Clinical Examination in the Diagnosis of Anterior Cruciate Ligament Injury: A Blinded, Cross-sectional Evaluation.

OBJECTIVE: This study was conducted to compare the effectiveness of clinical tests for anterior cruciate ligament (ACL) injury. METHODS: This study prospectively evaluated the effectiveness of the Lachman test, anterior drawer test, and lever test in diagnosing ACL injury in 133 patients with knee pathology. The examiner was blinded to the patient's history, symptoms, and laterality of the pain at the time of examination. One hundred twenty-three patients in the study underwent MRI, and 90 went on to arthroscopy. The performance of the examination maneuvers and MRI was calculated. RESULTS: This study showed notable differences in sensitivity and specificity between the Lachman test and the lever test and in specificity between the anterior drawer test and the lever test. The Lachman test was also found to be more sensitive than the anterior drawer. All ACL tears diagnosed by a composite of the physical examination maneuvers were confirmed by MRI. MRI findings were concordant with arthroscopic findings in all cases. CONCLUSIONS: The Lachman test and the anterior draw test demonstrated clinical utility, but the results of the lever test should be interpreted with caution. Clinical examination was found to be highly specific but less sensitive than MRI.

Effects of acute and chronic heat stress on feed sorting behaviour of lactating dairy cows.

Nutritional strategies to mitigate the negative effects of heat stress on animal welfare and productivity often involve changes in ration formulation. However, cattle commonly sort their ration in favour of certain components, and it is not clear how feed sorting responds to heat stress. This study investigated the association between heat stress and feed sorting behaviour. Lactating Holstein dairy cows (n = 32; parity = 2.8±1.2; mean±SD) were housed in a free stall barn and milked 3×/day. Cows were fed individually using the Calan Broadbent Feeding System and offered ad libitum access to a total mixed ration (containing on a dry matter basis: 3.3% ryegrass hay, 16.5% ryegrass baleage, 24.7% corn silage, 11.1% brewers grains, 19.7% ground corn, 19.8% concentrate and 4.9% protein/mineral supplement), provided 1×/day. Beginning at 186±60 days in milk, cows were exposed to either: heat stress conditions (HT; n = 15) (average temperature-humidity index: 77.6), or evaporative cooling (CL; n = 17), consisting of misters and fans over the freestall and feed bunks. Data were collected during a 4-day baseline period, and two 4-day experimental periods: starting at 10 days after implementing treatments (defined as acute heat stress for HT cows), and at 62 days after implementing treatments (defined as chronic heat stress for HT cows). Daily feed intake and physiological responses to heat stress (body temperature, respiration rate) were recorded. Samples of fresh and refused feed were collected daily from individual cows for particle size analysis. The particle size separator had three screens (19, 8 and 1.18 mm) and a bottom pan, resulting in 4 fractions (long, medium, short and fine particles). Feed sorting was calculated as the actual intake of each particle size fraction expressed as a percentage of the predicted intake of that fraction. During both heat stress periods, HT cows sorted for long particles more than CL cows (105.0% v. 100.6%; SE = 1.1). During acute heat stress, HT cows sorted to a greater extent than CL cows against medium and short particles, whereas sorting of these fractions did not differ during chronic heat stress. Body temperature and respiration rate were associated across treatments with the extent of sorting for long particles and against short particles during acute heat stress. These results suggest that feed sorting is particularly influenced during acute heat stress, and that sorting for longer particles may increase in heat stress.

Life expectancy after spinal cord injury: a 50-year study.

STUDY DESIGN: Cohort of incident cases from 1955 to 2006. OBJECTIVES: To analyse acute and long-term mortality, estimate life expectancy and identify survival patterns of individuals experiencing traumatic spinal cord injury (SCI). SETTING: Specialised SCI unit in Australia. METHODS: Data for patients with traumatic SCI admitted to a spinal unit in Sydney, Australia between January 1955 and June 2006 were collated and deaths confirmed. Cumulative survival probability was estimated using life-table techniques and mortality rates were calculated from the number of deaths and aggregate years of exposure. Standardised mortality ratios (SMRs) were estimated from the ratio of observed to expected number of deaths. Life expectancy was then estimated using adjusted attained age-specific mortality rates. RESULTS: From 2014 persons, 88 persons with tetraplegia (8.2%) and 38 persons with paraplegia (4.1%) died within 12 months of injury, most often with complete C1-4 tetraplegia. Among first-year survivors, overall 40-year survival rates were 47 and 62% for persons with tetraplegia and paraplegia, respectively. The most significant increases in mortality were seen in those with tetraplegia and American Spinal Injury Association Impairment Scale (AIS) grades A-C lesions, with SMRs between 5.4 and 9.0 for people <50 years, reducing with advancing attained age. Estimated life expectancies from 25 to 65 years ranged between 69-64%, 74-65%, 88-91% and 97-96% for C1-4 AIS A-C, C5-8 A-C, T1-S5 A-C and all AIS D lesions, respectively. CONCLUSION: Survival related strongly to extent of neurological impairment. Future research should focus on identifying contextual factors, personal or environmental, that may contribute to the reduced life expectancy after SCI.

In vitro evaluation of the protective role of human antibodies to West Nile virus (WNV) produced during natural WNV infection.

BACKGROUND: West Nile virus (WNV) is endemic in the United States and transmissible by transfusion. Since 2003, the US blood supply has been screened by nucleic-acid tests (NAT) for WNV in minipools (MP-NAT) of 6 or 16 specimens. WNV infection begins with low-level viremia detectable only by individual testing (ID-NAT) and no detectable WNV antibodies. Viremia then increases to levels detectable by MP-NAT, and antibodies become detectable; later, viremia decays to levels detectable only by ID-NAT before becoming undetectable. All but 1 documented WNV transmission by transfusion involved blood components negative for WNV antibodies, raising the question whether WNV antibody-positive blood components with low levels of WNV RNA are infectious. METHODS: Specimens from 102 viremic donors with and without WNV antibodies were used to investigate infectivity in cultures of Vero cells and human monocyte-derived macrophages (MDMs). RESULTS: In Vero cell culture, 54 (74%) of 73 WNV antibody-negative specimens and 10 (36%) of 28 WNV antibody-positive specimens were infectious. In a random subset of 20 specimens tested in MDM culture, 7 (88%) of 8 WNV antibody-positive specimens and 12 (100%) of 12 WNV antibody-negative specimens were infectious. CONCLUSION: WNV antibodies do not always protect susceptible cells from WNV infection in vitro. RNA positivity in the presence of antibody cannot be ignored as a theoretical risk for blood recipients and needs further investigation.

World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines for evaluating the efficacy of anticoccidial drugs in chickens and turkeys.

These guidelines have been written to aid in the design, implementation and interpretation of studies for the assessment of drug efficacy against Eimeria species in chickens and turkeys. The information provided deals with many aspects of how to conduct controlled studies in battery cages (dose determination), floor pens (dose confirmation), and commercial facilities (field effectiveness studies), the selection of birds, housing, feeding, preparation of medicated rations, record keeping, diagnostic techniques, and methods for the preparation, maintenance and use of parasites. These guidelines are also intended to assist investigators in conducting specific studies, provide specific information for registration authorities involved in the decision-making process, assist in the approval and registration of new anticoccidial drugs, and facilitate the world-wide adoption of standard procedures.

Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV.

BACKGROUND: Toward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons. RESULTS: We cloned into a Deltapre-M/E dengue virus replicon genes for either green fluorescent protein (GFP), HIV gp160 or HIV gp120 and tested the ability of these constructs to express dengue virus proteins as well as the heterologous proteins in tissue culture after transfection of replicon RNA. CONCLUSIONS: Heterologous proteins were readily expressed from these constructs. GFP and gp120 demonstrated minimal or no toxicity. Gp160 expressing replicons were found to express proteins abundantly at 36 hours post transfection, but after 50 hrs of transfection, few replicon positive cells could be found despite the presence of cellular debris positive for replicon proteins. This suggested that gp160 expressed from dengue virus replicons is considerably more toxic than either GFP or gp120. The successful expression of heterologous proteins, including HIV gp120 for long periods in culture suggests this vector system may be useful as a vaccine vector, given appropriate delivery methods.

Development of Dengue virus type 2 replicons capable of prolonged expression in host cells.

BACKGROUND: As part of a program to develop a Dengue virus vaccine which avoids the deleterious effects of antibody dependent enhancement (ADE) of infection mediated by antibodies to Dengue virus structural proteins, we have begun to investigate the possibility of designing Dengue vaccines based on non-structural proteins. RESULTS: Dengue constructs which lack major structural proteins replicate intracellularly in tissue culture. These replicons are capable of prolonged expression of Dengue virus non-structural proteins for at least seven days in culture. CONCLUSIONS: Dengue virus genomes lacking major structural proteins can, like other flaviviruses, replicate intracellularly and express virus non-structural proteins with minimal toxicity to host cells. These findings pave the way for the development of dengue virus replicons as a form of live, attenuated virus vaccine.

Identification of a potential HIV-induced source of bystander-mediated apoptosis in T cells: upregulation of trail in primary human macrophages by HIV-1 tat.

The induction of apoptosis in T cells by bystander cells has been repeatedly implicated as a mechanism contributing to the T cell depletion seen in HIV infection. It has been shown that apoptosis could be induced in T cells from asymptomatic HIV-infected individuals in a Fas-independent, TNF-related apoptosis-inducing ligand (TRAIL)-dependent manner if the cells were pretreated with anti-CD3. It has also been shown that T cells from HIV-infected patients were even more sensitive to TRAIL induction of apoptosis than they were to Fas induction. Recently, it has been reported that in an HIV-1 SCID-Hu model, the vast majority of the T cell apoptosis is not associated with p24 and is therefore produced by bystander effects. Furthermore, few apoptotic cells were associated with neighboring cells which were positive for either Fas ligand or TNF. However, most of the apoptotic cells were associated with TRAIL-positive cells. The nature of these TRAIL-positive cells was undetermined. Here, we report that HIV infection of primary human macrophages switches on abundant TRAIL production both at the RNA and protein levels. Furthermore, more macrophages produce TRAIL than are infected by HIV, indicating that a bystander mechanism may, at least in part, upregulate TRAIL. Exogenously supplied HIV-1 Tat protein upregulates TRAIL production by primary human macrophages to an extent indistinguishable from infection. The results suggest a model in which HIV-1-infected cells produce extracellular Tat protein, which in turn upregulates TRAIL in macrophages which then can induce apoptosis in bystander T cells.

Effect of vaccination on experimental infection with Bordetella bronchiseptica in dogs.

OBJECTIVE: To determine comparative efficacy of vaccines administered IM and intranasally, used alone or sequentially, to protect puppies from infection with Bordetella bronchiseptica and determine whether systemic or mucosal antibody response correlated with protection. DESIGN: Randomized controlled trial. ANIMALS: 50 specific-pathogen-free Beagle puppies. PROCEDURE: In 2 replicates of 25 dogs each, 14-week-old puppies that were vaccinated against canine distemper virus and parvovirus were vaccinated against B bronchiseptica via intranasal, IM, intranasal-IM, or IM-intranasal administration or were unvaccinated controls. Puppies were challenge exposed via aerosol administration of B bronchiseptica 2 weeks after final vaccination. Clinical variables and systemic and mucosal antibody responses were monitored for 10 days after challenge exposure. Puppies in replicate 1 were necropsied for histologic and immunohistochemical studies. RESULTS: Control puppies that were seronegative before challenge exposure developed paroxysmal coughing, signs of depression, anorexia, and fever. Vaccinated puppies (either vaccine) that were seronegative before challenge exposure had fewer clinical signs. Puppies that received both vaccines had the least severe clinical signs and fewest lesions in the respiratory tract. Vaccinated dogs had significantly higher concentrations of B bronchiseptica-reactive antibodies in serum saliva before and after challenge. Antibody concentrations were negatively correlated with bacterial growth in nasal cavity and pharyngeal samples after challenge exposure. CONCLUSIONS AND CLINICAL RELEVANCE: Parenterally and intranasally administered vaccines containing B bronchiseptica may provide substantial protection from clinical signs of respiratory tract disease associated with infection by this bacterium. Administration of both types of vaccines in sequence afforded the greatest degree of protection against disease.

Comparative testing of anticoccidials in broiler chickens: the role of coccidial lesion scores.

The relationship between oocyst dose and lesion score was evaluated in trials involving five field isolates each of Eimeria acervulina, Eimeria maxima, and Eimeria tenella. Each trial included an uninfected, unmedicated treatment, and at least three treatments of unmedicated birds given different doses of oocysts from a single isolate. In four trials each with E. acervulina and E. tenella, and all five trials with E. maxima, infected, salinomycin-medicated (60 ppm) treatments were included. Each treatment consisted of five cages with eight male broiler birds per cage using a randomized complete block design. The relationship between oocyst dose and lesion score was examined within each coccidial species using the linear model: Y = beta0 + beta1(log(n) oocyst dose + 1). The results demonstrated that in unmedicated birds, low oocyst doses caused mean lesion scores up to 2.0, but the numbers required to cause higher mean scores were many times greater. Second, the estimated oocyst dose in salinomycin-medicated birds for any given mean lesion score was substantially more than the corresponding estimate for unmedicated birds. These results indicated that there could be wide differences in levels of oocyst dose between unmedicated and medicated birds that lesion scores failed to measure. If lesion scores are used in trials comparing anticoccidial drugs, an alternative design may be to include three infected, unmedicated treatments each given a different level of inoculum (e.g., low, medium, and high). Medicated treatments, given the highest oocyst dose only, would then be compared to each of the infected, unmedicated treatments.

Comparison of arithmetic and geometric means as measures of a central tendency in cattle nematode populations.

Efficacy calculations in anthelmintic studies require estimates of the central tendency for the nematode populations. Confusion exists among practitioners regarding which measures of central tendency are most appropriate; although the arithmetic mean is frequently used, there are theoretical reasons for preferring the geometric mean. To investigate this controversy, arithmetic and geometric means were compared for their suitability for use in measuring efficacy. Arithmetic and geometric means were compared as measures of central tendency for skewed distributions. The following criteria were developed to facilitate the comparison: (1) probability around the parameter, (2) influence of extreme values, and (3) proximity to the median. Under log-normality, theoretical results demonstrated the superiority of the geometric mean. Modified-bootstrap simulations using empirical data from cattle were used to confirm theoretical expectations. Simulations on log-normal data supported the geometric mean as the better indicator of the central tendency. Additionally, for data not confirmed as log-normal, the superiority of geometric means was demonstrated. In a comparison of precision, it was shown that mean squared error was always smaller for sample geometric means than for arithmetic means when n> or =2. Simulation results added support to that conclusion.

Tolerance of diverse amino acid substitutions at conserved positions in the nuclear export signal (NES) of HIV-1 Rev.

The effector domain of the Rev protein is a nuclear export signal (NES) that is responsible for transporting Rev and its bound congeners out of the nucleus and into the cytoplasm. Previous work has identified several critical residues in the NES and has led to the belief that NESs of the Rev type are necessarily leucine rich. Here we present the sequences of a large number of functional Rev molecules with NES mutations. The data indicate a previously unreported diversity in allowable residues at a number of positions, including each of the leucine residues previously considered essential.

Targeting to the HIV-1 RRE of the Influenza Virus NS1 Protein Effector Domain as a Potent, Specific Anti-HIV Agent.

The Rev axis of HIV autoregulation is one of two critical viral regulatory pathways required for expression of viral genomic and mRNA and for replication. Consequently it is an attractive therapeutic target. Previous studies have investigated the anti-HIV efficacy of targeting to the RRE (the viral RNA target sequence of the Rev axis) a trans-dominant negative inhibitor mutant Rev, M10. In this study we have fused a portion of the influenza virus NS1 protein (which normally inhibits polyA(+) mRNA transport and splicing) to the Rev M10 gene while deleting the NS1 poly(A) binding region. The resulting chimera demonstrates specific and enhanced inhibition of viral-RRE-containing RNA expression. Copyright 1997 S. Karger AG, Basel

Development of a Nuclear Export Signal Trapping Method for Isolating Genes with HIV Rev Activity.

We have developed a method for nuclear export signal trapping (NEST) to isolate functional Rev clones from various types of libraries such as libraries of Rev mutants. The expression libraries are cotransfected into COS cells together with a novel Rev-dependent immunoselectable CD28 expression plasmid, pCMV128-CD28. CD28-positive cells are recovered by FACS or by immune precipitation with magnetic beads, and the low-molecular-weight extra chromosomal DNA is recovered, amplified for Rev-containing DNA by PCR and recloned into expression plasmids. The resulting clones are enriched for functional Rev clones. These can be recovered efficiently after several repetitive NEST cycles. This technique may be usefully applied to study various regions of Rev, such as the RNA binding domain and the nuclear export signal, or effector domain and potentially to the isolation of cellular factors with nuclear export capabilities.

Two secondary structures for the RRE of HIV-1?

The RRE, the target sequence for the Rev protein of HIV-1, is a highly structured RNA sequence characterized by multiple stem loops. Although agreement on the stem/loop organization outside the high-affinity site was reached some time ago by several laboratories, recent work has suggested an alternative structure in which sequences from two of the stem/loops (IV and V) pair to form one long stem/loop (IV/V) when enough HIV material is present to allow the formation of an extended central stem structure (I/I'). Here we address the contribution of the original and alternative structures to function in RRE constructs with short and extended I'I' regions. We confirm that extended I/I' structures improve RRE function and may stabilize the overall structure. However, we find no evidence that an extended I/I' structure preferentially stabilizes either alternative structure with respect to the other. The two alternative structures are approximately functionally equivalent, and both are probably present in an in vivo population of RREs.

The Rev Axis of HIV-1 and Its Associated Host Cofactors: A Viral Window onto the Workings of Eukaryotic Posttranscriptional RNA Processing.

The Rev axis of HIV is one of two key autoregulatory pathways required for viral replication and pathogenesis. The viral Rev protein interacts with its RNA target sequence, the RRE, to overcome the inhibitory effects of constitutive repressor sequences and promote nucleocytoplasmic transport and expression of viral RNAs. The Rev axis is the subject of intense scrutiny not only because it plays a central role in the viral life cycle, but also because it offers a window onto the workings of key mechanisms of posttranscriptional regulation, including splicing, polyadenylation, degradation, transport, and translation. Recent reports have conclusively demonstrated a central role for transport in the Rev mechanism and have identified cellular factors that are good candidates for mediating the transport phenomena. Other potentially involved cellular factors are being investigated. Much of the apparent heterogeneity in the observed effects of Rev may actually derive from heterogeneity in the constitutive repressor sequences rather than from heterogeneity in the mechanism of action of Rev per se. Copyright 1996 S. Karger AG, Basel

Sequence specificity in the higher-order interaction of the Rev protein of HIV-1 with its target sequence, the RRE.

The Rev protein of human immunodeficiency virus type 1 (HIV-1) multimerizes along RNAs containing the Rev target sequence, the RRE. Although sequence-specific information is recognized in the high affinity or initial interaction, it is not known what role RNA-contained information plays in higher-order binding events. We have quantitatively studied the binding of Rev protein to the primary Rev binding domain (II + III) of wild-type and mutant RREs. RRE mutations that retain the basic secondary structure of wild type can separately and differentially alter the Kds for formation of the first, second, and third Rev/RRE complexes (C1, C2, and C3). The data suggest that Rev recognizes sequence-specific information in the RRE when it forms higher-order complexes. However, the formation of higher-order complexes is not as dependent on sequence-specific information as the first or lowest order binding interaction, which involves recognition of the high-affinity site.

The RRE of human immunodeficiency virus type 1 contributes to cell-type-specific viral tropism.

As part of a general program investigating the mechanism of the Rev axis of human immunodeficiency virus type 1 (HIV-1) autoregulation, a series of proviral HIV-1 mutants which differ from the parental HXB2 strain at selected positions within the RRE were constructed. All of the mutations were designed to perturb the RRE by introducing local helix disruptions without altering the coding potential of the overlapping envelope open reading frame. Viral replication in various cell types was monitored by a cell supernatant reverse transcriptase assay and Northern (RNA blot) analysis. All proviral RRE mutants displayed at least some impairment in replication. However, the relative impairment varied drastically among the various cell types tested. This suggests that the RRE may contribute to cell-type-specific viral tropism.