HLA-DQB1*05 subtypes and not DRB1*10:01 mediates risk in anti-IgLON5 disease.

Anti-IgLON5 disease is a rare and likely underdiagnosed subtype of autoimmune encephalitis. The disease displays a heterogeneous phenotype that includes sleep, movement and bulbar-associated dysfunction. The presence of IgLON5-antibodies in CSF/serum, together with a strong association with HLA-DRB1*10:01∼DQB1*05:01, supports an autoimmune basis. In this study, a multicentric human leukocyte antigen (HLA) study of 87 anti-IgLON5 patients revealed a stronger association with HLA-DQ than HLA-DR. Specifically, we identified a predisposing rank-wise association with HLA-DQA1*01:05∼DQB1*05:01, HLA-DQA1*01:01∼DQB1*05:01 and HLA-DQA1*01:04∼DQB1*05:03 in 85% of patients. HLA sequences and binding cores for these three DQ heterodimers were similar, unlike those of linked DRB1 alleles, supporting a causal link to HLA-DQ. This association was further reflected in an increasingly later age of onset across each genotype group, with a delay of up to 11 years, while HLA-DQ-dosage dependent effects were also suggested by reduced risk in the presence of non-predisposing DQ1 alleles. The functional relevance of the observed HLA-DQ molecules was studied with competition binding assays. These proof-of-concept experiments revealed preferential binding of IgLON5 in a post-translationally modified, but not native, state to all three risk-associated HLA-DQ receptors. Further, a deamidated peptide from the Ig2-domain of IgLON5 activated T cells in two patients, compared with one control carrying HLA-DQA1*01:05∼DQB1*05:01. Taken together, these data support a HLA-DQ-mediated T-cell response to IgLON5 as a potentially key step in the initiation of autoimmunity in this disease.

Different Genetic Signatures of Small-Cell Lung Cancer Characterize Anti-GABAB R and Anti-Hu Paraneoplastic Neurological Syndromes.

OBJECTIVE: Small-cell lung cancer (SCLC) is the malignancy most frequently associated with paraneoplastic neurological syndromes (PNS) and can trigger different antibody responses against intracellular (Hu) or neuronal surface (GABAB R) antigens. Our aim was to clarify whether the genomic and transcriptomic features of SCLC are different in patients with anti-GABAB R or anti-Hu PNS compared with SCLC without PNS. METHODS: A total of 76 SCLC tumor samples were collected: 34 anti-Hu, 14 anti-GABAB R, and 28 SCLC without PNS. The study consisted of 4 steps: (1) pathological confirmation; (2) next generation sequencing using a panel of 98 genes, including those encoding the autoantibodies targets ELAVL1-4, GABBR1-2, and KCTD16; (3) genome-wide copy number variation (CNV); and (4) whole-transcriptome RNA sequencing. RESULTS: CNV analysis revealed that patients with anti-GABAB R PNS commonly have a gain in chromosome 5q, which contains KCTD16, whereas anti-Hu and control patients often harbor a loss. No significantly different number of mutations regarding any onconeural genes was observed. Conversely, the transcriptomic profile of SCLC was different, and the differentially expressed genes allowed effective clustering of the samples into 3 groups, reflecting the antibody-based classification, with an overexpression of KCTD16 specific to anti-GABAB R PNS. Pathway analysis revealed that tumors of patients with anti-GABAB R encephalitis were enriched in B-cell signatures, as opposed to those of patients with anti-Hu, in which T-cell- and interferon-γ-related signatures were overexpressed. INTERPRETATION: SCLC genetic and transcriptomic features differentiate anti-GABAB R, anti-Hu, and non-PNS tumors. The role of KCTD16 appears to be pivotal in the tumor immune tolerance breakdown of anti-GABAB R PNS. ANN NEUROL 2023;94:1102-1115.

Detection of High-Risk Paraneoplastic Antibodies against TRIM9 and TRIM67 Proteins.

OBJECTIVE: Co-occurring anti-tripartite motif-containing protein 9 and 67 autoantibodies (TRIM9/67-IgG) have been reported in only a very few cases of paraneoplastic cerebellar syndrome. The value of these biomarkers and the most sensitive methods of TRIM9/67-IgG detection are not known. METHODS: We performed a retrospective, multicenter study to evaluate the cerebrospinal fluid and serum of candidate TRIM9/67-IgG cases by tissue-based immunofluorescence, peptide phage display immunoprecipitation sequencing, overexpression cell-based assay (CBA), and immunoblot. Cases in which TRIM9/67-IgG was detected by at least 2 assays were considered TRIM9/67-IgG positive. RESULTS: Among these cases (n = 13), CBA was the most sensitive (100%) and revealed that all cases had TRIM9 and TRIM67 autoantibodies. Of TRIM9/67-IgG cases with available clinical history, a subacute cerebellar syndrome was the most common presentation (n = 7/10), followed by encephalitis (n = 3/10). Of these 10 patients, 70% had comorbid cancer (7/10), 85% of whom (n = 6/7) had confirmed metastatic disease. All evaluable cancer biopsies expressed TRIM9 protein (n = 5/5), whose expression was elevated in the cancerous regions of the tissue in 4 of 5 cases. INTERPRETATION: TRIM9/67-IgG is a rare but likely high-risk paraneoplastic biomarker for which CBA appears to be the most sensitive diagnostic assay. ANN NEUROL 2023;94:1086-1101.

Discriminating Aroma Compounds in Five Cocoa Bean Genotypes from Two Brazilian States: White Kerosene-like Catongo, Red Whisky-like FL89 (Bahia), Forasteros IMC67, PA121 and P7 (Pará).

Fine-grade cocoa beans are characterized by their great organoleptic quality. Brazilian cocoa producers increasingly privilege organoleptic quality over yield. Catongo and FL89, fine Brazilian cocoa genotypes from Bahia, are characterized by particular flavors (respectively, kerosene and whisky). The beans IMC67, PA121, and P7 from the state of Pará are genotypes that have high resistance to diseases. Solvent-assisted flavor evaporation (SAFE) aroma extraction was used here to identify and quantify the volatile compounds discriminating these genotypes. The results show that the kerosene aroma of Catongo is likely due to the presence, at high levels, of ethyl acetate and isobutyl acetate. On the other hand, ethyl benzoate, heptanoate, and octanoate, trans-2-nonenal, 1-octen-3-ol, and 3-methylbutanol could play a key role in the whisky notes of FL89. Heptan-2-ol, heptan-2-one, nonan-2-one, linalool (although still more concentrated in IMC67 from Pará state), benzaldehyde, and phenylacetaldehyde also discriminate these beans. Other compounds, although not discriminating, appear important in determining their aromatic quality. The PCA showed that cocoa from Pará state formed a cluster due to similar aromas, while FL89 was the most distinct among the genotypes. Beans from Brazil show great potential and diversity for the fine cocoa market.

The impact of essential oils on antibiotic use in animal production regarding antimicrobial resistance - a review.

Population growth directly affects the global food supply, demanding a higher production efficiency without farmland expansion - in view of limited land resources and biodiversity loss worldwide. In such scenario, intensive agriculture practices have been widely used. A commonly applied method to maximize yield in animal production is the use of subtherapeutic doses of antibiotics as growth promoters. Because of the strong antibiotic selection pressure generated, the intense use of antibiotic growth promoters (AGP) has been associated to the rise of antimicrobial resistance (AMR). Also, cross-resistance can occur, leading to the emergence of multidrug-resistant pathogens and limiting treatment options in both human and animal health. Thereon, alternatives have been studied to replace AGP in animal production. Among such alternatives, essential oils and essential oil components (EOC) stand out positively from others due to, besides antimicrobial effectiveness, improving zootechnical indexes and modulating genes involved in resistance mechanisms. This review summarizes recent studies in essential oils and EOC for zoonotic bacteria control, providing detailed information about the molecular-level effects of their use in regard to AMR, and identifying important gaps to be filled within the animal production area.

Distinctive clinical presentation and pathogenic specificities of anti-AK5 encephalitis.

Limbic encephalitis with antibodies against adenylate kinase 5 (AK5) has been difficult to characterize because of its rarity. In this study, we identified 10 new cases and reviewed 16 previously reported patients, investigating clinical features, IgG subclasses, human leucocyte antigen and CSF proteomic profiles. Patients with anti-AK5 limbic encephalitis were mostly male (20/26, 76.9%) with a median age of 66 years (range 48-94). The predominant symptom was severe episodic amnesia in all patients, and this was frequently associated with depression (17/25, 68.0%). Weight loss, asthenia and anorexia were also highly characteristic, being present in 11/25 (44.0%) patients. Although epilepsy was always lacking at disease onset, seizures developed later in a subset of patients (4/25, 16.0%). All patients presented CSF abnormalities, such as pleocytosis (18/25, 72.0%), oligoclonal bands (18/25, 72.0%) and increased Tau (11/14, 78.6%). Temporal lobe hyperintensities were almost always present at disease onset (23/26, 88.5%), evolving nearly invariably towards severe atrophy in subsequent MRIs (17/19, 89.5%). This finding was in line with a poor response to immunotherapy, with only 5/25 (20.0%) patients responding. IgG1 was the predominant subclass, being the most frequently detected and the one with the highest titres in nine CSF-serum paired samples. A temporal biopsy from one of our new cases showed massive lymphocytic infiltrates dominated by both CD4+ and CT8+ T cells, intense granzyme B expression and abundant macrophages/microglia. Human leucocyte antigen (HLA) analysis in 11 patients showed a striking association with HLA-B*08:01 [7/11, 63.6%; odds ratio (OR) = 13.4, 95% confidence interval (CI): 3.8-47.4], C*07:01 (8/11, 72.7%; OR = 11.0, 95% CI: 2.9-42.5), DRB1*03:01 (8/11, 72.7%; OR = 14.4, 95% CI: 3.7-55.7), DQB1*02:01 (8/11, 72.7%; OR = 13.5, 95% CI: 3.5-52.0) and DQA1*05:01 (8/11, 72.7%; OR = 14.4, 95% CI: 3.7-55.7) alleles, which formed the extended haplotype B8-C7-DR3-DQ2 in 6/11 (54.5%) patients (OR = 16.5, 95% CI: 4.8-57.1). Finally, we compared the CSF proteomic profile of five anti-AK5 patients with that of 40 control subjects and 10 cases with other more common non-paraneoplastic limbic encephalitis (five with antibodies against leucine-rich glioma inactivated 1 and five against contactin-associated protein-like 2), as well as 10 cases with paraneoplastic neurological syndromes (five with antibodies against Yo and five against Ma2). These comparisons revealed 31 and seven significantly upregulated proteins in anti-AK5 limbic encephalitis, respectively mapping to apoptosis pathways and innate/adaptive immune responses. These findings suggest that the clinical manifestations of anti-AK5 limbic encephalitis result from a distinct T cell-mediated pathogenesis, with major cytotoxicity-induced apoptosis leading to a prompt and aggressive neuronal loss, likely explaining the poor prognosis and response to immunotherapy.

Anti-CASPR2 clinical phenotypes correlate with HLA and immunological features.

OBJECTIVE: Antibodies against contactin-associated protein-like 2 (CASPR2-Abs) have been described in acquired neuromyotonia, limbic encephalitis (LE) and Morvan syndrome (MoS). However, it is unknown whether these constitute one sole spectrum of diseases with the same immunopathogenesis or three distinct entities with different mechanisms. METHODS: A cluster analysis of neurological symptoms was performed in a retrospective cohort of 56 CASPR2-Abs patients. In parallel, immunological features and human leucocyte antigen (HLA) were studied. RESULTS: Cluster analysis distinguished patients with predominant limbic symptoms (n=29/56) from those with peripheral nerve hyperexcitability (PNH; n=27/56). In the limbic-prominent group, limbic features were either isolated (LE/-; 18/56, 32.1%), or combined with extralimbic symptoms (LE/+; 11/56, 19.6%). Those with PNH were separated in one group with severe PNH and extralimbic involvement (PNH/+; 16/56, 28.6%), resembling historical MoS descriptions; and one group with milder and usually isolated PNH (PNH/-; 11/56, 19.6%). LE/- and LE/+ patients shared immunogenetic characteristics demonstrating a homogeneous entity. HLA-DRB1*11:01 was carried more frequently than in healthy controls only by patients with LE (94.1% vs 18.3%; p=1.3×10-10). Patients with LE also had serum titres (median 1:40 960) and rates of cerebrospinal fluid positivity (93.1%) higher than the other groups (p<0.05). Conversely, DRB1*11:01 association was absent in PNH/+ patients, but only they had malignant thymoma (87.5%), serum antibodies against leucine-rich glioma-inactivated 1 protein (66.7%) and against netrin-1 receptor deleted in colorectal carcinoma (53.8%), and myasthenia gravis (50.0%). INTERPRETATION: Symptoms' distribution supports specific clinical phenotypes without overlap between LE and MoS. The distinct immunogenetic characteristics shared by all patients with LE and the particular oncological and autoimmune associations of MoS suggest two very different aetiopathogenesis.

Transient Neurological Symptoms Preceding Cerebellar Ataxia with Glutamic Acid Decarboxylase Antibodies.

A prompt diagnosis and treatment of patients with autoimmune cerebellar ataxia (CA) with antibodies against glutamic acid decarboxylase (GAD-Abs) may lead to a better prognosis. Herein, we report prodromal transient neurological symptoms that should raise clinical suspicion of CA with GAD-Abs. We initially identified a 70-year-old man who presented a first acute episode of vertigo, diplopia, and ataxia lasting 2 weeks. Two months later, he experienced a similar episode along with new-onset gaze-evoked nystagmus. After 4 months, downbeat nystagmus, left limb dysmetria, and gait ataxia progressively appeared, and an autoimmune CA was diagnosed based on the positivity of GAD-Abs in serum and cerebrospinal fluid (CSF). We searched retrospectively for similar presentations in a cohort of 31 patients diagnosed with CA and GAD-Abs. We found 11 (35.4%) patients (all women, median age 62 years; 8/11 [72.7%] with autoimmune comorbidities) with transient neurological symptoms antedating CA onset by a median of 3 months, including vertigo in 9 (81.8%; described as paroxysmal in 8) and fluctuating diplopia in 3 (27.3%) patients. The identification of transient neurological symptoms of unknown etiology, such as paroxysmal vertigo and fluctuating diplopia, should lead to GAD-Abs testing in serum and CSF, especially in patients with autoimmune comorbidities.

Evaluation of gaseous allyl isothiocyanate against the growth of mycotoxigenic fungi and mycotoxin production in corn stored for 6 months.

BACKGROUND: Brazil produces approximately 63 million tons of corn kernels annually, which is commonly contaminated with fungi and mycotoxins. The objective of this study was to evaluate the efficacy of gaseous allyl isothiocyanate (AITC) to inhibit the growth of Aspergillus parasiticus and Fusarium verticillioides, and mycotoxin production (aflatoxins B1 , B2 , G1 and G2 , fumonisins B1 and B2 ) in corn during 180 days of storage. RESULTS: AITC at 50 µL L-1 resulted in a significant reduction of the fungal population (P < 0.05) after 180 days, decreasing 3.17 log(CFU g-1 ) and 3.9 log(CFU g-1 ) of A. parasiticus and F. verticillioides respectively in comparison with the control. In addition, 10 and 50 µL L-1 treatments prevented the production of fumonisin B1 for the whole period. Aflatoxins were not detected in either control or treated groups. Residual levels of AITC in corn treated with 10 µL L-1 and 50 µL L-1 were detected up to 14 days and 30 days respectively. CONCLUSION: Prophylactic treatment with AITC reduced the fungal population and inhibited fumonisin B1 production in stored corn, exhibiting great potential to be applied in corn silos to prevent fungi contamination and minimize mycotoxin levels. © 2018 Society of Chemical Industry.

Whole-genome optical mapping reveals a mis-assembly between two rRNA operons of Corynebacterium pseudotuberculosis strain 1002.

BACKGROUND: Studies have detected mis-assemblies in genomes of the species Corynebacterium pseudotuberculosis. These new discover have been possible due to the evolution of the Next-Generation Sequencing platforms, which have provided sequencing with accuracy and reduced costs. In addition, the improving of techniques for construction of high accuracy genomic maps, for example, Whole-genome mapping (WGM) (OpGen Inc), have allow high-resolution assembly that can detect large rearrangements. RESULTS: In this work, we present the resequencing of Corynebacterium pseudotuberculosis strain 1002 (Cp1002). Cp1002 was the first strain of this species sequenced in Brazil, and its genome has been used as model for several studies in silico of caseous lymphadenitis disease. The sequencing was performed using the platform Ion PGM and fragment library (200 bp kit). A restriction map was constructed, using the technique of WGM with the enzyme KpnI. After the new assembly process, using WGM as scaffolder, we detected a large inversion with size bigger than one-half of genome. A specific analysis using BLAST and NR database shows that the inversion occurs between two homology RNA ribosomal regions. CONCLUSION: In conclusion, the results showed by WGM could be used to detect mismatches in assemblies, providing genomic maps with high resolution and allow assemblies with more accuracy and completeness. The new assembly of C. pseudotuberculosis was deposited in GenBank under the accession no. CP012837.

Differential transcriptional profile of Corynebacterium pseudotuberculosis in response to abiotic stresses.

BACKGROUND: The completion of whole-genome sequencing for Corynebacterium pseudotuberculosis strain 1002 has contributed to major advances in research aimed at understanding the biology of this microorganism. This bacterium causes significant loss to goat and sheep farmers because it is the causal agent of the infectious disease caseous lymphadenitis, which may lead to outcomes ranging from skin injury to animal death. In the current study, we simulated the conditions experienced by the bacteria during host infection. By sequencing transcripts using the SOLiDTM 3 Plus platform, we identified new targets expected to potentiate the survival and replication of the pathogen in adverse environments. These results may also identify possible candidates useful for the development of vaccines, diagnostic kits or therapies aimed at the reduction of losses in agribusiness. RESULTS: Under the 3 simulated conditions (acid, osmotic and thermal shock stresses), 474 differentially expressed genes exhibiting at least a 2-fold change in expression levels were identified. Important genes to the infection process were induced, such as those involved in virulence, defence against oxidative stress, adhesion and regulation, and many genes encoded hypothetical proteins, indicating that further investigation of the bacterium is necessary. The data will contribute to a better understanding of the biology of C. pseudotuberculosis and to studies investigating strategies to control the disease. CONCLUSIONS: Despite the veterinary importance of C. pseudotuberculosis, the bacterium is poorly characterised; therefore, effective treatments for caseous lymphadenitis have been difficult to establish. Through the use of RNAseq, these results provide a better biological understanding of this bacterium, shed light on the most likely survival mechanisms used by this microorganism in adverse environments and identify candidates that may help reduce or even eradicate the problems caused by this disease.

Reference genes for RT-qPCR studies in Corynebacterium pseudotuberculosis identified through analysis of RNA-seq data.

Reference genes presenting stable expression profiles over a wide variety of conditions are required in relative expression studies of specific bacterial genes by quantitative reverse transcription PCR (RT-qPCR). High-throughput sequencing of bacterial transcriptomes using the RNA-seq methodology now provides a wealth of data that may be searched for identification of the most stably expressed genes of a given bacterium. Herein, we searched a RNA-seq dataset from various experiments with the pathogenic bacterium Corynebacterium pseudotuberculosis, grown under different stress conditions, in order to select appropriate candidate reference genes for this species. Nineteen genes involved in maintenance of basic cellular functions, so-called housekeeping genes, were chosen for study and their expression profiles in C. pseudotuberculosis were evaluated throughout all growth conditions. Eight of these genes (atpA, dnaG, efp, fusA, gyrA, gyrB, rpoB, and rpoC), mostly participating in DNA replication and transcription, matched the defined criteria to be included as candidate reference genes. Transcriptional levels of these genes were quantified by RT-qPCR assays after growth of C. pseudotuberculosis under two additional conditions. Expression stability analysis by NormFinder indicated the combination of genes encoding DNA gyrase subunit A (gyrA) and elongation factor P (fusA) as the most suitable for normalization of RT-qPCR studies in C. pseudotuberculosis.

HLA-DR3 ~ DQ2 associates with sensory neuropathy in paraneoplastic neurological syndromes with Hu antibodies.

OBJECTIVES: To investigate the association between human leukocyte antigen (HLA) and paraneoplastic neurological syndromes (PNS) with Hu antibodies, and potential specificities according to clinical presentation and cancer status. METHODS: HLA genotypes at four-digit resolution were imputed from available genome-wide association data. Allele carrier frequencies were compared between patients (whole cohort, n = 100, and according to clinical presentation and cancer status) and matched healthy controls (n = 508) using logistic regression controlled by the three main principal components. RESULTS: The clinical presentation of 100 anti-Hu patients involved the central nervous system (28, 28%), the peripheral nervous system (36, 36%) or both combined (36, 36%). Cancer diagnosis was certain in 75 (75%). HLA association analyses revealed that anti-Hu PNS patients were more frequently carriers of DQA1*05:01 (39% vs. 19%, OR = 2.8 [1.74-4.49]), DQB1*02:01 (39% vs. 18%, OR = 2.88 [1.79-4.64]) and DRB1*03:01 (41% vs. 19%, OR = 2.92 [1.80-4.73]) than healthy controls. Remarkably, such DR3 ~ DQ2 association was absent in patients with pure central involvement, but more specific to those manifesting with peripheral involvement: DQA1*05:01 (OR = 3.12 [1.48-6.60]), DQB1*02:01 (OR = 3.35 [1.57-7.15]) and DRB1*03:01 (OR = 3.62 [1.64-7.97]); being even stronger in cases with sensory neuropathy, DQA1*05:01 (OR = 4.41 [1.89-10.33]), DQB1*02:01 (OR = 4.85 [2.04-11.53]) and DRB1*03:01 (OR = 5.79 [2.28-14.74]). Similarly, DR3 ~ DQ2 association was only observed in patients with cancer. DISCUSSION: Patients with anti-Hu PNS show different HLA profiles according to clinical presentation and, probably, cancer status, suggesting pathophysiological differences.

Diagnostic and prognostic biomarkers in immune checkpoint inhibitor-related encephalitis: a retrospective cohort study.

Background: Immune checkpoint inhibitor-related encephalitis (ICI-encephalitis) is not well characterised and diagnostic and prognostic biomarkers are lacking. We aimed to comprehensively characterise ICI-encephalitis and identify diagnostic biomarkers and outcome predictors. Methods: This retrospective observational study included all patients with ICI-encephalitis studied in the French Reference Centre on Paraneoplastic Neurological Syndromes (PNS) and Autoimmune Encephalitis (2015-2023). ICI encephalitis was considered definite in case of inflammatory findings at paraclinical tests and/or well-characterised neural antibodies. Predictors of immune-related adverse event (irAE) treatment response, defined as a Common Terminology Criteria for Adverse Events v5.0 grade < 3 at any time after therapeutic intervention, were assessed by logistic regression analysis, and predictors of mortality by Cox regression analysis. Neurofilament light chain (NfL) was measured by enzyme-linked immunosorbent assay. Findings: Sixty-seven patients with definite encephalitis were identified (median age, 69 years; 66% male). A focal syndrome was observed in 43/67 patients (64%; limbic encephalitis, cerebellar ataxia, and/or brainstem encephalitis), while 24/67 (36%) had meningoencephalitis, a non-focal syndrome with altered mental status (22/24 patients, 92%) and pleocytosis (24/24 patients, 100%). Patients with focal encephalitis more frequently had abnormal brain MRI (26/42, 62% versus 8/24, 33%, p = 0.025), PNS-related antibodies (36/43, 84% versus 1/24, 4%, p < 0.001), and neuroendocrine cancers (22/43, 51% versus 1/24, 4%; p < 0.001) than patients with meningoencephalitis. Focal encephalitis patients had a lower rate of irAE treatment response (7/39, 18%) and higher mortality (27/43, 63%) compared to meningoencephalitis patients (12/22, 77% and 5/24, 21%, respectively, p < 0.001 each). PNS-related antibodies were associated with less irAE treatment response, independently of age, sex, and baseline severity (adjusted OR 0.05; 95%CI [0.01; 0.19]; p < 0.001) as well as higher mortality, independently of age and cancer type (adjusted HR 5.07; 95% CI [2.12; 12.12]; p < 0.001). Serum NfL discriminated patients with definite ICI-encephalitis (n = 27) from cancer-matched controls (n = 16; optimal cut-off >273.5 pg/mL, sensitivity 81%, specificity 88%, AUC 0.87, 95% CI [0.76; 0.98]) and irAE treatment responders (n = 10) from non-responders (n = 17, optimal cut-off >645 pg/mL, sensitivity 90%, specificity 65%; AUC 0.75, 95% CI [0.55; 0.94]). Interpretation: ICI-encephalitis corresponds to a set of clinically-recognisable syndromes. Patients with focal encephalitis, PNS-related antibodies, and/or higher serum NfL have low irAE treatment response rates. Research is needed on the underlying immunopathogenesis to foster therapeutic innovations. Funding: Agence Nationale de la Recherche.

HLA and KIR genetic association and NK cells in anti-NMDAR encephalitis.

Introduction: Genetic predisposition to autoimmune encephalitis with antibodies against N-methyl-D-aspartate receptor (NMDAR) is poorly understood. Given the diversity of associated environmental factors (tumors, infections), we hypothesized that human leukocyte antigen (HLA) and killer-cell immunoglobulin-like receptors (KIR), two extremely polymorphic gene complexes key to the immune system, might be relevant for the genetic predisposition to anti-NMDAR encephalitis. Notably, KIR are chiefly expressed by Natural Killer (NK) cells, recognize distinct HLA class I allotypes and play a major role in anti-tumor and anti-infection responses. Methods: We conducted a Genome Wide Association Study (GWAS) with subsequent control-matching using Principal Component Analysis (PCA) and HLA imputation, in a multi-ethnic cohort of anti-NMDAR encephalitis (n=479); KIR and HLA were further sequenced in a large subsample (n=323). PCA-controlled logistic regression was then conducted for carrier frequencies (HLA and KIR) and copy number variation (KIR). HLA-KIR interaction associations were also modeled. Additionally, single cell sequencing was conducted in peripheral blood mononuclear cells from 16 cases and 16 controls, NK cells were sorted and phenotyped. Results: Anti-NMDAR encephalitis showed a weak HLA association with DRB1*01:01~DQA1*01:01~DQB1*05:01 (OR=1.57, 1.51, 1.45; respectively), and DRB1*11:01 (OR=1.60); these effects were stronger in European descendants and in patients without an underlying ovarian teratoma. More interestingly, we found increased copy number variation of KIR2DL5B (OR=1.72), principally due to an overrepresentation of KIR2DL5B*00201. Further, we identified two allele associations in framework genes, KIR2DL4*00103 (25.4% vs. 12.5% in controls, OR=1.98) and KIR3DL3*00302 (5.3% vs. 1.3%, OR=4.44). Notably, the ligands of these KIR2DL4 and KIR3DL3, respectively, HLA-G and HHLA2, are known to act as immune checkpoint with immunosuppressive functions. However, we did not find differences in specific KIR-HLA ligand interactions or HLA-G polymorphisms between cases and controls. Similarly, gene expression of CD56dim or CD56bright NK cells did not differ between cases and controls. Discussion: Our observations for the first time suggest that the HLA-KIR axis might be involved in anti-NMDAR encephalitis. While the genetic risk conferred by the identified polymorphisms appears small, a role of this axis in the pathophysiology of this disease appears highly plausible and should be analyzed in future studies.

Comparative Study of Paraneoplastic and Nonparaneoplastic Autoimmune Encephalitis With GABABR Antibodies.

BACKGROUND AND OBJECTIVES: While patients with paraneoplastic autoimmune encephalitis (AE) with gamma-aminobutyric-acid B receptor antibodies (GABABR-AE) have poor functional outcomes and high mortality, the prognosis of nonparaneoplastic cases has not been well studied. METHODS: Patients with GABABR-AE from the French and the Dutch Paraneoplastic Neurologic Syndromes Reference Centers databases were retrospectively included and their data collected; the neurologic outcomes of paraneoplastic and nonparaneoplastic cases were compared. Immunoglobulin G (IgG) isotyping and human leukocyte antigen (HLA) genotyping were performed in patients with available samples. RESULTS: A total of 111 patients (44/111 [40%] women) were enrolled, including 84 of 111 (76%) paraneoplastic and 18 of 111 (16%) nonparaneoplastic cases (cancer status was undetermined for 9 patients). Patients presented with seizures (88/111 [79%]), cognitive impairment (54/111 [49%]), and/or behavioral disorders (34/111 [31%]), and 54 of 111 (50%) were admitted in intensive care unit (ICU). Nonparaneoplastic patients were significantly younger (median age 54 years [range 19-88] vs 67 years [range 50-85] for paraneoplastic cases, p < 0.001) and showed a different demographic distribution. Nonparaneoplastic patients more often had CSF pleocytosis (17/17 [100%] vs 58/78 [74%], p = 0.02), were almost never associated with KTCD16-abs (1/16 [6%] vs 61/70 [87%], p < 0.001), and were more frequently treated with second-line immunotherapy (11/18 [61%] vs 18/82 [22%], p = 0.003). However, no difference of IgG subclass or HLA association was observed, although sample size was small (10 and 26 patients, respectively). After treatment, neurologic outcome was favorable (mRS ≤2) for 13 of 16 (81%) nonparaneoplastic and 37 of 84 (48%) paraneoplastic cases (p = 0.03), while 3 of 18 (17%) and 42 of 83 (51%) patients had died at last follow-up (p = 0.008), respectively. Neurologic outcome no longer differed after adjustment for confounding factors but seemed to be negatively associated with increased age and ICU admission. A better survival was associated with nonparaneoplastic cases, a younger age, and the use of immunosuppressive drugs. DISCUSSION: Nonparaneoplastic GABABR-AE involved younger patients without associated KCTD16-abs and carried better neurologic and vital prognoses than paraneoplastic GABABR-AE, which might be due to a more intensive treatment strategy. A better understanding of immunologic mechanisms underlying both forms is needed.

Recent advances in the use of bacterial probiotics in animal production.

Animal husbandry is increasingly under pressure to meet world food demand. Thus, strategies are sought to ensure this productivity increment. The objective of this review was to gather advances in the use of bacterial probiotics in animal production. Lactobacilli correspond to the most used bacterial group, with several beneficial effects already reported and described, as well as the Enterococcus and Pediococcus genera - being the latter expressively used in aquaculture. Research on the Bifidobacterium genus is mostly focused on human health, which demonstrates great effects on blood biochemical parameters. Such results sustain the possibility of expanding its use in veterinary medicine. Other groups commonly assessed for human medicine but with prospective expansion to animal health are the genera Leuconostoc and Streptococcus, which have been demonstrating interesting effects on the prevention of viral diseases, and in dentistry, respectively. Although bacteria from the genera Bacillus and Lactococcus also have great potential for use in animal production, a complete characterization of the candidate strain must be previously made, due to the existence of pathogenic and/or spoilage variants. It is noteworthy that a growing number of studies have investigated the genus Propionibacterium, but still in very early stages. However, the hitherto excellent results endorse its application. In this way, in addition to the fact that bacterial probiotics represent a promising approach to promote productivity increase in animal production, the application of other strains than the traditionally employed genera may allow the exploitation of novel mechanisms and enlighten unexplored possibilities.

SOX1 antibody-related paraneoplastic neurological syndromes: clinical correlates and assessment of laboratory diagnostic techniques.

OBJECTIVE: To describe the clinical associations of SOX1 antibodies (SOX1-Abs), determine the accuracy of various detection techniques, and propose laboratory criteria to identify definite paraneoplastic neurological syndromes (PNS) associated with SOX1-Abs. METHODS: Single-center, retrospective study of patients referred to the French Reference Center between 2009 and 2019 for confirmation of SOX1-Ab positivity, without concurrent neural antibodies. Patients were classified according to the updated diagnostic PNS criteria; biological samples were systematically retested with three distinct techniques (line blot, cell-based assay, indirect immunofluorescence). RESULTS: Among 77 patients with isolated SOX1-Ab positivity, 23 (29.9%) fulfilled the criteria for definite PNS; all of them had lung cancer (mostly small-cell) and presented mainly with Lambert-Eaton myasthenic syndrome (10/23) and rapidly progressive cerebellar ataxia (6/23). SOX1-Ab positivity varied depending on the laboratory methods which were used, and a single technique was not sufficient to draw conclusions about the PNS diagnosis. The combination of an antigen-specific test (line blot and/or cell-based assay) and immunofluorescence showed the highest accuracy (81.5%, 95% CI 70.0-90.1) in identifying definite PNS. Moreover, when the PNS-Care score was recalculated assigning three points at the laboratory-level only to patients with positive "antigenic-specific test + immunofluorescence" and 0 points to the remaining cases, a higher certainty for definite and non-PNS was achieved (from 41/77, 53.2%, to 60/77, 77.9%; p < 0.001). CONCLUSION: SOX1-Abs should be considered high-risk antibodies only when detected with a positive antigenic-specific test and immunofluorescence. Other laboratory results and clinical associations different from Lambert-Eaton myasthenic syndrome and rapidly progressive cerebellar ataxia should be carefully reassessed to rule out false positivity and alternative diagnoses.

Early-Stage Contactin-Associated Protein-like 2 Limbic Encephalitis: Clues for Diagnosis.

BACKGROUND AND OBJECTIVES: Previous studies suggested that autoimmune limbic encephalitis with antibodies against contactin-associated protein-like 2 (CASPR2-encephalitis) is clinically heterogeneous and progresses slowly, preventing its early recognition. We aimed to describe the onset and progression of CASPR2-encephalitis and to assess long-term outcomes. METHODS: We retrospectively analyzed the medical records of all patients whose CSF tested positive for anti-CASPR2 antibodies in our center between 2006 and 2020. Standardized telephone interviews of all available patients and relatives were conducted, assessing long-term functional independence using the Functional Activity Questionnaire (FAQ) and quality of life using the 36-Item Short-Form Survey (SF36). RESULTS: Forty-eight patients were included (98% males; median age 64 years), and 35 participated in telephone interviews (73%). At onset, 81% had at least 1 neurologic symptom among the following: limbic (54%), peripheral nerve hyperexcitability (PNH; 21%), and/or cerebellar symptoms (17%). Most of the patients (75%) had initially symptoms of only one of these categories. Limbic symptoms at onset included mostly seizures (33%), while memory disturbances were less frequent (10%). PNH signs were mostly neuropathic pain (9/10 patients). Other symptoms seen at onset included asthenia (33%), mood disorders (25%), and insomnia (21%); 19% of patients did not show any limbic, peripheral, or cerebellar symptom at onset but only asthenia (15%), mood disorders (6%), weight loss (8%), dysautonomia (4%), and/or insomnia (2%). The peak of the disease was attained in median 16.7 months after onset. Over the study period (median follow-up, 58.8 months, range 10.6-189.1), 77% of patients developed ≥3 core CASPR2 symptoms and 42% fulfilled the diagnostic criteria for autoimmune limbic encephalitis, although all patients ultimately developed limbic symptoms. At the last visit, most interviewed patients (28/35 patients, 80%; median, 5 years after onset) had recovered functional independence (FAQ <9) while only the vitality subscore of the SF36 was lower than normative data (mean 49.9 vs 58.0, p = 0.0369). DISCUSSION: CASPR2-encephalitis has a progressive course and is highly heterogeneous at the early stage. In men older than 50 years, otherwise unexplained seizures, cerebellar ataxia, and/or neuropathic pain are suggestive of early-stage CASPR2-encephalitis, especially if they coincide with recent asthenia, mood disorders, or insomnia.

Genome sequence of the Corynebacterium pseudotuberculosis Cp316 strain, isolated from the abscess of a Californian horse.

The bacterium Corynebacterium pseudotuberculosis is of major veterinary importance because it affects livestock, particularly sheep, goats, and horses, in several countries, including Australia, Brazil, the United States, and Canada, resulting in significant economic losses. In the present study, we describe the complete genome of the Corynebacterium pseudotuberculosis Cp316 strain, biovar equi, isolated from the abscess of a North American horse.