The ALPHA phase 1 study: pulmonary ArteriaL hypertension treated with CardiosPHere-Derived allogeneic stem cells.

BACKGROUND: Pulmonary Arterial Hypertension (PAH) is a progressive condition with no cure. Even with pharmacologic advances, survival remains poor. Lung pathology on PAH therapies still shows impressive occlusive arteriolar remodelling and plexiform lesions. Cardiosphere-derived cells (CDCs) are heart-derived progenitor cells exhibiting anti-inflammatory and immunomodulatory effects, are anti -fibrotic, anti-oxidative and anti-apoptotic to potentially impact several aspects of PAH pathobiology. In preclinical trials CDCs reduced right ventricular (RV) systolic pressure, RV hypertrophy, pulmonary arteriolar wall thickness and inflammation. METHODS: The ALPHA study was a Phase 1a/b study in which CDCs were infused into patients with Idiopathic (I)PAH, Heritable (H) HPAH, PAH-connective tissue disease (CTD) and PAH-human immunodeficiency virus (HIV). The study was IRB approved and DSMB monitored. Phase 1a, was an open label study (n = 6). Phase 1b was a double-blind placebo-controlled study (n = 20) in which half received 100 million CDCs (the maximum feasible dose from manufacturing perspective) and half placebo (PLAC) infusions. Right heart catheterization (RHC) and cardiac MR imaging (cMR) were performed at baseline and at 4 months post infusion. Patients were followed over a year. FINDINGS: No short-term clinical safety adverse events (AE) were related to the IP, the primary outcome measure. There were no adverse hemodynamic, gas exchange, rhythm or other clinical events following infusion and in the 1st 23 h monitored in hospital. There were no long-term AEs over 12 months noted, including unrelated limited hospitalizations. No immunologic short or long-term AEs were noted. We examined exploratory outcomes across multiple domains to determine encouraging signals to motivate future advanced phase testing. Phase 1a data showed encouraging observations for both 50 and 100 million CDC doses. Several encouraging findings favouring CDCs (n = 16) compared to placebo (n = 10) were noted. On cMR, the RV end diastolic volume (RVEDV) and index (RVEDVI) decreased with CDCs with a rise in the PLAC group. The 6-min walk distance was increased 2 months post infusion in the CDC group compared with PLAC. With PLAC, diffusing capacity (DLCO) decreased at 4 months but was unchanged with CDCs. Serum creatinine decreased with CDCs at 4 months. Encouraging observations favouring CDCs were also noted for RV fractional area change on echo and RV ejection fraction (RVEF) on cMR at 4 months. No differences were observed for mean pulmonary artery pressures or pulmonary vascular resistance. Review of long-term data to 12 months showed continued decline in DLCO for the PLAC cohort at 6 months with no change through 12 months. By contrast, CDC subjects showed an unchanged DLCO over 12-months. For parameters exhibiting early encouraging exploratory findings in CDC subjects, no further improvement was noted in long-term follow up through 12 months. INTERPRETATION: Intravenous CDCs were safe in both the short and long term in PAH subjects and thus may be safe in larger cohorts, in line with our extensive track record of safety in clinical trials for other conditions. Further, CDCs exhibited encouraging exploratory findings across several domains. Repeat dosing (quarterly, over one year) of intravenous CDCs has been reported to yield highly significant sustained disease-modifying bioactivity in subjects with advanced Duchenne muscular dystrophy. Because only single CDC doses were used here, the findings represent a lower limit estimate of CDC's potential in PAH. Upcoming phase 2 studies would logically use a repeat dosing paradigm. FUNDING: California Institute for Regenerative Medicine (CIRM). Project Number: CLIN2-09444.

Severe Nonspecific Interstitial Pneumonia (NSIP) in an Adolescent.

Childhood interstitial lung disease (chILD) is remarkably rare with a reported prevalence from 0.13 per 100,000 children under 17 years to 16.2 per 100,000 children under 15 years of age (Kornum et al., 2008). Here, we present a case of a 15-year-old with subacute hypoxemic respiratory failure, admitted to the critical care unit. Her imaging on admission showed bilateral interstitial infiltrates; her laboratory workup, including autoimmune serologies, was unrevealing. A bronchoscopy revealed the diagnosis of nonspecific interstitial pneumonia. She had a partial recovery after a course of steroids.

Chronic Thromboembolic Pulmonary Hypertension: the Bench.

PURPOSE OF REVIEW: Chronic thromboembolic pulmonary hypertension (CTEPH) is an uncommon complication of acute pulmonary embolism (PE), in which the red, platelet-rich thrombus does not resolve but forms into an organized yellow, fibrotic scar-like obstruction in the pulmonary vasculature. Here we review the pathobiology of CTEPH. RECENT FINDINGS: Our current knowledge has predominantly been informed by studies of human samples and animal models that are inherently limited in their ability to recapitulate all aspects of the disease. These studies have identified alterations in platelet biology and inflammation in the formation of a scar-like thrombus that comprised endothelial cells, myofibroblasts, and immune cells, along with a small vessel pulmonary arterial hypertension-like vasculopathy. The development of CTEPH-specific therapies is currently hindered by a limited knowledge of its pathobiology. The development of new CTEPH medical therapies will require new insights into its pathobiology that bridge the gap from bench to bedside.

Chronic Thromboembolic Pulmonary Hypertension: the Bedside.

PURPOSE OF REVIEW: Chronic thromboembolic pulmonary hypertension (CTEPH), included in group 4 PH, is an uncommon complication of acute pulmonary embolism (PE), in which emboli in the pulmonary vasculature do not resolve but rather form into an organized scar-like obstruction which can result in right ventricular (RV) failure. Here we provide an overview of current diagnosis and management of CTEPH. RECENT FINDINGS: CTEPH management is complex with treatments that range from surgery, percutaneous interventions, to medical therapies. Current CTEPH medical therapies have largely been repurposed from pulmonary arterial hypertension (PAH). The diagnosis of CTEPH can be challenging, requiring a multimodality approach to differentiate from disease mimics. While these treatments improve symptoms, they may not reverse the underlying pathology of CTEPH.

Evaluating the Right Ventricle in Acute and Chronic Pulmonary Embolism: Current and Future Considerations.

The right ventricle (RV), due to its morphologic and physiologic differences, is susceptible to sudden increase in RV afterload, as noted in patients with acute pulmonary embolism (PE). Functional impairment of RV function is a stronger presage of adverse outcomes in acute PE than the location or burden of emboli. While current iterations of most clinical prognostic scores do not incorporate RV dysfunction, advancements in imaging have enabled more granular and accurate assessment of RV dysfunction in acute PE. RV enlargement and dysfunction on imaging is noted only in a subset of patients with acute PE and is dependent on underlying cardiopulmonary reserve and clot burden. Specific signs like McConnell's and "60/60" sign are noted in less than 20% of patients with acute PE. About 2% of patients with acute PE develop chronic thromboembolic pulmonary hypertension, characterized by continued deterioration in RV function in a subset of patients with a continuum of RV function from preserved to overt right heart failure. Advances in molecular and other imaging will help better characterize RV dysfunction in this population and evaluate the response to therapies.

Disease-modifying bioactivity of intravenous cardiosphere-derived cells and exosomes in mdx mice.

Dystrophin deficiency leads to progressive muscle degeneration in Duchenne muscular dystrophy (DMD) patients. No known cure exists, and standard care relies on the use of antiinflammatory steroids, which are associated with side effects that complicate long-term use. Here, we report that a single intravenous dose of clinical-stage cardiac stromal cells, called cardiosphere-derived cells (CDCs), improves the dystrophic phenotype in mdx mice. CDCs augment cardiac and skeletal muscle function, partially reverse established heart damage, and boost the regenerative capacity of skeletal muscle. We further demonstrate that CDCs work by secreting exosomes, which normalize gene expression at the transcriptome level, and alter cell signaling and biological processes in mdx hearts and skeletal muscle. The work reported here motivated the ongoing HOPE-2 clinical trial of systemic CDC delivery to DMD patients, and identifies exosomes as next-generation cell-free therapeutic candidates for DMD.

Clinical trial design and new therapies for pulmonary arterial hypertension.

Until 20 years ago the treatment of pulmonary arterial hypertension (PAH) was based on case reports and small series, and was largely ineffectual. As a deeper understanding of the pathogenesis and pathophysiology of PAH evolved over the subsequent two decades, coupled with epidemiological studies defining the clinical and demographic characteristics of the condition, a renewed interest in treatment development emerged through collaborations between international experts, industry and regulatory agencies. These efforts led to the performance of robust, high-quality clinical trials of novel therapies that targeted putative pathogenic pathways, leading to the approval of more than 10 novel therapies that have beneficially impacted both the quality and duration of life. However, our understanding of PAH remains incomplete and there is no cure. Accordingly, efforts are now focused on identifying novel pathogenic pathways that may be targeted, and applying more rigorous clinical trial designs to better define the efficacy of these new potential treatments and their role in the management scheme. This article, prepared by a Task Force comprised of expert clinicians, trialists and regulators, summarises the current state of the art, and provides insight into the opportunities and challenges for identifying and assessing the efficacy and safety of new treatments for this challenging condition.

Therapeutic benefits of intravenous cardiosphere-derived cell therapy in rats with pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is a progressive condition characterized by occlusive pulmonary arteriopathy, in which survival remains poor despite pharmacologic advances. The aim of this study was to evaluate the ability of cardiosphere-derived cells (CDCs), cardiac progenitor cells with potent anti-inflammatory and immunomodulatory properties, to attenuate hemodynamic and morphometric remodeling of the right ventricle (RV) and pulmonary arterioles in rats with established monocrotaline (MCT)-induced PAH. Animals were divided into 3 groups: 1) Control (CTL), 2) PAH in which CDCs were centrally infused (CDC) and 3) PAH in which saline was given (Sham). Significant increments in RV systolic pressure (RVSP) and RV hypertrophy were noted in Sham animals compared to CTL. In CDC rats at day 35, RSVP fell (- 38%; p< 0.001) and RV hypertrophy decreased (-26%; p< 0.01). TAPSE and cardiac output were preserved in all 3 groups at day 35. Pulmonary arteriolar wall thickness was greater in Sham rats compared to CTL, and reduced in CDC animals for vessels 20-50 µm (P<0.01; back to CTL levels) and 50-80µm (P<0.01) in diameter. The macrophage population was increased in Sham animals compared to CTL (P< 0.001), but markedly reduced in CDC rats. In conclusion, infusion of CDCs markedly attenuated several key pathophysiologic features of PAH. As adjunctive therapy to PAH-specific agents, CDCs have the potential to impact on the pathobiology of adverse pulmonary arteriolar remodeling, by acting on multiple mechanisms simultaneously.

Effect of endurance and/or strength training on muscle fiber size, oxidative capacity, and capillarity in hemodialysis patients.

We previously reported reduced limb muscle fiber succinate dehydrogenase (SDH) activity and capillarity density and increased cross-sectional areas (CSAs) of all fiber types in maintenance hemodialysis (MHD) patients compared with matched controls that may contribute to their effort intolerance and muscle weakness. This study evaluated whether endurance training (ET), strength training (ST), or their combination (EST) alters these metabolic and morphometric aberrations as a mechanism for functional improvement. Five groups were evaluated: 1) controls; 2) MHD/no training; 3) MHD/ET; 4) MHD/ST; and 5) MHD/EST. Training duration was 21.5 ± 0.7 wk. Vastus lateralis muscle biopsies were obtained after HD at baseline and at study end. Muscle fibers were classified immunohistochemically, and fiber CSAs were computed. Individual fiber SDH activity was determined by a microdensitometric assay. Capillaries were identified using antibodies against endothelial cells. Type I and IIA fiber CSAs decreased significantly (10%) with EST. In the ET group, SDH activity increased 16.3% in type IIA and 19.6% in type IIX fibers. Capillary density increased significantly by 28% in the EST group and 14.3% with ET. The number of capillaries surrounding individual fiber type increased significantly in EST and ET groups. Capillary-to-fiber ratio increased significantly by 11 and 9.6% in EST and ET groups, respectively. We conclude that increments in capillarity and possibly SDH activity in part underlie improvements in endurance of MHD patients posttraining. We speculate that improved specific force and/or neural adaptations to exercise underlie improvements in limb muscle strength of MHD patients.

Metabolic and morphometric profile of muscle fibers in chronic hemodialysis patients.

Muscle weakness and effort intolerance are common in maintenance hemodialysis (MHD) patients. This study characterized morphometric, histochemical, and biochemical properties of limb muscle in MHD patients compared with controls (CTL) with similar age, gender, and ethnicity. Vastus lateralis muscle biopsies were obtained from 60 MHD patients, 1 day after dialysis, and from 21 CTL. Muscle fiber types and capillaries were identified immunohistochemically. Individual muscle fiber cross-sectional areas (CSA) were quantified. Individual fiber oxidative capacities were determined (microdensitometric assay) to measure succinate dehydrogenase (SDH) activity. Mean CSAs of type I, IIA, and IIX fibers were 33, 26, and 28% larger in MHD patients compared with CTL. SDH activities for type I, IIA, and IIX fibers were reduced by 29, 40, and 47%, respectively, in MHD. Capillary to fiber ratio was increased by 11% in MHD. The number of capillaries surrounding individual fiber types were also increased (type I: 9%; IIA: 10%; IIX: 23%) in MHD patients. However, capillary density (capillaries per unit muscle fiber area) was reduced by 34% in MHD patients, compared with CTL. Ultrastuctural analysis revealed swollen mitochondria with dense matrix in MHD patients. These results highlight impaired oxidative capacity and capillarity in MHD patients. This would be expected to impair energy production as well as substrate and oxygen delivery and exchange and contribute to exercise intolerance. The enlarged CSA of muscle fibers may, in part, be accounted for by edema. We speculate that these changes contribute to reduce limb strength in MHD patients by reducing specific force.

Effects of malnutrition with or without eicosapentaenoic acid on proteolytic pathways in diaphragm.

Attenuation of muscle wasting has been reported with eicosapentaenoic acid (EPA) use in cachectic states. Pathways mediating muscle proteolysis with severe short-term nutritional deprivation (ND)±EPA were evaluated, including diaphragm fiber-specific cross-sectional areas, mRNA (real-time PCR) and protein expression (Western blot). Rats were divided into three groups: (1) free-eating controls, (2) ND and (3) ND+EPA. ND significantly influenced multiple proteolytic pathways. EPA significantly reduced mRNA abundances for most genes to control levels with ND. However, discordant muscle protein expression of many genes was noted with the use of EPA, as protein levels failed to fall. EPA had no impact on diaphragm muscle atrophy, despite the impressive mRNA and some protein results. We conclude that EPA does not attenuate diaphragm muscle atrophy with severe levels of ND. Postulated mechanisms include reduction in muscle protein synthesis and persistent ongoing stimuli for proteolysis. Our study provides unique data on proteolytic signals with ND and has important implications for future studies using EPA.

Testosterone and resistance training effects on muscle nitric oxide synthase isoforms in COPD men.

BACKGROUND: Skeletal muscle dysfunction contributes to exercise limitation in COPD. The role of the nitric oxide synthase (NOS) system in muscle dysfunction is ill defined. Reduced levels of endothelial NOS (eNOS) and elevated levels of inducible NOS (iNOS) in the skeletal muscle of COPD patients have been recently reported. We hypothesized that resistance exercise training (R) and/or testosterone supplementation (T) would alter the transcription and expression of the NOS isoenzymes in COPD skeletal muscle. METHODS: Vastus lateralis biopsies were obtained before and after a 10-week intervention in 40 men with severe COPD(age 67.7 ± 8.3, FEV(1) 41.4 ± 12.6% predicted): placebo + no training (P) (n = 11), placebo + resistance training (PR) (n = 8), testosterone + no training (T) (n = 11) and testosterone + resistance training (TR) (n = 10) groups. eNOS, nNOS and iNOS mRNA and protein levels were measured in each sample. mRNA and protein levels were measured using real-time PCR and enzyme-linked immunosorbant assay, respectively. RESULTS: eNOS mRNA increased in the TR group compared to P and T groups (P < 0.001). eNOS protein was increased in TR and T groups after intervention (P < 0.05) but not in the PR group. nNOS protein increased in the PR, T, and TR groups (P < 0.05). iNOS protein decreased only in the TR group (P = 0.01). CONCLUSION: Resistance training and testosterone supplementation increased eNOS and nNOS proteins and decreased iNOS protein in the skeletal muscles of men with COPD. These changes in NO system might explain some of the favorable effects of these therapies.

Time course expression of Foxo transcription factors in skeletal muscle following corticosteroid administration.

Increased expression of forkhead box O (Foxo) transcription factors were reported in cultured myotubes and mouse limb muscle with corticosteroid (CS) treatment. We previously reported that administration of CS to rats resulted in muscle fiber atrophy only by day 7. The aim of this study, therefore, was to evaluate the time-course changes in the expression of Foxo transcription factors and muscle-specific ubiquitin E3 ligases in rat limb muscle following CS administration. Triamcinolone (TRI; 1 mg x kg(-1) x day(-1) im) was administered for 1, 3, or 7 days. Control (CTL) rats were given saline. Muscle mRNA was analyzed by real-time RT-PCR. Compared with CTL, body weights of TRI-treated animals decreased by 3, 12, and 21% at days 1, 3, and 7, respectively. Muscle IGF-1 mRNA levels decreased by 33, 65, and 58% at days 1, 3, and 7 in TRI-treated rats compared with CTL. Levels of phosphorylated Akt were 28, 50, and 36% lower in TRI animals at these time points. Foxo1 mRNA increased progressively by 1.2-, 1.4-, and 2.5-fold at days 1, 3, and 7 in TRI animals. Similar changes were noted in the expression of Foxo3a mRNA (1.3-, 1.4-, and 2.6-fold increments). By contrast, Foxo4 mRNA was not significantly changed in TRI animals. With TRI, muscle atrophy F box/Atrogin-1 increased by 1.8-, 4.1-, and 7.5-fold at days 1, 3, and 7 compared with CTL rats. By contrast, muscle RING finger 1 increased only from day 7 (2.7-fold). Gradual reduction in IGF-I expression with TRI over the time series paralleled that of Akt. These findings are consistent with a progressive stimulus to muscle protein degradation and the need to process/remove disassembled muscle proteins via the ubiquitin-proteasome system. Elucidating the dynamic catabolic responses to CS challenge is important in understanding the mechanisms underlying muscle atrophy and therapeutic measures to offset this.

Do faculty intensivists have better outcomes when caring for patients directly in a closed ICU versus consulting in an open ICU?

BACKGROUND: Intensivists have been associated with decreased mortality in several studies, but in one major study, centers with intensivist-staffed units reported increased mortality compared with controls. We hypothesized that a closed unit, in which a unit-based intensivist directly provides and coordinates care on all cases, has improved mortality and utilization compared with an open unit, in which individual attendings and consultants provide care, while intensivists serve as supervising consultants. METHODS: We undertook the retrospective study of outcomes in 2 intensive care units (ICUs)-a traditional open unit managed by faculty intensivists and a second closed unit overseen by the same faculty intensivists who coordinated the care on all patients in a large community hospital. PRIMARY OUTCOME: In-hospital mortality. SECONDARY OUTCOMES: Hospital length of stay (LOS), ICU LOS, and relative costs of hospitalization. RESULTS: From January 2006 to December 2007, we identified 2602 consecutive admissions to the 2 medical ICUs. Of all patients admitted to the closed and open units, 19.2% and 24.7%, respectively, did not survive (P < 0.001, adjusted for severity). Median hospital LOS was 10 days for the closed unit and 12 days for the open unit (P < 0.001). Median ICU LOS was 2.2 days for the closed unit and 2.4 days for the open unit (P = NS). The unadjusted cost index for the open unit was 1.11 relative to the closed unit (1.0) (P < 0.001). However, after adjusting for disease severity, cost differences were not significantly different. CONCLUSIONS: We observed significant reductions in mortality and hospital LOS for patients initially admitted to a closed ICU versus an open unit. We did not observe a significant difference in ICU LOS or total cost after adjustment for severity.

Skeletal muscle adaptations to testosterone and resistance training in men with COPD.

We recently reported increased leg lean mass and strength in men with chronic obstructive pulmonary disease (COPD) receiving 10 wk of testosterone (T) and leg resistance training (R) (Casaburi R, Bhasin S, Cosentino L, Porszasz J, Somfay A, Lewis M, Fournier M, Storer T. Am J Respir Crit Care Med 170: 870-878, 2004). The present study evaluates the role of muscle IGF and related factors as potential mechanisms for our findings, using quadriceps muscle biopsies from the same cohort. Patient groups were 1) weekly placebo (P) injections + no R; 2) P and R; 3) weekly injections of T + no R; and 4) T + R (TR). Muscle fibers were classified histochemically, and their cross-sectional areas (CSAs) and fiber density (number of fibers per unit area) were determined. Gene transcripts were determined by real-time PCR and protein expression by RIA. While no significant changes in fiber CSAs were noted across groups, increased trends were observed after 10 wk, and significant decrements in muscle fiber density were noted in all treated groups. A global increase in all myosin heavy chain (MyHC) mRNA isoforms was observed in TR patients. Muscle IGF-IEa and IGF-IEc mRNAs were significantly increased with TR group. Muscle IGF-I protein was increased in all intervention groups (greatest in TR). While TR IGF-II mRNA was increased, protein levels were unaltered. IGF binding protein-4 mRNA was increased with TR. Myogenin mRNA was increased in both T groups, while MyoD and myostatin were unchanged. Muscle atrophy F-box mRNA tended to increase with TR. Our data suggest that the combined interventions produced an enhanced local anabolic milieu driven in large part by the muscle IGF system, despite potentially negative biochemical influences present in COPD patients.

Effect of severe short-term malnutrition on diaphragm muscle signal transduction pathways influencing protein turnover.

The aim of this study was to evaluate the effect of nutritional deprivation (ND) on signal transduction pathways influencing the translational apparatus in the diaphragm muscle. Male rats were divided into two groups: 1) 20% of usual food intake for 4 days (ND) with water provided at libitum and 2) free-eating control (Ctl). Total protein and RNA were extracted from the diaphragm. Insulin-like growth factor I mRNA was analyzed by RT-PCR. Protein analyses of key cytoplasmic proteins for three signaling pathways deemed important in influencing protein turnover [phosphatidylinositol 3-kinase- Akt-mammalian target of rapamycin, P13K/Akt/glycogen synthase kinase (GSK)-3, and MAPK-ERK] were performed by Western blot. Body weight decreased 30% in ND and increased 17% in Ctl animals. Diaphragm mass decreased 29% in ND animals. Muscle insulin-like growth factor I mRNA abundance was reduced 63% in ND animals. ND resulted in a 55% reduction in phosphorylated (Ser473) Akt. Phosphorylation of mammalian target of rapamycin at Ser2448 was reduced by 85% in ND animals. Downstream effectors important in translation initiation were also affected by ND. Phosphorylated (Thr389) 70-kDa ribosomal protein S6 kinase was significantly reduced (35%) by ND. ND also resulted in significant dephosphorylation of the translational repressor initiation factor 4E-binding protein 1. Phosphorylation of GSK-3alpha (Ser21) and GSK-3beta (Ser9) was increased 55 and 45%, respectively, with ND. Phosphorylation of ERK1 (Thr202) and ERK2 (Tyr204), p44 and p42, respectively, was reduced 64 and 55%, respectively, with ND. Total protein concentration for all signaling intermediates of the three pathways was preserved. We conclude that short-term ND altered the phosphorylation states of key proteins of several pathways involved in protein turnover. This forms the framework for future studies aimed at identifying therapeutic targets in the management of short-term nutritionally induced cachectic states.

Tumor necrosis factor-alpha and malnutrition-induced inhibition of diaphragm fiber growth in young rats.

Tumor necrosis factor (TNF)-alpha has been implicated in several muscle-wasting disorders, with increased levels of the cytokine reported in malnourished children. The role of TNF-alpha in mediating malnutrition-induced inhibition of diaphragm (DIA) muscle growth in young growing rats was evaluated. Three groups of rats were studied: 1) control (CTL); 2) nutritional deprivation (ND; 50% of normal food intake for 7 days); and 3) ND + rat specific anti-TNF-alpha antibody. DIA fiber cross-sectional areas were determined. Serum and muscle TNF-alpha levels were measured by real-time PCR, ELISA, and immunohistochemistry. Body weights decreased 20% in ND rats and increased 46% in CTL animals. Anti-TNF-alpha had no effect on body weight or on DIA mass in ND animals. ND significantly reduced cross-sectional areas of all fiber types (33-46%). Anti-TNF-alpha failed to attenuate ND-induced inhibition of DIA fiber growth. Serum TNF-alpha levels increased 2.6-fold in ND animals, with levels suppressed to below CTL values with anti-TNF-alpha. DIA TNF-alpha mRNA and protein levels increased two- to threefold in ND rats. Anti-TNF-alpha antibodies suppressed muscle levels of the cytokine in ND animals to near CTL values. TNF-alpha immunoreactivity in all DIA fibers revealed similar directions of change in both ND groups. Direction and magnitude of change in DIA phosphorylated p38 MAPK (a likely second messenger of TNF-alpha) tracked those of TNF-alpha. Muscle levels of IGF-I mRNA and phosphorylated Akt were markedly reduced in ND animals with no change following anti-TNF-alpha therapy. Thus rat anti-TNF-alpha at a dose known to neutralize the cytokine failed to attenuate or reverse ND-induced inhibition of DIA fiber growth in our model.