Influence of lactic acid on the proliferation,metabolism, and differentiation of rabbit mesenchymal stem cells

Lactic acid, originated from degradation of biomaterials, cell cultures, and so on, would be a toxic compound in acute states. The present study was undertaken to ascertain whether the proliferation, metabolism, and differentiation of rabbit mesenchymal stem cells (rMSCs) were affected by additional lactic acid. Furthermore, this study aimed to determine whether this influence was due to decreasing pH, increasing osmotic pressure, or chemical action of lactate ion. It was shown that the proliferation and metabolism of MSCs were inhibited by decreasing pH or increasing lactate. However, when osmolarity was adjusted to the same level as that of sodium lactate using sodium chloride, cell proliferation was little affected by osmotic pressure. We also concluded that colony-forming potential and osteogenic differentiation capacity were significantly depressed by decreasing pH or increasing lactate. As was shown, this inhibition of lactate was not only due to osmotic pressure, but also mainly due to chemical action of lactate ion. However, we observed that acidifying extracellular medium and lactate ion promoted the retention of adipogenic differentiation potential of MSCs during in vitro expansion, which suggested that growth arrest and the decrease of osteogenic differentiation potential did not affect the adipogenic conversion of MSCs.     

Feeding lactate for CHO cell culture processes: impact on culture metabolism and performance

Lactate has long been regarded as one of the key metabolites of mammalian cell cultures. High levels of lactate have clear negative impacts on cell culture processes, and therefore, a great amount of efforts have been made to reduce lactate accumulation and/or to induce lactate consumption in the later stage of cultures. However, there is virtually no report on the impact of lactate depletion after initial accumulation. In this work, we observed that glucose uptake rate dropped over 50% at the onset of lactate consumption, and that catabolism of alanine due to lactate depletion led to ammonium accumulation. We explored the impact of feeding lactate as well as pyruvate to the cultures. In particular, a strategy was employed where CO(2) was replaced by lactic acid for culture pH control, which enabled automatic lactate feeding. The results demonstrated that lactate or pyruvate can serve as an alternative or even preferred carbon source during certain stage of the culture in the presence of glucose, and that by feeding lactate or pyruvate, very low levels of ammonia can be achieved throughout the culture. In addition, low levels of pCO(2) were also maintained in these cultures. This was in strong contrast to the control cultures where lactate was depleted during the culture, and ammonia and pCO(2) build-up were significant. Culture growth and productivity were similar between the control and lactate-fed cultures, as well as various product quality attributes. To our knowledge, this work represents the first comprehensive study on lactate depletion and offers a simple yet effective strategy to overcome ammonia and pCO(2) accumulation that could arise in certain cultures due to early depletion of lactate.     

Feeding tricarboxylic acid cycle intermediates improves lactate consumption and antibody production in Chinese hamster ovary cell cultures

Media components play an important role in modulating cell metabolism and improving product titer in mammalian cell cultures. To sustain cell productivity, highly active oxidative metabolism is desired. Here we explored the effect of tricarboxylic acid (TCA) cycle intermediates supplementation on lactate metabolism and productivity in Chinese hamster ovary fed-batch cultures. Direct addition of 5 mM alpha-ketoglutarate (α-KG), malic acid, or succinic acid in the basal medium did not have any significant impact on culture performance. On the other hand, feeding α-KG, malic acid, and succinic acid in the stationary phase, either as a single solution or as a mixture, significantly improved lactate consumption, reduced ammonium accumulation, and led to higher cell specific productivity and antibody titer (~35% increase for the best condition). Delivering those intermediates as an acidic solution for pH control eliminated CO₂ sparging and accumulation. Feeding TCA cycle intermediates was also demonstrated to be superior to feeding lactic acid or pyruvic acid in titer improvement. Taken together, feeding TCA cycle intermediates was effective in improving lactate consumption and increasing product titer, which is likely due to enhanced oxidative metabolism in an extended duration.     

Endogenous regulation of endothelial cell proliferation

Bovine aortic endothelial cells (BAEC) in culture have the ability to regulate their own proliferation. We have found that a fraction below 100,000 daltons obtained from the media of confluent cultures of BAEC inhibits tritiated thymidine [3H]TdR incorporation as well as their proliferation. The inhibition is dose- and time-dependent; maximum inhibition of [3H]TdR incorporation occurs 8 hr after cells are released from synchronization and the inhibitory fraction is added. Inhibition is evident at concentrations as low as 50 micrograms/ml and reaches a maximum at 600 micrograms/ml. The blockage of [3H]TdR incorporation is reflected in the inhibition of cell proliferation. In the presence of 400 micrograms of endogenous inhibitor per ml of media, added at the time of plating, the average population doubling time increases from 19 to 41 hr. These findings indicate that, in culture, BAEC can regulate their own proliferation by synthesizing an endogenous inhibitor(s) of proliferation.     

High viable cell concentration fed-batch cultures of hybridoma cells through on-line nutrient feeding

A hybridoma cell line was cultivated in fed-batch cultures using a low-protein, serum-free medium. On-line oxygen uptake rate (OUR) measurement was used to adjust the nutrient feeding rate based on glucose consumption, which was estimated on-line using the stoichiometric relations between glucose and oxygen consumption. Through on-line control of the nutrient feeding rate, not only sufficients were supplied for cell growth and antibody production, but also the concentrations of glucose and other important nutrients such as amino acids were maintained at low levels during the cell growth phase. During the cultivation, cell metabolism changed from high lactate production and low oxygen consumption to low lactate production and high oxygen consumption. As a result the accumulation of lactate was reduced and the growth phase was extended. In comparison with the batch cultures, in which cells reached a concentration of approximately 2 x 10(6) cells/mL, a very high concentration of 1.36 x 10(7) cells/mL with a high cell viability (>90%) was achieved in the fed-batch culture. By considering the consumption of glucose and amino acids, as well as the production of cell mass, metabolites, and antibodies, a well-closed material balance was established. Our results demonstrate the value of coupling on-line OUR measurement and the stoichiometric realations for dynamic nutrient feeding in high cell concentration fed batch cultures. (c) 1995 John Wiley & Sons, Inc.     

Real-time method for determining the colony-forming cell content of human hematopoietic cell cultures

Glucose and lactate metabolic rates were evaluated for cultures of cord blood (CB) mononuclear cell (MNC), peripheral blood (PB) MNC, and PB CD34(+) cell cultures carried out in spinner flasks and in T-flasks in both serum-containing and serum-free media. Specific glucose uptake rates (q(gluc), in micromoles per cell per hour) and lactate generation rates (q(lac)) correlated with the percentage of colony-forming cells (CFC) present in the culture for a broad range of culture conditions. Specifically, the time of maximum CFC percentage in each culture coincided with the time of maximum q(gluc) and q(lac) in cultures with different seeding densities and cytokine combinations. A two-population model (Q(lac) = alpha[CFC] + beta([TC] - [CFC ]), where [TC] is total cell concentration; Q(lac) is volumetric lactate production rate in micromoles per milliliter per hour; alpha is q(lac) for an average CFC; and beta is q(lac) for an average non-CFC) was developed to describe lactate production. The model described lactate production well for cultures carried out in both T-flasks and spinner flasks and inoculated with either PB or CB MNC or PB CD34(+) cells. The values for alpha and beta that were derived from the model varied with both the inoculum density and the cytokine combination. However, preliminary results indicate that cultures carried out under the same conditions from different samples with similar initial CD34(+) cell content have similar values for beta and beta. These findings suggest that it should be possible to use lactate production data to predict the harvest time that corresponds to the maximum number of CFC in culture. The ability to harvest ex vivo hematopoietic cultures for transplantation when CFC are at a maximum has the potential to speed the rate at which immunocompromised patients recover. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 693-700, 1997.     

Hyaluronan accumulation is elevated in cultures of low density lipoprotein receptor-deficient cells and is altered by manipulation of cell cholesterol content

The extracellular matrix molecule hyaluronan (HA) accumulates in human atherosclerotic lesions. Yet the reasons for this accumulation have not been adequately addressed. Because abnormalities in lipid metabolism promote atherosclerosis, we have asked whether disrupted cholesterol homeostasis alters HA accumulation in low density lipoprotein receptor-deficient cell cultures. Cultured aortic smooth muscle cells (ASMC) from Watanabe heritable hyperlipidemic (WHHL) rabbits and skin fibroblasts from homozygous patients with familial hypercholesterolemia accumulated 2-4-fold more HA than corresponding cells from age- and sex-matched normolipidemic rabbits and individuals. This occurred in both cell-associated and secreted HA fractions and was independent of cell density or medium serum concentration. WHHL ASMC cultures synthesized twice the proportion of high molecular mass HA (>2x10(6) Da) as normal rabbit ASMC but showed a lower capacity to degrade exogenous [3H]HA. Most importantly, cholesterol depletion or blocking cholesterol synthesis markedly reduced HA accumulation in WHHL ASMC cultures, whereas cholesterol replenishment or stimulation of cholesterol synthesis restored elevated HA levels. We conclude the following: 1) maintaining normal HA levels in cell cultures requires normal cell cholesterol homeostasis; 2) HA degradation may contribute to but is not the predominant mechanism to increase high molecular mass HA accumulation in low density lipoprotein receptor-deficient WHHL ASMC cultures; and 3) elevated accumulation of HA depends on cellular or membrane cholesterol content and, potentially, intact cholesterol-rich microdomains.     

Alteration of mammalian cell metabolism by dynamic nutrient feeding

The metabolism of hybridoma cells was controlled to reduce metabolic formation in fed-batch cultures by dynamically feeding a salt-free nutrient concentrate. For this purpose, on-line oxygen uptake rate (OUR) measurement was used to estimate the metabolic demand of hybridoma cells and to determine the feeding rate of a concentrated solution of salt-free DMEM/F12 medium supplemented with other medium components. The ratios among glucose, glutamine and other medium components in the feeding nutrient concentrate were adjusted stoichiometrically to provide balanced nutrient conditions for cell growth. Through on-line control of the feeding rate of the nutrient concentrate, both glucose and glutamine concentrations were maintained at low levels of 0.5 and 0.2 mM respectively during the growth stage. The concentrations of the other essential amino acids were also maintained without large fluctuations. The cell metabolism was altered from that observed in batch cultures resulting in a significant reduction of lactate, ammonia and alanine production. Compared to a previously reported fed-batch culture in which only glucose was maintained at a low level and only a reduced lactate production was observed, this culture has also reduced the production of other metabolites, such as ammonium and alanine. As a result, a high viable cell concentration of more than 1.0 × 10⁷ cells/mL was achieved and sustained over an extended period. The results demonstrate an efficient nutrient feeding strategy for controlling cell metabolism to achieve and sustain a high viable cell concentration in fed-batch mammalian cell cultures in order to enhance the productivity. This revised version was published online in July 2006 with corrections to the Cover Date.     

Extracellular matrix alters the relationship between tritiated thymidine incorporation and proliferation of MC3T3-E1 cells during osteogenesis in vitro

Bone cells in vivo exist in direct contact with extracellular matrix, which regulates their basic biological processes including metabolism, development, growth and differentiation. Thus, the in vitro activity of cells cultured on tissue culture treated plastic could be different from the activity of cells cultured on their natural substrate. We selected MC3T3-E1 pre-osteoblastic cells to study the effect of extracellular matrix on cell proliferation because these cells undergo a progressive developmental sequence of proliferation and differentiation. MC3T3-E1 cells were cultured on plastic or plastic coated with ECM, fibronectin, collagen type I, BSA or poly l-lysine and their ability to proliferate was assessed by incorporation of [3H]dT or by enumeration of cells. Our results show that (1) ECM inhibits incorporation of [3H]dT by MC3T3-E1 cells; (2) collagen type I, but not BSA, poly l-lysine or fibronectin also inhibits incorporation of [3H]dT; (3) the level of ECM inhibition of [3H]dT incorporation is directly related to the number of cells cultured, but unrelated to the cell cycle distribution or endogenous thymidine content; (4) the kinetic profile of [3H]dT uptake suggest that ECM inhibits transport of [3H]dT from the extracellular medium, and (5) cell counts are similar in cultures whether cells are grown on plastic or ECM. These results suggest that decreased incorporation of [3H]dT by cells cultured on ECM is not reflective of bone cell proliferation.     

Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line

Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.     

High-end pH-controlled delivery of glucose effectively suppresses lactate accumulation in CHO fed-batch cultures

A simple method for control of lactate accumulation in suspension cultures of Chinese hamster ovary (CHO) cells based on the culture's pH was developed. When glucose levels in culture reach a low level (generally below 1 mM) cells begin to take up lactic acid from the culture medium resulting in a rise in pH. A nutrient feeding method has been optimized which delivers a concentrated glucose solution triggered by rising pH. We have shown that this high-end pH-controlled delivery of glucose can dramatically reduce or eliminate the accumulation of lactate during the growth phase of a fed-batch CHO cell culture at both bench scale and large scale (2,500 L). This method has proven applicable to the majority of CHO cell lines producing monoclonal antibodies and other therapeutic proteins. Using this technology to enhance a 12-day fed-batch process that already incorporated very high initial cell densities and highly concentrated medium and feeds resulted in an approximate doubling of the final titers for eight cell lines. The increase in titer was due to additional cell growth and higher cell specific productivity.     

Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes

The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO₂ concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO₂ in batch and fed‐batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO₂ values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO₂. The presented results are especially interesting for large‐scale and perfusion processes where increased pCO₂ concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO₂ accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO₂ might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.     

Differential metabolism of guanine nucleosides by human lymphoid cell lines

Deficiency of the enzyme purine nucleoside phosphorylase is associated with a specific depletion of T cells which is presumably mediated by its substrate, 2'-deoxyguanosine. Inhibitors of this enzyme are therefore being developed as potential immunosuppressive agents. We have compared the effects of 8-aminoguanosine, a competitive inhibitor of purine nucleoside phosphorylase, on the metabolism of 2'-deoxyguanosine by human T lymphoblasts, B lymphoblasts, and mature T-cell lines. 8-Aminoguanosine markedly potentiates the accumulation of dGTP in T lymphoblasts, but results in increased GTP levels in B lymphoblasts and mature T cells. GTP accumulation is associated with ATP depletion of a magnitude similar to that seen with an inhibitor of de novo purine biosynthesis, but does not result in inhibition of either DNA or RNA synthesis. In contrast, direct inhibition of de novo purine biosynthesis sharply decreased the incorporation of [3H]uridine into both DNA and RNA. We conclude that the mechanism of cell damage resulting from prolonged accumulation of GTP appears to involve more than inhibition of de novo purine biosynthesis and consequent ATP depletion. Perturbations in guanine nucleotide pools resulting from partial inhibition of purine nucleoside phosphorylase activity in vivo could result in cellular toxicity not limited to the target T cell population.     

Comparative metabolic analysis of lactate for CHO cells in glucose and galactose

t-PA producing CHO cells have been shown to undergo a metabolic shift when the culture medium is supplemented with a mixture of glucose and galactose. This metabolic change is characterized by the reincorporation of lactate and its use as an additional carbon source. The aim of this work is to understand lactate metabolism. To do so, Chinese hamster ovary cells were grown in batch cultures in four different conditions consisting in different combinations of glucose and galactose. In experiments supplemented with glucose, only lactate production was observed. Cultures with glucose and galactose consumed glucose first and produced lactate at the same time, after glucose depletion galactose consumption began and lactate uptake was observed. Comparison of the metabolic state of cells with and without the shift by metabolic flux analysis show that the metabolic fluxes distribution changes mostly in the reactions involving pyruvate metabolism. When not enough pyruvate is being produced for cells to support their energy requirements, lactate dehydrogenase complex changes the direction of the reaction yielding pyruvate to feed the TCA cycle. The slow change from high fluxes during glucose consumption to low fluxes in galactose consumption generates intracellular conditions that allow the influx of lactate. Lactate consumption is possible in cell cultures supplemented with glucose and galactose due to the low rates at which galactose is consumed. Evidence suggests that an excessive production and accumulation of pyruvate during glucose consumption leads to lactate production and accumulation inside the cell. Other internal conditions such as a decrease in internal pH, forces the flow of lactate outside the cell. After metabolic shift the intracellular pool of pyruvate, lactate and H⁺ drops permitting the reversal of the monocarboxylate transporter direction, therefore leading to lactate uptake. Metabolic analysis comparing glucose and galactose consumption indicates that after metabolic shift not enough pyruvate is produced to supply energy metabolism and lactate is used for pyruvate synthesis. In addition, MFA indicates that most carbon consumed during low carbon flux is directed towards maintaining energy metabolism.     

Diffusion chamber colony-forming unit (CFU-d): a primitive stem cell

Assay of hematopoietic precursor cells in diffusion chambers (DCs) implanted intraperitoneally in experimental animals provides a powerful tool for studying stem cell kinetics in vivo. In this system, the effect of cell migration (which complicates whole animal studies) is eliminated because the membranes utilized in the construction of the chambers are impermeable for cells, while permitting free passage of molecules present in the humoral phase of the host. As judged by light microscopy, conditions in the DC cultures primarily favor macrophage and granulocyte growth. However, the use of in vitro and in vivo subculture to further analyze chamber contents has demonstrated that the system supports proliferation of early hematopoietic progenitors. Additionally, cells capable of rescuing lethally irradiated mice proliferate in DC cultures. Development of the plasma clot DC technique has revealed that most of the growth occurs in colonies which are derived from single cells (CFU-d). Characterization of these cells indicates that they are at least as primitive as other colony-forming cells and, also based on subculture studies, can differentiate along several hematopoietic lineages. In addition to normal CFU-d, both embryonal and leukemic cells can give rise to granulocytes, macrophages, megakaryocytes and erythroid cells in the DC cultures. Evaluation of the effects of humoral factors on hematopoietic cell proliferation and differentiation in the system has led to the identification of both stimulators and inhibitors that may be different from the well-characterized cytokines. Thus, the system seems to be useful for detecting molecules controlling the most primitive stages of hematopoiesis. We believe that the DC culture technique holds enormous potential in the study of stem cell proliferation and differentiation in vivo.     

Acetate accumulation and regulation by process parameters control in Chinese hamster ovary cell culture

Chinese hamster ovary (CHO) cells represent a group of predominantly used mammalian hosts for producing recombinant therapeutic proteins. Known for their rapid proliferation rates, CHO cells undergo aerobic glycolysis that is characterized by fast glucose consumption, that ultimately gives rise to a group of small-molecule organic acids. However, only the function of lactate has been extensively studied in CHO cell culture. In this study, we observed the accumulation of acetate from the late exponential phase to harvest day, potentially contributing to the pH decline in late culture stage regardless of lactate consumption. In addition, we evaluated the acidification of the fresh media and the cell culture suspension, and the data revealed that acetate presented a lower acidification capacity compared to lactate and exhibited limited inhibitory effect on cells with less than 20 mM supplemented in the media. This study also explored the ways to control acetate accumulation in CHO cell culture by manipulating the process parameters such as temperature, glucose, and pH control. The positive correlation between the specific glucose consumption rate and acetate generation rate provides evidence of the endogenous acetate generation from overflow metabolism. Reducing these parameters (temperature, glucose consumption) and HCl-controlled low pH ultimately suppress acetate build-up. In addition, the specific acetate generation rate and relevant glucose consumption rate are found to be a metabolic trait associated with specific cell lines. Taken together, the results presented in these experiments provide a means to advance industrial CHO cell culture process control and development.     

Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures

At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the glycolysis pathway, being lactate the main by-product. However, HEK293 cells are also able to metabolize lactate depending on the environmental conditions. It has been recently observed that one of the most important modes of lactate metabolization is the cometabolism of lactate and glucose, observed even during the exponential growth phase. Extracellular lactate concentration and pH appear to be the key factors triggering the metabolic shift from glucose consumption and lactate production to lactate and glucose concomitant consumption. The hypothesis proposed for triggering this metabolic shift to lactate and glucose concomitant consumption is that HEK293 cells metabolize extracellular lactate as a response to both extracellular protons and lactate accumulation, by means of cotransporting them (extracellular protons and lactate) into the cytosol. At this point, there exists a considerable controversy about how lactate reaches the mitochondrial matrix: the first hypothesis proposes that lactate is converted into pyruvate in the cytosol, and afterward, pyruvate enters into the mitochondria; the second alternative considers that lactate enters first into the mitochondria, and then, is converted into pyruvate. In this study, lactate transport and metabolization into mitochondria is shown to be feasible, as evidenced by means of respirometry tests with isolated active mitochondria, including the depletion of lactate concentration of the respirometry assay. Although the capability of lactate metabolization by isolated mitochondria is demonstrated, the possibility of lactate being converted into pyruvate in the cytosol cannot be excluded from the discussion. For this reason, the calculation of the metabolic fluxes for an HEK293 cell line was performed for the different metabolic phases observed in batch cultures under pH controlled and noncontrolled conditions, considering both hypotheses. The main objective of this study is to evaluate the redistribution of cellular metabolism and compare the differences or similarities between the phases before and after the metabolic shift of HEK293 cells (shift observed when pH is not controlled). That is from a glucose consumption/lactate production phase to a glucose-lactate coconsumption phase. Interestingly, switching to a glucose and lactate cometabolization results in a better-balanced cell metabolism, with decreased glucose and amino acids uptake rates, affecting minimally cell growth. This behavior could be applied to further develop new approaches in terms of cell engineering and to develop improved cell culture strategies in the field of animal cell technology.     

Effects of cis-4-hydroxy-L-proline, and inhibitor of Schwann cell differentiation, on the secretion of collagenous and noncollagenous proteins by Schwann cells

The proline analog cis-4-hydroxy-L-proline (CHP) was previously shown to inhibit both Schwann cell (SC) differentiation and extracellular matrix (ECM) formation in cultures of rat SCs and dorsal root ganglion neurons. We confirmed that CHP inhibits basal lamina formation by immunofluorescence with antibodies to laminin, type IV collagen, and heparan sulfate proteoglycan. In order to test the hypothesis that CHP inhibits SC differentiation by specifically inhibiting the secretion of collagen, cultures grown in the presence or absence of CHP were metabolically labeled with [3H]leucine and the media were analyzed for relative amounts of (a) collagenous and noncollagenous proteins by assay with bacterial collagenase and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), or (b) triple-helical collagen by pepsin digestion followed by SDS-PAGE. The results indicate that although CHP inhibited the accumulation of secreted collagen in the culture medium and disrupted collagen triple-helix formation, it also significantly inhibited the accumulation of secreted noncollagenous proteins in the medium. CHP had no significant effect on either total protein synthesis (medium plus cell layer) or cell number. We conclude that CHP does not act as a specific inhibitor of collagen secretion in this system, and thus data from these experiments cannot be used to relate SC collagen production to other aspects of SC differentiation. We discuss the evidence for and against specificity of CHP action in other systems.     

Deoxyadenosine modulates human suppressor T cell function and B cell differentiation stimulated by Staphylococcus aureus protein A

Adenosine deaminase (ADA) deficiency and the resultant accumulation of deoxyadenosine (AdR) are associated with profound T cell dysfunction and variable B cell dysfunction in vivo. We examined the effects of AdR on the in vitro function of normal human peripheral blood B and T lymphocytes whose ADA activity was inhibited by 2'-deoxycoformycin. We found that OKT8+ T cell-mediated suppression of SPA-induced Ig production was markedly reduced by concentrations of AdR (3 to 10 microM) that did not affect helper T cell function. Because the lectin-induced proliferative responses of OKT8+ T cells and OKT8- T cells were equally susceptible to AdR, modulation of in vitro immune responses by low-dose AdR probably reflected different proliferative requirements for the expression of T cell helper or suppressor functions. Although low doses of AdR did not inhibit Ig production in SPA-stimulated cultures, we found that T cell-dependent, SPA-stimulated B cell proliferation was blocked by 3 to 10 microM AdR. Therefore, it appeared that B cell proliferation was not required for the induction of Ig synthesis in this system. Higher doses (30 to 100 microM) of AdR did block the induction of Ig synthesis, presumably by interfering with T-helper functions via a mechanism other than inhibition of proliferation and/or by inhibiting B cell differentiation events.     

Impact of pH on lactate formation and utilization by human fecal microbial communities

The human intestine harbors both lactate-producing and lactate-utilizing bacteria. Lactate is normally present at <3 mmol liter(-1) in stool samples from healthy adults, but concentrations up to 100 mmol liter(-1) have been reported in gut disorders such as ulcerative colitis. The effect of different initial pH values (5.2, 5.9, and 6.4) upon lactate metabolism was studied with fecal inocula from healthy volunteers, in incubations performed with the addition of dl-lactate, a mixture of polysaccharides (mainly starch), or both. Propionate and butyrate formation occurred at pH 6.4; both were curtailed at pH 5.2, while propionate but not butyrate formation was inhibited at pH 5.9. With the polysaccharide mix, lactate accumulation occurred only at pH 5.2, but lactate production, estimated using l-[U-(13)C]lactate, occurred at all three pH values. Lactate was completely utilized within 24 h at pH 5.9 and 6.4 but not at pH 5.2. At pH 5.9, more butyrate than propionate was formed from l-[U-(13)C]lactate in the presence of polysaccharides, but propionate, formed mostly by the acrylate pathway, was the predominant product with lactate alone. Fluorescent in situ hybridization demonstrated that populations of Bifidobacterium spp., major lactate producers, increased approximately 10-fold in incubations with polysaccharides. Populations of Eubacterium hallii, a lactate-utilizing butyrate-producing bacterium, increased 100-fold at pH 5.9 and 6.4. These experiments suggest that lactate is rapidly converted to acetate, butyrate, and propionate by the human intestinal microbiota at pH values as low as 5.9, but at pH 5.2 reduced utilization occurs while production is maintained, resulting in lactate accumulation.