Impact of peripheral immune status on central molecular responses to facial nerve axotomy.

When facial nerve axotomy (FNA) is performed on immunodeficient recombinase activating gene-2 knockout (RAG-2-/-) mice, there is greater facial motoneuron (FMN) death relative to wild type (WT) mice. Reconstituting RAG-2-/- mice with whole splenocytes rescues FMN survival after FNA, and CD4+ T cells specifically drive immune-mediated neuroprotection. Evidence suggests that immunodysregulation may contribute to motoneuron death in amyotrophic lateral sclerosis (ALS). Immunoreconstitution of RAG-2-/- mice with lymphocytes from the mutant superoxide dismutase (mSOD1) mouse model of ALS revealed that the mSOD1 whole splenocyte environment suppresses mSOD1 CD4+ T cell-mediated neuroprotection after FNA. The objective of the current study was to characterize the effect of CD4+ T cells on the central molecular response to FNA and then identify if mSOD1 whole splenocytes blocked these regulatory pathways. Gene expression profiles of the axotomized facial motor nucleus were assessed from RAG-2-/- mice immunoreconstituted with either CD4+ T cells or whole splenocytes from WT or mSOD1 donors. The findings indicate that immunodeficient mice have suppressed glial activation after axotomy, and cell transfer of WT CD4+ T cells rescues microenvironment responses. Additionally, mSOD1 whole splenocyte recipients exhibit an increased astrocyte activation response to FNA. In RAG-2-/- + mSOD1 whole splenocyte mice, an elevation of motoneuron-specific Fas cell death pathways is also observed. Altogether, these findings suggest that mSOD1 whole splenocytes do not suppress mSOD1 CD4+ T cell regulation of the microenvironment, and instead, mSOD1 whole splenocytes may promote motoneuron death by either promoting a neurotoxic astrocyte phenotype or inducing Fas-mediated cell death pathways. This study demonstrates that peripheral immune status significantly affects central responses to nerve injury. Future studies will elucidate the mechanisms by which mSOD1 whole splenocytes promote cell death and if inhibiting this mechanism can preserve motoneuron survival in injury and disease.

Optimization and critical evaluation of decellularization strategies to develop renal extracellular matrix scaffolds as biological templates for organ engineering and transplantation.

The ability to generate patient-specific cells through induced pluripotent stem cell (iPSC) technology has encouraged development of three-dimensional extracellular matrix (ECM) scaffolds as bioactive substrates for cell differentiation with the long-range goal of bioengineering organs for transplantation. Perfusion decellularization uses the vasculature to remove resident cells, leaving an intact ECM template wherein new cells grow; however, a rigorous evaluative framework assessing ECM structural and biochemical quality is lacking. To address this, we developed histologic scoring systems to quantify fundamental characteristics of decellularized rodent kidneys: ECM structure (tubules, vessels, glomeruli) and cell removal. We also assessed growth factor retention--indicating matrix biofunctionality. These scoring systems evaluated three strategies developed to decellularize kidneys (1% Triton X-100, 1% Triton X-100/0.1% sodium dodecyl sulfate (SDS) and 0.02% Trypsin-0.05% EGTA/1% Triton X-100). Triton and Triton/SDS preserved renal microarchitecture and retained matrix-bound basic fibroblast growth factor and vascular endothelial growth factor. Trypsin caused structural deterioration and growth factor loss. Triton/SDS-decellularized scaffolds maintained 3 h of leak-free blood flow in a rodent transplantation model and supported repopulation with human iPSC-derived endothelial cells and tubular epithelial cells ex vivo. Taken together, we identify an optimal Triton/SDS-based decellularization strategy that produces a biomatrix that may ultimately serve as a rodent model for kidney bioengineering.

Declining sexually transmitted infections among female sex workers: the results of an HIV and sexually transmitted infection prevention strategy in Honduras, 2006-08.

In 2006, we implemented an HIV and sexually transmitted infection (STI) prevention programme for female sex workers (FSWs) in three Honduran cities. All FSW attending STI clinics underwent regular examinations and STI testing. Information on condom use with different partners was collected at each visit. After three years, we detected a significant decline in the prevalence of syphilis from 2.3% at the first screening to 0.0% at the third screening (P = 0.05), and of chlamydia, from 6.1% to 3.3% (P = 0.01). No changes were observed in the prevalence of gonorrhoea or trichomoniasis. The cumulative HIV prevalence remained constant (P = 0.44). Reports of condom use with clients increased from 93.8% to 98.9% (P < 0.001). The implementation of an HIV/STI prevention programme in FSW has contributed to increases in condom use with clients and the reduction in syphilis and chlamydia prevalence. The intervention should be strengthened and considered as part of a national health policy strategy.

Jarisch-Herxheimer reaction among syphilis patients in Rio de Janeiro, Brazil.

The Jarisch-Herxheimer reaction (JHR) is a syndrome observed after antimicrobial treatment of some infectious diseases. The syndrome has clinical characteristics of an inflammatory reaction to antibiotic treatment. A prospective study of patients with a clinical and laboratory diagnosis of syphilis was conducted at a sexually transmitted diseases clinic in Rio de Janeiro, Brazil. Patients were treated with benzathine penicillin and observed for the JHR. A total of 115 patients were included in this study. Fifty-one patients (44%) had secondary syphilis; 37 (32%), primary; 26 (23%), latent; and one (1%), tertiary syphilis. Ten patients (9%) developed the JHR. All JHRs occurred in patients with secondary and latent syphilis. No patients experienced an allergic reaction to penicillin. The JHR occurred less frequently than in previous studies. It is important that health-care professionals recognize the clinical characteristics of the JHR so that it is not misinterpreted as an allergic reaction to penicillin.

Higher pH promotes megakaryocytic maturation and apoptosis.

Megakaryocytic (Mk) cells mature adjacent to bone marrow (BM) sinus walls and subsequently release platelets within the sinusoidal space or in lung capillaries. In contrast, primitive stem and Mk progenitor cells reside the furthest away from the BM sinus walls. The existence of pH gradients in the BM raises the question of whether pH affects Mk maturation and differentiation. We generated Mk cells from peripheral blood CD34(+) cells in a serum-free medium at different pH levels (7.2, 7.4, and 7.6) and found that higher pH resulted in an earlier and higher polyploidization of CD41(+) Mk cells and an earlier onset of Mk-cell apoptosis. The peak day of high ploidy was correlated well with the onset day of Mk apoptosis, thus suggesting that a decline in the fraction of high-ploidy Mk cells at the late culture stage is caused by Mk-cell apoptosis. We further explored the relationship between Mk-cell maturation and apoptosis by employing an antiapoptotic agent Z-Val-Ala-Asp(Ome)-FMK (zVAD). Addition of zVAD led to an average 30% higher and 2.8-day delayed polyploidization, while apoptosis was delayed by 2.4 days. Faster depletion of CD34(+) cells and an earlier peak in the fraction of larger colony-forming Mk cells (BFU-Mks) were also observed at higher pH. Taken together, these data suggest that higher pH promotes Mk-cell differentiation, maturation, and apoptosis.

Oxygen tension modulates the expression of cytokine receptors, transcription factors, and lineage-specific markers in cultured human megakaryocytes.

OBJECTIVE: We have recently reported that 20% O2 significantly enhances total megakaryocyte (Mk) number, polyploidy, and proplatelet formation compared to 5% O2 in culture. In order to further elucidate the regulatory role of pO2 on megakaryocytopoiesis, we conducted a kinetic study of the expression of surface markers CD41a and CD42a; receptors for thrombopoietin (TPO), interleukin-3 (IL-3), and Flt3-ligand; the glutamate receptor of the N-methyl-D-aspartate subtype 1 (NMDAR1); and transcription factors GATA-1, NF-E2, and E2F-1. MATERIALS AND METHODS: Mks were generated from mobilized peripheral blood (PB) CD34+ cells from normal donors in serum-free medium with TPO, IL-3, and Flt3-ligand at 20% and 5% O2. Quantitative assessment of Mk surface receptors and nuclear transcription factors was performed using multiparameter flow cytometry. mRNA levels of the nuclear transcription factors GATA-1 and NF-E2 were evaluated using RT-PCR. RESULTS: The proportions of cells expressing the early Mk marker CD41a and the late Mk marker CD42a at day 15 were 4 and 5 times higher, respectively, at 20% O2. CD41a and CD42a protein levels per cell were also higher at 20% O2. After day 5, c-Mpl (TPO receptor) generally followed similar kinetics as CD41a. The proportion of IL-3 receptor (IL-3R)++ Mks at day 5 was 1.5 times higher at 5% O2. The NMDAR1 protein previously known to be expressed by neuronal cells has recently been identified in Mks. NMDAR1 and the transcription factors were studied on days 6, 9, and 11. NMDAR1 was expressed at a 1.5- to 1.8-fold higher level at 5% O2. Twenty percent O2 supported higher expression of the Mk-early and -late-maturation-specific transcription factors GATA-1 (1.2- to 2.2-fold higher) and NF-E2 (1.1- to 2.8-fold higher). This was consistent with RT-PCR data indicating the presence of higher levels of GATA-1 and NF-E2 mRNA at 20% O2. E2F-1, a ubiquitously expressed cell cycle transcription factor, was expressed at a 1.5-fold higher level at 20% O2 on day 6, but this difference did not persist by day 9. CONCLUSION: These findings demonstrate that cytokine receptors c-Mpl and IL-3R, and Mk differentiation-specific surface receptors CD41a, CD42a, and NMDAR1, are significantly modulated by pO2, and suggest that one of the mechanisms of enhanced maturation at 20% O2 may involve regulation of transcription factors GATA-1 and NF-E2.

Modeling pO(2) distributions in the bone marrow hematopoietic compartment. I. Krogh's model.

Human bone marrow (BM) is a tissue of complex architectural organization, which includes granulopoietic loci, erythroblastic islets, and lymphocytic nodules. Oxygen tension (pO(2)) is an important determinant of hematopoietic stem and progenitor cell proliferation and differentiation. Thus, understanding the impact of the BM architectural organization on pO(2) levels in extravascular hematopoietic tissue is an important biophysical problem. However, currently it is impossible to measure pO(2) levels and their spatial variations in the BM. Homogeneous Kroghian models were used to estimate pO(2) distribution in the BM hematopoietic compartment (BMHC) and to conservatively simulate pO(2)-limited cellular architectures. Based on biophysical data of hematopoietic cells and characteristics of BM physiology, we constructed a tissue cylinder solely occupied by granulocytic progenitors (the most metabolically active stage of the most abundant cell type) to provide a physiologically relevant limiting case. Although the number of possible cellular architectures is large, all simulated pO(2) profiles fall between two extreme cases: those of homogeneous tissues with adipocytes and granulocytic progenitors, respectively. This was illustrated by results obtained from a parametric criterion derived for pO(2) depletion in the extravascular tissue. Modeling results suggest that stem and progenitor cells experience a low pO(2) environment in the BMHC.

Modeling pO(2) distributions in the bone marrow hematopoietic compartment. II. Modified Kroghian models.

Hematopoietic cells of various lineages are organized in distinct cellular architectures in the bone marrow hematopoietic compartment (BMHC). The homogeneous Kroghian model, which deals only with a single cell type, may not be sufficient to accurately describe oxygen transfer in the BMHC. Thus, for cellular architectures of physiological significance, more complex biophysical-transport models were considered and compared against simulations using the homogeneous Kroghian model. The effects of the heterogeneity of model parameters on the oxygen tension (pO(2)) distribution were examined using the multilayer Kroghian model. We have also developed two-dimensional Kroghian models to simulate several cellular architectures in which a cell cluster (erythroid cluster) or an individual cell (megakaryocyte or adipocyte) is located in the BMHC predominantly occupied by mature granulocytes. pO(2) distributions in colony-type cellular arrangements (erythroblastic islets, granulopoietic loci, and lymphocytic nodules) in the BMHC were also evaluated by modifying the multilayer Kroghian model. The simulated results indicate that most hematopoietic progenitors experience low pO(2) values, which agrees with the finding that low pO(2) promotes the expansion of various hematopoietic progenitors. These results suggest that the most primitive stem cells, which are located even further away from BM sinuses, are likely located in a very low pO(2) environment.

Model-based estimation of myeloid hematopoietic progenitor cells in ex vivo cultures for cell and gene therapies.

Ex vivo production of hematopoietic progenitor cells has potential applications for cell therapy to alleviate cytopenias associated with chemotherapy and for gene therapy. In both therapies, progenitor and stem cells are considered crucial factors for therapeutic success. Assays for progenitor cells, however, take 2 weeks to complete, which is similar to the length of a typical culture. Therefore, a real-time estimation of the percentage or number of progenitor cells, based on rapid measurements, would be useful for optimization of feeding and harvest decisions. In this study, metabolic activity assays and flow cytometric analysis were used to estimate the content of progenitor cells. The measured metabolic activities are a collective contribution from all types of cells. Cells in granulomonocytic cultures have been lumped into six cell types and metabolic rates have been modeled as a linear function of cell composition and growth rate and as a nonlinear function of cell density. Data from 24 experiments were utilized to determine the model parameters in a calibration step. These data include flow cytometric analysis of more mature hematopoietic cells, progenitor cell colony assays, total cell content, and metabolite concentrations, and cover a wide range of cell composition, cell density, and growth rate. After calibration, the model is able to deliver good predictions of progenitor cell content for cultures with higher percentages of progenitor cells, as well as the peak progenitor cell content, based only on parameters that can be rapidly measured. With the aid of those predictions a harvest strategy was developed that will allow optimizing the harvest time based on the culture kinetics of each patient or donor inoculum, rather than using retrospective analysis to determine a uniform harvest time.

Oxygen tension influences the differentiation, maturation and apoptosis of human megakaryocytes.

Megakaryocytes (Mks) mature adjacent to bone marrow (BM) sinus walls and subsequently release platelets within the sinusoidal space or in lung capillaries. As the sites for platelet release have higher levels of oxygen tension (pO(2)) than the core of the BM where stem and progenitor cells reside, we investigated whether pO(2) influences Mk maturation. Mks were generated from CD34(+) cells (from mobilized peripheral blood from cancer patients) under 5% and 20% O(2). At day 15, CD41(+) Mk expansion in 20% and 5% O(2) cultures was 85-fold and 31-fold respectively. Twenty percent O(2) cultures also had higher levels of high ploidy (> or = 8N, eightfold higher) and proplatelet-forming (fivefold higher) Mks. At day 21, 20% O(2) cultures had a fivefold higher number of apoptotic Mks. In contrast, 5% O(2) promoted Mk colony-forming unit (CFU-Mk) generation and maintenance. Similar results were observed in cultures initiated with CD41(+) Mks, indicating that pO(2) directly affects Mks. The change from 20% to 5% O(2) on day 5 and day 7 delayed both maturation and apoptosis, suggesting that these two processes are closely linked. These results were confirmed in CD34(+) cultures from normal BM samples. These data may provide insights into in vivo Mk maturation, such as an explanation for hypoxia-induced thrombocytopenia in animals.

The lactate issue revisited: novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures.

It is well established that cell proliferation in batch (unfed) hematopoietic cell cultures is greatly inhibited relative to that in cultures with feeding. What is not known, however, is the nature of this inhibition. On the basis of our observations in hematopoietic cultures that cell proliferation ceases when the lactate concentration ([lactate]) exceeds 20 mM (accompanied by a decrease in culture pH), we investigated the effect of lactate accumulation on cell proliferation, metabolism, and differentiation. We differ in our approach from previous efforts in that we have tried to more accurately recreate the manner in which lactate accumulates in culture by employing a daily feeding protocol in which [lactate] and/or pH in the fresh medium was adjusted to match the conditions prior to feeding. We conclude that the decrease in pH associated with lactate accumulation significantly inhibits both cell proliferation and metabolism. Although inhibition in cultures with high [lactate] and low pH is similar to that in unfed cultures, pH control in unfed cultures does not alleviate the inhibition, indicating that other inhibitory factors are also present. Thus, pH control is necessary, but not sufficient, to eliminate inhibition of cell growth and metabolism in unfed hematopoietic cell cultures. We also conclude that high [lactate] and low pH have little effect on cell differentiation in fed cultures, although there is evidence to suggest that low pH may play a role in monocyte differentiation in unfed cultures.

Cell science and protein crystal growth research for the International Space Station.

The recent National Research Council report, Future Biotechnology Research on the International Space Station, evaluates NASA's plans for research in cell science and protein crystal growth to be conducted on the International Space Station. This report concludes that the NASA biotechnology programs have the potential to significantly impact relevant scientific fields and to increase understanding and insight into fundamental biological issues. In order to realize the potential impacts, NASA must focus its research programs by selecting specific questions related to gravitational forces' role in cell behavior and by using the microgravity environment as a tool to determine the structure of macromolecules with important biological implications. Given the time and volume constraints associated with space-based experiments, instrumentation to be used on the space station must be designed to maximize the productivity of researchers, and NASA's recruitment of investigators and support for space station experiments should aim to encourage and facilitate cutting-edge research.

Dynamic model of ex vivo granulocytic kinetics to examine the effects of oxygen tension, pH, and interleukin-3.

OBJECTIVE: Evaluating kinetics in hematopoietic cultures is complicated by the distribution of cells over various stages of differentiation and by the presence of cells from different lineages. Thus, an observed response is an integral response from distributed cell populations. Growth factors and other parameters can greatly affect the lineage and maturation stage of the culture outcome. To resolve the kinetics and more clearly define the differential effects of O(2) tension (pO(2)), pH, and interleukin-3 (IL-3) on granulopoiesis, a mathematical model-based approach was undertaken. MATERIALS AND METHODS: Granulocytic differentiation is described within a continuous, deterministic framework in which cells develop from primitive granulocytic progenitors to mature neutrophils. The model predicts two distributed populations-quiescent and cycling cells-by incorporating rates of growth, death, differentiation, and transition between quiescence and active cycling. The response of these four model processes to changes in the culture environment was examined. RESULTS: Model simulations of experimental data revealed the following: 1) pO(2) effects are exerted only on the growth rate but not maturation times. 2) pH effects between pH 7.25 and 7.4 on growth and differentiation are coupled; however, with increasing pH values, especially at pH 7. 6, the death rate for cells in the early stages of differentiation becomes increasingly significant. 3) The absence of IL-3 increases the death rate for primitive cells only minimally but markedly enhances the rate of differentiation through the myeloblast window in the differentiation pathway. The combined effects of these environmental factors can be predicted based on changes in the model parameters derived from the individual effects. CONCLUSIONS: Experimental data combined with mathematical modeling can elucidate the mechanisms underlying the regulation of granulopoiesis by pO(2), pH, and IL-3. The model also can be readily adapted to evaluate the effects of other culture conditions. The increased understanding of experimental results gained with this approach can be used to modify culture conditions to optimize ex vivo production of neutrophil precursors.

Phosphate feeding improves high-cell-concentration NS0 myeloma culture performance for monoclonal antibody production.

Phosphorus depletion was identified in high-cell-concentration fed-batch NS0 myeloma cell cultures producing a humanized monoclonal antibody (MAb). In these cultures, the maximum viable and total cell concentration was generally ca. 5 x 10(9) and 7 x 10(9) cells/L, respectively, without phosphate feeding. Depletion of essential amino acids, such as lysine, was initially thought to cause the onset of cell death. However, further improvement of cell growth was not achieved by feeding a stoichiometrically balanced amino acid solution, which eliminated depletion of amino acids. Even though a higher cell viability was maintained for a longer period, no increase in total cell concentration was observed. Afterwards, phosphorus was found to be depleted in these cultures. By also feeding a phosphate solution to eliminate phosphorus depletion, the cell growth phase was prolonged significantly, resulting in a total cell concentration of ca. 17 x 10(9) cells/L, which is much greater than ca. 7 x 10(9) cells/L without phosphate feeding. The maximum viable cell concentration reached about 10 x 10(9) cells/L, twice as high as that without phosphate feeding. Apoptosis was also delayed and suppressed with phosphate feeding. A nonapoptotic viable cell population of 6.5 x 10(9) cells/L, as compared with 3 x 10(9) cells/L without phosphate feeding, was obtained and successfully maintained for about 70 h. These results are consistent with the knowledge that phosphorus is an essential part of many cell components, including phospholipids, DNA, and RNA. As a result of phosphate feeding, a much higher integral of viable cell concentration over time was achieved, resulting in a correspondingly higher MAb titer of ca. 1.3 g/L. It was also noted that phosphate feeding delayed the cell metabolism shift from lactate production to lactate consumption typically observed in recombinant NS0 cultures. The results highlight the importance of phosphate feeding in high-cell-concentration NS0 cultures.

A kinetic analysis of hybridoma growth and metabolism in batch and continuous suspension culture: effect of nutrient concentration, dilution rate, and pH. Reprinted from Biotechnology and Bioengineering, Vol. 32, Pp 947-965 (1988).

Hybridomas are finding increased use for the production of a wide variety of monoclonal antibodies. Understanding the roles of physiological and environmental factors on the growth and metabolism of mammalian cells is a prerequisite for the development of rational scale-up procedures. An SP2/0-derived mouse hybridoma has been employed in the present work as a model system for hybridoma suspension culture. In preliminary shake flask studies to determine the effect of glucose and glutaminE, it was found that the specific growth rate, the glucose and glutamine metabolic quotients, and the cumulative specific antibody production rate were independent of glucose concentration over the range commonly employed in cell cultures. Only the specific rate of glutamine uptake was found to depend on glutamine concentration. The cells were grown in continuous culture at constant pH and oxygen concentration at a variety of dilution rates. Specific substrate consumption rates and product formation rates were determined from the steady state concentrations. The specific glucose uptake rate deviated from the maintenance energy model(1) at low specific growth rates, probably due to changes in the metabolic pathways of the cells. Antibody production was not growth-associated; and higher specific antibody production rates were obtained at lower specific growth rates. The effect of pH on the metabolic quotients was also determined. An optimum in viable cell concentration was obtained between pH 7.1 and 7.4. The viable cell number and viability decreased dramatically at pH 6.8. At pH 7.7 the viable cell concentration initially decreased, but then recovered to values typical of pH 7.1-7.4. Higher specific nutrient consumption rates were found at the extreme pH values; however, glucose consumption was inhibited at low pH. The pH history also influenced the behavior at a given pH. Higher antibody metabolic quotients were obtained at the extreme pH values. Together with the effect of specific growth rate, this suggests higher antibody production under environmental or nutritional stress.

Physiologically significant effects of pH and oxygen tension on granulopoiesis.

OBJECTIVE: Granulocyte differentiation in the bone marrow (BM) takes place in regions with lower pH and O(2) tension (pO(2)) than those in the BM sinuses. This suggests that granulopoiesis will be enhanced at subvascular pH and pO(2). MATERIALS AND METHODS: The effects of pH AND pO2 on granulocyte proliferation, differentiation, and granulocyte colony-stimulating factor receptor (G-CSFR) expression were evaluated using mobilized peripheral blood CD34(+) cells directed down the granulocytic pathway with stem cell factor, interleukin 3, interleukin 6, and G-CSF. RESULTS: Cell expansion was enhanced at subvascular pH, with twice as many total cells and CD15(bright)/CD11b(+) late neutrophil precursors (myelocytes, metamyelocytes, bands) produced at pH 7.07 to 7.21 as was produced at pH 7.38. Low pH accelerated the rate of differentiation concomitant with this increase in proliferation. Also, total, CD15(bright)/CD11b(-) (promyelocytes, early myelocytes), and CD15(bright)/CD11b(+) cell expansion was enhanced at lower pO(2), with twice as many of each cell type produced at 5% O(2) as at 20% O(2). The effects of low pH and low pO(2) were additive, such that generation of total, CD15(bright)/CD11b(-), and CD15(bright)/CD11b(+) cells was 3.5-, 2.4-, and 4.0-fold greater at pH 7.21 and 5% O(2) than at the standard hematopoietic culture conditions of pH 7.38 and 20% O(2). Low pH resulted in faster upregulation of G-CSFR surface expression, whereas pO(2) had no effect on G-CSFR expression. CONCLUSION: These data provide compelling evidence that pH and pO(2) gradients within the BM play significant roles in regulating hematopoiesis. More rapid granulocytic cell proliferation and differentiation at low pH may be explained in part by more rapid G-CSFR expression. The ability to alter cell development by manipulating pH and pO(2) has important implications for optimizing ex vivo production of neutrophil precursors.

Cell density-dependent proliferation in frequently-fed peripheral blood mononuclear cell cultures.

BACKGROUND: Our goal was to produce granulocyte progenitor (CFU-G) and post-progenitor (CD15(+)CD11b(+/-)) cells for subsequent transplantation. We hypothesized that increasing the feeding frequency and maintaining constant densities may overcome inhibitory growth conditions (i.e. low pH) in high-density cultures. METHODS: To study the effect of cell density on total cell expansion, differentiation and lactate production, 50% daily medium exchanges were used in cultures of peripheral blood mononuclear cells (PB MNC) maintained at constant densities (ranging from 5 x 10(4)cells/mL to 2.5 x 10(6)cells/mL). RESULTS: We observed a significant increase in total cell expansion when the density was increased from 5 x 10(4) cells/mL to 1 x 10(6) cells/mL, but a further increase to 2.5 x 10(6)cells/mL resulted in a decline in cell expansion. Increasing feeding to 90% daily exchange in cultures with 2.5 x 10(6) cells/mL did not enhance cell expansion; nor did reducing the extent of feeding in cultures with 5 x 10(4) cells/mL to 10% daily exchange. We did not observe a relationship between cell density and the percentage of granulocyte progenitor and post-progenitor (CD15(+)CD11b(-/+)) cells. While specific lactate production (q(lac)) in cultures with 2.5 x 10(6) cells/mL was approximately 60% of those observed in lower density cultures by Day 13, this difference was largely eliminated by increasing the extent of feeding in cultures with 2.5 x 10(6) cells/mL. DISCUSSION: Our results suggest that feeding rates must be adjusted according to cell density to maximize culture performance. They also suggest that cellular crowding on the culture surface can limit expansion in suspension (nonadherent) cultures.

Clinical-scale production of granulocyte progenitor and post-progenitor cells using daniplestim, leridistim, Progenipoietin, Promegapoietin and autologous plasma.

BACKGROUND: Supplementation of PBPC autografts with ex vivo expanded PBMC may significantly reduce or eliminate the period of neutropenia associated with high-dose chemotherapy. METHODS: Unmanipulated growth-factor mobilized PBMC were expanded in media containing daniplestim, leridistim, Promegapoietin, and Progenipoietin (DLPP) and 2% autologous plasma at 4 x 10(5) PBMC/mL, first in 25 cm(2) T-flasks, with sampling on Days 7, 10, 13 and 15, and then in 1264 cm(2) Nunclon Cell Factories, with sampling on Days 7 and 13. RESULTS: In T25-flasks, maximal CFU-GM expansion ([38.2 +/- 9.5]-fold) occurred on Day 10, whereas maximal total cell expansion ([6.7 +/- 1.1]-fold) occurred on Day 15. Production of CD15(+)CD11b(-) and CD15(+)CD11b(+) granulocytic post-progenitors (3.0 +/- 0.4 x 10(6) and 3.7 +/- 0.9 x 10(6), respectively) was also maximal at Day 15. Compared with the previously studied combination of Flt3L, PIXY321, G-CSF, GM-CSF and Epo, the DLPP cocktail performed similarly, with the exception of yielding larger GM colonies at Day 10 and fewer granulocyte post-progenitors on Day 15. In Cell Factories, CFU-GM were expanded (31.6 +/- 14.5)-fold, while total nonadherent cells were expanded (2.6 +/- 0.5)-fold. The two stack Cell Factory cultures seeded with 1.0 x 10(8) unselected PBMC produced approximately 3.3 x 10(6) CFU-GM and 1.3 x 10(8) myeloid post-progenitors. DISCUSSION: Whereas expansion of cell numbers, CFU-GM and granulocytic post-progenitors in Cell Factories mirrored that achieved in T25-flasks, future preclinical studies with the DLPP cytokine combination may be performed in small volumes, with subsequent translation to the larger volume Cell Factories. Sufficient expansion can be achieved using the DLPP cytokine combination in the Cell Factories to provide the numbers of progenitors required for clinical trials.

Considerations for osmolality measurement under elevated pCO(2): comparison of vapor pressure and freezing point osmometry.

Osmolality increases with pCO(2) in bioreactors with pH control, and it has been shown that osmolality compensation by decreasing the basal NaCl concentration partially mitigates the adverse effects of elevated pCO(2) on animal cell growth, protein production, and glycosylation. Thus, measurement of osmolality is important for a complete characterization of the culture environment under elevated pCO(2). However, osmolality measurement may be compromised by CO(2) evolution. Freezing point depression and vapor pressure depression osmometry were directly compared for the measurement of osmolality in samples at elevated pCO(2) (up to 250 mmHg) and at a variety of pH values (6.7-7.5). More extensive degassing may be expected with the vapor pressure osmometer due to the smaller sample volume and larger surface area employed. However, both types of osmometer yielded similar results for all pCO(2) and pH values studied. Moreover, the measured values agreed with osmolality values calculated using a semi-empirical model. Further analysis showed that, while sample degassing may result in a large decrease in pCO(2), there is little associated decrease in osmolality. The great majority of total CO(2) in solution is present as bicarbonate (HCO(3)(-)). Although a small amount of HCO(3)(-) is converted to CO(2) to compensate for CO(2) evolution, further depletion of HCO(3)(-) is inhibited by the associated increase in medium pH and by the need for HCO(3)(-) to maintain charge neutrality in solution. This explanation is consistent with the observed similarity in osmolality values for the two types of osmometer. It was also observed that osmolality did not change in samples that were frozen at -20 degrees C for up to 1 year.

Detection of high grade squamous intraepithelial lesions and tumors using the AutoPap System: results of a primary screening clinical trial.

BACKGROUND: The AutoPap System for the initial screening and quality control of conventional cervical cytology slides previously has shown superior performance for the detection of abnormal slides of low grade squamous intraepithelial lesions and above. This report presents data regarding the important category of high grade squamous intraepithelial lesions and above (HSIL+). METHODS: All slides were run through a Current Practice (CP) arm of manual screening with 10% random quality control followed by an AutoPap (AP) arm of device initial screening with approximately 25% of slides receiving no further review; 75% received a manual rescreen with AP ranking and QC rescreening of 15% of the top ranking negative manual screen slides was performed. Detection performance for truth-determined HSIL+ cases was compared between the two study arms. Available follow-up biopsy results were correlated for cases determined to be HSIL+. RESULTS: Of 25,124 analyzed slides, 70 slides had truth-determination at the HSIL+ level (67 HSILs, 1 adenocarcinoma in situ, and 2 invasive tumors). The AP arm identified 68 of 70 cases (including both invasive tumors). Neither false-negative HSIL+ was found in the 25% "No Further Review" population. The CP arm identified 65 of 70 cases (missing both invasive tumors). The results showed statistical equivalence between the two arms (P = 0.0129). Biopsy follow-up was available in 27 of 70 HSIL+ cases identified by AP and showed abnormality at some level in 100% of cases. CONCLUSIONS: The AP arm was statistically equivalent and showed numeric superiority to the CP arm for the category of HSIL+. Follow-up data confirmed the true-positive nature of these findings. These results are significant because any overall improvement in the performance of an initial screening device must be paralleled by at least equivalence in the clinically important subset category of HSIL+. Cancer (Cancer Cytopathol)