Characterization of cryobiological responses in TF-1 cells using interrupted freezing procedures

Cryopreservation currently is the only method for long-term preservation of cellular viability and function for uses in cellular therapies. Characterizing the cryobiological response of a cell type is essential in the approach to designing and optimizing cryopreservation protocols. For cells used in therapies, there is significant interest in designing cryopreservation protocols that do not rely on dimethyl sulfoxide (Me₂SO) as a cryoprotectant, since this cryoprotectant has been shown to have adverse effects on hematopoietic stem cell (HSC) transplant patients. This study characterized the cryobiological responses of the human erythroleukemic stem cell line TF-1, as a model for HSC. We measured the osmotic parameters of TF-1 cells, including the osmotically-inactive fraction, temperature-dependent membrane hydraulic conductivity and the membrane permeability to 1 M Me₂SO. A two-step freezing procedure (interrupted rapid cooling with hold time) and a graded freezing procedure (interrupted slow cooling without hold time) were used to characterize TF-1 cell recovery during various phases of the cooling process. One outcome of these experiments was high recovery of TF-1 cells cryopreserved in the absence of traditional cryoprotectants. The results of this study of the cryobiology of TF-1 cells will be critical for future understanding of the cryobiology of HSC, and to the design of cryopreservation protocols with specific design criteria for applications in cellular therapies.     

Compression or tension? The stress distribution in the proximal femur

Background  

Questions regarding the distribution of stress in the proximal human femur have never been adequately resolved. Traditionally, by considering the femur in isolation, it has been believed that the effect of body weight on the projecting neck and head places the superior aspect of the neck in tension. A minority view has proposed that this region is in compression because of muscular forces pulling the femur into the pelvis. Little has been done to study stress distributions in the proximal femur. We hypothesise that under physiological loading the majority of the proximal femur is in compression and that the internal trabecular structure functions as an arch, transferring compressive stresses to the femoral shaft.     

Temporal Variation in Seed Predation by Insects in a Population of Syagrus romanzoffiana (Arecaceae) in Santa Catarina Island, SC, Brazil

Insect seed predation may vary depending on seed production. The present study considers the hypothesis that the rates of seed predation tend to be smaller in years of higher fruit production. Thus, we monitored the production of fruits and predation of seeds of the palm Syagrus romanzoffiana over 2?years in the Atlantic Forest (Parque Municipal da Lagoa do Peri, Florianópolis, SC, Brazil), between July 2006 and June 2008. Plots of 0.25?m² were fitted under 20 mother plants and fruits were monthly collected for assessment of abundance and seed predation. There was variation in fruit production between the 2?years and among reproductive plants. Predation rates were high and occurred in the predispersal phase by the Curculionidae Revena rubiginosa Boheman, Anchylorhynchus aegrotus Fahraeus, and Anchylorhynchus variabilis Gyllenhal. Seed predation by these species of Anchylorhynchus is first registered in the present study. In average, about 60% of the seeds monthly produced in the population tend to escape insect predation in year of high or low production, becoming available for recruitment. The predation rate was not related to the amount of fruits produced per reproductive plant. Also, different than expected, there was a positive relation between the rates of seed predation and the total of fruits produced monthly on the plots. Thus, no evidence for the satiation of insect seed predators was found in this study with S. romanzoffiana.     

Infectious (Non)tolerance—Frustrated Commensalism Gone Awry?

Despite advances in medicine, infectious diseases remain major causes of death and disability worldwide. Acute or chronic infectious agents mediate host tissue damage and cause a spectrum of disease as diverse as overwhelming sepsis and shock within hours to persistent tissue inflammation causing organ failure or even cancer over years. Although pathogen exposure can cause disease via host-derived inflammation, pathogens share recognized elements with harmless human commensals. Mouse models and organisms with simpler flora are revealing the dialogue between multicellular hosts and commensal flora. In some instances the persistent inflammation associated with pathogens can be interpreted within a framework of frustrated commensalism in which the host and pathogen cannot complete the requisite dialogue that establishes homeostasis. In contrast, coevolved commensals interact cooperatively with the host immune system, resulting in immunotolerance. Attempts to more thoroughly understand the molecular nature of the dialogue may uncover novel approaches to the control of inflammation and tissue damage.Trends over the past century have shown both the power of humankind to conquer disease and the limitations of that power. In the developed world, advances in sanitation, antimicrobial therapeutics, and vaccination campaigns have dramatically reduced the rates of disability and death due to infectious diseases. In 2008 the World Health Organization announced that for the first time, noncommunicable diseases such as heart disease and stroke caused more deaths than infections (WHO 2008). However, in too many parts of the world political and economic challenges of the 21st century result in public health conditions more reminiscent of the pre–antibiotic era, and more than one-quarter of deaths worldwide are still caused by infections like pneumonia, diarrheal disease, tuberculosis, and malaria (Khabbaz et al. 2010).In both the developed and the developing world, ecological and social factors have conspired with host and pathogen factors to promote the emergence and reemergence of infectious diseases (Smolinski et al. 2003). The confluence of the human immunodeficiency virus (HIV) and tuberculosis epidemics and the rise of multidrug-resistant tuberculosis illustrate how culture, politics, and economics can fuel a public health crisis. In the United States, which spends more money on healthcare and biomedical discovery than any other country, pneumonia remains one of the leading causes of death in the elderly (National Center for Health Statistics 2011). Chemotherapy, antimicrobials, catheters, and ventilators have increased life expectancy and reduced morbidity, but have also brought increasing rates of healthcare-associated antibiotic resistance and opportunistic infections (Klevens et al. 2007; Hidron et al. 2008; Scott 2009).What do these statistics reveal about our evolution? Throughout human history social and ecological factors have given rise to endemic and pandemic infections (Morens et al. 2008), although humans and pathogens coevolved in relative equilibrium without antibiotics or vaccines. Pneumonia, malaria, and diarrheal diseases have killed countless children, and malaria continues to cause nearly 2000 childhood deaths per day (Mulholland 2007). High birth rates have compensated for high child mortality, and natural selection by childhood infections contributed to our immune evolution (Greenwood et al. 2005; Finch 2010). Indeed, genes related to immunity are among the most polymorphic in the mammalian genome, and more human gene mutations have been linked to resistance to malaria than to any other single infectious disease (Murphy 1993; Hill 2010).From a microbial parasite’s perspective, pathogenicity can be a detriment. When “parasite” is broadly defined to include viruses, mathematical models and empirical evidence suggest that virulence is only beneficial inasmuch as it maximizes parasite survival within a host and/or transmission to a new niche (Anderson and May 1982). Over time host and parasite populations coevolve to balance tolerance and transmissibility such that the most injurious pathogens tend to be recent arrivals to a new host species, whereas in their native host they cause mild or no disease (Read 1994). As extreme examples, Ebola hemorrhagic virus and avian influenza, which have only recently been described in humans, have case-fatality rates of >60%; in contrast, human herpesvirus 6, which has an ancient relationship with humans, latently infects >95% of adults and results in little pathology (Braun et al. 1997; Nichol et al. 2000). In highly pathogenic infections, host and microbial factors trigger overwhelming inflammation, whereas milder infections are controlled and tolerated. Experiments in mice suggest that highly adapted latent DNA viruses can even protect hosts from subsequent infectious challenges by altering the thresholds for activation in response to microbial invasion (Barton et al. 2009). In this setting latent lifelong infection may in fact enhance the host’s fitness.Understanding the evolutionary drive from pathogenicity to latency is essential because insights into contrasting patterns of host–microbial interaction may uncover new treatments for human disease. Specifically, microbial products evolved to induce immunotolerance could be used to treat conditions characterized by inappropriately robust inflammation. In this review we first describe immune recognition pathways responsible for host morphogenic changes that are conserved across beneficial and pathogenic host–bacterial interactions in plants, invertebrates, and mammals. We then hypothesize that because bacterial mutualism implies that symbionts provide their hosts a selective advantage, a key difference between human pathogens and commensals is our immune recognition of commensal factors that provide a homeostatic and/or metabolic benefit.     

Application of the osmotic virial equation in cryobiology

The multisolute osmotic virial equation is the only multisolute thermodynamic solution theory that has been derived from first principles and can make predictions of multisolute solution behaviour in the absence of multisolute solution data. Other solution theories either (i) include simplifying assumptions that do not take into account the interactions between different types of solute molecules or (ii) require fitting to multisolute data to obtain empirical parameters. The osmotic virial coefficients, which are obtained from single-solute data, can be used to make predictions of multisolute solution osmolality. The osmotic virial coefficients for a range of solutes of interest in cryobiology are provided in this paper, for use with concentration units of both molality and mole fraction, along with an explanation of the background and theory necessary to implement the multisolute osmotic virial equation.     

Therapeutic dendritic cell vaccine preparation using tumor RNA transfection: A promising approach for the treatment of prostate cancer

Background

Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery.

Methods

Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model.

Results

Experiments confirm that electroporation alone is more effective in enhancing reporter gene expression than plasmid injection alone, plasmid conjugation with liposomes followed by injection, or than the combination of liposomes and electroporation. However, with two time courses of multiple electrically-mediated plasmid deliveries, neither the levels nor duration of transgene expression are significantly increased. Tissue damage may increase following a second treatment, no further damage is observed after a third treatment. When electroporation conditions utilized in a mouse model are tested in thicker rat skin, only higher field strengths or longer pulses were as effective in plasmid delivery.

Conclusion

Electroporation enhances reporter plasmid delivery to the skin to a greater extent than the liposome conjugation method tested. Multiple deliveries do not necessarily result in higher or longer term expression. In addition, some impact on tissue integrity with respect to surface damage is observed. Pulsing conditions should be optimized for the model and for the expression profile desired.