Characterization of Nipah virus infection in a model of human airway epithelial cells cultured at an air-liquid interface.

Nipah virus (NiV) is an emerging paramyxovirus that can cause lethal respiratory illness in humans. No vaccine/therapeutic is currently licensed for humans. Human-to-human transmission was previously reported during outbreaks and NiV could be isolated from respiratory secretions, but the proportion of cases in Malaysia exhibiting respiratory symptoms was significantly lower than that in Bangladesh. Previously, we showed that primary human basal respiratory epithelial cells are susceptible to both NiV-Malaysia (M) and -Bangladesh (B) strains causing robust pro-inflammatory responses. However, the cells of the human respiratory epithelium that NiV targets are unknown and their role in NiV transmission and NiV-related lung pathogenesis is still poorly understood. Here, we characterized NiV infection of the human respiratory epithelium using a model of the human tracheal/bronchial (B-ALI) and small airway (S-ALI) epithelium cultured at an air-liquid interface. We show that NiV-M and NiV-B infect ciliated and secretory cells in B/S-ALI, and that infection of S-ALI, but not B-ALI, results in disruption of the epithelium integrity and host responses recruiting human immune cells. Interestingly, NiV-B replicated more efficiently in B-ALI than did NiV-M. These results suggest that the human tracheal/bronchial epithelium is favourable to NiV replication and shedding, while inducing a limited host response. Our data suggest that the small airways epithelium is prone to inflammation and lesions as well as constituting a point of virus entry into the pulmonary vasculature. The use of relevant models of the human respiratory tract, such as B/S-ALI, is critical for understanding NiV-related lung pathogenesis and identifying the underlying mechanisms allowing human-to-human transmission.

Photokinetic Drug Delivery: Light-Enhanced Permeation in an In Vitro Eye Model.

PURPOSE: To investigate light-enhanced molecular movement as a potential technology for drug delivery. To do this, we developed an in vitro eye model while representing similar concentration gradient conditions and compositions found in the eye. METHODS: The eye model unit was fabricated by inserting a cross-linked type I collagen membrane in a spectrophotometer cuvette with 1% hyaluronic acid as the drug recipient medium. Photokinetic delivery was studied by illuminating 1 mg/mL methotrexate (MTX) placed in the drug donor compartment on top of the membrane, with noncoherent 450 nm light at 8.2 mW from an LED source pulsed at 25 cycles per second, placed in contact with the solution. A modified UV-visual spectrophotometer was employed to rapidly determine the concentration of MTX, at progressive 1 mm distances away from the membrane, within the viscous recipient medium of the model eye after 1 h. RESULTS: A defined, progressive concentration gradient was observed within the nonagitated drug recipient media, diminishing with greater distances from the membrane. Transport of MTX through the membrane was significantly enhanced (ranging from 2 to 3 times, P < 0.05 to P ≤ 0.001) by photokinetic methods compared with control conditions by determining drug concentrations at 4 defined distances from the membrane. According to scanning electron microscopy images, no structural damage or shunts were created on the surface of the cross-linked gelatin membrane. CONCLUSION: The application of pulsed noncoherent visible light significantly enhances the permeation of MTX through a cross-linked collagen membrane and hyaluronic acid recipient medium without causing structural damage to the membrane.

Volume density and distribution of mitochondria in harbor seal (Phoca vitulina) skeletal muscle.

Recent studies have shown that harbor seals (Phoca vitulina) have an increased skeletal muscle mitochondrial volume density that may be an adaptation for maintaining aerobic metabolism during diving. However, these studies were based on single samples taken from locomotory muscles. In this study, we took multiple samples from a transverse section of the epaxial (primary locomotory) muscles and single samples from the m. pectoralis (secondary locomotory) muscle of five wild harbor seals. Average mitochondrial volume density of the epaxial muscles was 5.6%, which was 36.6% higher than predicted for a terrestrial mammal of similar mass, and most (82.1%) of the mitochondria were interfibrillar, unlike athletic terrestrial mammals. In the epaxial muscles, the total mitochondrial volume density was significantly greater in samples collected from the deep (6.0%) compared with superficial (5.0%) regions. Volume density of mitochondria in the pectoralis muscle was similar (5.2%) to that of the epaxial muscles. Taken together, these adaptations reduce the intracellular distance between mitochondria and oxymyoglobin and increase the mitochondrial diffusion surface area. This, in combination with elevated myoglobin concentrations, potentially increases the rate of oxygen diffusion into mitochondria and prevents diffusion limitation so that aerobic metabolism can be maintained under low oxygen partial pressure that develops during diving.

Ultrastructural evidence of the ehrlichial developmental cycle in naturally infected Ixodes persulcatus ticks in the course of coinfection with Rickettsia, Borrelia, and a flavivirus.

Ehrlichiae are small gram-negative obligately intracellular bacteria that multiply within vacuoles of their host cells and are associated for a part of their life cycle with ticks, which serve as vectors for vertebrate hosts. Two morphologically and physiologically different ehrlichial cell types, reticulate cells (RC) and dense-cored cells (DC), are observed during experimental infection of cell cultures, mice, and ticks. Dense-cored cells and reticulate cells in vertebrate cell lines alternate in a developmental cycle. We observed ultrastructure of RC and DC of Ehrlichia muris in morulae in salivary gland cells and coinfection with Borrelia burgdorferi sensu lato (sl), "Candidatus Rickettsia tarasevichiae," and a flavivirus (presumably, tick-borne encephalitis virus [TBEV]) of Ixodes persulcatusticks collected in the Cis-Ural region of Russia. Polymerase chain reaction revealed 326 (81.5%) of 400 ticks carrying at least one infectious agent, and 41.5% (166 ticks) were coinfected with two to four agents. Ehrlichiae and rickettsiae were identified by sequencing of 359 bp of the 16S rRNA gene of E. muris and of 440 bp of the 16S rRNA gene and 385 bp of the gltA gene of "R. tarasevichiae." Different organs of the same tick harbored different microorganisms: TBEV in salivary gland and borreliae in midgut; E. muris in salivary gland; and "R. tarasevichiae" in midgut epithelium. Salivary gland cells contained both RC and DC, a finding that confirmed the developmental cycle in naturally infected ticks. Dense-cored cells in tick salivary glands were denser and of more irregular shape than DC in cell cultures. Ehrlichia-infected salivary gland cells had lysed cytoplasm, suggesting pathogenicity of E. muris for the tick host at the cellular level, as well as potential transmission during feeding. Rickettsiae in the midgut epithelial cells multiplied to significant numbers without altering the host cell ultrastructure. This is the first demonstration of E. muris, "R. tarasevichiae," and the ehrlichial developmental cycle in naturally infected I. persulcatus sticks.