Enhanced levels of fractalkine and HSP60 in cerebrospinal fluid of sporadic amyotrophic lateral sclerosis patients.

Amyotrophic Lateral Sclerosis (ALS) is a multifactorial neurodegenerative disorder with a significant contribution of non-cell autonomous mechanisms to motor neuronal degeneration. Amongst a plethora of molecules, fractalkine (C-X3-C motif chemokine ligand 1), and Heat Shock Protein 60 (HSP60), are key modulators of microglial activation. The contribution of these molecules in Sporadic ALS (SALS) remains unexplored. To investigate this, fractalkine levels were estimated in Cerebrospinal fluid (CSF) of SALS patients (ALS-CSF; n = 44) by Enzyme-linked Immunosorbent Assay (ELISA) and correlated with clinical parameters including disease severity and duration. CSF HSP60 levels were estimated by Western blotting (ALS-CSF; n = 19). Also, CSF levels of Chitotriosidase-1 (CHIT-1), a microglia-specific neuroinflammatory molecule, were measured and its association, if any, with fractalkine and HSP60 was investigated. Both fractalkine and HSP60 levels were significantly elevated in ALS-CSF. Similar to our earlier observation, CHIT-1 levels were also upregulated. Fractalkine showed a moderate negative correlation with the ALS-Functional Rating Scale (ALSFRS) score indicating its significant rise in mild cases which plateaued in cases with high disease severity. However, no obvious correlation was found between fractalkine, HSP60, and CHIT-1. Our study hints that high fractalkine levels in mild cases might be conferring neuroprotection by combating microglial activation and highlights its importance as a novel therapeutic target for SALS. On the other hand, significantly enhanced levels of HSP60, a pro-inflammatory molecule, hint towards its role in accentuating microgliosis, although, it doesn't act synergistically with CHIT-1. Our study suggests that fractalkine and HSP60 act independently of CHIT-1 to suppress and accentuate neuroinflammation, respectively.

Antimicrobial Efficacy of Essential Oils and Their Combination Against Microorganisms Associated With Postradiation Therapy in Patients With Head and Neck Cancer: An In Vitro Study.

Background Head and neck cancer ranks as the sixth most common cancer globally. Reduced saliva production brought on by postradiation therapy upsets the delicate balance between bacterial load and a weakened immune system. Oral hygiene is commonly neglected in patients who have undergone radiotherapy and they often develop dry mouth, mucositis due to radiation therapy, etc., as side effects. Despite being a part of the current standard, chlorhexidine carries numerous disadvantages such as taste alteration, teeth staining, and dry mouth. An extensive review of the literature demonstrates the antibacterial properties of essential oils (EOs) derived from plant materials, which may be able to prevent the development of such opportunistic microorganisms in the oral cavity. Methodology The cinnamon bark EO and Cajeput EO were procured and checked for their solubility. The final ratio at which the oils were found to be soluble was the 1:1 (w/v) ratio. The minimum inhibitory concentration (MIC) of cinnamon bark oil (Cinnamomum verum) and Cajeput oil (Melaleuca leucadendron) against Staphylococcus aureus, Enterococcus faecalis, and Candida albicans was determined by serial dilution method using Resazurin dye, and the minimum bactericidal concentration (MBC) was done by a spread plating method. The polyherbal mouthwash was subjected to cytotoxicity assay against human gingival fibroblasts. All the experiments were performed in triplicates. Results The overall results showed that cinnamon bark EO had the strongest efficacy against S. aureus (0.33 ± 0.14 mg/mL) and E. faecalis (0.41 ± 0.14 mg/mL), but not against C. albicans (2.85 ± 2.11 mg/mL). Cajeput EO showed the least efficacy against all the groups; whereas the combination of EOs proved to be the most efficacious and showed good antimicrobial activity against these most commonly encountered microorganisms in head and neck cancer postradiotherapy. Conclusions Cinnamon and Cajeput EOs in combination proved to be effective in this in vitro study against the most common microorganisms encountered in patients with head and neck cancer postradiotherapy and are comparable to 0.2% chlorhexidine.

Impact of dental caries and periodontal disease on oral health-related quality of life among the Karnataka State Reserve Police Personnel in Belagavi, India.

Introduction: The Karnataka State Reserve Police (KSRP) is a state-level police force in India. Good Oral Health-Related Quality of Life (OHRQoL) among them is quintessential for the welfare of the society. The aim of the study was to assess the impact of dental caries and periodontal disease on the OHRQoL among the Karnataka State Reserve Police (KSRP) stationed in Belagavi, India. Methods: A cross-sectional design was used with a total sample size of 720. The personnel were recruited by simple random sampling. The Oral Health Impact Profile 14 (OHIP 14) was used to assess OHRQoL in 7 domains. The intra-examiner reliability for World Health Organisation (WHO) oral assessment form 2013 was assessed using Kappa statistics and was found to be 0.86. Dentition and periodontal status were recorded using the same. Statistical analysis was performed using descriptive statistics, Analysis of variance (ANOVA), Pearson's coefficient of correlation and multiple linear regression analysis. Results: Physical pain and psychological discomfort had the highest mean scores among the seven domains of OHIP-14. Constables had higher mean OHIP-14 scores among the study population. A significant positive correlation was found between oral health parameters with the domains of OHIP-14. The highest dependence on the socio-demographic and oral health predictors were found in the domains of physical pain (44.2%), psychological discomfort (38.3%), and physical disability (30.5%). Conclusions: The study revealed that dental caries and periodontal disease had a significant impact on OHRQoL among reserve police personnel and the OHRQoL was poor particularly among the lower ranked personnel.

Oligodendroglia Confer Neuroprotection to NSC-34 Motor Neuronal Cells Against the Toxic Insults of Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients.

Amyotrophic lateral sclerosis (ALS) is a complex neurodegenerative disorder with multifactorial pathomechanisms affecting not only motor neurons but also glia. Both astrocytes and microglia get activated and contribute significantly to neurodegeneration. The role of oligodendroglia in such a situation remains obscure, especially in the sporadic form of ALS (SALS), which contributes to 90% of cases. Here, we have investigated the role of oligodendroglia in SALS pathophysiology using a human oligodendroglial cell line, MO3.13, by exposing the cells to cerebrospinal fluid from SALS patients (ALS-CSF; 10% v/v for 48 h). ALS-CSF significantly reduced the viability of MO3.13 cells and down-regulated the expression of oligodendroglia-specific proteins, namely, CNPase and Olig2. Furthermore, to investigate the effect of the observed oligodendroglial changes on motor neurons, NSC-34 motor neuronal cells were co-cultured/supplemented with conditioned/spent medium of MO3.13 cells upon exposure to ALS-CSF. Live cell imaging experiments revealed protection to NSC-34 cells against ALS-CSF toxicity upon co-culture with MO3.13 cells. This was evidenced by the absence of neuronal cytoplasmic vacuolation and beading of neurites, which instead resulted in better neuronal differentiation. Enhanced lactate levels and increased expression of its transporter, MCT-1, with sustained expression of trophic factors, namely, GDNF and BDNF, by MO3.13 cells hint towards metabolic and trophic support provided by the surviving oligodendroglia. Similar metabolic changes were seen in the lumbar spinal cord oligodendroglia of rat neonates intrathecally injected with ALS-CSF. The findings indicate that oligodendroglia are indeed rescuer to the degenerating motor neurons when the astrocytes and microglia turn topsy-turvy.

Abnormalities in the migration of neural precursor cells in familial bipolar disorder.

Cellular migration is a ubiquitous feature that brings brain cells into appropriate spatial relationships over time; and it helps in the formation of a functional brain. We studied the migration patterns of induced pluripotent stem cell-derived neural precursor cells (NPCs) from individuals with familial bipolar disorder (BD) in comparison with healthy controls. The BD patients also had morphological brain abnormalities evident on magnetic resonance imaging. Time-lapse analysis of migrating cells was performed, through which we were able to identify several parameters that were abnormal in cellular migration, including the speed and directionality of NPCs. We also performed transcriptomic analysis to probe the mechanisms behind the aberrant cellular phenotype identified. Our analysis showed the downregulation of a network of genes, centering on EGF/ERBB proteins. The present findings indicate that collective, systemic dysregulation may produce the aberrant cellular phenotype, which could contribute to the functional and structural changes in the brain reported for bipolar disorder. This article has an associated First Person interview with the first author of the paper.

Donor-derived cell-free DNA as a composite marker of acute lung allograft dysfunction in clinical care.

BACKGROUND: As a marker of underlying lung allograft injury, donor-derived cell-free DNA (dd-cfDNA) may be used to identify episodes of acute allograft injury in lung transplant recipients. We investigated the utility of dd-cfDNA to monitor subjects at risk of acute rejection or infection in routine clinical practice. METHODS: This multicenter, retrospective cohort study collected data from lung transplant recipients within 3 years of transplant at 4 centers between March 24, 2020 and September 1, 2020. During this period, as part of routine care during the COVID-19 pandemic, these centers implemented a home-based surveillance program using plasma dd-cfDNA in preference to surveillance bronchoscopy. Dd-cfDNA was used to detect acute lung allograft dysfunction (ALAD) - a composite endpoint of acute rejection and infection. dd-cfDNA levels in patients with ALAD were compared to stable patients. The performance characteristics of dd-cfDNA ≥ 1.0% to detect ALAD were estimated. RESULTS: A total of 175 patients underwent 380 dd-cfDNA measurements, of which 290 were for routine surveillance purposes. dd-cfDNA was higher in patients with ALAD than stable patients (Median (IQR) 1.7% (0.63, 3.1) vs 0.35% (0.22, 0.79), p < 0.001). As an indication of underlying ALAD during surveillance testing, the estimated sensitivity of dd-cfDNA ≥1% was 73.9%, specificity of 87.7%, positive predictive value of 43.4% and negative predictive value of 96.5%. CONCLUSIONS: dd-cfDNA identified acute lung allograft dysfunction in asymptomatic lung transplant patients that may not have been identified by using a clinically indicated biopsy strategy alone. dd-cfDNA <1.0% may be useful in ruling out acute rejection and infection, supporting its use as a potential noninvasive marker for surveillance monitoring.

Biological Variation of Donor-Derived Cell-Free DNA in Stable Lung Transplant Recipients.

BACKGROUND: Prior studies demonstrate that donor-derived cell-free DNA (dd-cfDNA) in lung transplant recipients may serve as a marker of allograft injury for detecting allograft rejection and infection. Clinical interpretation of dd-cfDNA requires understanding its biological variation in stable lung transplant patients in order to identify abnormal results suggesting underlying allograft dysfunction. This study establishes the biological variation and reference change values (RCV) of dd-cfDNA in stable lung transplant recipients using an analytically validated assay with an established analytic coefficient of variation (CVA). METHODS: The AlloSure® assay, a targeted, sequencing-based approach, was used to measure plasma dd-cfDNA in a cohort of lung transplant patients at 4 centers that used dd-cfDNA to monitor for allograft dysfunction in preference to surveillance transbronchial biopsy. Patients with stable allograft function and ≥3 dd-cfDNA samples were included. Intraindividual coefficient of variation (CVI), interindividual CV (CVG), index of individuality (II) and the RCV were calculated. RESULTS: Thirty-five patients with a combined 124 dd-cfDNA samples were included in the final analysis. The median dd-cfDNA was 0.31% (interquartile range 0.18%-0.68%), the 97.5th percentile and 95th percentile were 1.3% and 1.0%, respectively. In 30 stable patients with an average of 3.7 tests, the CVI was 25%, CVG 19%, II 1.33, and RCV 70%. CONCLUSION: In stable lung transplant patients, fluctuations in dd-cfDNA levels of up to 70% or levels less than 1% are within normal biological variation. With further validation, these thresholds may be incorporated into surveillance monitoring algorithms to identify potentially abnormal results indicating allograft dysfunction.

An effective method for saliva stabilization and magnetic nanoparticles based DNA extraction for genomic applications.

Introduction of magnetisable solid phase extraction procedures have provided various advantages over spin-column based extraction techniques. Although certain methods for magnetic bead based extraction of DNA from human saliva already exist, there is still a need to address the inadequate purity profile and low yield which occur due to the inefficiency of extraction methods. Hence, an improved method for DNA extraction from human saliva using uncoated magnetic nanoparticles (MNPs) intended to resolve the issues mentioned above is described here. The uncoated magnetic nanoparticles used in this study facilitate reversible binding of DNA and due to the absence of surface coating the particle size remains small thereby providing higher surface area to volume ratio for binding DNA. Another objective of this study was to develop a saliva preservation buffer (SPB) to solve the major challenges associated with storage and easy transportation of saliva sample at room temperature. Human saliva samples stored in the saliva preservation buffer were stable up to 160 days at room temperature without any bacterial or fungal growth and the quality of genomic DNA was intact.

Meningococcal carriage in periods of high and low invasive meningococcal disease incidence in the UK: comparison of UKMenCar1-4 cross-sectional survey results.

BACKGROUND: The incidence of invasive meningococcal disease in the UK decreased by approximately four times from 1999 to 2014, with reductions in serogroup C and serogroup B disease. Lower serogroup C invasive meningococcal disease incidence was attributable to implementation of the meningococcal serogroup C conjugate vaccine in 1999, through direct and indirect protection, but no vaccine was implemented against serogroup B disease. UK Meningococcal Carriage surveys 1-3 (UKMenCar1-3), conducted in 1999, 2000, and 2001, were essential for understanding the impact of vaccination. To investigate the decline in invasive meningococcal disease incidence, we did a large oropharyngeal carriage survey in 2014-15, immediately before the changes to meningococcal vaccines in the UK national immunisation schedule. METHODS: UKMenCar4 was a cross-sectional survey in adolescents aged 15-19 years who were enrolled from schools and colleges geographically local to one of 11 UK sampling centres between Sept 1, 2014, and March 30, 2015. Participants provided an oropharyngeal swab sample and completed a questionnaire on risk factors for carriage, including social behaviours. Samples were cultured for putative Neisseria spp, which were characterised with serogrouping and whole-genome sequencing. Data from this study were compared with the results from the UKMenCar1-3 surveys (1999-2001). FINDINGS: From the 19 641 participants (11 332 female, 8242 male, 67 not stated) in UKMenCar4 with culturable swabs and completed risk-factor questionnaires, 1420 meningococci were isolated, with a carriage prevalence of 7·23% (95% CI 6·88-7·60). Carriage prevalence was substantially lower in UKMenCar4 than in the previous surveys: carriage prevalence was 16·6% (95% CI 15·89-17·22; 2306/13 901) in UKMenCar1 (1999), 17·6% (17·05-18·22; 2873/16 295) in UKMenCar2 (2000), and 18·7% (18·12-19·27; 3283/17 569) in UKMenCar3 (2001). Carriage prevalence was lower for all serogroups in UKMenCar4 than in UKMenCar1-3, except for serogroup Y, which was unchanged. The prevalence of carriage-promoting social behaviours decreased from 1999 to 2014-15, with individuals reporting regular cigarette smoking decreasing from 2932 (21·5%) of 13 650 to 2202 (11·2%) of 19 641, kissing in the past week from 6127 (44·8%) of 13 679 to 7320 (37·3%) of 19 641, and attendance at pubs and nightclubs in the past week from 8436 (62·1%) of 13 594 to 7662 (39·0%) of 19 641 (all p<0·0001). INTERPRETATION: We show that meningococcal carriage prevalence in adolescents sampled nationally during a low incidence period (2014-15) was less than half of that in an equivalent population during a high incidence period (1999-2001). Disease and carriage caused by serogroup C was well controlled by ongoing vaccination. The prevalence of behaviours associated with carriage declined, suggesting that public health policies aimed at influencing behaviour might have further reduced disease. FUNDING: Wellcome Trust, UK Department of Health, and National Institute for Health Research.

Chitotriosidase, a biomarker of amyotrophic lateral sclerosis, accentuates neurodegeneration in spinal motor neurons through neuroinflammation.

BACKGROUND: Cerebrospinal fluid from amyotrophic lateral sclerosis patients (ALS-CSF) induces neurodegenerative changes in motor neurons and gliosis in sporadic ALS models. Search for identification of toxic factor(s) in CSF revealed an enhancement in the level and enzyme activity of chitotriosidase (CHIT-1). Here, we have investigated its upregulation in a large cohort of samples and more importantly its role in ALS pathogenesis in a rat model. METHODS: CHIT-1 level in CSF samples from ALS (n = 158), non-ALS (n = 12) and normal (n = 48) subjects were measured using ELISA. Enzyme activity was also assessed (ALS, n = 56; non-ALS, n = 10 and normal-CSF, n = 45). Recombinant CHIT-1 was intrathecally injected into Wistar rat neonates. Lumbar spinal cord sections were stained for Iba1, glial fibrillary acidic protein and choline acetyl transferase to identify microglia, astrocytes and motor neurons respectively after 48 h of injection. Levels of tumour necrosis factor-α and interleukin-6 were measured by ELISA. FINDINGS: CHIT-1 level in ALS-CSF samples was increased by 20-fold and it can distinguish ALS patients with a sensitivity of 87% and specificity of 83.3% at a cut off level of 1405.43 pg/ml. Enzyme activity of CHIT-1 was also 15-fold higher in ALS-CSF and has a sensitivity of 80.4% and specificity of 80% at cut off value of 0.1077989 µmol/µl/min. Combining CHIT-1 level and activity together gave a positive predictive value of 97.78% and negative predictive value of 100%. Administration of CHIT-1 increased microglial numbers and astrogliosis in the ventral horn with a concomitant increase in the levels of pro-inflammatory cytokines. Amoeboid-shaped microglial and astroglial cells were also present around the central canal. CHIT-1 administration also resulted in the reduction of motor neurons. CONCLUSIONS: CHIT-1, an early diagnostic biomarker of sporadic ALS, activates glia priming them to attain a toxic phenotype resulting in neuroinflammation leading to motor neuronal death.

Why are people dying due to tuberculosis? A study from Alappuzha District, Kerala, India.

BACKGROUND: Tuberculosis (TB)is a major killer disease worldwide. It is the ninth leading cause of death worldwide and the leading cause from a single infectious agent. In India also, TB kills about 480,000 persons every year and more than 1400 every day. Vision of the National TB Control Programme is TB-Free India with zero deaths, disease and poverty due to TB. Specific targets set in the End TB strategy include a 90% reduction in TB deaths and an 80% reduction in TB incidence by 2030, compared with 2015. Understanding about real cause of death is important to plan strategies to further prevent TB deaths. In the above circumstances we conducted a study, the objective of which was to find out the cause of deaths among patients registered in RNTCP unit of Alappuzha district of Kerala, India. METHODS: In RNTCP a patient who died during the course of treatment regardless of cause is declared as 'Died' due to TB. During the year 2015, 1618 cases were registered in RNTCP of Alappuzha district of which 90 patients died, showing a case fatality rate of 5.56%. Verbal autopsy can be considered as an essential public health tool for studying reasonable estimate of the cause of death at a community level even though not an accurate method at individual level. As part of the study, we visited the 4 RNTCP units of the district and collected the address of the TB patients who died in the area. With the help of the field staff we visited their houses and filled the death audit form of RNTCP along with the additional details. Verbal autopsy was conducted using WHO verbal autopsy format 2012 with immediate house hold contacts. RESULTS: Out of 90 deaths which occurred, three addresses could not be traced and another 15 patient relatives could not be contacted as they migrated out or were not available at their homes on two visits. Among them, mean age was found to be 62.6 years (SD+12.9). Males were 67 (77%) and rest 20 (23%)were females. Cause of death was analysed after Verbal autopsy for 72 deaths. Among 72 deaths, it was found that 29 (40.3%) had nothing other than TB, where as cause of death for 13 (18.1%) patients was myocardial infarction, 11 (15.3%) had cancer, 2 (2.8%) stroke and 17 (23.7%) other causes which include bronchiectasis, COPD, chicken pox, hepatitis, renal failure, and suicide. Only in 35 cases nothing other than TB could be suggested as a cause of death. Thus in 52 out of 87 (60%) cases, the causes of death were diseases other than TB. CONCLUSION: Among the TB deaths in Alappuzha district, 60% of deaths were due to diseases other than TB. Along with early diagnosis of all TB cases, screening for co-morbidity, appropriate management of co-morbidity and periodic clinical review of TB patients should also be part of the major strategies to prevent TB related deaths.

Antenatal vaccination against Group B streptococcus: attitudes of pregnant women and healthcare professionals in the UK towards participation in clinical trials and routine implementation.

INTRODUCTION: Maternal vaccination is increasingly part of antenatal care in the UK and worldwide. Trials of Group B streptococcus vaccines are ongoing. This study investigated the attitudes of pregnant women and healthcare professionals towards antenatal vaccination, both in routine care and a clinical trial setting. MATERIAL AND METHODS: Survey of 269 pregnant women, 273 midwives/obstetricians and 97 neonatal doctors across seven sites in the UK assessing attitudes towards antenatal vaccinations, knowledge of Group B streptococcus, a hypothetical Group B streptococcus vaccine, and participation in clinical vaccine trials. RESULTS: 68% of pregnant women intended to receive a vaccine during their current pregnancy (183/269) and 43% (of all respondents, 115/269) reported they would be very/fairly likely to accept a vaccine against Group B streptococcus despite only 29% (55/269) knowing what Group B streptococcus was. This increased to 69% after additional information about Group B streptococcus was provided. Twenty-four percent of pregnant women reported they would be likely to take part in a clinical trial of an unlicensed Group B streptococcus vaccine. Fifty-nine percent of maternity professionals and 74% of neonatologists would be likely to recommend participation in a Group B streptococcus vaccine trial to women, with the vast majority (>99%) willing to be involved in such a study. Incentives to take part cited by pregnant women included extra antenatal scans and the opportunity to be tested for Group B streptococcus. CONCLUSION: Pregnant women and healthcare professionals were open to the idea of an antenatal Group B streptococcus vaccine and involvement in clinical trials of such a vaccine. Education and support from midwives would be key to successful implementation.

Brain-Derived Neurotrophic Factor Facilitates Functional Recovery from ALS-Cerebral Spinal Fluid-Induced Neurodegenerative Changes in the NSC-34 Motor Neuron Cell Line.

BACKGROUND: The survival of motor neurons is dependent upon neurotrophic factors both during childhood and adolescence and during adult life. In disease conditions, such as in patients with amyotrophic lateral sclerosis (ALS), the mRNA levels of trophic factors like brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1), fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor are downregulated. This was replicated in our in vivo experimental system following the injection of cerebral spinal fluid (CSF) of sporadic ALS (ALS-CSF) patients. OBJECTIVE: To evaluate the protective role of BDNF in a model of sporadic ALS patients. METHODS: The expressions of endogenous BDNF, its receptor TrkB, the enzyme choline acetyl transferase (ChAT), and phosphorylated neurofilaments were studied in NSC-34 cells. The calcium-buffering and proapoptotic effects were assessed by calbindin-D28K and caspase-3 expression, respectively. RESULTS: ALS-CSF considerably depleted the endogenous BDNF protein, while its effect on IGF-1 and FGF-2 was inconsequential; this indirectly indicates a key role for BDNF in supporting motor neuronal survival. The exogenous supplementation of BDNF reversed autocrine expression; however, it may not be completely receptor mediated, as the TrkB levels were not restored. BDNF completely revived ChAT expression. It may inhibit apoptosis by restoring Ca2+ homeostasis, since caspase-3 and calbindin-D28K expression was back to normal. The organellar ultrastructural changes were only partially reversed. CONCLUSION: Our study provides evidence that BDNF supplementation ameliorates most but not all degenerative changes. The incomplete revival at the ultrastructural level signifies the requirement of factors other than BDNF for near-total protection of motor neurons, and, to an extent, it explains why only a partial success is achieved in clinical trials with BDNF in ALS patients.

Lymphocyte subpopulations in premature infants: an observational study.

BACKGROUND AND OBJECTIVES: The infant's immune system evolves over the first months and years of life. Strong correlation exists between lymphocyte count, lymphocyte subpopulations and gestational age at birth. Associations with antenatal and postnatal steroid treatment, infection and chronic lung disease have also been described. Few published studies report the effect of increasing postnatal age (PNA) and comorbidities on lymphocyte subpopulations in premature infants beyond the first 4 months of life. This study aimed to describe changes in lymphocyte subpopulations in preterm infants up to 13 months PNA. METHODS: Premature infants (23-34 weeks completed gestation) from five centres had lymphocyte subpopulations measured at 2, 5 or 7, 12 and 13 months PNA alongside their vaccine responses in a vaccination trial. RESULTS: 393 blood samples from 151 babies were analysed. There was an increase in absolute numbers of total lymphocytes (median cell count 6.21×109/L at 13 months compared with 4.9×109/L at 2 months PNA) and CD3+, CD4+, CD8+, natural killer and B cells with increasing age. At 2 months PNA, there was a positive correlation between gestation and CD3+ and CD4+ counts (r=0.32 and 0.46, respectively) and proportions (r=0.22 and 0.41, respectively), and CD4+:CD8+ ratios (r=0.57), but a negative correlation with CD8+ proportions (r=-0.32). CONCLUSIONS: This longitudinal study describes the distribution of lymphocyte subpopulations in premature infants and provides reference ranges for the major lymphocyte subsets to help guide clinicians when assessing premature infants for immunodeficiency in the first year of life. TRIAL REGISTRATION NUMBER: EudraCT 2007-007535-23.

Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients Induces Mitochondrial and Lysosomal Dysfunction.

In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS.

Micro-economic impact of congenital heart surgery: results of a prospective study from a limited-resource setting.

INTRODUCTION: The microeconomic impact of surgery for congenital heart disease is unexplored, particularly in resource limited environments. We sought to understand the direct and indirect costs related to congenital heart surgery and its impact on Indian households from a family perspective. METHODS: Baseline and first follow-up data of 644 consecutive children admitted for surgery for congenital heart disease (March 2013 - July 2014) in a tertiary referral hospital in Central Kerala, South India was collected prospectivelyfrom parents through questionnaires using a semi-structured interview schedule. RESULTS: The median age was 8.2 months (IQR: 3.0- 36.0 months). Most families belonged to upper middle (43.0%) and lower middle (35.7%) socioeconomic class. Only 3.9% of families had some form of health insurance. The median expense for the admission and surgery was INR 201898 (IQR: 163287-266139) [I$ 11989 (IQR: 9696-15804)], which was 0.93 (IQR: 0.52-1.49) times the annual family income of affected patients. Median loss of man-days was 35 (IQR: 24-50) and job-days was 15 (IQR: 11-24). Surgical risk category and hospital stay duration significantly predicted higher costs. One in two families reported overwhelming to high financial stress during admission period for surgery. Approximately half of the families borrowed money during the follow up period after surgery. CONCLUSION: Surgery for congenital heart disease results in significant financial burden for majority of families studied. Efforts should be directed at further reductions in treatment costs without compromising the quality of care together with generating financial support for affected families.

Role of VEGF and VEGFR2 Receptor in Reversal of ALS-CSF Induced Degeneration of NSC-34 Motor Neuron Cell Line.

Vascular endothelial growth factor (VEGF), the well-known angiogenic factor is both neurotrophic and neuroprotective. Altered VEGF signalling is implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal degenerative disease of motor neurons. We have shown earlier that VEGF protects NSC-34 motor neuronal cell line, when exposed to cerebrospinal fluid (CSF) from sporadic ALS patients (ALS-CSF). Here, we have investigated the consequences of ALS-CSF and VEGF supplementation on the VEGFR2 receptor and endogenous VEGF expression. ALS-CSF caused significant down-regulation of VEGFR2 as well as the Calbindin-D28K levels, but not endogenous VEGF. Exogenous supplementation restored the depletion of VEGFR2 and Calbindin-D28K with a concomitant up-regulation of endogenous VEGF. The up-regulated caspase 3 in the ALS-CSF group was reinstated to basal levels along with a significant reduction in the number of TUNEL-positive cells. Electron photomicrographs of ALS-CSF-exposed cells divulged presence of cytoplasmic vacuoles alongside severe damage to organelles like mitochondria, endoplasmic reticulum, etc. Substantial recovery of most of the damaged organelles was noted in response to VEGF supplementation. While the enhancement in endogenous VEGF levels highlights the autocrine functions, the up-regulation of VEGFR2 receptor emphasizes the paracrine functions of VEGF in modulating its neuroprotective effect against ALS-CSF. The revival of cellular organellar structure, increased calbindin expression and enhanced survival in response to VEGF supplementation consolidates the opinion that VEGF indeed has a therapeutic potential in sporadic ALS.

Complications of Reduced Intensity Conditioning HSCT for XIAP Deficiency (Alloimmune Cytopenias and HLH) Successfully Managed With Donor Lymphocyte Infusion.

X-linked inhibitor of apoptosis protein deficiency is a rare illness and although stem cell transplant is curative, full intensity conditioning is associated with high mortality rates. We describe a child with unusual complications associated with residual host lymphocytes following reduced intensity stem cell transplant. Recipient derived, donor directed, antigranulocyte antibodies led to life-threatening and prolonged neutropenia and residual recipient lymphocytes reestablished hemophagocytic lymphohistiocytosis after withdrawal of immune suppression despite high levels of whole blood chimerism. Hemophagocytic lymphohistiocytosis was abolished following specific improvement in donor T-cell chimerism after donor lymphocyte infusions, and alloimmune cytopenias were no longer evident.

Chitotriosidase - a putative biomarker for sporadic amyotrophic lateral sclerosis.

BACKGROUND: Potential biomarkers to aid diagnosis and therapy need to be identified for Amyotrophic Lateral Sclerosis, a progressive motor neuronal degenerative disorder. The present study was designed to identify the factor(s) which are differentially expressed in the cerebrospinal fluid (CSF) of patients with sporadic amyotrophic lateral sclerosis (SALS; ALS-CSF), and could be associated with the pathogenesis of this disease. RESULTS: Quantitative mass spectrometry of ALS-CSF and control-CSF (from orthopaedic surgical patients undergoing spinal anaesthesia) samples showed upregulation of 31 proteins in the ALS-CSF, amongst which a ten-fold increase in the levels of chitotriosidase-1 (CHIT-1) was seen compared to the controls. A seventeen-fold increase in the CHIT-1 levels was detected by ELISA, while a ten-fold elevated enzyme activity was also observed. Both these results confirmed the finding of LC-MS/MS. CHIT-1 was found to be expressed by the Iba-1 immunopositive microglia. CONCLUSION: Elevated CHIT-1 levels in the ALS-CSF suggest a definitive role for the enzyme in the disease pathogenesis. Its synthesis and release from microglia into the CSF may be an aligned event of neurodegeneration. Thus, high levels of CHIT-1 signify enhanced microglial activity which may exacerbate the process of neurodegeneration. In view of the multifold increase observed in ALS-CSF, it can serve as a potential CSF biomarker for the diagnosis of SALS.