Effects of controlled ovarian stimulation on vascular barrier and endothelial glycocalyx: a pilot study.

PURPOSE: Controlled ovarian stimulation significantly amplifies the number of maturing and ovulated follicles as well as ovarian steroid production. The ovarian hyperstimulation syndrome (OHSS) increases capillary permeability and fluid extravasation. Vascular integrity intensely is regulated by an endothelial glycocalyx (EGX) and we have shown that ovulatory cycles are associated with shedding of EGX components. This study investigates if controlled ovarian stimulation impacts on the integrity of the endothelial glycocalyx as this might explain key pathomechanisms of the OHSS. METHODS: Serum levels of endothelial glycocalyx components of infertility patients (n=18) undergoing controlled ovarian stimulation were compared to a control group of healthy women with regular ovulatory cycles (n=17). RESULTS: Patients during luteal phases of controlled ovarian stimulation cycles as compared to normal ovulatory cycles showed significantly increased Syndecan-1 serum concentrations (12.6 ng/ml 6.1125th-19.1375th to 13.9 ng/ml 9.625th-28.975th; p=0.026), indicating shedding and degradation of the EGX. CONCLUSION: A shedding of EGX components during ovarian stimulation has not yet been described. Our study suggests that ovarian stimulation may affect the integrity of the endothelial surface layer and increasing vascular permeability. This could explain key features of the OHSS and provide new ways of prevention of this serious condition of assisted reproduction.

Hibernating astronauts-science or fiction?

For long-duration manned space missions to Mars and beyond, reduction of astronaut metabolism by torpor, the metabolic state during hibernation of animals, would be a game changer: Water and food intake could be reduced by up to 75% and thus reducing payload of the spacecraft. Metabolic rate reduction in natural torpor is linked to profound changes in biochemical processes, i.e., shift from glycolysis to lipolysis and ketone utilization, intensive but reversible alterations in organs like the brain and kidney, and in heart rate control via Ca2+. This state would prevent degenerative processes due to organ disuse and increase resistance against radiation defects. Neuro-endocrine factors have been identified as main targets to induce torpor although the exact mechanisms are not known yet. The widespread occurrence of torpor in mammals and examples of human hypometabolic states support the idea of human torpor and its beneficial applications in medicine and space exploration.

PlanHab study: assessment of psycho-neuroendocrine function in male subjects during 21 d of normobaric hypoxia and bed rest.

Immobilization and hypoxemia are conditions often seen in patients suffering from severe heart insufficiency or primary pulmonary diseases (e.g. fibrosis, emphysema). In future planned long-duration and exploration class space missions (including habitats on the moon and Mars), healthy individuals will encounter such a combination of reduced physical activity and oxygen tension by way of technical reasons and the reduced gravitational forces. These overall unconventional extraterrestrial conditions can result in yet unknown consequences for the regulation of stress-permissive, psycho-neuroendocrine responses, which warrant appropriate measures in order to mitigate foreseeable risks. The Planetary Habitat Simulation Study (PlanHab) investigated these two space-related conditions: bed rest as model of reduced gravity and normobaric hypoxia, with the aim of examining their influence on psycho-neuroendocrine responses. We hypothesized that both conditions independently increase measures of psychological stress and enhance neuroendocrine markers of stress, and that these effects would be exacerbated by combined treatment. The cross-over study composed of three interventions (NBR, normobaric normoxic horizontal bed rest; HBR, normobaric hypoxic horizontal bed rest; HAMB, normobaric hypoxic ambulatory confinement) with 14 male subjects during three sequential campaigns separated by 4 months. The psychological state was determined through three questionnaires and principal neuroendocrine responses were evaluated by measuring cortisol in saliva, catecholamine in urine, and endocannabinoids in blood. The results revealed no effects after 3 weeks of normobaric hypoxia on psycho-neuroendocrine responses. Conversely, bed rest induced neuroendocrine alterations that were not influenced by hypoxia.

Hyperbaric hyperoxia alters innate immune functional properties during NASA Extreme Environment Mission Operation (NEEMO).

BACKGROUND: Spaceflight is associated with immune dysregulation which is considered as risk factor for the performance of exploration-class missions. Among the consequences of confinement and other environmental factors of living in hostile environments, the role of different oxygen concentrations is of importance as either low (e.g. as considered for lunar or Martian habitats) or high (e.g. during extravehicular activities) can trigger immune dysfunction. The aim of this study was to investigate the impact of increased oxygen availability--generated through hyperbaricity--on innate immune functions in the course of a 14 days NEEMO mission. METHODS: 6 male subjects were included into a 14 days undersea deployment at the Aquarius station (Key Largo, FL, USA). The underwater habitat is located at an operating depth of 47 ft. The 2.5 times higher atmospheric pressure in the habitat leads to hyperoxia. The collection of biological samples occurred 6 days before (L-6), at day 7 (MD7) and 11/13 (MD11/13) during the mission, and 90 days thereafter (R). Blood analyses included differential blood cell count, ex vivo innate immune activation status and inhibitory competences of granulocytes. RESULTS: The absolute leukocyte count showed an increase during deployment as well as the granulocyte and monocyte count. Lymphocyte count was decreased on MD7. The assessments of native adhesion molecules on granulocytes (CD11b, CD62L) indicated a highly significant cellular activation (L-6 vs. MD7/MD13) during mission. In contrast, granulocytes were more sensitive towards anti-inflammatory stimuli (adenosine) on MD13. CONCLUSION: Living in the NEEMO habitat for 14 days induced significant immune alterations as seen by an activation of adhesion molecules and vice versa higher sensitivity towards inhibition. This investigation under hyperbaric hyperoxia is important especially for Astronauts' immune competence during extravehicular activities when exposed to similar conditions.

520-d Isolation and confinement simulating a flight to Mars reveals heightened immune responses and alterations of leukocyte phenotype.

During interplanetary exploration, chronic stress caused by long term isolation and confinement in the spacecraft is one of the major concerns of physical and psychological health of space travelers. And for human on Earth, more and more people live in an isolated condition, which has become a common social problem in modern western society. Collective evidences have indicated prolonged chronic stress could bring big influence to human immune function, which may lead to a variety of health problems. However, to what extent long-term isolation can affect the immune system still remains largely unknow. A simulated 520-d Mars mission provided an extraordinary chance to study the effect of prolonged isolation. Six healthy males participated in this mission and their active neuroendocrine and immune conditions were studied with saliva and blood samples from all participants on chosen time points during the isolation period. As a typical neuroendocrine parameter, stress hormone cortisol was measured in the morning saliva samples. Immune phenotype changes were monitored through peripheral leukocyte phenotype analysis. Using an ex vivo viral infection simulation assay we assessed the immune response changes characterized by the ability to produce representative endogenous pro-inflammatory cytokines. The results of this study revealed elevated cortisol levels, increased lymphocyte amount and heightened immune responses, suggesting that prolonged isolation acting as chronic stressors are able to trigger leukocyte phenotype changes and poorly controlled immune responses.

A corticoid-sensitive cytokine release assay for monitoring stress-mediated immune modulation.

The human immune system is orchestrated in a complex manner and protects the host against invading organisms and controls adequate immune responses to different antigen challenges in an endo-, auto- and paracrine-regulated fashion. The variety and intensity of immune responses are known to be dependent on stress-sensitive neural, humoral and metabolic pathways. The delayed-type hypersensitivity (DTH) skin test was a validated and standardized measure applied in clinical studies to monitor the integral function of cellular immune responses in vivo. The DTH skin test was, however, phased out in 2002. To obtain insight into the mechanisms of stress-sensitive immune reactions, we have developed an alternative in-vitro assay which allows the evaluation of antigen-dependent cellular immune responses triggered by T lymphocytes. The change in the concentration of proinflammatory cytokines in supernatant of the blood-antigen mixture is of particular interest to mirror the degree and adequacy of cellular immune responses. In this study we report that the proinflammatory cytokines interleukin (IL)-2, interferon (IFN)-γ and tumour necrosis factor (TNF)-α show a time-dependent increase upon ex-vivo bacterial, viral and fungal antigen stimulations. Furthermore, evidence is provided that this assay is sensitive to mirror stress hormone-mediated immune modulation in humans as shown either after hydrocortisone injection or after acute stress exposure during free fall in parabolic flight. This in-vitro test appears to be a suitable assay to sensitively mirror stress hormone-dependent inhibition of cellular immune responses in the human. Because of its standardization and relatively simple technical handling, it may also serve as an appropriate research tool in the field of psychoneuroendocrinology in clinical as in field studies.

A new cytokine release assay: a simple approach to monitor the immune status of HIV-infected patients.

PURPOSE: To test a new assay based on an ex vivo cytokine release from whole blood for the monitoring of immune changes in human immunodeficiency virus (HIV)-infected patients. METHODS: A pilot study of outpatients with HIV infection (n = 9) at a large academic hospital who were divided into three groups: HIV-infected patients on highly active antiretroviral therapy (HAART) with a CD4(+) cell count >350/µL (group I) or a CD4(+) cell count <350/µL (group II) and HIV-infected HAART-naïve subjects with a CD4(+) cell count >350/µL (group III). All groups were compared with healthy volunteers (n = 3). The ex vivo cytokine release assay was performed in a three-step process: (1) blood collection, (2) whole-blood ex vivo incubation over 48 h without or with a standard set of well-defined recall antigens as comparable to those used formerly in the skin delayed-type hypersensitivity (DTH) test, (3) cytokine determination from the assay supernatant. RESULTS: Under stimulated conditions, untreated HIV-infected patients with a CD4(+) count >350/µL had similar interleukin-2 (IL-2) levels in the supernatant of the whole-blood incubation to HIV-infected patients on HAART with a low CD4(+) count. Both groups revealed lower IL-2 levels in the supernatant than HIV-infected patients on HAART and with a CD4(+) count >350/µL or healthy volunteers. The determination of interferon-γ and tumour necrosis factor-α in the supernatant showed a similar arrangement of cytokines between groups. CONCLUSIONS: Our results suggest that this cytokine release assay could be a suitable tool to mirror the immunological responsiveness of patients with HIV infection in a gradual manner; further studies are required in order to assess its value in HAART monitoring.

Psychoneuroendocrine alterations during 5 days of head-down tilt bed rest and artificial gravity interventions.

This study aimed to investigate psychological stress and endocrine responses during 5 days of head-down tilt bed rest (HDTBR) with or without the impact of artificial gravity (AG). Participants were assigned to one of three bed-rest-protocols either with (i) no centrifugation, (ii) continuous 30 min (AG1) or (iii) discontinuous 6 × 5 min (AG2) centrifugation periods at 1G in the center of mass periods. Centrifugations were performed daily in one session. Questionnaires for assessing psychological stress and the corresponding biological sample collection were performed before, during and after HDTBR or centrifugation. Overall, questionnaires showed no significant changes of anxiety or emotional stress during HDTBR. In the AG1-group, salivary cortisol levels were significantly higher after centrifugation irrespective of the progress of the HDTBR and day of intervention. The AG2-group showed higher cortisol concentrations after centrifugation only on the first days of head-down tilt but no more on day 5 of HDTBR. During bed rest, urine epinephrine excretion increased in all groups, but showed the highest day concentrations in the AG1-group, which were also significantly higher when compared with AG2. These results indicate that 5 days of HDT alone is not a major stressor and accordingly resulted only in moderate changes of neuroendocrine responses over time. However, daily centrifugation for a continuous duration of 30 min induced a significant neuroendocrine response, which was not subject to a habituation as compared with daily but intermittent centrifugation for 6 × 5 min. Discontinuous centrifugation is better tolerated and associated with lower adrenocortical stress responses during HDTBR.

Effects of exercise stress on the endocannabinoid system in humans under field conditions.

The effects of physical exercise stress on the endocannabinoid system in humans are almost unexplored. In this prospective study, we investigated in a crossover design and under field conditions at different altitudes the effects of physical exercise on the endocannabinoid system (ECS) in 12 trained healthy volunteers. For determination of alterations on the ECS three different protocols were analyzed: Protocol A (physical exercise at lower altitude) involved strenuous hiking below 2,100 m, whereas Protocol B (physical exercise by active ascent to high altitude) involved hiking up to 3,196 m, an accommodation at the cottage and a descent the next day. Protocol C (passive ascent) included a helicopter ascent to 3,196 m, an overnight stay at this altitude and a flight back to the base camp the following day. The cumulative hiked altitude in Protocol A and B was comparable (~1,650 m). The blood EC concentrations of anandamide increased significantly in Protocol A/B from baseline (T0) 0.12 ± 0.01/0.16 ± 0.02 (mean ± SEM) to 0.27 ± 0.02/0.42 ± 0.02 after exercise (T1) (p < 0.05). Anandamide levels in Protocol C remained stable at 0.20 ± 0.02. We conclude that the ECS is activated upon strenuous exercise whereas the combination with hypoxic stress further increases its activity. The reduced partial pressure of oxygen at high altitude alone did not affect this system. In summary, physical exercise activates the endocannabinoid system, whereas the combination with high altitude enhances this activation. This discloses new perspectives to adaptation mechanisms to physical exercise.

Effects of cryopreservation with polyethylene glycol on the expression of CD11b and CD62L on the surface of polymorphonuclear leukocytes.

In experimental and clinical studies, expression of surface adhesion molecules such as ß2-integrine (CD11b) and L-selectin (CD62L) on polymorphonuclear leukocyte (PMNL) are investigated to assess certain crucial innate immune functions. Because the expression of CD11b and CD62L on PMNL can alter they cannot be quantified reliably when the time between blood draw and measurements is prolonged. Goals of this study were to test effects of cryopreservation on the expression of CD11b and CD62L on human PMNLs either under native conditions as well as after stimulation-dependant adhesion molecules´ expression pattern. CD11b and CD62L expression on PMNL can be cryopreserved with 10% of PEG-solution for at least one month at -60 degree C. This was observed in native, unstimulated as well as in stimulated cell-preparations. CD11b is very stable in contrast to CD62L expression which appears to be more susceptible to alteration due to freezing-thawing. However, the relative stimulus-dependant changes of activation can still be reflected.

Parabolic flight primes cytotoxic capabilities of polymorphonuclear leucocytes in humans.

BACKGROUND: Previously performed in vitro studies suggested that gravitational stress may alter functions of immune cells. This study investigated the in vivo effects of parabolic flight manoeuvres as a short-term model of micro- and hypergravity on the cytotoxic and microbicidal polymorphonuclear leucocyte (PMN) functions as the key element of innate immunity. MATERIAL AND METHODS: Twenty-one healthy male volunteers underwent 30 subsequent parabolic flight manoeuvres. Each manoeuvre produced 22-s periods of nearly weightlessness close to <<0g>>, with each parabola starting with a pull-up and ending with a pull-out (hypergravity) at 1.8 g for about 20 s each. Blood samples were drawn 24 h prior to take off (T0), after 25-30 parabolas (T1), and 24 h (T2) and 48 h (T3) after flight for determination of (i) leucocyte number and subpopulations, (ii) PMNs' capabilities to produce hydrogen peroxide (H(2)O(2)) and to adhere and phagocytose particles and (iii) plasma cytokines known to prime PMN functions [interleukin-8 (IL-8), tumour necrosis factor-alpha (TNF-alpha), granulocyte-colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF)]. RESULTS: Parabolic flight induced an increase in leucocyte number with a significant elevation of the PMN fraction. The spontaneous H(2)O(2) production by PMNs did not change; however, the capability of PMNs to produce H(2)O(2) in response to soluble stimuli [N-formyl-methionyl-leucyl-phenylalanine (fMLP), fMLP and TNF-alpha, calcium ionophore (A23187), phorbol myristate acetate (PMA)] was increased. Adhesive and phagocytic properties of PMNs were not altered. Regarding priming cytokines, IL-8 and G-CSF were significantly elevated. CONCLUSIONS: Our data indicate that parabolic flight induces priming of the cytotoxic capabilities of PMNs without affecting microbicidal functions.

Enhanced anandamide plasma levels in patients with complex regional pain syndrome following traumatic injury: a preliminary report.

The complex regional pain syndrome (CRPS) is a disabling neuropathic pain condition that may develop following injuries of the extremities. The pathogenesis of this syndrome is not clear; however, it includes complex interactions between the nervous and the immune system resulting in chronic inflammation, pain and trophic changes. This interaction may be mediated by chronic stress which is thought to activate the endogenous cannabinoid (endocannabinoid) system (ECS). We conducted an open, prospective, comparative clinical study to determine plasma level of the endocannabinoid anandamide by high-performance liquid chromatography and a tandem mass spectrometry system in 10 patients with CRPS type I versus 10 age- and sex-matched healthy controls. As compared to healthy controls, CRPS patients showed significantly higher plasma concentrations of anandamide. These results indicate that the peripheral ECS is activated in CRPS. Further studies are warranted to evaluate the role of the ECS in the limitation of inflammation and pain.

Sex differences in stress and immune responses during confinement in Antarctica.

BACKGROUND: Antarctica challenges human explorers by its extreme environment. The effects of these unique conditions on the human physiology need to be understood to best mitigate health problems in Antarctic expedition crews. Moreover, Antarctica is an adequate Earth-bound analogue for long-term space missions. To date, its effects on human physiology have been studied mainly in male cohorts though more female expeditioners and applicants in astronaut training programs are selected. Therefore, the identification of sex differences in stress and immune reactions are becoming an even more essential aim to provide a more individualized risk management. METHODS: Ten female and 16 male subjects participated in three 1-year expeditions to the German Antarctic Research Station Neumayer III. Blood, saliva, and urine samples were taken 1-2 months prior to departure, subsequently every month during their expedition, and 3-4 months after return from Antarctica. Analyses included cortisol, catecholamine and endocannabinoid measurements; psychological evaluation; differential blood count; and recall antigen- and mitogen-stimulated cytokine profiles. RESULTS: Cortisol showed significantly higher concentrations in females than males during winter whereas no enhanced psychological stress was detected in both sexes. Catecholamine excretion was higher in males than females but never showed significant increases compared to baseline. Endocannabinoids and N-acylethanolamides increased significantly in both sexes and stayed consistently elevated during the confinement. Cytokine profiles after in vitro stimulation revealed no sex differences but resulted in significant time-dependent changes. Hemoglobin and hematocrit were significantly higher in males than females, and hemoglobin increased significantly in both sexes compared to baseline. Platelet counts were significantly higher in females than males. Leukocytes and granulocyte concentrations increased during confinement with a dip for both sexes in winter whereas lymphocytes were significantly elevated in both sexes during the confinement. CONCLUSIONS: The extreme environment of Antarctica seems to trigger some distinct stress and immune responses but-with the exception of cortisol and blood cell counts-without any major relevant sex-specific differences. Stated sex differences were shown to be independent of enhanced psychological stress and seem to be related to the environmental conditions. However, sources and consequences of these sex differences have to be further elucidated.

Cells´ Flow and Immune Cell Priming under alternating g-forces in Parabolic Flight.

Gravitational stress in general and microgravity (µg) in particular are regarded as major stress factors responsible for immune system dysfunction in space. To assess the effects of alternating µg and hypergravity (hyper-g) on immune cells, the attachment of peripheral blood mononuclear cells (PBMCs) to adhesion molecules under flow conditions and the antigen-induced immune activation in whole blood were investigated in parabolic flight (PF). In contrast to hyper-g (1.8 g) and control conditions (1 g), flow and rolling speed of PBMCs were moderately accelerated during µg-periods which were accompanied by a clear reduction in rolling rate. Whole blood analyses revealed a "primed" state of monocytes after PF with potentiated antigen-induced pro-inflammatory cytokine responses. At the same time, concentrations of anti-inflammatory cytokines were increased and monocytes displayed a surface molecule pattern that indicated immunosuppression. The results suggest an immunologic counterbalance to avoid disproportionate immune responses. Understanding the interrelation of immune system impairing and enhancing effects under different gravitational conditions may support the design of countermeasures to mitigate immune deficiencies in space.

Microvascular perfusion measured by orthogonal polarization spectral imaging is well maintained during exposure to high altitude in trained mountaineers.

The objective of this study was to examine microvascular perfusion during hypobaric hypoxia and physical exercise. We used orthogonal polarization spectral imaging for the non-invasive visualization and assessment of the sublingual mucosal microcirculation in twelve healthy altitude acclimatized mountaineers. Red blood cell velocity (RCV), microvascular diameter (Dia), functional capillary density (FCD) and the number of rolling leukocytes were studied at baseline and after (I) a climb to an altitude of 3196 m, (II) a passive ascent to the same altitude by helicopter and (III) an exercise program at an altitude below 2100 m in the European Alps. Exposure to high altitude and exercise resulted in an increased heart rate (Trial I: 64 (54-66) vs. 95 (84-100); median (interquartile range); P<0.05) and decreased oxygen saturation (Trial I: 98 (98-99) vs. 90 (88-92); P<0.05). However, RCV, Dia and FCD did not change significantly. Furthermore, no enhanced rolling of leukocytes in postcapillary venules could be observed (Trial I: 6.2 (4.4-6.8) vs. 7.8 (4.3-6.7)). In the pooled data of all three trials of this study we could show a significant positive correlation between oxygen saturation and red blood cell velocity (r = 0.25; P = 0.02). These results indicate that orthogonal polarization spectral imaging can be a useful tool for the microcirculatory assessment of man under hypoxic conditions. We could show that in trained, acclimatized subjects microvascular perfusion is well maintained during hypobaric hypoxia at an altitude of 3196 m and no evidence for an increased postcapillary leukocyte adhesion was seen.

Early immune anergy towards recall antigens and mitogens in patients at onset of septic shock.

The pathology of sepsis is typically characterized by an infection and excessive initial inflammation including a cytokine storm, followed by a state of immune suppression or paralysis. This classical view of a two peak kinetic immune response is currently controversially discussed. This study was a sub-study of the randomized clinical Trial SISPCT registered with www.clinicaltrials.gov (NCT00832039, Registration date: 29/01/2009). Blood samples from 76 patients with severe sepsis and septic shock were incubated for 48 h at 37 °C in vitro with bacterial or fungal recall-antigens or specific mitogen antigens within 24 hours of sepsis onset. Recall-antigen stimulation led to a severe dampening of normal cytokine release. This immunologic anergy was similarly observed after mitogen stimulation. Moreover, patients under hydrocortisone therapy or with lowered arterial oxygen tension had further reductions in cytokine levels upon B- and T-cell mitogen stimulation. This investigation reveals an early onset of immunoparalysis during sepsis. This immune incompetence in mounting an adequate response to further infections includes previously sensitized pathogens, as seen with recall-antigens. Also, the immune-suppressive role of hydrocortisone and low PaO2 is highlighted. Aside from early broad-spectrum antimicrobial therapy, our findings reinforce the need for maximal immunological support and protection against further infections at the onset of sepsis.

PlanHab Study: Consequences of combined normobaric hypoxia and bed rest on adenosine kinetics.

Adenosine plays a role in the energy supply of cells and provokes differential, hormone-like functions in circulating cells and various tissues. Its release is importantly regulated by oxygen tension. This renders adenosine and its kinetics interesting to investigate in humans subjected to low oxygen conditions. Especially for space exploration scenarios, hypoxic conditions - together with reduced gravity - represent two foreseen living conditions when planning manned long-duration space missions or planetary habitats. The PlanHab study investigated microgravity through inactivity in bed rest and normobaric hypoxia to examine their independent or combined effect on adenosine and its kinetics. Healthy male subjects (n = 14) completed three 21-day interventions: hypoxic bed rest (HBR); hypoxic ambulatory confinement (HAMB); normoxic bed rest (NBR). The interventions were separated by 4 months. Our hypothesis of a hypoxia-triggered increase in adenosine was confirmed in HAMB but unexpectedly also in NBR. However, the highest adenosine levels were noted following HBR. Furthermore, the percentage of hemolysis was elevated in HBR whereas endothelial integrity markers stayed low in all three interventions. In summary, these data suggest that neocytolysis accounts for these effects while we could reduce evidence for microcirculatory changes.

Effects of isolation and confinement on humans-implications for manned space explorations.

Human psychology and physiology are significantly altered by isolation and confinement. In light of planned exploration class interplanetary missions, the related adverse effects on the human body need to be explored and defined as they have a large impact on a mission's success. Terrestrial space analogs offer an excellent controlled environment to study some of these stressors during a space mission in isolation without the complex environment of the International Space Station. Participants subjected to these space analog conditions can encounter typical symptoms ranging from neurocognitive changes, fatigue, misaligned circadian rhythm, sleep disorders, altered stress hormone levels, and immune modulatory changes. This review focuses on both the psychological and the physiological responses observed in participants of long-duration spaceflight analog studies, such as Mars500 or Antarctic winter-over. They provide important insight into similarities and differences encountered in each simulated setting. The identification of adverse effects from confinement allows not only the crew to better prepare for but also to design feasible countermeasures that will help support space travelers during exploration class missions in the future.

Effect of adenosine on the expression of beta(2) integrins and L-selectin of human polymorphonuclear leukocytes in vitro.

Adenosine has been shown to inhibit the adhesion of polymorphonuclear leukocytes (PMNL) to the vascular endothelium. Because the underlying molecular mechanisms have not been fully understood, the present study characterizes the effect of adenosine on the expression of adhesion molecules of human PMNL. When PMNL were activated by N-formyl-methionyl-leucyl-phenylalanine the number of cell surface beta2 integrins increased fivefold, whereas L-selectin molecules were completely shed. Activation-dependent numerical up-regulation Of beta2 integrins and shedding of L-selectin were inhibited by exogenously applied adenosine receptor agonists in a concentration-dependent fashion. The rank order of potencies of adenosine receptor agonists, measured by the agonists' half-maximal inhibitory concentrations, revealed that adenosine inhibited the numerical up-regulation of beta2 integrins and shedding of L-selectin most likely via an A2(a) receptor site. When extracellular concentrations of endogenously formed adenosine were enhanced by the nucleoside uptake inhibitor dipyridamole, up-regulation of beta2 integrins, and shedding of L-selectin was again inhibited. Both effects were reversed by the enzyme adenosine deaminase, which degrades active adenosine to inactive inosine, suggesting that endogenously formed adenosine may play an important role in the regulation of beta2 integrins and L-selectin of human PMNL.

Effects of hypertonic saline on expression of human polymorphonuclear leukocyte adhesion molecules.

Hypertonic saline prevents vascular adherence of neutrophils and ameliorates ischemic tissue injury. We hypothesized that hypertonic saline attenuates N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated expression of adhesion molecules on human polymorphonuclear leukocytes (PMNLs). fMLP-stimulated up-regulation of beta2-integrins was diminished by hypertonic saline but not by hypertonic choline chloride-, mannitol-, or sucrose-modified Hanks' buffered salt solution. Shedding of L-selectin was decreased by hypertonic saline and choline chloride but not by hypertonic mannitol or sucrose. When the effects of hypertonic sodium chloride- and choline chloride-modified media were compared, neither solution affected fMLP-receptor binding but both equally inhibited fMLP-stimulated increase in intracellular calcium, ionophore A23187, and phorbol myristate acetate (PMA)-stimulated numerical up-regulation of beta2-integrins. Analysis of mitogen-activated protein (MAP) kinases p38 and p44/42 for phosphorylation revealed that hypertonic solutions did not differ in preventing fMLP-stimulated increases in phospho-p38 and phospho-p44/42. Resting PMNLs shrunk by hypertonic saline increased their volume during incubation and further during chemotactic stimulation. Addition of amiloride further enhanced inhibition of up-regulation of beta2-integrins. No fMLP-stimulated volume changes occurred in PMNLs exposed to hypertonic choline chloride, resulting in significant cell shrinkage. Results suggest a sodium-specific inhibitory effect on up-regulation of beta2-integrins of fMLP-stimulated PMNLs, which is unlikely to be caused by alterations of fMLP receptor binding, decrease in cytosolic calcium, attenuation of calcium or protein kinase C-dependent pathways, suppression of p38- or p44/42 MAP kinase-dependent pathways, or cellular ability to increase or decrease volumes.