Varicella-zoster DNA in saliva of patients with meningoencephalitis: a preliminary study.

OBJECTIVES: Since the routine use of polymerase chain reaction testing (PCR) in diagnosing herpes infections, varicella-zoster virus is increasingly recognized as a cause of varicella-zoster meningoencephalitis (VZV ME) among immunocompetent patients. We were interested to determine whether patients with VZV ME had VZV DNA in their saliva during the acute phase of the illness. MATERIALS AND METHODS: Forty-five consecutive patients who underwent a lumbar puncture for diagnostic purposes were included in the study. The cerebrospinal fluid was examined for the presence of VZV DNA by PCR, and patients with positive findings were treated with acyclovir. The saliva was later analyzed in a blinded fashion for the presence of VZV DNA. RESULTS: VZV DNA was found in saliva in four of five (80%) patients with PCR confirmed VZV ME (sensitivity 0.8, specificity 0.84, and likelihood ratio 5). This was significantly more than in patients with non-zoster viral ME (0%, P = 0.009), parainfectious headache (12%, P = 0.03) and controls (9.5%, P = 0.007). In immunocompromised patients with systemic lymphoma and AIDS, VZV DNA was present at a similar rate (67%, P = 0.6). CONCLUSIONS: We have found VZV DNA in saliva of patients with PCR confirmed VZV ME at a higher proportion than in controls and patients with non-VZV viral ME. This finding might be of clinical importance, especially in immunocompetent individuals with suspected VZV ME where the results of genetic and immunological testing are not conclusive.

Multiple latent viruses reactivate in astronauts during Space Shuttle missions.

Latent virus reactivation and diurnal salivary cortisol and dehydroepiandrosterone were measured prospectively in 17 astronauts (16 male and 1 female) before, during, and after short-duration (12-16 days) Space Shuttle missions. Blood, urine, and saliva samples were collected during each of these phases. Antiviral antibodies and viral load (DNA) were measured for Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and cytomegalovirus (CMV). Three astronauts did not shed any virus in any of their samples collected before, during, or after flight. EBV was shed in the saliva in all of the remaining 14 astronauts during all 3 phases of flight. Seven of the 14 EBV-shedding subjects also shed VZV during and after the flight in their saliva samples, and 8 of 14 EBV-shedders also shed CMV in their urine samples before, during, and after flight. In 6 of 14 crewmembers, all 3 target viruses were shed during one or more flight phases. Both EBV and VZV DNA copies were elevated during the flight phase relative to preflight or post-flight levels. EBV DNA in peripheral blood was increased preflight relative to post-flight. Eighteen healthy controls were also included in the study. Approximately 2-5% of controls shed EBV while none shed VZV or CMV. Salivary cortisol measured preflight and during flight were elevated relative to post-flight. In contrast DHEA decreased during the flight phase relative to both preflight and post-flight. As a consequence, the molar ratio of the area under the diurnal curve of cortisol to DHEA with respect to ground (AUCg) increased significantly during flight. This ratio was unrelated to viral shedding. In summary, three herpes viruses can reactivate individually or in combination during spaceflight.

Reactivation of latent viruses is associated with increased plasma cytokines in astronauts.

Success of long duration space missions will depend upon robust immunity. Decreased immunity has been observed in astronauts during short duration missions, as evident by the reactivation of latent herpes viruses. Seventeen astronauts were studied for reactivation and shedding of latent herpes viruses before, during, and after 9-14 days of 8 spaceflights. Blood, urine, and saliva samples were collected 10 days before the flight (L-10), during the flight (saliva only), 2-3h after landing (R+0), 3 days after landing (R+3), and 120 days after landing (R+120). Values at R+120 were used as baseline levels. No shedding of viruses occurred before flight, but 9 of the 17 (designated "virus shedders") shed at least one or more viruses during and after flight. The remaining 8 astronauts did not shed any of the 3 target viruses (non-virus shedders). Virus-shedders showed elevations in 10 plasma cytokines (IL-1α, IL-6, IL-8, IFNγ, IL-4, IL-10, IL-12, IL-13, eotaxin, and IP-10) at R+0 over baseline values. Only IL-4 and IP-10 were elevated in plasma of non-virus shedders. In virus shedders, plasma IL-4 (a Th2 cytokine) was elevated 21-fold at R+0, whereas IFNγ (a Th1 cytokine) was elevated only 2-fold indicating a Th2 shift. The inflammatory cytokine IL-6 was elevated 33-fold at R+0. In non-shedding astronauts at R+0, only IL-4 and IP-10 levels were elevated over baseline values. Elevated cytokines began returning to normal by R+3, and by R+120 all except IL-4 had returned to baseline values. These data show an association between elevated plasma cytokines and increased viral reactivation in astronauts.

Space flight alters bacterial gene expression and virulence and reveals a role for global regulator Hfq.

A comprehensive analysis of both the molecular genetic and phenotypic responses of any organism to the space flight environment has never been accomplished because of significant technological and logistical hurdles. Moreover, the effects of space flight on microbial pathogenicity and associated infectious disease risks have not been studied. The bacterial pathogen Salmonella typhimurium was grown aboard Space Shuttle mission STS-115 and compared with identical ground control cultures. Global microarray and proteomic analyses revealed that 167 transcripts and 73 proteins changed expression with the conserved RNA-binding protein Hfq identified as a likely global regulator involved in the response to this environment. Hfq involvement was confirmed with a ground-based microgravity culture model. Space flight samples exhibited enhanced virulence in a murine infection model and extracellular matrix accumulation consistent with a biofilm. Strategies to target Hfq and related regulators could potentially decrease infectious disease risks during space flight missions and provide novel therapeutic options on Earth.

Three-dimensional growth of extravillous cytotrophoblasts promotes differentiation and invasion.

Human trophoblast research relies on a combination of in vitro models, including isolated primary cultures, explant cultures, and trophoblast cell lines. In the present study, we have utilized the rotating wall vessel (RWV) bioreactor to generate a three-dimensional (3-D) model of human placentation for the study of cytotrophoblast (CTB) invasion. The RWV supported the growth of the human CTB cell line SGHPL-4 and allowed for the formation of complex, multilayered 3-D aggregates that were morphologically, phenotypically, and functionally distinct from SGHPL-4 monolayers. The cells cultured three-dimensionally differentiated into an aggressively invasive cell population characterized by the upregulation of matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-9 and urokinase-type plasminogen activator (uPA) secretion and activation. Microarray analysis of the 3-D and 2-D cultured cells revealed increased expression in the 3-D cells of various genes that are known mediators of invasion, including MT1-MMP, PECAM-1 and L-selectin, as well as genes not previously associated with CTB differentiation such as MMP-13 and MT5-MMP. These results were verified by quantitative real-time PCR. These findings suggest that when cultured in 3-D, SGHPL-4 cells closely mimic differentiating in utero CTBs, providing a novel approach for the in vitro study of the molecular mechanisms that regulate CTB differentiation and invasion.

Epstein-Barr virus shedding by astronauts during space flight.

Patterns of Epstein-Barr virus (EBV) reactivation in 32 astronauts and 18 healthy age-matched control subjects were characterized by quantifying EBV shedding. Saliva samples were collected from astronauts before, during, and after 10 space shuttle missions of 5-14 days duration. At one time point or another, EBV was detected in saliva from each of the astronauts. Of 1398 saliva specimens from 32 astronauts, polymerase chain reaction analysis showed that 314 (23%) were positive for EBV DNA. Examination by flight phase showed that 29% of the saliva specimens collected from 28 astronauts before flight were positive for EBV DNA, as were 16% of those collected from 25 astronauts during flight and 16% of those collected after flight from 23 astronauts. The mean number of EBV copies from samples taken during the flights was 417 per mL, significantly greater (p<.05) than the number of viral copies from the preflight (40) and postflight (44) phases. In contrast, the control subjects shed EBV DNA with a frequency of 3.7% and mean number of EBV copies of 40 per mL of saliva. Ten days before flight and on landing day, titers of antibody to EBV viral capsid antigen were significantly (p<.05) greater than baseline levels. On landing day, urinary levels of cortisol and catecholamines were greater than their preflight values. In a limited study (n=5), plasma levels of substance P and other neuropeptides were also greater on landing day. Increases in the number of viral copies and in the amount of EBV-specific antibody were consistent with EBV reactivation before, during, and after space flight.

A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.

Microbial characterization during the early habitation of the International Space Station.

An evaluation of the microbiota from air, water, and surface samples provided a baseline of microbial characterization onboard the International Space Station (ISS) to gain insight into bacterial and fungal contamination during the initial stages of construction and habitation. Using 16S genetic sequencing and rep-PCR, 63 bacterial strains were isolated for identification and fingerprinted for microbial tracking. Of the bacterial strains that were isolated and fingerprinted, 19 displayed similarity to each other. The use of these molecular tools allowed for the identification of bacteria not previously identified using automated biochemical analysis and provided a clear indication of the source of several ISS contaminants. Strains of Bradyrhizobium and Sphingomonas unable to be identified using sequencing were identified by comparison of rep-PCR DNA fingerprints. Distinct DNA fingerprints for several strains of Methylobacterium provided a clear indication of the source of an ISS water supply contaminant. Fungal and bacterial data acquired during monitoring do not suggest there is a current microbial hazard to the spacecraft, nor does any trend indicate a potential health risk. Previous spacecraft environmental analysis indicated that microbial contamination will increase with time and will require continued surveillance.

Microbial characterization of free floating condensate aboard the Mir space station.

Three samples of humidity condensate that had accumulated behind panels aboard the Russian space station Mir were collected and returned to earth for analysis. As these floating masses of liquid come into contact with the astronauts and the engineering systems, they have the potential to affect both crew health and systems performance. Using a combination of culturing techniques, a wide variety of organisms were isolated included Escherichia coli, Serratia marcescens, and a presumed Legionella species. In addition, microscopic analysis indicated the presence of protozoa, dust mites, and spirochetes. These findings suggest the need for more comprehensive microbial analysis of the environment through the use of new methodologies to allow a more thorough risk assessment of spacecraft.

Elevated stress hormone levels relate to Epstein-Barr virus reactivation in astronauts.

OBJECTIVE: The objective of this study was to determine the effects of stress and spaceflight on levels of neuroendocrine hormones and Epstein-Barr virus (EBV)-specific antibodies in astronauts. METHODS: Antiviral antibody titers and stress hormones were measured in plasma samples collected from 28 astronauts at their annual medical exam (baseline), 10 days before launch (L-10), landing day (R+0), and 3 days after landing (R+3). Urinary stress hormones were also measured at L-10 and R+0. RESULTS: Significant increases (p <.01) in EBV virus capsid antigen antibodies were found at all three time points (L-10, R+0, and R+3) as compared with baseline samples. Anti-EBV nuclear antigen antibodies were significantly decreased at L-10 (p <.05) and continued to decrease after spaceflight (R+0 and R+3, p <.01). No changes were found in antibodies to the nonlatent measles virus. The 11 astronauts who showed evidence of EBV reactivation had significant increases in urinary epinephrine and norepinephrine as compared with astronauts without EBV reactivation. CONCLUSION: These findings indicate that physical and psychological stresses associated with spaceflight resulted in decreased virus-specific T-cell immunity and reactivation of EBV.

Immune responses and latent herpesvirus reactivation in spaceflight.

BACKGROUND: Increased frequency and severity of herpesvirus infections are common in individuals with impaired cellular immunity, a phenomenon observed in both the elderly and astronauts alike. This study investigated immune responses and latent herpesvirus reactivation during a 9-d spaceflight. In addition, adrenocortical and immune responses of an elderly astronaut (payload specialist-2, PS2; age 77) who flew on this mission were compared with that of younger crewmembers. HYPOTHESIS: Spaceflight and associated stresses will decrease cellular immunity and reactivate latent herpesviruses. METHODS: Blood and urine samples, collected from the seven crewmembers who flew on the Space Shuttle Discovery (STS-95), were analyzed for levels of neuroendocrine hormones, leukocyte and lymphocyte subsets, and evidence of herpes-virus reactivation. RESULTS: Prior to flight, increased antibody titers to latent Epstein-Barr virus were found. During flight, acute changes in dehydroepiandrosterone sulfate (DHEAS) and cortisol resulted in a pronounced decrease in the DHEAS/cortisol ratio by the end of the mission for PS2 and a younger crewmember. Shedding of cytomegalovirus (CMV) in urine and increased CMV antibody titers also occurred inflight. At landing, the percent increases in adrenocorticotropic hormone and cortisol were greatest for PS2 as compared with the other six crewmembers. A significant neutrophilia also was observed in all crewmembers. Notably, PS2 had large increases in monocytes and natural killer cells at landing while other crewmembers showed little change or a decrease. CONCLUSIONS: These findings indicate that spaceflight and associated stresses reactivate latent herpesviruses and suggest that acute changes in neuroendocrine hormones mediate these changes in part by downregulating cellular immunity. Moreover, the similarities between aging and spaceflight suggest that the study of the immune system in elderly subjects may be useful as a predictive model for astronauts enduring long-term spaceflights.

Three-dimensional tissue assemblies: novel models for the study of Salmonella enterica serovar Typhimurium pathogenesis.

The lack of readily available experimental systems has limited knowledge pertaining to the development of Salmonella-induced gastroenteritis and diarrheal disease in humans. We used a novel low-shear stress cell culture system developed at the National Aeronautics and Space Administration in conjunction with cultivation of three-dimensional (3-D) aggregates of human intestinal tissue to study the infectivity of Salmonella enterica serovar Typhimurium for human intestinal epithelium. Immunohistochemical characterization and microscopic analysis of 3-D aggregates of the human intestinal epithelial cell line Int-407 revealed that the 3-D cells more accurately modeled human in vivo differentiated tissues than did conventional monolayer cultures of the same cells. Results from infectivity studies showed that Salmonella established infection of the 3-D cells in a much different manner than that observed for monolayers. Following the same time course of infection with Salmonella, 3-D Int-407 cells displayed minimal loss of structural integrity compared to that of Int-407 monolayers. Furthermore, Salmonella exhibited significantly lower abilities to adhere to, invade, and induce apoptosis of 3-D Int-407 cells than it did for infected Int-407 monolayers. Analysis of cytokine expression profiles of 3-D Int-407 cells and monolayers following infection with Salmonella revealed significant differences in expression of interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, IL-1Ra, and tumor necrosis factor alpha mRNAs between the two cultures. In addition, uninfected 3-D Int-407 cells constitutively expressed higher levels of transforming growth factor beta1 mRNA and prostaglandin E2 than did uninfected Int-407 monolayers. By more accurately modeling many aspects of human in vivo tissues, the 3-D intestinal cell model generated in this study offers a novel approach for studying microbial infectivity from the perspective of the host-pathogen interaction.

Decreased non-MHC-restricted (CD56+) killer cell cytotoxicity after spaceflight.

Cytotoxic activity of non-major histocompatibility complex-restricted (CD56+) (NMHC) killer cells and cell surface marker expression of peripheral blood mononuclear cells were determined before and after spaceflight. Ten astronauts (9 men, 1 woman) from two space shuttle missions (9- and 10-day duration) participated in the study. Blood samples were collected 10 days before launch, within 3 h after landing, and 3 days after landing. All peripheral blood mononuclear cell preparations were cryopreserved and analyzed simultaneously in a 4-h cytotoxicity (51)Cr release assay using K562 target cells. NMHC killer cell lytic activity was normalized per 1,000 CD56+ cells. When all 10 subjects were considered as one study group, NMHC killer cell numbers did not change significantly during the three sampling periods, but at landing lytic activity had decreased by approximately 40% (P < 0.05) from preflight values. Nine of ten astronauts had decreased lytic activity immediately after flight. NMHC killer cell cytotoxicity of only three astronauts returned toward preflight values by 3 days after landing. Consistent with decreased NMHC killer cell cytotoxicity, urinary cortisol significantly increased after landing compared with preflight levels. Plasma cortisol and ACTH levels at landing were not significantly different from preflight values. No correlation of changes in NMHC killer cell function or hormone levels with factors such as age, gender, mission, or spaceflight experience was found. After landing, expression of the major lymphocyte surface markers (CD3, CD4, CD8, CD14, CD16, CD56), as determined by flow cytometric analysis, did not show any consistent changes from measurements made before flight.

Shear stress enhances microcin B17 production in a rotating wall bioreactor, but ethanol stress does not.

Stress, including that caused by ethanol, has been shown to induce or promote secondary metabolism in a number of microbial systems. Rotating-wall bioreactors provide a low stress and simulated microgravity environment which, however, supports only poor production of microcin B17 by Escherichia coli ZK650, as compared to production in agitated flasks. We wondered whether the poor production is due to the low level of stress and whether increasing stress in the bioreactors would raise the amount of microcin B17 formed. We found that applying shear stress by addition of a single Teflon bead to a rotating wall bioreactor improved microcin B17 production. By contrast, addition of various concentrations of ethanol to such bioreactors (or to shaken flasks) failed to increase microcin B17 production. Ethanol stress merely decreased production and, at higher concentrations, inhibited growth. Interestingly, cells growing in the bioreactor were much more resistant to the growth-inhibitory and production-inhibitory effects of ethanol than cells growing in shaken flasks.

Characteristics of human dendritic cells generated in a microgravity analog culture system.

Generation of an effective immune response requires that antigens be processed and presented to T lymphocytes by antigen-presenting cells, the most efficient of which are dendritic cells (DC). Because of their influence on both the innate and the acquired arms of immunity, a defect in DC would be expected to result in a broad impairment of immune function, not unlike that observed in astronauts during or after space flight. In the study reported here, we investigated whether DC generation and function are altered in a culture environment that models microgravity, i.e., the rotary-cell culture system (RCCS). We observed that RCCS supported the generation of DC identified by morphology, phenotype (HLA-DR+ and lacking lineage-associated markers), and function (high allostimulatory activity). However, the yield of DC from RCCS was significantly lower than that from static cultures. RCCS-generated DC were less able to phagocytose Aspergillus fumigatus conidia and expressed a lower density of surface HLA-DR. The proportion of DC expressing CD80 was also significantly reduced in RCCS compared to static cultures. When exposed to fungal antigens, RCCS-generated DC produced lower levels of interleukin-12 and failed to upregulate some costimulatory/adhesion molecules involved in antigen presentation. These data suggest that DC generation, and some functions needed to mount an effective immune response to pathogens, may be disturbed in the microgravity environment of space.

The release of alginate lyase from growing Pseudomonas syringae pathovar phaseolicola.

Pseudomonas syringae pathovar phaseolicola, which produces alginate during stationary growth phase, displayed elevated extracellular alginate lyase activity during both mid-exponential and late-stationary growth phases of batch growth. Intracellular activity remained below 22% of the total activity during exponential growth, suggesting that alginate lyase has an extracellular function for this organism. Extracellular enzyme activity in continuous cultures, grown in either nutrient broth or glucose-simple salts medium, peaked at 60% of the washout rate, although nutrient broth-grown cultures displayed more than twice the activity per gram of cell mass. These results imply that growth rate, nutritional composition, or both initiate a release of alginate lyase from viable P. syringae pv. phaseolicola, which could modify its entrapping biofilm.

Routine detection of Epstein-Barr virus specific T-cells in the peripheral blood by flow cytometry.

The ability to detect cytomegalovirus-specific T-cells (CD4(+)) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein-Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45(+), CD3(+), CD4(+), CD45RA(-), CD69(+), CD25(-). This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4(+) T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNgamma/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactivation.

Microbial contamination of spacecraft.

Spacecraft and space habitats supporting human exploration contain a diverse population of microorganisms. Microorganisms may threaten human habitation in many ways that directly or indirectly impact the health, safety, or performance of astronauts. The ability to produce and maintain spacecraft and space stations with environments suitable for human habitation has been established over 40 years of human space flight. An extensive database of environmental microbiological parameters has been provided for short-term (< 20 days) space flight by more than 100 missions aboard the Space Shuttle. The NASA Mir Program provided similar data for long-duration missions. Interestingly, the major bacterial and fungal species found in the Space Shuttle are similar to those encountered in the nearly 15-year-old Mir. Lessons learned from both the US and Russian space programs have been incorporated into the habitability plan for the International Space Station. The focus is on preventive measures developed for spacecraft, cargo, and crews. On-orbit regular housekeeping practices complete with visual inspections are essential, along with microbiological monitoring. Risks associated with extended stays on the Moon or a Mars exploration mission will be much greater than previous experiences because of additional unknown variables. The current knowledge base is insufficient for exploration missions, and research is essential to understand the effects of space flight on biological functions and population dynamics of microorganisms in spacecraft. Equally important is a better understanding of the immune response and of human-microorganism-environment interactions during long-term space habitation.

Reactivation and shedding of cytomegalovirus in astronauts during spaceflight.

The reactivation of cytomegalovirus (CMV) in 71 astronauts was investigated, using polymerase chain reaction. A significantly greater (P<.0001) shedding frequency was found in urine samples from astronauts before spaceflight (10.6%) than in urine from the healthy control subject group (1.2%). Two of 4 astronauts studied during spaceflight shed CMV in urine. A significant increase (P<.0001) in CMV antibody titer, compared with baseline values, was also found 10 days before spaceflight. CMV antibody titer was further increased (P<.001) 3 days after landing, compared with 10 days before the mission. Significant increases in stress hormones were also found after landing. These results demonstrate that CMV reactivation occurred in astronauts before spaceflight and indicate that CMV may further reactivate during spaceflight.

Growth of Steptomyces hygroscopicus in rotating-wall bioreactor under simulated microgravity inhibits rapamycin production.

Growth of Streptomyces hygroscopicus under conditions of simulated microgravity in a rotating-wall bioreactor resulted in a pellet form of growth, lowered dry cell weight, and inhibition of rapamycin production. With the addition of Teflon beads to the bioreactor, growth became much less pelleted, dry cell weight increased but rapamycin production was still markedly inhibited. Growth under simulated microgravity favored extracellular production of rapamycin, in contrast to a greater percentage of cell-bound rapamycin observed under normal gravity conditions.