The novel fungal CYP51 inhibitor VT-1598 displays classic dose-dependent antifungal activity in murine models of invasive aspergillosis.

Aspergillus spp. infections remain a global concern, with ∼30% attributable mortality of invasive aspergillosis (IA). VT-1598 is a novel fungal CYP51 inhibitor designed for exquisite selectivity versus human CYP enzymes to achieve a maximal therapeutic index and therefore maximal antifungal efficacy. Previously, its broad-spectrum in vitro antifungal activity was reported. We report here the pharmacokinetics (PK) and pharmacodynamics (PD) of VT-1598 in neutropenic mouse models of IA. The plasma area-under-the-curve (AUC) of VT-1598 increased nearly linearly between 5 and 40 mg/kg after 5 days of QD administration (155 and 1033 µg*h/ml, respectively), with a further increase with 40 mg/kg BID dosing (1354 µg*h/ml). When A. fumigatus isolates with in vitro susceptibilities of 0.25 and 1.0 µg/ml were used in a disseminated IA model, VT-1598 treatment produced no decrease in kidney fungal burden at QD 10 mg/kg, intermediate decreases at QD 20 mg/kg and maximum or near maximum decreases at 40 mg/kg QD and BID. The PK/PD relationships of AUCfree/MIC for 1-log killing for the two strains were 5.1 and 1.6 h, respectively, similar to values reported for approved CYP51 inhibitors. In a survival study where animals were observed for 12 days after the last treatment, survival was 100% at the doses tested (20 and 40 mg/kg QD), and fungal burden remained suppressed even though drug wash-out was complete. Similar dose-dependent reductions in lung fungal burden were observed in a pulmonary model of IA. These data strongly support further exploration of VT-1598 for the treatment of this lethal mold infection.

The effect of five proteins on stem cells used for osteoblast differentiation and proliferation: a current review of the literature.

Bone-tissue engineering is a therapeutic target in the field of dental implant and orthopedic surgery. It is therefore essential to find a microenvironment that enhances the growth and differentiation of osteoblasts both from mesenchymal stem cells (MSCs) and those derived from dental pulp. The aim of this review is to determine the relationship among the proteins fibronectin (FN), osteopontin (OPN), tenascin (TN), bone sialoprotein (BSP), and bone morphogenetic protein (BMP2) and their ability to coat different types of biomaterials and surfaces to enhance osteoblast differentiation. Pre-treatment of biomaterials with FN during the initial phase of osteogenic differentiation on all types of surfaces, including slotted titanium and polymers, provides an ideal microenvironment that enhances adhesion, morphology, and proliferation of pluripotent and multipotent cells. Likewise, in the second stage of differentiation, surface coating with BMP2 decreases the diameter and the pore size of the scaffold, causing better adhesion and reduced proliferation of BMP-MSCs. Coating oligomerization surfaces with OPN and BSP promotes cell adhesion, but it is clear that the polymeric coating material BSP alone is insufficient to induce priming of MSCs and functional osteoblastic differentiation in vivo. Finally, TN is involved in mineralization and can accelerate new bone formation in a multicellular environment but has no effect on the initial stage of osteogenesis.

The enhancement of osteogenesis through the use of dental pulp pluripotent stem cells in 3D.

The potential for osteogenic differentiation of dental pulp mesenchymal stem cells (DPMSCs) in vitro and in vivo has been well documented in a variety of studies. Previously, we obtained a population of cells from human dental pulp called dental pulp pluripotent stem cells (DPPSCs) that could differentiate into mesodermal, ectodermal and endodermal progenies. We compared the osteogenic capacity of DPPSCs and DPMSCs that had been isolated from the same donors (N=5) and cultivated in the same osteogenic medium in 3D (three dimensions) Cell Carrier glass scaffolds. We also compared the architecture of bone-like tissue obtained from DPPSCs and human maxillary bone tissue. Differentiation was evaluated by scanning electron microscopy, whereas the expression of bone markers such as ALP, Osteocalcin, COLL1 and Osteonectin was investigated by quantitative real time polymerase chain reaction (qRT-PCR). We also used calcium quantification, Alizarin red staining and alkaline phosphatase (ALP) activity to compare the two cell types. New bone tissue formed by DPPSCs was in perfect continuity with the trabecular host bone structure, and the restored bone network demonstrated high interconnectivity. Significant differences between DPPSCs and DPMSCs were observed for the expression of bone markers, calcium deposition and ALP activity during osteogenic differentiation; these criteria were higher for DPPSCs than DPMSCs. Both DPPSCs and differentiated tissue showed normal chromosomal dosage after being cultured in vitro and analysed using short-chromosome genomic hybridisation (short-CGH). This study demonstrates the stability and potential for the use of DPPSCs in bone tissue engineering applications.

Viability of maxillary bone harvesting by using different osteotomy techniques. A pilot study.

The use of autogenous grafts is still considered in bone regeneration surgeries. However, the bone cell viability of such grafts after being harvested from donor sites remains a matter of debate. The aim of the present study is to evaluate particulated and block bone cell viability, in terms of presence or absence of apoptosis and necrosis, obtained from different maxillary intra-oral harvesting methods: bone scraper, rotary carbide burs and piezoelectric device. Five healthy patients were enrolled in the study. The patients required sinus augmentation by lateral window approach. The bone was harvested by the bone scraper, piezoelectric device and rotary surgical instrument. The samples were processed with the Annexin V/FITC (fluorescein isothiocyanate stain) kit and were analyzed by means of Fluoresence-Activated Cell Sorted (FACS) technique. Within the limitations of this pilot study, the results indicated that autogenous bone chips collected from the three harvesting methods presented a large percentage of apoptotic cells, although large scale production of necrotic cells was not detected. In summary, although rotary surgical instrument and piezoelectric devices are frequently used instruments for oral osteotomy, fresh autogenous bone chips collected from them did not present a viable bone cell source.

Isolation of pluripotent stem cells from human third molar dental pulp.

Potent stem/progenitor cells have been isolated from normal human dental pulps, termed dental pulp stem cells (DPSCs). However, no study has described the presence of stem cell populations in human dental pulp from the third molar with embryonic phenotypes. The dental pulp tissue was cultured in media with the presence of LIF, EGF, and PDGF. In the present study, we describe a new population of pluripotent stem cells that were isolated from dental pulp (DPPSC). These cells are SSEA-4(+), Oct4(+), Nanog(+), FLK-1(+), HNF3beta(+), Nestin(+), Sox2(+), Lin28(+), c-Myc(+), CD13(+), CD105(+), CD3(-), CD45(-), CD90(low), CD29(+), CD73(low), STRO-1(low) and CD146(-). We have investigated by SEM analysis and q-RT-PCR the capacity of DPPSCs to 3D differentiate in vitro using the Cell Carrier 3D glass scaffold into tissues that have similar characteristics to embryonic mesoderm and endoderm layers. These data would support the use of these cells, which are derived from an easily accessible source and can be used in future regeneration protocols for many tissue types that differentiate from the three embryonic layers.

Kinetic modelling of the role of the aldehyde dehydrogenase enzyme and the breast cancer resistance protein in drug resistance and transport.

A compartmental model for the in vitro uptake kinetics of the anti-cancer agent topotecan (TPT) has been extended from a previously published model. The extended model describes the drug activity and delivery of the pharmacologically active form to the DNA target as well as the catalysis of the aldehyde dehydrogenase (ALDH) enzyme and the elimination of drug from the cytoplasm via the efflux pump. Verification of the proposed model is achieved using scanning-laser microscopy data from live human breast cancer cells. Before estimating the unknown model parameters from the experimental in vitro data it is essential to determine parameter uniqueness (or otherwise) from this imposed output structure. This is formally performed as a structural identifiability analysis, which demonstrates that all of the unknown model parameters are uniquely determined by the output structure corresponding to the experiment.