Sex-Differences and Associations Between Complement Activation and Synovial Vascularization in Patients with Late-Stage Knee Osteoarthritis.

Purpose: Synovial inflammation in knee osteoarthritis (OA) causes disorganized synovial angiogenesis and complement activation in synovial fluid, but links between complement and synovial microvascular pathology have not been established. Since complement causes vascular pathology in other diseases and since sex-differences exist in complement activation and in OA, we investigated sex differences in synovial fluid complement factors, synovial tissue vascular pathology, and associations between complement and synovial vascular pathology in patients with late-stage knee OA. Methods: Patients with symptomatic, late-stage radiographic knee OA undergoing total knee arthroplasty or high tibial osteotomy provided matched synovial fluid and tissue biopsies during surgery. Complement factors (C2, C5, adipsin, MBL, and CFI) and terminal complement complex (sC5b-C9) were measured in synovial fluid by multiplex or enzyme-linked immunosorbent assay, respectively. Features of synovial vascular pathology (vascularization, perivascular edema, and vasculopathy) were assessed by histopathology. Multivariate linear regression models were used to assess associations between synovial fluid complement factors and histopathological features of vascular pathology, with adjustment for age, sex, body mass index, and sex interaction. Sex-disaggregated comparisons were completed. Results: Synovial fluid biomarker and histopathology data were included from 97 patients. Most synovial fluid complement factors and synovial tissue histopathological features were similar between sexes. Synovial fluid C5 trended to lower levels in males (-20.93 ng/mL [95%CI -42.08, 0.23] p=0.05). Median vasculopathy scores (0.42 [95%CI 0.07, 0.77] p=0.02) were higher in males. In the full cohort, C5 concentration was associated with lower vascularization scores (-0.005 [95%CI -0.010, -0.0001] p=0.04) while accounting for sex*C5 interaction. In sex-disaggregated analyses, increased C5 concentration was associated with lower vascularization scores (-0.005 [95%CI -0.009, -0.0001] p=0.04) in male patients, but not in female patients. Males had higher sC5b-C9 compared to females. Additionally, males with high C5 had a higher synovial fluid concentration of sC5b-C9 compared to males with low C5. No differences were found in females. Conclusion: Higher synovial fluid C5 levels were associated with increased complement activation and decreased synovial vascularization in males but not in females with OA. Future studies should test whether synovial fluid complement activation suppresses synovial angiogenesis and identify mechanisms accounting for C5-related sex-differences in synovial fluid complement activation in patients with knee OA.

Effects of trypanocidal drugs on DNA synthesis: new insights into melarsoprol growth inhibition.

Trypanothione is the primary thiol redox carrier in Trypanosomatids whose biosynthesis and utilization pathways contain unique enzymes that include suitable drug targets against the human parasites in this family. Overexpression of the rate-limiting enzyme, γ-glutamylcysteine synthetase (GSH1), can increase the intracellular concentration of trypanothione. Melarsoprol directly inhibits trypanothione and has predicted the effects on downstream redox biology, including ROS management and dNTP synthesis that require further investigation. Thus, we hypothesized that melarsoprol treatment would inhibit DNA synthesis, which was tested using BrdU incorporation assays and cell cycle analyses. In addition, we analysed the effects of eflornithine, which interfaces with the trypanothione pathway, fexinidazole, because of the predicted effects on DNA synthesis, and pentamidine as an experimental control. We found that melarsoprol treatment resulted in a cell cycle stall and a complete inhibition of DNA synthesis within 24 h, which were alleviated by GSH1 overexpression. In contrast, the other drugs analysed had more subtle effects on DNA synthesis that were not significantly altered by GSH1 expression. Together these findings implicate DNA synthesis as a therapeutic target that warrants further investigation in the development of antitrypanosomal drugs.

A Trypanosoma brucei ORFeome-Based Gain-of-Function Library Identifies Genes That Promote Survival during Melarsoprol Treatment.

Trypanosoma brucei is an early branching protozoan parasite that causes human and animal African trypanosomiasis. Forward genetics approaches are powerful tools for uncovering novel aspects of trypanosomatid biology, pathogenesis, and therapeutic approaches against trypanosomiasis. Here, we have generated a T. brucei cloned ORFeome consisting of >90% of the targeted 7,245 genes and used it to make an inducible gain-of-function parasite library broadly applicable to large-scale forward genetic screens. We conducted a proof-of-principle genetic screen to identify genes whose expression promotes survival in melarsoprol, a critical drug of last resort. The 57 genes identified as overrepresented in melarsoprol survivor populations included the gene encoding the rate-limiting enzyme for the biosynthesis of an established drug target (trypanothione), validating the tool. In addition, novel genes associated with gene expression, flagellum localization, and mitochondrion localization were identified, and a subset of those genes increased melarsoprol resistance upon overexpression in culture. These findings offer new insights into trypanosomatid basic biology, implications for drug targets, and direct or indirect drug resistance mechanisms. This study generated a T. brucei ORFeome and gain-of-function parasite library, demonstrated the library's usefulness in forward genetic screening, and identified novel aspects of melarsoprol resistance that will be the subject of future investigations. These powerful genetic tools can be used to broadly advance trypanosomatid research.IMPORTANCE Trypanosomatid parasites threaten the health of more than 1 billion people worldwide. Because their genomes are highly diverged from those of well-established eukaryotes, conservation is not always useful in assigning gene functions. However, it is precisely among the trypanosomatid-specific genes that ideal therapeutic targets might be found. Forward genetics approaches are an effective way to identify novel gene functions. We used an ORFeome approach to clone a large percentage of Trypanosoma brucei genes and generate a gain-of-function parasite library. This library was used in a genetic screen to identify genes that promote resistance to the clinically significant yet highly toxic drug melarsoprol. Hits arising from the screen demonstrated the library's usefulness in identifying known pathways and uncovered novel aspects of resistance mediated by proteins localized to the flagellum and mitochondrion. The powerful new genetic tools generated herein are expected to promote advances in trypanosomatid biology and therapeutic development in the years to come.

The pancreas-specific form of secretory pathway calcium ATPase 2 regulates multiple pathways involved in calcium homeostasis.

Acinar cell exocytosis requires spatiotemporal Ca2+ signals regulated through endoplasmic reticulum (ER) stores, Ca2+ATPases, and store-operated Ca2+ entry (SOCE). The secretory pathway Ca2+ATPase 2 (SPCA2) interacts with Orai1, which is involved in SOCE and store independent Ca2+ entry (SICE). However, in the pancreas, only a C-terminally truncated form of SPCA2 (termed SPAC2C) exists. The goal of this study was to determine if SPCA2C effects Ca2+ homeostasis in a similar fashion to the full-length SPCA2. Using epitope-tagged SPCA2C (SPCA2CFLAG) expressed in HEK293A cells and Fura2 imaging, cytosolic [Ca2+] was examined during SICE, SOCE and secretagogue-stimulated signaling. Exogenous SPCA2C expression increased resting cytosolic [Ca2+], Ca2+ release in response to carbachol, ER Ca2+ stores, and store-mediated and independent Ca2+ influx. Co-IP detected Orai1-SPCA2C interaction, which was altered by co-expression of STIM1. Importantly, SPCA2C's effects on store-mediated Ca2+ entry were independent of Orai1. These findings indicate SPCA2C influences Ca2+ homeostasis through multiple mechanisms, some of which are independent of Orai1, suggesting novel and possibly cell-specific Ca2+ regulation.