Generation of an immortalized mesenchymal stem cell line producing a secreted biosensor protein for glucose monitoring.

Diabetes is a chronic disease characterized by high levels of blood glucose. Diabetic patients should normalize these levels in order to avoid short and long term clinical complications. Presently, blood glucose monitoring is dependent on frequent finger pricking and enzyme based systems that analyze the drawn blood. Continuous blood glucose monitors are already on market but suffer from technical problems, inaccuracy and short operation time. A novel approach for continuous glucose monitoring is the development of implantable cell-based biosensors that emit light signals corresponding to glucose concentrations. Such devices use genetically modified cells expressing chimeric genes with glucose binding properties. MSCs are good candidates as carrier cells, as they can be genetically engineered and expanded into large numbers. They also possess immunomodulatory properties that, by reducing local inflammation, may assist long operation time. Here, we generated a novel immortalized human MSC line co-expressing hTERT and a secreted glucose biosensor transgene using the Sleeping Beauty transposon technology. Genetically modified hMSCs retained their mesenchymal characteristics. Stable transgene expression was validated biochemically. Increased activity of hTERT was accompanied by elevated and constant level of stem cell pluripotency markers and subsequently, by MSC immortalization. Furthermore, these cells efficiently suppressed PBMC proliferation in MLR transwell assays, indicating that they possess immunomodulatory properties. Finally, biosensor protein produced by MSCs was used to quantify glucose in cell-free assays. Our results indicate that our immortalized MSCs are suitable for measuring glucose concentrations in a physiological range. Thus, they are appropriate for incorporation into a cell-based, immune-privileged, glucose-monitoring medical device.

The three Aspergillus fumigatus CFEM-domain GPI-anchored proteins (CfmA-C) affect cell-wall stability but do not play a role in fungal virulence.

Fungal cell-wall proteins containing the conserved fungal CFEM domain have been implicated in host-pathogen interactions and virulence. To determine the role of these proteins in the mold pathogen Aspergillus fumigatus, we deleted the entire family of three CFEM-containing genes (CfmA-C), singly and in all combinations. We found an additive increase in the susceptibility of the single, double and triple ΔCfm mutants towards the chitin/ß-glucan-microfibril destabilizing compounds Congo Red (CR) and Calcofluor White (CFW), indicating that the A. fumigatus CFEM proteins are involved in stabilizing the cell wall. No defects in growth or germination were observed, indicating that CFEM proteins do not have an essential role in the morphogenesis of A. fumigatus. Unlike in Candida albicans, the A. fumigatus CFEM proteins were not implicated in heme uptake or biofilm formation. The ΔTriple-Cfm deletion strain did not exhibit altered virulence in either insect or murine models of infection, suggesting that cell-wall proteins containing the conserved fungal CFEM domain are not a significant virulence factor in A. fumigatus.

Coding tandem repeats generate diversity in Aspergillus fumigatus genes.

Genes containing multiple coding mini- and microsatellite repeats are highly dynamic components of genomes. Frequent recombination events within these tandem repeats lead to changes in repeat numbers, which in turn alters the amino acid sequence of the corresponding protein. In bacteria and yeasts, the expansion of such coding repeats in cell wall proteins is associated with alterations in immunogenicity, adhesion, and pathogenesis. We hypothesized that identification of repeat-containing putative cell wall proteins in the human pathogen Aspergillus fumigatus may reveal novel pathogenesis-related elements. Here, we report that the genome of A. fumigatus contains as many as 292 genes with internal repeats. Fourteen of 30 selected genes showed size variation of their repeat-containing regions among 11 clinical A. fumigatus isolates. Four of these genes, Afu3g08990, Afu2g05150 (MP-2), Afu4g09600, and Afu6g14090, encode putative cell wall proteins containing a leader sequence and a glycosylphosphatidylinositol anchor motif. All four genes are expressed and produce variable-size mRNA encoding a discrete number of repeat amino acid units. Their expression was altered during development and in response to cell wall-disrupting agents. Deletion of one of these genes, Afu3g08990, resulted in a phenotype characterized by rapid conidial germination and reduced adherence to extracellular matrix suggestive of an alteration in cell wall characteristics. The Afu3g08990 protein was localized to the cell walls of dormant and germinating conidia. Our findings suggest that a subset of the A. fumigatus cell surface proteins may be hypervariable due to recombination events in their internal tandem repeats. This variation may provide the functional diversity in cell surface antigens which allows rapid adaptation to the environment and/or elusion of the host immune system.

The Aspergillus nidulans pkcA gene is involved in polarized growth, morphogenesis and maintenance of cell wall integrity.

The protein kinase C (PKC) family participates in maintaining integrity and growth of fungal cell walls. However, the precise molecular role of these proteins in the filamentous fungi remains unknown. In this work, pkcA, the gene encoding the PKC homolog in the filamentous fungus Aspergillus nidulans, was cloned and its function analyzed using a conditional alcA-PKC mutant strain. Repression of pkcA expression resulted in increased conidial swelling, decreased rates of hyphal growth, changes in the ultrastructure of the cell wall and increased sensitivity to antifungal agents. These results suggest that the protein encoded by pkcA is involved in key aspects of cell morphogenesis and cell wall integrity.

Disruption of the Aspergillus fumigatus ECM33 homologue results in rapid conidial germination, antifungal resistance and hypervirulence.

The ECM33/SPS2 family of glycosylphosphatidylinositol-anchored proteins plays an important role in maintaining fungal cell wall integrity and virulence. However, the precise molecular role of these proteins is unknown. In this work, AfuEcm33, the gene encoding the ECM33 homologue in the important pathogenic fungus Aspergillus fumigatus, has been cloned and its function analysed. It is shown that disruption of AfuEcm33 results in rapid conidial germination, increased cell-cell adhesion, resistance to the antifungal agent caspofungin and increased virulence in an immunocompromised mouse model for disseminated aspergillosis. These results suggest that the protein encoded by AfuEcm33 is involved in key aspects of cell wall morphogenesis and plays an important role in A. fumigatus virulence.

Anti-inflammatory effects of moxifloxacin on IL-8, IL-1beta and TNF-alpha secretion and NFkappaB and MAP-kinase activation in human monocytes stimulated with Aspergillus fumigatus.

OBJECTIVES: We have previously shown that moxifloxacin conferred protective anti-inflammatory effects against Candida pneumonia in immunosuppressed mice. Further in vitro studies showed anti-inflammatory effects of moxifloxacin in LPS and cytokine-stimulated monocytic and epithelial cells. In the present study, concentrating on a more challenging pathogen of immunosuppressed hosts, we studied the effect of moxifloxacin on cytokine secretion and signal transduction mechanisms in monocytic cells stimulated with Aspergillus fumigatus. METHODS: Human peripheral blood monocytes (PBMCs) and a human monocytic cell line (THP-1) were incubated with 1.5x10(6)/mL conidia of a clinical isolate of A. fumigatus. Cytokine secretion and activation of NFkappaB and the MAP-kinases ERK1/2 and p38 were measured with and without the addition of moxifloxacin (5-20 mg/L). RESULTS: Stimulation of PBMCs and THP-1 cells with A. fumigatus increased IL-8, IL-1beta and TNF-alpha secretion (4.1-, 8.3- and 7-fold, and 5.4-, 3.7- and 17.8-fold, respectively). Addition of moxifloxacin (5-20 mg/L) inhibited cytokine secretion up to 45.7+/-5%, 72+/-13% and 73+/-10% in PBMCs and up to 35.6+/-0.5%, 30+/-2.4% and 19+/-4% in THP-1 cells (P<0.05). Signal transduction studies showed that incubation of THP-1 cells with A. fumigatus increased ERK1/2 and p38 phosphorylation and p65-NFkappaB protein expression by 1.6-, 1.3- and 1.8-fold, respectively. Addition of moxifloxacin inhibited ERK1/2, p38 and p65-NFkappaB by up to 69+/-14%, 58+/-3% and 75+/-15%, respectively. CONCLUSIONS: Our results indicate that moxifloxacin acts as an anti-inflammatory agent in monocytic cells stimulated with A. fumigatus conidia. Whether these effects may be protective as in the Candida pneumonia model is unknown and merits in vivo studies in models of pulmonary aspergillosis.

Injections of the neuroprotective peptide NAP to newborn mice attenuate head-injury-related dysfunction in adults.

The prophylactic neuroprotective effects of NAP, a femtomolar-acting neuroprotective peptide were tested in a mouse model of head trauma. NAP was injected for the first 3 weeks of life and head injury was initiated at 4 months. After trauma, mice were tested for their performance by evaluating damaged motor ability, balance and alertness. Comparison of the performance 1 h and 1 week after injury indicated that NAP treatment resulted in faster and enhanced recovery. In a 5-day Morris water maze test with mice suffering moderate to severe injuries, only the NAP-treated group learned to find the hidden platform in the maze. Furthermore, NAP treatment resulted in decreased mRNA expression of the inflammation marker, Mac-1. Thus, a potentially new prophylactic treatment against neurodegeneration is suggested.

NAP, a femtomolar-acting peptide, protects the brain against ischemic injury by reducing apoptotic death.

BACKGROUND AND PURPOSE: We sought to determine the cerebroprotective potential of NAP, a synthetic octapeptide related to vasoactive intestinal peptide. Activity-dependent neuroprotective protein mediates some of the protective effects of vasoactive intestinal peptide. The neuroprotective NAP sequence is derived from activity-dependent neuroprotective protein. METHODS: Spontaneously hypertensive rats underwent permanent middle cerebral artery occlusion by craniotomy and electrocoagulation. After dose-response and time-course experiments, the animals were injected with NAP (3 microg/kg) or vehicle intravenously 1 hour after stroke onset. Another group of rats was injected with the D-amino acid isomer of NAP (D-NAP) and served as a negative control. Rats were examined for motor and behavioral deficits 24 hours to 30 days later, and infarct volumes were determined. The effect of NAP administration on apoptotic death was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and caspase-3 stainings. RESULTS: NAP significantly reduced motor disability and infarct volumes compared with vehicle or D-NAP when tested at 24 hours after stroke onset (9.67+/-1.4% versus 17.04+/-1.18% and 19.19+/-1.9% of hemispheric volume, respectively; P<0.05). NAP given 4 but not 6 hours after permanent middle cerebral artery occlusion still conferred significant neuroprotection (infarct volume 10.9+/-3.9% of hemispheric volume; P<0.05 versus vehicle). Long-term studies demonstrated that infarct volumes and disability scores remained significantly lower after 30 days in NAP-treated animals. NAP significantly reduced the number of apoptotic cells. CONCLUSIONS: Our results indicate that the durable cerebroprotection by NAP involves antiapoptotic mechanisms.

A single administration of the peptide NAP induces long-term protective changes against the consequences of head injury: gene Atlas array analysis.

The femtomolar-acting eight-amino-acid peptide (NAP), derived from activity-dependent neuroprotective protein (ADNP), provides long-term protection against the deleterious effects of closed head injury (CHI) in mice. Fifteen minutes after injury, mice were divided into two groups, control and NAP-treated and a single subcutaneous injection of NAP or vehicle was administered. A third group served as sham-treated (not subjected to head trauma). Each mouse was assessed for its clinical function, using neurological severity score, at various time intervals following CHI, up to 30-45 d. Total cerebral cortex RNA was prepared from the site of injury of CHI mice, and from parallel regions in peptide-treated and sham brains. RNA was then reversed transcribed to yield radioactive cDNA preparations that were hybridized to Atlas array membranes containing 1200 cDNAs spots. Comparison of sham-treated individual mice showed differential expression levels of at least 15 mRNA species. Furthermore, results indicated that one of the genes that did not change among individuals but specifically increased after CHI and decreased after NAP treatment was the cell surface glycoprotein Mac-1 (CD11B antigen). Thus, Mac-1 is suggested as a marker for the long-term outcome of head injury and as a potential target for NAP protective actions.

Unusual alternative splicing within the human kallikrein genes KLK2 and KLK3 gives rise to novel prostate-specific proteins.

Prostate-specific antigen (PSA) and human kallikrein 2 are closely related products of the human kallikrein genes KLK3 and KLK2, respectively. Both PSA and human kallikrein 2 are produced and secreted in the prostate and have important applications in the diagnosis of prostate cancer. We report here the identification of unusual mRNA splice variants of the KLK2 and KLK3 genes that result from inclusion of intronic sequences adjacent to the first exon. The novel proteins encoded by these transcripts, named PSA-linked molecule (PSA-LM) and hK2-linked molecule (K-LM), share only the signal peptide with the original protein product of the respective gene. The mature proteins are entirely different and bear no similarity to the kallikrein family or to other proteins in the databases. As is the case with PSA, PSA-LM is expressed in the secretory epithelial cells of the prostate and is up-regulated in response to androgenic stimulation. A similar pattern of expression is suggested for K-LM.