The relationship between sleep, pain,and musculoskeletal injuries in US Army Soldiers.

INTRODUCTION: The purpose of this study was to investigate the relationship between sleep and pain in military personnel and to determine if metrics of sleep and pain intensity differ between the injured and uninjured in this population. METHODS: Active-duty US Army Soldiers (n=308; 26.8±6.5 years, 82% male) from the 2nd Infantry Division, Joint Base Lewis-McChord, Washington, and 101st Airborne Division, Fort Campbell, Kentucky, completed the Pittsburgh Sleep Quality Index (PSQI), Epworth Sleepiness Scale (ESS), and questionnaires about current musculoskeletal injuries and pain intensity (0=no pain to 10=worst imaginable pain). Pearson correlation coefficients were used to assess the association between pain and sleep. Differences in sleep and pain between injured and uninjured participants were determined using an analysis of covariance. RESULTS: Pain intensity was positively correlated with sleep quality (global PSQI score, r=0.337, p<0.001) and daytime sleepiness (ESS score, r=0.163, p=0.005), and negatively associated with sleep duration (r=-0.118, p=0.039). Injured participants accounted for 37.7% (n=116) of the study population. Injured participants reported greater pain intensity (3.7±2.5 vs 1.3±1.9, p<0.001), were older (28.5±7.4 years vs 25.8±5.7 years, p=0.001) and in the service longer (6.3±6.3 years vs 4.6±4.7 years, p=0.013) than uninjured participants. Injured participants had higher global PSQI scores (9.0±4.1 vs 6.4±3.4, p<0.001), including each of the seven PSQI components (all p<0.050), and reported sleeping less per night than uninjured participants (5.7±1.3 hours vs 6.1±1.2 hours, p=0.026). CONCLUSION: These data demonstrate that pain intensity is associated with sleep in active-duty US Army Soldiers and that those who report a musculoskeletal injury, regardless of age and time in service, report poorer sleep quality, shorter sleep durations, and greater levels of pain than uninjured Soldiers.

Physical sleeping environment is related to insomnia risk and measures of readiness in US army special operations soldiers.

BACKGROUND: US military service members have characteristically poor sleep, even when 'in garrison' or at one's home base. The physical sleeping environment, which is often poor in military-provided housing or barracks, may contribute to poor sleep quality in soldiers. The current study aimed to assess whether the sleeping environment in garrison is related to sleep quality, insomnia risk and military readiness. METHODS: Seventy-four US army special operations soldiers participated in a cross-sectional study. Soldiers were queried on their sleeping surface comfort and the frequency of being awakened at night by excess light, abnormal temperatures and noise. Subjective sleep quality and insomnia symptoms were also queried, via the Pittsburgh Sleep Quality Index and Insomnia Severity Index, respectively. Lastly, measures of soldier readiness, including morale, motivation, fatigue, mood and bodily pain, were assessed. RESULTS: Soldiers reporting temperature-related and light-related awakenings had poorer sleep quality higher fatigue and higher bodily pain than soldiers without those disturbances. Lower ratings of sleeping surface comfort were associated with poorer sleep quality and lower motivation, lower morale, higher fatigue and higher bodily pain. Each 1-point increase in sleeping surface comfort decreased the risk for a positive insomnia screen by 38.3%, and the presence of temperature-related awakenings increased risk for a positive insomnia screen by 78.4%. Those living on base had a poorer sleeping environment than those living off base. CONCLUSION: Optimising the sleep environment-particularly in on-base, military-provided housing-may improve soldier sleep quality, and readiness metrics. Providers treating insomnia in soldiers should rule out environment-related sleep disturbances prior to beginning more resource-intensive treatment.

A novel DNA inversion causing severe hemophilia A.

Almost half of all cases of severe hemophilia A are caused by recurrent DNA inversions, which disrupt the factor VIII (FVIII) gene. These inversions generally occur between a region of intron 22 (int22h) and one of two homologous copies of this region, located 300 to 400 kb telomeric to the FVIII gene. They are routinely detected by a Bcl I Southern blot assay in which the sizes of two of the three normal hybridization bands are characteristically altered. However, atypical hybridization patterns have been observed, and this report describes the first detailed analysis of a hemophilia A patient with such a pattern. The abnormal result was found to be caused by a novel FVIII inversion involving an extra copy of int22h from a site only 70 to 200 kb telomeric of the FVIII gene. Polymerase chain reaction (PCR) allowed one of the inversion junctions to be analyzed, showing that the int22h sequence at this inversion junction was truncated. This patient and his novel inversion provide further evidence that int22h is associated with instability in Xq28.

Factor VIII gene inversions in severe hemophilia A: results of an international consortium study.

Twenty-two molecular diagnostic laboratories from 14 countries participated in a consortium study to estimate the impact of Factor VIII gene inversions in severe hemophilia A. A total of 2,093 patients with severe hemophilia A were studied; of those, 740 (35%) had a type 1 (distal) factor VIII inversion, and 140 (7%) showed a type 2 (proximal) inversion. In 25 cases, the molecular analysis showed additional abnormal or polymorphic patterns. Ninety-eight percent of 532 mothers of patients with inversions were carriers of the abnormal factor VIII gene; when only mothers of nonfamilial cases were studied, 9 de novo inversions in maternal germ cells were observed among 225 cases (approximately 1 de novo maternal origin of the inversion in 25 mothers of sporadic cases). When the maternal grandparental origin was examined, the inversions occurred de novo in male germ cells in 69 cases and female germ cells in 1 case. The presence of factor VIII inversions is not a major predisposing factor for the development of factor VIII inhibitors; however, slightly more patients with severe hemophilia A and factor VIII inversions develop inhibitors (130 of 642 [20%]) than patients with severe hemophilia A without inversions (131 of 821 [16%]).

Investigation of the factor VIII intron 22 repeated region (int22h) and the associated inversion junctions.

A region of intron 22 of the factor VIII gene, which contains factor VIII-associated gene A (F8A), is repeated twice more nearer the Xq telomere. It has been proposed that intrachromosomal homologous recombination occurs between the intron 22 repeat and either of the two extragenic copies, resulting in the recurrent inversions that cause almost half of all cases of severe haemophilia A. We have precisely defined the repeated region as 9.5 kb of DNA which we have termed int22h (intron 22 homologous region). The junctions of the inversions examined were shown to represent precise exchanges between the int22h repeats, thus providing conclusive evidence for homologous recombination. The three copies of int22h were compared along 8 kb of their length, using chemical mismatch analysis, and found to be 99.9% similar. The presence of such long, almost identical inverted repeats near the Xq telomere could account for the high frequency at which the inversions occur.

Analysis of factor VIII mRNA reveals defects in everyone of 28 haemophilia A patients.

Haemophilia A is a mutationally heterogeneous disease caused by defects in the large and complex factor VIII gene. Recent studies examining the putative promoter, all exons and most intron/exon boundaries have failed to detect mutations in half the patients with severe disease leading to hypotheses such as mutations in remote controlling regions or even in genes other than factor VIII. We have amplified the factor VIII gene (putative promotor, coding region and polyadenylation/cleavage signal region) in 8 fragments from reverse transcribed mRNA and genomic DNA. Any mutation is then located by chemical mismatch detection and characterised by direct sequencing. This rapid and efficient method has been fully successful and has revealed an unusual cluster of mutations causing severe disease. Of the 28 patients we have reported, 5 had mild or moderate disease and all had a missense mutation. Twenty-three patients were severely affected and 13 of these had different detrimental mutations that were fully characterised at the genomic DNA level. The remaining 10 patients all had mRNA with exon 22 not contiguous to exon 23. Since all exons were normal and so were the splice sites of intron 22, the mutation in these patients should be in the regions of intron 22 that were not screened. These results prove that all haemophilia A cases are due to mutations of the factor VIII gene where, unexpectedly, intron 22 seems to be the target of approximately 40% of the mutations causing severe haemophilia A.

Factor VIII gene explains all cases of haemophilia A.

Using an mRNA-based method to examine haemophilia A mutations we provide an explanation for the puzzling report that half of the mutations causing severe disease are not detected by analysis of the putative promoter, exons, and most exon/intron boundaries of the factor VIII gene. An unusual cluster of mutations involving regions of intron 22 not examined earlier leads to defective joining of exons 22 and 23 in the mRNA and caused haemophilia A in 10/24 severely affected UK patients.

Detection of three novel mutations in two haemophilia A patients by rapid screening of whole essential region of factor VIII gene.

In an attempt to replace the existing, DNA-based, 50% effective, carrier and prenatal diagnoses of haemophilia A with the 100% successful direct detection of defective genes, a new procedure was developed to screen and identify mutations in all the essential regions of the factor VIII gene (putative promoter, coding sequence, and the cleavage and polyadenylation region). Genomic DNA and cDNA obtained by reverse transcription of the "leaky" mRNA found in peripheral lymphocytes were amplified by means of the polymerase chain reaction to yield a set of eight segments comprising the essential gene sequences. The segments were then screened individually for mutations by the amplification mismatch detection method, which detects and locates any type of sequence discrepancy between the test DNA and the control probe by cleavage of the probe at the site of mismatches. Two haemophilia A patients were studied. The first showed two single-base changes: one (substitution of tryptophan 2229 by cysteine in the C2 domain) is the probable cause of the disease, since it affects a conserved residue of factor VIIIa, whereas the other (the conservative substitution of aspartic acid at position 1241 by glutamic acid) occurs in a domain (B) irrelevant to factor VIII activity. The second patient showed a complete failure of pre-mRNA splicing due to a single-base substitution that changes the obligatory AG acceptor splice site of intron 5 to GG. The method characterises the gene defect in 10 days or less and should lead to the rapid accumulation of information on the molecular biology of haemophilia A.

Reactivities of polyclonal and monoclonal antibodies raised to the major capsid protein of human papillomavirus type 16.

Polyclonal and monoclonal antibodies have been raised against a fusion protein containing beta-galactosidase and part of the major capsid protein L1 of the human papillomavirus (HPV) type 16. The polyclonal antibodies cross-reacted with the L1 protein of several HPV types including HPV-1, -2, -6 and -11 when reacted with virus-infected tissue sections, and with HPV-6 and -18 L1 fusion proteins on Western blotting. Monoclonal antibodies against the L1 fusion protein of HPV-16 reacted only with HPV-16 L1 fusion proteins on Western blots and with HPV-16-containing biopsy sections as assessed by in situ DNA-DNA hybridization. These antibodies did not detect HPV-6 L1 protein after Western blotting or in HPV-6-infected tissue sections, although one did react with an HPV-18 fusion protein after Western blotting. The monoclonal antibodies were able to detect HPV-16 antigens in routine formaldehyde-fixed, wax-embedded sections of cervical intraepithelial neoplasia sections. HPV-16 L1 proteins were seen in one-third of biopsies that were positive using the polyclonal cross-reacting antisera. Polyclonal antibodies to fusion proteins containing part of the minor capsid protein L2 of HPV-6 or -16 appeared to be more type-specific as no cross-reactivity was seen when these antibodies were reacted with HPV-1- and -2-infected tissue sections.

Measurement of monoclonal antibody concentrations in hybridoma cultures: comparison of competitive inhibition and antigen capture enzyme immunoassays.

Competitive inhibition and antigen capture enzyme immunoassays were compared for the measurement of mouse monoclonal IgGl antibody produced by a hybridoma culture. Both methods yielded standard curves that were linear over several orders of magnitude, and both were comparable in sensitivity (10 ng/ml). However, the slope of the antigen capture curve was flatter than the slope for competitive inhibition. This difference in slope, coupled with a larger average standard deviation for each point on the standard curve for antigen capture, resulted in a significantly larger range of variability in IgGl levels. It is concluded that the competitive inhibition enzyme immunoassay method is better suited to the precise quantification of mouse monoclonal antibodies in hybridoma culture supernatants.