Age-related macular degeneration is not macular process only--peripheral retina is attacked too

On a small island of Rab, geographical latitude 44 degrees 40' N, with the highest solar radiation among Adriatic islands, 1371 patients were examined in 2003-2005 period, with the aim of estimating the frequency of the patients number with Age-related Macular Degeneration (AMD), and further if the peripheral retina is damaged similarly as the macular region. In the first group of agriculturists and fishermen (n = 1300) we estimated the AMD, initial and middle stage of AMD, in 18% of population, but in urban population only by 2 patients. Perimetric analysis with computerized Kowa perimeter, estimated that the peripheral retina is affected similarly as the macular region. Author concludes that for this reason, the usual term of "Age-related Macular Degeneration" should necessarily be named with the suffix "Peripheral", or "Age-related Retinopathy".     

Isolation and characterization of a spontaneously immortalized bovine retinal pigmented epithelial cell line

Background  

The Retinal Pigmented Epithelium (RPE) is juxtaposed with the photoreceptor outer segments of the eye. The proximity of the photoreceptor cells is a prerequisite for their survival, as they depend on the RPE to remove the outer segments and are also influenced by RPE cell paracrine factors. RPE cell death can cause a progressive loss of photoreceptor function, which can diminish vision and, over time, blindness ensues. Degeneration of the retina has been shown to induce a variety of retinopathies, such as Stargardt's disease, Cone-Rod Dystrophy (CRD), Retinitis Pigmentosa (RP), Fundus Flavimaculatus (FFM), Best's disease and Age-related Macular Degeneration (AMD). We have cultured primary bovine RPE cells to gain a further understanding of the mechanisms of RPE cell death. One of the cultures, named tRPE, surpassed senescence and was further characterized to determine its viability as a model for retinal diseases.     

Biosynthesis,Characterization, and Efficacy in Retinal Degenerative Diseases of Lens Epithelium-derived Growth Factor Fragment (LEDGF1–326), a Novel Therapeutic Protein

For vision-threatening retinitis pigmentosa and dry age-related macular degeneration, there are no United States Food and Drug Administration (FDA)-approved treatments. We identified, biosynthesized, purified, and characterized lens epithelium-derived growth factor fragment (LEDGF_1–326) as a novel protein therapeutic. LEDGF_1–326 was produced at about 20 mg/liter of culture when expressed in the Escherichia coli system, with about 95% purity and aggregate-free homogeneous population with a mean hydrodynamic diameter of 9 ± 1 nm. The free energy of unfolding of LEDGF_1–326 was 3.3 ± 0.5 kcal mol⁻¹, and melting temperature was 44.8 ± 0.2 °C. LEDGF_1–326 increased human retinal pigment epithelial cell viability from 48.3 ± 5.6 to 119.3 ± 21.1% in the presence of P23H mutant rhodopsin-mediated aggregation stress. LEDGF_1–326 also increased retinal pigment epithelial cell FluoSphere uptake to 140 ± 10%. Eight weeks after single intravitreal injection in Royal College of Surgeons (RCS) rats, LEDGF_1–326 increased the b-wave amplitude significantly from 9.4 ± 4.6 to 57.6 ± 8.8 μV for scotopic electroretinogram and from 10.9 ± 5.6 to 45.8 ± 15.2 μV for photopic electroretinogram. LEDGF_1–326 significantly increased the retinal outer nuclear layer thickness from 6.34 ± 1.6 to 11.7 ± 0.7 μm. LEDGF_1–326 is a potential new therapeutic agent for treating retinal degenerative diseases.     

Development of tumor lysis syndrome (TLS): A potential risk factor in cancer patients receiving anticancer therapy

Tumor lysis syndrome (TLS) is characterized by hyperuricaemia, hyperphosphatemia, hyperkalaemia, as well as hypocalcaemia due to the breakdown of tumor cells undergoing cancer therapy (chemo/radio). Therefore it is of interest to evaluate oxidative stress using selective biological markers [Malondialdehyde (MDA), Superoxide Dismutase (SOD), Glutathione (GSH) and Catalase (CAT)] in TLS. We report the marked differences (statistically significant with control) observed among a selected set of biomarkers of oxidative stress (MDA = 8.66±1.37; SOD = 0.15±0.11; GSH = 2.25±.77; CAT = 0.76±.57) in TLS patients in addition to other conventional biomarkers. Moreover, correlation was investigated among the parameters of oxidative stress and other circulating biomarkers of TLS. Data suggest the use of SOD, MDA, and GSH as potential diagnostic biomarker for TLS with other biomarkers.     

Visual Pigments of Goldfish Cones : Spectral Properties and Dichroism

Freshly isolated retinal photoreceptors of goldfish were studied microspectrophotometrically. Absolute absorptance spectra obtained from dark-adapted cone outer segments reaffirm the existence of three spectrally distinct cone types with absorption maxima at 455 ± 3,530 ± 3, and 625 ± 5 nm. These types were found often recognizable by gross cellular morphology. Side-illuminated cone outer segments were dichroic. The measured dichroic ratio for the main absorption band of each type was 2–3:1. Rapidly bleached cells revealed spectral and dichroic transitions in regions near 400–410, 435–455, and 350–360 nm. These photoproducts decay about fivefold as fast as the intermediates in frog rods. The spectral maxima of photoproducts, combined with other evidence, indicate that retinene₂ is the chromophore of all three cone pigments. The average specific optical density for goldfish cone outer segments was found to be 0.0124 ± 0.0015/µm. The spectra of the blue-, and green-absorbing cones appeared to match porphyropsin standards with half-band width Δν = 4,832 ± 100 cm^–1. The red-absorbing spectrum was found narrower, having Δν = 3,625 ± 100 cm^–1. The results are consistent with the notion that visual pigment concentration within the outer segments is about the same for frog rods and goldfish cones, but that the blue-, and green-absorbing pigments possess molar extinctions of 30,000 liter/mol cm. The red-absorbing pigment was found to have extinction of 40,000 liter/mol cm, assuming invariance of oscillator strength among the three cone spectra.     

Multi-annual performance evaluation of laboratories in post-mortem diagnosis of animal rabies: Which techniques lead to the most reliable results in practice?

Rabies diagnosis proficiency tests on animal specimens using four techniques (FAT, RTCIT, conventional RT-PCR and real-time RT-PCR) were organised over 10 years (2009–2019). Seventy-three laboratories, of which 59% were from Europe, took part. As the panels were prepared with experimentally-infected samples, the error rate of laboratories on positive and negative samples was accurately estimated. Based on fitted values produced by mixed modelling including the variable “laboratory” as a random variable to take into account the longitudinal design of our dataset, the technique that provided the most concordant results was conventional RT-PCR (99.3%; 95% CI 99.0–99.6), closely followed by FAT (99.1%; 95% CI 98.7–99.4), real-time RT-PCR (98.7%; 95% CI 98.1–99.3) and then RTCIT (96.8%; 95% CI 95.8–97.7). We also found that conventional RT-PCR provided a better diagnostic sensitivity level (99.3% ±4.4%) than FAT (98.7% ±1.6%), real-time RT-PCR (97.9% ±0.8%) and RTCIT (95.3% ±5.1%). Regarding diagnostic specificity, RTCIT was the most specific technique (96.4% ±3.9%) followed closely by FAT (95.6% ±3.8%), real-time RT-PCR (95.0% ±1.8%) and conventional RT-PCR (92.9% ±0.5%). Due to multiple testing of the samples with different techniques, the overall diagnostic conclusion was also evaluated, and found to reach an inter-laboratory concordance level of 99.3%. The concordance for diagnostic sensitivity was 99.6% ±2.0% and for diagnostic specificity, 98.0% ±8.5%. Molecular biology techniques were, however, found to be less specific than expected. The potential reasons for such findings are discussed herein. The regular organisation of performance tests has contributed to an increase in the performance of participating laboratories over time, demonstrating the benefits of such testing. Maintaining a high-quality rabies diagnosis capability on a global scale is key to achieving the goal of eliminating dog-mediated human rabies deaths. The regular organisation of exercises on each continent using selected local strains to be tested according to the local epidemiological situation is one factor that could help increase reliable diagnosis worldwide. Rabies diagnosis capabilities could indeed be enhanced by providing adequate and sustainable proficiency testing on a large scale and in the long term     

Novel Pharmacologic Targeting of Tight Junctions and Focal Adhesions in Prostate Cancer Cells

Cancer cell resistance to anoikis driven by aberrant signaling sustained by the tumor microenvironment confers high invasive potential and therapeutic resistance. We recently generated a novel lead quinazoline-based Doxazosin® derivative, DZ-50, which impairs tumor growth and metastasis via anoikis. Genome-wide analysis in the human prostate cancer cell line DU-145 identified primary downregulated targets of DZ-50, including genes involved in focal adhesion integrity (fibronectin, integrin-α6 and talin), tight junction formation (claudin-11) as well as insulin growth factor binding protein 3 (IGFBP-3) and the angiogenesis modulator thrombospondin 1 (TSP-1). Confocal microscopy demonstrated structural disruption of both focal adhesions and tight junctions by the downregulation of these gene targets, resulting in decreased cell survival, migration and adhesion to extracellular matrix (ECM) components in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. Stabilization of cell-ECM interactions by overexpression of talin-1 and/or exposing cells to a fibronectin-rich environment mitigated the effect of DZ-50. Loss of expression of the intracellular focal adhesion signaling effectors talin-1 and integrin linked kinase (ILK) sensitized human prostate cancer to anoikis. Our findings suggest that DZ-50 exerts its antitumor effect by targeting the key functional intercellular interactions, focal adhesions and tight junctions, supporting the therapeutic significance of this agent for the treatment of advanced prostate cancer.     

The Electroretinogram of a Diurnal Gecko

Using the electroretinogram as the criterion of retinal activity the flicker fusion frequency, course of dark adaptation, and spectral sensitivity of the pure cone retina of the diurnal gecko, Phelsuma inunguis, were investigated. Both the curve relating flicker fusion frequency to stimulus intensity and that relating the amplitude of the flicker response to stimulus intensity showed a break as the intensity was increased. The dark adaptation curve was that typical of cone retinae; there was no break, adaptation was relatively rapid, and there was a total increase of sensitivity of only about 3 log units. The spectral sensitivity curve showed two maxima, a major one at about 560 mµ and another at about 460 mµ. Chromatic adaptation with red and blue lights demonstrated the presence of two independent mechanisms. Although red adaptation could not have had a direct effect on the pigment responsible for the "blue" mechanism the sensitivity of this mechanism was depressed by red adaptation. The possible relationships of the two mechanisms are discussed.     

Prednisolone neuroprotective therapy in age-related macular degeneration

134 patients with Age-related Macular Degeneration (AMD) (aging 47-75 years) were treated in therapy procedure with parabulbar injections of Methylprednisolone Acetate and Prednisolone Acetate. In the first group of patients with AMD (n = 71 patients) were treated with Methylprednisolone acetate, and second group (n = 63 patients) with Prednisolone acetate. Each patient was given doses of 60 mg, through two weeks, 10 mg every second day. It's estimated in all patients ameliorate in macular threshold, so that it's in the group with Methylprednisolone treatment, ameliorate effect begins after first week, than in second group, treated with Prednisolone, initial ameliorate effect is after second week. Complete effect in both groups is after 2 months. It can be concluded that the treatment of AMD with glucocorticoids has the ameliorate effect in vision loss and it is decided that earlier effect in the group treated with Methylprednisolone, is probably of higher affinity for glucocorticoid receptors.     

Evaluation of oxidative stress, 3-Nitrotyrosine,and HMGB-1 levels in patients with wet type Age-Related Macular Degeneration

BackgroundThis study aims to compare serum HMGB-1, 3-nitrotyrosine (3-NT), TAS, TOS, and OSI levels in Wettype Age-Related Macular Degeneration (wAMD) patients and healthy controls to determine the correlation of these parameters with each other.MethodsThirty patients with Wet-type Age-Related Macular Degeneration (wAMD) and 27 healthy adults, as controls were enrolled in the study. We determined the TAS and TOS levels in serum samples of both groups using commercial kits on a microplate reader. Serum HMGB-1 and 3-NT levels were measured with the enzyme-linked immunosorbent assay method.ResultsHMGB-1 levels were significantly higher in the patient group (137.51 pg/mL, p=0.001), while there was no difference between the two groups in serum 3-NT levels (p=0.428). A statistically significant difference found in the levels of TOS and OSI (p=0.001 and p=0.045, respectively) between the patients and controls, however, no significant difference was observed between the groups in terms of TAS levels (p=0.228).ConclusionsOxidative stress and HMGB-1 levels were increased in wAMD patients and enhanced oxidative stress may be associated with increased tissue necrosis and inflammation. Thus administration of antioxidant treatment in addition to routine therapy should be considered in wAMD.     

A Novel Electrocardiographic Index for the Diagnosis of Diastolic Dysfunction

Background

Although the assessment of diastolic dysfunction (DD) is an integral part of routine cardiologic examinations, little is known about associated electrocardiographic (ECG) changes. Our aim was to investigate a potential role of ECG indices for the recognition of patients with DD.

Methods and Results

ECG parameters correlating with echocardiographic findings of DD were retrospectively assessed in a derivation group of 172 individuals (83 controls with normal diastolic function, 89 patients with DD) and their diagnostic performance was tested in a validation group of 50 controls and 50 patients. The patient group with a DD Grade 1 and 2 showed longer QTc (422±24ms and 434±32ms vs. 409±25ms, p<0.0005) and shorter Tend–P and Tend–Q intervals, reflecting the electrical and mechanical diastole (240±78ms and 276±108ms vs. 373±110ms, p<0.0001; 409±85ms and 447±115ms vs. 526±119ms, p<0.0001). The PQ–interval was significantly longer in the patient group (169±28ms and 171±38ms vs. 153±22ms, p<0.005). After adjusting for possible confounders, a novel index (Tend–P/[PQxAge]) showed a high performance for the recognition of DD, stayed robust in the validation group (sensitivity 82%, specificity 93%, positive predictive value 93%, negative predictive value 82%, accuracy 88%) and proved a substantial added value when combined with the indexed left atrial volume (LAESVI, sensitivity 90%, specificity 92%, positive predictive value 95%, negative predictive value 86%, accuracy 91%).

Conclusions

A novel electrocardiographic index Tend–P/(PQxAge) demonstrates a high diagnostic accuracy for the diagnosis of DD and yields a substantial added value when combined with the LAESVI.     

Slow Cone Reflectance Changes during Bleaching Determined by Adaptive Optics Scanning Laser Ophthalmoscope in Living Human Eyes

To investigate the changes in the reflectance of human cone photoreceptors by an adaptive optics scanning laser ophthalmoscope (AO-SLO) during photobleaching. A custom-built AO-SLO with an observation light of 840-nm was used to measure the cone densities and the reflectance changes during bleaching by 630 nm red light emitting diodes. Measurements were made at 1° and 3° temporal to the fovea within an area of 1° × 1° in 8 eyes of 8 normal subjects. After dark-adaptation, images of the cone mosaics were recorded continuously for 5-min before, 5-min during, and after 5-min of light stimulation with a sampling rate of 5-Hz. The first positive peak (P1) was observed at 72.2 ± 15.0-s and a second positive peak (P2) at 257.5 ± 34.5-s at 1°. The increase of the reflectance of P1 was significantly larger at 1° (34.4 ± 13.9%) than at 3° (26.0 ± 10.5%; P = 0.03, Wilcoxon’s signed rank test). The average cone density at 1° (51123.13 ± 1401.23 cells/mm²) was significantly larger than that at 3° (30876.13 ± 1459.28 cells/mm²; P <0.001, Wilcoxon’s signed rank test). The changes in the reflectance of the cones during bleaching by red light had two peaks. The two peaks may be caused by regeneration of cone photopigment during bleaching.     

Treatment of macular degeneration (a controlled case)

Our controlled case describes the drug possibility to stabilize the exudative form of Age-related Macular Degeneration. The new approach of selective destruction of choroidal neovascularization (CNV) can be applied in patients with subfoveal lesions. Photodynamic therapy (PDT) is based on the reaction of photoactivable drug with the light of low-energy laser beam. So far only verteporfin [Visudyne, Novartis] as sensitizer is marketed and laser with wavelength of 689 nm is used. But it is questionable whether to apply this very costly treatment to extremely old people. An example of treatment of 90-year old woman with AMD with classic form of CNV is presented. Her BCVA was 0.05 OD. She underwent four session of PDT. At the last visit (eighteen months after initial and ten months after last session of PDT) the BCVA remained 0.05 OD. The patient is using a special magnifying lens for reading achieving near vision of 0.32. The presented example indicates the necessity of detailed study of every case to promote the therapeutic decisions for the benefit of progress in the field.     

Human cone light sensitivity and melatonin rhythms following 24-hour continuous illumination

This study investigates the possibility of an endogenous circadian rhythm in retinal cone function in humans. A full-field cone electroretinogram (ERG) was performed every 2 h for 24 h under continuous rod-saturating ambient white light (53 ± 30 lux; pupils dilated) in nine healthy subjects. Distinct circadian variations were superimposed upon a gradual decrease in cone responsiveness to light, demonstrated most reliably in the implicit times of b-wave and oscillatory potentials, and to a lesser extent in amplitude and a-wave implicit times. After mathematical correction of the linear trend, the cone response was found to be greatest around 20:00 h and least around 06:00 h. The phase of the ERG circadian rhythm was not synchronized with the phase of the salivary melatonin rhythm measured the previous evening. Melatonin levels measured under constant light on the day of ERG assessments were suppressed by 53% on average compared to melatonin profiles obtained previously under near-total darkness in seven participants. The progressive decline in cone responsiveness to light over the 24 h may reflect an adaptation of the cone-driven retinal system to constant light, although the mechanism is unclear. The endogenous rhythm of cone responsiveness to light may be used as an additional index of central or retinal circadian clock time.     

Repolarization Parameters Are Associated With Mortality In Chagas Disease Patients In The United States

Objective

The goal of this study was to examine the association between ECG repolarization parameters and mortality in Chagas disease (CD) patients living in the United States.

Methods

CD patients with cardiomyopathy (CM) and bundle branch block (BBB) or BBB alone were compared to age- and sex-matched controls. QT interval, QT dispersion (QTd), T wave peak to T wave end duration (Tp-Te) and T wave peak to T wave end dispersion ((Tp-Te)d) were measured. Presence of fractionated QRS (fQRS) was also assessed. The main outcome measure was the association between ECG parameters and mortality or need for cardiac transplant.

Results

A total of 18 CM and 13 BBB CD patients were studied with 97% originating from Mexico or Central America. QTd (60.0±15.0 ms vs 43.5±9.8 ms, P=0.0002), Tp-Te (102.6±29.3 ms vs 77.1±11.0 ms, P=0.0002) and (Tp-Te)d (39.5±9.4 ms vs 22.7±7.6 ms, P<0.0001) were prolonged in CD CM patients compared to CM controls. Chagas CM patients had more fQRS then controls (84.2±0.10% vs 33.3±0.11%, p=0.0005). QTd (59.9±15.0 ms vs 29.5±6.9 ms, P=0.0001) and (Tp-Te)d (40.0±15.9 ms vs 18.5±5.4 ms, p<0.0001) were longer in the CD BBB group compared to BBB controls. Univariate analysis showed QTd (56.9±15.0 ms vs 46.5±17.3 ms, p=0.0412) and (Tp-Te)d (36.8±13.5 ms vs 28.5±13.3 ms, p=0.0395) were associated with death and/or need for cardiac transplant.

Conclusion

Our results indicate that P-max and PD are useful electrocardiographic markers for identifying the β-TM-high-risk patients for AF onset, even when the cardiac function is conserved.     

Lipids,oxidized lipids,oxidation-specific epitopes,and Age-related Macular Degeneration

Age-related Macular Degeneration (AMD) is the leading cause of blindness among the elderly in western societies. While antioxidant micronutrient treatment is available for intermediate non-neovascular disease, and effective anti-vascular endothelial growth factor treatment is available for neovascular disease, treatment for early AMD is lacking due to an incomplete understanding of the early molecular events. The role of lipids, which accumulate in the macula, and their oxidation, has emerged as an important factor in disease development. These oxidized lipids can either directly contribute to tissue injury or react with amine on proteins to form oxidation-specific epitopes, which can induce an innate immune response. If inadequately neutralized, the inflammatory response from these epitopes can incite tissue injury during disease development. This review explores how the accumulation of lipids, their oxidation, and the ensuing inflammatory response might contribute to the pathogenesis of AMD. This article is part of a Special Issue entitled: Lipid modification and lipid peroxidation products in innate immunity and inflammation edited by Christoph J. Binder .     

Initial experience of a cohort of patients with hypertrophic cardiomyopathy undergoing biventricular pacing

Background

Dual chamber pacing improves functional status and reduces left ventricular outflow tract gradients in some, but not all patients with hypertrophic cardiomyopathy (HCM) by altering ventricular depolarisation. We investigated the use of biventricular (BIV) pacing in symptomatic patients with HCM.

Method

8 patients aged 58±7yrs with symptomatic HCM underwent BIV pacing. 5 patients had LVOT gradients >30mmHg. Ventricular electrodes were placed in the right ventricle (RV) and a branch of the coronary sinus. An atrial electrode was inserted to achieve BIV pacing with a short AV delay. The short-term effects of different pacing modalities were assessed using 2-D and Doppler echocardiography. Symptoms and exercise tolerance were assessed after a month of each pacing mode. Long-term follow up data was available for 5 years.

Results

Baseline EF was 67±14% and mean QRS duration was 132±26msecs. BIV pacing reduced QRS duration compared to RV pacing (129±46 vs. 205±54msecs, p<0.005). Five of the seven patients had baseline LVOT gradients (mean 67±25mmHg) that decreased to 41±15mm Hg with RV pacing (p<0.01) and 25±15mmHg with BIV pacing (p<0.005). Improvements in exercise time with active pacing occurred in six out of eight patients (75%), three (37.5%) had optimal exercise times with RV pacing and three with BIV pacing. Of the three patients with short term improvements with BIV pacing, one died 4 years post implant, one deteriorated with LV dilatation and one had the system explanted for infection.

Conclusion

BIV pacing showed short-term beneficial effects in some patients over and above RV pacing alone.     

Neutral endopeptidase inhibitor suppresses the early phase of atrial electrical remodeling in a canine rapid atrial pacing model

Introduction

We examined the acute effects of neutral endopeptidase inhibitor on the hemodynamics and electrical properties of dogs subjected to rapid atrial pacing.

Methods

Ten beagle dogs were used and divided into two groups with and without candoxatril, a neutral endopeptidase inhibitor preadministration. Before and after the 6 hours rapid atrial pacing from the right atrial appendage, the hemodynamics, atrial effective refractory period, and monophasic action potential duration of the right atrial appendage were measured and blood samples were collected. Atrial tissue was also excised after the experiment.

Results

Candoxatril significantly increased plasma ANP levels (Control: 88.4 ± 50.25 vs. Candoxatril: 197.1 ± 32.09 pg/ml, p = 0.004) and prevented reductions in atrial effective refractory period and monophasic action potential duration. We further demonstrated that the treated animals exhibited significantly higher levels of atrial tissue cyclic GMP (Control: 28.1 ± 1.60 fmol/mg vs. Candoxatril: 44.5 ± 12.28 fmol/mg, p = 0.034) as well as that of plasma cyclic GMP (Control: 32 ± 5.5 vs. Candoxatril: 42 ± 7.1 pg/ml, p = 0.028).

Conclusion

Candoxatril suppressed the shortening of atrial effective refractory period and monophasic action potential duration in the rapid atrial pacing model. As plasma ANP and the atrial tissue levels of cyclic GMP were higher in the Candoxatril group than the control, this effect was considered to appear through the reduction of calcium overload caused by ANP and cyclic GMP.     

Synthesis of a Vpr-Binding Derivative for Use as a Novel HIV-1 Inhibitor

The emergence of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus type 1 (HIV-1) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. We previously identified a potential parent compound, hematoxylin, which suppresses the nuclear import of HIV-1 via the Vpr-importin α interaction and inhibits HIV-1 replication in a Vpr-dependent manner by blocking nuclear import of the pre-integration complex. However, it was unstable. Here, we synthesized a stable derivative of hematoxylin that bound specifically and stably to Vpr and inhibited HIV-1 replication in macrophages. Furthermore, like hematoxylin, the derivative inhibited nuclear import of Vpr in an in vitro nuclear import assay, but had no effect on Vpr-induced G2/M phase cell cycle arrest or caspase activity. Interestingly, this derivative bound strongly to amino acid residues 54–74 within the C-terminal α-helical domain (αH3) of Vpr. These residues are highly conserved among different HIV strains, indicating that this region is a potential target for drug-resistant HIV-1 infection. Thus, we succeeded in developing a stable hematoxylin derivative that bound directly to Vpr, suggesting that specific inhibitors of the interaction between cells and viral accessory proteins may provide a new strategy for the treatment of HIV-1 infection.     

Peripheral Blood Mitochondrial DNA as a Biomarker of Cerebral Mitochondrial Dysfunction following Traumatic Brain Injury in a Porcine Model

Background

Traumatic brain injury (TBI) has been shown to activate the peripheral innate immune system and systemic inflammatory response, possibly through the central release of damage associated molecular patterns (DAMPs). Our main purpose was to gain an initial understanding of the peripheral mitochondrial response following TBI, and how this response could be utilized to determine cerebral mitochondrial bioenergetics. We hypothesized that TBI would increase peripheral whole blood relative mtDNA copy number, and that these alterations would be associated with cerebral mitochondrial bioenergetics triggered by TBI.

Methodology

Blood samples were obtained before, 6 h after, and 25 h after focal (controlled cortical impact injury: CCI) and diffuse (rapid non-impact rotational injury: RNR) TBI. PCR primers, unique to mtDNA, were identified by aligning segments of nuclear DNA (nDNA) to mtDNA, normalizing values to nuclear 16S rRNA, for a relative mtDNA copy number. Three unique mtDNA regions were selected, and PCR primers were designed within those regions, limited to 25-30 base pairs to further ensure sequence specificity, and measured utilizing qRT-PCR.

Results

Mean relative mtDNA copy numbers increased significantly at 6 and 25 hrs after following both focal and diffuse traumatic brain injury. Specifically, the mean relative mtDNA copy number from three mitochondrial-specific regions pre-injury was 0.84 ± 0.05. At 6 and 25 h after diffuse non-impact TBI, mean mtDNA copy number was significantly higher: 2.07 ± 0.19 (P < 0.0001) and 2.37 ± 0.42 (P < 0.001), respectively. Following focal impact TBI, relative mtDNA copy number was also significantly higher, 1.35 ± 0.12 (P < 0.0001) at 25 hours. Alterations in mitochondrial respiration in the hippocampus and cortex post-TBI correlated with changes in the relative mtDNA copy number measured in peripheral blood.

Conclusions

Alterations in peripheral blood relative mtDNA copy numbers may be a novel biosignature of cerebral mitochondrial bioenergetics with exciting translational potential for non-invasive diagnostic and interventional studies.