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1.
Int J Tuberc Lung Dis ; 24(2): 202-206, 2020 02 01.
Article in English | MEDLINE | ID: mdl-32127105

ABSTRACT

BACKGROUND: Chronic obstructive lung disease (COPD) is the third most common cause of death in the world. Factors other than smoking, such as socio-economic status, could be involved in the development of COPD.OBJECTIVE: To investigate the association between chronic airflow obstruction and socio-economic status in Morocco.DESIGN: Questionnaires were administered and spirometry tests performed as part of the BOLD (Burden of Obstructive Lung Disease) Study carried out in Fez, Morocco. Socio-economic status was evaluated using a wealth score (0-10) based on household assets. The ratio of forced expiratory volume in 1 s (FEV1) to forced vital capacity (FVC) was used to measure airflow obstruction.RESULTS: A total of 760 subjects were included in the analysis. The mean age was 55.3 years (standard deviation [SD] 10.2); the average wealth score was 7.54 (SD 1.63). After controlling for other factors and potential confounders, FEV1/FVC increased by 0.4% (95%CI 0.01-0.78; P < 0.04) per unit increase in wealth score. Ageing, tobacco smoking, underweight, history of tuberculosis and asthma were also independently associated with a higher risk of airflow obstruction.CONCLUSION: Our findings suggest that airflow obstruction is associated with poverty in Morocco. Further investigations are needed to better understand the mechanisms of this association.


Subject(s)
Pulmonary Disease, Chronic Obstructive , Forced Expiratory Volume , Humans , Middle Aged , Morocco/epidemiology , Prevalence , Pulmonary Disease, Chronic Obstructive/diagnosis , Pulmonary Disease, Chronic Obstructive/epidemiology , Risk Factors , Spirometry , Vital Capacity
3.
Immunol Invest ; 47(1): 18-39, 2018 Jan.
Article in English | MEDLINE | ID: mdl-28891721

ABSTRACT

We have recently mapped the in vitro proliferative responses of T cells from botulinum neurotoxin type A (BoNT/A)-treated cervical dystonia (CD) patients with overlapping peptides encompassing BoNT/A heavy chain (residues 449-1296). In the present study, we determined the recognition profiles, by peripheral blood lymphocytes (PBL) from the same set of patients, of BoNT/A light (L) chain (residues 1-453) by using 32 synthetic overlapping peptides that encompassed the entire L chain. Profiles of the T-cell responses (expressed in stimulation index, SI; Z score based on transformed SI) to the peptides varied among the patients. Samples from 14 patients treated solely with BoNT/A recognized 3-13 (average 7.2) peptides/sample at Z > 3.0 level. Two peptide regions representing residues 113-131 and 225-243 were recognized by around 40% of these patients. Regarding treatment parameters, treatment history with current BOTOX® only group produced significantly lower average T-cell responses to the 32 L-chain peptides compared to treatments with mix of type A including original and current BOTOX®. Influence of other treatment parameters on T-cell recognition of the L-chain peptides was also observed. Results of the submolecular T-cell recognition of the L chain are compared to those of the H chain and the T-cell recognition profile of the entire BoNT/A molecule is discussed. Abbreviations used: BoNT/A, botulinum neurotoxin type A; BoNT/Ai, inactivated BoNT/A; BoNT/B, botulinum neurotoxin type B; CD, cervical dystonia; L chain, the light chain (residues 1-448) of BoNT/A; LNC, lymph node cells; H chain, the heavy chain (residues 449-1296) of BoNT/A; HC, C-terminal domain (residues 855-1296) of H chain; HN, N-terminal domain (residues 449-859) of H chain; MPA, mouse protection assay; SI, stimulation index (SI = cpm of 3H-thymidine incorporated by antigen-stimulated T cells/cpm incorporated by unstimulated cells); TeNT, tetanus neurotoxin; TeNTi, inactivated TeNT.


Subject(s)
Botulinum Toxins, Type A/metabolism , Epitopes, T-Lymphocyte/metabolism , Immunodominant Epitopes/metabolism , Peptides/metabolism , T-Lymphocytes/immunology , Torticollis/immunology , Aged , Animals , Botulinum Toxins, Type A/therapeutic use , Cell Proliferation , Cells, Cultured , Epitopes, T-Lymphocyte/therapeutic use , Female , Humans , Immunodominant Epitopes/therapeutic use , Male , Mice , Middle Aged , Peptides/chemical synthesis , Peptides/therapeutic use , Torticollis/drug therapy , Torticollis/therapy
4.
Appl Microbiol Biotechnol ; 101(22): 8165-8179, 2017 Nov.
Article in English | MEDLINE | ID: mdl-29034433

ABSTRACT

Myasthenia gravis (MG) is an autoimmune disease caused by sensitization of the immune system to self-antigens. We have previously shown that targeting MG-susceptible alleles can significantly inhibit proliferation of disease-specific T cells. In this work, we humanized a murine monoclonal antibody (mAb) LG11, capable of blocking MG-associated DQ beta 1 (DQB1) allele and reformatted it into single-chain fragment variable (scFv). A fully functional humanized scFv was obtained by optimizing variable domain orientations and linker lengths, along with the optimization of expression conditions and codons to suit Escherichia coli expression machinery. Characterization of humanized scFv (FL8) revealed that the reformatted scFv, despite recognizing the same epitope as the parent murine LG11 mAb, exhibited superior binding affinity (0.97 nM) compared to the LG11 mAb, towards the immunizing antigen (DQB1*0601/70-90) and was able to block the proliferation of T cells cultured from PBLs of MG-patients typed DQB1*0601. The scFv was also capable of binding a variant MG-associated allele (DQB1*0502/70-90) with moderate affinity (18.7 nM), a feature that was absent in the LG11. To our knowledge, this is the first report of humanizing a MG-associated human leukocyte antigen (HLA) scFv for preclinical studies.


Subject(s)
Alleles , Antibodies, Monoclonal, Humanized/immunology , HLA Antigens/genetics , Myasthenia Gravis/immunology , Single-Chain Antibodies/immunology , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibodies, Monoclonal, Humanized/genetics , Antibodies, Monoclonal, Humanized/isolation & purification , Antibodies, Monoclonal, Humanized/therapeutic use , Escherichia coli/genetics , HLA Antigens/immunology , Humans , Mice , Myasthenia Gravis/drug therapy , Myasthenia Gravis/genetics , Single-Chain Antibodies/genetics , Single-Chain Antibodies/therapeutic use , Young Adult
5.
Immunol Lett ; 190: 257-264, 2017 10.
Article in English | MEDLINE | ID: mdl-28823519

ABSTRACT

Previously, we have examined the proliferative responses of T-cells from 25 patients and 8 controls to 32 light chain (L1-L32) and 60 heavy chain peptides (N1-N29, C1-C31) representing the entire clostridium botulinum neurotoxin type A (BoNT/A)[OM1-OM3]. In the current work, these T-cell responses were analyzed in the context of the patients HLA-DRB1, DQA1 and DQB1 variation. There were strong associations between the DQA1*01:02 and its derived haplotypes and cumulative T-cell proliferative responses. With or without HLA based differentiation the responses showed marked correlation. Inter-epitope correlation of responses demonstrably associated with particular regions (peptides N1-N29) peaking in the region covered by of N18-29. A second region of higher correlation was observed close to the carboxyl terminal of the heavy chain. Region N15 to N29 was found to have a significantly lower Stimulation Indices when DRB1*01:01-DQA1*01:01-DQB1*05:01 was present. This pattern was also evident in the HLA analysis where DQA1*01:02 associations were collectively most significant in the N1-N13 & C16-C31 region. Responses in these regions correlated well with one another. HLA-specific correlation analysis revealed that DQA1*01:01 bearing haplotype had the strongest inter-epitope correlations despite having a generally negative association with simulation indices. Structural and immunogenic implications of these findings are discussed.


Subject(s)
Botulinum Toxins, Type A/immunology , Diabetes Mellitus, Type 1/genetics , Epitopes, T-Lymphocyte/immunology , HLA-DQ alpha-Chains/metabolism , HLA-DQ beta-Chains/metabolism , HLA-DRB1 Chains/metabolism , T-Lymphocytes/immunology , Cell Proliferation , Cells, Cultured , Epitope Mapping , Genetic Predisposition to Disease , Haplotypes , Humans , Immunity, Cellular , Lymphocyte Activation
6.
J Glob Oncol ; 3(4): 338-345, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28831442

ABSTRACT

PURPOSE: As Syria enters its fifth year of conflict, the number of civilians killed and injured continues to rise sharply. Along with this conflict comes the rapid decline of medical care, specifically cancer care. To determine physician and equipment availability, cancer screening and management, and possible solutions relative to various major cities, a survey was distributed to physicians inside Syria through the help of the humanitarian organization Syrian American Medical Society. METHODS: Online surveys were distributed to both certified oncologists who work in cancer clinics and general physicians who work in rural and mobile clinics inside Syria. Variables assessed were physician specialty, location, population, cost, regional situation (besieged versus government controlled), and resource availability and access. Results were stratified by location and physician specialty. RESULTS: Survey results revealed a large shortage of specialized physicians and inhibited accessibility to screening and management options in besieged areas compared with government-controlled regions. Physicians within both government-controlled and besieged cities reported limited or no targeted agents, radiation therapy, clinical trials, bone marrow transplantation, positron emission tomography scans, magnetic resonance imaging, and genetic testing. CONCLUSION: The Syrian civil war has resulted in suboptimal oncology care in the majority of the region. In consideration of specific deficiencies in cancer care, we recommend several solutions that may better the level of care in Syria: patient education on medical documentation and self-examination; online consultation; and cheap, effective screening methods. The implementation of these recommendations may change the course of cancer care in a country that has deteriorated into the worst humanitarian crisis of the century.

7.
Autoimmunity ; 50(6): 346-353, 2017 Sep.
Article in English | MEDLINE | ID: mdl-28850269

ABSTRACT

Experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis (MG), can be induced in C57BL/6 (B6, H-2 b) mice by 2-3 injections with Torpedo californica AChR (tAChR) in complete Freund's adjuvant. Some EAMG mice exhibit weight loss with muscle weakness. The loss in body weight, which is closely associated with bone structure, is particularly evident in EAMG mice with severe muscle weakness. However, the relationship between muscle weakness and bone loss in EAMG has not been studied before. Recent investigations on bone have shed light on association of bone health and immunological states. It is possible that muscle weakness in EAMG developed by anti-tAChR immune responses might accompany bone loss. We determined whether reduced muscle strength associates with decreased bone mineral density (BMD) in EAMG mice. EAMG was induced by two injections at 4-week interval of tAChR and adjuvants in two different age groups. The first tAChR injection was either at age 8 weeks or at 15 weeks. We measured BMD at three skeletal sites, including femur, tibia, and lumbar vertebrae, using dual energy X-ray absorptiometry. Among these bone areas, femur of EAMG mice in both age groups showed a significant decrease in BMD compared to control adjuvant-injected and to non-immunized mice. Reduction in BMD in induced EAMG at a later-age appears to parallel the severity of the disease. The results indicate that anti-tAChR autoimmune response alone can reduce bone density in EAMG mice. BMD reduction was also observed in adjuvant-injected mice in comparison to normal un-injected mice, suggesting that BMD decrease can occur even when muscle activity is normal. Decreased BMD observed in both tAChR-injected and adjuvant-injected mice groups were discussed in relation to innate immunity and bone-related immunology involving activated T cells and tumour necrosis factor-related cytokines that trigger osteoclastogenesis and bone loss.


Subject(s)
Bone Density/immunology , Bone Resorption/pathology , Muscle Weakness/pathology , Myasthenia Gravis, Autoimmune, Experimental/pathology , Absorptiometry, Photon , Age Factors , Animals , Bone Resorption/chemically induced , Bone Resorption/diagnostic imaging , Bone Resorption/immunology , Femur/diagnostic imaging , Femur/immunology , Femur/pathology , Fish Proteins/administration & dosage , Freund's Adjuvant/administration & dosage , Lumbar Vertebrae/diagnostic imaging , Lumbar Vertebrae/immunology , Lumbar Vertebrae/pathology , Male , Mice , Mice, Inbred C57BL , Muscle Weakness/chemically induced , Muscle Weakness/diagnostic imaging , Muscle Weakness/immunology , Myasthenia Gravis, Autoimmune, Experimental/chemically induced , Myasthenia Gravis, Autoimmune, Experimental/diagnostic imaging , Myasthenia Gravis, Autoimmune, Experimental/metabolism , Receptors, Cholinergic/administration & dosage , Severity of Illness Index , Tibia/diagnostic imaging , Tibia/immunology , Tibia/pathology , Time Factors , Torpedo/metabolism
8.
Autoimmunity ; 50(5): 293-305, 2017 Aug.
Article in English | MEDLINE | ID: mdl-28548588

ABSTRACT

An animal model of myasthenia gravis (MG), termed experimental autoimmune MG (EAMG), is an important tool for investigations of disease mechanisms and/or methods of treatment for this disease. EAMG can be induced in C57BL/6 (B6, H-2b) mice by 2-3 times injections at 4 weeks intervals with Torpedo californica (t) acetylcholine receptor (AChR) in complete Freund's adjuvant (CFA). However, the protocol especially with a two-injection schedule occasionally produces a poor incidence of EAMG. We have investigated the efficacy of the additional adjuvant, inactive organisms of Bordetella pertussis (iBP), on the induction with a two-injection schedule. In a group immunized with tAChR in CFA + iBP, 76% of mice developed EAMG (average grade in exercise test, 1.02). Whereas, 46% of mice were found EAMG-positive (average grade, 0.73) in a group injected with tAChR/CFA alone. Thus, the combined use of CFA and iBP significantly increased both the occurrence and severity of clinical MG in the immunized mice. This was accompanied by higher antibody (Ab) and T-cell responses to tAChR. The effect on disease occurrence of the iBP use in a three-injection protocol was also described.


Subject(s)
Bordetella pertussis/immunology , Freund's Adjuvant/immunology , Myasthenia Gravis, Autoimmune, Experimental/immunology , Receptors, Cholinergic/immunology , Torpedo/immunology , Amino Acid Sequence , Animals , Antibodies/immunology , Electrophysiological Phenomena , Female , Freund's Adjuvant/administration & dosage , Immunization , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Muscle Weakness/immunology , Muscle Weakness/physiopathology , Myasthenia Gravis, Autoimmune, Experimental/diagnosis , Peptide Fragments , Phenotype , Receptors, Cholinergic/administration & dosage , Receptors, Cholinergic/chemistry , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
9.
Protein J ; 36(3): 155-156, 2017 06.
Article in English | MEDLINE | ID: mdl-28429155
10.
J Neuroimmunol ; 306: 31-39, 2017 05 15.
Article in English | MEDLINE | ID: mdl-28385185

ABSTRACT

We have conducted a 26-month-long comparative study involving young patients (2-6years old) with a clinical diagnosis of spastic equinus secondary to cerebral palsy who have been treated with BoNT/A (BOTOX®, Allergan) tri-annually or annually. Serum samples were obtained to determine the presence or absence of blocking antibodies (Abs) by a mouse protection assay (MPA) and levels of anti-BoNT/A Abs by radioimmune assay (RIA). HLA DQ alleles were typed using blood samples to determine the possible association of certain HLA type(s) with the disease or with the Ab status. Blocking Abs were detected in only two out of 18 serum samples of the tri-annual group, but none were found in 20 samples of the annual group. The MPA-positive serum samples gave in RIA significantly higher anti-BoNT/A Ab-binding levels than the MPA-negative samples. On the other hand, when two MPA-positive sample data were excluded, serum samples from tri-annual and annual groups showed similar anti-BoNT/A Ab levels. Linkage of the disorder with a particular HLA DQA1 and DQB1 allele types was not observed due to the small sample size. However, by combining results with other studies on BoNT/A-treated Caucasian patients with cervical dystonia (CD), we found that, among Caucasian patients treated with BoNT/A, DQA1*01:02 and DQB1*06:04 were higher in Ab-positive than in Ab-negative patients. The genetic linkage was on the threshold of corrected significance.


Subject(s)
Antibody Formation/drug effects , Botulinum Toxins, Type A/immunology , Botulinum Toxins, Type A/therapeutic use , Cerebral Palsy/drug therapy , Muscle Spasticity/drug therapy , Acetylcholine Release Inhibitors , Animals , Antibodies, Blocking/immunology , Cerebral Palsy/blood , Cerebral Palsy/complications , Child , Child, Preschool , Drug Administration Schedule , Female , HLA-DQ alpha-Chains/genetics , HLA-DQ beta-Chains/genetics , Histocompatibility Testing , Humans , Male , Muscle Spasticity/blood , Muscle Spasticity/complications , Pharmacogenetics , Radioimmunoassay
11.
J Neuroimmunol ; 300: 36-46, 2016 11 15.
Article in English | MEDLINE | ID: mdl-27806874

ABSTRACT

We have recently reported the submolecular T-cell recognition profile of the C-terminal half (HC, residues 855-1296) of the heavy (H) chain of botulinum neurotoxin type A (BoNT/A) with peripheral blood lymphocytes (PBL) from 25 BoNT-treated cervical dystonia (CD) patients. In the current study, we describe the mapping of the T-cell responses of the patients to the N-terminal half (HN, residues 449-859) of the heavy chain by using 29 synthetic overlapping peptides encompassing the entire HN domain of BoNT/A. The profiles of the T-cell responses to the peptides varied among the patients. Samples from 14 patients treated solely with BoNT/A recognized 1-9 (average 3.7) peptides/sample at Z>3.0 level. Three peptide regions representing residues 631-649, 659-677 and 743-761 were frequently recognized by 29-64% of the patients. In patients with positive anti-BoNT/A antibody responses the overall positive T cell responses to the HN peptides were significantly increased compared to antibody-negative patients. Influence of treatment parameters on the T-cell recognition of the HN peptides was also observed. The results were compared with those of previously identified HC region.


Subject(s)
Botulinum Toxins, Type A/therapeutic use , Peptide Fragments/immunology , T-Lymphocytes/immunology , Torticollis/drug therapy , Torticollis/immunology , Adult , Aged , Amino Acid Sequence , Botulinum Toxins, Type A/pharmacology , Cohort Studies , Female , Follow-Up Studies , Humans , Male , Middle Aged , Peptide Fragments/genetics , Protein Domains/drug effects , Protein Domains/genetics , Protein Domains/immunology , T-Lymphocytes/drug effects , Torticollis/genetics , Treatment Outcome
12.
Biochim Biophys Acta ; 1864(12): 1678-1685, 2016 12.
Article in English | MEDLINE | ID: mdl-27596061

ABSTRACT

Binding behaviors of the HN and the HC domains of BoNT/A were investigated individually to identify if there exist any differences in their interaction with the cell membrane. Recombinant fragments corresponding to both BoNT/A HN and HC regions were prepared (HN519-845 and HC967-1296) and their binding to synaptic proteins was verified. The binding behaviors of these heavy-chain domains were analyzed by treating the Neuro 2a, a murine neuroblastoma cell line, with compounds known to alter membrane properties. Cholesterol depletion and lipid raft inhibition increased the binding of HN519-845 to Neuro 2a cells without affecting HC967-1296-cell interaction. Sphingolipid depletion decreased the binding of cells to both HC967-1296 and HN519-845 whereas, loading exogenous GD1a, on to the Neuro 2a cells, increased the binding of both the peptides to cells. Microtubule disruption of the Neuro 2a cells by nocodazole decreased the binding of both HC967-1296 and HN519-845 to the treated cells. Inhibition of the clathrin-mediated endocytosis using dynasore, chlorpromazine or potassium (K+) depletion buffer lowered the binding of both HC967-1296 and HN519-845 to the cells, but seemed to exert a more pronounced effect on the binding of HC967-1296 than on the binding of HN519-845. Results indicate that while both the HN and HC domains are involved in the binding of the toxin to neuronal cells there are differences in their behavior which probably stem from their respective amino acid composition and structural location in the toxin three-dimensional structure along with their intended role in translocation and internalization into the cells.


Subject(s)
Botulinum Toxins, Type A/chemistry , Botulinum Toxins, Type A/metabolism , Animals , Botulinum Toxins, Type A/genetics , Caveolae/metabolism , Cell Line , Cell Membrane/metabolism , Clathrin/metabolism , Endocytosis , Membrane Microdomains/metabolism , Mice , Microtubules/metabolism , Models, Molecular , Neurons/metabolism , Neurotoxins/chemistry , Neurotoxins/genetics , Neurotoxins/metabolism , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Binding , Protein Domains , Synaptic Vesicles/metabolism
13.
Immunobiology ; 221(4): 568-76, 2016 Apr.
Article in English | MEDLINE | ID: mdl-26701846

ABSTRACT

We determined the T-cell proliferative responses of the peripheral blood lymphocytes (PBL) from 25 botulinum neurotoxin (BoNT)-treated patients to 31 overlapping synthetic peptides encompassing the C-terminal half (residues 855-1296) of BoNT/A heavy chain. Responses of PBL to HC peptides varied among patients. Samples from 14 patients treated solely with BoNT/A recognized 2-13 (average 6.4) peptides/sample at Z>3.0 level. Six peptide regions representing residues 855-873, 1023-1041, 1051-1069, 1093-1111, 1135-1153 and 1247-1265 were frequently recognized by 36-57% of these PBLs. Influence of treatment parameters on T-cell recognition of the peptides was also investigated.


Subject(s)
Botulinum Toxins, Type A/chemistry , Clostridium botulinum/chemistry , Epitopes, T-Lymphocyte/chemistry , Peptides/chemistry , T-Lymphocytes/immunology , Torticollis/drug therapy , Adult , Aged , Amino Acid Sequence , Botulinum Toxins, Type A/immunology , Botulinum Toxins, Type A/therapeutic use , Clostridium botulinum/immunology , Epitope Mapping , Epitopes, T-Lymphocyte/immunology , Female , Humans , Male , Middle Aged , Molecular Sequence Data , Peptides/immunology , Primary Cell Culture , Protein Binding , Protein Structure, Tertiary , Sequence Alignment , T-Lymphocytes/cytology , Torticollis/immunology , Torticollis/pathology
14.
Crit Rev Immunol ; 35(3): 203-51, 2015.
Article in English | MEDLINE | ID: mdl-26559228

ABSTRACT

Autoimmune diseases (ADs), or autoinflammatoiy diseases, are growing in complexity as diagnoses improve and many factors escalate disease risk. Considerable genetic similarity is found among ADs, and they are frequently associated with major histocompatibility complex (MHC) genes. However, a given disease may be associated with more than one human leukocyte antigen (HLA) allotype, and a given HLA may be associated with more than one AD. The associations of non-MHC genes with AD present an additional problem, and the situation is further complicated by the role that other factors, such as age, diet, therapeutic drugs, and regional influences, play in disease. This review discusses some of the genetics and biochemistry of HLA-linked AD and inflammation, covering some of the best-studied examples and summarizing indicators for class I- and II-mediated disease. However, the scope of this review limits a detailed discussion of all known ADs.


Subject(s)
Autoimmune Diseases/immunology , HLA Antigens/genetics , Animals , Autoimmune Diseases/genetics , Genetic Association Studies , Genetic Predisposition to Disease , Humans , Polymorphism, Genetic
15.
Sci Rep ; 5: 15776, 2015 Oct 28.
Article in English | MEDLINE | ID: mdl-26508475

ABSTRACT

Botulinum neurotoxins (BoNTs) are the most toxic substances known. BoNT intoxicates cells in a highly programmed fashion initiated by binding to the cell surface, internalization and enzymatic cleavage of substrate, thus, inhibiting synaptic exocytosis. Over the past two decades, immunological significance of BoNT/A C-terminal heavy chain (HC) and light chain (LC) domains were investigated extensively leading to important findings. In the current work, we explored the significance of BoNT/A heavy chain N-terminal (HN) region as a vaccine candidate. Mice were immunized with recombinant HN519-845 generating antibodies (Abs) that were found to be protective against lethal dose of BoNT/A. Immuno-dominant regions of HN519-845 were identified and individually investigated for antibody response along with synthetic peptides within those regions, using in vivo protection assays against BoNT/A. Results were confirmed by patch-clamp analysis where anti-HN antibodies were studied for the ability to block toxin-induced channel formation. This data strongly indicated that HN519-593 is an important region in generating protective antibodies and should be valuable in a vaccine design. These results are the first to describe and dissect the protective activity of the BoNT/A HN domain.


Subject(s)
Antibodies, Bacterial/immunology , Antibody Formation/immunology , Botulinum Toxins, Type A/immunology , Neurotoxins/immunology , Toxins, Biological/immunology , Amino Acid Sequence/physiology , Animals , Immunization/methods , Mice , Peptide Fragments/immunology , Peptides/immunology , Protein Binding/immunology , Vaccination/methods
16.
J Fr Ophtalmol ; 38(9): 815-21, 2015 Nov.
Article in French | MEDLINE | ID: mdl-26456487

ABSTRACT

PURPOSE: This study was designed to evaluate and compare the efficacy of early treatment of CRVO with either hemodilution by erythrocytopheresis or intravitreal (IVT) ranibizumab, or both. MATERIAL AND METHODS: A multicentric prospective randomized study including patients with CRVO for less than 1 month was designed. Patients were randomized into 3 treatment groups: hemodilution (HD group), 3 monthly intravitreal injections of ranibizumab followed by PRN treatment (IVT group), or combined treatment (IVT+HD group). A monthly evaluation during a 6-month follow-up included best-corrected visual acuity (BCVA) and macular thickness measurements with OCT. Fluorescein angiography was performed at baseline, month 2 and month 6. Local and systemic tolerability of the different treatments were also compared. RESULTS: Forty-four CRVO patients were included between February 2010 and June 2013: 20 in the IVT group, 13 in the HD group and 11 in the HD+IVT group. The mean duration of CRVO at baseline was 10 days and 16 days at the time of treatment without any significant difference between groups. Retinal ischemia was present at baseline in 40% of eyes in each group. After a 6-month follow-up, no difference between the 3 groups was observed in BCVA (10.5 ETDRS letters, 14.6 and 14.1 in the IVT group, HD group and IVT+HD group respectively, P=0.726) or in macular thickness (398 µ, 440 µ and 379 µ respectively, P=0.465). The time until treatment from CRVO onset, ranging from 1 to 35 days, was not correlated to final outcomes. No significant difference in the mean number of IVT (3.2 in the IVT+HD group vs 3.7 in the IVT group) was observed at 6 months. CONCLUSION: No difference in BCVA nor in macular thickness was seen at M6 between the study groups. The duration of CRVO at the time of the initiation of the treatment was not correlated to better visual outcomes. Therefore, etiologic treatment with HD can still be proposed as a first-line treatment in young patients, which allows delaying or avoiding the IVT treatment and its potential side effects. Anti-VEGF IVT still remains an effective option in every case and can be started one month after the beginning of the CRVO.


Subject(s)
Angiogenesis Inhibitors/therapeutic use , Hemodilution , Ranibizumab/therapeutic use , Retinal Vein Occlusion/therapy , Aged , Combined Modality Therapy , Early Medical Intervention , Female , Humans , Intravitreal Injections , Male , Middle Aged , Prospective Studies
17.
Toxicon ; 107(Pt A): 50-8, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26086358

ABSTRACT

Intensive research in this laboratory over the last 19 years has aimed at understanding the molecular bases for immune recognition of botulinum neurotoxin, types A and B and the role of anti-toxin immune responses in defense against the toxin. Using 92 synthetic 19-residue peptides that overlapped by 5 residues and comprised an entire toxin (A or B) we determined the peptides' ability to bind anti-toxin Abs of human, mouse, horse and chicken. We also localized the epitopes recognized by Abs of cervical dystonia patients who developed immunoresistance to correlate toxin during treatment with BoNT/A or BoNT/B. For BoNT/A, patients' blocking Abs bound to 13 regions (5 on L and 8 on H subunit) on the surface and the response to each region was under separate MHC control. The responses were defined by the structure of the antigen and by the MHC of the host. The antigenic regions coincided or overlapped with synaptosomes (SNPS) binding regions. Antibody binding blocked the toxin's ability to bind to neuronal cells. In fact selected synthetic peptides were able to inhibit the toxin's action in vivo. A combination of three synthetic strong antigenic peptides detected blocking Abs in 88% of immunoresistant patients' sera. Administration of selected epitopes, pre-linked at their N(α) group to monomethoxyployethylene glycol, into mice with ongoing blocking anti-toxin Abs, reduced blocking Ab levels in the recipients. This may be suitable for clinical applications. Defined epitopes should also be valuable in synthetic vaccines design.


Subject(s)
Antigens, Bacterial/immunology , Botulinum Toxins/immunology , Animals , Antibodies, Bacterial/genetics , Antibodies, Bacterial/immunology , Antigens, Bacterial/genetics , Binding Sites, Antibody/genetics , Binding Sites, Antibody/immunology , Botulinum Toxins/genetics , Humans , Mice , Molecular Structure , Synaptosomes/immunology , T-Lymphocytes/immunology
18.
Infect Immun ; 83(4): 1465-76, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25624352

ABSTRACT

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (HC) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (HN) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the HC domain but also regions of the HN domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising HN domain residues 729 to 845 (HN729-845). HN729-845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide HN729-845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the HC domain. These findings strongly indicate that HN729-845 and, by extension, the HN domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin.


Subject(s)
Botulinum Toxins, Type A/metabolism , Neuroblastoma/metabolism , Neurons/metabolism , Peptide Fragments/metabolism , Peptide Fragments/pharmacology , Acetylcholine/metabolism , Animals , Binding Sites/genetics , Botulinum Toxins, Type A/genetics , Cell Line, Tumor , Clostridium botulinum/pathogenicity , Humans , Mice , Mice, Inbred ICR , Peptide Fragments/genetics , Protein Binding , Protein Structure, Tertiary , Protein Transport/physiology , Synaptosomes/metabolism , Torticollis/drug therapy
19.
Immunobiology ; 219(12): 950-7, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25151501

ABSTRACT

Lymph node cells (LNC) from SJL (H-2(s)) and BALB/c (H-2(d)) mice primed once with inactivated botulinum neurotoxin type A (BoNT/A) were examined for their T-cell responses to each of 32 synthetic overlapping peptides (19 residues each, L1-L32) that encompass the entire L chain (residues 1-448) of BoNT/A. LNC of SJL gave strong responses to 6 regions on, L2 (residues 15-23), L10/11/12 (127-173), L19 (253-271) and L21 (281-299), and moderate to weak responses to L9 (113-131), L14/15 (183-215) and L27 (365-383). In BALB/c, LNC gave a substantial T-cell response only against peptide L12 (residues 155-173), and responded very weakly to 9 other peptides. The results were compared with the recognition profiles determined previously in these two strains after multiple BoNT/A injections. Overall responses to the L-chain peptides of T cells in later profiles were found to be somewhat weakened in SJL and stayed essentially at a similar level in BALB/c, although responses to BoNT/A increased. In SJL, response to L10 (127-145) remained the highest in the later profile. Strong responses against L12 (155-173) observed in both strains at early stage were reduced to an insignificant level. Cross-reactivity to tetanus neurotoxin by BoNT/A-specific T cells was observed in SJL but not in BALB/c. Design of an effective synthetic peptide vaccine will require incorporation of both T cell- and Ab-recognition elements of the BoNT molecule. Significance and possible implications of these results on BoNT/A-specific T-cell responses of BoNT-treated patients are discussed.


Subject(s)
Botulinum Toxins, Type A/immunology , Epitopes, T-Lymphocyte/immunology , Lymphocyte Activation/immunology , T-Lymphocytes/immunology , Amino Acid Sequence , Animals , Antibodies, Bacterial/immunology , Bacterial Vaccines/immunology , Botulinum Toxins, Type A/administration & dosage , Botulinum Toxins, Type A/chemistry , Epitopes, T-Lymphocyte/chemistry , Female , Immunization , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Peptides/chemistry , Peptides/immunology , Protein Binding
20.
J Neuroimmunol ; 272(1-2): 29-34, 2014 Jul 15.
Article in English | MEDLINE | ID: mdl-24841626

ABSTRACT

In cervical dystonia, injection of botulinum neurotoxin (BoNT) A or B into affected neck muscle reduces symptoms but may elicit anti-toxin antibodies (Abs) that block responsiveness to treatment. Previously, we localized the BoNT/A and BoNT/B sites that bind mouse or human blocking Abs. We also reported that site-specific auto-Abs can be suppressed by a monomethoxypolyethylene glycol (mPEG)-epitope conjugate. So we elicited here anti-toxin Abs in outbred mice by immunization with sublethal-suboptimal doses of active BoNT/A and determined the efficacy of selected mPEG-epitopes in reducing established anti-BoNT/A Abs. We tested in outbred mice four synthetic mPEG-N(α)-epitopes [N8 (residues 547-565), N25 (785-803), C15 (1051-1069), C31 (1275-1296)] of BoNT/A in tolerance against ongoing anti-toxin Abs. After short immunizations, tolerization with an mPEG-peptide reduced Abs to correlate peptide and caused varying Ab reductions to the other 3 peptides. Anti-N8 Abs were unaffected by mPEG-N25 tolerization, but mPEG-N8 and mPEG-N25 caused drop in anti-BoNT/A Abs. After long immunization with BoNT/A, tolerization with mPEG-N8 lessened anti-N8 Abs. Anti-C15 Abs decreased by tolerization with mPEG-C15 or any other mPEG-peptide. Anti-N25 Abs were not altered by mPEG-N25, but decreased after tolerization with mPEG-C15. Anti-C31 Abs disappeared on day 474 by tolerization with mPEG-C31 or mPEG-N8, mPEG-N25 or mPEG-C15. When an Ab response returns, a decrease can be re-established by re-administering the correlate mPEG-peptide. The method may be beneficial for extending BoNT treatment in immunoresistant patients.


Subject(s)
Antibodies/pharmacology , Botulinum Toxins, Type A/immunology , Immunodominant Epitopes/metabolism , Peptide Fragments/metabolism , Polyethylene Glycols/metabolism , Animals , Antibody Formation , Dose-Response Relationship, Drug , Humans , Immunodominant Epitopes/immunology , Mice , Mice, Inbred ICR , Peptide Fragments/administration & dosage , Peptide Fragments/chemical synthesis , Polyethylene Glycols/chemistry , Protein Binding/drug effects , Radioimmunoassay , Synaptosomes/drug effects , Synaptosomes/immunology , Time Factors
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