Rare variants and HLA haplotypes associated in patients with neuromyelitis optica spectrum disorders.

Neuromyelitis optica spectrum disorders (NMOSD) are rare, debilitating autoimmune diseases of the central nervous system. Many NMOSD patients have antibodies to Aquaporin-4 (AQP4). Prior studies show associations of NMOSD with individual Human Leukocyte Antigen (HLA) alleles and with mutations in the complement pathway and potassium channels. HLA allele associations with NMOSD are inconsistent between populations, suggesting complex relationships between the identified alleles and risk of disease. We used a retrospective case-control approach to identify contributing genetic variants in patients who met the diagnostic criteria for NMOSD and their unaffected family members. Potentially deleterious variants identified in NMOSD patients were compared to members of their families who do not have the disease and to existing databases of human genetic variation. HLA sequences from patients from Belgrade, Serbia, were compared to the frequency of HLA haplotypes in the general population in Belgrade. We analyzed exome sequencing on 40 NMOSD patients and identified rare inherited variants in the complement pathway and potassium channel genes. Haplotype analysis further detected two haplotypes, HLA-A*01, B*08, DRB1*03 and HLA-A*01, B*08, C*07, DRB1*03, DQB1*02, which were more prevalent in NMOSD patients than in unaffected individuals. In silico modeling indicates that HLA molecules within these haplotypes are predicted to bind AQP4 at several sites, potentially contributing to the development of autoimmunity. Our results point to possible autoimmune and neurodegenerative mechanisms that cause NMOSD, and can be used to investigate potential NMOSD drug targets.

Immune cell profiles of patients with interstitial cystitis/bladder pain syndrome.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a disorder characterized by bladder pain upon filling which severely affects quality of life. Clinical presentation can vary. Local inflammatory events typify the clinical presentation of IC/BPS patients with Hunner lesions (IC/BPS-HL). It has previously been proposed that B cells are more prevalent in HL, but understanding their exact role in this environment requires a more complete immunological profile of HL. We characterized immunological dysfunction specifically in HL using immunohistochemistry. We detected significantly more plasma cells (50× increase, p < 0.0001), B cells (28× increase, p < 0.0001), T cells (3× increase, p < 0.0001), monocytes/macrophages (6× increase, p < 0.0001), granulocytes (4× increase, p < 0.0001), and natural killer cells (2× increase, p = 0.0249) in IC/BPS patients with HL than in unaffected controls (UC). Patients with IC/BPS-HL also had significantly elevated urinary levels of IL-6 (p = 0.0054), TNF-α (p = 0.0064) and IL-13 (p = 0.0304) compared to patients with IC/BPS without HL (IC/BPS-NHL). In contrast, IL-12p70 levels were significantly lower in the patients with HL than in those without these lesions (p = 0.0422). Different cytokines were elevated in the urine of IC/BPS patients with and without HL, indicating that different disease processes are active in IC/BPS patients with and without HL. Elevated levels of CD138+, CD20+, and CD3+ cells in HL are consistent B and T-cell involvement in disease processes within HL.

Defining the role of T lymphocytes in the immunopathogenesis of neuromyelitis optica spectrum disorder.

Auto-reactive T cells are fundamental to many autoimmune processes, including neuromyelitis optica spectrum disorder (NMOSD). Several lines of evidence indicate that an antibody against aquaporin-4 (AQP4) is present in NMOSD patients. Further, this AQP4 antibody is pathogenic and can cause profound neurological damage. T cells are fundamental to many autoimmune processes, including NMOSD. Here we review work from animal models to discuss mechanisms by which auto-reactive T cells modulate the process by which antibodies cross the blood-brain barrier and orchestrate the local inflammatory milieu underlying NMOSD pathophysiology. We also examine clinical studies that document the presence of AQP4-specific T cells and the unique cytokine profile of NMOSD patients. This work encourages a renewed and broadened attention to the fundamental role of T cells in neuroautoimmune conditions which will hopefully lead to new therapies and better patients' outcomes.

Targeting DEC-205-DCIR2+ dendritic cells promotes immunological tolerance in proteolipid protein-induced experimental autoimmune encephalomyelitis.

BACKGROUND: Dendritic cells (DC) induce adaptive responses against foreign antigens, and play an essential role in maintaining peripheral tolerance to self-antigens. Therefore they are involved in preventing fatal autoimmunity. Selective delivery of antigens to immature DC via the endocytic DEC-205 receptor on their surface promotes antigen-specific T cell tolerance, both by recessive and dominant mechanisms. We provide evidence that the induction of antigen-specific T cell tolerance is not a unique property of CD11c+CD8+DEC-205+ DCs. METHODS: We employed a fusion between αDCIR2 antibodies and the highly encephalitogenic peptide 139-151 of myelin-derived proteolipid protein (PLP139-151), to target CD11c +CD8- DCs with a DEC-205-DCIR2+ phenotype in vivo, and to substantially improve clinical symptoms in the PLP139-151-induced model of experimental autoimmune encephalomyelitis (EAE). RESULTS: Consistent with previous studies targeting other cell surface receptors, EAE protection mediated by αDCIR2-PLP139-151 fusion antibody (Ab) depended on an immature state of targeted DCIR2+ DCs. The mechanism of αDCIR2-PLP139-151 mAb function included the deletion of IL-17- and IFN-γ-producing pathogenic T cells, as well as the enhancement of regulatory T (Treg) cell activity. In contrast to the effect of αDEC-205+ fusion antibodies, which involves extrathymic induction of a Foxp3+ Treg cell phenotype in naïve CD4+Foxp3- T cells, treatment of animals with DCIR2+ fusion antibodies resulted in antigen-specific activation and proliferative expansion of natural Foxp3+ Treg cells. CONCLUSIONS: These results suggest that multiple mechanisms can lead to the expansion of the Treg population, depending on the DC subset and receptor targeted.

Crosstalk between the immune system and neural pathways in interstitial cystitis/bladder pain syndrome.

Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS) is a condition causing intense pelvic pain and urinary symptoms. While it is thought to affect millions of people and significantly impair quality of life, difficulty with diagnosis and a lack of reliably effective treatment options leave much progress to be made in managing this condition. We describe what is currently known about the immunological and neurological basis of this disease, focusing on the interactions between the immune and nervous system. Evidence for immune involvement in IC/BPS comes from its high co-occurrence with known autoimmune diseases, altered cytokine profiles, and immune cell infiltration in patients. These cytokines have the ability to cross-talk with the nervous system via NGF signaling, resulting in hyper-sensitization of pain receptors, causing them to release substance P and creating a positive feedback loop of neuroinflammation. While it seems that the crosstalk between the immune and nervous system in IC is understood, much of the information comes from studying other diseases or from animal models, and it remains to be confirmed in patients with the disease. Identifying biomarkers and confirming the mechanism of IC/BPS are ultimately important for selecting drug targets and for improving the lives of patients with this disease.

Endoscopic Injection of Low Dose Triamcinolone: A Simple, Minimally Invasive, and Effective Therapy for Interstitial Cystitis With Hunner Lesions.

OBJECTIVE: To investigate the efficacy of low dose triamcinolone injection for effectiveness and durability in patients with interstitial cystitis/bladder pain syndrome (IC/BPS) with Hunner lesions (HL). MATERIALS AND METHODS: Clinical data from patients with HL who underwent endoscopic submucosal injection of triamcinolone were reviewed. Demographics, pre- and postoperative pain and nocturia scores, and long-term clinical outcomes were assessed. Duration of response was estimated by time to repeat procedure. Kaplan-Meier estimator was used to evaluate time to repeat procedure. RESULTS: A total of 36 patients who received injections of triamcinolone between 2011 and 2015 were included. Median age ± standard deviation of patients was 61.5 ± 12.0 years; 28 (77.8%) were female patients and 8 (22.2%) were male patients. Twenty six patients (72.2%) received only 1 set of injections, 8 (22.2%) received 2 sets of injections, and 2 (5.56%) received 3 or more sets of injections. Average time between injections in those receiving more than 1 set of injections was 344.9 days (median: 313.5, range: 77-714). Preprocedural pain scores were 8.3 ± 1.2 (mean ± standard deviation) on Likert pain scale (0-10), and mean postprocedural pain scores at approximately 1 month were 3.8 ± 2.2, P <.001. Mean preprocedural nocturia bother scores was 7.5 ± 2.0 and mean postprocedural nocturia bother scores was 5.1 ± 2.5, P <.001. CONCLUSION: Endoscopic submucosal injection of low dose triamcinolone in patients with IC/BPS with HL is an effective and durable adjunct to existing treatment modalities. This approach is associated with low morbidity and can be performed on an outpatient basis.

The role of B cells in multiple sclerosis: more than antibodies.

Multiple sclerosis (MS) is a multicomponent disease that is marked by continual inflammation, demyelination and irreparable damage to the central nervous system. While it was long thought to be mediated by T cells, B cells are now understood to be a central component of MS pathology. Dysfunction and aberrant activity of antigen presenting cells, T cells and B cells are all part of the pathophysiology of the disease. B cells and plasma cells contribute to disease progression through multiple mechanisms, including cytokine secretion, antibody production and antigen presentation. More recent evidence suggests that B cells may play a larger role than previously thought in driving acute episodes of MS. In this review we explore the classical understanding of MS, the evidence and current understanding of B cells in the central nervous system in health and disease, and the interactions present between B cells in the central nervous system and the peripheral nervous system. Lastly, we explore targeted immunological treatments which affect B cells and how this has informed our understanding of MS.

Effect of alginate microencapsulation on the catalytic efficiency and in vitro enzyme-prodrug therapeutic efficacy of cytosine deaminase and of recombinant E. coli expressing cytosine deaminase.

Cytosine deaminase (CD) catalyses the enzymatic conversion of the non-toxic prodrug 5-fluorocytosine (5-FC) to the potent chemotherapeutic form, 5-fluorouracil (5-FU). Intratumoral delivery of CD localises chemotherapy dose while reducing systemic toxicity. Encapsulation in biocompatible microcapsules immunoisolates CD and protects it from degradation. We report on the effect of alginate encapsulation on the catalytic and functional activity of isolated CD and recombinant E. coli engineered to express CD (E. coli(CD)). Alginate microcapsules containing either CD or Escherichia coli(CD) were prepared using ionotropic gelation. Conversion of 5-FC to 5-FU was quantitated in unencapsulated and encapsulated CD/E. coli(CD) using spectrophotometry, with a slower rate of conversion observed following encapsulation. Both encapsulated CD/5-FC and E. coli(CD)/5-FC resulted in cell kill and reduced proliferation of 9 L rat glioma cells, which was comparable to direct 5-FU treatment. Our results show that encapsulation preserves the therapeutic potential of CD and E. coli(CD) is equally effective for enzyme-prodrug therapy.

Novel markers of male infertility.

Diagnostic tests should detect disease, have prognostic value, and aid in clinical decision making. Nowhere else in laboratory medicine does one have to interpret a subject's results within the dynamic of a couple as in reproductive medicine. Abnormal markers of male reproduction do not necessarily mean sterility, but instead indicate problems with spermatogenesis, sperm maturation, transport through epididymis and ejaculatory duct, or abnormal ejaculatory function. Decades of research suggest that one test will never fit all scenarios and a battery of assays evaluating different aspects of male reproduction will likely have the best prognostic value. There is a strong need for standardization and harmonization of evolving assays to establish their clinical relevance. Next-generation genome sequencing and the discovery of small noncoding RNAs in sperm already are changing the field and permit further insight into the biology of male reproduction as well as offer new diagnostic tests.

Activation of GPER-1 estradiol receptor downregulates production of testosterone in isolated rat Leydig cells and adult human testis.

PURPOSE: Estradiol (E2) modulates testicular functions including steroidogenesis, but the mechanisms of E2 signaling in human testis are poorly understood. GPER-1 (GPR30), a G protein-coupled membrane receptor, mediates rapid genomic and non-genomic response to estrogens. The aim of this study was to evaluate GPER-1 expression in the testis, and its role in estradiol dependent regulation of steroidogenesis in isolated rat Leydig cells and human testis. MATERIALS AND METHODS: Isolated Leydig cells (LC) from adult rats and human testicular tissue were used in this study. Expression and localization studies of GPER-1 were performed with qRT-PCR, immunofluorescence, immunohistochemistry and Western Blot. Luteinizing Hormone (LH) -stimulated, isolated LC were incubated with estradiol, G-1 (GPER-1-selective agonist), and estrogen receptor antagonist ICI 182,780. Testosterone production was measured with radioimmunoassay. LC viability after incubation with G-1 was measured using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay. RESULTS: GPER-1 mRNA is abundantly expressed in rat LC and human testis. Co-localization experiments showed high expression levels of GPER-1 protein in LC. E2-dependent activation of GPER-1 lowers testosterone production in isolated rats LCs and in human testis, with statistically and clinically significant drops in testosterone production by 20-30% as compared to estradiol-naïve LC. The exposure to G-1 does not affect viability of isolated LCs. CONCLUSIONS: Our results indicate that activation of GPER-1 lowers testosterone levels in the rat and human testis. The expression of GPER-1 in human testis, which lack ERα, makes it an exciting target for developing new agents affecting testosterone production in men.

A novel sorting technology allows for highly efficient selection of sperm without chromatin damage.

Sperm chromatin damage has been associated with male infertility, increased risk for spontaneous abortion, and poor embryo development. Available methods for detecting chromatin damage render the sperm no longer suitable for clinical use. Early apoptotic events resulting in chromatin damage are associated with increased permeability of the cell membrane to large ions. We propose the use of a large fluorescent organic cation, proprietary fluorochrome (PF-1), for fluorescence-activated cell sorting (FACS) for negative selection of sperm without chromatin damage. Sperm with chromatin damage are PF-1 positive. Performance of cell sorting by PF-1 was verified with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) after FACS on PF-1(+) and PF-1(-) subpopulations. Whereas 19.5% of PF-1 positive sperm were TUNEL positive only 1.5% sperm in the PF-1(-) fraction were TUNEL positive (p < 0.00001). TUNEL values below 1.9% were considered background fluorescence. Post-sorting motility and vitality were 49.4% (SD: 12.5) and 65.0% (SD: 14.99), respectively. Proprietary fluorochrome activated sperm sorting may decrease or most likely eliminate all of TUNEL positive sperm without adverse effects on viability, providing a new therapeutic avenue for men with a high percentage of TUNEL positive sperm. Further research is needed to determine if the reduction in TUNEL positive sperm using PF-1 will improve in vitro fertilization (IVF) outcomes.

Flow cytometric characterization of apoptosis and chromatin damage in spermatozoa.

Apoptosis has been implicated in sperm chromatin damage; it is unclear whether apoptosis occurs through cytoplasmic or mitochondrial pathways. Sperm has minimal volume of cytoplasm but prominent mitochondria. Propidium iodide (PI), annexin V (AV), DiIC1(5) and proprietary fluorochrome (PF-1) were used to investigate apoptosis activation in human sperm using multichannel flow cytometry. There was a time-dependent increase in staining of spermatozoa with both AV and PF-1 and decrease in mitochondrial staining with DiIC1(5). These results strongly suggest that the drop in mitochondrial potential precedes changes in membrane phospholipids, and thus suggest apoptotic activation through mitochondrial pathway in human spermatozoa.