Improving screening and management of latent tuberculosis infection: development and evaluation of latent tuberculosis infection primary care model.

BACKGROUND: In Australia, demand for specialist infectious diseases services exceeds capacity to provide timely management of latent tuberculosis infection (LTBI) in areas of high refugee and asylum seeker settlement. A model for treating LTBI patients in primary care has been developed and piloted in a refugee-focused primary health service (Monash Health Refugee Health and Wellbeing [MHRHW]) and a universal primary care clinic. This study reports on the development and evaluation of the model, focusing on the model feasibility, and barriers and enablers to its success. METHODS: A convergent mix-methods design was used to evaluate the model for treating LTBI patients in primary care, where a prospective cohort study of patients commencing treatment either at MHRHW or the universal primary care clinic determined the model feasibility, while focus groups with clinicians directly involved in treating these patients explored barriers and enablers to sustainability and success of the model. RESULTS: From January 2017 to April 2018, 65 patients with confirmed LTBI presented at participating clinics. Treatment was accepted by 31 (48%) patients, of whom 15(48%) were treated at MHRHW and 16 (52%) at the universal primary care clinic. The 6-months' treatment completion rate was higher at MHRHW compared to the universal primary care clinic (14 (93%) compared to 9 (56%) respectively, p = 0.0373). Reasons for non-completion included adverse reaction, opting out and relocation. At the completion of the pilot, 15 clinicians participated in two focus groups. Clinicians identified barriers and enablers for successful LTBI management at patient, provider, organisational and clinical levels. While barriers for treatment completion and adherence were consistent across the two pilot sites, enablers, such as resources to facilitate patient education and follow-up, were available only at MHRHW. CONCLUSION: Screening and management of LTBI patients can be achieved within the primary care setting, considerate of barriers and enablers at patient, provider, organisational and clinical levels. Upscaling of a primary care response to the management of LTBI will require supporting primary care clinics with resources to employ dedicated clinical staff for patient education, follow-up communication and monitoring medication adherence.

Prototheca wickerhamii breast implant infection after reconstructive surgery: a new level of complexity.

We report the first published case of Prototheca wickerhamii breast implant infection. This occurred after mastectomy, chemotherapy, radiotherapy, breast reconstruction, implant revisions and breast seroma aspirations and was preceded by polymicrobial infection. Definitive treatment required implant removal and intravenous liposomal amphotericin B. The management of breast prosthesis infections is discussed.

The myotubularin MTMR4 regulates phagosomal phosphatidylinositol 3-phosphate turnover and phagocytosis.

Macrophage phagocytosis is required for effective clearance of invading bacteria and other microbes. Coordinated phosphoinositide signaling is critical both for phagocytic particle engulfment and subsequent phagosomal maturation to a degradative organelle. Phosphatidylinositol 3-phosphate (PtdIns(3)P) is a phosphoinositide that is rapidly synthesized and degraded on phagosomal membranes, where it recruits FYVE domain- and PX motif-containing proteins that promote phagosomal maturation. However, the molecular mechanisms that regulate PtdIns(3)P removal from the phagosome have remained unclear. We report here that a myotubularin PtdIns(3)P 3-phosphatase, myotubularin-related protein-4 (MTMR4), regulates macrophage phagocytosis. MTMR4 overexpression reduced and siRNA-mediated Mtmr4 silencing increased levels of cell-surface immunoglobulin receptors (i.e. Fcγ receptors (FcγRs)) on RAW 264.7 macrophages, associated with altered pseudopodal F-actin. Furthermore, MTMR4 negatively regulated the phagocytosis of IgG-opsonized particles, indicating that MTMR4 inhibits FcγR-mediated phagocytosis, and was dynamically recruited to phagosomes of macrophages during phagocytosis. MTMR4 overexpression decreased and Mtmr4-specific siRNA expression increased the duration of PtdIns(3)P on phagosomal membranes. Macrophages treated with Mtmr4-specific siRNA were more resistant to Mycobacterium marinum-induced phagosome arrest, associated with increased maturation of mycobacterial phagosomes, indicating that extended PtdIns(3)P signaling on phagosomes in the Mtmr4-knockdown cells permitted trafficking of phagosomes to acidic late endosomal and lysosomal compartments. In conclusion, our findings indicate that MTMR4 regulates PtdIns(3)P degradation in macrophages and thereby controls phagocytosis and phagosomal maturation.

The PTEN and Myotubularin phosphoinositide 3-phosphatases: linking lipid signalling to human disease.

Two classes of lipid phosphatases selectively dephosphorylate the 3 position of the inositol ring of phosphoinositide signaling molecules: the PTEN and the Myotubularin families. PTEN dephosphorylates PtdIns(3,4,5)P(3), acting in direct opposition to the Class I PI3K enzymes in the regulation of cell growth, proliferation and polarity and is an important tumor suppressor. Although there are several PTEN-related proteins encoded by the human genome, none of these appear to fulfill the same functions. In contrast, the Myotubularins dephosphorylate both PtdIns(3)P and PtdIns(3,5)P(2), making them antagonists of the Class II and Class III PI 3-kinases and regulators of membrane traffic. Both phosphatase groups were originally identified through their causal mutation in human disease. Mutations in specific myotubularins result in myotubular myopathy and Charcot-Marie-Tooth peripheral neuropathy; and loss of PTEN function through mutation and other mechanisms is evident in as many as a third of all human tumors. This chapter will discuss these two classes of phosphatases, covering what is known about their biochemistry, their functions at the cellular and whole body level and their influence on human health.

Silencer of death domains (SODD) inhibits skeletal muscle and kidney enriched inositol 5-phosphatase (SKIP) and regulates phosphoinositide 3-kinase (PI3K)/Akt signaling to the actin cytoskeleton.

Phosphoinositide 3-kinase (PI3K) regulates cell polarity and migration by generating phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P(3)) at the leading edge of migrating cells. The serine-threonine protein kinase Akt binds to PI(3,4,5)P(3), resulting in its activation. Active Akt promotes spatially regulated actin cytoskeletal remodeling and thereby directed cell migration. The inositol polyphosphate 5-phosphatases (5-ptases) degrade PI(3,4,5)P(3) to form PI(3,4)P(2), which leads to diminished Akt activation. Several 5-ptases, including SKIP and SHIP2, inhibit actin cytoskeletal reorganization by opposing PI3K/Akt signaling. In this current study, we identify a molecular co-chaperone termed silencer of death domains (SODD/BAG4) that forms a complex with several 5-ptase family members, including SKIP, SHIP1, and SHIP2. The interaction between SODD and SKIP exerts an inhibitory effect on SKIP PI(3,4,5)P(3) 5-ptase catalytic activity and consequently enhances the recruitment of PI(3,4,5)P(3)-effectors to the plasma membrane. In contrast, SODD(-/-) mouse embryonic fibroblasts exhibit reduced Akt-Ser(473) and -Thr(308) phosphorylation following EGF stimulation, associated with increased SKIP PI(3,4,5)P(3)-5-ptase activity. SODD(-/-) mouse embryonic fibroblasts exhibit decreased EGF-stimulated F-actin stress fibers, lamellipodia, and focal adhesion complexity, a phenotype that is rescued by the expression of constitutively active Akt1. Furthermore, reduced cell migration was observed in SODD(-/-) macrophages, which express the three 5-ptases shown to interact with SODD (SKIP, SHIP1, and SHIP2). Therefore, this study identifies SODD as a novel regulator of PI3K/Akt signaling to the actin cytoskeleton.

The myotubularin phosphatase MTMR4 regulates sorting from early endosomes.

Phosphatidylinositol 3-phosphate [PtdIns(3)P] regulates endocytic trafficking and the sorting of receptors through early endosomes, including the rapid recycling of transferrin (Tfn). However, the phosphoinositide phosphatase that selectively opposes this function is unknown. The myotubularins are a family of eight catalytically active and six inactive enzymes that hydrolyse PtdIns(3)P to form PtdIns. However, the role each myotubularin family member plays in regulating endosomal PtdIns(3)P and thereby endocytic trafficking is not well established. Here, we identify the myotubularin family member MTMR4, which localizes to early endosomes and also to Rab11- and Sec15-positive recycling endosomes. In cells with MTMR4 knockdown, or following expression of the catalytically inactive MTMR4, MTMR4(C407A), the number of PtdIns(3)P-decorated endosomes significantly increased. MTMR4 overexpression delayed the exit of Tfn from early endosomes and its recycling to the plasma membrane. By contrast, expression of MTMR4(C407A), which acts as a dominant-negative construct, significantly accelerated Tfn recycling. However, in MTMR4 knockdown cells Tfn recycling was unchanged, suggesting that other MTMs might also contribute to recycling. MTMR4 regulated the subcellular distribution of Rab11 and, in cells with RNAi-mediated knockdown of MTMR4, Rab11 was directed away from the pericentriolar recycling compartment. The subcellular distribution of VAMP3, a v-SNARE protein that resides in recycling endosomes and endosome-derived transport vesicles, was also regulated by MTMR4. Therefore, MTMR4 localizes at the interface of early and recycling endosomes to regulate trafficking through this pathway.

Regulation of PI(3)K/Akt signalling and cellular transformation by inositol polyphosphate 4-phosphatase-1.

Akt is a crucial phosphoinositide 3-kinase (PI(3)K) effector that regulates cell proliferation and survival. PI(3)K-generated signals, PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2), direct Akt plasma membrane engagement. Pathological Akt plasma membrane association promotes oncogenesis. PtdIns(3,4)P(2) is degraded by inositol polyphosphate 4-phosphatase-1 (4-ptase-1) forming PtdIns(3)P; however, the role of 4-ptase-1 in regulating the activation and function of Akt is unclear. In mouse embryonic fibroblasts lacking 4-ptase-1 ((-/-)MEFs), the Akt-pleckstrin homology (PH) domain was constitutively membrane-associated both in serum-starved and agonist-stimulated cells, in contrast to (+/+)MEFs, in which it was detected only at the plasma membrane following serum stimulation. Epidermal growth factor (EGF) stimulation resulted in increased Ser(473) and Thr(308)-Akt phosphorylation and activation of Akt-dependent signalling in (-/-)MEFs, relative to (+/+)MEFs. Significantly, loss of 4-ptase-1 resulted in increased cell proliferation and decreased apoptosis. SV40-transformed (-/-)MEFs showed increased anchorage-independent cell growth and formed tumours in nude mice. This study provides the first evidence, to our knowledge, that 4-ptase-1 controls the activation of Akt and thereby cell proliferation, survival and tumorigenesis.

Characterisation of PfRON6, a Plasmodium falciparum rhoptry neck protein with a novel cysteine-rich domain.

The pathological consequences of malaria infection are the result of parasite replication within red blood cells (RBCs). Invasion into RBCs is mediated by a large repertoire of parasite proteins that are distributed on the parasite surface and within specialised apical secretory organelles. As invasion is an essential step in the parasite life-cycle, targeting invasion-related molecules provides an avenue for therapeutic intervention. We have used genome and transcriptome data available for Plasmodium falciparum to identify proteins likely to be involved in RBC invasion. Of these candidates, we selected a protein which we have dubbed PfRON6 for detailed characterisation. PfRON6 contains a novel cysteine-rich domain that is conserved in other Apicomplexan parasites. We show that PfRON6 is localised in the rhoptry neck of merozoites and is transferred to the newly formed parasitophorous vacuole during invasion. Transfection experiments indicate that the gene which encodes PfRON6 is refractory to integration that disrupts the coding sequence, suggesting its absence is incompatible with the parasite life-cycle. Further, the cysteine-rich domain appears to be functionally important as it cannot be truncated. Taken together, these data identify PfRON6 as a novel and potentially important component of the Plasmodium invasion machinery.