The acute and long-term effects of Middle East sand particles on the rat airway following a single intratracheal instillation.

Military personnel deployed in the Middle East have emphasized concerns regarding high levels of dust generated from blowing desert sand and the movement of troops and equipment. Airborne particulate matter levels (PM(10); PM < 10 µm) in the region may exceed 1500 µg/m(3), significantly higher than the military exposure guideline (MEG) of 50 µg/m(3). Increases in PM(10) have been linked to a rise in incidences of asthma, obstructive pulmonary disease, lung cancer, and cardiovascular diseases. Male Sprague-Dawley rats received a single intratracheal (IT) instillation of 1, 5, or 10 mg of Middle East PM(10) collected at a military occupied site in Kuwait, silica (positive control), or titanium dioxide (TiO(2); negative control) suspended in 400 µl sterile saline, or saline alone (vehicle control). Twenty-four hours, 3 d, 7 d and 6 mo postexposure (n = 15/group), organs including lung were evaluated for histopathological changes and for particle contaminants. Bronchoalveolar fluid (BALF) was also analyzed for cellular and biochemical parameters, including cytokines and chemokines. Instillation of silica resulted in early, pronounced, sustained inflammation indicated by significant increases in levels of total protein and neutrophils, and activities of lactate dehydrogenase activity and ß-glucuronidase activity. Lower magnitude and transient changes using the same markers were observed in animals exposed to TiO(2) and Middle East PM(10). The results suggest that for acute exposures, this Middle East PM(10) is a nuisance-type dust with relatively low toxicity. However, since average deployment of military personnel to the Middle East is 180 d with potential for multiple follow-on tours, chronic exposure studies are needed to fully understand the pulmonary effects associated with Middle East PM exposure.

Two-generation reproductive toxicity study of implanted depleted uranium (DU) in CD rats.

Depleted uranium (DU) munitions and armor plating have been used in several conflicts over the last 17 yr, including the Persian Gulf War and the Iraq War. Because of its effectiveness and availability, DU will continue to be used in military applications into the foreseeable future. There is much controversy over the use of DU in weapons and equipment because of its potential radiological and toxic hazards, and there is concern over the chronic adverse health effects of embedded DU shrapnel in war veterans and bystanders. This study evaluated the effects of long-term implantation of DU on the reproductive success of F0 generation adults and development and survival of subsequent F1 and F2 generations in a two-generation reproductive toxicity study. F0 generation Sprague-Dawley rats, 8 wk of age, were surgically implanted with 0, 4, 8, 12, or 20 DU pellets (1 x 2 mm). Inert implant control animals were implanted with 12 or 20 tantallum (Ta) pellets. The F0 generation was then mated at 120 d post DU implantation. In the F0 generation, when measured on postimplantation d 27 and 117, uranium was present in the urine of DU-implanted animals in a dose-dependent manner. F0 reproductive success was similar across treatment groups and the maternal retrieval test revealed no changes in maternal behavior. DU implantation exerted no effect on the survival, health, or well-being of the F0 generation. Necropsy results of F0 animals were negative with the exception of a marked inflammatory response surrounding the implanted DU pellets. For the F1 generation, measures of F1 development through postnatal day (PND) 20 were unremarkable and no gross abnormalities were observed in F1 offspring. No uranium was detected in whole-body homogenates of PND 4 or PND 20 pups. Necropsy findings of F1 PND 20 pups were negative and no instances of ribcage malformation were observed in F1 PND 20 pups. Body weight and body weight gain of F1 rats through PND 120 were similar across treatment groups. Eight of 414 F1 animals observed from PND 20 to 120 died of unknown causes; 7 were from litters of DU-implanted F0 mating pairs. F1 mating success at 10 wk of age was an overall 70% compared with 91% for F0 mating pairs. Mating success was similar between F1 animals derived from DU-implanted F0 adults and those derived from F0 implant control adults suggesting that the comparatively low mating success was not due to F1 DU exposure. The gestational index of F1 animals derived from mid-dose F0 mating pairs was found to be lower compared with F1 controls. The average gestation duration of F1 animals derived from high-dose F0 mating pairs was found to be significantly longer than F1 controls. F1 sperm motility analyses did not differ among experimental groups and no gross abnormalities were identified at necropsy among surviving F1 animals at PND 120. Histopathology of kidneys, spleen, thymus, bone marrow, ovaries, and testes of F1 high-dose animals did not differ from F1 controls. F1 high-dose females had significantly higher mean relative liver and heart weights compared with F1 controls; the biological relevance of this finding could not be determined. For the F2 generation, measures of F2 development through PND 20 were unremarkable and no gross abnormalities were observed in F2 offspring. Necropsy findings of F2 PND 20 pups were negative and no instances of ribcage malformation were observed in F2 PND 20 pups. Body weight and body weight gain of F2 rats through PND 90 were similar across treatment groups. Mean relative heart weights of males derived from high-dose F0 parents were significantly lower compared with F2 controls. Sperm motility and concentration analysis of F2 males at PND 90 were similar across F2 groups. Overall, the consistent absence of positive findings in this study seems to suggest that DU is not a significant reproductive or developmental hazard, particularly when one considers that mid- and high-dose rats were implanted with the equivalent of 0.3 and 0.5 lb of DU in a 70-kg human, respectively. However, the findings that seven of eight F1 adults that died postweaning were from DU-implanted F0 mating pairs, and that mean relative heart weights were elevated in high-dose F1 and F2 pups, suggest conservatism is warranted in characterizing the reproductive and teratogenic hazards of embedded DU until further studies are completed.

Evaluation of the effect of implanted depleted uranium (DU) on adult rat behavior and toxicological endpoints.

In 2002, the Naval Health Research Center Toxicology Detachment began a study to determine the effects of surgically implanted depleted uranium (DU) pellets on adult rat (e.g., P1 generation) health and reproduction. In this report, the effect of implanted DU on adult rat behavior and health is described. Adult Sprague-Dawley (SD) rats, 8 wk of age, were surgically implanted with 0, 4, 8, 12, or 20 DU pellets (1 x 2 mm); 20 DU pellets of size 1 x 2 mm approximates to 0.22 kg (0.5 lb) of DU in a 70-kg (154 lb) person. Control animals were implanted with 12 or 20 tantallum (Ta) pellets. The animals were then housed for up to 150 d postimplantation or 20% of an assumed 2-yr life span for rats. The concentration of uranium in urine directly correlated with the number of implanted DU pellets, indicating that DU was migrating into the body from the implanted pellets. Three male and 4 female animals died during the 150-d period of causes apparently not related to DU implantation. Behavioral testing found no definitive evidence of neurobehavioral perturbations associated with DU implantation. Uranium translocated to tissues known to sequester uranium (bone, teeth, and kidneys), but uranium concentrations varied considerably within each dose group and did not follow a dose-response pattern as anticipated. Serum chemistry values were within normal ranges for the SD rat. However, alanine aminotransferase measurements were significantly lower for rats implanted with 20 DU pellets as compared to sham surgery controls but not when compared to animals implanted with Ta pellets only. Phosphate measurements were significantly lower for female rats implanted with 20 DU pellets as compared to both sham surgery controls and animals implanted with Ta pellets only. Monocyte ratios were higher in adult rats implanted with 20 DU pellets as compared to sham surgery controls but not when compared to animals implanted with 20 Ta pellets. Mean platelet volume was found to be significantly lower for rats implanted with 20 DU pellets as compared to sham surgery controls but not when compared to animals implanted with 20 Ta pellets. Gross necropsy found no obvious tissue abnormalities in implanted rats, and the weights of major tissues did not differ between Ta- and DU-implanted animals. Histopathologic analysis of major tissues from animals implanted with 0 pellets, 20 Ta pellets, or 20 DU pellets found no differences between treatment groups. The findings of this study indicate that implantation of up to 20 DU pellets in adult rats did not have a significant negative impact on their general health and neurobehavioral capacities when assessed after 150 d of pellet implantation. However, the growing body of data on the potential health effects associated with DU exposure warrants further studies involving higher embedded DU body burdens in conjunction with longer surveillance periods postimplantation.

Acute and subacute dermal toxicity of Break-Free CLP: a weapons cleaning and maintenance compound.

Break-Free CLP((R)) is a commercial cleaning, lubricating and preserving compound used in both the military and civilian sectors for maintenance of small- and large-caliber weapons. Like many commercial mixtures, there is very little information available on the toxicity of Break-Free CLP. Studies were conducted to characterize the biological effects of single or repeat dermal application of Break-Free CLP to the clipped backs of CD-1 mice. Break-Free CLP was applied neat, 50 microl three times of week for up to 2 weeks. Foci of epithelial ulceration were observed in skin sections from 22% of Break-Free CLP-treated animals in conjunction with markedly thickened epithelium suggesting that robust epithelial regeneration was occurring in these animals. Skin histopathology of Break-Free CLP-treated animals closely matched the histopathology from mice treated repeatedly with 2% croton oil in acetone (dermal irritation positive control). Serum alkaline phosphatase activity was significantly (P < 0.05) lower for mice treated with Break-Free CLP, 2% croton oil or 7,12-dimethylbenz[a]anthracene (DMBA) compared with negative and vehicle control mice. Skin nitric oxide (NO) levels were not significantly elevated for mice treated with Break-Free CLP but were significantly elevated for mice treated with dermal irritation positive control compound DMBA. The cumulative skin changes in Break-Free CLP-treated animals support conducting a subchronic dermal application study. The observed decreases in serum alkaline phosphatase activity suggest that future studies should include the liver and bone as possible target organs. Additionally, dermal penetration studies could provide key health risk assessment information for characterizing the potential health risks associated with chronic dermal exposure to Break-Free CLP.

Study of the reproductive effects in rats surgically implanted with depleted uranium for up to 90 days.

In 2001, the Naval Health Research Center Toxicology Detachment was funded by the U.S. Army Medical Research Acquisition Activity (USAMRAA) to conduct a study of the effects of surgically implanted depleted uranium (DU) pellets on adult rat reproductive success and development across two successive generations. This article presents some of the findings for the group of offspring from adult rats mated at 30 d post surgical implantation of DU pellets. Adult male and female Sprague-Dawley rats (P1 generation) were surgically implanted with 0, 4, 8, or 12 DU pellets (1 x 2 mm). The P1 generation was then cross-mated at 30 d post surgical implantation. Urine collected from P1 animals at 27 d post surgical implantation showed that DU was excreted in the urine of DU-implanted animals in a dose-dependent manner. DU surgical implantation did not have a negative impact on P1 reproductive success, survival, or body weight gain through post surgical implantation d 90. There were no statistically significant differences in F1 birth weight, survival, and litter size at postnatal day (PND) 0, 5, and 20. No gross physical abnormalities identified in the offspring were attributable to neonatal DU exposure. A series of neurodevelopment and immune function assessments were also conducted on F1 offspring. No group differences were observed that were related to parental DU exposure. Studies are ongoing on the impact of leaving DU embedded in soft tissue for 120 d on rat reproduction and subsequent offspring survival and development.

Effects of dietary supplementation with L-carnitine and fat on blood acid-base responses to handling in slaughter weight pigs.

Blood acid-base responses to handling were evaluated in slaughter weight pigs fed diets supplemented with l-carnitine and fat. The study was carried out as a randomized block design with a 2 x 2 factorial arrangement of treatments: 1) dietary L-carnitine supplementation (0 vs. 150 ppm, as-fed basis); and 2) dietary fat supplementation (0 vs. 5%, as-fed basis). Sixty pigs (91.1 +/- 5.14 kg BW) were housed in mixed-gender groups of five and had ad libitum access to test diets (0.68% true ileal digestible lysine, 3,340 kcal of ME/kg, as-fed basis) for 3 wk. At the end of the feeding period (110.3 +/- 7.52 kg BW), pigs were subjected to a standard handling procedure, which consisted of moving individual animals through a facility (12.2 m long x 0.91 m wide) for eight laps (up and down the facility), using electric prods (two times per lap). There was no interaction between dietary L-carnitine and fat supplementation for any measurement. Pigs fed 150 ppm of supplemental L-carnitine had lower baseline blood glucose (P < 0.05) and higher baseline blood lactate (P < 0.05) concentrations than the nonsupplemented pigs. After handling, pigs fed L-carnitine-supplemented diets had a higher (P < 0.05) blood pH and showed a smaller (P < 0.05) decrease in blood pH and base excess than those fed the nonsupplemental diets. Baseline plasma FFA concentrations were higher (P < 0.01) in pigs fed the 5% fat diet. After the handling procedure, blood glucose, lactate, and plasma FFA were higher (P < 0.05) in pigs fed the 5 vs. 0% fat diets, but blood pH, bicarbonate, and base excess were not affected by dietary fat. The handling procedure decreased (P < 0.01) blood pH, bicarbonate, base excess, and total carbon dioxide and increased (P < 0.01) blood lactate, partial pressure of oxygen, and glucose, and also increased (P < 0.01) rectal temperature. Free fatty acid concentrations were increased by handling in pigs fed both 0 and 5% fat and 150 ppm L-carnitine. In conclusion, dietary L-carnitine supplementation at the level and for the feeding period evaluated in the current study had a relatively small but positive effect on decreasing blood pH changes in finishing pigs submitted to handling stress; however, dietary fat supplementation had little effect on blood acid-base balance.

Effects of stachyose on performance, diarrhoea incidence and intestinal bacteria in weanling pigs.

A three-week trial was conducted to evaluate the effects of the oligosaccharide stachyose on performance, diarrhoea incidence and intestinal bacterial populations in weaned pigs (7.96 +/- 0.2kg). A total of 144 crossbred (Landrace x Large White x Duroc) pigs weaned at 28 days were allotted to one of four treatments with six pens per treatment and six pigs per pen. The experimental diets were based on maize, dried whey and whole-fat milk and were supplemented with 0, 1 or 2% stachyose. A fourth diet contained no milk but instead contained 20% soybean meal to provide 0.78% stachyose and 0.21% raffinose. Inclusion of 1 or 2% stachyose in the diet depressed growth compared with pigs fed the control diet. Pigs fed the soybean meal diet gained weight at a rate similar to pigs fed the diet containing 1% added stachyose. Diarrhoea incidence was highest for pigs fed the soybean meal diet and lowest for pigs fed the control diet, with pigs fed the diets containing stachyose being intermediate. Pigs fed 1% stachyose had more lactobacilli in the ileum as well as more bifidobacteria in the caecum and colon than control pigs. They also had fewer enterobacteria in the colon. In contrast, pigs fed the diet containing 2% stachyose had fewer lactobacilli and bifidobacteria in the jejunum, ileum and caecum than did control pigs. Volatile fatty acids in the ileum, caecum and colon were highest for pigs fed 1% stachyose and lowest for pigs fed 2% stachyose. Volatile fatty acid concentrations were not significantly different between pigs fed the soybean meal diet and those fed the control. The overall results of this experiment indicate that the oligosaccharide stachyose had a negative effect on pig performance and its presence may partially explain the poorer performance observed when soybean meal is used as the sole source of supplemental protein in cereal-based diets fed to weaned pigs.

A spectrum of ABCC6 mutations is responsible for pseudoxanthoma elasticum.

To better understand the pathogenetics of pseudoxanthoma elasticum (PXE), we performed a mutational analysis of ATP-binding cassette subfamily C member 6 (ABCC6) in 122 unrelated patients with PXE, the largest cohort of patients yet studied. Thirty-six mutations were characterized, and, among these, 28 were novel variants (for a total of 43 PXE mutations known to date). Twenty-one alleles were missense variants, six were small insertions or deletions, five were nonsense, two were alleles likely to result in aberrant mRNA splicing, and two were large deletions involving ABCC6. Although most mutations appeared to be unique variants, two disease-causing alleles occurred frequently in apparently unrelated individuals. R1141X was found in our patient cohort at a frequency of 18.8% and was preponderant in European patients. ABCC6del23-29 occurred at a frequency of 12.9% and was prevalent in patients from the United States. These results suggested that R1141X and ABCC6del23-29 might have been derived regionally from founder alleles. Putative disease-causing mutations were identified in approximately 64% of the 244 chromosomes studied, and 85.2% of the 122 patients were found to have at least one disease-causing allele. Our results suggest that a fraction of the undetected mutant alleles could be either genomic rearrangements or mutations occurring in noncoding regions of the ABCC6 gene. The distribution pattern of ABCC6 mutations revealed a cluster of disease-causing variants within exons encoding a large C-terminal cytoplasmic loop and in the C-terminal nucleotide-binding domain (NBD2). We discuss the potential structural and functional significance of this mutation pattern within the context of the complex relationship between the PXE phenotype and the function of ABCC6.

ACTH receptor distribution and modulation among murine mononuclear leukocyte populations.

Murine mononuclear leukocytes express adrenocorticotropin (ACTH) receptors that were recognized by a monospecific antiserum to the ACTH receptor on Y-1 adrenal cells. The antiserum was utilized in an immunofluorescence (IF) assay to characterize the distribution of ACTH receptors on resting murine mononuclear leukocyte populations. Forty-seven percent of spleen cells, 32% of lymph node cells, and 1% of thymocytes constitutively expressed ACTH receptors. Separation of lymphocytes into purified B cell and T cell populations, followed by IF analysis revealed that 47% of B cells and 23% of T cells possessed ACTH receptors. Helper T cells (CD4+ T cells) constituted the majority of ACTH receptor-positive T lymphocytes. Furthermore, 47% of resident peritoneal macrophages, purified by adherence to plastic, expressed ACTH receptors. The T-lymphocyte mitogen, concanavalin A, interferon gamma, and ACTH enhanced ACTH receptor expression. The differential distribution of ACTH receptor-positive cells among specific leukocyte populations explains in part why differential cellular responses are observed and implies important regulatory functions for these receptors in the generation or regulation of immune responses.

Immunomagnetic isolation and long-term culture of mouse type A spermatogonia.

In the mammalian testis, type A spermatogonia proliferate and differentiate into sperm under the tight control of both endocrine and paracrine factors. In order to study the complex process of spermatogenesis at the molecular level, an in vitro system must be devised in which type A spermatogonia can be cultured for a prolonged period of time. Therefore, cocultures including type A spermatogonia and Sertoli cells, which act as nurse cells to the developing germ cells, are desirable. We have developed a method for the specific isolation of type A spermatogonia using magnetic beads and antibodies that recognize the c-kit receptor or the homophilic adhesion molecule, Ep-CAM. Purified spermatogonia could survive for a period of 25 days when cocultivated on Sertoli cell monolayers. Moreover, we recently established Sertoli cell lines that produce growth factors that are essential for the maintenance of spermatogonia in a proliferative state. Some of these Sertoli cell lines are able to reorganize into tubular structures when cultivated on a layer of Matrigel as extracellular matrix. We show here that type A spermatogonia associate specifically with the Sertoli cell tubules, and are able to replicate their DNA in this environment. Thus, these in vitro culture systems could be used for the long-term culture of primary, nonimmortalized type A spermatogonia.

H reflex and F wave latencies to soleus normal values and side-to-side differences.

OBJECTIVE: Electromyographers must reliably differentiate between H reflexes and F waves when recording from the soleus muscle in the evaluation of S1 radiculopathy. The use of F waves in root-level injuries is questioned, whereas H reflexes have shown value in the evaluation of S1 radiculopathy. We studied the relationship between the tibial H reflex and F wave latencies in the limbs of 40 subjects. DESIGN: After recording the H wave latency, we changed the gain to 200 microV/cm and increased the stimulation to supramaximal for ten additional responses without moving the recording or stimulating electrodes. We also calculated the predicted H wave latency with the standard formula. Forty subjects, mean age 32 yr, consented and participated. RESULTS: The mean of the average F wave was 1.76 ms longer than the ipsilateral H reflex latency. The mean side-to-side difference of the average F wave was 0.56 ms. The H reflex latency side-to-side difference was 0.36 ms. CONCLUSION: The findings suggest that the average F wave latencies have a predictive value in the clinical context similar to the H reflex.

Out-patient parenteral antibiotic therapy (OPAT): clinical outcomes and adverse events.

A chart review was conducted of patients in a program featuring self directed, home infusion of antibiotics for serious infections utilizing an out-patient medical office for teaching, mixing of drugs, and monitoring of patients. 302 courses of out-patient parenteral antibiotic therapy (OPAT) were administered to 221 patients. Therapy was successful in 94% of the episodes. Objective adverse events were noted in 25% of patients. To maximize the chance for a successful outcome, treatment plans should be individualized and structured to include systematic monitoring for adverse effects.

Coronary artery bypass performed without the use of cardiopulmonary bypass is associated with reduced cerebral microemboli and improved clinical results.

STUDY OBJECTIVES: Strokes and neurocognitive dysfunction have been correlated with cerebral microemboli produced during cardiopulmonary bypass (CPB). The purpose of this study was to determine whether, and to what extent, off-pump coronary artery bypass (OPCAB) reduces the occurrence of cerebral microemboli compared with traditional coronary artery bypass grafting (CABG) with CPB and to compare clinical results. DESIGN AND PATIENTS: A retrospective review of 137 patients undergoing elective CABG was performed, 70 of whom underwent traditional CABG and 67 of whom underwent OPCAB. Using transcranial Doppler ultrasonography, 40 patients (20 CABG, 20 OPCAB) were continuously monitored intraoperatively for the occurrence and pattern of cerebral microemboli. SETTING: Private, university-affiliated tertiary care hospitals. RESULTS: There was no statistical difference in the age, sex, or underlying comorbidities between those patients undergoing CABG and OPCAB. CABG patients did have a slightly lower preoperative ejection fraction (50.9% vs 55.5%, p = 0.03). Despite these similar preoperative characteristics, the OPCAB group experienced significant reductions in cerebral microemboli (27 vs 1,766, p = 0.003), transfusion requirements (29.9% vs 47.1%, p = 0.04), intubation time (3.3 vs 9.5 h, p < 0.001), ICU length of stay (1.5 vs 2.8 days, p = 0.02), and overall hospitalization (4.9 vs 6.6 days, p = 0.01) without an increase in mortality. Fewer strokes and deaths were observed in the OPCAB group, but these trends failed to reach statistical significance. CONCLUSIONS: In similar patient populations, OPCAB was associated with significantly fewer cerebral microemboli and improved clinical results without an increase in mortality. We believe that these early results support OPCAB as a viable and potentially safer alternative to traditional CABG.

Computational and experimental analyses reveal previously undetected coding exons of the KRIT1 (CCM1) gene.

A notable difficulty in annotating genomic sequence is identifying the correct start codon in a gene. An important such case has been found with KRIT1, the cerebral cavernous malformation type 1 (CCM1) gene. Analysis of human and mouse genomic sequence encompassing the region containing KRIT1/Krit1 using exon/gene-prediction and comparative alignment programs revealed putative exons upstream of the previously described first exon. These additional candidate exons show significant matches to mouse and human ESTs that are contiguous with and extend upstream from the previously designated 5' end of the KRIT1 cDNA sequence. RT-PCR and 5'RACE experiments confirm the presence of four additional upstream coding exons that encode an additional 207 amino acids. Importantly, a novel frameshift mutation in one of these newly identified KRIT1 exons has been found in a CCM1 family. These data establish the authentic KRIT1 amino acid sequence and suggest that the additional KRIT1 exons may harbor mutations in other CCM1 families. In addition, these results provide another example of the utility of rigorous computational and comparative sequence analysis for refining gene structure.