Epiphytic lichens on Juniperus communis – an unexplored component of biodiversity in threatened alvar grassland

Semi‐natural grasslands, among them thin‐soil calcareous grasslands (alvars), have great conservation value but have become increasingly rare in Europe. The main threat to alvar grasslands is the encroachment by juniper Juniperus communis and therefore it is usually removed during the restoration practice. Juniper can also be a host plant for many epiphytic lichens, but its role as a phorophyte is poorly known. We studied epiphytic lichen diversity on 126 junipers in 17 sites in western Estonia and found 140 lichenized taxa including several rare and red‐listed species. Using indirect and direct multivariate analyses (DCA, pCCA) and general linear models we revealed that both habitat and phorophyte properties affect lichen assemblies on juniper. Lichen species richness per site showed a unimodal relationship with compound factors of site productivity and juniper characteristics (stem circumference and juniper width). Lichen species richness per phorophyte was increasing with its size and with the proportion of dead branches, and was twice higher in plate alvars than in ryhk alvars. Also, the species composition in plate alvars differed from ryhk alvars by having 42 characteristic lichen species in plate alvars vs three indicators of ryhk alvars. The composition of lichens was significantly influenced by encroachment of alvars, e.g. by high juniper cover and shrub layer height, as well as by the proportion of dead branches and stem circumference of juniper. We conclude that the epiphytic lichen assemblies on junipers are threatened by grassland encroachment similarly to ground layer lichen assemblies. We suggest that some old and scencent junipers should be preserved during the restoration of alvar grasslands.     

The comparison of susceptibility patterns of Gram-negative invasive and non-invasive pathogens in Estonian hospitals

A total of 560 invasive and 1062 non-invasive isolates were collected. The antimicrobial susceptibility of invasive versus non-invasive Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae isolates were evaluated using the E-tests. The equal domination of Gram-negative among both invasive and non-invasive pathogens was estimated in our study if contaminants were excluded. The emergence trend of Gram-positive microbes especially of coagulase negative staphylococci may be proved only after application of exclusive algorithms. Due to similar susceptibility, the data of non-invasive Gram-negative pathogens can be useful to predict resistance of invasive ones. Also, the surveillance of invasive pathogens provides useful information about the general susceptibility of pathogens.     

Empirical models of the proliferative response of cytokine-dependent hematopoietic cell lines

There is an expanding need for predictive mathematical models to accelerate the optimization of cell therapy culture processes. Here we demonstrate the ability of simple mathematical models to describe quantitatively the cytokine growth-rate dependence of two human hematopoietic cell lines, TF-1 and MO7e. These cells are immortal but depend on either interleukin-3 (IL-3) or granulocyte-macrophage colony stimulating factor (GM-CSF) for their continued survival and maximal proliferation. They are also responsive to interleukin-6 (IL-6) and exhibit saturation kinetics when these cytokines are limiting. A Monod-type relationship consistently failed to fit measured cytokine dose-proliferation response curves while a Hill-type relationship showed a good fit. Cytokine interactions were first modeled by modifying the Hill-function to include an interaction parameter, gamma. This model did not indicate either synergistic or even additive effects between IL-3 and GM-CSF. Based on the reported competition between IL-3 and GM-CSF for their common receptor (beta(c)) subunit, a competitive model was also developed. This model had no new parameters beyond those obtained from single cytokine cultures and provided improved prediction of the growth rates for both cell lines exposed to combinations of IL-3 and GM-CSF over a wide range of concentrations. As expected, the competitive model failed to fit the data for IL-6 in combination with either IL-3 or GM-CSF, since IL-6 signaling does not involve the beta(c) chain of the IL-3/GM-CSF receptors. Interestingly, the cell-specific rates of GM-CSF uptake and cell proliferation were found to be uncoupled processes. Taken together, these results illustrate the utility of appropriately designed empirical models to describe the proliferative responses of hematopoietic cells to cytokine stimulation.     

Controlled release process to recover heterologous glycosylphosphatidylinositol membrane anchored proteins from CHO cells

A semicontinuous process has been developed to recover heterologous proteins at increased concentrations and purities. Proteins attached to mammalian cell membranes by glycosylphosphatidylinositol (GPI) anchors can be selectively released into the supernatant by the enzyme phosphatidylinositol-phospholipase C (PI-PLC). Chinese hamster ovary (CHO) cells, genetically engineered to express the GPI anchored, human melanoma antigen (p97), were used as a model system. These cells were grown in protein containing growth medium. During a brief harvesting phase the medium was replaced by phosphate buffered saline (PBS) containing 10 mU/mL of PI-PLC and the GPI anchored protein was cleaved from the cell surface and recovered in soluble form at up to 30% purity. After harvesting, the cells were returned to growth medium where the protein was re-expressed within 40 h. The growth rate, viability, and protein production of cells, repeatedly harvested over a 44-day period, were not adversely affected. This continuous cyclic harvesting process allowed recovery of a heterologous protein at high purity and concentrations and could be applied to the recovery of other GPI anchored proteins and genetically engineered GPI anchored fusion proteins. (c) 1993 John Wiley & Sons, Inc.     

Accelerating perfusion process optimization by scanning non-steady-state responses

Perfusion processes provide consistent culture conditions, high productivity and low product residence times. However, process development can be slow due to the 1 week or more required to reach each steady state. The objective of this work was to accelerate process development in perfusion cultures by scanning non-steady-state transient responses to qualitatively predict steady-state performance. The method was tested using a shift in temperature every 3 days, scanned down by steps of 2 degrees C from 37 degrees C to 31 degrees C, then scanned up to 37 degrees C. Higher t-PA concentrations were predicted at lower temperatures, confirmed by subsequent pseudo-steady-state results. In most cases, transient values on the 3rd day were in close concordance with pseudo-steady-state values. To further accelerate process development, transient scanning was applied to small-scale, non-instrumented cultures. Similar results were obtained, although quantitative t-PA values were 15-30 times lower than in high cell density perfusion cultures. The method was further explored by investigating 1 day transient shifts in temperature where more variability was observed, suggesting that the cells were still adapting to the new environment. Nonetheless, the overall response again qualitatively predicted the pseudo-steady-state temperature response. Use of transient scanning in conjunction with pseudo-steady-state verification and refinement of optimal results could reduce process development time to a third or less of comparable steady-state-based optimization.     

Common and distinct features of cytokine effects on hematopoietic stem and progenitor cells revealed by dose-response surface analysis

Recent studies have identified thrombopoietin (TPO), flt-3 ligand (FL), Steel factor (SF), and interleukin-11 (IL-11) as cytokines able to stimulate amplification of the most primitive murine hematopoietic cells in vitro. However, dose-response and interaction parameters that predict how to optimize mixtures of these cytokines have not been previously defined. To obtain this information, Sca-1(+)lin(-) and c-kit(+)Sca-1(+)lin(-) adult mouse bone marrow cells were cultured for 10 and 14 days, respectively, in serum-free medium with varying concentrations of these cytokines. Quantitative assays were performed to determine the influences of the cytokine combinations tested on changes in long-term repopulating hematopoietic stem cells (HSCs), in vitro colony-forming cells (CFCs), and total cell numbers. A two-level factorial design was first used to screen the effects of TPO, SF, FL, and IL-11 as well as two different incubation temperatures. IL-11 and SF were found to be the most significant stimulators of murine HSC expansion. More detailed analyses of the effects on c-kit(+)Sca-1(+)lin(-) cells of IL-11, SF, and FL concentrations and their interactions using response surface methodology showed IL-11 to have a maximal stimulatory effect on HSC expansion at 20 ng/mL with higher concentrations being inhibitory. In contrast, not even high concentration saturation of the effects of either SF or FL was observed as the stimulatory effect of both SF and FL increased beyond 300 ng/mL. A negative interaction between SF and FL on HSCs was discovered. Interestingly, a generally similar pattern of cytokine effects was found to influence the 14-day output of CFCs and total cells from the same c-kit(+)Sca-1(+)lin(-) starting cell population. However, compared with HSCs, the cytokine requirements for maximizing the generation of CFCs and total cells were at much lower cytokine doses. From the information provided by the factorial analysis, mathematical models based on Monod kinetics for inhibitory substrates were developed that allow total cell, CFC, and HSC expansion to be predicted as a function of the IL-11, SF, and FL concentrations in terms of more widely recognized parameters. Overall, these methods should also serve as a guide for the future design and testing of other ex vivo stem cell expansion systems.     

Immobilized mammalian cell cultivation in hollow fiber bioreactors

Cultivation of animal cells for the production of recombinant proteins is an important method for manufacturing complex proteins requiring posttranslational processing. One of the often considered methods for cultivation is by immobilization of the cells in hollow fiber bioreactors (HFBRs). These systems allow the cells to grow to high densities in a shear protected environment; furthermore the product can be accumulated in high concentration in the case of ultrafiltration HFBRs. Operation and scale-up are constrained by nutrient and product transport with oxygen transfer to growing cells being the most critical parameter. Mathematical models describing HFBRs have proved to be useful in quantitating and understanding the constraints and guiding the scale-up of this approach to animal cell cultivation.