Cryopreservation of osteoblast-like cells: viability and differentiation with replacement of fetal bovine serum in vitro

In reconstructive medicine, the clinical use of cryopreservation techniques depends on the absence of infectious agents such as prions. Therefore, we investigated the viability and differentiation of human osteoblast-like cells during replacement of fetal bovine serum in vitro. The aim of the present study is to replace the potentially infectious supplement fetal bovine serum during the cryopreservation procedure in order to perform future clinical trials. We used a cryopreservation technique with Me(2)SO for human osteoblast-like cells of iliac cancellous bone. In the cell culture of cryopreserved and fresh osteoblast-like cells, we substituted Dulbecco's modification of Eagle's medium (DMEM)/Ham's F12 plus 1% penicillin/streptomycin with autologous serum, human serum albumin and Biseko for fetal bovine serum. For the fourth treatment group, we removed fetal bovine serum without replacing it. DMEM/Ham's F12 plus 1% penicillin/streptomycin with fetal bovine serum served as the control group. After 4, 7, 14 and 21 days of culture for the cryopreserved and noncryopreserved cells, we performed cell counting, a WST-1 test, ELISA for collagen type I, and osteocalcin. The activity of alkaline phosphatase was also measured. The best results were obtained for the group with autologous serum as a supplement after thawing, exceeding the other groups with regard to proliferation rate. Most viable cells were observed with no replacement before freezing and after thawing of the cells. With regard to differentiation, the cultures with autologous serum after thawing of the cells showed little concentration of the differentiation markers, probably due to early contact inhibition of the cells in vitro. With regard to effort and outcome, the most promising group for cryopreservation was the one with DMEM/Ham's F12 plus 1% penicillin/streptomycin alone before freezing, especially when osteoblast-like cells were cultured in medium with autologous serum after thawing. This is important, as this in vitro setting resembles the in vivo situation when cryopreserved bone is transplanted. These findings indicate that, for clinical purposes, fetal bovine serum can be removed for cryopreservation of iliac cancellous bone with minor loss of viability.     

People fifty years or older now account for the majority of AIDS cases in San Francisco,California, 2010

People aged 50 and older are an increasing proportion of the population of persons living with AIDS (PLWA) in the USA. We used San Francisco's population-based HIV/AIDS surveillance registry to examine trends in the age distribution of people diagnosed and living with AIDS in San Francisco, California. AIDS case reporting is highly complete. Death ascertainment is complete through 2009 and 95% complete for 2010. At the end of 2010, 9796 persons were living with AIDS in San Francisco. Of these, more than half (5112 or 52%) were 50-years old or older. This proportion has steadily increased since 1990 in San Francisco. Our data also indicate that age at AIDS diagnosis has increased in San Francisco during the years 1990–2010. The proportion of PLWA who are aged 50 years or older is now a majority among PLWA in San Francisco. We believe that San Francisco is the first local jurisdiction in the USA to reach this milestone. The growing population of older persons with AIDS presents new challenges for research, medical care and support services.     

Anthrax edema toxin impairs clearance in mice

The anthrax edema toxin (ET) of Bacillus anthracis is composed of the receptor-binding component protective antigen (PA) and of the adenylyl cyclase catalytic moiety, edema factor (EF). Uptake of ET into cells raises intracellular concentrations of the secondary messenger cyclic AMP, thereby impairing or activating host cell functions. We report here on a new consequence of ET action in vivo. We show that in mouse models of toxemia and infection, serum PA concentrations were significantly higher in the presence of enzymatically active EF. These higher concentrations were not caused by ET-induced inhibition of PA endocytosis; on the contrary, ET induced increased PA binding and uptake of the PA oligomer in vitro and in vivo through upregulation of the PA receptors TEM8 and CMG2 in both myeloid and nonmyeloid cells. ET effects on protein clearance from circulation appeared to be global and were not limited to PA. ET also impaired the clearance of ovalbumin, green fluorescent protein, and EF itself, as well as the small molecule biotin when these molecules were coinjected with the toxin. Effects on injected protein levels were not a result of general increase in protein concentrations due to fluid loss. Functional markers for liver and kidney were altered in response to ET. Concomitantly, ET caused phosphorylation and activation of the aquaporin-2 water channel present in the principal cells of the collecting ducts of the kidneys that are responsible for fluid homeostasis. Our data suggest that in vivo, ET alters circulatory protein and small molecule pharmacokinetics by an as-yet-undefined mechanism, thereby potentially allowing a prolonged circulation of anthrax virulence factors such as EF during infection.