Comparative differentiation analysis of distinct oral tissue-derived cells in response to osteogenic stimulation.

BACKGROUND: Mixed cell populations from oral tissues may be superior to pure stem cells for regenerative approaches. Therefore, the aim of the present study was to explore the osteogenic potential of mixed cells derived from oral connective tissues compared to alveolar osteoblasts. MATERIALS AND METHODS: Primary cells were isolated from the alveolar bone, periodontal ligament and gingiva. Following characterization by colony formation, growth capacity and flow cytometry, all cells were subjected to osteogenic differentiation induction and screened for a large panel of osteogenic markers using western blots, qPCR arrays, and matrix mineralization and alkaline phosphatase quantification. RESULTS: Non-induced mixed cells from gingiva showed higher colony formation efficiency but decreased proliferation compared to non-induced periodontal mixed cells, while both entities revealed similar surface markers tested in this setup. Following osteogenic induction, all cell populations individually expressed receptors with distinctively activated downstream effectors. Gene expression of induced periodontal mixed cells was similar to alveolar osteoblasts, but was differently modulated in gingival mixed cells. The latter failed to achieve osteogenic differentiation in terms of matrix mineralization and alkaline phosphatase activity, which was well observed in periodontal mixed cells and osteoblasts. CONCLUSION: Mixed cells from periodontal ligament but not from gingiva feature an inherent osteogenic capacity in vitro. From these results, it can be concluded that periodontal cells do not require further stem cell enrichment in order to qualify for bone regeneration. CLINICAL RELEVANCE: Our data contribute to the development of novel cell-based therapies using mixed cells from the periodontal ligament in regenerative periodontics.

Synthesis of plasmalemmal glycoproteins in intestinal epithelial cells. Separation of Golgi membranes from villus and crypt cell surface membranes; glycosyltransferase activity of surface membrane.

The relationship between Golgi and cell surface membranes of intestinal cells was studied. These membranes were isolated from intestinal crypt cells and villus cells. The villus cell membranes consisted of microvillus membrane, a Golgi-rich fraction, and two membrane fractions interpreted as representing lateral-basal membranes. The villus cell microvillus membrane was purified by previously published techniques while the other membranes were obtained from isolated cells by differential centrifugation and density gradient velocity sedimentation. The two membrane fractions obtained from villus cells and considered to be lateral-basal membranes were enriched for Na+,K+-ATPase activity, but one also showed enrichment in glycosyltransferase activity. The Golgi membrane fraction was enriched for glycosyltransferase activity and had low to absent Na+,K+-ATPase activity. Adenylate cyclase activity was present in all membrane fractions except the microvillus membrane but co-purified with Golgi rather than lateral-basal membranes. Electron microscopy showed that the Golgi fraction consisted of variably sized vesicles and cisternalike structures. The two lateral-basal membrane fractions showed only vesicles of smaller, more uniform size. After 125I labeling of isolated intact cells, radioactivity was found associated with the lateral-basal and microvillus membrane fractions and not with the Golgi fraction. Antibody prepared against lateral-basal membrane fractions reacted with the surface membrane of isolated villus cells. The membrane fractions from isolated crypt cells demonstrated that all had high glycosyltransferase activity. The data show that glycosyltransferase activity, in addition to its Golgi location, may be a significant property of the lateral-basal portion of the intestinal villus cell plasma membrane. Data obtained with crypt cells support earlier data and show that the crypt cell surface membrane possesses glycosyltransferase activity.

Venous pressure in man during weightlessness.

To determine whether the body fluid shift from the lower limbs toward the head that occurs during spaceflight leads to lasting increases of venous pressure in the upper body, venous pressure and hematocrit measurements were made on four astronauts before flight and 1 and 12 hours after recovery and compared with measurements in space. During the mission the hematocrit was elevated and the venous pressure lowered by 1 to 8 centimeters of water as compared with the preflight data. One hour after landing the hematocrit decreased, indicating a hemodilution, venous pressures were unexpectedly high, and a body weight loss of 4 to 5 percent was observed. Twelve hours later the venous pressures were the lowest recorded during the study. The fluid shift apparently takes place during the first several hours of spaceflight. Thereafter, the pressure in the peripheral veins and the central circulation is lower than that measured before flight.

Water immersion is associated with an increase in aquaporin-2 excretion in healthy volunteers.

Here, we report the alterations in renal water handling in healthy volunteers during a 6 h thermoneutral water immersion at 34 to 36 degrees C. We found that water immersion is associated with a reversible increase in total urinary AQP2 excretion.

Lymph chylomicron formation during the inhibition of protein synthesis. Studies of chylomicron apoproteins.

The effect of impaired intestinal protein synthesis on chylomicron apoprotein composition was studied in mesenteric lymph fistula rats. Lymph was obtained from animals with impaired protein synthesis given intraperitoneal acetoxycycloheximide (ACH), a potent inhibitor of protein synthesis. Lymph chylomicrons were then isolated by ultracentrifugation and purified on agarose columns. Purified chylomicrons from control and ACH-treated animals were delipidated, and their apoprotein pattern was examined on sodium dodecyl sulfate (SDS) polyacrylamide gels. Because we had previously demonstrated a markedly increased lymph chylomicron size during the inhibition of protein synthesis, it was first necessary to determine whether chylomicron apoprotein composition normally varied with chylomicron size. Chylomicrons of varying sizes were prepared by differential ultracentrifugation, and their apoprotein composition was determined densitometrically on SDS polyacrylamide gels. No significant difference in apoprotein composition was found normally with varying chylomicron size. In contrast, however, chylomicrons from ACH-treated animals showed a 50% decrease in a major apoprotein band with R(1) 0.67. Other chylomicron apoproteins were not decreased as a result of impaired protein synthesis, suggesting differing rates of synthesis of the various chylomicron apoproteins. In vivo incorporation studies of [(3)H]leucine into the various apoproteins of lymph chylomicrons demonstrated that this apoprotein (R(1) 0.67) had the most rapid synthesis rate and suggested that it seemed most affected by impaired intestinal protein synthesis. Immunologic studies indicated that this apoprotein was immunologically related to high-density lipoproteins (HDL) and was present in chylomicrons isolated directly from small intestinal mucosa. These studies demonstrate that impaired intestimal protein synthesis is associated with a deficiency in one of the major chylomicron apoproteins and may in part explain the impaired lipid absorption seen during states of impaired protein synthesis.

Fat absorption during inhibition of protein synthesis: studies of lymph chylomicrons.

The effect of protein synthesis inhibition on the absorption of oleic acid from micellar solution was studied in mesenteric lymph fistula rats. A micellar solution of oleic acid labeled with tracer doses of oleic acid-(14)C was administered by intraduodenal infusion to rats with indwelling mesenteric lymph cannulas. Protein synthesis was inhibited by intraperitoneal acetoxycycloheximide (ACH), 0.25 mg/kg, 1 hr before lipid infusion. Lymph chylomicrons labeled with oleic acid-(14)C were collected from control and protein inhibited animals at various times after lipid infusion and subjected to sucrose density gradient centrifugation to determine changes in size. In control animals there was a transient increase in chylomicron size during maximal triglyceride absorption; however, in protein-inhibited animals there was a marked and sustained increase in chylomicron size as late as 4 hr after lipid infusion. Triglyceride and phospholipid determinations on washed chylomicrons from both groups indicated a greater triglyceride/phospholipid ratio after protein synthesis inhibition supporting a greater chylomicron size. Electron microscopy of lymph from both groups further confirmed a markedly increased chylomicron size after protein synthesis inhibition. It is proposed that an increase in size conserves chylomicron surface components, i.e. apoprotein, during conditions of inhibition of protein synthesis. These studies clearly demonstrate that the intestinal inhibition of protein synthesis is associated with an increase in the size of intestinal lymph chylomicrons and support the concept that protein synthesis is important in the formation and transport of chylomicrons from the mucosal cell into the lymph.

Biological effects of c-Mer receptor tyrosine kinase in hematopoietic cells depend on the Grb2 binding site in the receptor and activation of NF-kappaB.

The c-Mer receptor tyrosine kinase (RTK) is most closely related to chicken c-Eyk and belongs to the Axl RTK subfamily. Although not detected in normal lymphocytes, c-Mer is expressed in B- and T-cell leukemia cell lines, suggesting an association with lymphoid malignancies. To gain an understanding of the role of this receptor in lymphoid cells, we expressed in murine interleukin-3 (IL-3)-dependent Ba/F3 pro-B-lymphocyte cells a constitutively active receptor, CDMer, formed from the CD8 extracellular domain and the c-Mer intracellular domain. Cells transfected with a plasmid encoding the CDMer receptor became IL-3 independent. When tyrosine (Y)-to-phenylalanine (F) mutations were introduced into c-Mer, only the Y867 change significantly reduced the IL-3-independent cell proliferation. The Y867 residue in the CDMer receptor mediated the binding of Grb2, which recruited the p85 phosphatidylinositol 3-kinase (PI 3-kinase). Despite the difference in promotion of proliferation, both the CDMer and mutant F867 receptors activated Erk in transfected cells. On the other hand, we found that both transcriptional activation of NF-kappaB and activation of PI 3-kinase were significantly suppressed with the F867 mutant receptor, suggesting that the activation of antiapoptotic pathways is the major mechanism for the observed phenotypic difference. Consistent with this notion, apoptosis induced by IL-3 withdrawal was strongly prevented by CDMer but not by the F867 mutant receptor.

Erythropoietin regulations in humans under different environmental and experimental conditions.

In the adult human, the kidney is the main organ for the production and release of erythropoietin (EPO). EPO is stimulating erythropoiesis by increasing the proliferation, differentiation and maturation of the erythroid precursors. In the last decades, enormous efforts were made in the purification, molecular encoding and description of the EPO gene. This led to an incredible increase in the understanding of the EPO-feedback-regulation loop at a molecular level, especially the oxygen-dependent EPO gene expression, a key function in the regulation loop. However, studies in humans at a systemic level are still very scanty. Therefore, it is the purpose of the present review to report on the main recent investigations on EPO production and release in humans under different environmental and experimental conditions, including: (i) studies on EPO circadian, monthly and even annual variations, (ii) studies in connection with short-, medium- and long-term exercise at sea-level which will be followed (iii) by studies performed at moderate and high altitude.

Beta 2-microglobulin gene is mutated in a human colon cancer cell line (HCT) deficient in the expression of HLA class I antigens on the cell surface.

The human colon cancer cell line HCT does not express any detectable HLA class I antigens on the cell surface. RNA blot analyses showed that HCT cells synthesize easily detectable levels of heavy chains as well as beta 2-microglobulin (beta 2m) transcripts. Experiments of immunoprecipitation revealed the presence of intracellular HLA heavy chains and the absence of beta 2m molecules. Sequencing studies, performed on polymerase chain reaction-mediated amplification of beta 2m-specific complementary DNAs, indicated that in HCT cells both beta 2m genes are mutated. The first mutation consists of an 11-base deletion, corresponding to the first 11 base pairs of the second exon of the beta 2m gene. This mutation alters the reading frame, starting from the third amino acid residue of the mature beta 2m protein, resulting in the synthesis of a 31-amino acid peptide with no remarkable homology to any of the sequences stored in the protein database. The second mutation is a point mutation (C----A), resulting in a UAA stop codon corresponding to the 10th amino acid residue of the mature beta 2m. Therefore, it would appear that in HCT cells the beta 2m genes have undergone two different mutational changes. This is the first molecular demonstration of beta 2m mutations in a human epithelial cell line.

Stabilization and productive positioning roles of the C2 domain of PTEN tumor suppressor.

PTEN is a tumor suppressor frequently inactivated in brain, prostate, and uterine cancer. It acts as a phosphoinositide phosphatase and consists of an amino-terminal phosphatase domain tightly linked to a COOH-terminal C2 domain involved in lipid membrane-binding. We investigated the functions of the C2 domain and their relevance for tumor growth. To discriminate between PTEN C2 domain ability to recruit or to position the active site to the membrane, we artificially membrane-targeted PTEN by a myristoylation signal. This modification increased wild-type PTEN growth inhibition but did not rescue a C2 mutant defective in lipid-binding, suggesting a model in which PTEN C2 domain positions the active site productively with respect to the membrane-bound phosphoinositide substrate. When tumor-derived mutations in the loops that connect the C2 beta-strands were analyzed, we found that these generally destabilized the protein but had variable effects on the phosphatase activity and tumor growth. The magnitude of these effects was dependent on the presence of the COOH-terminal PEST sequences and on the cell type where the mutant proteins were expressed, suggesting the existence of fluctuating structural defects of the mutant protein. One of the C2 loop mutants induced a total loss of PTEN tumor-suppressor function, most likely by affecting both the membrane binding and the protein stability. These data support a double role for PTEN C2 domain in protein stability and in productive orientation of the catalytic site.

Mader: a novel nuclear protein over expressed in human melanomas.

Mader is a novel delayed early response gene encoding a nuclear protein. Upregulation of the Mader 2.7 kb mRNA requires protein synthesis and can be induced in a variety of human cell lines by serum stimulation and in freshly isolated lymphocytes by mitogens. mRNA levels reach a maximum by 2 h and return to basal levels by 6 h. Mader is highly conserved as cross-hybridizing DNA sequences were observed in species as diverse as Rhesus and S. cerevisiae. The Mader protein of approximately 55 kD has two proline rich domains and contains 15 potential phosphorylation sites, a nuclear localization signal, and multiple S(T)PXX motifs that are characteristic of regulatory DNA binding proteins. Monoclonal antibodies produced against Mader confirm that it is localized to the nucleus. These features of Mader suggest that it may play a role in growth regulation. Although Mader mRNA can be detected in most cell lines, only occasional immunoreactive cells were detected in normal human tissues. In contrast, uniform strong nuclear staining was observed in all malignant melanomas examined. The fact that only one of six benign melanocytic nevi examined showed evidence of Mader expression suggests that over-expression of Mader protein may be associated with the malignant transformation of melanocytes.

Activation of the focal adhesion kinase signaling pathway by structural alterations in the carboxyl-terminal region of c-Crk II.

The Crk II adaptor protein encodes an SH2/SH3-domain containing adaptor protein with an SH2-SH3-SH3 domain structure that transmits signals from tyrosine kinases. The two SH3 domains are separated by a 54 amino acid linker region, whose length is highly conserved in xenopus, chicken, and mamalian Crk II proteins. To gain a better understanding into the role of the C-terminal region of Crk, we generated a series of C-terminal SH3 domain and SH3 linker mutants and examined their role in tyrosine kinase pathways. Expression of point mutations in the C-terminal SH3 domain (W276K Crk), at the tyrosine phosphorylation site (Y222F Crk II), or truncation of the entire C-terminus (Crk I or Crk Delta242), all increased c-Abl binding to the N-terminal SH3 domain of Crk and, where relevant, increased Tyr(222) phosphorylation. Deletion analysis of c-Crk II also revealed the presence of a C-terminal segment important for trans-activation of FAK. Such mutants, Crk Delta255 or Crk Delta242 Extended Linker (Crk Delta242([EL])), characterized by a disruption in the SH3 linker/C-terminal SH3 boundary, induced robust hyperphosphorylation of focal adhesion kinase (FAK) on Tyr(397), hyperphosphorylation of focal adhesion proteins p130(cas) and paxillin and increased focal adhesion formation in NIH3T3 cells. The effects of Crk Delta242([EL]) could be abrogated by co-expression of dominant negative c-Src or the protein tyrosine phosphatase PTP-PEST, but not by dominant negative Abl. Our results suggest that the C-terminal region of Crk contains negative regulatory elements important for both Abl and FAK dependent signal pathways, and offers a paradigm for an autoinhibitory region in the SH3 linker/C-terminal SH3 domain.

Islet cell antigens. Extraction studies and ELISA analysis.

Whole human and bovine pancreases were extracted in 20 mM Tris-HCl buffer without detergents and fractionated by high-speed centrifugation. The 80,000 x g supernatant was used to coat microtiter plates at a concentration of 5 micrograms protein/ml in phosphate-buffered saline. This solid-phase ELISA system was used for the detection of islet cell antigens defined by a series of monoclonal islet cell antibodies (HISL-1, -4, -5, -8, -14, and -19 and 4F2, 3G5, and A2B5). Both glycoprotein and glycolipid islet cell antigens in the total pancreatic extracts were detected by the monoclonal islet cell antibody in the ELISA system, indicating that epitope preservation had occurred during the extraction procedure. There was a good correlation between islet cell antigen quantitated by the ELISA system and the corresponding islet immunohistochemical reaction. Studies along these lines have the potential to facilitate the design of large-scale protocols for the purification of diabetes-related islet cell antigens to homogeneity.

A hypothesis: autonomic rhythms are reflected in growth lines of teeth in humans and extinct archosaurs.

A major determinant of tooth architecture is the arrangement of lines in dentin and in the enamel following the contour of the surface. Since the original description of these lines in the 19th century, they have been attributed to recurring events during tooth development. They have also attracted the attention of dental scientists and anthropologists; however, to date, studies of these structures have been largely theoretical and microscopic. We show here that the statistical properties of the spacing between the lines are similar in teeth from both ancient and modern humans and from extinct archosaurs, reptiles that lived tens or hundreds of millions of years ago-they also resemble heart rate variability of living humans. We propose that the deposition of these recurring structures is controlled by the autonomic nervous system. This control accounts for their regularity and recurrent nature and implies that the lines are an expression of a biologic rhythm which has been conserved throughout evolution. Details of the rhythms give clues to life styles in ancient civilizations and to the physiology of extinct archosaurs.

Recurrence rate and shift in histopathological differentiation of oral squamous cell carcinoma ­ A long-term retrospective study over a period of 13.5 years.

OBJECTIVE: Little information is available as to whether recurrences of oral squamous cell carcinoma (OSCC) show different histopathological grades than the primary tumor and whether postoperative radiotherapy (PORT) influences the grade of differentiation in the case of recurrence. The objective of this study was the evaluation of recurrence rates and change in differentiation. MATERIAL AND METHODS: This retrospective, single-institution cohort study included surgically treated OSCC patients over a 13-year period (2000-2013). The relationship among tumor size, lymph node metastases, and recurrence rate of OSCC was investigated. Primary tumor differentiation was compared with differentiation of recurrence. RESULTS: A total of 429 patients (277 men and 152 women) were included in this study. Of these, 124 (28.9%) received PORT. The incidence of primary cervical metastases increased significantly with tumor size (p < 0.001). Recurrence developed in 82 patients (19.1%). Stage T1/T2 showed a significantly lower recurrence rate than stage T3/T4 (16.3% vs. 30.2%) (p < 0.01). A total of 23 (30.7%) patients with recurrence showed a change in differentiation. CONCLUSION: Increasing primary tumor size correlates with incidence of cervical metastases and recurrence rate. Initial cervical metastases show no effect on recurrence rates. Differentiation of primary tumor does not correlate with the recurrence rate. The majority of recurrences show consistent histopathological grading.

Serum matrix metalloproteinase-2 in patients with malignant melanoma.

PURPOSE: Matrix metalloproteinases (MMPs) are a family of structurally related zinc-dependent endopeptidases that are able to degrade extracellular matrix components. MMPs play a role in tumor invasion and tumor metastasis. MMP-2 (also known as gelatinase A) is expressed in human melanoma cells. METHODS: In this study, we measured MMP-2 in 337 serum probes of 166 melanoma patients with a recently developed enzyme immunoassay and compared these data with the tumor stage, presence of metastases, and the levels of S100beta and soluble intracellular adhesion molecule-1 (sICAM-1) in serum. RESULTS: The mean levels were (189.2 +/- 50.8) ng/ml for MMP-2, (263.2 +/- 74.1) ng/ml for sICAM-1, and (0.424 +/- 1.568) U/ml for S100beta. There was a statistical significant correlation of MMP-2 with sICAM-1 (P=0.05) and Sl00beta (P=0.01). The mean MMP-2 levels (in ng/ml) in patients with metastatic melanoma were 196.4 +/- 54.0 versus 182.6 +/- 46.9 in non-metastasizing melanoma (P=0.037). However, there was no significant difference in MMP-2 levels between the different tumor stages. CONCLUSION: Determination of MMP-2 serum levels is of limited value as a tumor marker in melanoma, though there are higher levels in the more advanced disease.

Serum protein s100beta in patients with malignant melanoma detected by an immunoluminometric assay.

S100 protein is well established as a diagnostic tool in malignant melanoma immunohistology. In this study we measured S100beta in serum with a recently developed luminometric immunoassay with a detection limit of 0.02 microg/l. By measuring S-100beta in a group of apparently healthy individuals a mean value of 0.031 +/- 0.026 microg/l was found. In the reference group, serum S100beta was below 0.12 microg/l in all cases. To assess the sensitivity of the assay we investigated serum S-100beta levels in 371 serum samples of 315 patients with histological proven malignant melanoma at different disease stages. Staging was performed according to the German Society of Dermatology classification. Significant differences were observed between the control group and stages IIb (P = 0.01) and IV (P = 0.001). In tumour-bearing patients of stages IIIb and IV, the difference was highly significant (P < 0.0001). S100beta > 0.20 microg/l helps to distinguish between tumour-free and tumour-bearing patients with a specificity of 97.0% and a sensitivity of 64.6%. Our results demonstrate the serum S100beta is of limited value as a melanoma marker. However, it has clinical significance for identifying tumour-positive patients in advanced malignant melanoma stages III and IV.

Time course of erythropoietin, triiodothyronine, thyroxine, and thyroid-stimulating hormone at 2,315 m.

Erythropoietin (EPO), triiodothyronine, thyroxine, thyroid-stimulating hormone (TSH), hemoglobin (Hb), reticulocytes, packed cell volume, and plasma volume changes were studied in 29 male Austrian mountain rescue soldiers before and several days after an ascent from 744 to 2,315 m in the Alps. EPO concentrations showed a remarkable increase 48 h after the ascent (P < 0.01). No significant changes were found in triiodothyronine, thyroxine, and TSH. Reticulocytes increased only slightly. Hb decreased during the week (P < 0.01). Packed cell volume did not change, whereas plasma volume showed a slight decrease for 48 h after the ascent but then increased constantly (P < 0.01). It seems that thyroid hormones, TSH, and Hb play only a minor role in the regulation of EPO production and release under mild hypobaric-hypoxic conditions (2,315 m).

Effects of dehydration on the vasopressin response to immersion.

Nine healthy volunteers underwent three experimental procedures in random order. The protocols were 4 h of thermal dehydration followed by 2 h of head-out water immersion, 4 h of thermal dehydration followed by 2 h of chair rest, and 6 h of rest in the supine position. Four hours of heat exposure (50 degrees C) resulted in a body weight loss of approximately 3.5%. Plasma osmolality rose by approximately 5 mosmol/kg, mean arterial pressure (MAP) decreased from 85 to 78 mmHg, and body temperature increased from 36.8 to 38.6 degrees C. As a consequence of the combined action of hypertonicity, hypovolemia, hypotension, and hyperthermia, plasma arginine vasopressin (AVP) increased from 2.1 to 8.1 pg/ml after 4 h thermal dehydration. Changes in body weight, plasma osmolality, body temperature, and MAP were similar after either a subsequent 2 h of water immersion or 2 h of chair rest. However, during chair rest plasma AVP remained elevated (8.4 pg/ml), whereas during immersion plasma AVP decreased from 8.1 to 4.7 pg/ml. This was probably due to the central hypervolemia induced by immersion. Our results support the hypothesis that central hypervolemia rather than hypotonicity is the primary stimulus for AVP suppression during water immersion in dehydrated subjects. During the early immersion period hypoosmolality might contribute to the AVP suppression.

Erythropoietin under real and simulated microgravity conditions in humans.

The aim of this study was to analyze the time course of erythropoietin (EPO) during Earth-bound microgravity simulations such as bed rest, isolation and confinement (IC), head-down tilt (HDT; -6 degrees), and immersion to evaluate which factors could contribute to alterations in EPO under real microgravity conditions during and after short- (< 10 days) and long-term (> 6 mo) spaceflights. During bed rest (24h), no significant changes in EPO could be observed. Subjects confined in a diving chamber facility for 60 days showed a decrease in EPO. In the recovery period a slight increase was observed, but EPO concentrations did not reach the pre-IC control level. In the control period before HDT, subjects showed normal resting values for EPO, but on day 2 of HDT the EPO concentrations were decreased (P < 0.01). Later the EPO levels remained below the control value and were increased after HDT (P < 0.05). After immersion (24 h) increased EPO concentrations could be determined (P < 0.05). During a short-term spaceflight the astronauts showed in-flight (day 4) decreased and unchanged EPO concentrations. During a long-term spaceflight, 24 h after recovery, the cosmonaut showed slightly elevated EPO concentration, which increased markedly during the following days. It is concluded that 1) HDT (-6 degrees) causes a rapid decrease in EPO in humans, 2) IC per se leads to diminished EPO concentrations, 3) EPO regulation in humans during short- and long-term spaceflights might be different, 4) changes in central blood volume, i.e., central venous pressure, seem to be involved in the modulation of EPO production and release under simulated and real microgravity conditions, and 5) the HDT (-6 degrees) Earth-bound simulation reflects mostly the changes in EPO production and release observed under real microgravity conditions in humans.