Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.Download video file.^{(102M, mp4)}     

Prophylactic Treatment with a G Glycoprotein Monoclonal Antibody Reduces Pulmonary Inflammation in Respiratory Syncytial Virus (RSV)-Challenged Na?ve and Formalin-Inactivated RSV-Immunized BALB/c Mice

We examined whether prophylactically administered anti-respiratory syncytial virus (anti-RSV) G monoclonal antibody (MAb) would decrease the pulmonary inflammation associated with primary RSV infection and formalin-inactivated RSV (FI-RSV)-enhanced disease in mice. MAb 131-2G administration 1 day prior to primary infection reduced the pulmonary inflammatory response and the level of RSV replication. Further, intact or F(ab′)₂ forms of MAb 131-2G administered 1 day prior to infection in FI-RSV-vaccinated mice reduced enhanced inflammation and disease. This study shows that an anti-RSV G protein MAb might provide prophylaxis against both primary infection and FI-RSV-associated enhanced disease. It is possible that antibodies with similar reactivities might prevent enhanced disease and improve the safety of nonlive virus vaccines.Respiratory syncytial virus (RSV) infection in infants and young children causes substantial bronchiolitis and pneumonia (11, 27, 28, 40) resulting in 40,000 to 125,000 hospitalizations in the United States each year (27). RSV is also a prominent cause of respiratory illness in older children; those of any age with compromised cardiac, pulmonary, or immune systems; and the elderly (6, 7, 11, 17, 18, 39). Despite extensive efforts toward vaccine development (3, 5, 8, 20, 30, 38), none is yet available. Currently, only preventive measures are available that focus on infection control to decrease transmission and prophylactic administration of a humanized IgG monoclonal antibody (MAb) directed against the F protein of RSV (palivizumab) that is recommended for high-risk infants and young children (4, 7, 17). To date, no treatment has been highly effective for active RSV infection (17, 21).The first candidate vaccine, a formalin-inactivated RSV (FI-RSV) vaccine developed in the 1960s, not only failed to protect against disease but led to severe RSV-associated lower respiratory tract infection in young vaccine recipients upon subsequent natural infection (8, 16). The experience with FI-RSV has limited nonlive RSV vaccine development for the RSV-naïve infant and young child. Understanding the factors contributing to disease pathogenesis and FI-RSV vaccine-enhanced disease may identify ways to prevent such a response and to help achieve a safe and effective vaccine.The RSV G, or attachment, protein has been implicated in the pathogenesis of disease after primary infection and FI-RSV-enhanced disease (2, 26, 31). The central conserved region of the G protein contains four evolutionarily conserved cysteines in a cysteine noose structure, within which lies a CX3C chemokine motif (9, 29, 34). The G protein CX3C motif is also immunoactive, as suggested by studies with the mouse model that show that G protein CX3C motif interaction with CX3CR1 alters pulmonary inflammation (41), RSV-specific T-cell responses (12), FI-RSV vaccine-enhanced disease, and expression of the neurokinin substance P (14) and also depresses respiratory rates (32). Recent studies demonstrated that therapeutic treatment with a murine anti-RSV G protein monoclonal antibody (MAb 131-2G) which blocks binding to CX3CR1 can reduce pulmonary inflammation associated with primary infection (13, 23). These findings led us to hypothesize that prophylactic administration of this anti-RSV G monoclonal antibody may also diminish pulmonary inflammation associated with RSV infection in naïve and in FI-RSV-vaccinated mice. In this study, we evaluate the impact of prophylactic administration of MAb 131-2G on the pulmonary inflammatory response to primary infection and to RSV challenge following FI-RSV immunization in mice.     

Virulence-related <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp <Emphasis Type="Italic">hominissuis</Emphasis> MAV_2928 gene is associated with vacuole remodeling in macrophages

Background  

Mycobacterium avium subsp hominissuis (previously Mycobacterium avium subsp avium) is an environmental organism associated with opportunistic infections in humans. Mycobacterium hominissuis infects and replicates within mononuclear phagocytes. Previous study characterized an attenuated mutant in which the PPE gene (MAV_2928) homologous to Rv1787 was inactivated. This mutant, in contrast to the wild-type bacterium, was shown both to have impaired the ability to replicate within macrophages and to have prevented phagosome/lysosome fusion.     

Organization and adhesive properties of the hyaluronan pericellular coat of chondrocytes and epithelial cells

Hyaluronan is a megadalton glycosaminoglycan composed of repeating units of D-N-acetylglucosamine-beta-D-Glucuronic acid. It is known to form a highly hydrated pericellular coat around chondrocytes, fibrosarcoma, and smooth muscle cells. Using environmental scanning electron microscopy we detected fully hydrated hyaluronan pericellular coats around rat chondrocytes (RCJ-P) and epithelial cells (A6). Hyaluronan mediates early adhesion of both chondrocytes and A6 cells to glass surfaces. We show that chondrocytes in suspension establish early "soft contacts" with the substrate through a thick, hyaluronidase-sensitive coat (4.4 +/- 0.7 microm). Freshly-attached cells drift under shear stress, leaving hyaluronan "footprints" on the surface. This suggests that chondrocytes are surrounded by a multilayer of entangled hyaluronan molecules. In contrast, A6 cells have a 2.2 +/- 0.4- microm-thick hyaluronidase-sensitive coat, do not drift under shear stress, and remain firmly anchored to the surface. We consider the possibility that in A6 cells single hyaluronan molecules, spanning the whole thickness of the pericellular coat, mediate these tight contacts.     

Cosmid cloning and sample sequencing of the genome of the uncultivable mollicute,Western X-disease phytoplasma,using DNA purified by pulsed-field gel electrophoresis

Western X-disease (WX) phytoplasma is an uncultivable, intracellular pathogen of plants and insects with an AT-rich genome of 670 kb. As part of the genome sequencing project of WX phytoplasma, we have cloned approximately 50% of its genome into the pcosRW2 cosmid vector using DNA purified from pulsed-field gels. One of the cosmid clones with an insert of 24.6 kb was sequenced, which along with the cosmid end sequences, represents 60 kb of unique sequence from the WX phytoplasma genome. The putative genes identified in this sequence represent a wide variety of functions and many had not been previously identified in a phytoplasma.