Saliva Fatigue Biomarker Index As a Marker for Severe Myalgic Encephalomyelitis/Chronic Fatigue Syndrome in a Community Based Sample.

OBJECTIVE: The prevalence of pediatric Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) has been estimated from an ethnically and sociodemographically diverse community-based random sample of 10,119 youth aged 5-17. A team of physicians made a final diagnosis of ME/CFS if the participants met criteria for up to three selected case definitions following medical and psychiatric evaluations. We assessed whether a salivary biomarker of fatigue could identify youth with ME/CFS. STUDY DESIGN: We examined the ratio of the concentrations of 2 peptide fragments in saliva, referred to as the Fatigue Biomarker Index (FBI), in participants from our study diagnosed with ME/CFS (n=59) and matched controls (n=39). RESULTS: Significant overall differences were found in the FBI between those participants with severe ME/CFS and those with ME/CFS and the controls. CONCLUSIONS: If confirmed in other populations, the FBI could serve as an objective test to aid in the diagnosis of severe ME/CFS.

Increased circulating stem cells and better cognitive performance in traumatic brain injury subjects following hyperbaric oxygen therapy.

Traumatic brain injury (TBI) may cause persistent cognitive dysfunction. A pilot clinical study was performed to determine if hyperbaric oxygen (HBO2) treatment improves cognitive performance. It was hypothesized that stem cells, mobilized by HBO2 treatment, are recruited to repair damaged neuronal tissue. This hypothesis was tested by measuring the relative abundance of stem cells in peripheral blood and cognitive performance during this clinical trial. The subject population consisted of 28 subjects with persistent cognitive impairment caused by mild to moderate TBI suffered during military deployment to Iraq or Afghanistan. Fluorescence-activated cell sorting (FACS) analysis was performed for stem cell markers in peripheral blood and correlated with variables resulting from standard tests of cognitive performance and post-traumatic stress disorder: ImPACT, BrainCheckers and PCL-M test results. HBO2 treatment correlated with stem cell mobilization as well as increased cognitive performance. Together these results support the hypothesis that stem cell mobilization may be required for cognitive improvement in this population.

Salivary biomarkers of physical fatigue as markers of sleep deprivation.

STUDY OBJECTIVE: Determine whether a salivary biomarker of physical fatigue, referred to as the fatigue biomarker index (FBI), can discriminate a control group from a sleep deprived group when saliva is collected under controlled conditions. The study expands on previous work examining changes in the composition of saliva during periods of prolonged exercise. METHODS: Thirty (30) young adults (14 Control [CON]; 16 Sleep Deprived [SDEP]) were monitored for mood state (Profile of Mood States [POMS]), cognitive performance (Stroop Color-Conflict Tests), and salivary biomarkers of physical fatigue over a 48-h period with sampling at 3-h intervals. Trials lasted from 06:00 on day 1 (time = -3 h) to 09:00 on day 3 (time = 48 h). Levels of salivary biomarkers were calculated from liquid chromatography-mass spectrometry (LC-MS) data. Statistical comparisons were made using Wilcoxon rank sum tests with a Bonferroni correction to limit type 1 error. Receiver-operator characteristic (ROC) analysis was used to evaluate the ability of the various parameters to distinguish the SDEP population from the CON population. RESULTS: Longitudinal analysis demonstrated significant between-group differences in all three parameters. ROC analysis demonstrated that cognitive performance tests and salivary biomarkers of physical fatigue distinguish the SDEP population from the CON population. CONCLUSIONS: A previously identified salivary biomarker of physical fatigue may provide an alternative method for discriminating sleep deprived from rested individuals. The salivary biomarker of physical fatigue holds promise as an objective measure of sleep deprivation, perhaps eventually removing the reliance on self-reported sleep diaries and/or repeated polysomnographs for longitudinal tracking of sleep quality and/or diagnosis of sleep disorders.

Fatigue biomarker index: an objective salivary measure of fatigue level.

Fatigue changed the composition of the small-molecular weight (sMW) proteome of saliva during a 10h session of moderate (70% of maximum ventilatory threshold) physical exertion. Saliva samples were collected from nine recreationally trained cyclists participating in a cross-over study designed to simulate prolonged manual labor, a military operation or wildfire-suppression work. During each hour of the study, participants performed an exercise program that included upper and lower body exercises separated by short periods of recovery. Over the course of the study, fatigue level increased as suggested by a significant increase in the participants' relative perceived exertion. The composition of the sMW proteome was investigated using reversed-phase liquid chromatography with mass-spectrometric detection. Isotopes of acetic anhydride were used for mass-specific labeling of samples and subsequent identification of ions with significant changes in intensity. Cluster analysis was used to identify a pair of peptides with concentrations that changed in opposite directions with fatigue level, i.e. concentration of one peptide increased while concentration of the other decreased. The sequences of the two peptides were determined by high-resolution mass spectrometry. The ratio of the ion intensities of these two peptides, referred to as the fatigue biomarker index, was calculated for subjects throughout the study. The FBI values from the start of the study likely arose from a different distribution than the FBI values measured at the end of the study (Mann-Whitney test, P<.05). While this study is restricted to a small population of recreationally trained cyclists performing exercise under controlled conditions, it holds promise for the development of an objective salivary measurement of fatigue that is applicable to a much broader population performing in uncontrolled environments.

Hyperbaric oxygen therapy in a mouse model of implant-associated osteomyelitis.

Implant associated osteomyelitis (OM) is difficult to treat with antibiotics, and outcomes remain poor. Some reports suggest that hyperbaric oxygen treatment is a safe and effective means of treating OM. We tested this hypothesis in a murine model. Clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, and Klebsiella pneumoniae were used. The mice were infected with each of the three pathogens, treated with 100% oxygen at high pressure, hyperbaric oxygen (HBO), and monitored for the ability of HBO to prevent and/or clear the OM infection. Assessments included bacterial burden of the tibias and lesion scores, as well as receptor activator of NF-κB ligand (RANKL) and myeloperoxidase (MPO) concentrations. HBO resulted in more severe lesion scores and higher RANKL and MPO concentrations for MRSA. A significant positive correlation was found between RANKL concentration and lesion score. No significant difference was found with HBO in P. aeruginosa infections and K. pneumoniae seems to either not infect bone well or get cleared before establishing an infection. The model is useful for studying OM infections caused by MRSA and P. aeruginosa, but HBO does not appear to be an efficacious treatment of an implant-associated OM infection.

Linear mixed-effects modeling of the relationship between heart rate variability and fatigue arising from sleep deprivation.

INTRODUCTION: Fatigue degrades cognitive performance, yet there is no universally accepted objective measure of fatigue. We tested whether fatigue arising from sleep deprivation can be quantified objectively using heart rate variability (HRV). METHODS: There were 35 male subjects (mean +/- SD; age = 21.4 +/- 2.6 yr) who were assigned to one of two experimental groups: (1) control (N = 16), or (2) 48-h sleep-deprived (N=19). Using 3-h sampling intervals, we simultaneously tracked fatigue level, cognitive performance, and HRV. Linear mixed-effects (LME) models were used to evaluate linear relationships between fatigue level and cognitive performance, as well as between fatigue level and HRV. RESULTS: Significant negative slopes were observed in LME models of cognitive performance and fatigue level. Of the several HRV parameters examined during standing and supine rest, the ratio of low-frequency to high-frequency R-R interval in the supine position had the clearest significant relationship when modeled against fatigue level. DISCUSSION: In summary, our results suggest that HRV tracks fatigue arising from sleep deprivation. This noninvasive, objective tool can quantify fatigue in real time.

Laboratory evaluation of 10 heat and moisture exchangers using simulated aeromedical evacuation conditions.

Heat and moisture exchangers (HMEs) are used for airway humidification in mechanically ventilated patients and have been evaluated only under hospital conditions. U.S. Air Force aeromedical evacuation transports are performed under rugged conditions further complicated by the cold and dry environment in military aircrafts, and HMEs are used to provide airway humidification for patients. This study evaluated 10 commercial HMEs using a test system that simulated aeromedical evacuation conditions. Although the American National Standards Institute recommends inspired air to be at an absolute humidity value of > or = 30 mg/L for mechanically ventilated patients, the highest absolute humidity by any HME was approximately 20 mg/L. Although none of the HMEs were able to maintain a temperature high enough to achieve the humidity standard of the American National Standards Institute, the clinical significance of this standard may be less important than the relative humidity maintained in the respired air, especially on evacuation flights of short duration.

Predicting success in the tactical air combat party training pipeline.

OBJECTIVES: To develop a statistical model that predicts the likelihood of success or failure of military training candidates using tests administered before initial skill training as inputs. METHODS: Data were acquired from candidates before the start of U.S. Air Force Tactical Air Control Party training, including (1) demographic, (2) psychological composition evaluated using Emotional Quotient Inventory, (3) physical performance capability, (4) a physical activity questionnaire, and (5) salivary fatigue biomarker index. A total of 126 candidates were tracked until they either passed or failed the training, and a total of 55 variables were used as inputs for creation of the model. RESULTS: Clustering analysis of the data revealed that only 4 of 55 variables were useful for predicting success or failure. The variables in the order of their importance are as follows: run time, number of miles run per week in the last year, level of salivary fatigue biomarker, and height. CONCLUSIONS: The results suggest that simple testing methods can identify candidates at high risk of failure.

Extremity compartment syndrome in pigs during hypobaric simulation of aeromedical evacuation.

INTRODUCTION: Extremity compartment syndrome (ECS) is diagnosed when the pressure within a muscle compartment increases to within 45 mmHg of mean arterial pressure. Increased pressure limits perfusion and eventually produces tissue necrosis. This can result in disability or loss of the affected limb. Hypobaric pressure during aeromedical evacuation (AE) has been hypothesized to increase the incidence of ECS. This was tested in a threshold model of ECS in swine. METHODS: Injury was induced by placing an angioplasty balloon between the tibia and the anterior muscle compartment and inflating the balloon to produce an intracompartmental pressure (ICP) 30 mmHg greater than mean arterial pressure for either 5 h or 6 h. Afterwards, animals were maintained either at ground level pressure or at a pressure equivalent to a simulated altitude of 2135 m above sea level for 8 h. ICP was monitored for signs of ECS development. At the end of the period the muscle was collected and evaluated for pathological changes and expression of various molecules associated with inflammation and tissue injury. RESULTS: Simulated altitude did not increase incidence of ECS, peak intracompartmental pressures, or time to onset of ECS. However, muscle degeneration and formation of microvascular thrombi were reduced by exposure to altitude. TNF, IL-1 b, IL-6, IGFBP5, and TGFB2 were increased (P < 0.05) by exposure to altitude, whereas FGF, IGF1, IGFBP4, BMP4, nitrotyrosine, and nitrate were unchanged (P > 0.05). DISCUSSION: Simulated altitude did not increase incidence of ECS. Inflammatory protein expression was increased in muscle, but some aspects of pathology were less severe following altitude exposure.

Threshold model for extremity compartment syndrome in swine.

BACKGROUND: Extremity compartment syndrome occurs when swelling develops within a muscle compartment to such an extent that the microvasculature is compressed and tissue perfusion is compromised. Untreated, this condition can result in widespread tissue destruction and loss of the affected limb. METHODS: Swine were subjected to diffuse muscle compression injury using a balloon catheter inserted between the anterior muscle compartment of the hind limb and the anterior face of the tibia. Balloons were inflated with saline to produce a sustained intramuscular pressure (IMP) of approximately 30 mmHg greater than mean arterial pressure. Following injury the IMP was monitored for up to 8 h. At the end of the monitoring period, the tibialis anterior muscle was collected and examined for injury. RESULTS: One animal receiving 6 h injury dislodged the implanted pressure transducers and was dropped from the data analysis. In all other limbs (n = 8) receiving 6 h injury, significant spontaneous increases in IMP were observed following injury. The tibialis anterior in all of the 6 h injury limbs also showed extensive tissue damage. In the limbs injured for 5 h (n = 10), only three showed a significant increase in IMP. The magnitude and duration of this increase closely resembled that seen following 6 h injury. Tissue damage was reduced in comparison with 6 h injury. CONCLUSIONS: The injury technique described here provides a potential useful threshold model for studying extremity compartment syndrome and the influence of related factors on the progression of this condition.

Development and characterization of a rat model of nonpenetrating liver trauma.

The aim of this study was to develop and characterize a rodent model of liver trauma suitable for preclinical evaluation of new treatments and diagnostic technologies. Liver trauma was created by dropping a steel cylinder through a plastic tube onto the abdomen of supine, anesthetized rats. Internal hemorrhage in the absence of liver trauma was simulated by instilling fresh blood into the peritoneum. Platelet counts were elevated significantly after liver trauma but not simulated hemorrhage. Liver trauma and simulated internal hemorrhage both increased blood levels of the factor growth-regulated oncogene-Kupffer cell. Transcription of plasminogen activator inhibitor 1, heat shock protein 70, and suppressor of cytokine syntheses 3 was increased 77-, 22-, and 27-fold, respectively, 2 h after liver trauma but was unaltered by simulated internal hemorrhage. Levels returned to pretrauma levels by 24 h after trauma. Transcript levels for hypoxia-inducible transcription factor 1alpha were increased 2.8-fold at 24 h but not 2 h after trauma and were not affected by simulated hemorrhage. Production of heat shock protein 70 and inducible nitric oxide synthase in liver was limited to a penumbra surrounding areas of necrosis associated with trauma. The rat model described produces lesions similar to those that occur in humans after blunt trauma.

A comparison of cognitive performance decreases during acute, progressive fatigue arising from different concurrent stressors.

Fatigue is known to impair cognitive performance, but it remains unclear whether concurrent common stressors affect cognitive performance similarly. We used the Stroop Color-Word Conflict Test to assess cognitive performance over 24 hours for four groups: control, sleep-deprived (SD), SD + energy deficit, and SD + energy deficit + fluid restricted. Fatigue levels were quantified using the Profile of Mood States (POMS) survey. Linear mixed-effects (LME) models allowed for testing of group-specific differences in cognitive performance while accounting for subject-level variation. Starting fatigue levels were similar among all groups, while 24-hour fatigue levels differed significantly. For each cognitive performance test, results were modeled separately. The simplest LME model contained a significant fixed-effects term for slope and intercept. Moreover, the simplest LME model used a single slope coefficient to fit data from all four groups, suggesting that loss in cognitive performance over a 24-hour duty cycle with respect to fatigue level is similar regardless of the cause.

Hydration status of Air Force military basic trainees after implementation of the back-mounted hydration system.

The Air Force makes an extraordinary effort to prevent heat-related illnesses associated with basic military training (BMT) in south Texas. However, inadequate hydration can still contribute to lost training time and qualified trainees leaving military service without completing BMT. The purpose of the present study was to determine whether equipping BMTs with back-mounted hydration systems (BM) is better than the standard-issue (SI) canteens with respect to hydration status. Male BMTs were randomly assigned to either BM (n = 40) or SI (n = 38) groups. Baseline values were assessed at week 0 before any physical readiness training (PRT). Subsequent data collection took place in the a.m. before PRT and in the p.m. before dinner the first 3 weeks, and during the 5 weeks of training. BMT total body water (TBW) and body composition were assessed by bioelectrical impedance. Saliva osmolality and total protein concentration were also determined. Hydration status increased daily in BM and SI and was well maintained over the duration of BMT. A significant hydration effect (p < 0.05) was observed for average daily increases in TBW and body weight with BM gaining more compared to SI. Average a.m. TBW was 0.3-0.8 L greater in SI versus BM (p < 0.05). Our findings demonstrate that adequate hydration status is maintained during Air Force BMT in a hot environment using either hydration mode and therefore do not support widespread issuance of the BM system on the premise of improved hydration during USAF BMT military training.

Evaluation of intraosseous delivery of factor VIIa during hemorraghic shock in the pig.

The purpose of this investigation is to determine if intraosseous infusion (IO) is a suitable method for the delivery of recombinant human factor VIIa (rFVIIa) during hemorrhagic shock. The measures that were used to evaluate IO delivery suitability included: (1) determination of clinically significant local or systemic toxicity and (2) demonstration that systemic blood levels of rFVIIa increased rapidly following administration. Our results indicate that there was no evidence of significant local or systemic toxicity following infusion and that the systemic blood concentration of rFVIIa peaks immediately after the end of infusion. This result suggests that the systemic blood level profiles of rFVIIa delivered by IO infusion are similar to those that could be produced by intravenous (IV) administration. Furthermore, in all 25 test animals, access to the systemic circulation during shock was achieved as evidenced by rapid increase in a marker dye (flourescein) or rFVIIa in the blood. We conclude that administration of rFVIIa via IO infusion is a reasonable safe method and is likely to produce blood levels required for improved hemostasis during shock.

Heart rate variability to assess combat readiness.

Chronic fatigue/physical exhaustion (FPE) impacts combat readiness but is difficult to identify. We tested the hypothesis that resting heart rate variability (HRV), including both time- and frequency-domain assessments, would correlate with hydration status and aerobic capacity in military recruit-age men and women with varying fitness levels. Cardiac interbeat intervals were recorded using a heart R-R monitor during 20 minutes of quiet, supine rest with paced breathing (0.25 Hz). HRV metrics included average R-R interval (RRIavg), R-R interval standard deviation (RRISD), the percentage of adjacent R-R intervals varying by > or = 50 ms (pNN50), and integrated areas of R-R interval spectral power at the high (0.15-0.4 Hz) (RRIHF) and low (0.04-0.15 Hz) (RRILF) frequencies. Treadmill maximal oxygen uptake (VO2 max), segmental bioimpedance estimates of total body water (TBW), and urine specific gravity (USG) were also assessed. All dependent variables of interest were within expected ranges, although absolute ranges of individual values were considerable. RRI correlated with VO2 max (r = 0.49; p < 0.001), with TBW (r = 0.38; p < 0.001), and inversely with USG (r = -0.23; p = 0.02). RRISD correlated with VO2 max (r = 0.21; p = 0.03), but not with TBW or USG. pNN50 correlated inversely with USG (r = -0.21; p = 0.03) but not with VO2 max or TBW. R-R interval spectral power at the high and low frequencies did not correlate with VO2 max, TBW, or USG. We have demonstrated that fitness level and hydration status may affect cardiac function via changes in autonomic tone, highlighting the potential of field-based assessment of heart rate variability metrics to identify FPE and other aspects of combat readiness.

Comparison of blood pressure and thermal responses in rats exposed to millimeter wave energy or environmental heat.

Electromagnetic fields at millimeter wave lengths are being developed for commercial and military use at power levels that can cause temperature increases in the skin. Previous work suggests that sustained exposure to millimeter waves causes greater heating of skin, leading to faster induction of circulatory failure than exposure to environmental heat (EH). We tested this hypothesis in three separate experiments by comparing temperature changes in skin, subcutis, and colon, and the time to reach circulatory collapse (mean arterial blood pressure, 20 mmHg) in male Sprague-Dawley rats exposed to the following conditions that produced similar rates of body core heating within each experiment: (1) EH at 42 degrees C, 35 GHz at 75 mW/cm, or 94 GHz at 75 mW/cm under ketamine and xylazine anesthesia; (2) EH at 43 degrees C, 35 GHz at 90 mW/cm, or 94 GHz at 90 mW/cm under ketamine and xylazine anesthesia; and (3) EH at 42 degrees C, 35 GHz at 90 mW/cm, or 94 GHz at 75 mW/cm under isoflurane anesthesia. In all three experiments, the rate and amount of temperature increase at the subcutis and skin surface differed significantly in the rank order of 94 GHz more than 35 GHz more than EH. The time to reach circulatory collapse was significantly less only for rats exposed to 94 GHz at 90 mW/cm, the group with the greatest rate of skin and subcutis heating of all groups in this study, compared with both the 35 GHz at 90 mW/cm and the EH at 43 degrees C groups. These data indicate that body core heating is the major determinant of induction of hemodynamic collapse, and the influence of heating of the skin and subcutis becomes significant only when a certain threshold rate of heating of these tissues is exceeded.

Thermal injury resulting from application of a granular mineral hemostatic agent.

BACKGROUND: Uncontrolled hemorrhage accounts for the majority of deaths in combat. Effective topical hemostatic agents suitable for use on the battlefield may be valuable in controlling hemorrhage until definitive surgical intervention is possible. In an effort to identify a hemostatic agent suitable for battlefield use, we evaluated several potential hemostatic agents in a swine injury model and noted thermal injury to tissues with a granular mineral hemostatic agent (QuikClot). METHODS: Anesthetized swine were maintained with a mean arterial pressure in excess of 60 mm Hg. Cutaneous, muscular, hepatic, splenic, venous, and arterial wounds were created in a standardized fashion. Topical hemostatic agents were immediately applied to the wounds and the amount of bleeding and time to hemostasis were noted. RESULTS: The results reported here are part of a larger study in which a variety of hemostatic agents were evaluated. Only the findings related to the granular mineral hemostatic agent are discussed here. Application of the agent resulted in elevated tissue surface temperatures in excess of 95 degrees C and internal tissue temperatures exceeding 50 degrees C, 3 mm deep to the bleeding surface. Necrosis of fat and muscle were noted as well as full and partial thickness cutaneous burns. CONCLUSIONS: Topical administration of a granular mineral hemostatic agent to a variety of wounds in an experimental swine model resulted in thermal tissue injury and necrosis. Suggestions for reducing the extent of injury with this product are offered.

A retrospective evaluation of digital wound imaging to predict response to hyperbaric oxygen treatment.

As new wound care treatments become available, correct initial treatment selection and dynamic modification of regimens, based on wound response to treatment, must be applied to improve outcomes and reduce cost. One alternative is wound morphometry using digital wound images to evaluate wound response to treatment in realtime. To determine whether wound area measurements taken during the first 3 weeks of hyperbaric oxygen treatment predict eventual treatment response and how demographic and disease factors impact hyperbaric oxygen treatment response, a retrospective study using digital wound images, demographic data, and available clinical laboratory values was conducted. Participants included 29 wound care patients with nonhealing wounds of the lower extremities receiving treatment at a hyperbaric wound care facility. Conventional wound care (ie, debridement, dressing changes, and topical agents) plus hyperbaric oxygen treatment (100% oxygen breathing at 2.4 atmospheres absolute for 90 minutes) given once every weekday for up to 20 weeks was provided. Graphical analysis of normalized wound area over time revealed two groups: minimal responders (n=13) and robust responders (n=16). Minimal response was characterized by delayed onset of wound area reduction and virtual cessation of reduction by week 3. Robust response was continuous, sustained, and resulted in average wound area reduction of 80% by end of treatment, compared to 47% in minimally responsive patients. Age, blood glucose, and serum creatinine significantly affected the wound healing response to hyperbaric oxygen treatment (P<0.05). Digital images obtained during the first 3 weeks of treatment predicted if a patient is minimally responsive to hyperbaric oxygen treatment with 100% accuracy. Area measurements obtained in this manner can be used to identify patients minimally responsive to hyperbaric oxygen treatment, enabling rapid assessment of treatment response to make timely changes in therapy in order to optimize treatment outcomes.

Hyperbaric oxygen treatment prevents up-regulation of angiogenesis following partial-thickness skin grafts in the pig.

We hypothesized that hyperbaric oxygen treatment (HBO) would reduce neovascularization following partial-thickness skin grafting in the Yucatan pig. Results show that capillary density 4 days post-graft is increased twofold in grafts not treated with HBO, compared to normal, ungrafted skin or skin grafts from pigs treated with HBO, p < 0.05. Similarly, the expression of vascular endothelial growth factor, a growth factor associated with neovascularization, was also reduced by HBO. Cell density in the graft boundary increased gradually after grafting, reaching a maximum 2.7-fold increase, relative to normal skin, at day 4, p < 0.05. Cell nuclei positive for proliferating cell nuclear antigen, a marker of proliferation, increased threefold by day 2, p < 0.05, and then declined to normal levels by day 7. HBO had no effect on cell density or proliferation in the boundary region or on shear strength of the graft. In the epidermis, proliferation declined 80% 2 days after grafting and then returned to levels observed in normal skin by day 4, p < 0.05; however, in pigs treated with HBO, we observed no decline in proliferation. These findings confirm the hypothesis that HBO reduces neovascularization in the partial-thickness skin graft while preserving regenerative capacity in the graft boundary and normal proliferative capacity of the epidermis.

Delayed treatment with doxycycline has limited effect on anthrax infection in BLK57/B6 mice.

Blk57/B6 mice were infected with LD90 dose of Sterne strain anthrax spores subcutaneously and then treated with doxycycline. Doxycycline at a dose of 1.5mg/kg, by intra-peritoneal injection, protected mice from death when given at the same time as spores. When doxycycline administration was delayed 4h survival is 90%. Delay of 24h increased survival time but had no impact on eventual mortality. When doxycycline was delayed 48h, mortality and time to death were comparable to sham injection. Peritoneal macrophages harvested from Blk57/B6 mice were examined for response to anthrax lethal toxin and are shown to be deficient in their ability to produce TNF-alpha and have increased expression of IL-6 compared to RAW 264.7 murine macrophage cell line. These findings suggest that antibiotic therapy has limited effects following lethal anthrax spore challenge, even when the host is of a phenotype that does not produce TNF-alpha in response to anthrax lethal toxin exposure.