Targeting necroptosis in muscle fibers ameliorates inflammatory myopathies.

Muscle cell death in polymyositis is induced by CD8+ cytotoxic T lymphocytes. We hypothesized that the injured muscle fibers release pro-inflammatory molecules, which would further accelerate CD8+ cytotoxic T lymphocytes-induced muscle injury, and inhibition of the cell death of muscle fibers could be a novel therapeutic strategy to suppress both muscle injury and inflammation in polymyositis. Here, we show that the pattern of cell death of muscle fibers in polymyositis is FAS ligand-dependent necroptosis, while that of satellite cells and myoblasts is perforin 1/granzyme B-dependent apoptosis, using human muscle biopsy specimens of polymyositis patients and models of polymyositis in vitro and in vivo. Inhibition of necroptosis suppresses not only CD8+ cytotoxic T lymphocytes-induced cell death of myotubes but also the release of inflammatory molecules including HMGB1. Treatment with a necroptosis inhibitor or anti-HMGB1 antibodies ameliorates myositis-induced muscle weakness as well as muscle cell death and inflammation in the muscles. Thus, targeting necroptosis in muscle cells is a promising strategy for treating polymyositis providing an alternative to current therapies directed at leukocytes.

An anti-ADAMTS1 treatment relieved muscle dysfunction and fibrosis in dystrophic mice.

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene, accompanied by aberrant extracellular matrix synthesis and muscle damage. ADAMTS1 metalloproteinase was reported increased in dystrophin-deficient mdx mouse. The aim of this study was to explore the role of ADAMTS1 in muscle function, fibrosis and damage, and respiratory function of mdx mice. 102 DMD patients and their mothers were included in this study. Multiplex ligation dependent probe amplification (MLPA) assay and Next-generation sequencing (NGS) were adopted to do genetic diagnosis. Dystrophin-deficient mdx mice were treated with anti-ADAMTS1 antibody (anti-ADAMTS1) for three weeks. The results showed that ADAMTS1 was increased in gastrocnemius muscle of mdx mice and serum of DMD patients. Anti-ADAMTS1 treatment increased Versican transcription but suppressed versican protein expression. Besides, we found anti-ADAMTS1 improved muscle strength, diaphragm and extensor digitorum longus muscles functions in mdx mice. Meanwhile, muscle fibrosis and damage were attenuated in anti-ADAMTS1 treated dystrophic mice. In summary, anti-ADAMTS1 antibody relieved muscle dysfunction and fibrosis in dystrophic mice. It is suggested that ADAMTS1 is a potential target for developing new biological therapies for DMD.

The impact of low muscle function on health-related quality of life in Indonesian women with systemic lupus erythematosus.

OBJECTIVE: There was no study aimed at evaluating the effect of muscle function on SLE patients' quality of life using the Sarcopenia Quality of Life (SarQoL) questionnaire. METHODS: This cross-sectional study recruited 61 women with SLE consecutively, muscle function was measured with Jamar handheld-dynamometer and 6-meter walk test, HRQoL was measured with Sarcopenia Quality of Life (SarQoL) questionnaire. The cut-off point for low muscle strength (<18 kg) and low gait speed (<1.0 m/s) was according to the Asian Working Group on Sarcopenia 2019 criteria. Statistical analysis was conducted with a t-test for mean difference, and linear regression was used to adjust confounders (age, protein intake, physical exercise, and disease activity). RESULTS: The subjects' mean muscle strength was 19.54 kg (6.94), and 44.3% (n = 27) was found to have low muscle strength. The subjects' mean gait speed was 0.77 m/s (0.20), and 90.3% (n = 55) was found to have low gait speed. The difference of total SarQoL score in subjects with normal and low muscle strength was found to be significant; 74.86 (9.48) vs. 65.49 (15.51) (p = 0.009), and still statistically significant after adjustments with age, protein intake, physical exercise level, and disease activity [B 0.56; 95% CI 0.08-1.03; p = 0.022]. The difference of total SarQoL score in subjects with normal and low physical performance was found to be not significant, 70.67 (11.08) vs. 70.72 (13.56) (p = 0.993). CONCLUSION: There was a significant difference in SarQoL's total score in normal compared with low muscle strength groups of Indonesian women with SLE.

The immune system in sporadic inclusion body myositis patients is not compromised by blood-flow restricted exercise training.

BACKGROUND: Sporadic inclusion body myositis (sIBM) is clinically characterised by progressive proximal and distal muscle weakness and impaired physical function while skeletal muscle tissue displays abnormal cellular infiltration of T cells, macrophages, and dendritic cells. Only limited knowledge exists about the effects of low-load blood flow restriction exercise in sIBM patients, and its effect on the immunological responses at the myocellular level remains unknown. The present study is the first to investigate the longitudinal effects of low-load blood flow restriction exercise on innate and adaptive immune markers in skeletal muscle from sIBM patients. METHODS: Twenty-two biopsy-validated sIBM patients were randomised into either 12 weeks of low-load blood flow restriction exercise (BFRE) or no exercise (CON). Five patients from the control group completed 12 weeks of BFRE immediately following participation in the 12-week control period leading to an intervention group of 16 patients. Muscle biopsies were obtained from either the m. tibialis anterior or the m. vastus lateralis for evaluation of CD3-, CD8-, CD68-, CD206-, CD244- and FOXP3-positive cells by three-colour immunofluorescence microscopy and Visiopharm-based image analysis quantification. A linear mixed model was used for the statistical analysis. RESULTS: Myocellular infiltration of CD3-/CD8+ expressing natural killer cells increased following BFRE (P < 0.05) with no changes in CON. No changes were observed for CD3+/CD8- or CD3+/CD8+ T cells in BFRE or CON. CD3+/CD244+ T cells decreased in CON, while no changes were observed in BFRE. Pronounced infiltration of M1 pro-inflammatory (CD68+/CD206-) and M2 anti-inflammatory (CD68+/CD206+) macrophages were observed at baseline; however, no longitudinal changes in macrophage content were observed for both groups. CONCLUSIONS: Low-load blood flow restriction exercise elicited an upregulation in CD3-/CD8+ expressing natural killer cell content, which suggests that 12 weeks of BFRE training evokes an amplified immune response in sIBM muscle. However, the observation of no changes in macrophage or T cell infiltration in the BFRE-trained patients indicates that patients with sIBM may engage in this type of exercise with no risk of intensified inflammatory activity.

In vivo pathogenicity of IgG from patients with anti-SRP or anti-HMGCR autoantibodies in immune-mediated necrotising myopathy.

OBJECTIVES: In autoimmunity, autoantibodies (aAb) may be simple biomarkers of disease or true pathogenic effectors. A form of idiopathic inflammatory myopathy associated with anti-signal recognition particle (SRP) or anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) aAb has been individualised and is referred to as immune-mediated necrotising myopathy (IMNM). The level of aAb correlates with IMNM activity and disease may respond to immunosuppression, suggesting that they are pathogenic. We aimed to evaluate the pathogenicity of IgG from patients with anti-SRP or anti-HMGCR aAb in vivo by developing the first mouse model of IMNM. METHODS: IgG from patients suffering from anti-SRP or anti-HMGCR associated IMNM were passively transferred to wild-type, Rag2-/- or complement C3-/- mice. Muscle deficiency was evaluated by muscle strength on electrostimulation and grip test. Histological analyses were performed after haematoxylin/eosin staining or by immunofluorescence or immunohistochemistry analysis. Antibody levels were quantified by addressable laser bead assay (ALBIA). RESULTS: Passive transfer of IgG from patients suffering from IMNM to C57BL/6 or Rag2-/- mice provoked muscle deficiency. Pathogenicity of aAb was reduced in C3-/- mice while increased by supplementation with human complement. Breakage of tolerance by active immunisation with SRP or HMGCR provoked disease. CONCLUSION: This study demonstrates that patient-derived anti-SRP+ and anti-HMGCR+ IgG are pathogenic towards muscle in vivo through a complement-mediated mechanism, definitively establishing the autoimmune character of IMNM. These data support the use of plasma exchanges and argue for evaluating complement-targeting therapies in IMNM.

Engineered DNA plasmid reduces immunity to dystrophin while improving muscle force in a model of gene therapy of Duchenne dystrophy.

In gene therapy for Duchenne muscular dystrophy there are two potential immunological obstacles. An individual with Duchenne muscular dystrophy has a genetic mutation in dystrophin, and therefore the wild-type protein is "foreign," and thus potentially immunogenic. The adeno-associated virus serotype-6 (AAV6) vector for delivery of dystrophin is a viral-derived vector with its own inherent immunogenicity. We have developed a technology where an engineered plasmid DNA is delivered to reduce autoimmunity. We have taken this approach into humans, tolerizing to myelin proteins in multiple sclerosis and to proinsulin in type 1 diabetes. Here, we extend this technology to a model of gene therapy to reduce the immunogenicity of the AAV vector and of the wild-type protein product that is missing in the genetic disease. Following gene therapy with systemic administration of recombinant AAV6-microdystrophin to mdx/mTRG2 mice, we demonstrated the development of antibodies targeting dystrophin and AAV6 capsid in control mice. Treatment with the engineered DNA construct encoding microdystrophin markedly reduced antibody responses to dystrophin and to AAV6. Muscle force in the treated mice was also improved compared with control mice. These data highlight the potential benefits of administration of an engineered DNA plasmid encoding the delivered protein to overcome critical barriers in gene therapy to achieve optimal functional gene expression.

The dual CCR2/CCR5 chemokine receptor antagonist Cenicriviroc reduces macrophage infiltration and disease severity in Duchenne muscular dystrophy (Dmdmdx-4Cv) mice.

Duchenne muscular dystrophy (DMD) is characterized by progressive muscle weakness which is ultimately fatal, most often due to involvement of the diaphragm. Macrophage infiltration of dystrophic muscles has been strongly linked to muscle damage and fibrosis in DMD. We hypothesized that cenicriviroc (CVC), a dual chemokine receptor (CCR2/CCR5) antagonist currently under clinical evaluation for other diseases, could prevent macrophage accumulation and blunt disease progression in the diaphragms of mdx mice (genetic homologue of DMD). Treatment with CVC (20 mg/kg/day intraperitoneally) or vehicle was initiated in mdx mice at 2 weeks of age (prior to the onset of muscle necrosis) and continued for 4 weeks. Flow cytometry to assess inflammatory cell subsets as well as histological and force generation parameters were determined in mdx diaphragms at the conclusion of the treatment. CVC therapy induced a major (3.9-fold) reduction in total infiltrating macrophages, whereas total numbers of neutrophils and T lymphocytes (CD4+ and CD8+) were unaffected. No changes in macrophage polarization status (inflammatory versus anti-inflammatory skewing based on iNOS and CD206 expression) were observed. Muscle fiber size and fibrosis were not altered by CVC, whereas a significant reduction in centrally nucleated fibers was found suggesting a decrease in prior necrosis-regeneration cycles. In addition, maximal isometric force production by the diaphragm was increased by CVC therapy. These results suggest that CVC or other chemokine receptor antagonists which reduce pathological macrophage infiltration may have the potential to slow disease progression in DMD.

Induction of Anti-agrin Antibodies Causes Myasthenia Gravis in Mice.

Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction (NMJ). Most cases of MG are caused by autoantibodies against the acetylcholine receptor (AChR), muscle-specific kinase (MuSK) and low-density lipoprotein receptor-related protein 4 (LRP4). Recent studies have identified anti-agrin antibodies in MG patients lacking these three antibodies (i.e., triple negative MG). Agrin is a basal lamina protein that has two isoforms. Neural agrin (N-agrin) binds to LRP4 to activate MuSK to induce AChR clusters and is thus critical for NMJ formation. We demonstrate that mice immunized with N-agrin showed MG-associated symptoms including muscle weakness, fragmented and distorted NMJs. These effects were not observed in mice injected with muscle agrin (M-agrin), an isoform that is inactive in inducing AChR clusters. Treatment with anti-N-agrin, but not anti-M-agrin, antibodies reduced agrin-induced AChR clusters in muscle cells. Together, these observations suggest that agrin antibodies may be play a role in MG pathogenesis.

Differences in Physical, Physiological and Motor Performance Traits between Volleyball and Basketball Athletes in a University in Ghana.

Physical, physiological and motor performance traits play substantial role in both volleyball (VB) and basketball(BB) competitions. The differences in these traits among University athletes in Ghana have not been reported. Hence, thisstudy documents and compares the physical, physiological and motor performance traits of VB and BB university athletes.Purposive sampling technique involving thirty-five university athletes (24 males and 11 females) with mean age of 21.77 ±2.03years was used. Height, weightn, waist and hip circumferences, waist to hip ratio (WHR), body mass index (BMI), heartrate (HR), systolic blood pressure (SBP) and diastolic blood pressure (DBP), shoulder muscular endurance (SME), abdominal muscular endurance (AME), left and right Arm Strength, Leg Power, Reaction time, Agility and Speed traits were measured.Descriptive statistics was used while analysis of variable was by paired t-test and significance was at p<0.05. Volleyballplayers significantly have better WHR, AMS and agility while Basketball athletes possess better SBP, SME and reactiontime. Gender influence was significant in height, WHR, HR, SBP, SME, AME, LAS, RAS, speed, reaction time, power andagility. Athletes in both games do not have similar physical, physiological and motor performance traits. Volley Ball playershad better abdominal muscular endurance, right-hand muscular strength, speed, power and agility while BB players hadbetter shoulder muscular endurance and reaction time traits. These differences in traits should inform volleyball andbasketball coaches in their selection.

More severe disease and slower recovery in younger patients with anti-3-hydroxy-3-methylglutaryl-coenzyme A reductase-associated autoimmune myopathy.

Objective: To study disease severity and response to therapy in a large cohort of patients with anti-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR)-associated myositis. Methods: Muscle strength, creatine kinase levels and treatments were assessed in anti-HMGCR-positive patients at each clinical visit. Univariate and multivariate analyses were used to analyse the influence of clinical characteristics on strength and the change in strength over time. Whole exome sequencing was performed in a subset of patients. Results: . Among 50 patients followed for ⩾2 years, only 22 (44%) reached full strength with immunosuppressive therapy; even among those with full strength, 55% continued to have CK levels in excess of 500 IU/l and only three could be tapered off immunosuppressive therapy. Both univariate and multivariate analysis showed that patients who were older at disease onset were stronger at all time points (P < 0.001) and improved faster (P < 0.008) than younger patients; a history of statin exposure was not independently associated with the improvement rate. Younger patients were more likely to have refractory disease (P = 0.02) than older patients. Among eight refractory patients with DNA available for testing, whole exome sequencing did not reveal pathogenic mutations in known dystrophy genes. The risk of cancer was not increased in anti-HMGCR myositis patients compared with the general population. Conclusions: Anti-HMGCR myositis is usually a chronic disease requiring long-term immunosuppression. Although younger patients had more severe disease and a worse prognosis than older patients, they did not have evidence of a known co-existing muscular dystrophy to explain their persistent, and sometimes progressive, muscle weakness.

Interleukin-6 -174G/C gene polymorphism affects muscle damage response to acute eccentric resistance exercise in elderly obese women.

The IL-6 gene polymorphism has been associated with disease prevalence and different physiological responses to exercise. Eccentric resistance exercise (ERE) is considered a nonpharmacological tool to prevent the chronic degenerative profile associated with aging and obesity. Consequently, the aim of the present study was to investigate the influence of IL-6 -174G/C polymorphism on acute interleukin-6 (IL-6) and creatine kinase (CK) temporal response to ERE in elderly obese women. Ninety women completed seven sets of ten repetitions (eccentric only) of an acute ERE session at 110% of the ten repetitions maximum (10RM). IL-6 genotypes displayed no difference at baseline. ERE induced changes in CK concentration over time occurred only in the GG group, F(2.619, 136.173)=5.199, p=0.003, with CK activity increased from 106.8±6.9 U/l pre-intervention to 122.7±11.2 U/l at 24 h and 131.9±14.4 U/l at 48 h post-exercise. IL-6 concentration in the GG group was lower than the CC/CG group only at 0 h post-exercise (3.78±0.58 pg/ml versus 6.51±1.91 pg/ml, p=0.030). Only the GG genotype group had higher CK activity 24-48 h following ERE and greater CK integral values, while IL-6 activity over 48 h was higher in the CC/CG genotype group. In conclusion, IL-6 genotype affects CK and IL-6 in response to ERE. It is of interest that the ERE protocol induced an elevation in CK, indicating possible muscle damage without exacerbating IL-6 and CK for the GG genotype.

Inflammatory markers in skeletal muscle of older adults.

Older adults have an increase in circulating markers of inflammation. The current study examined whether there is an increase in the expression of inflammatory markers within the vastus lateralis, a major locomotive muscle, of older adults, and if so, whether the reduction in muscle strength and aerobic capacity in older adults is related to increased muscle inflammation. Skeletal muscle biopsies were taken from older adults (n = 17, 67 ± 1.6 years) and young individuals (n = 16, 24 ± 0.6 years) under resting and fasting conditions. Muscle was analyzed for mRNA levels of intracellular inflammatory molecules (MCP1, TNFα and IL-1ß) and total cellular protein abundance of cytokines, chemokines and kinases (IL-6, IL-8, MCP1, TNFα, p65 (NF-κB), JNK1/2 and STAT3). MCP1 expression was significantly higher (p < 0.05; 50 %, mRNA and 40 %, protein) in elderly than younger participants, as was IL-8 (4 %). No detectable difference in kinase protein expression was observed for STAT3, JNK or p65 (NF-κB), TNFα or IL-6. Muscle strength was lower in the elderly compared to the young group (1.55 ± 0.17 vs. 2.56 ± 0.13 Nm/kg, p < 0.001). The elderly group also had a significantly lower VO(2peak) compared to the young group (24.9 ± 1.9 vs. 39.3 ± 1.9, p < 0.001), but muscle strength and VO(2peak) were not correlated with the examined inflammatory markers. Older adults have increased MCP1 (mRNA and protein abundance) and IL-8 (protein abundance) and also reduced muscle strength and VO(2peak). However, the reduction in muscle strength and VO(2peak) was not related to the increase in muscle inflammatory markers in this cohort.

Immunological parameters in elderly women: correlations with aerobic power, muscle strength and mood state.

PURPOSE: Our objective was to relate immunological data for healthy but sedentary elderly women to aerobic power, strength, and mood state. METHODS: We measured peak aerobic power and one-repetition maximum strength along with mood (depression and fatigue), quality of life and carbohydrate intake on 42 women aged 60-77 years. Standard immunological techniques determined natural killer cell count and cytotoxic activity (NKCA), proliferative responses to phytohemaglutinin and OKT(3), various lymphocyte subpopulations (CD3(+), CD3(-)CD19(+), CD56(+), CD4(+), CD8(+), CD56(dim) and CD56(bright)), and markers of activation, maturation, down-regulation and susceptibility to apoptosis (CD25(+), CD28(+), CD45RA(+), CD45RO(+), CD69(+), CD95(+), HLA-DR(+)). RESULTS: Correlations of immune parameters with aerobic power and strength were very similar for absolute and relative immunological data. In the group as a whole, the only correlation with aerobic power was -0.35 (relative CD4(+)CD69(+) count), but in subjects with values <22.6 mL kg(-1)min(-1) correlations ranged from -0.57 (relative CD4(+)CD45RO(+)) to 0.92 (absolute CD56(dim)HLA-DR(+)). In terms of muscle strength, univariate correlation coefficients ranged from -0.34 (relative and absolute CD3(+)CD4(+)CD8(+)) to +0.48 (absolute CD3(+)HLA-DR(+)) and +0.50 (absolute CD8(+)CD45RA(+)CD45RO(+)). Neither NKCA nor lymphocyte proliferation were correlated with aerobic power or muscle strength. Although mood state and quality of life can sometimes be influenced by an individual's fitness level, our multivariate analyses suggested that depression, fatigue and quality of life were more important determinants of immune profile than our fitness measures. CONCLUSIONS: Psychological changes associated with aging may have a substantial adverse effect upon the immune system, and immunological function may be enhanced more by addressing these issues than by focusing upon aerobic or resistance training.

Combined exercise training reduces IFN-γ and IL-17 levels in the plasma and the supernatant of peripheral blood mononuclear cells in women with multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disorder in which lymphocytic infiltration mediated mainly by pro-inflammatory cytokines. In this study, we examined the effect of combined exercise training on the levels of IFN-γ, IL-4 and IL-17 in the plasma and the supernatant of peripheral blood lymphocytes in women with multiple sclerosis. Expanded Disability Status Scale (EDSS), VO(2)max, muscle strength, and balance tests were obtained at baseline and post-treatment follow-up. Combined exercises training was designed for 24 sessions during 8 weeks. Each session was started with 5 min warm-up and was followed by 10 min stretch training, 20 min aerobic exercises and 20 min resistance-endurance training. The disability score was significantly decreased in test MS subjects after 8 weeks combined exercise training. Muscle strength and balance were increased significantly after the training program in test group. In this study, plasma, and peripheral blood mononuclear cell (PBMC) IL-17 and IFN-γ production was significantly decreased after 8 weeks combined training. Our findings suggest that combined training has useful anti-inflammatory effects by decrease in PBMC and plasma IL-17 production.

Progressive resistance training in elderly HIV-positive patients: does it work?

BACKGROUND: Elderly people present alterations in body composition and physical fitness, compromising their quality of life. Chronic diseases, including HIV/AIDS, worsen this situation. Resistance exercises are prescribed to improve fitness and promote healthier and independent aging. Recovery of strength and physical fitness is the goal of exercise in AIDS wasting syndrome. OBJECTIVE: This study describes a case series of HIV-positive elderly patients who participated in a progressive resistance training program and evaluates their body composition, muscular strength, physical fitness and the evolution of CD4+ and CD8+ cell counts. METHODS: Subjects were prospectively recruited for nine months. The training program consisted of three sets of 8-12 repetitions of leg press, seated row, lumbar extension and chest press, performed with free weight machines hts, twice/week for one year. Infectious disease physicians followed patients and reported all relevant clinical data. Body composition was assessed by anthropometric measures and dual-energy x-ray absorptiometry before and after the training program. RESULTS: Fourteen patients, aged 62-71 years old, of both genders, without regular physical activity who had an average of nine years of HIV/AIDS history were enrolled. The strengths of major muscle groups increased (74%-122%, p=0.003-0.021) with a corresponding improvement in sit-standing and walking 2.4 m tests (p=0.003). There were no changes in clinical conditions and body composition measures, but triceps and thigh skinfolds were significantly reduced (p=0.037). In addition, there were significant increases in the CD4+ counts (N=151 cells; p=0.008) and the CD4+/CD8+ ratio (0.63 to 0.81, p=0.009). CONCLUSION: Resistance training increased strength, improved physical fitness, reduced upper and lower limb skinfolds, and were associated with an improvement in the CD4+ and CD4+/CD8+ counts in HIV positive elderly patients without significant side effects.

Progressive resistance training in elderly hiv-positive patients: does it work?

BACKGROUND: Elderly people present alterations in body composition and physical fitness, compromising their quality of life. Chronic diseases, including HIV/AIDS, worsen this situation. Resistance exercises are prescribed to improve fitness and promote healthier and independent aging. Recovery of strength and physical fitness is the goal of exercise in AIDS wasting syndrome. OBJECTIVE: This study describes a case series of HIV-positive elderly patients who participated in a progressive resistance training program and evaluates their body composition, muscular strength, physical fitness and the evolution of CD4+ and CD8+ cell counts. METHODS: Subjects were prospectively recruited for nine months. The training program consisted of three sets of 8-12 repetitions of leg press, seated row, lumbar extension and chest press, performed with free weight machines hts, twice/week for one year. Infectious disease physicians followed patients and reported all relevant clinical data. Body composition was assessed by anthropometric measures and dual-energy x-ray absorptiometry before and after the training program. RESULTS: Fourteen patients, aged 62-71 years old, of both genders, without regular physical activity who had an average of nine years of HIV/AIDS history were enrolled. The strengths of major muscle groups increased (74 percent-122 percent, p=0.003-0.021) with a corresponding improvement in sit-standing and walking 2.4 m tests (p=0.003). There were no changes in clinical conditions and body composition measures, but triceps and thigh skinfolds were significantly reduced (p=0.037). In addition, there were significant increases in the CD4+ counts (N=151 cells; p=0.008) and the CD4+/CD8+ ratio (0.63 to 0.81, p=0.009). CONCLUSION: Resistance training increased strength, improved physical fitness, reduced upper and lower limb skinfolds, and were associated with an improvement in the CD4+ and CD4+/CD8+ counts in HIV positive elderly patients without ...