Neural-hematopoietic-inflammatory axis in nonsmokers, electronic cigarette users, and tobacco smokers.

Amygdala activity in context of the splenocardiac model has not been investigated in healthy, young adults and has not been compared between nonsmokers, electronic cigarette users, and smokers. The purpose of the current study was to determine whether fluorodeoxyglucose positron emission tomography/computer tomography (FDG PET/CT) scans would demonstrate positively correlated metabolic activity in the amygdala, bone marrow, spleen, and aorta, elucidating activation of the splenocardiac axis in otherwise healthy young people who use tobacco products compared to nonusers. Moreover, the study was conducted to evaluate whether electronic cigarette users and tobacco smokers have similar levels of inflammation compared to nonusers. In 45 healthy adults (mean age = 25 years), including nonsmoker (n = 15), electronic cigarette user (n = 16), and smoker (n = 14) groups, metabolic activity in the amygdala, spleen, aorta, bone marrow of thoracic vertebrae, and adjacent erector spinae skeletal muscle was quantified through visualization of radioactive glucose (18 FDG) uptake by FDG-PET/CT. The maximum standardized uptake value for each region was calculated for correlation analyses and comparisons between groups. In correlation analyses, metabolic activity of the amygdala correlated with metabolic activity in the aorta (r = 0.757), bone marrow (r = 0.750), and spleen (r = 0.665), respectively. Metabolic activity in the aorta correlated with 18 FDG uptake in the thoracic vertebrae (r = 0.703) and spleen (r = 0.594), respectively. Metabolic activity in the spleen also correlated with 18 FDG uptake in the bone marrow (r = 0.620). Metabolic activity in the adjacent erector spinae skeletal muscle (our control tissue) was not positively correlated with any other region of interest. Finally, there were no statistically significant mean differences in metabolic activity between the three groups: nonsmokers, electronic cigarette users, and smokers in any target tissue. Amygdala metabolic activity, as measured by 18 FDG uptake in FDG-PET/CT scans, positively correlated with inflammation in the splenocardiac tissues, including: the aorta, bone marrow, and spleen, underscoring the existence of a neural-hematopoietic-inflammatory axis in healthy, young adults.

Reduced Satb1 expression predisposes CD4+ T conventional cells to Treg suppression and promotes transplant survival.

Limiting CD4+ T cell responses is important to prevent solid organ transplant rejection. In a mouse model of costimulation blockade-dependent cardiac allograft tolerance, we previously reported that alloreactive CD4+ conventional T cells (Tconvs) develop dysfunction, losing proliferative capacity. In parallel, induction of transplantation tolerance is dependent on the presence of regulatory T cells (Tregs). Whether susceptibility of CD4+ Tconvs to Treg suppression is modulated during tolerance induction is unknown. We found that alloreactive Tconvs from transplant tolerant mice had augmented sensitivity to Treg suppression when compared with memory T cells from rejector mice and expressed a transcriptional profile distinct from these memory T cells, including down-regulated expression of the transcription factor Special AT-rich sequence-binding protein 1 (Satb1). Mechanistically, Satb1 deficiency in CD4+ T cells limited their expression of CD25 and IL-2, and addition of Tregs, which express higher levels of CD25 than Satb1-deficient Tconvs and successfully competed for IL-2, resulted in greater suppression of Satb1-deficient than wild-type Tconvs in vitro. In vivo, Satb1-deficient Tconvs were more susceptible to Treg suppression, resulting in significantly prolonged skin allograft survival. Overall, our study reveals that transplantation tolerance is associated with Tconvs' susceptibility to Treg suppression, via modulated expression of Tconv-intrinsic Satb1. Targeting Satb1 in the context of Treg-sparing immunosuppressive therapies might be exploited to improve transplant outcomes.

Non-clinical assessment of cell therapy products: the perspective from five Asian countries/regions based on regulatory guidelines and the underpinning rationales.

BACKGROUND AIMS: Cell-based regenerative medicine is an innovative field that can potentially alter the overall survival and quality of life of patients with devastating diseases. Several cell therapy products (CTPs) have been approved within the last two decades, and more are under development. The establishment of an effective developmental strategy in accordance with the regulatory bodies of each country/region is crucial for fast delivery of each respective CTP. In particular, facilitating investigational new drug (IND) approval is important for accelerating the transition from non-clinical to clinical research/trial phases. METHODS: Here the authors compared the non-clinical prerequisites for initiating clinical studies in five Asian countries/regions (India, China, Korea, Taiwan and Japan) from an industry viewpoint. The authors first identified the differences and tried to clarify the perspectives/considerations underpinning the different requirements. RESULTS: The authors' findings revealed that differences in regulations and development experiences, especially with CTPs, have led to clear differences in the non-clinical study package and its corresponding study design. CONCLUSIONS: By sharing experiences of the research and development of CTPs among Asian countries/regions and including not only industry but also regulatory authorities, we will be able to expedite cross-border IND approval and eventually contribute to the early delivery of innovative CTPs to many Asian patients.

Investigating the genetic susceptibility to exertional heat illness.

BACKGROUND: We aimed to identify rare (minor allele frequency ≤1%), potentially pathogenic non-synonymous variants in a well-characterised cohort with a clinical history of exertional heat illness (EHI) or exertional rhabdomyolysis (ER). The genetic link between malignant hyperthermia (MH) and EHI was investigated due to their phenotypic overlap. METHODS: The coding regions of 38 genes relating to skeletal muscle calcium homeostasis or exercise intolerance were sequenced in 64 patients (mostly military personnel) with a history of EHI, or ER and who were phenotyped using skeletal muscle in vitro contracture tests. We assessed the pathogenicity of variants using prevalence data, in silico analysis, phenotype and segregation evidence and by review of the literature. RESULTS: We found 51 non-polymorphic, potentially pathogenic variants in 20 genes in 38 patients. Our data indicate that RYR1 p.T3711M (previously shown to be likely pathogenic for MH susceptibility) and RYR1 p.I3253T are likely pathogenic for EHI. PYGM p.A193S was found in 3 patients with EHI, which is significantly greater than the control prevalence (p=0.000025). We report the second case of EHI in which a missense variant at CACNA1S p.R498 has been found. Combinations of rare variants in the same or different genes are implicated in EHI. CONCLUSION: We confirm a role of RYR1 in the heritability of EHI as well as ER but highlight the likely genetic heterogeneity of these complex conditions. We propose defects, or combinations of defects, in skeletal muscle calcium homeostasis, oxidative metabolism and membrane excitability are associated with EHI.

The pursuit of transplantation tolerance: new mechanistic insights.

Donor-specific transplantation tolerance that enables weaning from immunosuppressive drugs but retains immune competence to non-graft antigens has been a lasting pursuit since the discovery of neonatal tolerance. More recently, efforts have been devoted not only to understanding how transplantation tolerance can be induced but also the mechanisms necessary to maintain it as well as how inflammatory exposure challenges its durability. This review focuses on recent advances regarding key peripheral mechanisms of T cell tolerance, with the underlying hypothesis that a combination of several of these mechanisms may afford a more robust and durable tolerance and that a better understanding of these individual pathways may permit longitudinal tracking of tolerance following clinical transplantation to serve as biomarkers. This review may enable a personalized assessment of the degree of tolerance in individual patients and the opportunity to strengthen the robustness of peripheral tolerance.

Malignant hyperthermia.

Malignant hyperthermia (MH) is a form of heat illness caused by increased heat generation exceeding the body's capacity for heat loss. It is classified separately from other forms of heat illness as the latter require assessment of mental function for differential diagnosis. This is not possible with MH which occurs during general anesthesia when mental function cannot be assessed. MH occurs in genetically predisposed individuals exposed to inhalation anesthetics or succinylcholine. The genetic defects identified so far cause perturbation of skeletal muscle excitation-contraction coupling resulting in myoplasmic calcium dysregulation. The most commonly involved gene is RYR1. Increased myoplasmic calcium leads to hypermetabolism and sustained muscle contractile activity with consequent increased oxygen consumption, carbon dioxide production, sympathetic stimulation, muscle rigidity, heat production, rhabdomyolysis, and disseminated intravascular coagulation. Untreated reactions are fatal. In this chapter we summarize clinical features and management and review current understanding of the pathophysiology and molecular genetics of MH.

Quantitative bone scan lesion area as an early surrogate outcome measure indicative of overall survival in metastatic prostate cancer.

A clinical validation of the bone scan lesion area (BSLA) as a quantitative imaging biomarker was performed in metastatic castration-resistant prostate cancer (mCRPC). BSLA was computed from whole-body bone scintigraphy at baseline and week 12 posttreatment in a cohort of 198 mCRPC subjects (127 treated and 71 placebo) from a clinical trial involving a different drug from the initial biomarker development. BSLA computation involved automated image normalization, lesion segmentation, and summation of the total area of segmented lesions on bone scan AP and PA views as a measure of tumor burden. As a predictive biomarker, treated subjects with baseline BSLA [Formula: see text] had longer survival than those with higher BSLA ([Formula: see text] and [Formula: see text]). As a surrogate outcome biomarker, subjects were categorized as progressive disease (PD) if the BSLA increased by a prespecified 30% or more from baseline to week 12 and non-PD otherwise. Overall survival rates between PD and non-PD groups were statistically different ([Formula: see text] and [Formula: see text]). Subjects without PD at week 12 had longer survival than subjects with PD: median 398 days versus 280 days. BSLA has now been demonstrated to be an early surrogate outcome for overall survival in different prostate cancer drug treatments.

Administration of Adult Human Bone Marrow-Derived, Cultured, Pooled, Allogeneic Mesenchymal Stromal Cells in Critical Limb Ischemia Due to Buerger's Disease: Phase II Study Report Suggests Clinical Efficacy.

Critical limb ischemia (CLI) due to Buerger's disease is a major unmet medical need with a high incidence of morbidity. This phase II, prospective, nonrandomized, open-label, multicentric, dose-ranging study was conducted to assess the efficacy and safety of i.m. injection of adult human bone marrow-derived, cultured, pooled, allogeneic mesenchymal stromal cells (BMMSC) in CLI due to Buerger's disease. Patients were allocated to three groups: 1 and 2 million cells/kg body weight (36 patients each) and standard of care (SOC) (18 patients). BMMSCs were administered as 40-60 injections in the calf muscle and locally, around the ulcer. Most patients were young (age range, 38-42 years) and ex-smokers, and all patients had at least one ulcer. Both the primary endpoints-reduction in rest pain (0.3 units per month [SE, 0.13]) and healing of ulcers (11% decrease in size per month [SE, 0.05])-were significantly better in the group receiving 2 million cells/kg body weight than in the SOC arm. Improvement in secondary endpoints, such as ankle brachial pressure index (0.03 [SE, 0.01] unit increase per month) and total walking distance (1.03 [SE, 0.02] times higher per month), were also significant in the group receiving 2 million cells/kg as compared with the SOC arm. Adverse events reported were remotely related or unrelated to BMMSCs. In conclusion, i.m. administration of BMMSC at a dose of 2 million cells/kg showed clinical benefit and may be the best regimen in patients with CLI due to Buerger's disease. However, further randomized controlled trials are required to confirm the most appropriate dose. Stem Cells Translational Medicine 2017;6:689-699.

Th17 cells are not required for maintenance of IL-17A-producing γδ T cells in vivo.

γδ T cells producing interleukin-17A (γδT17) are thought to develop spontaneously in the thymus and to be maintained in the periphery. Previous studies suggested a role for T-helper 17 (Th17) cells in the maintenance of γδT17 via the expression of transforming growth factor-ß1 (TGFß1). However, we have previously found that Th17 cells were not required for expansion of γδT17 cells after lung transplant in a mouse model. Using mice deficient in signal transducer and activator of transcription 3 (STAT3) in CD4+ T cells, which are unable to develop Th17 cells, we investigated the requirement for Th17 cells and TGFß1 to maintain γδT17 cells in the lung and lymphoid tissues. At steady state, we found no defect in γδT17 cells in the thymus or periphery of these mice. Further, STAT3-deficient CD4+ T cells produced significantly higher levels of TGFß1 than wild-type CD4+ T cells under Th17 differentiation conditions in vitro. To determine whether STAT3-deficient CD4+ T cells could expand γδT17 cells in vivo, we used TCRß-/- mice, which are known to have a defect in γδT17 cells that can be rescued by Th17 cells. However, adoptive transfer of wild-type Th17 cells or bulk CD4+ T cells did not expand γδT17 cells in TCRß-/- mice. In contrast, interferon-γ+ γδ T cells preferentially expanded, particularly in the lungs. Interestingly, we found in vivo and in vitro that TGFß1 may negatively regulate the pool of γδT17 cells. Our data suggest that Th17 cells and TGFß1 are not required for the maintenance of γδT17 cells.

Skeletal Muscle Microalterations in Patients Carrying Malignant Hyperthermia-Related Mutations of the e-c Coupling Machinery.

We have compared the ultrastructure of skeletal muscle biopsies from patients that have survived a [Malignant Hyperthermia, MH] episode and siblings that test positive for MH susceptibility with those from siblings that tested negatives. The aim is to establish whether life long exposure to the MH-related mutation effects may result in subtle abnormalities even in the absence of active episodes and/or clinically detectable deficiencies. Although a specific ultrastructural signature for MH mutants cannot be demonstrated, an MH related pattern of minor alterations does exist. These include the tendency for micro damage to the contractile apparatus and a higher than normal level of mitochondrial abnormalities.

Next-generation Sequencing of RYR1 and CACNA1S in Malignant Hyperthermia and Exertional Heat Illness.

BACKGROUND: Variants in RYR1 are associated with the majority of cases of malignant hyperthermia (MH), a form of heat illness pharmacogenetically triggered by general anesthetics, and they have also been associated with exertional heat illness (EHI). CACNA1S has also been implicated in MH. The authors applied a targeted next-generation sequencing approach to identify variants in RYR1 and CACNA1S in a cohort of unrelated patients diagnosed with MH susceptibility. They also provide the first comprehensive report of sequencing of these two genes in a cohort of survivors of EHI. METHODS: DNA extracted from blood was genotyped using a "long" polymerase chain reaction technique, with sequencing on the Illumina GAII or MiSeq platforms (Illumina Inc., USA). Variants were assessed for pathogenicity using bioinformatic approaches. For further follow-up, DNA from additional family members and up to 211 MH normal and 556 MH-susceptible unrelated individuals was tested. RESULTS: In 29 MH patients, the authors identified three pathogenic and four novel RYR1 variants, with a further five RYR1 variants previously reported in association with MH. Three novel RYR1 variants were found in the EHI cohort (n = 28) along with two more previously reported in association with MH. Two other variants were reported previously associated with centronuclear myopathy. The authors found one and three rare variants of unknown significance in CACNA1S in the MH and EHI cohorts, respectively. CONCLUSIONS: Targeted next-generation sequencing proved efficient at identifying diagnostically useful and potentially implicated variants in RYR1 and CACNA1S in MH and EHI.

Analysis of smoking and LPO in ALS.

Smoking has been suggested as one of the risk factor for amyotrophic lateral sclerosis (ALS) development. In order to investigate whether adverse effects of cigarette smoke in ALS have any association with increase in oxidative stress, disease severity, lipid hydroperoxides (LPO) and superoxide dismutase-1 (SOD1) levels were measured in biofluids of smoker and never smoker ALS patients and clinically correlated. Serum and CSF from sporadic ALS patients (n=50) diagnosed with El Escorial criteria were collected in the study. Serum (n=50) and CSF (n=42) were also collected from normal healthy controls. The LPO levels were estimated using commercially available kits. Enzyme-linked immunosorbent assays (ELISAs) were used to quantitate SOD1. Their levels were further analyzed among smoker and never smoker subjects. Significantly elevated LPO in sera and CSF of ALS patients were observed (p<0.05). There was considerably increased LPO in sera and CSF of smoker ALS subjects matched with disease severity as compared to never smoker ALS (p<0.05). ALS group did not show any alteration in SOD1 when compared to controls (p>0.05). In addition, no change has been observed in SOD1 levels in ALS subjects who smoke (p>0.05). Increased LPO and unaltered SOD1 in ALS patients may suggest the neuro-pathological association of LPO with ALS disease independent of SOD1. With current findings, it may be proposed that LPO levels might constitute as probable biomarker for smoker ALS patients, however, it cannot be concluded without larger gender matched studies. Additional investigations are needed to determine whether LPO upregulation is primary or secondary to motor neuron degeneration in ALS.

Does DcR1 (TNF-related apoptosis-inducing-ligand Receptor 3) have any role in human AMD pathogenesis?

It has been postulated that there is a link between age related degenerative diseases and cancer. The TNF-related apoptosis-inducing ligand (TRAIL) has been shown to selectively kill tumor cells by binding to pro-apoptotic and anti-apoptotic receptors. Our aim was to study the levels of anti-apoptotic receptor (DcR1) in age related macular degeneration (AMD) and controls. AMD patients (115) were classified into two groups: Dry and Wet AMD. Wet AMDs were further classified into occult, predominant classic and minimal classic. 61 healthy individuals were recruited as normal controls. After normalization with total protein, DcR1 levels were analyzed by ELISA. Mann Whitney U-statistic was used for analysis of DcR1 ELISA results. We have observed DcR1 levels in serum sample which were significantly lower in AMD patients as compared to controls (p = 0.001). On the other hand, we did not find difference in DcR1 levels between wet and dry AMD. The present study defines the plausible role of DcR1 in AMD pathology signifying a new therapeutic target for AMD.

A double blind randomized placebo controlled phase I/II study assessing the safety and efficacy of allogeneic bone marrow derived mesenchymal stem cell in critical limb ischemia.

BACKGROUND: Peripheral vascular disease of the lower extremities comprises a clinical spectrum that extends from no symptoms to presentation with critical limb ischemia (CLI). Bone marrow derived Mesenchymal Stem Cells (BM- MSCs) may ameliorate the consequences of CLI due to their combinatorial potential for inducing angiogenesis and immunomodulatory environment in situ. The primary objective was to determine the safety of BM- MSCs in patients with CLI. METHODS: Prospective, double blind randomized placebo controlled multi-center study was conducted in patients with established CLI as per Rutherford classification in category II-4, III-5, or III-6 with infra-inguinal arterial occlusive disease and were not suitable for or had failed revascularization treatment. The primary end point was incidence of treatment - related adverse events (AE). Exploratory efficacy end points were improvement in rest pain, increase in Ankle Brachial Pressure Index (ABPI), ankle pressure, healing of ulcers, and amputation rates. Twenty patients (BM-MSC: Placebo = 1:1) were administered with allogeneic BM-MSCs at a dose of 2 million cells/kg or placebo (PlasmaLyte A) at the gastrocnemius muscle of the ischemic limb. RESULTS: Improvement was observed in the rest pain scores in both the arms. Significant increase in ABPI and ankle pressure was seen in BM-MSC arm compared to the placebo group. Incidence of AEs in the BM-MSC arm was 13 vs. 45 in the placebo arm where as serious adverse events (SAE) were similar in both the arms (5 in BM-MSC and 4 in the placebo group). SAEs resulted in death, infected gangrene, amputations in these patients. It was observed that the SAEs were related to disease progression and not related to stem cells. CONCLUSION: BM-MSCs are safe when injected IM at a dose of 2 million cells/kg body weight. Few efficacy parameters such as ABPI and ankle pressure showed positive trend warranting further studies. TRIAL REGISTRATION: NIH website (http://www.clinicaltrials.gov/ct2/show/NCT00883870).

Mesenchymal stem cells for cartilage repair in osteoarthritis.

Osteoarthritis (OA) is a degenerative disease of the connective tissue and progresses with age in the older population or develops in young athletes following sports-related injury. The articular cartilage is especially vulnerable to damage and has poor potential for regeneration because of the absence of vasculature within the tissue. Normal load-bearing capacity and biomechanical properties of thinning cartilage are severely compromised during the course of disease progression. Although surgical and pharmaceutical interventions are currently available for treating OA, restoration of normal cartilage function has been difficult to achieve. Since the tissue is composed primarily of chondrocytes distributed in a specialized extracellular matrix bed, bone marrow stromal cells (BMSCs), also known as bone marrow-derived 'mesenchymal stem cells' or 'mesenchymal stromal cells', with inherent chondrogenic differentiation potential appear to be ideally suited for therapeutic use in cartilage regeneration. BMSCs can be easily isolated and massively expanded in culture in an undifferentiated state for therapeutic use. Owing to their potential to modulate local microenvironment via anti-inflammatory and immunosuppressive functions, BMSCs have an additional advantage for allogeneic application. Moreover, by secreting various bioactive soluble factors, BMSCs can protect the cartilage from further tissue destruction and facilitate regeneration of the remaining progenitor cells in situ. This review broadly describes the advances made during the last several years in BMSCs and their therapeutic potential for repairing cartilage damage in OA.

Possible association between expression of chemokine receptor-2 (CCR2) and amyotrophic lateral sclerosis (ALS) patients of North India.

BACKGROUND AND OBJECTIVES: We earlier reported elevated chemokine ligand-2 (CCL2) in Indian amyotrophic lateral sclerosis (ALS) patients. We now analysed chemokine receptor-2 (CCR2), the receptor of CCL2, in these ALS patients. METHODS: Indian sporadic ALS patients (n=50) were included on the basis of El Escorial criteria. Percentage (%) of CCR2 expressing peripheral blood mononuclear cells (PBMCs) was evaluated using Flow Cytometry. Real Time Polymerase Chain Reaction (PCR) was used to quantitate CCR2 mRNA expression in PBMCs. Normal controls (n = 40) were also included for comparison. RESULTS: Flow Cytometry revealed significantly reduced CCR2 expressing PBMCs in the ALS patients. We also found a significant decline in number of CCR2 expressing PBMCs in limb onset ALS when compared to bulbar onset ALS. PBMCs from ALS patients showed substantial down-regulation of CCR2 mRNA. CCR2 mRNA expression was found to be decreased among limb ALS patients as compared to bulbar onset ALS. Further, the count of CCR2+ PBMCs and CCR2 mRNA transcript in PBMCs was significantly lower in severe and moderate ALS as compared to ALS patients with mild impairments. CONCLUSIONS: Downregulation of PBMCs CCR2 may indicate its etio-pathological relevance in ALS pathogenesis. Reduced PBMCs CCR2 may result in decreased infiltration of leukocytes at the site of degeneration as a compensatory response to ALS. CCR2 levels measurements in hematopoietic stem cells and estimation of comparative PBMCs count among ALS, disease controls and normal controls can unveil its direct neuroprotective role. However, the conclusions are restricted by the absence of neurological/non-neurological disease controls in the study.

Vascular endothelial growth factor-A and chemokine ligand (CCL2) genes are upregulated in peripheral blood mononuclear cells in Indian amyotrophic lateral sclerosis patients.

BACKGROUND: We have earlier shown that protein levels of vascular endothelial growth factor-A (VEGF-A) and chemokine ligand-2 (CCL2) were elevated in Indian amyotrophic lateral sclerosis (ALS) patients. Here, we report the mRNA levels of VEGF-A and CCL2 in Indian ALS patients since they display extended survival after disease onset. METHODS: VEGF-A and CCL2 mRNA levels were measured in peripheral blood mononuclear cells (PBMCs) of 50 sporadic Indian ALS patients using Real Time Polymerase Chain Reaction (PCR) and compared with normal controls (n = 50). Their levels were adjusted for possible confounders like cigarette smoking, alcohol and meat consumption. RESULTS: VEGF-A and CCL2 mRNA levels were found to be significantly elevated in PBMCs in ALS patients as compared to controls. PBMCs from definite ALS revealed higher VEGF-A mRNA expression as compared to probable and possible ALS. CCL2 mRNA levels were found to be unaltered when definite, probable and possible ALS were compared. PBMCs from patients with respiratory dysfunction showed much higher VEGF-A and CCL2 elevation when compared to patients without respiratory dysfunction. No association of smoking, alcohol and meat consumption with VEGF-A and CCL2 was observed after analyzing the data with univariate and multivariate analysis. CONCLUSION: VEGF-A and CCL2 mRNA upregulation in PBMCs may have a clinico-pathological/etiological/epidemiological association with ALS pathogenesis. The cross-cultural and cross-ethnic investigations of these molecules could determine if they have any role in enhancing the mean survival time unique to Indian ALS patients.

Vascular endothelial growth factor-A (VEGF-A) and chemokine ligand-2 (CCL2) in amyotrophic lateral sclerosis (ALS) patients.

BACKGROUND: Vascular endothelial growth factor-A (VEGF-A) and chemokne ligand-2 (CCL2) levels have been examined in Amyotrophic Lateral Sclerosis (ALS) patients in Western countries. We measured these values in North Indian ALS patients, since these patients display considerably enhanced survival duration. METHODS: Sporadic ALS patients were included on the basis of El Escorial criteria. VEGF-A and CCL2 levels were analyzed in serum and cerebrospinal fluid (CSF) of 50 ALS patients using enzyme linked immunosorbent assay (ELISA) and compared with normal controls. Their levels were adjusted for possible confounders like cigarette smoking, alcohol and meat consumption. RESULTS: Contrary to previous studies, VEGF-A was found to be elevated significantly in serum and CSF in ALS patient population studied. We also found an increase in CCL2 levels in CSF of these ALS patients. Serum and CSF from definite ALS revealed higher VEGF-A as compared to probable and possible ALS. CCL2 was unaltered between definite, probable and possible ALS. Univariate and multivariate analysis revealed a lack of association of smoking, alcohol and meat consumption with VEGF-A and CCL2 levels. CONCLUSIONS: VEGF-A upregulation may indicate an activation of compensatory responses in ALS which may reflect or in fact account for increased survival of North Indian ALS patients after disease onset. The intrathecal synthesis of CCL2 suggests the involvement of adult neural stem cells and microglial activation in ALS pathogenesis which needs further investigation.