Assessment of porphyrogenicity of drugs and chemicals in selected hepatic cell culture models through a fluorescence-based screening assay.

Compounds that induce 5-aminolevulinic acid [ALA] synthase-1 and/or cytochromes P-450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias [AHPs]. Currently, there is no simple, robust model used to assess and predict the porphyrogenicity of drugs and chemicals. Our aim was to develop a fluorescence-based in vitro assay for this purpose. We studied four different hepatic cell culture models: HepG2 cells, LMH cells, 3D HepG2 organoids, and 3D organoids of primary liver cells from people without known disease [normal human controls]. We took advantage of the fluorescent properties of protoporphyrin IX [PP], the last intermediate of the heme biosynthesis pathway, performing fluorescence spectrometry to measure the intensity of fluorescence emitted by these cells treated with selected compounds of importance to patients with AHPs. Among the four cell culture models, the LMH cells produced the highest fluorescence readings, suggesting that these cells retain more robust heme biosynthesis enzymes or that the other cell models may have lost their inducibility of ALA synthase-1 [ALAS-1]. Allyl isopropyl acetamide [AIA], a known potent porphyrogen and inducer of ALAS-1, was used as a positive control to help predict porphyrogenicity for tested compounds. Among the tested compounds (acetaminophen, acetylsalicylic acid, ß-estradiol, hydroxychloroquine sulfate, alpha-methyldopa, D (-) norgestrel, phenobarbital, phenytoin, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan), concentrations greater than 0.314 mM for norgestrel, phenobarbital, phenytoin, and sodium valproate produced fluorescence readings higher than the reading produced by the positive AIA control. Porphyrin accumulation was also measured by HPLC to confirm the validity of the assay. We conclude that LMH cell cultures in multi-well plates are an inexpensive, robust, and simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.

Changes in standing stability with balance-based torso-weighting with cerebellar ataxia: A pilot study.

OBJECTIVES: People with cerebellar ataxia have few options to improve the standing stability they need for function. Strategic placement of light weights on the torso using the balance-based torso-weighting (BBTW) method has improved stability and reduced falls in people with multiple sclerosis, but has not been tested in cerebellar ataxia. We examined whether torso-weighting increased standing stability and/or functional movement in people with cerebellar ataxia. METHODS: Ten people with cerebellar ataxia and 10 matched controls participated in this single-session quasi-experimental pilot study. People with ataxia performed the Scale for the Assessment and Rating of Ataxia (SARA) prior to clinical testing. All participants donned inertial sensors that recorded postural sway; stopwatches recorded duration for standing and mobility tasks. All participants stood for up to 30 s on firm and foam surfaces with eyes open then eyes closed, and performed the Timed Up and Go (TUG) test. Light weights (0.57-1.25 kg) were strategically applied to a vest-like garment. Paired t tests compared within-group differences with and without BBTW weights. Independent t tests assessed differences from controls. All t tests were one-tailed with alpha set at .05. RESULTS: Duration of standing for people with ataxia was significantly longer with weighting (p = .004); all controls stood for the maximum time of 120 s with and without weights. More severe ataxia according to SARA was moderately correlated with greater improvement in standing duration with BBTW (Pearson r = .54). Tasks with more sensory challenges (eyes closed, standing on firm surface) showed less body sway with weighting. Duration for the TUG was unchanged by torso-weighting in people with ataxia. CONCLUSION: Strategic weighting improved standing stability but not movement speed in people with ataxia. BBTW has potential for improving stability and response to challenging sensory conditions in this population. Future studies should further examine gait stability measures along with movement speed.

Four Months of Wearing a Balance Orthotic Improves Measures of Balance and Mobility Among a Cohort of Community-Living Older Adults.

BACKGROUND AND PURPOSE: The Centers for Disease Control and Prevention estimated that there were 29 million falls and 7 million injuries in 2014 in the United States. Falls, decreased balance, and mobility disability are common in older adults and often result in loss of independence. Finding interventions to address these issues is important, as this age group is growing exponentially. Prior studies indicate balance and mobility can be improved by the balance-based torso-weighting (BBTW) assessment implemented through wear of a balance orthotic (BO). This study sought to determine the impact of wearing a BO on balance, mobility, and fall risk over time. METHODS: This quasiexperimental, 1-group pre-/posttest study investigated the effect of 4 months of daily wear (4 hours per day) of a BO on mobility, balance, and falls efficacy in 30 older adults living in a retirement community with limited mobility defined by a Short Physical Performance Battery (SPPB) score range between 4 and 9 out of a maximum of 12 points. Pre- and posttreatment tests included the Timed Up and Go (TUG), Functional Gait Assessment (FGA), Falls Efficacy Scale (FES), and SPPB. Participants received the BBTW assessment, consisting of individualized assessment of 3-dimensional balance loss, and treatment with a strategically weighted and fitted BO to control balance loss. The BO was worn twice a day for 2 hours (4 hours per day) for 4 months. Participants continued regular activity and no other interventions were provided. All posttests were conducted after 4 months and at least 8 hours after removal of the BO. Subitems from the SPPB (gait speed [GS], 5-time sit-to-stand [FTSST], and tandem stance time [TST]) were analyzed as separate outcome measures. Data were analyzed with paired t tests with a Bonferroni correction (SPPB, GS, FGA, and FES) when statistical assumptions were met. Data that did not meet the statistical assumptions of the paired t test (FTSST, TST, and TUG) were analyzed with Wilcoxon signed rank tests with a Bonferroni correction. RESULTS AND DISCUSSION: Twenty-four participants, average age 87 (5.7) years, completed the study. Paired t tests indicated that mean group scores on the SPPB, GS, and FGA significantly improved from pre- to posttests. The SPPB improved by 1.3 points (P = .001). GS improved by 0.09 m/s (P = .004) and both mean values improved beyond fall risk cutoffs. The FGA also improved by 2.6 points (P = .001). There were no significant changes in FES scores (P = .110). Wilcoxon signed rank tests indicated median group scores of the FTSST significantly improved from pre- to posttests by 7.4 seconds (P = .002) and median TUG times improved by 3.5 seconds (P = .004). There were no changes in TST (P = .117). CONCLUSIONS: This study suggests that wearing a BO for 4 hours per day for 4 months results in improvements in functional assessments related to fall risk (SPPB, GS, FGA, TUG, and FTSST) in a group of older adult participants with limited mobility.

Transgenic Nicotiana tabacum seeds expressing the Mycobacterium tuberculosis Alanine- and Proline-rich antigen.

The glycoprotein APA (Alanine- and Proline-rich Antigen, a 45/47 kDa antigen complex, Rv1860) is considered as a major immunodominant antigen secreted by M. tuberculosis. This antigen has proved to be highly immunogenic in experimental models and humans, presenting a significant potential for further development of a new vaccine for tuberculosis. Glycosylation plays a key role in the immunogenicity of the APA protein. Because plants are known to promote post-translational modification such as glycosylation and to be one of the most economic and safe hosts for recombinant protein expression, we have over expressed the APA protein in transgenic tobacco plants aiming to produce a glycosylated version of the protein. Seeds are known to be a well-suited organ to accumulate recombinant proteins, due to low protease activity and higher protein stability. We used a seed-specific promoter from sorghum, a signal peptide to target the protein to the endoplasmic reticulum and ultimately in the protein storage vacuoles. We show that the recombinant protein accumulated in the seeds had similar isoelectric point and molecular weight compared with the native protein. These findings demonstrate the ability of tobacco plants to produce glycosylated APA protein, opening the way for the development of secure, effective and versatile vaccines or therapeutic proteins against tuberculosis.

Effects of Torso-Weighting on Standing Balance and Falls During the Sensory Organization Test in People with Multiple Sclerosis.

BACKGROUND: In people with multiple sclerosis (MS), common gait and balance impairments can lead to falls, fear of falling, activity restriction, and social isolation. Sensory augmentation in the form of torso-weighting has resulted in improvement in gait and balance, but research on its effect on falls in MS is lacking. METHODS: 60 people with MS and 10 bin-matched controls completed the Sensory Organization Test (SOT) while nonweighted and again while weighted using the Balance-Based Torso-Weighting assessment method. This was a quasi-experimental pre-post intervention study. The SOT composite scores, equilibrium scores, and number of falls occurring across six SOT conditions were compared between and within groups using 2-way analysis of variance, α = .05 with planned t test analyses of weighting effects. RESULTS: A significant increase in composite score of 9.14 points nonweighted to weighted occurred in the MS group (P < .001) but not in controls (P = .626). Equilibrium scores were significantly higher with weights in the MS group (P < .001) but not in controls (P = .5). Falls during the SOT were reduced by 35% with weights in the MS group versus without weights (P < .001), with the greatest number of falls occurring in the most challenging SOT conditions. CONCLUSIONS: During a single testing session, torso-weighting produced significant improvements in postural stability and fall reduction during the SOT for people with MS but no change in controls. Further research is needed to determine whether torso-weighting has the potential to reduce falls in MS during real-world activities.

Transgenic Nicotiana tabacum seeds expressing the Mycobacterium tuberculosis Alanine- and Proline-rich antigen

The glycoprotein APA (Alanine- and Proline-rich Antigen, a 45/47 kDa antigen complex, Rv1860) is considered as a major immunodominant antigen secreted by M. tuberculosis. This antigen has proved to be highly immunogenic in experimental models and humans, presenting a significant potential for further development of a new vaccine for tuberculosis. Glycosylation plays a key role in the immunogenicity of the APA protein. Because plants are known to promote post-translational modification such as glycosylation and to be one of the most economic and safe hosts for recombinant protein expression, we have over expressed the APA protein in transgenic tobacco plants aiming to produce a glycosylated version of the protein. Seeds are known to be a well-suited organ to accumulate recombinant proteins, due to low protease activity and higher protein stability. We used a seed-specific promoter from sorghum, a signal peptide to target the protein to the endoplasmic reticulum and ultimately in the protein storage vacuoles. We show that the recombinant protein accumulated in the seeds had similar isoelectric point and molecular weight compared with the native protein. These findings demonstrate the ability of tobacco plants to produce glycosylated APA protein, opening the way for the development of secure, effective and versatile vaccines or therapeutic proteins against tuberculosis.

Transgenic Nicotiana tabacum seeds expressing the Mycobacterium tuberculosis Alanine- and Proline-rich antigen

The glycoprotein APA (Alanine- and Proline-rich Antigen, a 45/47 kDa antigen complex, Rv1860) is considered as a major immunodominant antigen secreted by M. tuberculosis. This antigen has proved to be highly immunogenic in experimental models and humans, presenting a significant potential for further development of a new vaccine for tuberculosis. Glycosylation plays a key role in the immunogenicity of the APA protein. Because plants are known to promote post-translational modification such as glycosylation and to be one of the most economic and safe hosts for recombinant protein expression, we have over expressed the APA protein in transgenic tobacco plants aiming to produce a glycosylated version of the protein. Seeds are known to be a well-suited organ to accumulate recombinant proteins, due to low protease activity and higher protein stability. We used a seed-specific promoter from sorghum, a signal peptide to target the protein to the endoplasmic reticulum and ultimately in the protein storage vacuoles. We show that the recombinant protein accumulated in the seeds had similar isoelectric point and molecular weight compared with the native protein. These findings demonstrate the ability of tobacco plants to produce glycosylated APA protein, opening the way for the development of secure, effective and versatile vaccines or therapeutic proteins against tuberculosis.

Liver BCATm transgenic mouse model reveals the important role of the liver in maintaining BCAA homeostasis.

Unlike other amino acids, the branched-chain amino acids (BCAAs) largely bypass first-pass liver degradation due to a lack of hepatocyte expression of the mitochondrial branched-chain aminotransferase (BCATm). This sets up interorgan shuttling of BCAAs and liver-skeletal muscle cooperation in BCAA catabolism. To explore whether complete liver catabolism of BCAAs may impact BCAA shuttling in peripheral tissues, the BCATm gene was stably introduced into mouse liver. Two transgenic mouse lines with low and high hepatocyte expression of the BCATm transgene (LivTg-LE and LivTg-HE) were created and used to measure liver and plasma amino acid concentrations and determine whether the first two BCAA enzymatic steps in liver, skeletal muscle, heart and kidney were impacted. Expression of the hepatic BCATm transgene lowered the concentrations of hepatic BCAAs while enhancing the concentrations of some nonessential amino acids. Extrahepatic BCAA metabolic enzymes and plasma amino acids were largely unaffected, and no growth rate or body composition differences were observed in the transgenic animals as compared to wild-type mice. Feeding the transgenic animals a high-fat diet did not reverse the effect of the BCATm transgene on the hepatic BCAA catabolism, nor did the high-fat diet cause elevation in plasma BCAAs. However, the high-fat-diet-fed BCATm transgenic animals experienced attenuation in the mammalian target of rapamycin (mTOR) pathway in the liver and had impaired blood glucose tolerance. These results suggest that complete liver BCAA metabolism influences the regulation of glucose utilization during diet-induced obesity.

Skeletal Muscle Mitochondrial Content, Oxidative Capacity, and Mfn2 Expression Are Reduced in Older Patients With Heart Failure and Preserved Ejection Fraction and Are Related to Exercise Intolerance.

OBJECTIVES: The aim of this study was to examine skeletal muscle mitochondria content, oxidative capacity, and the expression of key mitochondrial dynamics proteins in patients with heart failure with preserved ejection fraction (HFpEF), as well as to determine potential relationships with measures of exercise performance. BACKGROUND: Multiple lines of evidence indicate that severely reduced peak exercise oxygen uptake (peak VO2) in older patients with HFpEF is related to abnormal skeletal muscle oxygen utilization. Mitochondria are key regulators of skeletal muscle metabolism; however, little is known about how these organelles are affected in HFpEF. METHODS: Both vastus lateralis skeletal muscle citrate synthase activity and the expression of porin and regulators of mitochondrial fusion were examined in older patients with HFpEF (n = 20) and healthy, age-matched control subjects (n = 17). RESULTS: Compared with age-matched healthy control subjects, mitochondrial content assessed by porin expression was 46% lower (p = 0.01), citrate synthase activity was 29% lower (p = 0.01), and Mfn2 (mitofusin 2) expression was 54% lower (p <0.001) in patients with HFpEF. Expression of porin was significantly positively correlated with both peak VO2 and 6-min walk distance (r = 0.48, p = 0.003 and r = 0.33, p = 0.05, respectively). Expression of Mfn2 was also significantly positively correlated with both peak VO2 and 6-min walk distance (r = 0.40, p = 0.02 and r = 0.37, p = 0.03 respectively). CONCLUSIONS: These findings suggest that skeletal muscle oxidative capacity, mitochondrial content, and mitochondrial fusion are abnormal in older patients with HFpEF and might contribute to their severe exercise intolerance.

Blood-cell bioenergetics are associated with physical function and inflammation in overweight/obese older adults.

BACKGROUND: Physical function and strength decline with age and lead to limited mobility and independence in older adults. Alterations in mitochondrial function are thought to underlie numerous age-related changes, including declining physical ability. Recent studies suggest that systemic changes in bioenergetic capacity may be reported by analyzing mitochondrial function in circulating cells. The objective of this study was to determine whether the bioenergetic capacity of peripheral blood mononuclear cells (PBMCs) is related to differences in physical function among older, overweight/obese, adults. To address this, we tested the hypothesis that greater PBMC respirometric capacity would be associated with better physical function, muscular strength, leg lean mass, and muscle quality. Furthermore, we tested whether the respirometric capacity of PBMCs is related to cellular composition and inflammatory status reported by interleukin-6 (IL-6). METHODS: Fasted PBMC respiration (pmol/min/500,000 cells), expanded short physical performance battery (Ex-SPPB), peak knee extensor (KE) strength (Nm), grip strength (kg), leg lean mass (kg, via dual energy X-ray absorptiometry [DXA]), muscle quality (Nm/kg), and plasma IL-6 (pg/mL) were analyzed in 15 well-functioning, community-dwelling, sedentary overweight/obese older men (n=9) and women (n=6) aged 65 to 78 (mean 68.3 ± 3.5 years). Pearson and partial correlations were calculated to determine associations between PBMC respiration and these variables. RESULTS: Higher maximal respiration of PBMCs was associated with better Ex-SPPB (r=0.58, p=0.02), greater KE strength (r=0.60, p=0.02), greater grip strength (r=0.52, p=0.05) and lower IL-6 (r=-0.58, p=0.04). Higher spare respiratory capacity was associated with better Ex-SPPB (r=0.59, p=0.02), greater KE strength (r=0.60, p=0.02), greater grip strength (r=0.54, p=0.04), greater leg muscle quality (r=0.56, p=0.04), and lower IL-6 (r=-0.55, p=0.05). Monocyte and lymphocyte counts were not related to PBMC respiratory capacity. CONCLUSIONS: Our results indicate that respirometric profiles of readily obtainable blood cells are associated with physical function and strength. Future studies should be undertaken in order to determine whether blood-based bioenergetic profiling can provide an objective index of systemic mitochondrial health.

Respirometric Profiling of Muscle Mitochondria and Blood Cells Are Associated With Differences in Gait Speed Among Community-Dwelling Older Adults.

BACKGROUND: Gait speed provides an integrated measure of physical ability that is predictive of morbidity, disability, and mortality in older adults. Energy demands associated with walking suggest that mitochondrial bioenergetics may play a role in gait speed. Here, we examined the relationship between gait speed and skeletal muscle mitochondrial bioenergetics, and further evaluated whether blood-based bioenergetic profiling might have similar associations with gait speed. METHODS: Participants in this study were comprised of two subsets (n = 17 per subset) and were overweight/obese (body mass index, 30.9 ± 2.37), well-functioning, community-dwelling older adults (69.1 ± 3.69 years) without major comorbidity. Gait speeds were calculated from a fast-paced 400 m walk test. Respiratory control ratios were measured from mitochondria isolated from leg skeletal muscle biopsies from one subset. Maximal respiration and spare respiratory capacity were measured from peripheral blood mononuclear cells from the other subset. RESULTS: Individual differences in gait speed correlated directly with respiratory control ratio of mitochondria isolated from skeletal muscle (r = .536, p = .027) and with both maximal respiration and spare respiratory capacity of peripheral blood mononuclear cells (r = .585 and p = .014; r = .609 and p = .009, respectively). CONCLUSIONS: The bioenergetic profile of mitochondria isolated from skeletal muscle is associated with gait speed in older adults. Blood-based bioenergetic profiling is also associated with gait speed and may provide an alternative measure of mitochondrial function.

Gait changes with balance-based torso-weighting in people with multiple sclerosis.

BACKGROUND AND PURPOSE: People with multiple sclerosis (PwMS) commonly have mobility impairments that may lead to falls and limitations in activities. Physiotherapy interventions that might improve mobility typically take several weeks. Balance-based torso-weighting (BBTW), a system of strategically placing light weights to improve response to balance perturbations, has resulted in immediate small improvements in clinical measures in PwMS, but changes in spatiotemporal gait parameters are unknown. The purpose was to investigate the effects of BBTW on gait parameters in PwMS and healthy controls. METHODS DESIGN: This study is a non-randomized controlled experiment. PARTICIPANTS: This study included 20 PwMS and 20 matched healthy controls PROCEDURES: People with multiple sclerosis walked on an instrumented mat at their fastest speed for three trials each in two conditions: without BBTW then with BBTW. Healthy controls walked in both conditions at two speeds: their fastest speed and at velocities equivalent to their matched PwMS. RESULTS: Averaged gait trials showed that, with BBTW, PwMS had significantly increased velocity (p = 0.002), cadence (p = 0.007) and time spent in single-limb support (p = 0.014), with decreased time in double-limb support (p = 0.004). Healthy controls increased velocity (p = 0.012) and cadence (p = 0.015) and decreased support base (p = 0.014) in fast trials with BBTW; at matched velocities, step length (p = 0.028) and support base (p = 0.006) were significantly different from PwMS. All gait variables in healthy controls at fast speeds were significantly different from PwMS walking at their fastest speeds. DISCUSSION: All participants showed increases in gait velocity and cadence during fast walk with BBTW. Improvements in time spent in single-limb and double-limb support by PwMS with BBTW may reflect greater stability in gait. Future research might ascertain if these immediate improvements could enhance effectiveness of longer-term physiotherapy on functional mobility in PwMS.

In-home measurement of the effect of strategically weighted vests on ambulation.

Strategically weighted vests are currently being used to treat patients with Parkinson's, Multiple Sclerosis, and ataxia. While studies have been conducted to demonstrate the effectiveness of these vests, there has been very little research into the mechanisms that give rise to the vest's results. This study demonstrates the ability to capture gait parameters from depth images[1] in the home with sufficient sensitivity to support future investigation of the weighted vest intervention. The study also explores multiple metrics, using in-home gait sensing, to study a subject's ambulatory ability including gait mechanics, uncertainty in motion, and gait cadence. We then investigate the effects of these vests on a subject's ambulation by examining these metrics both before and after the vest is worn. While only four subjects were used, results are promising, showing a statistically significant and clinically significant change in many of these metrics as a result of the vest. The cases presented here concern two subjects, one with a "tight" gait caused by Progressive Supranuclear Palsy, and the second with an excessively "loose" gait due to Parkinson's disease. We show that in both subjects, using the vest immediately moved the metrics in a direction beneficial to the subject's clinical condition. This result concurs with clinical observations as measured using various clinical fall risk instruments.

A single dose of a DNA vaccine encoding apa coencapsulated with 6,6'-trehalose dimycolate in microspheres confers long-term protection against tuberculosis in Mycobacterium bovis BCG-primed mice.

Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6'-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB.

Protection conferred by heterologous vaccination against tuberculosis is dependent on the ratio of CD4(+) /CD4(+) Foxp3(+) cells.

CD4(+) Foxp3(+) regulatory T cells inhibit the production of interferon-γ, which is the major mediator of protection against Mycobacterium tuberculosis infection. In this study, we evaluated whether the protection conferred by three different vaccines against tuberculosis was associated with the number of spleen and lung regulatory T cells. We observed that after homologous immunization with the 65 000 molecular weight heat-shock protein (hsp 65) DNA vaccine, there was a significantly higher number of spleen CD4(+) Foxp3(+) cells compared with non-immunized mice. Heterologous immunization using bacillus Calmette-Guérin (BCG) to prime and DNA-hsp 65 to boost (BCG/DNA-hsp 65) or BCG to prime and culture filtrate proteins (CFP)-CpG to boost (BCG/CFP-CpG) induced a significantly higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells compared with non-immunized mice. In addition, the protection conferred by either the BCG/DNA-hsp 65 or the BCG/CFP-CpG vaccines was significant compared with the DNA-hsp 65 vaccine. Despite the higher ratio of spleen CD4(+) /CD4(+) Foxp3(+) cells found in BCG/DNA-hsp 65-immunized or BCG/CFP-CpG-immunized mice, the lungs of both groups of mice were better preserved than those of DNA-hsp 65-immunized mice. These results confirm the protective efficacy of BCG/DNA-hsp 65 and BCG/CFP-CpG heterologous prime-boost vaccines and the DNA-hsp 65 homologous vaccine. Additionally, the prime-boost regimens assayed here represent a promising strategy for the development of new vaccines to protect against tuberculosis because they probably induce a proper ratio of CD4(+) and regulatory (CD4(+) Foxp3(+) ) cells during the immunization regimen. In this study, this ratio was associated with a reduced number of regulatory cells and no injury to the lungs.

IFN-γ-mediated efficacy of allergen-free immunotherapy using mycobacterial antigens and CpG-ODN.

Epidemiological and experimental evidence supports the notion that microbial infections that are known to induce Th1-type immune responses can suppress Th2 immune responses, which are characteristics of allergic disorders. However, live microbial immunization might not be feasible for human immunotherapy. Here, we evaluated whether induction of Th1 immunity by the immunostimulatory sequences of CpG-oligodeoxynucleotides (CpG-ODN), with or without culture filtrate proteins (CFP), from Mycobacterium tuberculosis would suppress ongoing allergic lung disease. Presensitized and ovalbumin (OVA)-challenged mice were treated subcutaneously with CpG, or CpG in combination with CFP (CpG/CFP). After 15 days of treatment, airway inflammation and specific T- and B-cell responses were determined. Cell transfer experiments were also performed. CpG treatment attenuated airway allergic disease; however, the combination CpG/CFP treatment was significantly more effective in decreasing airway hyperresponsiveness, eosinophilia and Th2 response. When an additional intranasal dose of CFP was given, allergy was even more attenuated. The CpG/CFP therapy also reduced allergen-specific IgG1 and IgE antibodies and increased IgG2a. Transfer of spleen cells from mice immunized with CpG/CFP also reduced allergic lung inflammation. CpG/CFP treatment induced CFP-specific production of IFN-γ and IL-10 by spleen cells and increased production of IFN-γ in response to OVA. The essential role of IFN-γ for the therapeutic effect of CpG/CFP was evidenced in IFN-γ knockout mice. These results show that CpG/CFP treatment reverses established Th2 allergic responses by an IFN-γ-dependent mechanism that seems to act both locally in the lung and systemically to decrease allergen-specific Th2 responses.

Leucine and alpha-ketoisocaproic acid, but not norleucine, stimulate skeletal muscle protein synthesis in neonatal pigs.

The branched-chain amino acid, leucine, acts as a nutrient signal to stimulate protein synthesis in skeletal muscle of young pigs. However, the chemical structure responsible for this effect has not been identified. We have shown that the other branched-chain amino acids, isoleucine and valine, are not able to stimulate protein synthesis when raised in plasma to levels within the postprandial range. In this study, we evaluated the effect of leucine, alpha-ketoisocaproic acid (KIC), and norleucine infusion (0 or 400 micromol kg(-1) h(-1) for 60 min) on protein synthesis and activation of translation initiation factors in piglets. Infusion of leucine, KIC, and norleucine raised plasma levels of each compound compared with controls. KIC also increased (P < 0.01) and norleucine reduced (P < 0.02) plasma levels of leucine compared with controls. Administration of leucine and KIC resulted in greater (P < 0.006) phosphorylation of eukaryotic initiation factor (eIF) 4E binding protein-1 (4E-BP1) and eIF4G, lower (P < 0.04) abundance of the inactive 4E-BP1.eIF4E complex, and greater (P < 0.05) active eIF4G.eIF4E complex formation in skeletal muscle compared with controls. Protein synthesis in skeletal muscle was greater (P < 0.02) in leucine- and KIC-infused pigs than in those in the control group. Norleucine infusion did not affect muscle protein synthesis or translation initiation factor activation. In liver, neither protein synthesis nor activation of translation initiation factors was affected by treatment. These results suggest that the ability of leucine to act as a nutrient signal to stimulate skeletal muscle protein synthesis is specific for leucine and/or its metabolite, KIC.

Mycobacterium tuberculosis culture filtrate proteins plus CpG Oligodeoxynucleotides confer protection to Mycobacterium bovis BCG-primed mice by inhibiting interleukin-4 secretion.

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-gamma) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-gamma-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-gamma and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-gamma and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.

Randomized clinical trial of balance-based torso weighting for improving upright mobility in people with multiple sclerosis.

BACKGROUND: Torso weighting has sometimes been effective for improving upright mobility in people with multiple sclerosis, but parameters for weighting have been inconsistent. OBJECTIVE: To determine whether balance-based torso weighting (BBTW) has immediate effects on upright mobility in people with multiple sclerosis. METHODS: This was a 2-phase randomized clinical trial. In phase 1, 36 participants were randomly assigned to experimental and control groups. In phase 2, the control group was subsequently randomized into 2 groups with alternate weight-placement. Tests of upright mobility included: timed up and go (TUG), sharpened Romberg, 360-degree turns, 25-foot walk, and computerized platform posturography. Participants were tested at baseline and again with weights placed according to group membership. In both phases, a physical therapist assessed balance for the BBTW group and then placed weights to decrease balance loss. In phase 1, the control group had no weights placed. In phase 2, the alternate treatment group received standard weight placement of 1.5% body weight. RESULTS: People with BBTW showed a significant improvement in the 25-foot walk (P = .01) over those with no weight, and the TUG (P = .01) over those with standard weight placement. BBTW participants received an average of 0.5 kg, less than 1.5% of any participant's body weight. CONCLUSION: BBTW can have immediate advantages over a nonweighted condition for gait velocity and over a standardized weighted condition for a functional activity in people with multiple sclerosis (MS) who are ambulatory but have balance and mobility abnormalities.

Balance-based torso-weighting may enhance balance in persons with multiple sclerosis: preliminary evidence.

OBJECTIVE: To determine whether weight placed on the trunk in response to directional balance loss would enhance function and stability in people with multiple sclerosis (MS). DESIGN: Quasi-experimental study in which subjects served as their own controls. SETTING: Research laboratory. PARTICIPANTS: Subjects (N=16) age 20 to 65 years with MS recruited through the Northern California Chapter of the National Multiple Sclerosis Society. INTERVENTIONS: Balance-based torso-weighting where up to 1.5% body weight was placed in a garment on the trunk. Subjects were tested at baseline and then in randomly ordered balance-based torso-weighting and nonweighted garment conditions. MAIN OUTCOME MEASURES: Sharpened Romberg, eyes open (SREO) and Sharpened Romberg, eyes closed, computerized dynamic platform posturography (CDPP), Timed Up & Go (TUG), and 25-foot timed walk. RESULTS: Significant improvement (P<.014) was found with SREO in the balance-based torso-weighting compared with nonweighted conditions. CDPP eyes open and TUG showed improvements (P<.03) from baseline to balance-based torso-weighting and nonweighted conditions. CONCLUSIONS: Improved performance in a group of adults with MS was seen when light weights were placed on the torso to counteract balance loss. Placement of weights may have the potential to produce immediate improvements in balance in this population.