Infectious and inflammatory diseases have repeatedly shown strong genetic associations within the major histocompatibility complex (MHC); however, the basis for these associations remains elusive. To define host genetic effects on the outcome of a chronic viral infection, we performed genome-wide association analysis in a multiethnic cohort of HIV-1 controllers and progressors, and we analyzed the effects of individual amino acids within the classical human leukocyte antigen (HLA) proteins. We identified >300 genome-wide significant single-nucleotide polymorphisms (SNPs) within the MHC and none elsewhere. Specific amino acids in the HLA-B peptide binding groove, as well as an independent HLA-C effect, explain the SNP associations and reconcile both protective and risk HLA alleles. These results implicate the nature of the HLA–viral peptide interaction as the major factor modulating durable control of HIV infection.
The diagnosis and treatment of breast cancer can adversely affect quality of life. Here the aim was to determine the effects of reflexology on host defences and endocrine function in women with early breast cancer. Six weeks after surgery for early breast cancer, 183 women were randomly assigned to self-initiated support (SIS), SIS plus foot reflexology, or SIS plus scalp massage. Peripheral blood mononuclear cells and serum were isolated at T1 (6 weeks post surgery; baseline), T2 and T3 (4 and 10 weeks post completion of intervention, respectively). Lymphocyte phenotyping found that CD25+ cells were significantly higher in the massage group compared with the SIS group at T3. The percentage of T cells, and more specifically the T helper subset expressing IL4, decreased significantly in the massage group compared with the SIS group at T3. This change was accompanied by an increase in the percentage of CD8+ T cytotoxic cells expressing IFNγ in the massage group. Natural killer and lymphokine activated killer cell cytotoxicity measurements, serum levels of cortisol, prolactin and growth hormone, and flow cytometric assessment of their corresponding receptors all revealed no significant differences between the three groups of patients. This study provides evidence that the immunological balance of patients can be altered in a potentially beneficial manner by massage. The original trial was registered with the International Standard Randomised Controlled Trial Registry (ISRCTN87652313).
breast cancer; complementary and alternative therapies; stress; immune response; neuroendocrine
Psychosocial support services are an important component of modern cancer treatment. A major challenge for all psychosocial services is the achievement of equity of use. Previous studies in the UK have found that women of higher socio-economic status with breast cancer were over-represented amongst those accessing support services. People with other cancer diagnoses, those from socio-economically deprived areas, and men, were under-represented.
The Oncology Health Service, Kingston Upon Hull, UK, delivers fully integrated psychosocial support and interventions. To assess equity of access in this service, a cross-sectional study of all patients with cancer accessing the service during a 5 day period was carried out. One hundred and forty-five patients attended. Forty four percent were male, and the types of cancer were broadly in the proportions expected on the basis of population prevalence (breast cancer 22%, colorectal cancer 21%, lung cancer 16%). Sixty six percent came from the three most deprived quintiles of the Townsend deprivation Index.
The fully integrated Oncology Health Service in Hull is accessed by a more diverse range of patients than previously reported for other services, and is an example of a model of service by which socially equitable use of psychosocial support in the National Health Service might be achieved.
In a series of South African populations, mean faecal pH values were found to be: rural and urban blacks, 6.12 and 6.15; Indians 6.21; coloureds (Eur-African-Malay), 6.29; these are significantly lower (p less than 0.01) than that of whites, 6.88. Apart from that of the coloureds, mean values for series of children and adults did not differ significantly. In the populations mentioned, corresponding mean dietary fibre intakes of children's mothers (or associates of mothers) were all relatively low, namely, roughly 25 g, 18 g, 20 g, 21 g, 23 g, respectively. Frequency of colon cancer (also other non-infective bowel diseases, e.g. appendicitis) is very low in rural and urban blacks, is low in Indians and coloureds, yet much higher in whites. Thus, in these different ethnic populations, rarity or low frequency of colon cancer is associated more with low faecal pH than with level of dietary fibre intake, suggesting that components additional to fibre have a role in determining the milieu intérieur of the bowel and its proneness to disease.
Despite numerous attempts over many years to develop an HIV vaccine based on classical strategies, none has convincingly succeeded to date. A number of approaches are being pursued in the field, including building upon possible efficacy indicated by the recent RV144 clinical trial, which combined two HIV vaccines. Here, we argue for an approach based, in part, on understanding the HIV envelope spike and its interaction with broadly neutralizing antibodies (bnAbs) at the molecular level and using this understanding to design immunogens as possible vaccines. BnAbs can protect against virus challenge in animal models and many such antibodies have been isolated recently. We further propose that studies focused on how best to provide T cell help to B cells that produce bnAbs are crucial for optimal immunization strategies. The synthesis of rational immunogen design and immunization strategies, together with iterative improvements, offers great promise for advancing toward an HIV vaccine.
Background The extent to which adult height, a biomarker of the interplay of genetic endowment and early-life experiences, is related to risk of chronic diseases in adulthood is uncertain.
Methods We calculated hazard ratios (HRs) for height, assessed in increments of 6.5 cm, using individual–participant data on 174 374 deaths or major non-fatal vascular outcomes recorded among 1 085 949 people in 121 prospective studies.
Results For people born between 1900 and 1960, mean adult height increased 0.5–1 cm with each successive decade of birth. After adjustment for age, sex, smoking and year of birth, HRs per 6.5 cm greater height were 0.97 (95% confidence interval: 0.96–0.99) for death from any cause, 0.94 (0.93–0.96) for death from vascular causes, 1.04 (1.03–1.06) for death from cancer and 0.92 (0.90–0.94) for death from other causes. Height was negatively associated with death from coronary disease, stroke subtypes, heart failure, stomach and oral cancers, chronic obstructive pulmonary disease, mental disorders, liver disease and external causes. In contrast, height was positively associated with death from ruptured aortic aneurysm, pulmonary embolism, melanoma and cancers of the pancreas, endocrine and nervous systems, ovary, breast, prostate, colorectum, blood and lung. HRs per 6.5 cm greater height ranged from 1.26 (1.12–1.42) for risk of melanoma death to 0.84 (0.80–0.89) for risk of death from chronic obstructive pulmonary disease. HRs were not appreciably altered after further adjustment for adiposity, blood pressure, lipids, inflammation biomarkers, diabetes mellitus, alcohol consumption or socio-economic indicators.
Conclusion Adult height has directionally opposing relationships with risk of death from several different major causes of chronic diseases.
Height; cardiovascular disease; cancer; cause-specific mortality; epidemiological study; meta-analysis
Histone deacetylase (HDAC) inhibitors either alone or in combination with hypomethylating agents have limited clinical effect in acute myeloid leukemia (AML). Previously we demonstrated that AML patients with higher miR-29b expression had better response to the hypomethylating agent decitabine. Therefore, an increase in miR-29b expression preceding decitabine treatment may provide a therapeutic advantage. We previously showed that miR-29b expression is suppressed by a repressor complex that includes HDACs. Thus, HDAC inhibition may increase miR-29b expression. We hypothesized that priming AML cells with the novel HDAC inhibitor (HDACI) AR-42 would result in increased response to decitabine treatment via upregulation of miR-29b. Here we show that AR-42 is a potent HDACI in AML, increasing miR-29b levels and leading to downregulation of known miR-29b targets (i.e., SP1, DNMT1, DNMT3A, and DNMT3B). We then demonstrated that the sequential administration of AR-42 followed by decitabine resulted in a stronger anti-leukemic activity in vitro and in vivo than decitabine followed by AR-42 or either drug alone. These preclinical results with AR-42 priming before decitabine administration represents a promising, novel treatment approach and a paradigm shift with regard to the combination of epigenetic-targeting compounds in AML, where decitabine has been traditionally given before HDAC inhibitors.
acute myeloid leukemia; HDACI; AR-42; decitabine; miR-29b
Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication1. Here, using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focussed on protein truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, we show that rare PTVs in the p53 inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and to ovarian cancer. PPM1D PTV mutations were present in 25/7781 cases vs 1/5861 controls; P=1.12×10−5, which included 18 mutations in 6,912 individuals with breast cancer; P = 2.42×10−4 and 12 mutations in 1,121 individuals with ovarian cancer; P = 3.10×10−9. Notably, all the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370 bp region in the final exon of the gene, C-terminal to the phosphatase catalytic domain. Functional studies demonstrated that the mutations result in enhanced suppression of p53 in response to ionising radiation exposure, suggesting the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function typically associated with this class of variant, but instead likely have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the utility of sequencing in their identification.
The pathological progression of hypertrophic cardiomyopathy (HCM) is sex dimorphic such that male HCM mice develop phenotypic indicators of cardiac disease well before female HCM mice. Here, we hypothesized that alterations in myofilament function underlies, in part, this sex dimorphism in HCM disease development. Firstly, 10–12 month female HCM (harboring a mutant [R403Q] myosin heavy chain) mice presented with proportionately larger hearts than male HCM mice. Next, we determined Ca2+-sensitive tension development in demembranated cardiac trabeculae excised from 10–12 month female and male HCM mice. Whereas HCM did not impact Ca2+-sensitive tension development in male trabeculae, female HCM trabeculae were more sensitive to Ca2+ than wild-type (WT) counterparts and both WT and HCM males. We hypothesized that the underlying cause of this sex difference in Ca2+-sensitive tension development was due to changes in Ca2+ handling and sarcomeric proteins, including expression of SR Ca2+ ATPase (2a) (SERCA2a), β-myosin heavy chain (β-MyHC) and post-translational modifications of myofilament proteins. Female HCM hearts showed an elevation of SERCA2a and β-MyHC protein whereas male HCM hearts showed a similar elevation of β-MyHC protein but a reduced level of cardiac troponin T (cTnT) phosphorylation. We also measured the distribution of cardiac troponin I (cTnI) phosphospecies using phosphate-affinity SDS–PAGE. The distribution of cTnI phosphospecies depended on sex and HCM. In conclusion, female and male HCM mice display sex dimorphic myofilament function that is accompanied by a sex- and HCM-dependent distribution of sarcomeric proteins and cTnI phosphospecies.
Hypertrophic cardiomyopathy; SR Ca2+ ATPase (2a) (SERCA2a); cTnI; Site-specific phosphorylation; Phosphate-affinity SDS–PAGE
Diabetes mellitus affects virtually every organ system in the body and the degree of organ involvement depends on the duration and severity of the disease, and other co-morbidities. Gastrointestinal (GI) involvement can present with esophageal dysmotility, gastro-esophageal reflux disease (GERD), gastroparesis, enteropathy, non alcoholic fatty liver disease (NAFLD) and glycogenic hepatopathy. Severity of GERD is inversely related to glycemic control and management is with prokinetics and proton pump inhibitors. Diabetic gastroparesis manifests as early satiety, bloating, vomiting, abdominal pain and erratic glycemic control. Gastric emptying scintigraphy is considered the gold standard test for diagnosis. Management includes dietary modifications, maintaining euglycemia, prokinetics, endoscopic and surgical treatments. Diabetic enteropathy is also common and management involves glycemic control and symptomatic measures. NAFLD is considered a hepatic manifestation of metabolic syndrome and treatment is mainly lifestyle measures, with diabetes and dyslipidemia management when coexistent. Glycogenic hepatopathy is a manifestation of poorly controlled type 1 diabetes and is managed by prompt insulin treatment. Though GI complications of diabetes are relatively common, awareness about its manifestations and treatment options are low among physicians. Optimal management of GI complications is important for appropriate metabolic control of diabetes and improvement in quality of life of the patient. This review is an update on the GI complications of diabetes, their pathophysiology, diagnostic evaluation and management.
Gastrointestinal complications; Diabetes mellitus; Esophageal complications; Nonalcoholic fatty liver disease; Diabetic gastroparesis; Diabetic enteropathy; Glycogenic hepatopathy
Use of genetically modified mice enhances our understanding of molecular mechanisms underlying several neurological disorders such as a spinal cord injury (SCI). Freehand manual control used to produce a laceration model of SCI creates inconsistent injuries often associated with a crush or contusion component and, therefore, a novel technique was developed. Our model of cervical laceration SCI has resolved inherent difficulties with the freehand method by incorporating 1) cervical vertebral stabilization by vertebral facet fixation, 2) enhanced spinal cord exposure, and 3) creation of a reproducible laceration of the spinal cord using an oscillating blade with an accuracy of ±0.01 mm in depth without associated contusion. Compared to the standard methods of creating a SCI laceration such as freehand use of a scalpel or scissors, our method has produced a consistent lesion. This method is useful for studies on axonal regeneration of corticospinal, rubrospinal, and dorsal ascending tracts.
Medicine; Issue 75; Neurobiology; Anatomy; Physiology; Neuroscience; Immunology; Infection; Surgery; Nervous System Diseases; Diagnosis; Therapeutics; Surgical Procedures; Operative; Investigative Techniques; spine; spinal cord injury; SCI; mouse; laceration; stabilization; axonal regeneration; injury; mice; animal model; surgical techniques
To determine prevalence and incidence of bacterial vaginosis (BV) and risk factors in young sexually-active Australian women.
1093 women aged 16–25 years were recruited from primary-care clinics. Participants completed 3-monthly questionnaires and self-collected vaginal smears 6-monthly for 12-months. The primary endpoint was a Nugent Score = 7–10 (BV) and the secondary endpoint was a NS = 4–10 (abnormal flora [AF]). BV and AF prevalence estimates and 95% confidence intervals (95%CI) were derived, and adjusted odds ratios (AOR) calculated to explore epidemiological associations with prevalent BV and AF. Proportional-hazards regression models were used to examine factors associated with incident BV and AF.
At baseline 129 women had BV [11.8% (95%CI: 9.4–14.2)] and 188 AF (17.2%; 15.1–19.5). Prevalent BV was associated with having a recent female partner [AOR = 2.1; 1.0–4.4] and lack of tertiary-education [AOR = 1.9; 1.2–3.0]; use of an oestrogen-containing contraceptive (OCC) was associated with reduced risk [AOR = 0.6; 0.4–0.9]. Prevalent AF was associated with the same factors, and additionally with >5 male partners (MSP) in 12-months [AOR = 1.8; 1.2–2.5)], and detection of C.trachomatis or M.genitalium [AOR = 2.1; 1.0–4.5]. There were 82 cases of incident BV (9.4%;7.7–11.7/100 person-years) and 129 with incident AF (14.8%; 12.5–17.6/100 person-years). Incident BV and AF were associated with a new MSP [adjusted rate ratio (ARR) = 1.5; 1.1–2.2 and ARR = 1.5; 1.1–2.0], respectively. OCC-use was associated with reduced risk of incident AF [ARR = 0.7; 0.5–1.0].
This paper presents BV and AF prevalence and incidence estimates from a large prospective cohort of young Australian women predominantly recruited from primary-care clinics. These data support the concept that sexual activity is strongly associated with the development of BV and AF and that use of an OCC is associated with reduced risk.
Tuberculosis incidence in the UK has risen in the past decade. Disease control depends on epidemiological data, which can be difficult to obtain. Whole-genome sequencing can detect microevolution within Mycobacterium tuberculosis strains. We aimed to estimate the genetic diversity of related M tuberculosis strains in the UK Midlands and to investigate how this measurement might be used to investigate community outbreaks.
In a retrospective observational study, we used Illumina technology to sequence M tuberculosis genomes from an archive of frozen cultures. We characterised isolates into four groups: cross-sectional, longitudinal, household, and community. We measured pairwise nucleotide differences within hosts and between hosts in household outbreaks and estimated the rate of change in DNA sequences. We used the findings to interpret network diagrams constructed from 11 community clusters derived from mycobacterial interspersed repetitive-unit–variable-number tandem-repeat data.
We sequenced 390 separate isolates from 254 patients, including representatives from all five major lineages of M tuberculosis. The estimated rate of change in DNA sequences was 0·5 single nucleotide polymorphisms (SNPs) per genome per year (95% CI 0·3–0·7) in longitudinal isolates from 30 individuals and 25 families. Divergence is rarely higher than five SNPs in 3 years. 109 (96%) of 114 paired isolates from individuals and households differed by five or fewer SNPs. More than five SNPs separated isolates from none of 69 epidemiologically linked patients, two (15%) of 13 possibly linked patients, and 13 (17%) of 75 epidemiologically unlinked patients (three-way comparison exact p<0·0001). Genetic trees and clinical and epidemiological data suggest that super-spreaders were present in two community clusters.
Whole-genome sequencing can delineate outbreaks of tuberculosis and allows inference about direction of transmission between cases. The technique could identify super-spreaders and predict the existence of undiagnosed cases, potentially leading to early treatment of infectious patients and their contacts.
Medical Research Council, Wellcome Trust, National Institute for Health Research, and the Health Protection Agency.
Unloading a failing heart with a left ventricular assist device (LVAD) can improve ejection fraction (EF) and left ventricular (LV) size; however, recovery with LVAD explantation is rare. We hypothesized that evaluation of myocyte contractility and biochemistry at the sarcomere level before and after LVAD may explain organ level changes.
Methods and Results
Paired LV tissue samples were frozen from 8 patients with nonischemic cardiomyopathy at LVAD implantation (Before LVAD) and prior to transplant (After LVAD). These were compared to 8 nonfailing hearts. Isolated skinned myocytes were purified, attached to a force transducer, and dimensions, maximal calcium saturated force (Fmax), calcium sensitivity, and myofilament cooperativity were assessed. Relative isoform abundance and phosphorylation levels of sarcomeric contractile proteins were measured. With LVAD support, the unloaded EF improved (10.0±1.0 to 25.6±11.0%, p=0.007), LV size decreased (LVIDd 7.6±1.2 to 4.9±1.4cm, p<0.001), and myocyte dimensions decreased (cross-sectional area 1247±346 to 638±254μm2, p=0.001). Fmax improved after LVAD (3.6±0.9 to 7.3±1.8mN/mm2, p<0.001), but was still lower than nonfailing (7.3±1.8 vs. 17.6±1.8mN/mm2, p<0.001). An increase in troponin I (TnI) phosphorylation after LVAD was noted, but protein kinase C phosphorylation of TnI decreased. Biochemical changes of other sarcomeric proteins were not observed after LVAD.
There is significant improvement in LV and myocyte size with LVAD, but there is only partial recovery of EF and myocyte contractility. LVAD support was only associated with biochemical changes in TnI. This suggests that alternate mechanisms might contribute to contractile changes after LVAD and that additional interventions may be needed to alter biochemical remodeling of the sarcomere to further enhance myofilament and organ level recovery.
mechanical circulatory support; remodeling heart failure; mechanical unloading; heart failure
This study aimed to estimate rates of chlamydia incidence and re-infection and to investigate the dynamics of chlamydia organism load in prevalent, incident and re-infections among young Australian women.
1,116 women aged 16 to 25 years were recruited from primary care clinics in Australia. Vaginal swabs were collected at 3 to 6 month intervals for chlamydia testing. Chlamydia organism load was measured by quantitative PCR.
There were 47 incident cases of chlamydia diagnosed and 1,056.34 person years of follow up with a rate of 4.4 per 100 person years (95% CI: 3.3, 5.9). Incident infection was associated with being aged 16 to 20 years [RR = 3.7 (95%CI: 1.9, 7.1)], being employed [RR = 2.4 (95%CI: 1.1, 4.9)] and having two or more new sex partners [RR = 5.5 (95%CI: 2.6, 11.7)]. Recent antibiotic use was associated with a reduced incidence [RR:0.1 (95%CI: 0.0, 0.5)]. There were 14 re-infections with a rate of 22.3 per 100 person years (95%CI: 13.2, 37.6). The median time to re-infection was 4.6 months. Organism load was higher for prevalent than incident infections (p<0.01) and for prevalent than re-infections (p<0.01).
Chlamydia is common among young women and a high proportion of women are re-infected within a short period of time, highlighting the need for effective partner treatment and repeat testing. The difference in organism load between prevalent and incident infections suggests prevalent infection may be more important for ongoing transmission of chlamydia.
This study investigated the role of parental diagnosis of alcohol abuse/dependence and perceived family norms for adolescent drinking on alcohol use and alcohol-related problems among urban American Indian youth. A total of 251 urban, American Indian youth and their parents/caregivers were followed from age 13 to age 18. Perceived family norms against alcohol decreased and alcohol use increased from age 13 to age 18. Relative to no parental diagnosis, youth with one or two parents diagnosed with alcohol abuse/dependence were less likely to perceive family norms against alcohol use. Youth with two parents diagnosed were more likely to report alcohol-related problems at age 18 compared to no parental diagnosis. Faster rates of decrease in perceived family norms against alcohol use were associated with faster increases in alcohol use over time. Higher rates of perceived family norms against alcohol use protected youth from high rates of use at age 13, but higher rates of alcohol use at age 13 predicted more alcohol-related problems at age 18. These results suggest that both family history and family behaviors in the form of communication of norms for adolescent alcohol use are likely to impact both rates of use and eventual alcohol-related problems.
Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained regions of the virus in order to ensure the maintenance of immunodominant CD8 responses and the sustained decline of early viremia.
The ability of HIV-1 and other highly variable pathogens to rapidly mutate to escape vaccine-induced immune responses represents a major hurdle to the development of effective vaccines to these highly persistent pathogens. Application of next-generation or deep sequencing technologies to the study of host pathogens could significantly improve our understanding of the mechanisms by which these pathogens subvert host immunity, and aid in the development of novel vaccines and therapeutics. Here, we developed a 454 deep sequencing approach to enable the sensitive detection of low-frequency viral variants across the entire HIV-1 genome. When applied to the acute phase of HIV-1 infection we observed that the majority of early, low frequency mutations represented viral adaptations to host cellular immune responses, evidence of strong host immunity developing during the early decline of peak viral load. Rapid viral escape from the most dominant immune responses however correlated with loss of this initial viral control, suggestive of the importance of mounting immune responses against more conserved regions of the virus. These data provide a greater understanding of the early evolutionary events subverting the ability of host immune responses to control early HIV-1 replication, yielding important insight into the design of more effective vaccine strategies.
Secondary damage following primary spinal cord injury extends pathology beyond the site of initial trauma, and effective management is imperative for maximizing anatomical and functional recovery. Bisperoxovanadium compounds have proven neuroprotective effects in several central nervous system injury/disease models, however, no mechanism has been linked to such neuroprotection from bisperoxovanadium treatment following spinal trauma. The goal of this study was to assess acute bisperoxovanadium treatment effects on neuroprotection and functional recovery following cervical unilateral contusive spinal cord injury, and investigate a potential mechanism of the compound's action. Two experimental groups of rats were established to 1) assess twice-daily 7 day treatment of the compound, potassium bisperoxo (picolinato) vanadium, on long-term recovery of skilled forelimb activity using a novel food manipulation test, and neuroprotection 6 weeks following injury and 2) elucidate an acute mechanistic link for the action of the drug post-injury. Immunofluorescence and Western blotting were performed to assess cellular signaling 1 day following SCI, and histochemistry and forelimb functional analysis were utilized to assess neuroprotection and recovery 6 weeks after injury. Bisperoxovanadium promoted significant neuroprotection through reduced motorneuron death, increased tissue sparing, and minimized cavity formation in rats. Enhanced forelimb functional ability during a treat-eating assessment was also observed. Additionally, bisperoxovanadium significantly enhanced downstream Akt and mammalian target of rapamycin signaling and reduced autophagic activity, suggesting inhibition of the phosphatase and tensin homologue deleted on chromosome ten as a potential mechanism of bisperoxovanadium action following traumatic spinal cord injury. Overall, this study demonstrates the efficacy of a clinically applicable pharmacological therapy for rapid initiation of neuroprotection post-spinal cord injury, and sheds light on the signaling involved in its action.
Investigations into the physiological mechanisms of sleep control require an animal psychomotor vigilance task (PVT) with fast response times (<300ms). Rats provide a good PVT model since whisker stimulation produces a rapid and robust cortical evoked response, and animals can be trained to lick following stimulation. Our prior experiments used deprivation-based approaches to maximize motivation for operant conditioned responses. However, deprivation can influence physiological and neurobehavioral effects. In order to maintain motivation without water deprivation, we conditioned rats for immobilization and head restraint, then trained them to lick for a 10% sucrose solution in response to whisker stimulation. After approximately 8 training sessions, animals produced greater than 80% correct hits to the stimulus. Over the course of training, reaction times became faster and correct hits increased. Performance in the PVT was examined after 3, 6 and 12 hours of sleep deprivation achieved by gentle handling. A significant decrease in percent correct hits occurred following 6 and 12 hours of sleep deprivation and reaction times increased significantly following 12 hours of sleep deprivation. While behaviorally the animals appeared to be awake, we observed significant increases in EEG delta power prior to misses. The rat PVT with fast response times allows investigation of sleep deprivation effects, time on task and pharmacological agents. Fast response times also allow closer parallel studies to ongoing human protocols.
PVT; restraint; conditioned learning; lick; somatosensory cortex; electrophysiology; sleep deprivation; reaction time
The success of nucleoside reverse transcriptase inhibitors (NRTIs) in treating HIV-1 infection and reducing mother-to-child transmission of the virus during pregnancy is accompanied by evidence that NRTIs cause long-term health risks for cancer and mitochondrial disease. Thus, agents that mitigate toxicities of the current combination drug therapies are needed. Previous work had shown that the NRTI-drug pair zidovudine (AZT)–didanosine (ddI) was highly cytotoxic and mutagenic; thus, we conducted preliminary studies to investigate the ability of the active moiety of amifostine, WR1065, to protect against the deleterious effects of this NRTI-drug pair. In TK6 cells exposed to 100 μM AZT-ddI (equimolar) for 3 days with or without 150 μM WR1065, WR1065 enhanced long-term cell survival and significantly reduced AZT-ddI-induced mutations. Follow-up studies were conducted to determine if coexposure to AZT and WR1065 abrogated the antiretroviral efficacy of AZT. In human T-cell blasts infected with HIV-1 in culture, inhibition of p24 protein production was observed in cells treated with 10 μM AZT in the absence or presence of 5–1,000 μM WR1065. Surprisingly, WR1065 alone exhibited dose-related inhibition of HIV-1 p24 protein production. WR1065 also had antiviral efficacy against three species of adenovirus and influenza A and B. Intracellular levels of unbound WR1065 were measured following in vitro/in vivo drug exposure. These pilot study results indicate that WR1065, at low intracellular levels, has cytoprotective and antimutagenic activities against the most mutagenic pair of NRTIs and has broad spectrum anti-viral effects. These findings suggest that the activities have a possible common mode of action that merits further investigation.
antimutagenesis; antiviral; amifostine; WR1065; cytoprotection
A sensitive vertical denaturing gradient gel electrophoresis (DGGE) method, using 13 unipolar psoralen-clamped PCR primer pairs, was developed for detecting sequence variants in the 22 tRNA genes and flanking regions (together spanning ~21%) of the human mitochondrial genome. A study was conducted to determine (i) if mitochondrial DNA (mtDNA) polymorphisms and/or mutations were detectable in healthy newborns and (ii) if prepartum 3′-azido-2′,3′-dideoxythymidine (AZT) based HIV-1 prophylaxis was associated with significant increases in mtDNA mutations and changes in the degree of heteroplasmy of sequence variants in uninfected infants born to HIV-1-infected mothers. DGGE analysis of umbilical cord tissue (where vascular endothelium and smooth muscle cells are the major source of mtDNA) showed that mtDNA sequence variants were significantly elevated by threefold in AZT-treated infants compared with unexposed controls (P < 0.001), with 24 changes observed in 19/52 (37%) treated newborns (averaging 0.46 changes/subject) versus only eight changes found in 7/55 (13%) unexposed newborns (averaging 0.15 changes/subject). Six distinct sequence variants occurring in unexposed controls were predominately synonymous and homoplasmic, representing previously reported polymorphisms. Uninfected infants exposed to a combination of AZT and 2′,3′-dideoxy-3′-thiacytidine and “maternal HIV-1” had a significant shift in the spectrum of mutations (P = 0.04) driven by increases in nonsynonymous heteroplasmic sequence variants at polymorphic sites (10 distinct variants) and novel sites (four distinct variants). While the weight of evidence suggests that prepartum AZT-based prophylaxis produces mtDNA mutations, additional research is needed to determine the degree to which fetal responses to maternal HIV-1 infection, in the absence of antiretroviral treatment, contribute to prenatal mtDNA mutagenesis.
antiretrovirals; AZT; DGGE; mitochondrial DNA mutation; nucleoside analogs; 3TC; tRNA genes; transplacental exposure
To delineate temporal changes in the integrity and function of mitochondria/cardiomyocytes in hearts from mice exposed in utero to commonly used nucleoside analogs (NRTIs), CD-1 mice were exposed in utero to 80 mg AZT/kg, 40 mg 3TC/kg, 80 mg AZT/kg plus 40 mg 3TC/kg, or vehicle alone during days 12–18 of gestation and hearts from female mouse offspring were examined at 13 and 26 weeks postpartum. Alterations in cardiac mitochondrial DNA (mtDNA) content, oxidative phosphorylation (OXPHOS) enzyme activities, mtDNA mutations, and echocardiography of NRTI-exposed mice were assessed and compared with findings in vehicle-exposed control mice. A hybrid capture-chemiluminescence assay showed significant twofold increases in mtDNA levels in hearts from AZT- and AZT/3TC-exposed mice at 13 and 26 weeks postpartum, consistent with near doubling in mitochondrial numbers over time compared with vehicle-exposed mice. Echocardiographic measurements at 13 and 26 weeks postpartum indicated progressive thinning of the left ventricular posterior wall in NRTI-exposed mice, relative to controls, with differences becoming statistically significant by 26 weeks. Overall, progressive functional changes occurred in mouse mitochondria and cardiac tissue several months after in utero NRTI exposures; AZT and 3TC acted in concert to cause additive cardiotoxic effects of AZT/3TC compared with either drug alone.
AZT; 3TC; Cardiotoxicity; Echocardiography Mitochondrial; Mitochondrial DNA content; Mitochondrial DNA mutation; Mitochondrial dysfunction; Mitochondrial toxicity; OXPHOS; Transplacental exposure