Thermoneutral Housing and a Western Diet Combination Exacerbates Dysferlin-Deficient Muscular Dystrophy.

INTRODUCTION/AIMS: Most mouse models of muscular dystrophy (MD) show mild phenotypes, which limits the translatability of experimental therapies to patients. A growing body of evidence suggests that MD is accompanied by metabolic abnormalities that could potentially exacerbate the primary muscle wasting process. Since thermoneutral (TN) housing of mice (~30°C) has been shown to affect many metabolic parameters, particularly when combined with a Western diet (WD), our aim was to determine whether the combination of TN and WD exacerbates muscle wasting in dysferlin-deficient BLAJ mice, a common model of limb-girdle MD type 2b (LGMD2b). METHODS: The 2-mo-old wild-type (WT) and BLAJ mice were housed at TN or room temperature (RT) and fed a WD or regular chow for 9 mo. Ambulatory function, muscle histology, and protein immunoblots of skeletal muscle were assessed. RESULTS: BLAJ mice at RT and fed a chow diet showed normal ambulation function similar to WT mice, whereas 90% of BLAJ mice under WD and TN combination showed ambulatory dysfunction (p < 0.001), and an up to 4.1-fold increase in quadriceps and gastrocnemius fat infiltration. Western blotting revealed decreased autophagy marker microtubules-associated protein 1 light chain 3-B (LC3BII/LC3BI) ratio and up-regulation of protein kinase B/AKT and ribosomal protein S6 phosphorylation, suggesting inefficient cellular debris and protein clearance in TN BLAJ mice fed a WD. Male and female BLAJ mice under TN and WD combination showed heterogenous fibro-fatty infiltrate composition. DISCUSSION: TN and WD combination exacerbates rodent LGMD2b without affecting WT mice. This improves rodent modeling of human MD and helps elucidate how metabolic abnormalities may play a causal role in muscle wasting.

Cholesterol absorption blocker ezetimibe prevents muscle wasting in severe dysferlin-deficient and mdx mice.

BACKGROUND: Muscular dystrophy (MD) causes muscle wasting and is often lethal in patients due to a lack of proven therapies. In contrast, mouse models of MD are notoriously mild. We have previously shown severe human-like muscle pathology in mdx [Duchenne MD (DMD)] and dysferlin-deficient limb-girdle MD type 2B (LGMD2B) mice by inactivating the gene encoding for apolipoprotein E (ApoE), a lipid transporter synthesized by the liver, brain and adipocytes to regulate lipid and fat metabolism. Having recently established that human DMD is a novel type of primary genetic dyslipidaemia with elevated cholesterol, we sought to determine whether cholesterol could exacerbate the muscle wasting process observed in severe rodent MD. METHODS: Severe mdx and dysferlin knock-out mice lacking ApoE were treated with ezetimibe (15 mg/kg/day), a clinically approved drug exhibiting few pleiotropic effects. In separate studies, dietary cholesterol was raised (from 0.2% to 2% cholesterol) in combination with experimental micro-injury and direct cholesterol injection assays. Muscles were assessed histologically for changes in collagen and adipocyte infiltration and both transcriptomic and cellular changes by RNA-seq and fluorescence-activated cell sorting analysis. RESULTS: Treatment of severe DMD and LGMD2B mice with ezetimibe completely prevented clinical signs of ambulatory dysfunction (0% incidence vs. 33% for vehicle treatment; P < 0.05). Histological analyses revealed that ezetimibe-reduced fibro-fatty infiltration up to 84% and 63% in severely affected triceps (P ≤ 0.0001) and gastrocnemius (P ≤ 0.003) muscles, resulting in a respective 1.9-fold and 2.2-fold retention of healthy myofibre area (P ≤ 0.0001). Additionally, raising dietary cholesterol and thus concentrations of plasma low-density lipoprotein-associated cholesterol (by 250%; P < 0.0001) reduced overall survivability (by 100%; P < 0.001) and worsened muscle damage in the LGMD2B triceps by 767% (P < 0.03). Micro-pin-induced mechanical injury in LGMD2B mice fed a high cholesterol diet exacerbated muscle damage by 425% (P < 0.03) and increased macrophage recruitment (by 98%; P = 0.03) compared with those injured on a chow diet. Parallel RNA-seq analyses revealed that injury in cholesterol-fed mice also modulated the expression of 3671 transcripts (1953 up-regulated), with fibrogenic, inflammatory and programmed cell death-associated pathways among the most enriched. Mice lacking dysferlin also displayed heightened muscle necrosis (by 123%; P < 0.0001) following a direct intramuscular injection of cholesterol compared with control mice. CONCLUSIONS: Cholesterol exacerbates rodent MD. Specific inhibition of cholesterol absorption with ezetimibe may safely attenuate human MD severity and delay death.

Radiofrequency therapy improves exercise capacity of mice with emphysema.

Emphysema is a common phenotype of chronic obstructive pulmonary disease (COPD). Although resection of emphysematous tissue can improve lung mechanics, it is invasive and fraught with adverse effects. Meanwhile, radiofrequency (RF) treatment is an extracorporeal method that leads to tissue destruction and remodeling, resulting in "volume reduction" and overall improvement in lung compliance of emphysematous lungs. Whether these changes lead to improved exercise tolerance is unknown. Here, we investigated the effectiveness of RF treatment to improve the exercise capacity of mice with emphysema. Fifty-two mice (7 weeks of age) were used in this experiment. A bilateral emphysema model was created by intratracheally instilling porcine pancreatic elastase (PPE) (1.5U/100 g body weight). RF treatment (0.5 W/ g body weight) was administered extracorporeally 14 days later and mice were sacrificed after another 21 days. The exercise capacity of mice was measured using a treadmill. Treadmill runs were performed just before PPE instillation (baseline), before RF treatment and before sacrifice. Following sacrifice, lung compliance and mean linear intercept (Lm) were measured and fibrosis was assessed using a modified Ashcroft score. There were 3 experimental groups: controls (instilled with saline, n = 12), emphysema (instilled with porcine pancreatic elastase, PPE, n = 11) and emphysema + treatment (instilled with PPE and given RF, n = 9). At endpoint, the maximum velocity of the emphysema + treatment group was significantly higher than that of the emphysema group, indicating improved exercise tolerance (86.29% of baseline vs 61.69% of baseline, p = 0.01). Histological analysis revealed a significant reduction in emphysema as denoted by Lm between the two groups (median 29.60 µm vs 35.68 µm, p = 0.03). The emphysema + treatment group also demonstrated a higher prevalence of lung fibrosis (≧Grade 3) compared with the emphysema group (11.7% vs 5.4%, p < 0.01). No severe adverse events from RF were observed. RF treatment improved the exercise capacity of mice with emphysema. These data highlight the therapeutic potential of RF treatment in improving the functional status of patients with COPD.

Voluntary resistance wheel exercise from mid-life prevents sarcopenia and increases markers of mitochondrial function and autophagy in muscles of old male and female C57BL/6J mice.

BACKGROUND: There is much interest in the capacity of resistance exercise to prevent the age-related loss of skeletal muscle mass and function, known as sarcopenia. This study investigates the molecular basis underlying the benefits of resistance exercise in aging C57BL/6J mice of both sexes. RESULTS: This study is the first to demonstrate that long-term (34 weeks) voluntary resistance wheel exercise (RWE) initiated at middle age, from 15 months, prevents sarcopenia in selected hindlimb muscles and causes hypertrophy in soleus, by 23 months of age in both male and female C57BL/6J mice. Compared with 23-month-old sedentary (SED) controls, RWE (0-6 g of resistance) increased intramuscular mitochondrial density and oxidative capacity (measured by citrate synthase and NADH-TR) and increased LC3II/I ratios (a marker of autophagy) in exercised mice of both sexes. RWE also reduced mRNA expression of Gadd45α (males only) and Runx1 (females only) but had no effect on other markers of denervation including Chrng, Chrnd, Musk, and Myog. RWE increased heart mass in all mice, with a more pronounced increase in females. Significant sex differences were also noted among SED mice, with Murf1 mRNA levels increasing in male, but decreasing in old female mice between 15 and 23 months. CONCLUSIONS: Overall, long-term RWE initiated from 15 month of age significantly improved some markers of the mitochondrial and autophagosomal pathways and prevented age-related muscle wasting.

A prospective longitudinal study of polyomavirus shedding in lung-transplant recipients.

BACKGROUND: Polyomavirus infection causes renal dysfunction after kidney transplantation, but it has not been thoroughly investigated in nonrenal solid-organ transplantation. METHODS: Fifty lung-transplant recipients provided prospective urine and blood samples over the course of 17 months. Samples were analyzed for BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40) using conventional polymerase chain reaction (PCR), sequence analysis, and quantitative real-time PCR. RESULTS: Thirty-one (62%) of 50 patients had polyomavirus detected in at least 1 urine specimen, including 16 (32%) for BKV, 12 (24%) for JCV, and 6 (12%) for SV40. Mean BKV loads (5.0 log(10) copies/mL) did not differ from those of JCV (5.7 log(10) copies/mL; P=.38), but SV40 loads (2.5 log(10) copies/mL) were lower than those of BKV (P=.006) and JCV (P=.002). Blood samples were negative. Infection with individual polyomaviruses or polyomavirus infection in aggregate was not associated with reduced creatinine clearance. Patients not shedding polyomavirus had better survival than patients shedding polyomavirus (P=.049). CONCLUSIONS: Polyomaviruses BKV and JCV were commonly detected in urine from lung-transplant recipients. SV40 was found in 12% of patients but was shed at a lower frequency and with lower viral loads than the other viruses. Polyomavirus infection was not associated with renal dysfunction.

Specific and quantitative detection of human polyomaviruses BKV, JCV, and SV40 by real time PCR.

BACKGROUND: The polyomaviruses that infect humans, BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40), typically establish subclinical persistent infections. However, reactivation of these viruses in immunocompromised hosts is associated with renal nephropathy and hemorrhagic cystitis (HC) caused by BKV and with progressive multifocal leukoencephalopathy (PML) caused by JCV. Additionally, SV40 is associated with several types of human cancers including primary brain and bone cancers, mesotheliomas, and non-Hodgkin's lymphoma. Advancements in detection of these viruses may contribute to improved diagnosis and treatment of affected patients. OBJECTIVE: To develop sensitive and specific real time quantitative polymerase chain reaction (RQ-PCR) assays for the detection of T-antigen DNA sequences of the human polyomaviruses BKV, JCV, and SV40 using the ABI Prism 7000 Sequence Detection System. STUDY DESIGN: Assays for absolute quantification of the viral T-ag sequences were designed and the sensitivity and specificity were evaluated. A quantitative assay to measure the single copy human RNAse P gene was also developed and evaluated in order to normalize viral gene copy numbers to cell numbers. RESULTS: Quantification of the target genes is sensitive and specific over a 7 log dynamic range. Ten copies each of the viral and cellular genes are reproducibly and accurately detected. The sensitivity of detection of the RQ-PCR assays is increased 10- to 100-fold compared to conventional PCR and agarose gel protocols. The primers and probes used to detect the viral genes are specific for each virus and there is no cross reactivity within the dynamic range of the standard dilutions. The sensitivity of detection for these assays is not reduced in human cellular extracts; however, different DNA extraction protocols may affect quantification. CONCLUSION: These assays provide a technique for rapid and specific quantification of polyomavirus genomes per cell in human samples.

Detection of BK virus and simian virus 40 in the urine of healthy children.

Seroprevalence studies indicate that most primary infections with BK virus (BKV) and JC virus (JCV) occur in the first and second decades of life, respectively. Relatively little is known about the transmission of these agents, including the primary source of human exposure, the portal of entry, and the pathophysiology of life-long viral persistence. We sought to determine if simian virus 40 (SV40) excretion could be detected in the urine of healthy children and to define the age-related prevalence of polyomavirus shedding in this population. A point prevalence study of polyomavirus shedding was conducted in healthy children using rigorous enrollment criteria. Urine samples were collected from healthy children, age from 3 to 18 years, during routine evaluation at two urban pediatric clinics. Qualitative PCR analysis was performed using primers that detect a conserved region of the T-antigen gene of BKV, JCV, and SV40. The identity of polyomaviruses detected was determined by DNA sequence analysis and/or PCR amplification of other regions of the viral genomes. Seven of 72 (9.7%) urine samples were positive for polyomaviruses: three with BKV (ages 4, 6, 13), two with SV40 (ages 6, 16), two with BKV and SV40 co-excretion (ages 6, 15), and none with JCV. DNA sequence analysis confirmed the identity of viruses detected. These results suggest that the timing of SV40 infections in humans may be similar to that of BKV and that urine from healthy children could contribute to the ubiquity of BKV infection early in life.

Phylogenetic analysis of polyomavirus simian virus 40 from monkeys and humans reveals genetic variation.

A phylogenetic analysis of 14 complete simian virus 40 (SV40) genomes was conducted in order to determine strain relatedness and the extent of genetic variation. This analysis included infectious isolates recovered between 1960 and 1999 from primary cultures of monkey kidney cells, from contaminated poliovaccines and an adenovirus seed stock, from human malignancies, and from transformed human cells. Maximum-parsimony and distance methods revealed distinct SV40 clades. However, no clear patterns of association between genotype and viral source were apparent. One clade (clade A) is derived from strain 776, the reference strain of SV40. Clade B contains isolates from poliovaccines (strains 777 and Baylor), from monkeys (strains N128, Rh911, and K661), and from human tumors (strains SVCPC and SVMEN). Thus, adaptation is not essential for SV40 survival in humans. The C terminus of the T-antigen (T-ag-C) gene contains the highest proportion of variable sites in the SV40 genome. An analysis based on just the T-ag-C region was highly congruent with the whole-genome analysis; hence, sequencing of just this one region is useful in strain identification. Analysis of an additional 16 strains for which only the T-ag-C gene was sequenced indicated that further SV40 genetic diversity is likely, resulting in a provisional clade (clade C) that currently contains strains associated with human tumors and human strain PML-1. Four other polymorphic regions in the genome were also identified. If these regions were analyzed in conjunction with the T-ag-C region, most of the phylogenetic signal could be captured without complete genome sequencing. This report represents the first whole-genome approach to establishing phylogenetic relatedness among different strains of SV40. It will be important in the future to develop a more complete catalog of SV40 variation in its natural monkey host, to determine if SV40 strains from different clades vary in biological or pathogenic properties, and to identify which SV40 strains are transmissible among humans.

Epstein-Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy.

Patients with human immunodeficiency virus type 1 (HIV-1) infection are at high risk of developing Epstein-Barr virus (EBV)-associated lymphoma. However, little is known of the EBV DNA loads in patients receiving highly active antiretroviral therapy (HAART). Using a real-time quantitative polymerase chain reaction assay, we demonstrated that significantly more HIV-1-infected patients receiving HAART than HIV-1-uninfected volunteers had detectable EBV DNA in blood (57 [81%] of 70 vs. 11 [16%] of 68 patients; P=.001) and saliva (55 [79%] of 68 vs. 37 [54%] of 68 patients; P=.002). The mean EBV loads in blood and saliva samples were also higher in HIV-1-infected patients than in HIV-1-uninfected volunteers (P=.001). The frequency of EBV detection in blood was associated with lower CD4+ cell counts (P=.03) among HIV-1-infected individuals, although no differences were observed in the EBV DNA loads in blood or saliva samples in the HIV-1-infected group. Additional studies are needed to determine whether EBV-specific CD4+ and CD8+ cells play a role in the pathogenesis of EBV in HIV-1-infected patients receiving HAART.

The dynamics of herpesvirus and polyomavirus reactivation and shedding in healthy adults: a 14-month longitudinal study.

Humans are infected with viruses that establish long-term persistent infections. To address whether immunocompetent individuals control virus reactivation globally or independently and to identify patterns of sporadic reactivation, we monitored herpesviruses and polyomaviruses in 30 adults, over 14 months. Epstein-Barr virus (EBV) DNA was quantitated in saliva and peripheral blood mononuclear cells (PBMCs), cytomegalovirus (CMV) was assayed in urine, and JC virus (JCV) and BK virus (BKV) DNAs were assayed in urine and PBMCs. All individuals shed EBV in saliva, whereas 67% had >or=1 blood sample positive for EBV. Levels of EBV varied widely. CMV shedding occurred infrequently but occurred more commonly in younger individuals (P<.03). JCV and BKV virurias were 46.7% and 0%, respectively. JCV shedding was age dependent and occurred commonly in individuals >or=40 years old (P<.03). Seasonal variation was observed in shedding of EBV and JCV, but there was no correlation among shedding of EBV, CMV, and JCV (P>.50). Thus, adults independently control persistent viruses, which display discordant, sporadic reactivations.

Polyomavirus JCV excretion and genotype analysis in HIV-infected patients receiving highly active antiretroviral therapy.

OBJECTIVE: To assess the frequency of shedding of polyomavirus JC virus (JCV) genotypes in urine of HIV-infected patients receiving highly active antiretroviral therapy (HAART). METHODS: Single samples of urine and blood were collected prospectively from 70 adult HIV-infected patients and 68 uninfected volunteers. Inclusion criteria for HIV-infected patients included an HIV RNA viral load < 1000 copies, CD4 cell count of 200-700 x 106 cells/l, and stable HAART regimen. PCR assays and sequence analysis were carried out using JCV-specific primers against different regions of the virus genome. RESULTS: JCV excretion in urine was more common in HIV-positive patients but not significantly different from that of the HIV-negative group [22/70 (31%) versus 13/68 (19%); P = 0.09]. HIV-positive patients lost the age-related pattern of JCV shedding (P = 0.13) displayed by uninfected subjects (P = 0.01). Among HIV-infected patients significant differences in JCV shedding were related to CD4 cell counts (P = 0.03). Sequence analysis of the JCV regulatory region from both HIV-infected patients and uninfected volunteers revealed all to be JCV archetypal strains. JCV genotypes 1 (36%) and 4 (36%) were the most common among HIV-infected patients, whereas type 2 (77%) was the most frequently detected among HIV-uninfected volunteers. CONCLUSION: These results suggest that JCV shedding is enhanced by modest depressions in immune function during HIV infection. JCV shedding occurred in younger HIV-positive persons than in the healthy controls. As the common types of JCV excreted varied among ethnic groups, JCV genotypes associated with progressive multifocal leukoencephalopathy may reflect demographics of those infected patient populations.

Association between simian virus 40 and non-Hodgkin lymphoma.

BACKGROUND: Non-Hodgkin lymphoma has increased in frequency over the past 30 years, and is a common cancer in HIV-1-infected patients. Although no definite risk factors have emerged, a viral cause has been postulated. Polyomaviruses are known to infect human beings and to induce tumours in laboratory animals. We aimed to identify which one of the three polyomaviruses able to infect human beings (simian virus 40 [SV40], JC virus, and BK virus) was associated with non-Hodgkin lymphoma. METHODS: We analysed systemic non-Hodgkin lymphoma from 76 HIV-1-infected and 78 HIV-1-uninfected patients, and non-malignant lymphoid samples from 79 HIV-1-positive and 107 HIV-1-negative patients without tumours; 54 colon and breast carcinoma samples served as cancer controls. We used PCR followed by Southern blot hybridisation and DNA sequence analysis to detect DNAs of polyomaviruses and herpesviruses. FINDINGS: Polyomavirus T antigen sequences, all of which were SV40-specific, were detected in 64 (42%) of 154 non-Hodgkin lymphomas, none of 186 non-malignant lymphoid samples, and none of 54 control cancers. This difference was similar for HIV-1-infected patients and HIV-1-uninfected patients alike. Few tumours were positive for both SV40 and Epstein-Barr virus. Human herpesvirus type 8 was not detected. SV40 sequences were found most frequently in diffuse large B-cell and follicular-type lymphomas. INTERPRETATION: SV40 is significantly associated with some types of non-Hodgkin lymphoma. These results add lymphomas to the types of human cancers associated with SV40.

Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma.

Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.