The development of high-throughput assays to screen for potential anticancer and antimalarial compounds that target ADP-ribosylation factor 6 and its signalling machineries

ADP-ribosylation factors (Arfs) are small GTP-binding proteins that cycle between active GTP-bound forms and inactive GDP-bound forms. GDP/GTP cycling is regulated by large families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). ArfGEFs activate Arfs by mediating the exchange of GDP for GTP, while ArfGAPs terminate Arf function by stimulating the hydrolysis of the terminal phosphate group of GTP. Arf6 is a major regulator of endocytic trafficking and reorganization of the actin cytoskeleton in eukaryotic organisms. Owing to its participation in wide range of fundamentally distinct cellular processes, Arf6 may be a drug target for cancer and malaria amongst other diseases. As with cancer cells, rapid growth and viability of eukaryotic pathogens likely places a heavy burden on their endocytic pathways and a critical reliance on Arf6 activity. A putative malarial homolog of Arf6 (PfArf6) localises to numerous puncta along the periphery of the parasite in the mature trophozoite life stage of the parasite (T. Swart, MSc dissertation). Owing to highly inefficient parasite transfection procedures and a relative shortage of well described and validated parasite organelle markers, the possible functions of PfArf6 were explored using HeLa cells as a surrogate model for parasites by fluorescence microscopy of cells transfected with GFP-tagged PfArf6. Partial co-localisation was observed with the mammalian markers HsArf6 and LC3, which suggested possible roles in Arf6-dependent endocytosis and autophagy, respectively. While these possible roles are currently under investigation in parasites, an overall long-term goal which was initiated in this study was to determine whether PfArf6 is a valid drug target. To chemically validate PfArf6 as a drug target, a potent inhibitor needs to be identified. This requires the development of assays that may be employed for high-throughput screening of compound libraries. To support this goal, a novel plate-based assay was developed using human Arf6. The assay relies on the selective binding of an Arf effector protein domain (GGA3) fused to glutathione-S-transferase (GST), to His-tagged Arf6 immobilised on a nickel-coated plate. The assay format was developed and could robustly distinguish HsArf6-GDP (inactive) from HsArf6-GTP (active). Furthermore, it could be employed to detect the deactivation of Arf6 by ArfGAP1-stimualted GTP hydrolysis, but not Arf6 activation by ARNO-stimulated GDP/GTP exchange (ARNO is an ArfGEF). The ArfGAP1 deactivation assay was chemically validated using a known ArfGAP inhibitor, QS11. An improved assay was developed that employs JIP4 as an Arf6-specific binding partner instead of GGA3. In addition to superior performance, the alternative assay format could potentially be exploited for cancer drug discovery, since Arf6-JIP4 interaction has been implicated in cancer cell invasion and metastasis. Both assays may be employed to explore alternative ArfGEFs and ArfGAPs that act on Arf6 and contribute to the advancement of cancer. In parallel experiments, where development of PfArf6 assays was the focus, several issues arose. Firstly, we could not prepare GDP- and GTP-bound forms of PfArf6 since EDTA-mediated nucleotide exchange appeared to irreversibly destabilise the protein. However, PfArf6 activation (i.e. the preparation of PfArf6-GTP) was possible when mediated by ARNO and assessed by tryptophan fluorescence kinetic assays, suggesting that PfArf6 may be expressed in GDP-bound form in E. coli. As with human Arf6, ARNO-mediated GDP/GTP exchange on PfArf6 was not detectable in the immobilised PfArf6-GGA interaction GST assay format. However, a more sensitive assay was developed which relies on the use of nickel-horseradish peroxidase to detect the binding of His-tagged PfArf6 to JIP4-GST immobilised on glutathione plates and could detect ARNO-mediated PfArf6 activation. Since we could not prepare PfArf6-GTP (that did not rely on the presence of the ArfGEF, ARNO), malarial ArfGAP deactivation studies were conducted using PfArf1 instead of PfArf6 in the GGA-GST interaction assay. Both PfArfGAP1and PfArfGAP2 stimulated GTP hydrolysis by PfArf1, but only the former was inhibited by the standard human ArfGAP inhibitor, QS11. The development of these simple, cost-effective assays can be used in the high-throughput screening of novel anticancer and antimalarial compounds that target Arf signalling machineries. In theory, the assay could be extended as a tool to identify novel inhibitors of the multitude of Arfs, ArfGEFs and ArfGAPs originating from any organism and hence has broad clinical significance

High sensitivity of ultrasound for the diagnosis of tuberculosis in adults in South Africa: A proof-of-concept study.

BACKGROUND: There are limited data on the performance characteristics of ultrasound for the diagnosis of pulmonary tuberculosis in both HIV-positive and HIV-negative persons. The objective of this proof-of-concept study was to determine the sensitivity and specificity of ultrasound for the diagnosis of tuberculosis in adults. METHODS: Comprehensive thoracic and focused abdominal ultrasound examinations were performed by trained radiologists and pulmonologists on adults recruited from a community multimorbidity survey and a primary healthcare clinic in KwaZulu-Natal Province, South Africa. Sputum samples were systematically collected from all participants. Sensitivity and specificity of ultrasound to detect tuberculosis were calculated compared to a reference standard of i) bacteriologically-confirmed tuberculosis, and ii) either bacteriologically-confirmed or radiologic tuberculosis. RESULTS: Among 92 patients (53 [58%] male, mean age 41.9 [standard deviation 13.7] years, 49 [53%] HIV positive), 34 (37%) had bacteriologically-confirmed tuberculosis, 8 (9%) had radiologic tuberculosis with negative bacteriologic studies, and 50 (54%) had no evidence of active tuberculosis. Ultrasound abnormalities on either thoracic or abdominal exams were detected in 31 (91%) participants with bacteriologic tuberculosis and 27 (54%) of those without tuberculosis. Sensitivity and specificity of any ultrasound abnormality for bacteriologically-confirmed tuberculosis were 91% (95% confidence interval [CI] 76%-98%) and 46% (95% CI 32%-61%). Sensitivity and specificity of any ultrasound abnormality for either bacteriologically-confirmed or radiologic tuberculosis were 86% (95% CI 71%-95%) and 46% (95% CI 32%-61%). Overall performance did not appear to differ markedly between participants with and without HIV. CONCLUSION: A comprehensive ultrasound scanning protocol in adults in a high TB burden setting had high sensitivity but low specificity to identify bacteriologically-confirmed tuberculosis

Sensitivity and specificity of ultrasound findings for bacteriologic TB in participants with and without HIV.

(XLSX)</p

MR1-Restricted MAIT Cells From The Human Lung Mucosal Surface Have Distinct Phenotypic, Functional, and Transcriptomic Features That Are Preserved in HIV Infection

Mucosal associated invariant T (MAIT) cells are a class of innate-like T cells that utilize a semi-invariant αβ T cell receptor to recognize small molecule ligands produced by bacteria and fungi. Despite growing evidence that immune cells at mucosal surfaces are often phenotypically and functionally distinct from those in the peripheral circulation, knowledge about the characteristics of MAIT cells at the lung mucosal surface, the site of exposure to respiratory pathogens, is limited. HIV infection has been shown to have a profound effect on the number and function of MAIT cells in the peripheral blood, but its effect on lung mucosal MAIT cells is unknown. We examined the phenotypic, functional, and transcriptomic features of major histocompatibility complex (MHC) class I-related (MR1)-restricted MAIT cells from the peripheral blood and bronchoalveolar compartments of otherwise healthy individuals with latent Mycobacterium tuberculosis (Mtb) infection who were either HIV uninfected or HIV infected. Peripheral blood MAIT cells consistently co-expressed typical MAIT cell surface markers CD161 and CD26 in HIV-negative individuals, while paired bronchoalveolar MAIT cells displayed heterogenous expression of these markers. Bronchoalveolar MAIT cells produced lower levels of pro-inflammatory cytokine IFN-γ and expressed higher levels of co-inhibitory markers PD-1 and TIM-3 than peripheral MAIT cells. HIV infection resulted in decreased frequencies and pro-inflammatory function of peripheral blood MAIT cells, while in the bronchoalveolar compartment MAIT cell frequency was decreased but phenotype and function were not significantly altered. Single-cell transcriptomic analysis demonstrated greater heterogeneity among bronchoalveolar compared to peripheral blood MAIT cells and suggested a distinct subset in the bronchoalveolar compartment. The transcriptional features of this bronchoalveolar subset were associated with MAIT cell tissue repair functions. In summary, we found previously undescribed phenotypic and transcriptional heterogeneity of bronchoalveolar MAIT cells in HIV-negative people. In HIV infection, we found numeric depletion of MAIT cells in both anatomical compartments but preservation of the novel phenotypic and transcriptional features of bronchoalveolar MAIT cells.</jats:p

Demographic and clinical characteristics of study population (N = 92).

Demographic and clinical characteristics of study population (N = 92).</p

Ultrasound findings in all participants stratified by TB status.

Ultrasound findings in all participants stratified by TB status.</p

Ultrasound findings in participants with bacteriologic TB compared to healthy participants.

(DOCX)</p

Classification of ultrasound findings.

Classification of ultrasound findings.</p

Typical thoracic ultrasound findings.

Panel A demonstrates the appearance of normal air-filled lung, characterized by a bright white horizontal pleural line interrupted by rib shadows, and repeating horizontal reverberation artifacts known as A-lines. Panel B shows lung consolidation, characterized by subpleural echo-poor region greater than 10 mm in depth or length. Panel C demonstrates a small subpleural consolidation (SPC), characterized by a hypoechoic subpleural region less than 10 mm in depth and length, with distinct borders and a trailing comet-tail artifact.</p