Adityasai Ambati

All Publications

• Autoimmunity to hypocretin and molecular mimicry to flu in type 1 narcolepsy. Proceedings of the National Academy of Sciences of the United States of America Luo, G., Ambati, A., Lin, L., Bonvalet, M., Partinen, M., Ji, X., Maecker, H. T., Mignot, E. J. 2018

  Abstract

  Type 1 narcolepsy (T1N) is caused by hypocretin/orexin (HCRT) neuronal loss. Association with the HLA DQB1*06:02/DQA1*01:02 (98% vs. 25%) heterodimer (DQ0602), T cell receptors (TCR) and other immune loci suggest autoimmunity but autoantigens are unknown. Onset is seasonal and associated with influenza A, notably pandemic 2009 H1N1 (pH1N1) infection and vaccination (Pandemrix). Peptides derived from HCRT and influenza A, including pH1N1, were screened for DQ0602 binding and presence of cognate DQ0602 tetramer-peptide-specific CD4+ T cells tested in 35 T1N cases and 22 DQ0602 controls. Higher reactivity to influenza pHA273-287 (pH1N1 specific), PR8 (H1N1 pre-2009 and H2N2)-specific NP17-31 and C-amidated but not native version of HCRT54-66 and HCRT86-97 (HCRTNH2) were observed in T1N. Single-cell TCR sequencing revealed sharing of CDR3beta TRBV4-2-CASSQETQGRNYGYTF in HCRTNH2 and pHA273-287-tetramers, suggesting molecular mimicry. This public CDR3beta uses TRBV4-2, a segment modulated by T1N-associated SNP rs1008599, suggesting causality. TCR-alpha/beta CDR3 motifs of HCRT54-66-NH2 and HCRT86-97-NH2 tetramers were extensively shared: notably public CDR3alpha, TRAV2-CAVETDSWGKLQF-TRAJ24, that uses TRAJ24, a chain modulated by T1N-associated SNPs rs1154155 and rs1483979. TCR-alpha/beta CDR3 sequences found in pHA273-287, NP17-31, and HCRTNH2 tetramer-positive CD4+ cells were also retrieved in single INF-gamma-secreting CD4+ sorted cells stimulated with Pandemrix, independently confirming these results. Our results provide evidence for autoimmunity and molecular mimicry with flu antigens modulated by genetic components in the pathophysiology of T1N.

  View details for PubMedID 30541895

• Neural network analysis of sleep stages enables efficient diagnosis of narcolepsy. Nature communications Stephansen, J. B., Olesen, A. N., Olsen, M., Ambati, A., Leary, E. B., Moore, H. E., Carrillo, O., Lin, L., Han, F., Yan, H., Sun, Y. L., Dauvilliers, Y., Scholz, S., Barateau, L., Hogl, B., Stefani, A., Hong, S. C., Kim, T. W., Pizza, F., Plazzi, G., Vandi, S., Antelmi, E., Perrin, D., Kuna, S. T., Schweitzer, P. K., Kushida, C., Peppard, P. E., Sorensen, H. B., Jennum, P., Mignot, E. 2018; 9 (1): 5229

  Abstract

  Analysis of sleep for the diagnosis of sleep disorders such as Type-1 Narcolepsy (T1N) currently requires visual inspection of polysomnography records by trained scoring technicians. Here, we used neural networks in approximately 3,000 normal and abnormal sleep recordings to automate sleep stage scoring, producing a hypnodensity graph-a probability distribution conveying more information than classical hypnograms. Accuracy of sleep stage scoring was validated in 70 subjects assessed by six scorers. The best model performed better than any individual scorer (87% versus consensus). It also reliably scores sleep down to 5 s instead of 30 s scoring epochs. A T1N marker based on unusual sleep stage overlaps achieved a specificity of 96% and a sensitivity of 91%, validated in independent datasets. Addition of HLA-DQB1*06:02 typing increased specificity to 99%. Our method can reduce time spent in sleep clinics and automates T1N diagnosis. It also opens the possibility of diagnosing T1N using home sleep studies.

  View details for PubMedID 30523329

• Autoimmunity in narcolepsy. Current opinion in pulmonary medicine Bonvalet, M., Ollila, H. M., Ambati, A., Mignot, E. 2017; 23 (6): 522–29

  Abstract

  Summarize the recent findings in narcolepsy focusing on the environmental and genetic risk factors in disease development.Both genetic and epidemiological evidence point towards an autoimmune mechanism in the destruction of orexin/hypocretin neurons. Recent studies suggest both humoral and cellular immune responses in the disease development.Narcolepsy is a severe sleep disorder, in which neurons producing orexin/hypocretin in the hypothalamus are destroyed. The core symptoms of narcolepsy are debilitating, extreme sleepiness, cataplexy, and abnormalities in the structure of sleep. Both genetic and epidemiological evidence point towards an autoimmune mechanism in the destruction of orexin/hypocretin neurons. Importantly, the highest environmental risk is seen with influenza-A infection and immunization. However, how the cells are destroyed is currently unknown. In this review we summarize the disease symptoms, and focus on the immunological findings in narcolepsy. We also discuss the environmental and genetic risk factors as well as propose a model for disease development.

  View details for PubMedID 28991006

  View details for PubMedCentralID PMC5773260

• Increased β-haemolytic group A streptococcal M6 serotype and streptodornase B-specific cellular immune responses in Swedish narcolepsy cases. Journal of internal medicine Ambati, A., Poiret, T., Svahn, B. M., Valentini, D., Khademi, M., Kockum, I., Lima, I., Arnheim-Dahlström, L., Lamb, F., Fink, K., Meng, Q., Kumar, A., Rane, L., Olsson, T., Maeurer, M. 2015

  Abstract

  Type 1 narcolepsy is a neurological disorder characterized by excessive daytime sleepiness and cataplexy associated with the HLA allele DQB1*06:02. Genetic predisposition along with external triggering factors may drive autoimmune responses, ultimately leading to the selective loss of hypocretin-positive neurons.The aim of this study was to investigate potential aetiological factors in Swedish cases of postvaccination (Pandemrix) narcolepsy defined by interferon-gamma (IFNγ) production from immune cells in response to molecularly defined targets.Cellular reactivity defined by IFNγ production was examined in blood from 38 (HLA-DQB1*06:02(+) ) Pandemrix-vaccinated narcolepsy cases and 76 (23 HLA-DQB1*06:02(+) and 53 HLA-DQB1*06:02(-) ) control subjects, matched for age, sex and exposure, using a variety of different antigens: β-haemolytic group A streptococcal (GAS) antigens (M5, M6 and streptodornase B), influenza (the pandemic A/H1N1/California/7/09 NYMC X-179A and A/H1N1/California/7/09 NYMC X-181 vaccine antigens, previous Flu-A and -B vaccine targets, A/H1N1/Brisbane/59/2007, A/H1N1/Solomon Islands/3/2006, A/H3N2/Uruguay/716/2007, A/H3N2/Wisconsin/67/2005, A/H5N1/Vietnam/1203/2004 and B/Malaysia/2506/2004), noninfluenza viral targets (CMVpp65, EBNA-1 and EBNA-3) and auto-antigens (hypocretin peptide, Tribbles homolog 2 peptide cocktail and extract from rat hypothalamus tissue).IFN-γ production was significantly increased in whole blood from narcolepsy cases in response to streptococcus serotype M6 (P = 0.0065) and streptodornase B protein (P = 0.0050). T-cell recognition of M6 and streptodornase B was confirmed at the single-cell level by intracellular cytokine (IL-2, IFNγ, tumour necrosis factor-alpha and IL-17) production after stimulation with synthetic M6 or streptodornase B peptides. Significantly, higher (P = 0.02) titres of serum antistreptolysin O were observed in narcolepsy cases, compared to vaccinated controls.β-haemolytic GAS may be involved in triggering autoimmune responses in patients who developed narcolepsy symptoms after vaccination with Pandemrix in Sweden, characterized by a Streptococcus pyogenes M-type-specific IFN-γ cellular immune response.

  View details for DOI 10.1111/joim.12355

  View details for PubMedID 25683265

• The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight SCIENCE Garrett-Bakelman, F. E., Darshi, M., Green, S. J., Gur, R. C., Lin, L., Macias, B. R., McKenna, M. J., Meydan, C., Mishra, T., Nasrini, J., Piening, B. D., Rizzardi, L. F., Sharma, K., Siamwala, J. H., Taylor, L., Vitaterna, M., Afkarian, M., Afshinnekoo, E., Ahadi, S., Ambati, A., Arya, M., Bezdan, D., Callahan, C. M., Chen, S., Choi, A. K., Chlipala, G. E., Contrepois, K., Covington, M., Crucian, B. E., De Vivo, I., Dinges, D. F., Ebert, D. J., Feinberg, J. I., Gandara, J. A., George, K. A., Goutsias, J., Grills, G. S., Hargens, A. R., Heer, M., Hillary, R. P., Hoofnagle, A. N., Hook, V. H., Jenkinson, G., Jiang, P., Keshavarzian, A., Laurie, S. S., Lee-McMullen, B., Lumpkins, S. B., MacKay, M., Maienschein-Cline, M. G., Melnick, A. M., Moore, T. M., Nakahira, K., Patel, H. H., Pietrzyk, R., Rao, V., Saito, R., Salins, D. N., Schilling, J. M., Sears, D. D., Sheridan, C. K., Stenger, M. B., Tryggvadottir, R., Urban, A. E., Vaisar, T., Van Espen, B., Zhang, J., Ziegler, M. G., Zwart, S. R., Charles, J. B., Kundrot, C. E., Scott, G. I., Bailey, S. M., Basner, M., Feinberg, A. P., Lee, S. C., Mason, C. E., Mignot, E., Rana, B. K., Smith, S. M., Snyder, M. P., Turek, F. W. 2019; 364 (6436): 144-+

  View details for DOI 10.1126/science.aau8650

  View details for Web of Science ID 000464620000031

• PROTEOMIC BIOMARKERS OF CIRCADIAN TIME Ambati, A., Lin, L., Zitting, K., Duffy, J. F., Zeitzer, J., Spiegel, D., Czeisler, C. A., Mignot, E. OXFORD UNIV PRESS INC. 2019

  View details for Web of Science ID 000471071000044

• AUTOIMMUNITY TO HYPOCRETIN AND MOLECULAR MIMICRY TO FLU IN TYPE 1 NARCOLEPSY Luo, G., Ambati, A., Lin, L., Bonvalet, M., Partinen, M., Ji, X., Maecker, H., Mignot, E. OXFORD UNIV PRESS INC. 2019

  View details for Web of Science ID 000471071000018

• The NASA Twins Study: A multidimensional analysis of a year-long human spaceflight. Science (New York, N.Y.) Garrett-Bakelman, F. E., Darshi, M., Green, S. J., Gur, R. C., Lin, L., Macias, B. R., McKenna, M. J., Meydan, C., Mishra, T., Nasrini, J., Piening, B. D., Rizzardi, L. F., Sharma, K., Siamwala, J. H., Taylor, L., Vitaterna, M. H., Afkarian, M., Afshinnekoo, E., Ahadi, S., Ambati, A., Arya, M., Bezdan, D., Callahan, C. M., Chen, S., Choi, A. M., Chlipala, G. E., Contrepois, K., Covington, M., Crucian, B. E., De Vivo, I., Dinges, D. F., Ebert, D. J., Feinberg, J. I., Gandara, J. A., George, K. A., Goutsias, J., Grills, G. S., Hargens, A. R., Heer, M., Hillary, R. P., Hoofnagle, A. N., Hook, V. Y., Jenkinson, G., Jiang, P., Keshavarzian, A., Laurie, S. S., Lee-McMullen, B., Lumpkins, S. B., MacKay, M., Maienschein-Cline, M. G., Melnick, A. M., Moore, T. M., Nakahira, K., Patel, H. H., Pietrzyk, R., Rao, V., Saito, R., Salins, D. N., Schilling, J. M., Sears, D. D., Sheridan, C. K., Stenger, M. B., Tryggvadottir, R., Urban, A. E., Vaisar, T., Van Espen, B., Zhang, J., Ziegler, M. G., Zwart, S. R., Charles, J. B., Kundrot, C. E., Scott, G. B., Bailey, S. M., Basner, M., Feinberg, A. P., Lee, S. M., Mason, C. E., Mignot, E., Rana, B. K., Smith, S. M., Snyder, M. P., Turek, F. W. 2019; 364 (6436)

  Abstract

  To understand the health impact of long-duration spaceflight, one identical twin astronaut was monitored before, during, and after a 1-year mission onboard the International Space Station; his twin served as a genetically matched ground control. Longitudinal assessments identified spaceflight-specific changes, including decreased body mass, telomere elongation, genome instability, carotid artery distension and increased intima-media thickness, altered ocular structure, transcriptional and metabolic changes, DNA methylation changes in immune and oxidative stress-related pathways, gastrointestinal microbiota alterations, and some cognitive decline postflight. Although average telomere length, global gene expression, and microbiome changes returned to near preflight levels within 6 months after return to Earth, increased numbers of short telomeres were observed and expression of some genes was still disrupted. These multiomic, molecular, physiological, and behavioral datasets provide a valuable roadmap of the putative health risks for future human spaceflight.

  View details for PubMedID 30975860

• Response to "H1N1 hemagglutinin-specific HLA-DQ6-restricted CD4+ T cells can be readily detected in narcolepsy type 1 patients and healthy controls". Journal of neuroimmunology Mignot, E., Ambati, A., Luo, G. 2019; 333: 476959

  View details for DOI 10.1016/j.jneuroim.2019.04.019

  View details for PubMedID 31108400

• Neural network analysis of sleep stages enables efficient diagnosis of narcolepsy NATURE COMMUNICATIONS Stephansen, J. B., Olesen, A. N., Olsen, M., Ambati, A., Leary, E. B., Moore, H. E., Carrillo, O., Lin, L., Han, F., Yan, H., Sun, Y. L., Dauvilliers, Y., Scholz, S., Barateau, L., Hogl, B., Stefani, A., Hong, S., Kim, T., Pizza, F., Plazzi, G., Vandi, S., Antelmi, E., Perrin, D., Kuna, S. T., Schweitzer, P. K., Kushida, C., Peppard, P. E., Sorensen, H. D., Jennum, P., Mignot, E. 2018; 9

  View details for DOI 10.1038/s41467-018-07229-3

  View details for Web of Science ID 000452283400001

• In vitro and in vivo evaluation of cephalosporins for the treatment of Lyme disease DRUG DESIGN DEVELOPMENT AND THERAPY Pothineni, V., Parekh, M. B., Babar, M., Ambati, A., Maguire, P., Inayathullah, M., Kim, K., Tayebi, L., Potula, H. K., Rajadas, J. 2018; 12: 2915–21

  View details for DOI 10.2147/DDDT.S164966

  View details for Web of Science ID 000444298700004

• In vitro and in vivo evaluation of cephalosporins for the treatment of Lyme disease. Drug design, development and therapy Pothineni, V. R., Parekh, M. B., Babar, M. M., Ambati, A., Maguire, P., Inayathullah, M., Kim, K. M., Tayebi, L., Potula, H. S., Rajadas, J. 2018; 12: 2915–21

  Abstract

  Lyme disease accounts for >90% of all vector-borne disease cases in the United States and affect ~300,000 persons annually in North America. Though traditional tetracycline antibiotic therapy is generally prescribed for Lyme disease, still 10%-20% of patients treated with current antibiotic therapy still show lingering symptoms.In order to identify new drugs, we have evaluated four cephalosporins as a therapeutic alternative to commonly used antibiotics for the treatment of Lyme disease by using microdilution techniques like minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). We have determined the MIC and MBC of four drugs for three Borrelia burgdorferi s.s strains namely CA8, JLB31 and NP40. The binding studies were performed using in silico analysis.The MIC order of the four drugs tested is cefoxitin (1.25 µM/mL) > cefamandole (2.5 µM/mL), > cefuroxime (5 µM/mL) > cefapirin (10 µM/mL). Among the drugs that are tested in this study using in vivo C3H/HeN mouse model, cefoxitin effectively kills B. burgdorferi. The in silico analysis revealed that all four cephalosporins studied binds effectively to B. burgdorferi proteins, SecA subunit penicillin-binding protein (PBP) and Outer surface protein E (OspE).Based on the data obtained, cefoxitin has shown high efficacy killing B. burgdorferi at concentration of 1.25 µM/mL. In addition to it, cefoxitin cleared B. burgdorferi infection in C3H/HeN mice model at 20 mg/kg.

  View details for PubMedID 30254421

• IL-7d5 protein is expressed in human tissues and induces expression of the oxidized low density lipoprotein receptor 1 (OLR1) in CD14+ monocytes. International journal of infectious diseases Rane, L., Rahman, S., Magalhaes, I., Ambati, A., Andersson, J., Zumla, A., Brighenti, S., Maeurer, M. J. 2017; 59: 29-36

  Abstract

  The 6 exon spanning ́canonical' Interleukin-7 (IL-7c) is a non-redundant cytokine in human T-cell homeostasis that undergoes extensive alternative pre-mRNA splicing. The IL-7 gene variant lacking, exon 5 (IL-7δ5), exhibits agonistic effects as compared to IL-7c. We studied in this report for the first time the protein expression of IL-7δ5 variant in tissues and its role in monocyte activation.We visualized the expression of IL-7δ5 protein by immunohistochemistry in both healthy and malignant (human) tissues and investigated the impact of IL-7δ5 stimulation on CD14+ monocytes using gene expression analysis and flow cytometry.IL-7≏5 is largely expressed by human epithelial cells, yet also by stromal cells in malignant lesions. Gene expression analysis in CD14+ monocytes, induced by the 6-exon spanning IL-7 or IL-7δ5 showed similar changes resulting in a pro-inflammatory phenotype and increased expression of genes involved in lipid metabolism. IL7≏5 was superior in inducing upregulation of the oxidised low density lipoprotein receptor (OLR), measured by flow cytometry, in CD14+ cells.IL-7≏5, produced from non-transformed and transformed cells, may contribute to chronic inflammatory responses and development of 'foamy' cells by increased OLR1 expression that mediates increased oxLDL uptake.

  View details for DOI 10.1016/j.ijid.2017.03.001

  View details for PubMedID 28279736

• Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer. Journal of immunotherapy (Hagerstown, Md. : 1997) Meng, Q., Liu, Z., Rangelova, E., Poiret, T., Ambati, A., Rane, L., Xie, S., Verbeke, C., Dodoo, E., Del Chiaro, M., Löhr, M., Segersvärd, R., Maeurer, M. J. ; 39 (2): 81–89

  Abstract

  Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-γ production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR Vβ was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8 T cells. CD3CD8, CD3CD4, and CD3CD4CD8 [double-negative (DN) T cells] resided predominantly in central (CD45RACCR7) and effector (CD45RACCR7) memory subsets. CD8 TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4 TILs showed only up to 12% LAG-3 staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-α and IFN-γ production. Twelve of 17 of CD8 TILs showed preferential expansion of certain TCR Vβ families (eg, 99.2% Vβ13.2 in CD8 TILs, 77% in the Vβ1, 65.9% in the Vβ22, and 63.3% in the Vβ14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8 Vβ13.2) reacting strongly against autologous tumor defined by INF-γ production or by cytotoxicity. We have optimized methods for generating pancreatic cancer-specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.

  View details for DOI 10.1097/CJI.0000000000000111

  View details for PubMedID 26849077

• Antibody Affinity Against 2009 A/H1N1 Influenza and Pandemrix Vaccine Nucleoproteins Differs Between Childhood Narcolepsy Patients and Controls. Viral immunology Lind, A., Freyhult, E., Ramelius, A., Olsson, T., Arnheim-Dahlström, L., Lamb, F., Khademi, M., Ambati, A., Maeurer, M., Lima Bomfim, I., Fink, K., Fex, M., Törn, C., Elding Larsson, H., Lernmark, Å. 2017

  Abstract

  Increased narcolepsy incidence was observed in Sweden following the 2009 influenza vaccination with Pandemrix(®). A substitution of the 2009 nucleoprotein for the 1934 variant has been implicated in narcolepsy development. The aims were to determine (a) antibody levels toward wild-type A/H1N1-2009[A/California/04/2009(H1N1)] (NP-CA2009) and Pandemrix-[A/Puerto Rico/8/1934(H1N1)] (NP-PR1934) nucleoproteins in 43 patients and 64 age-matched controls; (b) antibody affinity in reciprocal competitive assays in 11 childhood narcolepsy patients compared with 21 age-matched controls; and (c) antibody levels toward wild-type A/H1N1-2009[A/California/04/2009(H1N1)] (H1N1 NS1), not a component of the Pandemrix vaccine. In vitro transcribed and translated (35)S-methionine-labeled H1N1 influenza A virus proteins were used in radiobinding reciprocal competition assays to estimate antibody levels and affinity (Kd). Childhood patients had higher NP-CA2009 (p = 0.0339) and NP-PR1934 (p = 0.0246) antibody levels compared with age-matched controls. These childhood controls had lower NP-CA2009 (p = 0.0221) and NP-PR1934 (p = 0.00619) antibodies compared with controls 13 years or older. In contrast, in patients 13 years or older, the levels of NP-PR1934 (p = 0.279) and NP-CA2009 (p = 0.0644) antibodies did not differ from the older controls. Childhood antibody affinity (Kd) against NP-CA2009 was comparable between controls (68 ng/mL) and patients (74 ng/mL; p = 0.21) with NP-CA2009 and NP-PR1934 displacement (controls: 165 ng/mL; patients: 199 ng/mL; p = 0.48). In contrast, antibody affinity against NP-PR1934 was higher in controls with either NP-PR1934 (controls: 9 ng/mL; patients: 20 ng/mL; p = 0.0031) or NP-CA2009 (controls: 14 ng/mL; patients: 23 ng/mL; p = 0.0048). A/H1N1-NS1 antibodies were detected in 0/43 of the narcolepsy patients compared with 3/64 (4.7%) controls (p = 0.272). Similarly, none (0/11) of the childhood patients and 1/21 (4.8%) of the childhood controls had A/H1N1-NS1 antibodies. The higher antibody affinities against NP-PR1934 in controls suggest better protection against wild-type virus. In contrast, the reduced NP-PR1934 antibody affinities among childhood narcolepsy patients suggest poor protection from the wild-type A/H1N1 virus and possibly increased risk for viral damage.

  View details for DOI 10.1089/vim.2017.0066

  View details for PubMedID 28796576

• Absence of anti-hypocretin receptor 2 autoantibodies in post pandemrix narcolepsy cases. PloS one Luo, G., Lin, L., Jacob, L., Bonvalet, M., Ambati, A., Plazzi, G., Pizza, F., Leib, R., Adams, C. M., Partinen, M., Mignot, E. J. 2017; 12 (12): e0187305

  Abstract

  A recent publication suggested molecular mimicry of a nucleoprotein (NP) sequence from A/Puerto Rico/8/1934 (PR8) strain, the backbone used in the construction of the reassortant strain X-179A that was used in Pandemrix® vaccine, and reported on anti-hypocretin (HCRT) receptor 2 (anti-HCRTR2) autoantibodies in narcolepsy, mostly in post Pandemrix® narcolepsy cases (17 of 20 sera). In this study, we re-examined this hypothesis through mass spectrometry (MS) characterization of Pandemrix®, and two other pandemic H1N1 (pH1N1)-2009 vaccines, Arepanrix® and Focetria®, and analyzed anti-HCRTR2 autoantibodies in narcolepsy patients and controls using three independent strategies.MS characterization of Pandemrix® (2 batches), Arepanrix® (4 batches) and Focetria® (1 batch) was conducted with mapping of NP 116I or 116M spectrogram. Two sets of narcolepsy cases and controls were used: 40 post Pandemrix® narcolepsy (PP-N) cases and 18 age-matched post Pandemrix® controls (PP-C), and 48 recent (≤6 months) early onset narcolepsy (EO-N) cases and 70 age-matched other controls (O-C). Anti-HCRTR2 autoantibodies were detected using three strategies: (1) Human embryonic kidney (HEK) 293T cells with transient expression of HCRTR2 were stained with human sera and then analyzed by flow cytometer; (2) In vitro translation of [35S]-radiolabelled HCRTR2 was incubated with human sera and immune complexes of autoantibody and [35S]-radiolabelled HCRTR2 were quantified using a radioligand-binding assay; (3) Optical density (OD) at 450 nm (OD450) of human serum immunoglobulin G (IgG) binding to HCRTR2 stably expressed in Chinese hamster ovary (CHO)-K1 cell line was measured using an in-cell enzyme-linked immunosorbent assay (ELISA).NP 116M mutations were predominantly present in all batches of Pandemrix®, Arepanrix® and Focetria®. The wild-type NP109-123 (ILYDKEEIRRIWRQA), a mimic to HCRTR234-45 (YDDEEFLRYLWR), was not found to bind to DQ0602. Three or four subjects were found positive for anti-HCRTR2 autoantibodies using two strategies or the third one, respectively. None of the post Pandemrix® narcolepsy cases (0 of 40 sera) was found positive with all three strategies.Anti-HCRTR2 autoantibody is not a significant biological feature of narcolepsy or of post Pandemrix® autoimmune responses.

  View details for PubMedID 29220370

  View details for PubMedCentralID PMC5722318

• Selenite promotes all-trans retinoic acid-induced maturation of acute promyelocytic leukemia cells. Oncotarget Misra, S., Selvam, A. K., Wallenberg, M., Ambati, A., Matolcsy, A., Magalhaes, I., Lauter, G., Björnstedt, M. 2016

  Abstract

  Selective targeting of the PML/RARα oncoprotein demonstrates a successful molecular targeted therapy in acute promyelocytic leukemia (APL) with a typical t(15:17) chromosomal translocation. The zinc-thiolate coordination is critical for structural stability of zinc finger proteins, including the PML moiety of PML/RARα. Based on the known interaction of redox-active selenium compounds with thiolate ligands of zinc, we herein have investigated the abrogatory effects of selenite alone or in combination with all-trans retinoic acid on PML/RARα and the possible effects on differentiation in these cells. At pharmacological concentrations, selenite inhibited the proliferation and survival of APL originated NB4 cells. In combination with ATRA, it potentiated the differentiation of NB4 cells without any differentiating effects of its own as a single agent. Concordant with our hypothesis, PML/RARα oncoprotein expression was completely abrogated by selenite. Increased expression of RAR, PU.1 and FOXO3A transcription factors in the combined treatment suggested the plausible basis for increased differentiation in these cells. We show that selenite at clinically achievable dose targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemic cells in combination with ATRA. The present investigation reveals the hitherto unknown potential of selenite in targeted abrogation of PML/RARα in APL cells with prospective therapeutic value.

  View details for DOI 10.18632/oncotarget.12531

  View details for PubMedID 27732960

• T-Cell Therapy: Options for Infectious Diseases. Clinical infectious diseases Parida, S. K., Poiret, T., Zhenjiang, L., Meng, Q., Heyckendorf, J., Lange, C., Ambati, A. S., Rao, M. V., Valentini, D., Ferrara, G., Rangelova, E., Dodoo, E., Zumla, A., Maeurer, M. 2015; 61: S217-24

  Abstract

  The emergence of drug-resistant tuberculosis is challenging tuberculosis control worldwide. In the absence of an effective vaccine to prevent primary infection with Mycobacterium tuberculosis and tuberculosis disease, host-directed therapies may offer therapeutic options, particularly for patients with multidrug-resistant and extensively drug-resistant tuberculosis where prognosis is often limited. CD8(+) and CD4(+) T cells mediate antigen-specific adaptive cellular immune responses. Their use in precision immunotherapy in clinical conditions, especially in treating cancer as well as for prevention of life-threatening viral infections in allogeneic transplant recipients, demonstrated safety and clinical efficacy. We review key achievements in T-cell therapy, including the use of recombinant immune recognition molecules (eg, T-cell receptors and CD19 chimeric antigen receptors), and discuss its potential in the clinical management of patients with drug-resistant and refractory tuberculosis failing conventional therapy.

  View details for DOI 10.1093/cid/civ615

  View details for PubMedID 26409284

• Evaluation of pretransplant influenza vaccination in hematopoietic SCT: a randomized prospective study. Bone marrow transplantation AMBATI, A., Boas, L. S., Ljungman, P., Testa, L., de Oliveira, J. F., AOUN, M., Colturato, V., Maeurer, M., Machado, C. M. 2015; 50 (6): 858-864

  Abstract

  Pretransplant influenza vaccination of the donor or allogeneic hematopoietic SCT (HSCT) candidate was evaluated in a randomized study. One hundred and twenty-two HSCT recipients and their donors were assigned to three randomization groups: no pretransplant vaccination (n=38), donor pretransplant vaccination (n=44) or recipient pretransplant vaccination (n=40). Specific IgG was assessed by both hemagglutinin inhibition (HI) and, in 57 patients, by an indirect influenza-specific ELISA at specified times after HSCT. Vaccinated donors had seroprotective HI titers for Ags H1 and H3 (P<0.001) compared with the other groups at the time of donation. The titers against H1 (P=0.028) and H3 (P<0.001) were highest in the pretransplant recipient vaccination group until day 180 after transplantation. A significant difference was found in the specific Ig levels against pandemic H1N1 at 6 months after SCT (P=0.02). The mean IgG levels against pandemic H1N1 and generic H1N1 and H3N2 were highest in the pretransplant recipient vaccination group. We conclude that pretransplant recipient vaccination improved the influenza-specific seroprotection rates.

  View details for DOI 10.1038/bmt.2015.47

  View details for PubMedID 25798680

• H1N1 viral proteome peptide microarray predicts individuals at risk for H1N1 infection and segregates infection versus Pandemrix(®) vaccination. Immunology Ambati, A., Valentini, D., Montomoli, E., Lapini, G., Biuso, F., Wenschuh, H., Magalhaes, I., Maeurer, M. 2015; 145 (3): 357–66

  Abstract

  A high content peptide microarray containing the entire influenza A virus [A/California/08/2009(H1N1)] proteome and haemagglutinin proteins from 12 other influenza A subtypes, including the haemagglutinin from the [A/South Carolina/1/1918(H1N1)] strain, was used to gauge serum IgG epitope signatures before and after Pandemrix(®) vaccination or H1N1 infection in a Swedish cohort during the pandemic influenza season 2009. A very narrow pattern of pandemic flu-specific IgG epitope recognition was observed in the serum from individuals who later contracted H1N1 infection. Moreover, the pandemic influenza infection generated IgG reactivity to two adjacent epitopes of the neuraminidase protein. The differential serum IgG recognition was focused on haemagglutinin 1 (H1) and restricted to classical antigenic sites (Cb) in both the vaccinated controls and individuals with flu infections. We further identified a novel epitope VEPGDKITFEATGNL on the Ca antigenic site (251-265) of the pandemic flu haemagglutinin, which was exclusively recognized in serum from individuals with previous vaccinations and never in serum from individuals with H1N1 infection (confirmed by RNA PCR analysis from nasal swabs). This epitope was mapped to the receptor-binding domain of the influenza haemagglutinin and could serve as a correlate of immune protection in the context of pandemic flu. The study shows that unbiased epitope mapping using peptide microarray technology leads to the identification of biologically and clinically relevant target structures. Most significantly an H1N1 infection induced a different footprint of IgG epitope recognition patterns compared with the pandemic H1N1 vaccine.

  View details for DOI 10.1111/imm.12448

  View details for PubMedID 25639813

• Immunogenicity of virosomal adjuvanted trivalent influenza vaccination in allogeneic stem cell transplant recipients. Transplant infectious disease : an official journal of the Transplantation Society Ambati, A., Einarsdottir, S., Magalhaes, I., Poiret, T., Bodenstein, R., LeBlanc, K., Brune, M., Maeurer, M., Ljungman, P. 2015; 17 (3): 371–79

  Abstract

  Influenza vaccination is generally recommended to hematopoietic stem cell transplant (HSCT) recipients. However, the seasonal subunit vaccination response is frequently suboptimal, and alternate more efficient vaccination systems must be examined. We compared the immunogenicity of an adjuvanted virosomal influenza and subunit vaccine in HSCT recipients.The immunogenicity after a single dose (0.5 mL) of adjuvanted trivalent virosomal vaccination was evaluated in a study cohort of 21 HSCT recipients and compared to a control cohort of 30 HSCT recipients who received a single dose (0.5 mL) of non-adjuvanted seasonal trivalent subunit vaccination over 4 seasons from 2010 to 2014. Whole blood interferon-gamma (IFN-γ) release assays were tested, both before and 30 days after vaccination, in response to influenza pandemic (pdm) H1N1, H3N2, and B antigens. HLA-A*02 dextramers, to gauge for the absolute number of antigen-specific CD8(+) T-cells, and pdm 2009 hemagglutinin inhibition (HI) assays, to test for neutralizing antibodies, were used as immunological readouts.The pdm HI titers were poor in both cohorts with only 23% (5/21) after virosomal vaccination and 13.3% (4/30) in the seasonal vaccine cohort having protective titers (≥40). The delta change of IFN-γ production in response to influenza pdm H1N1 (P = 0.005) and influenza B antigens (P = 0.01) were significantly elevated in blood from individuals who received the virosomal as compared to the seasonal vaccine. The IFN-γ response to pdm H1N1 was stronger (P < 0.001), as compared to seasonal vaccination, in patients vaccinated >6 month post HSCT. We detected a significant increase in the frequency of matrix 1 (GILGFVTL) dextramer-specific CD8(+) T-cells after the virosomal vaccine (P = 0.01). No differences were seen in the hemagglutinin-specific CD8(+) T-cells between the 2 cohorts.Vaccination using a virosomal delivery system is beneficial in eliciting robust cellular immune responses to pdm H1N1 influenza in SCT recipients.

  View details for DOI 10.1111/tid.12382

  View details for PubMedID 25817044

• TCR(+)CD4(-)CD8(-) T cells in Antigen-specific MHC Class I-restricted T-cell Responses After Allogeneic Hematopoietic Stem Cell Transplantation JOURNAL OF IMMUNOTHERAPY Ahmed, R. K., Poiret, T., Ambati, A., Rane, L., Remberger, M., Omazic, B., Vudattu, N. K., Winiarski, J., Ernberg, I., Axelsson-Robertson, R., Magalhaes, I., Castelli, C., Ringden, O., Maeurer, M. 2014; 37 (8): 416-425

  Abstract

  Human TCRαβ(+) CD4(-)CD8(-) double-negative (DN) T cells represent a minor subset in peripheral blood, yet are important in infectious diseases and autoimmune responses. We examined the frequency of DN T cells in 17 patients after allogeneic hematopoietic stem cell transplantation (aHSCT) at 1, 2, 3, 6, and 12 months post-aHSCT and show that these cells increase early after aHSCT and decrease with time after aHSCT. DN T cells reside in the terminally differentiated effector (CD45RA(+)CCR7(-)) T-cell population and are polyclonal, determined by T-cell receptor Vβ CDR3 analysis. Gene expression analysis of ex vivo sorted DN T cells showed a distinct set of gene expression, including interleukin-8, as compared with CD4(+) or CD8(+) T cells. DN T cells contributed to MHC class I-restricted EBV-directed immune responses, defined by antigen-specific cytokine production and by detection of HLA-A*02:01-restricted EBV BMLF-1 (GLCTLVAML), LMP-2A (CLGGLLTMV), and HLA-A*24:02-restricted EBV BRLF-1 (DYCNVLNKEF) and EBNA3 (RYSIFFDY)-specific T cells. We created retroviral-transfected Jurkat cell lines with a Melan-A/MART-1-specific TCR(+) and the CD8α chain to study TCR(+) DN T cells in response to their nominal MHC class I/peptide ligand. We show that DN T cells exhibit increased TCRζ chain phosphorylation as compared with the TCR(+)CD8(+) transgenic T-cell line. DN T cells contribute to antigen-specific T-cell responses and represent an effector T-cell population that may be explored in immunotherapeutic approaches against viral infections or transformed cells.

  View details for Web of Science ID 000341965500004

  View details for PubMedID 25198529

• Difference in immune response in vaccinated and unvaccinated Swedish individuals after the 2009 influenza pandemic BMC INFECTIOUS DISEASES Magalhaes, I., Eriksson, M., Linde, C., Muhammad, R., Rane, L., Ambati, A., Axelsson-Robertson, R., Khalaj, B., Alvarez-Corrales, N., Lapini, G., Montomoli, E., Linde, A., Pedersen, N. L., Maeurer, M. 2014; 14

  Abstract

  Previous exposures to flu and subsequent immune responses may impact on 2009/2010 pandemic flu vaccine responses and clinical symptoms upon infection with the 2009 pandemic H1N1 influenza strain. Qualitative and quantitative differences in humoral and cellular immune responses associated with the flu vaccination in 2009/2010 (pandemic H1N1 vaccine) and natural infection have not yet been described in detail. We designed a longitudinal study to examine influenza- (flu-) specific immune responses and the association between pre-existing flu responses, symptoms of influenza-like illness (ILI), impact of pandemic flu infection, and pandemic flu vaccination in a cohort of 2,040 individuals in Sweden in 2009-2010.Cellular flu-specific immune responses were assessed by whole-blood antigen stimulation assay, and humoral responses by a single radial hemolysis test.Previous seasonal flu vaccination was associated with significantly lower flu-specific IFN-γ responses (using a whole-blood assay) at study entry. Pandemic flu vaccination induced long-lived T-cell responses (measured by IFN-γ production) to influenza A strains, influenza B strains, and the matrix (M1) antigen. In contrast, individuals with pandemic flu infection (PCR positive) exhibited increased flu-specific T-cell responses shortly after onset of ILI symptoms but the immune response decreased after the flu season (spring 2010). We identified non-pandemic-flu vaccinated participants without ILI symptoms who showed an IFN-γ production profile similar to pandemic-flu infected participants, suggesting exposure without experiencing clinical symptoms.Strong and long-lived flu-M1 specific immune responses, defined by IFN-γ production, in individuals after vaccination suggest that M1-responses may contribute to protective cellular immune responses. Silent flu infections appeared to be frequent in 2009/2010. The pandemic flu vaccine induced qualitatively and quantitatively different humoral and cellular immune responses as compared to infection with the 2009 H1N1 pandemic H1N1 influenza strain.

  View details for DOI 10.1186/1471-2334-14-319

  View details for Web of Science ID 000338728900001

  View details for PubMedID 24916787

• A/H1N1 antibodies and TRIB2 autoantibodies in narcolepsy patients diagnosed in conjunction with the Pandemrix vaccination campaign in Sweden 2009-2010 JOURNAL OF AUTOIMMUNITY Lind, A., Ramelius, A., Olsson, T., Arnheim-Dahlstrom, L., Lamb, F., Khademi, M., Ambati, A., Maeurer, M., Nilsson, A., Bomfim, I. L., Fink, K., Lernmark, A. 2014; 50: 99-106

  Abstract

  Narcolepsy is a lifelong sleep disorder related to hypocretin deficiency resulting from a specific loss of hypocretin-producing neurons in the lateral hypothalamic area. The disease is thought to be autoimmune due to a strong association with HLA-DQB1*06:02. In 2009 the World Health Organization (WHO) declared the H1N1 2009 flu pandemic (A/H1N1PDM09). In response to this, the Swedish vaccination campaign began in October of the same year, using the influenza vaccine Pandemrix(®). A few months later an excess of narcolepsy cases was observed. It is still unclear to what extent the vaccination campaign affected humoral autoimmunity associated with narcolepsy. We studied 47 patients with narcolepsy (6-69 years of age) and 80 healthy controls (3-61 years of age) selected after the Pandemrix vaccination campaign. The first aim was to determine antibodies against A/H1N1 and autoantibodies to Tribbles homolog 2 (TRIB2), a narcolepsy autoantigen candidate as well as to GAD65 and IA-2 as disease specificity controls. The second aim was to test if levels and frequencies of these antibodies and autoantibodies were associated with HLA-DQB1*06:02. In vitro transcribed and translated [(35)S]-methionine and -cysteine-labeled influenza A virus (A/California/04/2009/(H1N1)) segment 4 hemagglutinin was used to detect antibodies in a radiobinding assay. Autoantibodies to TRIB2, GAD65 and IA-2 were similarly detected in standard radiobinding assays. The narcolepsy patients had higher median levels of A/H1N1 antibodies than the controls (p = 0.006). A/H1N1 antibody levels were higher among the <13 years old (n = 12) compared to patients who were older than 30 years (n = 12, p = 0.014). Being HLA-DQB1*06:02 positive was associated with higher A/H1N1 antibody levels in both patients and controls (p = 0.026). Serum autoantibody levels to TRIB2 were low overall and high binders did not differ between patients and controls. We observed an association between levels of A/H1N1 antibodies and TRIB2 autoantibody levels particularly among the youngest narcolepsy patients (r = 0.819, p < 0.001). In conclusion, following the 2009 influenza pandemic vaccination, A/H1N1 antibody levels were associated with young age-at-onset narcolepsy patients positive for HLA-DQB1*06:02. The possibility that TRIB2 is an autoantigen in narcolepsy remains to be clarified. We could verify autoantibody responses against TRIB2 which needs to be determined in larger patient cohorts and control populations.

  View details for DOI 10.1016/j.jaut.2014.01.031

  View details for Web of Science ID 000336114600013

  View details for PubMedID 24485154