REPEATED COURSES OF GRANULOCYTE COLONY-STIMULATING FACTOR IN AMYOTROPHIC LATERAL SCLEROSIS: CLINICAL AND BIOLOGICAL RESULTS FROM A PROSPECTIVE MULTICENTER STUDY ` , MD,1 GABRIELE MORA, MD,2 VINCENZO LA BELLA, MD,3 CLAUDIA CAPONNETTO, MD,4 ADRIANO CHIO GIANLUIGI MANCARDI, MD,4 MARIO SABATELLI, MD,5 GABRIELE SICILIANO, MD,6 VINCENZO SILANI, MD,7 MASSIMO CORBO, MD,8 CRISTINA MOGLIA, MD,1 ANDREA CALVO, MD, PhD,1 ROBERTO MUTANI, MD,1 ` , MD,12 SERGIO RUTELLA, MD,9 FRANCESCA GUALANDI, MD,10 MARIO MELAZZINI, MD,8,11 ROSANNA SCIME MARIO PETRINI, MD,13 PAOLA BONDESAN, BS,14 SILVIA GARBELLI, PhD,15 STEFANIA MANTOVANI, PhD,15 CATERINA BENDOTTI, PhD,16 CORRADO TARELLA, MD,17 and the STEMALS Study Group 1
Dipartimento di Neuroscienze, SC Neurologia I & Centro per la Sclerosi Laterale Amiotroﬁca, Torino, Italy SC Neuroriabilitazione, IRCCS Fondazione Salvatore Maugeri, Milano, Italy 3 SC Neurologia e Centro Regionale di Riferimento Malattie del Motoneurone, Universita` degli Studi di Palermo, Palermo, Italy 4 Clinica Neurologica II, Universita` degli Studi di Genova, Genova, Italy 5 SC Neurologia, Universit`Cattolica del Sacro Cuore, Roma, Italy 6 SC Neurologia, Universita` degli Studi di Pisa, Pisa, Italy 7 UO Neurologia e Laboratorio Neuroscienze, Centro ‘‘Dino Ferrari,’’ Universita` degli Studi di Milano, IRCCS Istituto Auxologico Italiano, Milano, Italy 8 NEuroMuscular Omnicentre (NEMO), Milano, Italy 9 SC Ematologia, Universit` Cattolica del Sacro Cuore, Roma and IRCCS San Raffaele Pisana, Roma, Italy 10 Centro Trapianti di Midollo, II Divisione di Ematologia, Azienda Ospedale Universit` San Martino, Genova, Italy 11 SC Oncoematologia, Fondazione Salvatore Maugeri, Pavia, Italy 12 Centro Trapianti di Midollo Osseo, Ospedale Cervello, Palermo, Italy 13 Divisione di Ematologia, Dipartimento di Oncologia, dei Trapianti e delle Nuove Tecnologie in Medicina, Universit` degli Studi di Pisa, Pisa, Italy 14 SC Ematologia, AOU Giovanni Battista, Torino, Italy 15 Laboratorio per le Malattie Neurodegenerative, IRCCS Fondazione Salvatore Maugeri, Pavia, Italy 16 Dipartimento di Neuroscienze, Istituto di Ricerche Farmacologiche ‘‘Mario Negri,’’ Milano, Italy 17 Dipartimento Medicina–Oncologia Sperimentale, SCU Ematologia e Terapie Cellulari, A.O. Mauriziano and Molecular Biotechnology Center, Torino, Italy 2
Accepted 16 July 2010 ABSTRACT: Granulocyte colony-stimulating factor (G-CSF) induces a transient mobilization of hematopoietic progenitor cells from bone marrow to peripheral blood. Our aim was to evaluate safety of repeated courses of G-CSF in patients with amyotrophic lateral sclerosis (ALS), assessing disease progression and changes in chemokine and cytokine levels in serum and cerebrospinal fluid (CSF). Twenty-four ALS patients entered an open-label, multicenter trial in which four courses of G-CSF and mannitol were administered at 3-month intervals. Levels of G-CSF were increased after treatment in the serum and CSF. Few and transitory adverse events were observed. No significant reduction of the mean monthly decrease in ALSFRS-R score and forced vital capacity was observed. A significant reduction in CSF levels of monocyte chemoattractant protein-1
Abbreviations: ALS, amyotrophic lateral sclerosis; ALSFRS-R, ALS Functional Rating Scale–Revised; bFGF, basic fibroblast growth factor; BM, bone marrow; BMC, bone marrow cell; BSA, bovine serum albumin; CNS, central nervous system; CSF, cerebrospinal fluid; DVT, deep venous thrombosis; ECG, electrocardiogram; FVC, forced vital capacity; G-CSF, granulocyte-colony stimulating factor; GM-CSF, granulocyte macrophage colony-stimulating factor; IFN-c, interferon-gamma; IL, interleukin; IL-1ra, interleukin-1 receptor antagonist; IP-10, interferon-induced protein-10; MCP-1, monocyte chemoattractant protein-1; MIP-1a, macrophage inflammatory protein-1a PBS, phosphate-buffered saline; PDGF, plateletderived growth factor; RANTES, regulated-on-activation normal T-cell expressed and secreted; SOD1, superoxide dismutase 1; TNF-a, tumor necrosis factor-alpha; VEGF, vascular endothelial growth factor; WBC, white blood cells Key words: amyotrophic lateral sclerosis, clinical trial, granulocyte colonystimulating factor, hematopoietic stem cells, neuroinflammation Correspondence to: A. Chio`; MD, ALS Centre, Department of Neuroscience, University of Turin, via Cherasco15, 10126 Torino, Italy; e-mail: [email protected]
C 2011 Wiley Periodicals, Inc. V
Published online 15 January 2011 in Wiley (wileyonlinelibrary.com). DOI 10.1002/mus.21851
G-CSF in a Multicenter ALS Study
(MCP-1) and interleukin-17 (IL-17) was observed. G-CSF treatment was safe and feasible in a multicenter series of ALS patients. A decrease in the CSF levels of proinflammatory cytokines MCP-1 and IL-17 was found, indicating a G-CSF–induced central anti-inflammatory response. Muscle Nerve 43: 189–195, 2011
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of lower and upper motor neurons at the spinal and bulbar levels. At present, the only approved therapy for ALS is riluzole, an inhibitor of neuronal glutamate release, which prolongs survival by 9%.1 Recently, stem cell transplantation has been proposed as a promising therapeutic strategy in neurological disorders.2,3 In superoxide dismutase 1 (SOD1) mutant ALS mice, this approach, using bone marrow cells (BMCs), has been found to modify the microenvironment of the nervous system by generating microglia and glial cells,4–6 slowing disease progression and increasing survival.7 Granulocyte colony-stimulating factor (G-CSF) is a growth factor that stimulates the proliferation, differentiation, and survival of hematopoietic progenitor cells. It induces the transient mobilization of hematopoietic progenitors from the bone marrow (BM) to the peripheral blood. G-CSF is being used extensively to both accelerate recovery from MUSCLE & NERVE
neutropenia after cytotoxic therapy and mobilize and collect BMCs for hematopoietic stem cell transplantation.8 G-CSF mobilizes, among other cells, a population of early CD34þ hematopoietic stem cells from the marrow into the peripheral blood. During G-CSF–induced mobilization, circulating BMCs may spread into the injured brain and contribute to brain repair.9–12 G-CSF may also have signiﬁcant neuroprotective effects on motor neuron cell lines in ALS11,13 and in SOD1 mutant mice.14 Indeed, several observations suggest that GCSF may affect neural cells and the central nervous system in general. Therefore, we decided to use GCSF to mobilize BMCs into the peripheral circulation in patients with ALS. The aims of this study were to: (1) assess safety and tolerability of repeated cycles of BMC mobilization with G-CSF in ALS patients; (2) assess clinical progression during the 12-month treatment with G-CSF, compared to a 4-month lead-in period; and (3) assess the changes in chemokine and cytokine levels in serum and cerebrospinal ﬂuid (CSF) before and after treatment with G-CSF as markers of neuroinﬂammation. METHODS
The STEMALS trial was a multicenter, open-label, pilot study with a phase I–II design consisting of a 4-month lead-in phase and a 12-month treatment period. The study was performed in seven Italian ALS centers with hematological laboratories and with experience in the use of G-CSF. Patients. Inclusion criteria were: deﬁnite, probable, or probable-laboratory-supported ALS15; age between 40 and 65 years; disease duration 12 months; moderate disability (score 3 on the ALS Functional Rating Scale–Revised [ALSFRS-R] for swallowing, cutting food, and walking); forced vital capacity (FVC) 80% of predicted; documented disease progression in the last 3 months; use of riluzole (50 mg twice daily); and a negative pregnancy test for premenopausal women. Exclusion criteria were: positive family history for ALS; serious medical conditions; current or past cancer; current or past malignant myeloproliferative disorders, secondary polycythemia; thrombophilic state; spleen hypertrophy (diameter 18 cm); or frontotemporal dementia. All patients gave written informed consent. Before entering the study, patients underwent diagnostic lumbar puncture, electrocardiogram (ECG), chest X-ray, ultrasonography of the upper and lower abdomen with spleen volume assessment and evaluation of nodal sites in the neck and axillae; spirometry; and bone marrow aspiration with morphological and cytogenetic evaluation and in vitro cultures. 190
G-CSF in a Multicenter ALS Study
FIGURE 1. STEMALS protocol. Patients were followed for 4 months (lead-in period), after which G-CSF was administered subcutaneously at a dose of 5 lg/kg every 12 hours for 4 consecutive days. In addition, 18% mannitol was administered four times per day for 5 consecutive days starting on the third day of G-CSF administration. Four G-CSF courses were delivered to each patient at 3-month intervals (months 0, 3, 6, and 9); the final assessment was scheduled for month 12 (treatment period). Treatment Plan. Patients were hospitalized during each cycle of G-CSF administration. G-CSF (Myelostim; kindly provided free of charge by Italfarmaco) was administered subcutaneously at a dose of 5 lg/kg, twice daily, for 4 consecutive days (Fig. 1). This treatment was repeated every 3 months (T0, T3, T6, and T9), for a total of four courses. For safety reasons, G-CSF administration was interrupted if the leukocyte count was 50,000/ll. From the third day of G-CSF administration, mannitol 18%, given in doses of 125 ml four times a day for 5 days, was administered intravenously to permeabilize the brain–blood barrier. Clinical Assessments. Patients were enrolled in the protocol and were followed for 4 months (lead-in phase, T 4 to T0), without any speciﬁc treatment other than riluzole and supportive care measures. At each scheduled visit, patients were subjected to the following: the ALSFRS-R; FVC assessment; and the McGill Quality of Life Questionnaire.16 Adverse events (AEs) were assessed at each visit. Evaluation of BMC Mobilization into Peripheral Blood
Complete blood cell counts and evaluation of CD34þ cells were performed daily throughout the cycle of G-CSF administration and for at least 2 days after drug interruption. In some cases, characterization of progenitors was performed by evaluating speciﬁc cell surface antigens. The presence of BMCs in the CSF was assessed with a lumbar puncture 2 days after the end of the administration of G-CSF, at T0 and T6, and compared with a basal evaluation performed at T 4. and CSF.
Multiplexed Fluorescent Bead–Based Immunoassay for Determination of Multiple Cytokines in CSF and Serum Samples. CSF samples were collected before the ﬁrst treatment (T0) and after the third treatment MUSCLE & NERVE
(T6) and immediately stored at 80 C until the cytokine assay. Thawed CSF samples were centrifuged at 400 g at 4 C for 5 minutes, then loaded onto the plate (Bio-Rad Laboratories) by diluting 1 volume of the CSF sample with 3 volumes of phosphate-buffered saline (PBS, pH 7.4) supplemented with 0.8% bovine serum albumin (BSA; SigmaAldrich) and with protease inhibitors (MiniComplete, Roche). Serum samples were collected at T0 and after the last treatment (T9) and immediately frozen at 80 C until cytokine assay. Human cytokine/chemokine panel kits (Bio-Rad Laboratories and Millipore Corp.) were used for simultaneous quantiﬁcation of the following human cytokines and chemokines: interleukin (IL)-1b, IL-1 receptor antagonist (IL-1ra), IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, eotaxin, basic ﬁbroblast growth factor (bFGF), FGF-2, transforming growth factor-alpha (TGF-a), G-CSF, granulocyte macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-c), interferon-induced protein-10 (IP-10), MCP-1 (MCAF), macrophage inﬂammatory protein-1&alpha (MIP1a), MIP-1b, platelet-derived growth factor (PDGF)-BB, regulated-on-activation normal T-cell expressed and secreted (RANTES), tumor necrosis factor-alpha (TNF-a), and vascular endothelial growth factor (VEGF). The Bio-Rad cytokine/chemokine panel kits were used for analysis of CSF and serum samples, according to the manufacturer’s instructions. Brieﬂy, all buffers and diluents were warmed to room temperature prior to use. Lyophilized cytokine standard was reconstituted with 500 ll of PBS/0.8% BSA to a master standard stock. Fourfold serial dilutions of the master standard stock provided eight concentrations used to determine a standard curve. Five hundred microliters of diluted CSF sample was added to 50 ll of anti–cytokineconjugated beads (Bio-Rad) on a 96-well ﬁlter plate (Bio-Rad) and incubated at room temperature for 4 hours, with shaking at 300 rpm. The plate was washed, and 25 ll of the detection biotinylated antibody solution (Bio-Rad) was added to each well, followed by incubation at room temperature for 1 hour, with shaking at 300 rpm. The plate was then washed, and 50 ll of streptavidinconjugated phycoerythrin (Bio-Rad) was added to each well and incubated for 10 minutes. Following a ﬁnal wash, the contents of each well were resuspended in 125 ll of Bio-Plex assay buffer (BioRad), shaken at 1100 rpm for 30 seconds, and immediately read on an array reader (Bio-Plex 200 System; Bio-Rad) calibrated with a high PMT setting. The data were analyzed using Bio-Plex Manager 4.1 software with ﬁve-parameter logistic regression curve-ﬁtting. Cytokine concentrations G-CSF in a Multicenter ALS Study
were calculated by reference to a standard curve for each cytokine assayed in the same manner as the samples. Each point on the standard curve was within 70–130% of actual levels, and intra-assay variability, expressed as a coefﬁcient of variation, was 70% recovery were set as the lower detection limits. All samples were run in triplicate. Study Power and Statistical Methods. As the primary endpoints were treatment safety and tolerability, we did not calculate the power of the study. We compared the difference in the mean monthly variation of ALSFRS-R and FVC scores between the lead-in (T 4 to T0) and treatment (T0 to T12) periods. For example, the mean monthly variation of ALSFRS-R score was calculated as follows: [(ALSFRS-R at T 4) (ALSFRS-R score at T0)]/4 and [(ALSFRS-R at T0) (ALSFRS-R score at T12)]/12.17 For the patients who did not complete the 12-month trial period the last available observation was considered. Differences were compared with the two-tailed Student’s t-test. The analysis was performed on the whole group of patients who underwent at least one cycle of treatment (intent-to-treat population). Changes in the levels of single cytokines in the CSF and serum samples before and after treatment were analyzed by paired Student’s t-test. A multiple comparison analysis was not applicable, because, in some cases, the levels of cytokines either before or post-treatment, especially in the CSF, were out of the detectable range. P < 0.05 was considered signiﬁcant.
The study was approved by the ethics committees of the participating centers. The trial was registered with the public database of the Italian Agency for Drugs (AIFA; http://oss-sper-clin. agenziafarmaco.it/) (EudraCT Number 2005-00324875), and was performed according to the revised consolidated standards of reporting trials (CONSORT) guidelines.
Role of the Study Sponsor. The trial was sponsored by the Italian National Health System and was initiated and undertaken by the investigators. The study sponsor approved the design and protocol but had no involvement in the study design; collection, analysis, and interpretation of data; writing of the report; or the decision to submit the paper for publication. RESULTS Patients’ Characteristics. A total of 26 patients were enrolled (17 deﬁnite, 7 probable, and 2 probable-laboratory-supported ALS). One patient with MUSCLE & NERVE
Table 1. Mean monthly decline in ALSFRS-R score and FVC.
ALSFRS-R score FVC
T 4 to T0
T0 to T12
1.45 (0.96) 2.61 (2.75)
1.15 (0.99) 1.91 (1.97)
*Lead-in phase vs. treatment phase.
deﬁnite ALS died during the lead-in phase, and 1 patient with probable ALS withdrew his consent before starting the treatment. Thus, 24 patients (12 men and 12 women; 9 bulbar and 15 spinal onset) were started on the G-CSF treatment protocol and were considered for the study. At the time of enrollment, the mean age at onset was 54.6 years (SD ¼ 6.6, range 40–64 years), the mean disease duration was 10 months (SD ¼ 2.6, range 4– 12 months), the mean ALSFRS-R score was 43.0 (SD ¼ 3.4, range 32–47), and the mean FVC was 95.8% (SD ¼ 12.9%, range 81–108%). Overall Outcome and Adverse Effects. Eighteen patients completed all cycles of treatment and the 12-month follow-up. Four patients died at months 5, 8, 9, and 11, respectively. One patient had died suddenly the day before starting the third cycle, likely due to gastroesophageal aspiration. The remaining 3 patients died at home due to respiratory failure. None of these patients had autopsy. Two patients underwent elective tracheotomy, both at month 6, due to severe respiratory failure. On November 30, 2009, 12 patients (50%) were still alive and tracheostomy-free, at a median follow-up of 2.6 years since enrollment. The overall median tracheostomy-free survival from symptom onset of the whole series was 3.6 years. There were 2 severe AEs. One patient developed hyperprolactinemia, manifested by asthenia and decrease of libido, and 1 patient developed deep vein thrombosis (DVT), requiring anti-coagulation therapy for 4 months. Neither patient
stopped the study treatment. Both AEs were likely to be unrelated to the study drug. Other minor adverse events were ﬂu-like symptoms, nausea, and asthenia, reported by 8 patients over the total course of 12 treatment cycles, and effectively treated with acetaminophen (500 mg twice daily). Clinical Response. No signiﬁcant reduction of the mean monthly decline of ALSFRS-R score and FVC between the lead-in and the treatment phase was observed in the whole patient series (Table 1). Quality of life did not show any modiﬁcation during the treatment phase. Hematological Response. Hematological results have been reported in detail elsewhere.18 Brieﬂy, all patients consistently displayed a rapid and marked increase in white blood cells (WBC) and circulating CD34þ cells following G-CSF administration. In the four courses, the median peak values of WBC/ll ranged between 40,290 and 43,425 and those of CD34þ cells/ll ranged between 41 and 57. There were no signiﬁcant differences in peak values of either WBC or CD34þ cells among the four G-CSF courses. Peak values were usually reached between days 3 and 4 of stimulation, with a rapid decrease to pretreatment values within a few days of G-CSF discontinuation. In 2 patients, administration of G-CSF was discontinued due to a rapid increase in leukocytes to a level of 50,000/ ll. CD34þ cells were not found in the CSF of patients at either basal or 2 days after the end of the ﬁrst and third courses of G-CSF. Multiple Cytokines in CSF and Serum. Twenty-nine cytokines were evaluated in the serum and CSF of patients during the lead-in phase and following GCSF treatment. Herein we report only the data for the cytokines that showed signiﬁcant changes following treatment. As shown in Figure 2, the mean G-CSF concentration was signiﬁcantly increased in CSF (P ¼ 0.007) and serum (P ¼ 0.031) after G-
FIGURE 2. Cytokine concentrations in CSF and serum of ALS patients after G-CSF administration. 192
G-CSF in a Multicenter ALS Study
MUSCLE & NERVE
CSF treatment, compared with the lead-in phase. In the CSF, a signiﬁcant reduction in the levels of MCP-1 (P ¼ 0.029) and IL-17 (P ¼ 0.021) was observed following G-CSF treatment, compared with the basal level. Serum levels of MCP-1 were also signiﬁcantly decreased in ALS patients after treatment, whereas the concentration of IP-10, a member of the chemokine family induced by IFNc, was signiﬁcantly increased compared with the basal level (P ¼ 0.009). DISCUSSION
In this study we found that G-CSF administration in four successive courses is well tolerated in ALS patients. The hematological response is similar to that usually observed in healthy bone marrow donors.18 We recorded only 2 serious adverse events, namely transient hyperprolactinemia in 1 case and DVT in 1 case. A total of 6 patients (25%) did not complete the follow-up: 4 patients died and 2 were tracheotomized. These ﬁgures are in line with those expected in ALS, especially considering that we enrolled patients with a very short time from onset to diagnosis, one of the most relevant negative prognostic factors in ALS.19,20 However, we cannot exclude the possibility that the treatment hastened the respiratory disturbances in these patients. This concern should be accurately considered in future studies of G-CSF in ALS. Although our study was not designed speciﬁcally to assess the effect of G-CSF and mannitol on the course of ALS, we compared the progression of the disease between the 4-month lead-in phase and the 12-month treatment phase. With regard to ALSFRS-R and FVC we found no signiﬁcant reduction in the monthly rate of decline. Our trial has several limitations. As already stated, it was designed to assess G-CSF safety and not to assess its efﬁcacy in ALS patients. Moreover, the trends were determined using a lead-in methodology. The appropriateness of comparing the decline rate in two consecutive time periods is still being debated. It is supported by the notion that the decline in ALS, measured with ALSFRS-R and FVC, is linear, at least in the ﬁrst 2 years after onset, which was the case for the patients included in our trial.17,21 Accordingly, in ALS the lead-in method for clinical trials is considered to be more effective than the use of historical controls.22 Therefore, the observed clinical trends should be considered with caution. Our ﬁndings parallel those of a recent study on the treatment of SOD1 transgenic mice with G-CSF by continuous subcutaneous delivery,14 which reported that the drug given at the stage of the disease where muscle denervation is already eviG-CSF in a Multicenter ALS Study
dent leads to signiﬁcant improvement in motor performance, delays the onset of severe motor impairment, and prolongs overall survival. This effect was interpreted in part as a direct neuroprotective action of G-CSF on motor neurons. In ALS patients the levels of G-CSF were increased in both serum and CSF. Thus, we cannot exclude the possibility that G-CSF may have a direct neuroprotective role.14 In ALS patients, two phase I trials on the use of G-CSF have been published. In the ﬁrst trial, no adverse events were found during the 6-month follow-up of 8 patients who underwent a single 5– 6-day course of G-CSF stimulation, at a dose of 300–600 lg.23 In the second trial, a reduction in the ALSFRS-R decline was observed in 13 ALS patients in a 6-month period following a single course of G-CSF (administered for 5 consecutive days at 2 lg/kg).24 A small, placebo-controlled trial of G-CSF in ALS has also been published.25 In that study G-CSF was administered in four courses at 3-month intervals, at a dose of 5 lg/kg/day for 4 consecutive days. No major side-effects were observed. A trend toward a better outcome in the drug-treated group was reported in the ﬁrst 6 months of treatment, but the results at 12 months were not signiﬁcant. However, this trial was underpowered, and the dose of the study drug was half that usually applied in the hematological setting and in our study. This likely accounted for the outcome difference observed between this trial and ours. G-CSF is thought to exert its neuroprotective actions through the inhibition of neuroinﬂammation and stimulation of neurogenesis. We found that treatment with G-CSF signiﬁcantly reduced the levels of MCP-1 in both CSF and serum of ALS patients. MCP-1 is a chemokine that plays an important role in the recruitment of immune/inﬂammatory cells into the central nervous system (CNS) and is likely involved in the neurodegenerative process of a variety of CNS diseases. In ALS patients, MCP-1 levels were signiﬁcantly increased in the serum26 and in CSF26,27 with respect to healthy controls. The overexpression of MCP-1 in the CSF after G-CSF treatment is possibly related to the potential effect of this chemokine on the recruitment of dendritic cells and reactive microglia/macrophages in ALS spinal cord tissue.27 We also observed a signiﬁcant reduction in IL-17, a T-cell–derived proinﬂammatory cytokine, after G-CSF treatment. IL-17 is well known for its proinﬂammatory effects,28 and it has been reported to play an important role in many autoimmune and inﬂammatory diseases.29–33 IL-17 is produced by a variety of cell types, in particular CD4þ and CD8þ T cells. However, IL-17 MUSCLE & NERVE
expression has been demonstrated in the brain, particularly in microglia and astrocytes.34–36 Both CD4þ and CD8þ T cells, besides reactive astrocytes and microglia, have been detected in the vulnerable neural regions of postmortem ALS tissues.37,38 The effect G-CSF in reducing this cytokine could thus be mediated through modulation of T lymphocytes or microglia. Taken together, these data suggest that G-CSF exhibits an anti-inﬂammatory response in the CNS of ALS patients, and this could contribute to the trend of slowing disease progression reported in this study. The treatment with G-CSF in ALS patients also signiﬁcantly increased the serum levels of IP-10. IP10 is a chemokine secreted by several cell types in response to IFN-c, which plays a role in the accumulation of T cells. The signiﬁcance of this peripheral change is unknown. Last, G-CSF is able to induce BMC mobilization. In a previous report, we documented that large quantities of BMC are consistently mobilized following repeated G-CSF administration.18 Thus, one cannot exclude the possibility that circulating early BMC might migrate to the damaged CNS and somehow contribute to neural repair. In conclusion, we have observed that the repeated use of G-CSF to induce BMC mobilization is safe, well tolerated, and feasible in ALS and causes few and reversible adverse effects. Interestingly, the treatment was associated with a decrease in the levels of proinﬂammatory cytokines in the CSF, such as MCP-1 and IL-17, indicating a central anti-inﬂammatory response induced by G-CSF. A placebo-controlled trial is needed to verify the possible beneﬁt of this procedure in ALS and to further investigate the hypothesis of an anti-inﬂammatory strategy aimed at inhibiting these cytokines as a potential therapeutic approach. The other members of the STEMALS study group include: Paolo Ghiglione (Torino); Paola Omede´ (Torino); Marco Ulla (Torino); Maria Mascolo (Genova); Cecilia Carlesi (Pisa); Pietro Attilio Tonali (Roma); Amelia Conte (Roma); Marco Luigetti (Roma); Dario Alimonti (Milano, Bergamo); Francesco Onida (Milano); Patrizia Bossolasco (Milano); Giorgio Lambertenghi Deliliers (Milano); and Francesca Valentino (Palermo). The study was sponsored by the Italian National Health System. Other funding was provided by Istituto Superiore di Sanita`, Neurodegenerative Disorders Grant, 2005 (to A.C.); Ministero Universita` e Ricerca, Programmi di Ricerca Scientiﬁca di Rilevante Interesse Nazionale (PRIN), 2006 (to A.C. and C.T.); and Fondazione Vialli e Mauro per la Ricerca e lo Sport ONLUS (to C.B., A.C., and G.M.). G-CSF (Myelostim 34) was kindly provided free of charge by Italfarmaco.
REFERENCES 1. Miller RG, Mitchell JD, Lyon M, Moore DH. Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane Database Syst Rev 2007;1:CD001447. 2. Vora N, Jovin T, Kondziolka D. Cell transplantation for ischemic stroke. Neurodegen Dis 2006;3:101–105.
G-CSF in a Multicenter ALS Study
3. Newman MB, Bakay RA. Therapeutic potentials of human embryonic stem cells in Parkinson’s disease. Neurotherapeutics 2008;5:237–251. 4. Beers DR, Henkel JS, Xiao Q, Zhao W, Wang J, Yen AA, et al. Wildtype microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis. Proc Natl Acad Sci USA 2006;103: 16021–16026. 5. Riley J, Sweeney W, Boulis N. Shifting the balance: cell-based therapeutics as modiﬁers of the amyotrophic lateral sclerosis-speciﬁc neuronal microenvironment. Neurosurg Focus 2008;24:E10. 6. Einstein O, Ben-Hur T. The changing face of neural stem cell therapy in neurologic diseases. Arch Neurol 2008;65:452–456. 7. Corti S, Locatelli F, Donaoni C, Guglieri M, Papadimitriou D, Strazzer S, et al. Wild-type bone marrow cells ameliorate the phenotype of SOD1–G93A mice and contribute to the CNS, heart and skeletal muscle tissues. Brain 2004;127:2518–2532. 8. Nervi B, Link DC, DiPersio JF. Cytokines and hematopoietic stem cell mobilization. J Cell Biochem 2006;99:690–705. 9. Solaroglu I, Jadhav V, Zhang JH. Neuroprotective effect of granulocyte-colony stimulating factor. Frontiers Biosci 2007;12:712–724. 10. Scha¨bitz WR, Schneider A. New targets for established proteins: exploring G-CSF for the treatment of stroke. Trends Pharmacol Sci 2007;28:157–161. 11. Six I, Gasan G, Mura E, Bordet R. Beneﬁcial effect of pharmacological mobilization of bone marrow in experimental cerebral ischemia. Eur J Pharmacol 2003;458:327–328. 12. Hennemann B, Ickenstein G, Sauerbruch S, Luecke K, Haas S, Horn M, et al. Mobilization of CD34þ hematopoietic cells, colony-forming cells and long-term culture-initiating cells into the peripheral blood of patients with an acute cerebral ischemic insult. Cytotherapy 2008; 10:303–311. 13. Tanaka M, Kikuchi H, Ishizu T, Minohara M, Osoegawa M, Motomura K, et al. Intrathecal upregulation of granulocyte colony stimulating factor and its neuroprotective actions on motor neurons in amyotrophic lateral sclerosis. J Neuropathol Exp Neurol 2006;65: 816–825. 14. Pitzer C, Kru¨ger C, Plaas C, Kirsch F, Dittgen T, Mu¨ller R, et al. Granulocyte-colony stimulating factor improves outcome in a mouse model of amyotrophic lateral sclerosis. Brain 2008;131:3335–3347. 15. Brooks BR, Miller RG, Swash M, Munsat TL. World Federation of Neurology Research Group on Motor Neuron Diseases. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph Lateral Scler Other Motor Neuron Disord 2000;1:293–299. 16. Cohen SR, Mount BM, Strobel MG, Bui FI. The McGill Quality of Life Questionnaire: a measure of quality of life appropriate for people with advanced diseases. A preliminary study of validity and acceptability. Pall Med 1995;9:207–219. 17. Armon C, Graves MC, Moses D, Forte DK, Sepulveda L, Darby SM, et al. Linear estimates of disease progression predict survival in patients with amyotrophic lateral sclerosis. Muscle Nerve 2000;23: 874–882. 18. Tarella C, Rutella S, Gualandi F, Melazzini M, Scime´ R, Petrini M, et al. Consistent bone marrow–derived cell mobilization following repeated short courses of G-CSF in patients with amyotrophic lateral sclerosis: results from a multicenter prospective trial. Cytotherapy 2010;12:50–59. 19. Del Aguila MA, Longstreth WT Jr, McGuire V, Koepsell TD, van Belle G. Prognosis in amyotrophic lateral sclerosis. A population-based study. Neurology 2003;60:813–819. 20. Chio` A, Logroscino G, Hardiman O, Swingler R, Mitchell D, Beghi E, et al. Prognostic factors in ALS: a critical review. Amyotroph Lateral Scler 2009;10:310–323. 21. Kimura F, Fujimura C, Ishida S, Nakajima H, Furutama D, Uehara H, et al. Progression rate of ALSFRS-R at time of diagnosis predicts survival time in ALS. Neurology 2006;66:265–267. 22. Moore DH Jr, Miller RG. Improving efﬁciency of ALS clinical trials using lead-in designs. Amyotroph Lateral Scler Other Motor Neuron Disord 2004;5(suppl 1):57–60. 23. Cashman N, Tan LY, Krieger C, Ma¨dler B, Mackay A, Mackenzie I, et al. Pilot study of granulocyte colony stimulating factor (G-CSF)mobilized peripheral blood stem cells in amyotrophic lateral sclerosis (ALS). Muscle Nerve 2008;37:620–625. 24. Zhang Y, Wang L, Fu Y, Song H, Zhao H, Deng M, et al. Preliminary investigation of effect of granulocyte colony stimulating factor on amyotrophic lateral sclerosis. Amyotroph Lateral Scler 2008;10: 430–431. 25. Nefussy B, Artamonov I, Deutsch V, Naparstek E, Nagler A, Drory V. Recombinant human granulocyte-colony stimulating factor administration for treating amyotrophic lateral sclerosis: a pilot study. Amyotroph Lateral Scler 2010;11:187–193. 26. Baron P, Bussini S, Cardin V, Corbo M, Conti G, Galimberti D, et al. Production of monocyte chemoattractant protein-1 in amyotrophic lateral sclerosis. Muscle Nerve 2005;32:541–544. 27. Henkel JS, Engelhardt JI, Siklo´s L, Simpson EP, Kim SH, Pan T, et al. Presence of dendritic cells, MCP-1, and activated microglia/
MUSCLE & NERVE
28. 29. 30.
macrophages in amyotrophic lateral sclerosis spinal cord tissue. Ann Neurol 2004;55:221–235. Kolls JK, Linde´n A. Interleukin-17 family members and inﬂammation. Immunity 2004;21:467–476. Wang YH, Liu YJ. The IL-17 cytokine family and their role in allergic inﬂammation. Curr Opin Immunol 2008;20:697–702. Ivanov II, Frutos Rde L, Manel N, Yoshinaga K, Rifkin DB, Sartor RB, et al. Speciﬁc microbiota direct the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine. Cell Host Microbe 2008;4:337–49. Kramer JM, Gaffen SL. Interleukin-17: a new paradigm in inﬂammation, autoimmunity, and therapy. J Periodontol 2007;78:1083– 1093. Pongcharoen S, Ritvirool PN, Sanguansermsri D, Chanchan P, Jienmongkol P, Butkhamchot P, et al. Reduced interleukin-17 expression of Burkholderia pseudomallei–infected peripheral blood mononuclear cells of diabetic patients. Asian Pac J Allergy Immunol 2008;26: 63–69.
G-CSF in a Multicenter ALS Study
33. Suryani S, Sutton I. An interferon-gamma-producing Th1 subset is the major source of IL-17 in experimental autoimmune encephalitis. J Neuroimmunol 2007;183:96–103. 34. Li GZ, Zhong D, Yang LM, Sun B, Zhong ZH, Yin YH, et al. Expression of interleukin-17 in ischemic brain tissue. Scand J Immunol 2005;62:481–486. 35. Kawanokuchi J, Shimizu K, Nitta A, Yamada K, Mizuno T, Takeuchi H, et al. Production and functions of IL-17 in microglia. J Neuroimmunol 2008;194:54–61. 36. Meeuwsen S, Persoon-Deen C, Bsibsi M, Bajramovic JJ, Ravid R, De Bolle L, et al. Modulation of the cytokine network in human adult astrocytes by human herpesvirus-6A. J Neuroimmunol 2005;164: 37–47. 37. Engelhardt JI, Tajti J, Appel SH. Lymphocytic inﬁltrates in the spinal cord in amyotrophic lateral sclerosis. Arch Neurol 1993;50:30–36. 38. Kawamata T, Akiyama H, Yamada T, McGeer PL. Immunologic reactions in amyotrophic lateral sclerosis brain and spinal cord tissue. Am J Pathol 1992;140:691–707.
MUSCLE & NERVE