A comparative analysis of blood and faecal-based laboratory methods in the diagnosis of extraintestinal microsporidia infection.

Diagnosis of extraintestinal microsporidiosis is always hampered due to non-specific symptoms and difficulty in diagnosis. This study aimed to compare the diagnostic utility of blood and faecal-based polymerase chain reaction (PCR) to detect microsporidiosis in immunocompromised patients. A total of 42 immunocompromised patients consisting of HIV-infected and chemotherapy-treated patients were enrolled. Paired faecal and blood samples were collected and subjected to PCR to detect Enterocytozoon bieneusi and Encephalitozoon spp. Faecal samples were microscopically screened for microsporidia spores. Overall, 42.9% (18/42) of patients were positive for microsporidiosis. Of this, 19.0% (8/42) and 4.8% (2/42) were positive by blood and stool PCR respectively. Meanwhile, 33.3% (14/42) of the faecal specimens were microscopically positive. Among the positive patients, 22.2% (4/18) had microsporidia confirmed by blood PCR and stool microscopy, suggestive of dissemination. Interestingly, the stool specimen in which microsporidia spores were detected via microscopy is not positive via PCR method. This highlights the limitation of the faecal-based detection method and the important use of blood samples for diagnosing extraintestinal microsporidiosis. Only E. bieneusi species were detected in all PCR-positive samples. This study highlights the diagnostic value of blood PCR in diagnosing extraintestinal microsporidiosis infections.

Disseminated microsporidiosis: An underdiagnosed and emerging opportunistic disease.

Disseminated microsporidiosis is a life-threatening disease resulting from the haematogenous spread of microsporidia species. The diagnosis is challenging owing to its subtle nonspecific clinical presentation, which usually reflects the underlying organ involved. Therefore, a high index of suspicion is required for early diagnosis. Besides, tools for confirmatory laboratory diagnosis are limited. Currently, there is no direct diagnostic method that can detect the infection without involving invasive procedures. Clinical confirmation of disseminated microsporidiosis is usually based on light and transmission electron microscopy of infected tissue specimens. These are then followed by species detection using polymerase chain reaction (PCR). Disseminated microsporidiosis shows the potential to be cleared up by albendazole or fumagillin if they are detected and treated early. Based on a series of case reports, this review aims to present a current update on disseminated microsporidiosis with emphasis on the clinical manifestations based on the organ system infected, diagnostic approach and treatment of this devastating condition.

Sprouty proteins: modified modulators, matchmakers or missing links?

Sprouty proteins are involved in organogenesis, particularly during the branching of endothelial tubes, and existing evidence suggests that Sprouty's point of action lies downstream of receptor signaling to inhibit the activation of the central Ras/Erk pathway. How Sprouty proteins accomplish their inhibitory action and whether they interact with other signaling pathways are significant questions. Sprouty proteins are devoid of any recognizable protein interaction domain, and clues as to how they function have been mainly derived from screening for interacting partners. Conserved across all the Sprouty proteins are three sequences: a Cbl-tyrosine kinase-binding (TKB) binding motif centered on an obligatorily phosphorylated tyrosine (Y55 in Sprouty2), a serine-rich motif (SRM) and a cysteine-rich domain (CRD). With the exception of a handful of proteins that bind to the N-terminus, most of the binding to Sprouty occurs via the CRD, predominantly by serine/threonine kinases that target sites within the SRM on Sprouty. Some of the resultant increase in phosphorylation is opposed by activated protein phosphatase 2A that binds to the N-terminal Cbl-TKB binding motif. Significantly, two ubiquitin E3 ligases also bind to the N-terminus of Sprouty: c-Cbl binds with high affinity to the TKB binding motif and SIAH2 binds constitutively to a different site; both proteins are able to direct the ubiquitination of Sprouty proteins and its destruction. The collective evidence points to Sprouty proteins as being substantially covalently-modified to control its location, stability, association, and destruction. With such stringent control of the Sproutys, the main question is what key proteins does this facilitator bring together?

Phospholipase D is activated by phorbol ester but not CSF-1 in murine bone marrow-derived macrophages.

Phospholipase D activity was measured in murine bone marrow-derived macrophages (BMM) treated with either colony stimulating factor-1 (CSF-1) or phorbol myristyl acetate (PMA) by measuring formation of phosphatidylbutanol (PtBut) in cells preloaded with n-butanol. Addition of 10(-7) M PMA for 15 min stimulated the amount of PtBut formed in growth arrested cells by 3-4 fold whereas no stimulation was observed with 5000 units mL-1 CSF-1 for 0.5, 2 or 15 min. Protein kinase C activity was determined in growth-arrested BMM by phosphorylation of Myristoylated Alanine-Rich C Kinase Substrate (MARCKS). PMA stimulation for 5 min increased protein kinase C activity 5-6 fold whereas CSF-1 treatment for 5 min or 15 min did not. Contrary to earlier reports, CSF-1 did not stimulate diradyl glycerol formation in BMM. These results show that stimulation of protein kinase C and the activation of phospholipase D are not involved in the early events of CSF-1-stimulated signal transduction pathways in BMM.

Differences in the kinetics of activation of protein kinases and extracellular signal-related protein kinase 1 in colony-stimulating factor 1-stimulated and lipopolysaccharide-stimulated macrophages.

To determine the relevance of mitogen-activated protein kinase activity to macrophage proliferation, we measured the stimulation of myelin basic protein (MBP) kinase and extracellular signal-related protein kinase (ERK) activity in a macrophage cell line (BAC1.2F5), bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM). By using an 'ingel' MBP kinase assay the activities of renaturable MBP kinases were detected, including several with molecular masses similar to those of ERK-1 and ERK-2. These represented a minor fraction of total activity and were not activated to an appreciable extent by colony-stimulating factor 1 (CSF-1). By using a sensitive and specific immune-complex kinase assay, activation of ERK-1 by CSF-1 and lipopolysaccharide (LPS) was demonstrated. Two kinetically distinct pathways of ERK-1 activation by CSF-1 were resolved, with peak activations occurring at 5 and 15 min. The kinetics and degree of activation were similar in BMM, BAC1.2F5 cells and RPM. LPS activated ERK-1 with a single peak at 10-15 min, corresponding to the later peak of activation by CSF-1. Thus there was no strict correlation between ERK activation and macrophage proliferation.

Haematopoietic colony stimulating factors CSF-1 and GM-CSF increase phosphatidylinositol 3-kinase activity in murine bone marrow-derived macrophages.

The activity of phosphatidylinositol (PI) 3-kinase was examined in murine bone marrow-derived macrophages (BMM) stimulated with the haematopoietic growth factors colony stimulating factor-1 (CSF-1) and granulocyte/macrophage-CSF (GM-CSF). PI 3-kinase was immunoprecipitated from cell lysates using anti-phosphotyrosine antibody or an antibody directed against the 85K subunit of PI 3-kinase, and the activity assayed by the phosphorylation of PI in the presence of [gamma 32P]-ATP. The results demonstrate that CSF-1 increases the activity of PI 3-kinase, as compared to the non-stimulated control, in murine macrophages. Maximum activity was seen after 10 min of stimulation with CSF-1 at 3000-5000 U/ml. The dose-response of CSF-1 is consistent with other biochemical effects of CSF-1 seen in the BMM. GM-CSF also stimulated PI 3-kinase activity although to a lesser extent than CSF-1, correlating well with their degree of mitogenic activity on the BMM. Non-mitogenic macrophage activating agents, such as the phorbol myristate acetate, lipopolysaccharide, concanavalin A and formyl-methionyl-leucyl-phenylalanine, did not significantly increase the PI 3-kinase activity. Furthermore, CSF-1 failed to stimulate PI 3-kinase activity in resident peritoneal macrophages, a population of macrophages with poor proliferative capacity. These results suggest that the PI 3-kinase activity may be involved in the haemopoietic growth factor signalling pathways regulating macrophage growth.

Sprouty proteins are targeted to membrane ruffles upon growth factor receptor tyrosine kinase activation. Identification of a novel translocation domain.

Sprouty (Spry) was first identified in a genetic screen in Drosophila to be an antagonist of fibroblast growth factor and epidermal growth factor (EGF) signaling, seemingly by inhibiting the Ras/MAP kinase pathway. Data base searches lead to the identification and cloning of, to date, four mammalian sprouty genes. The primary sequences of the mammalian sprouty gene products share a well conserved cysteine-rich C-terminal domain with the Drosophila protein. The N-terminal regions, however, do not exhibit significant homology. This study aimed at determining the disposition of Spry proteins in intact cells before and after stimulation of the EGF receptor tyrosine kinase. Full-length or deletion mutants of Spry, tagged at the N termini with the FLAG-epitope, were expressed in COS-1 cells by transient transfection and analyzed by immunofluorescence microscopy before and after EGF stimulation of the cells. In unstimulated cells, the Spry proteins were distributed throughout the cytosol except for human Sprouty2 (hSpry2), which, although generally located in the cytosol, co-localized with microtubules. In all cases, the Spry proteins underwent rapid translocation to membrane ruffles following EGF stimulation. The optimal translocation domain was identified by deletion and immunofluorescence analysis to be a highly conserved 105-amino acid domain in the C-terminal half of the hSpry2 protein. The translocation of this conserved domain, based on hSpry2 data, was independent of the activation of phosphatidylinositol-3 kinase.