Effect of primer selection on estimates of GB virus C (GBV-C) prevalence and response to antiretroviral therapy for optimal testing for GBV-C viremia.

GB virus C (GBV-C; also called hepatitis G virus) is a common cause of infection associated with prolonged survival among HIV-infected individuals. The prevalences of GBV-C viremia vary widely in different studies, and there has been poor agreement among different laboratories performing GBV-C RNA detection in quality control studies. To determine the optimal method of measuring GBV-C RNA in clinical samples, samples obtained from 939 HIV-infected subjects were studied using reverse transcription (RT)-PCR methods amplifying four separate regions of the GBV-C genome. Primers amplifying the E2 coding region were 100% specific; however, their sensitivity was only 76.6%. In contrast, primers amplifying three additional conserved regions of the GBV-C genome (the 5' nontranslated region and the nonstructural protein-coding regions 3 and 5A) were more sensitive but produced higher rates of false-positive results. Using low-specificity primer sets influenced the significance of association between GBV-C viremia and response to antiretroviral therapy. Using a quantitative GBV-C RNA method, the GBV-C RNA concentration did not correlate with baseline or set point HIV RNA levels; however, a correlation between negative, low, and high GBV-C RNA levels and increasing reduction in HIV RNA following antiretroviral therapy was observed. Subjects with both GBV-C E2 antibody and viremia had significantly lower GBV-C RNA levels than did viremic subjects without E2 antibody. These studies demonstrate that accurate detection of GBV-C RNA by nested RT-PCR requires the use of primers representing multiple genome regions. Analyses based on testing with single primers do not lead to reliable conclusions about the association between GBV-C infection and clinical outcomes.

Sailing and sports medicine: a literature review.

Sailing medicine has been mainly addressed by healthcare professionals who happen to sail. Although there has been an increase in the number of studies of various aspects of sailing over the last 15 years, efforts to advance evidence based knowledge of sailing and sports medicine face unique obstacles. Recent interest in research by groups such as Olympic and America's Cup teams has produced beneficial changes.

Nitration of manganese superoxide dismutase during ocular inflammation.

Reactive nitrogen species, in particular, peroxynitrite (ONOO(-)) have been proposed to play an important role in the pathogenesis of endotoxin-induced uveitis (EIU). Tyrosine nitration by ONOO(-) has been shown in other model systems to inhibit the activity of the superoxide anion quenching enyzme, manganese superoxide dismutase (MnSOD), perhaps contributing to progression of disease. In this study, it is confirmed through immunoanalysis that nitrated proteins are produced during EIU, and furthermore, that MnSOD is a target of nitration during the inflammatory response. In addition, through microsequencing analyses, nitrated albumin--apparent in both control and EIU eyes--was identified. Positive immunostaining of nitrated proteins was seen in the ciliary epithelium, inflammatory cells, and protein exudate of eyes from rats injected with endotoxin. Incubation of nitrotyrosine immunoprecipitates from the iris and ciliary body (ICB) with a polyclonal antibody against MnSOD revealed that nitrated MnSOD was present only in the ICB of EIU rats. When the total activity of the enzyme was examined, it was observed that despite the presence of nitrated MnSOD, activity was increased relative to control. Analysis of MnSOD mRNA and protein from the ICB of both groups demonstrated an increase in mRNA expression and consequently a three- to five-fold increase in MnSOD protein in EIU rats as compared to control rats. Further examination of MnSOD protein expression through immunohistochemistry noted enhanced immunostaining in the ciliary epithelium of eyes of EIU rats. Additional investigation of a 70 kDa band apparent in nitrotyrosine immunoprecipitates from the ICB of control and EIU rats revealed that the plasma protein albumin is nitrated as well. This protein is present as a result of the breakdown of the blood-aqueous barrier during inflammation. In summary, two endogenous nitration targets, albumin and MnSOD, were identified. Nitrated MnSOD appears to be specifically targeted to the ICB during inflammation, underscoring the importance of the interface in EIU. Furthermore, the expression and activity of the enzyme is increased in the ICB during EIU, perhaps regulating reactive nitrogen species produced within the cells. This study implicates ONOO(-) in the pathogenesis of EIU and imparts the putative role MnSOD plays in disease resolution.

Warfarin resistance and enteral feedings: 2 case reports and a supporting in vitro study.

This study investigated whether an interaction between common enteral feeding products and warfarin exists. Two clinical cases of apparent interaction spurred a supporting in vitro study. Both the clinical cases and the in vitro study showed that several enteral feeding products bind warfarin, reducing the bioavailability of the drug. The binding appears to occur between warfarin and the protein component of the feeding product. This clinically important interaction is likely when warfarin and enteral feeding products are used concurrently. Clinicians should be aware of this potential interaction and monitor the therapy closely, particularly when enteral feeding is discontinued.

Balance and mobility following stroke: effects of physical therapy interventions with and without biofeedback/forceplate training.

BACKGROUND AND PURPOSE: Visual biofeedback/forceplate systems are often used for treatment of balance disorders. In this study, the researchers investigated whether the addition of visual biofeedback/forceplate training could enhance the effects of other physical therapy interventions on balance and mobility following stroke. SUBJECTS: The study included a sample of convenience of 13 outpatients with hemiplegia who ranged in age from 30 to 77 years (mean=60.4, SD=15.4) and were 15 to 538 days poststroke. METHODS: Subjects were assigned randomly to either an experimental group or a control group when the study began, and their cognitive and visual-perceptual skills were tested by a psychologist. Subjects were also assessed using the Berg Balance Scale and the Timed "Up & Go" Test before and after 4 weeks of physical therapy. Both groups received physical therapy interventions designed to improve balance and mobility 2 to 3 times per week. The experimental group trained on the NeuroCom Balance Master for 15 minutes of each 50-minute treatment session. The control group received other physical therapy for 50 minutes. RESULTS: Following intervention, both groups scored higher on the Berg Balance Scale and required less time to perform the Timed "Up & Go" Test. These improvements corresponded to increased independence of balance and mobility in the study population. However, a comparison of mean changes revealed no differences between groups. DISCUSSION AND CONCLUSION: Although both groups demonstrated improvement following 4 weeks of physical therapy interventions, no additional effects were found in the group that received visual biofeedback/forceplate training combined with other physical therapy.

Use of an intravitreal sustained-release cyclosporine delivery device for treatment of equine recurrent uveitis.

OBJECTIVE: To evaluate the use of an intravitreal sustained-release cyclosporine (CsA) delivery device for treatment of horses with naturally occurring recurrent uveitis. ANIMALS: 16 horses with recurrent uveitis. PROCEDURES: Horses with frequent recurrent episodes of uveitis or with disease that was progressing despite appropriate medication were selected for this study. Additional inclusion criteria included adequate retinal function as determined by use of electroretinography, lack of severe cataract formation, and no vision-threatening ocular complications (eg, retinal detachment, severe retinal degeneration, and posterior synechia). Sustained-release CsA delivery devices (4 microg of CsA/d) were implanted into the vitreous through a sclerotomy at the pars plana. Reexaminations were performed 1, 3, 6, and 12 months after implantation, then continued annually. Ophthalmic changes, number of recurrent episodes of uveitis, and vision were recorded. RESULTS: The rate of recurrent episodes after device implantation (0.36 episodes/y) was less than prior to surgery (75 episodes/y). In addition, only 3 horses developed episodes of recurrent uveitis after surgery. Vision was detected in 14 of 16 affected eyes at a mean follow-up time of 13.8 months (range, 6 to 24 months). CONCLUSIONS AND CLINICAL RELEVANCE: This intravitreal sustained-release CsA delivery device may be a safe and important tool for long-term treatment of horses with chronic recurrent uveitis.

Effect of an intravitreal cyclosporine implant on experimental uveitis in horses.

The purpose of this study was to determine the effects of an intravitreal device releasing cyclosporine A (CsA) on recurrent inflammatory episodes in experimental uveitis. Nine normal horses were immunized peripherally with H37RA-mTB antigen twice, and then received 25 microg of H37RA-mTB antigen intravitreally in the right eye and an equal volume of balanced salt solution intravitreally in the left eye. Two weeks later, the animals randomly received either a CsA or a polymer implant (without CsA) in both eyes 1 week following implantation of the devices, 25 microg of H37RA-mTB antigen was reinjected into the right eye of each animal. Clinical signs of ophthalmic inflammation were graded following injections and implantation. The animals from each group were euthanized at 3, 14, and 28 days following the second injection. Aqueous and vitreous humor protein concentrations were measured. The presence, number, and type (CD4, 5 and 8) of infiltrating inflammatory cells and amount of tissue destruction were determined. Total RNA was isolated and quantitative reverse transcriptase-polymerase chain reaction was performed for equine specific interleukin (IL) 2 and 4, interferon-gamma (IFN gamma) and beta-actin. In addition, aqueous and vitreous humor and peripheral blood were collected at the termination of the experiments and analyzed for CsA concentration by HPLC. Within 4h of the first intravitreal H37RA-mTB antigen injection, each animal developed epiphora, blepharospasm, mild corneal edema, aqueous flare, myosis, and vitreous opacity. The severity of signs peaked 48 to 72 h after injection and subsequently decreased back to normal within 14 days. Following the second injection, clinical signs in the eyes with the CsA device were less severe and significantly shorter in duration than signs with the polymer only implant eyes. Aqueous and vitreous humor protein levels, infiltrating cell numbers, total number of T-lymphocytes, and levels of IL-2 and IFN gamma-mRNA were significantly less in eyes with the CsA implant compared to eyes with the polymer only. CsA implants did not completely eliminate the development of a second ('recurrent') experimental inflammatory episode in these horses. However, the duration and severity of inflammation, cellular infiltration, tissue destruction, and pro-inflammatory cytokines RNA transcript levels were significantly less in those eyes implanted with the CsA device.

Long-term effect on the equine eye of an intravitreal device used for sustained release of cyclosporine A.

OBJECTIVE: To determine the long-term toxicity of an intravitreal device releasing continuous cyclosporinee A (CsA) in normal eyes of horses by evaluating clinical signs, electroretinography, and histopathology. Animals Studied Ten adult horses with normal ophthalmic examinations were used in this study Procedure(s) Four horses had one eye implanted with a CsA device, and six horses had the right eye implanted with a CsA-containing device (10 eyes with CsA in total) and the left eye (six eyes in total) with the device without drug (control). The implants were placed in the vitreous of the eyes through a sclerotomy 1 cm posterior to the limbus in the dorso-temporal quadrant of the eye. Scotopic electroretinograms were performed prior to implantation and at 1 week, and at 1, 3, 6, 9, and 12 months postimplantation. Two of the unilaterally implanted horses were euthanized at 1 weeks postimplantation, and two at 6 weeks postimplantation. Two of the bilaterally implanted horses were euthanized at 6 months, two at 9 months, and two at 12 months postimplantation. At euthanasia, the eyes were removed, aqueous and vitreous humor aspirated, and tissues fixed in 10% buffered formalin and processed for histopathology. CsA concentrations were measured by high pressure liquid chromatography in the aqueous and vitreous humors, and in peripheral blood. RESULTS: The devices were tolerated well in 14 of 16 eyes. There was minimal postoperative inflammation in most eyes, with a normal appearance within 7 days. In two eyes implanted with the CsA device, severe inflammation resulted in phthisis bulbi by 28 days. One of these eyes exhibited suspected bacterial endophthalmitis, and one had a sterile endophthalmitis and cataract presumably from trauma to the lens during implantation. In the other 14 eyes, no change was observed in the scotopic electroretinograms (ERG) from preoperative results, and no significant differences between the right (CsA) and left (control device) eyes were observed. CsA levels in the aqueous and vitreous humor, and peripheral blood were below the detection limit of the HPLC. Histologic findings revealed only a mild lymphoplasmacytic cellular infiltrate in the ciliary body and pars plana near the implantation site. CONCLUSIONS: The CsA devices were well tolerated with no long-term complications from the implants themselves. However, complications may occur from inadvertent implantation trauma or contamination during surgery. The long-term safety of the device may make it useful for delivery of CsA in the control of equine recurrent uveitis.

Characterization of T-lymphocytes in the anterior uvea of eyes with chronic equine recurrent uveitis.

Equine recurrent uveitis (ERU), a chronic, recurrent inflammation primarily of the anterior uveal tract, is the most common cause of blindness in horses. Recently, T-lymphocytes have been found to be the most numerous cell type to infiltrate the anterior uveal of horses with ERU. In the present study, we characterized the T-lymphocyte population in the anterior uveal tract of eyes of horses with chronic ERU by evaluating the microscopic appearance (histopathologic features), the T-lymphocyte subsets, and the relative levels and amounts of T-lymphocyte cytokine mRNA in the anterior uvea. Seven inflamed eyes (from six horses with chronic ERU) and 5 normal eyes (from five horses with nonocular problems) were studied. After clinical examination, the eyes were removed, ocular fluids were aspirated, and anterior uveal tissues (iris and ciliary body) were processed for histologic and molecular (RNA isolation) analyses. Histologic examination by hematoxylin and eosin (H and E) staining and immunohistochemistry evaluating T-lymphocyte subsets (anti-CD4, CD8, CD5) were performed for each sample. RNA samples were analyzed for levels of messenger (m) RNA specific for interleukin (IL)-2, 4, and interferon-gamma (IFNgamma) by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). Eyes with ERU exhibited characteristic clinical signs, including corneal edema, aqueous flare, posterior synechia, corpora nigra degeneration, and cataract formation. Histologically, infiltration of the uveal tract with lymphocytes, plasma cells, and macrophages was most evident in the ciliary body and base of the iris. Loss of tissue structure (destruction) was most evident in the ciliary processes. Infiltrating lymphocytes were predominantly CD4+ T-cells (e.g. 48% CD4+ and 18% CD8+ in the ciliary body stroma), as determined by immunohistochemistry. Few inflammatory cells were observed in the normal eyes. The QRT-PCR results revealed increased transcription of IL-2 and IFNgamma and low IL-4 mRNA expression in eyes with chronic ERU compared to normal eyes, demonstrating a Thelper (Th) 1-like inflammatory response in eyes with ERU.

Sports medicine and sailing.

Although there is little epidemiologic data in the sport of sailing, the identification of important trends can assist the clinician in successful evaluation, treatment, and rehabilitation of the individual. It appears that like other sports, the majority of injuries encountered are of the microtraumatic or overuse type. An understanding of biomechanics, the overload injury, and the sport of sailing will allow the development of a comprehensive rehabilitation program to ensure the optimal performance and safety of the sailor.

Suppression of NF-kappaB-dependent proinflammatory gene expression in human RPE cells by a proteasome inhibitor.

PURPOSE: To determine whether nuclear transcription factor-kappaB (NF-kappaB) is activated in human retinal pigment epithelial (hRPE) cells in response to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) alone or in combination and if so, whether expression of proinflammatory genes induced by these agents can be blocked by a proteasome inhibitor, MG-132, which inhibits the degradation of I kappaB, an NF-kappaB inhibitor, thereby preventing nuclear translocation of NF-kappaB. METHODS: Cultured hRPE were pretreated for 60 minutes with medium alone or medium containing the proteasome inhibitor MG-132 (20 microM) and then exposed to TNF-alpha (1.1 x 10(3) U/ml), IL-1beta (5 U/ml), or IFN-gamma (7.5 x 10(3) U/ml) alone or in combination (TII). Nuclear translocation of NF-kappaB was determined by fluorescence staining of the NF-kappaB Rel A (p65) subunit. Cytoplasmic I kappaB protein was measured by western blot analysis. Nuclear extract binding to kappaB DNA motifs was measured by electrophoretic mobility shift assay and antibody supershift assay. Steady state mRNA expression of the chemokines melanoma growth stimulating activity (MGSA)/gro-alpha, regulated on activation normal T-cell expression and secreted (RANTES), and monocyte chemoattractant protein (MCP-1), the cytokines IL-1beta and macrophage colony stimulating factor (M-CSF) and intercellular adhesion molecule-1 (ICAM-1) was evaluated by semiquantitative reverse transcription-polymerase chain reaction. Chemokine and cytokine protein secretion was measured by enzyme-linked immunosorbent assay. Cell-surface ICAM-1 expression was determined by flow cytometry. RESULTS: TNF-alpha, IL-1beta, and TII but not IFN-gamma alone caused degradation of I kappaB, Rel A nuclear translocation, and increased NF-kappaB DNA binding activity, effects that were blocked by pretreatment with MG-132. MG-132 suppressed MGSA/gro-alpha, RANTES, MCP-1, IL-1beta, M-CSF, and ICAM-1 mRNA expression and secreted RANTES, MCP-1, and M-CSF protein, and cell-surface ICAM-1 that were induced by IL-1beta, TNF-alpha, and TII. CONCLUSIONS: TNF-alpha, IL-1beta, and TII induce expression of proinflammatory cytokines and ICAM-1 in hRPE cells through an NF-kappaB-dependent signal transduction pathway. This effect is blocked by MG-132, a proteasome inhibitor that prevents I kappaB degradation. Inhibition of NF-kappaB may be a useful strategy to treat proliferative vitreoretinopathy and uveitis, ocular diseases initiated and perpetuated by cytokine activation.

Measurements and model of the cat middle ear: evidence of tympanic membrane acoustic delay.

In order to better understand the mechanics of tympanic membrane (TM) transduction at frequencies above a few kHz, the middle-ear (ME) impedance measured near the tympanic membrane is studied for three anesthetized cat ears after widely opening the ME cavities (MEC). Three conditions were measured: intact ossicles, drained cochlea, and disarticulated stapes. When the cochlear load is removed from the ME by disarticulating the stapes, the impedance magnitude varies by about +/- 25 dB in the 5- to 30-kHz range, with peaks and valleys at intervals of approximately 5 kHz. These measurements suggest middle-ear standing waves. It is argued that these standing waves reside in the TM. In contrast, the magnitude of the impedance for the intact case varies by less than +/- 10 dB, indicating that for this case the standing waves are damped by the cochlear load. Since the measurements were made within 2 mm of the TM, standing waves in the ear canal can be ruled out at these frequencies. Although the ME cavities were widely opened, reflections from the ME cavity walls or surrounding structures could conceivably result in standing waves. However, this possibility is ruled out by model predictions showing that such large standing waves in the ME cavity space would also be present in the intact case, in disagreement with the observation that standing waves are damped by cochlear loading. As a first-order approximation, the standing waves are modeled by representing the TM as a lossless transmission line with a frequency-independent delay of 36 microseconds. The delay was estimated by converting the impedance data to reflectance and analyzing the reflectance group delay. In the model the ossicles are represented as lumped-parameter elements. In contrast to previous models, the distributed and lumped parameter model of the ME is consistent with the measured impedance for all three conditions in the 200-Hz to 30-kHz region. Also in contrast with previous models, the ear-canal impedance is not mass dominated for frequencies above a few kHz. Finally, the present model is shown to be consistent, at high frequencies, with widely accepted transfer functions between (i) the stapes displacement and ear-canal pressure, (ii) the vestibule pressure and ear-canal pressure, and (iii) the umbo velocity and ear-canal volume velocity. An improved understanding of TM mechanics is important to improve hearing aid transducer design, ear-plug design, as well as otoacoustic emissions research.

Nitric oxide and peroxynitrite production in ocular inflammation.

Recent studies have implicated nitric oxide and peroxynitrite in the pathogenesis of many diseases, such as septic shock, arthritis, lung disease, and atherosclerosis. Nitric oxide (.NO) exerts many diverse effects on vascular tone, affecting neurotransmission and cellular cytotoxicity/communication. Our laboratory and others have documented a proinflammatory role for .NO in ocular inflammation. Uveitis, which is an inflammation of the highly vascular uveal tract in the eye, is a debilitating condition that can lead to visual impairment and blindness. It is characterized by acute, recurrent, or persistent inflammation with disruption of the blood-aqueous barrier and is accompanied by protein leakage and leukocyte infiltration into the aqueous humor and anterior chamber. Systemic injection of endotoxin into mice and rats, or intraocular injection of endotoxin into mice, rats, and rabbits induces acute uveitis, which clinically and histologically resembles acute anterior uveitis in humans. These models facilitate the study of pathogenic mechanisms that contribute to ocular inflammation. In addition to .NO, superoxide anion radicals (O2.-), and peroxynitrite (ONOO-), the products of the reaction between .NO and O2.-, are also implicated in uveitis. The role of peroxynitrite in ocular inflammation is still largely unknown. Characterization of the roles of these important uveitic mediators in the ocular inflammatory response will provide information critical to the understanding of the pathogenesis of intraocular inflammation so that more effective therapeutic intervention(s) can be developed.

The lens influences aqueous humor levels of transforming growth factor-beta 2.

BACKGROUND: Transforming growth factor-beta 2 (TGF-beta 2) is a pluripotent cytokine which has been suggested to play a number of roles in ocular physiologic and pathologic states. Intraocular fluid (i.o.f.) levels of TGF-beta 2 are quite high. Although the sources of ocular TGF-beta are not completely defined, the retinal pigment epithelium, the epithelium of the ciliary body and trabecular meshwork cells all secrete it. In this study we utilized canine lens and rabbit ciliary pigmented epithelial cell cultures to quantitate the in vitro secretion of TGF-beta 2. In addition, the effects of aphakia or the presence of cataractous lenses on IOF TGF-beta 2 levels were determined. METHODS: Lens and ciliary body epithelial cell culture supernatants and aqueous humors were assayed for total TGF-beta 2 levels by ELISA and bioassay. RESULTS: TGF-beta 2 accumulated in the media bathing lens epithelial cell cultures (0.7 +/- 0.03 ng/ml at day 2) and ciliary pigmented epithelial cell cultures (0.8 +/- 0.06 ng/ml at day 2) in a time-dependent manner. Surprisingly, aqueous humor from aphakic rabbit eyes contained significantly higher levels of TGF-beta 2 than their contralateral phakic controls. Furthermore, aqueous humor from canine eyes with cataracts also contained significantly higher levels of TGF-beta 2 than normal eyes. CONCLUSIONS: These results suggest that the lens secretes TGF-beta 2 and that the presence and status of the lens may influence IOF TGF-beta 2 levels.

Measurement of distortion product phase in the ear canal of the cat.

Amplitudes of odd order distortion products (DPs) that are detected in animal ear canals have been used to probe cochlear health, to search for cochlear amplification, and to measure aspects of cochlear mechanical frequency response. Like the DP amplitude, DP phase is also an important measure of the cochlear mechanical response. Reported here are measurements of DP phase in the ear canal of the cat. The phase data show frequency-dependent time delays. One of these delays is a function of f2, the frequency of the higher-frequency primary. Hence the DP phase phi d is of the form phi d = phi 0 + omega d tau, where omega d is the DP angular frequency and tau is a fixed time delay. Our results show that phi d is independent of input level a2 as long as the ratio a2/a1 < or = 2, where a2 and a1 are the amplitudes of the input tones. As a2/a1 becomes greater than two, the fixed time delays increase for DPs whose frequencies are less than the frequencies of the input tones. When both levels are varied together the delay increases as the levels decrease. There can be phase changes as large as pi associated with deep nulls in the DP magnitude for the two lower-frequency DPs. Features of the nulls may be modeled assuming that there is partial reflection of the DP wave from the DP place. The assumption of energy remitted from the DP place also explains amplitude-ratio-dependent time delays and 2 pi level-dependent bifurcations in phase. The DP phase shows different dependencies for f2 < 1 kHz compared to f2 > 2 kHz.

The Saccharomyces cerevisiae MEC1 gene, which encodes a homolog of the human ATM gene product, is required for G1 arrest following radiation treatment.

The Saccharomyces cerevisiae gene MEC1 represents a structural homolog of the human gene ATM mutated in ataxia telangiectasia patients. Like human ataxia telangiectasia cell lines, mec1 mutants are defective in G2 and S-phase cell cycle checkpoints in response to radiation treatment. Here we show an additional defect in G1 arrest following treatment with UV light or gamma rays and map a defective arrest stage at or upstream of START in the yeast cell cycle.