCA were measured by indirect immunofluorescence microscopy (IIF) and/or proteinase 3 (PR3)-ANCA and myeloperoxidase (MPO)-ANCA by enzyme-linked immunosorbent assay (ELISA). IIF patterns were classified as perinuclear (P-ANCA), cytoplasmic (C-ANCA) and atypical (X-ANCA). We compared the ANCA-positive versus the ANCA-negative IgG4-RD group. Results Out of 69 patients, 31 IgG4-RD patients had an ANCA determination. Four patients with concomitant systemic autoimmune diseases were excluded. We found positive ANCA by IIF in 14 (56%) of 25 patients tested. The most common IIF pattern was C-ANCA in eight (57.1%), followed by dual C-ANCA/X-ANCA in four (28.6%) and P-ANCA and dual C-ANCA/P-ANCA in one each (7.1%). Of the 20 patients with ANCA determination by both IIF and ELISA, four have positive ANCA by ELISA (three for MPO-ANCA and one for PR3-ANCA). Of the two patients with only ELISA determination, one was positive for MPO-ANCA. The prevalence of ANCA positivity by ELISA was 22.7% (5 out of 22 patients). ANCA was more frequent in the Mikulizc/systemic phenotype (42.9%) compared to other phenotypes (p = 0.04). ANCA-positive IgG4-RD patients had more frequently lymph node and kidney involvement, high IgG1 levels and erythrocyte sedimentation rate, and positive antinuclear antibodies. Conclusion ANCA are found in a significant number of patients with IgG4-RD and differed from the ANCA-negative group in terms of clinical and serological features.

Validation of Plasmodium falciparum dUTPase as the target of 5′-tritylated deoxyuridine analogues with anti-malarial activity

Background Malaria remains as a major global problem, being one of the infectious diseases that engender highest mortality across the world. Due to the appearance of resistance and the lack of an effective vaccine, the search of novel anti-malarials is required. Deoxyuridine 5′-triphosphate nucleotido-hydrolase (dUTPase) is responsible for the hydrolysis of dUTP to dUMP within the parasite and has been proposed as an essential step in pyrimidine metabolism by providing dUMP for thymidylate biosynthesis. In this work, efforts to validate dUTPase as a drug target in Plasmodium falciparum are reported. Methods To investigate the role of PfdUTPase in cell survival different strategies to generate knockout mutants were used. For validation of PfdUTPase as the intracellular target of four inhibitors of the enzyme, mutants overexpressing PfdUTPase and HsdUTPase were created and the IC50 for each cell line with each compound was determined. The effect of these compounds on dUTP and dTTP levels from P. falciparum was measured using a DNA polymerase assay. Detailed localization studies by indirect immunofluorescence microscopy and live cell imaging were also performed using a cell line overexpressing a Pfdut-GFP fusion protein. Results Different attempts of disruption of the dut gene of P. falciparum were unsuccessful while a 3′ replacement construct could recombine correctly in the locus suggesting that the enzyme is essential. The four 5′-tritylated deoxyuridine analogues described are potent inhibitors of the P. falciparum dUTPase and exhibit antiplasmodial activity. Overexpression of the Plasmodium and human enzymes conferred resistance against selective compounds, providing chemical validation of the target and confirming that indeed dUTPase inhibition is involved in anti-malarial activity. In addition, incubation with these inhibitors was associated with a depletion of the dTTP pool corroborating the central role of dUTPase in dTTP synthesis. PfdUTPase is mainly localized in the cytosol. Conclusion These results strongly confirm the pivotal and essential role of dUTPase in pyrimidine biosynthesis of P. falciparum intraerythrocytic stages.

Germs within Worms: Localization of Neorickettsia sp. within Life Cycle Stages of the Digenean Plagiorchis elegans

ABSTRACTNeorickettsiaspp. are bacterial endosymbionts of parasitic flukes (Digenea) that also have the potential to infect and cause disease (e.g., Sennetsu fever) in the vertebrate hosts of the fluke. One of the largest gaps in our knowledge ofNeorickettsiabiology is the very limited information available regarding the localization of the bacterial endosymbiont within its digenean host. In this study, we used indirect immunofluorescence microscopy to visualizeNeorickettsiasp. within several life cycle stages of the digeneanPlagiorchis elegans. Individual sporocysts, cercariae, metacercariae, and adults ofP. elegansnaturally infected withNeorickettsiasp. were obtained from our laboratory-maintained life cycle, embedded, sectioned, and prepared for indirect immunofluorescence microscopy using anti-Neorickettsiaristiciihorse serum as the primary antibody.Neorickettsiasp. was found within the tegument of sporocysts, throughout cercarial embryos (germ balls) and fully formed cercariae (within the sporocysts), throughout metacercariae, and within the tegument, parenchyma, vitellaria, uteri, testes, cirrus sacs, and eggs of adults. Interestingly,Neorickettsiasp. was not found within the ovarian tissue. This suggests that vertical transmission ofNeorickettsiawithin adult digeneans occurs via the incorporation of infected vitelline cells into the egg rather than direct infection of the ooplasm of the oocyte, as has been described for other bacterial endosymbionts of invertebrates (e.g.,RickettsiaandWolbachia).