The application of local hypobaric pressure - A novel means to enhance macromolecule entry into the skin.

The local application of controlled hypobaric stress represents a novel means to facilitate drug delivery into the skin. The aims of this work were to understand how hypobaric stress modified the properties of the skin and assess if this penetration enhancement strategy could improve the percutaneous penetration of a macromolecule. Measurements of skin thickness demonstrated that the topical application of hypobaric stress thinned the tissue (p<0.05), atomic force microscopy showed that it shrunk the corneocytes in the stratum corneum (p<0.001) and the imaging of the skin hair follicles using multiphoton microscopy showed that it opened the follicular infundibula (p<0.001). Together, these changes contributed to a 19.6-fold increase in in vitro percutaneous penetration of a 10,000 molecular weight dextran molecule, which was shown using fluorescence microscopy to be localized around the hair follicles, when applied to the skin using hypobaric stress. In vivo, in the rat, a local hemodynamic response (i.e. a significant increase in blood flow, p<0.001) was shown to contribute to the increase in follicular transport of the dextran to produce a systemic absorption of 7.2±2.81fg·mL(-1). When hypobaric stress was not applied to the rat there was no detectable absorption of dextran and this provided evidence that this novel penetration enhancement technique can improve the percutaneous penetration of macromolecules after topical application to the skin.

Joint-dependent response to impact and implications for post-traumatic osteoarthritis.

OBJECTIVE: The prevalence of osteoarthritis (OA) varies between joints. Cartilage in eight different joints was evaluated to elucidate the disparate susceptibilities between joints to post-traumatic OA (PTOA) and provide evidence for joint-specific clinical treatments. The hypothesis was that cartilage in different joints would have varying cell death and anabolic gene expression profiles after injury. METHODS: Adult equine cartilage explants were harvested from shoulder (SH), elbow (EL), carpal (CA), metacarpophalangeal (MC), patellofemoral (FP), tarsal (TA), metatarsophalangeal (MT), and proximal interphalangeal (PP) joints, and injured by loading with 30 MPa within 1 s. Fractional dissipated energy, cell density, cell death, and gene expression were quantified. RESULTS: PP had the highest fractional dissipated energy (94%, 95% confidence interval [CI] 88 to 101%). Cell density was highest in the superficial zone in all samples, with MC and MT having the highest peak density. Injured samples had significantly increased cell death (13.5%, 95% CI 9.1 to 17.9%) than non-injured samples (6.8%, 95% CI 2.5 to 11.1%, P = 0.016); however, cell death after injury was not significantly different between joints. Gene expression was significantly different between joints. CD-RAP expression in normal cartilage was lowest in FP (Cp = 21, 95% CI -80 to 122). After injury, the change in CD-RAP expression increased and was highest in FP (147% relative increase after injury, 95% CI 64 to 213). CONCLUSION: Different joints have different baseline characteristics, including cell density and gene expression, and responses to injury, including energy dissipation and gene expression. These unique characteristics may explain differences in OA prevalence and suggest differences in susceptibility to PTOA. CLINICAL RELEVANCE: Understanding differences in the response to injury and potential susceptibility to OA can lead to the development of preventative or treatment strategies. KEY TERMS: Gene expression, cartilage injury, chondrocyte, multiphoton microscopy, cartilage biomechanical properties, PTOA. WHAT IS KNOWN ABOUT THE SUBJECT: The prevalence of OA is variable among joints; however, most laboratory studies are performed on a single joint - most commonly the knee, and extrapolated to other joints such as the ankle or shoulder. A small number of studies have compared knee and ankle cartilage and reported differences in mechanical properties and gene expression. WHAT THIS STUDY ADDS TO EXISTING KNOWLEDGE: There are differences in baseline cell density and gene expression, and differences in response to injury, including gene expression and cell death. This suggests that there are inherent differences leading to varying susceptibilities in OA prevalence among joints. Joint-specific treatments may improve OA therapies.

Advanced fluorescence lifetime imaging algorithms for CMOS single-photon sensor based multi-focal multi-photon microscopy.

We have developed a fast hardware friendly bi-exponential fluorescence lifetime algorithm suitable for 2D CMOS single-photon avalanche diode (SPAD) arrays. The performance of the proposed algorithm against other techniques is demonstrated on the data from a plant specimen (Convallaria) by using 0.13µm CMOS SPAD arrays mounted on a multi-beam multi-photon microscopy system.

Investigation of dental samples using a 35MHz focussed ultrasound piezocomposite transducer.

Dental erosion and decay are increasingly prevalent but as yet there is no quantitative monitoring tool. Such a tool would allow earlier diagnosis and treatment and ultimately the prevention of more serious disease and pain. Despite ultrasound having been demonstrated as a method of probing the internal structures of teeth more than 40 years ago, development of a clinical tool has been slow. The aim of the study reported here was to investigate the use of a novel high frequency ultrasound transducer and validate it using a known dental technique. A tooth extracted for clinical reasons was sectioned to provide a sample that contained an enamel and dentine layer such that the enamel-dentine junction (EDJ) was of a varying depth. The sample was then submerged in water and a B-scan recorded using a custom-designed piezocomposite ultrasound transducer with a centre frequency of 35 MHz and a -6 dB bandwidth of 24 MHz. The transducer has an axial resolution of 180 microm and a spatial resolution of 110 microm, a significant advance on previous work using lower frequencies. The depth of the EDJ was measured from the resulting data set and compared to measurements from the sequential grinding and imaging (SGI) method. The B-scan showed that the EDJ was of varying depth. Subsequently, the EDJ measurements were found to have a correlation of 0.89 (p<0.01) against the SGI measurements. The results indicate that high frequency ultrasound is capable of measuring enamel thickness to an accuracy of within 10% of the total enamel thickness, whereas currently there is no clinical tool available to measure enamel thickness.

Mask-less ultraviolet photolithography based on CMOS-driven micro-pixel light emitting diodes.

We report on an approach to ultraviolet (UV) photolithography and direct writing where both the exposure pattern and dose are determined by a complementary metal oxide semiconductor (CMOS) controlled micro-pixellated light emitting diode array. The 370 nm UV light from a demonstrator 8 x 8 gallium nitride micro-pixel LED is projected onto photoresist covered substrates using two back-to-back microscope objectives, allowing controlled demagnification. In the present setup, the system is capable of delivering up to 8.8 W/cm2 per imaged pixel in circular spots of diameter approximately 8 microm. We show example structures written in positive as well as in negative photoresist.

Active focus locking in an optically sectioning microscope utilizing a deformable membrane mirror.

A significant challenge for in vivo imaging is to remove movement artifacts. These movements (typically due to either respiration and cardiac-related movement or surface chemical response) are normally limited to the axial direction, and hence features move in and out of the focal plane. This presents a real problem for high-resolution optically sectioned imaging techniques such as confocal and multiphoton microscopy. To overcome this we have developed an actively locked focus-tracking system based around a deformable membrane mirror. This has a significant advantage over more conventional focus-tracking techniques where the microscope objective is dithered, since the active element is not in direct, or indirect, contact with the sample. To examine the operational limits and to demonstrate possible applications for this form of focus locking, sample oscillation and movement are simulated for two different biological applications. We were able to track focus over a 400 microm range (limited by the range of the piezomounted objective) with a rms precision on the focal depth of 0.31 microm +/- 0.05 microm.

Development of fibre-optic confocal microscopy for detection and diagnosis of dental caries.

We report on the development of a fibre-optics-based confocal imaging system for the detection and potential diagnosis of early dental caries. A novel optical instrument, capable of recording axial profiles through caries lesions using single-mode optical fibres, has been developed. The practical study illustrates that miniature confocal devices based around single-mode optical fibres may provide additional diagnostic information for the general dental practitioner.

Adaptive optics for enhanced signal in CARS microscopy.

We report the use of adaptive optics with coherent anti-Stokes Raman scattering (CARS) microscopy for label-free deep tissue imaging based on molecular vibrational spectroscopy. The setup employs a deformable membrane mirror and a random search optimization algorithm to improve signal intensity and image quality at large sample depths. We demonstrate the ability to correct for both system and sample-induced aberrations in test samples as well as in muscle tissue in order to enhance the CARS signal. The combined system and sample-induced aberration correction increased the signal by an average factor of approximately 3x for the test samples at a depth of 700 microm and approximately 6x for muscle tissue at a depth of 260 microm. The enhanced signal and higher penetration depth offered by adaptive optics will augment CARS microscopy as an in vivo and in situ biomedical imaging modality.

Cytomegalovirus-caused release of collagenase IV from human cerebral microvascular endothelial cells.

BACKGROUND: Human cytomegalovirus (HCMV) in most normal individuals results in an asymptomatic infection, but under some circumstances, such as in the transplant setting, AIDS and intrauterine infection of the fetus, HCMV infection can lead to more serious consequences, including central nervous system infection. Recently it has been demonstrated that HCMV-infected endothelial cells can be detected in the circulation; however, no mechanism has been suggested. OBJECTIVE: To determine whether HCMV infection of confluent human microvascular endothelial cells (HMEC) in culture results in the induction of type IV collagenase. This would provide a mechanism by which HCMV-infected HMECs could be released into the circulation. STUDY DESIGN: Confluent cultures of brain-derived HMECs were infected with HCMV and culture supernatants were sampled for collagenase IV, general protease and viral titers. RESULTS: HCMV infection of HMECs stimulated a significant release of collagenase type IV activity which peaked early in the assay within 3-5 days and fell off rapidly thereafter. This stimulation of protease activity differed only slightly between non-infected and infected cultures. By day 6, viral cytopathic effects became evident and HCMV titers reached approximately 5 x 10(5) PFU/ml by day 9. CONCLUSIONS: These results demonstrate that HCMV infection of HMEC induces the release of collagenase type IV. This may lead to the degradation of the basement membrane and subsequently to the release of fully infected endothelial cells into the circulation resulting in further dissemination of the infection.

HIV-1 infection of human brain-derived microvascular endothelial cells in vitro.

We examined the ability of human immunodeficiency virus (HIV) type 1 (HIV-1) to infect in vitro, primary brain-derived human microvascular endothelial cells (HMEC) that constitute the blood-brain barrier (BBB). Immunofluorescence (IFA) and antigen capture assays failed to demonstrate p24 antigen from HIV inoculated endothelial cells and supernatants did not contain detectable levels of reverse transcriptase (RT). HIV could be rescued by cocultivation of infected HMEC with a susceptible T-lymphocyte line (CEM-SS), which were then shown to form syncytia and produce RT activity and p24 Ag (IFA, antigen captive assay). Polymerase chain reaction (PCR) was successfully used to amplify HIV-specific gag and env gene sequences from HMEC. CD4 expression was not identified on these cells by IFA. These results suggest that HIV infection of BBB endothelium occurs, but that viral replication is minimal. Infection of the BBB by HIV may give the virus a foothold in the CNS and suggests that the brain might be infected directly and may not be limited to just the passage of infected mononuclear cells.

The extent of human cytomegalovirus replication in primary neurons is dependent on host cell differentiation.

To study fetal brain infection with human cytomegalovirus (HCMV), an in vitro model was established using the human primary nontransformed neuronal cell line HCN-1A. On exposure to a mixture of factors promoting differentiation, HCN-1A cells differentiate into mature neurons. Both undifferentiated and differentiated neurons were permissive to HCMV replication as assessed by immunohistochemistry and in situ DNA hybridization. Infectious center assays revealed that the ratio of virus-infected differentiated cells to undifferentiated cells dropped from 11:1 to 2:1 7-21 days after infection. However, release of infectious progeny from the differentiated HCN-1A cells was greater by 100- to 1000-fold. Cytopathic effect appeared earlier and was more pronounced in differentiated cells. These results suggest that differentiation of HCN-1A cells dramatically affects the rate and amount of virus production from these cells. This model should be useful in the study of congenital HCMV disease and virus-host cell interaction.

New developments with herpesviruses and the nervous system.

Most of the human herpesviruses have a unique relationship with the central nervous system. In this review we summarize recent developments in the establishment of viral latency, and discuss the nervous system illnesses instigated by direct viral cytopathic effect, by immune activation, and by neoplastic events. Special consideration is given to herpesvirus infections in the context of acquired immunodeficiency syndrome, and in association with neonatal infections. New diagnostic techniques and treatments are also summarized.

Cytomegalovirus in the brain: in vitro infection of human brain-derived cells.

Models for human cytomegalovirus (HCMV) brain infection have been developed in a variety of brain-derived cells in which the factors governing virus infectivity might be studied in vitro. Studies were initiated with brain endothelial cells, the likely portal of entry for virus into the central nervous system. Primary explant cultures of brain endothelial cells, derived from homogenates of healthy human brain, supported complete viral gene expression and cytopathic effect (CPE). Endothelial cells do not appear to be a barrier for HCMV passage into the central nervous system. Astroglial lines (primary explant or tumor-derived) varied in their ability to support HCMV replication. Some (T98G) supported incomplete (immediate-early) gene expression while others (A-172) did not support any detectable gene expression. Some astroglial lines (HS-683) supported extensive virus replication with minimal viral CPE. Neuronal cell lines (SK-N-MC) were fully permissive. The more differentiated glial lines (astrocytoma) were fully permissive to HCMV infection; however, the less differentiated glial lines (glioblastoma) were partly or nonpermissive.

Cytomegalovirus and Rasmussen's encephalitis.

In-situ hybridisation with a biotinylated cytomegalovirus (CMV) DNA probe was done on brain biopsy specimens from 10 patients with Rasmussen's encephalitis (RE) and 46 age-matched control patients with other neurological diseases. All 10 patients with RE had intractable epilepsy and focal neurological deficits, and there was perivascular cuffing, microglial nodules, astrogliosis, and neuronal loss. CMV genomic material was demonstrated in 7 of the 10 patients with RE (in neurons, astrocytes, oligodendrocytes, and endothelial cells) and in 2 of the 46 control patients. Probes for herpes simplex virus and hepatitis B virus were negative in all patients and in fibroblast controls. The results suggest that CMV is a likely cause of Rasmussen's encephalitis.

Encephalopathy in liver transplantation: neuropathology and CMV infection.

The clinical histories and pathological findings of 27 autopsied cases of orthotopic liver transplantation (OLT) were reviewed. Fatal OLT was complicated in 93% of cases by neurological dysfunction, usually manifested by encephalopathy, with or without seizures. The etiology of the encephalopathy was largely multifactorial (44%) or undetermined (20%). Subarachnoid hemorrhage, central pontine myelinolysis, meningitis, brain infarction, polyclonal B cell lymphoma and spinal cord necrosis were common neuropathological findings. These diagnoses were often masked by other systemic illnesses. The role of cytomegalovirus (CMV) in neurologic dysfunction was explored with in situ hybridization and immunohistochemical techniques. OLT cases showed a significantly higher (89%) frequency of CMV genomic material in brain tissue compared to age-matched non immunocompromised (NIC) patients (23%). All OLT cases with encephalopathy of undetermined cause demonstrated usually prominent hybridization to the CMV probe. CMV may be an important cause of encephalopathy in such patients.