HLA-DR Expression on Monocytes and Sepsis Index Are Useful in Predicting Sepsis.

The reduction of mortality in patients with sepsis depends on the early identification and treatment of at-risk patients. The aim was to evaluate the HLA-DR expression on the surface of monocytes (MHLA-DR ratio), the sepsis index (CD64 expression on neutrophils/MHLA-DR ratio), and C-reactive protein (CRP) with the development of sepsis. We prospectively enrolled 77 critically ill patients, 59 with stroke and 18 with traumatic brain injuries. The biomarkers were tested at the baseline and 3, 6, 9, 12, and 15 days later. Most patients (71%) developed sepsis (4.2 ± 1.3 days after admission). On day 3, those subsequently developing sepsis had lower levels of MHLA-DR+ (81.7 ± 16.2% vs. 88.5 ± 12.1%, p < 0.05) and higher sepsis indexes (0.19 ± 0.19 vs. 0.08 ± 0.08, p < 0.01) than those not developing sepsis. The MHLA-DR ratio slowly recovered before day 6, while the sepsis index remained raised in septic patients up to day 9 (p < 0.05). To predict the development of sepsis, optimal cut-offs were CRP levels > 106.90 mg/mL (74.19% sensitivity, 69.49 specificity) and MHLA-DR expression rate < 72.80% (45.31% sensitivity, 89.47% specificity). The periodic monitoring of the MHLA-DR expression together with CRP and sepsis index may help to identify patients in the ICU at increased risk of developing sepsis.

Clinical and immunological control of experimental autoimmune encephalomyelitis by tolerogenic dendritic cells loaded with MOG-encoding mRNA.

BACKGROUND: Although effective in reducing relapse rate and delaying progression, current therapies for multiple sclerosis (MS) do not completely halt disease progression. T cell autoimmunity to myelin antigens is considered one of the main mechanisms driving MS. It is characterized by autoreactivity to disease-initiating myelin antigen epitope(s), followed by a cascade of epitope spreading, which are both strongly patient-dependent. Targeting a variety of MS-associated antigens by myelin antigen-presenting tolerogenic dendritic cells (tolDC) is a promising treatment strategy to re-establish tolerance in MS. Electroporation with mRNA encoding myelin proteins is an innovative technique to load tolDC with the full spectrum of naturally processed myelin-derived epitopes. METHODS: In this study, we generated murine tolDC presenting myelin oligodendrocyte glycoprotein (MOG) using mRNA electroporation and we assessed the efficacy of MOG mRNA-electroporated tolDC to dampen pathogenic T cell responses in experimental autoimmune encephalomyelitis (EAE). For this, MOG35-55-immunized C57BL/6 mice were injected intravenously at days 13, 17, and 21 post-disease induction with 1α,25-dihydroxyvitamin D3-treated tolDC electroporated with MOG-encoding mRNA. Mice were scored daily for signs of paralysis. At day 25, myelin reactivity was evaluated following restimulation of splenocytes with myelin-derived epitopes. Ex vivo magnetic resonance imaging (MRI) was performed to assess spinal cord inflammatory lesion load. RESULTS: Treatment of MOG35-55-immunized C57BL/6 mice with MOG mRNA-electroporated or MOG35-55-pulsed tolDC led to a stabilization of the EAE clinical score from the first administration onwards, whereas it worsened in mice treated with non-antigen-loaded tolDC or with vehicle only. In addition, MOG35-55-specific pro-inflammatory pathogenic T cell responses and myelin antigen epitope spreading were inhibited in the peripheral immune system of tolDC-treated mice. Finally, magnetic resonance imaging analysis of hyperintense spots along the spinal cord was in line with the clinical score. CONCLUSIONS: Electroporation with mRNA is an efficient and versatile tool to generate myelin-presenting tolDC that are capable to stabilize the clinical score in EAE. These results pave the way for further research into mRNA-electroporated tolDC treatment as a patient-tailored therapy for MS.

Optimal response to dimethyl fumarate is mediated by a reduction of Th1-like Th17 cells after 3 months of treatment.

AIM: Dimethyl fumarate (DMF) is one of the most promising therapies for relapsing-remitting multiple sclerosis (RRMS) patients since it has shown immunomodulatory and neuroprotective effects. However, a percentage of RRMS patients do not exhibit an optimal response to DMF. The objective of this study was to identify early biomarkers of treatment response by analyzing changes in peripheral leukocyte subpopulations directly in whole blood samples. METHODS: A longitudinal and prospective study analyzing peripheral blood leukocyte subpopulations in 22 RRMS patients before initiating DMF treatment (baseline) and at 1, 3, 6, and 12 months of follow-up was performed. Differences between no evidence of disease activity (NEDA) and ongoing disease activity (ODA) patients were analyzed. RESULTS: The beneficial effect of DMF was associated with a specific depletion of memory CD4+ and CD8+ T lymphocytes and B cells. Importantly, only NEDA patients showed (a) a shift from a pro- to an antiinflammatory profile, with an increase of Th2 cells and a decrease of Th1-like Th17 lymphocytes; and (b) an increase of regulatory CD56bright NK cells. CONCLUSION: The optimal response to DMF is mediated by a shift to antiinflammatory and immunoregulatory profile, which puts forward Th1-like Th17 lymphocytes as a potential early biomarker of treatment response.

Monitoring CD49d Receptor Occupancy: A Method to Optimize and Personalize Natalizumab Therapy in Multiple Sclerosis Patients.

BACKGROUND: In natalizumab-treated relapsing-remitting MS (RRMS) patients, various extended interval dosing strategies are under evaluation to minimize severe treatment-associated side effects, mainly progressive multifocal leukoencephalopathy development. Up to now, it has not been presented any approach, even in form of assay design, to determine the optimal percentage of CD49d receptor occupancy (RO) associated with a favorable clinical, radiological, and immunological response. METHODS: A multiparametric quantitative flow cytometry method was settled to measure CD49d RO on peripheral blood lymphocytes. The analytical protocol was tested in a 6-month follow-up from 19 RRMS patients treated with the natalizumab standard dosing of every 4 weeks or an extended-interval dosing of every 6 weeks. RESULTS: Extended natalizumab dose schedule promoted an increase of CD49d molecules per cell surface and a reduction of CD49d RO levels. The reduction observed on CD49d RO was not only depending on dose schedule but also on individual parameters such as body mass. Interestingly, individual clinical outcome was apparently the same between the different dose schedules or even better with the extended interval dosing. CONCLUSIONS: Following up CD49d RO levels with a well-regulated monitoring work scheme is crucial to further identify over-/under-treated patients and to define a safe, personalized natalizumab regimen. © 2017 International Clinical Cytometry Society.

Cryopreserved vitamin D3-tolerogenic dendritic cells pulsed with autoantigens as a potential therapy for multiple sclerosis patients.

BACKGROUND: Tolerogenic dendritic cells (tolDC) have been postulated as a potent immunoregulatory therapy for autoimmune diseases such as multiple sclerosis (MS). In a previous study, we demonstrated that the administration of antigen-specific vitamin D3 (vitD3) tolDC in mice showing clinical signs of experimental autoimmune encephalomyelitis (EAE; the animal model of MS) resulted in abrogation of disease progression. With the purpose to translate this beneficial therapy to the clinics, we have investigated the effectivity of vitD3-frozen antigen-specific tolDC pulsed with myelin oligodendrocyte glycoprotein 40-55 peptide (f-tolDC-MOG) since it would reduce the cost, functional variability and number of leukapheresis to perform to the patients. METHODS: Mice showing EAE clinical signs were treated with repetitive doses of f-tolDC-MOG. Tolerogenic mechanisms induced by the therapy were analysed by flow cytometry and T cell proliferation assays. RESULTS: Treatment with f-tolDC-MOG was effective in ameliorating clinical signs of mice with EAE, inhibiting antigen-specific reactivity and inducing Treg. In addition, the long-term treatment was well tolerated and leading to a prolonged maintenance of tolerogenicity mediated by induction of Breg, reduction of NK cells and activation of immunoregulatory NKT cells. CONCLUSIONS: The outcomes of this study show that the use of antigen-specific f-tolDC promotes multiple and potent tolerogenic mechanisms. Moreover, these cells can be kept frozen maintaining their tolerogenic properties, which is a relevant step for their translation to the clinic. Altogether, vitD3 f-tolDC-MOG is a potential strategy to arrest the autoimmune destruction in MS patients.

Baseline Differences in Minor Lymphocyte Subpopulations may Predict Response to Fingolimod in Relapsing-Remitting Multiple Sclerosis Patients.

AIMS: Fingolimod, oral treatment for relapsing-remitting multiple sclerosis (RRMS), is an agonist of sphingosine and its metabolite S1P that binds their receptors, blocking the egress of lymphocytes from lymph nodes. The aim of this study was immunomonitoring of minor peripheral lymphocyte subpopulations in RRMS patients under treatment with fingolimod and correlation with treatment response. METHODS: Prospective study. T- and B-cell subpopulations were analyzed using multiparametric flow cytometry in peripheral blood from 14 RRMS patients under treatment with fingolimod at baseline, +1, +3, +6, +9, and +12 months of follow-up. Response to therapy was assessed at month +12. RESULTS: Most changes in minor lymphocyte subpopulations occurred in the first month of treatment and were maintained until the end of follow-up. The basal percentages of recent thymic emigrants (RTEs) and transitional B cells were lower in responder patients than in nonresponders. After 1 month of follow-up, the percentages of late effector memory CD4(+) T cells in peripheral blood were higher in responder patients. CONCLUSION: If confirmed in a bigger cohort of patients, analysis of percentages of minor lymphocyte subpopulations in peripheral blood of patients with RRMS prior and after +1 month of treatment might predict clinical response to fingolimod.

Analysis of the cumulative changes in Graves' disease thyroid glands points to IFN signature, plasmacytoid DCs and alternatively activated macrophages as chronicity determining factors.

Graves' disease (GD) is a chronic autoimmune process in the thyroid gland and involves IFN and IFN driven immune activation. Assuming the thyroid gland is the main site stimulating the autoimmune response, we investigated the role of IFNs and other factors in the chronic evolution of GD by comparing the transcriptomic profiles of thyroid glands from short clinical course (SC), long clinical course (LC) cases, and control glands (C). Over 200 differentially expressed genes of the immune system were identified. Results were extensively analyzed bioinformatically and validated by qPCR in 31 glands. The analysis indicated that GD involved a progressive accumulation of changes with clearly distinct profiles in the SC and LC glands. Humoral response, antigen presentation and chemokines & cytokines were overall the most represented gene ontology categories in LC cases. Ingenuity Pathway Analysis pointed to a few inflammatory pathways in SC cases whereas LC cases involved numerous complex pathways, such us "communication between innate and adaptive immune cells" and "autoimmune thyroid signaling". A broad IFN signature consisted of the over-expression of 74 and 84 type I and type II IFN responsive genes respectively (overall 96 out of 211, 45%), but many of these genes can also be directly activated through cytoplasmic viral receptors. For the first time, plasmocytoid dendritic cells were identified in GD thyroid, but surprisingly, the main producers of IFN-alpha were cells with a myeloid cell phenotype. In addition, cells with the phenotype of alternatively activated macrophages were detected in abundance in GD thyroids, confirming data from the transcriptomic analysis. Collectively, these results confirmed the role of IFNs, suggested other natural immunity triggers, identified new cell types in the local disease process, and expanded our knowledge of the processes that may determine the chronicity of GD.

Undiagnosed thyroid dysfunction, thyroid antibodies, and iodine excretion in a Mediterranean population.

The prevalence of thyroid dysfunction varies in different populations. The aim of this cross-sectional study was to analyze the prevalence of undiagnosed thyroid dysfunction and thyroid antibodies and their relationship with urine iodine excretion in a representative sample of 1,124 (55.5% women; mean age: 44.8 ± 15.2 years) non-hospitalized Mediterranean adults, in Catalonia (Spain). Free thyroxine, thyroid-stimulating hormone, thyroperoxidase and thyroglobulin antibodies, and urine iodine were measured. Undiagnosed thyroid dysfunction was 5.3% (hypothyroidism 3.8%; 56.66% of these subjects were women). The total (diagnosed + undiagnosed) thyroid dysfunction was 8.9% (71.15% women). Thyroperoxidase antibodies were positive in 2.4% of men and 9.4% of women and thyroglobulin antibodies, in 1.3% of men and 3.8% of women. No differences were observed in urine iodine between groups with thyroid dysfunction and euthyroidism, or between subjects with positive or negative antibodies. In subjects over 60, undiagnosed thyroid dysfunction was 9.8% (hypothyroidism 6.9%, hyperthyroidism 3.3%; 36.36% women) and total thyroid dysfunction 13.61% (53.12% women). Women and men over 60 had similar thyroid dysfunction prevalence. Thus, aggressive case-finding should be recommended in both, over 60.

Regulatory T cells in type 1 diabetic patients with autoimmune chronic atrophic gastritis.

Type A chronic atrophic gastritis (CAG) is increased in type 1 diabetic patients (DM1). To address this issue, we determined and analyzed the number of peripheral blood regulatory T cells (Tregs) in 15 DM1-CAG patients, 15 DM1 patients without associated autoantibodies (DM1) and 15 healthy controls by flow cytometry and compared gastric Tregs expression (CD4+Foxp3+/CD4+) in DM1-CAG patients with that observed in 10 control Helicobacter pylori CAG-infected biopsies. The percentage of peripheral Tregs was higher in DM1-CAG patients compared to DM1 and controls (CD4+Foxp3+: 7.67 +/- 1.91% vs. 5.38 +/- 1.57% and 5.65 +/- 1.76%, P < 0.001, respectively), with no differences between DM1 and controls. Gastric mucosal Tregs were higher in H. pylori CAG than in DM1-CAG patients (31.31 +/- 5.52% vs. 7.68 +/- 3.70%; P < 0.001). Data suggest that Tregs are stimulated in patients with more than one autoimmune disease (DM1 + CAG) in an ineffectual attempt to control autoimmune response and that the number of Tregs in gastric mucosa implicated in the chronification of gastritis differs according to the etiology.

Plasma ghrelin concentrations in type 1 diabetic patients with autoimmune atrophic gastritis.

OBJECTIVE: Type 1 diabetes mellitus patients (DM1) show increased prevalence of pernicious anaemia, the histological substrate of which is type A chronic atrophic gastritis (CAG) in the stomach corpus, the main source of ghrelin. We aimed to compare plasma ghrelin concentrations in DM1 patients with type A CAG (DM1-CAG), DM1 patients without type A CAG and healthy controls and in DM1-CAG group, to ascertain a possible relationship between ghrelin and biochemical markers of gastric mucosa atrophy and/or neuroendocrine (NE) cell hyperplasia and histological gastric biopsy findings. DESIGN AND METHODS: Fifteen DM1-CAG patients were matched for age, sex and body mass index with 15 DM1 patients without type A CAG and 15 controls. Pepsinogen I, pepsinogen II, gastrin, parietal cell antibodies, chromogranin A (CgA) and ghrelin were determined in all subjects. In DM1-CAG patients, immunohistochemical analysis of gastric biopsies using antibodies to CgA and ghrelin was performed. RESULTS: Ghrelin concentrations differed among groups; however, paired comparisons between groups were not significant. In DM1-CAG, no correlation was found between ghrelin and gastric body atrophy markers, pepsinogen I and the pepsinogen I/II ratio. Immunohistochemical studies of DMI-CAG patients showed CgA staining in 12 and ghrelin staining in 6, which was confined to the foci of NE cell hyperplasia. Those patients who stained positive for ghrelin had higher ghrelin concentrations when compared with the negative patients. CONCLUSIONS: Ghrelin concentrations are not decreased in DM1-CAG patients; thus, our data suggest that ghrelin is not a good marker of gastric mucosa atrophy in these patients, given the possible ghrelin synthesis in hyperplastic gastric endocrine/enterochromaffin-like cells.

[Evaluation of splenic function by dynamic gammagraphy, study of pitted erythrocytes and submembranous vacuoles in patients with slight and severe splenic trauma receiving conservative treatment or splenectomy]. / Valoración de la función esplénica mediante gammagrafía dinámica y estudio de "pits" de la membrana eritrocitaria y de vacuolas submembranarias en los pacientes con traumatismo leve y grave de bazo tratados de forma conservadora o mediante esplenectomía.

BACKGROUND AND OBJECTIVE: The splenic function of patients followed by the Department of General and Digestive Surgery in the Hospital Universitari Germans Trias i Pujol (HUGTiP) from 1985 to 2003 for different degrees of splenic trauma according to the classification of the American Association for the Surgery of Trauma (AAST) 1994 was quantified and related to the treatment received (non surgical, total splenectomy with or without splenosis and splenectomy plus autotransplantation) to detect splenic dysfunction predisposing the development of postsplenectomy sepsis (PSS). PATIENTS AND METHOD: 43 patients underwent an isotopic study with dynamic splenic gammagraphy and pitted erythrocytes (Normarsky optics) and submembranous vacuoles (transmission electron microscopy) were evaluated. RESULTS: The non surgical group presented normal phagocytic and filtration function with the median speed of splenic enhancement being 3.46 Kcts/s2 (interval: 0.8-6.98). The percentage of pitted erythrocytes was 2% (0-8.8), the number of pits per erythrocyte was 0.03 (0-0.12) and the percentage of erythrocytes with 1, 2, 3 and 4 pits was 1.6%, 0.4%, 0% and 0%, respectively. The percentage of red cells with submembranous vacuoles was 2.55% (0-5.6), the number of vacuoles per red cell was 0.03 (0-0.06) and the percentage of red cells with 1, 2, 3 and 4 vacuoles was 2%, 0.2%, 0% and 0%, respectively. In the operated group, the splenic enhancement speed was 0.08 Kcts/s2 (0-1.75) (p < 0.0001). The percentage of pitted erythrocytes was 38% (0.2-64) (p < 0.0001), the number of pits per erythrocyte was 0.86 (0-1.8) (p < 0.0001) and the percentage of erythrocytes with 1,2,3 and 4 pits was 16.39%, 7.2%, 3.59% and 2.52%, respectively (p < 0.0001). The percentage of red cells with submembranous vacuoles was 11.2% (1.8-31.9) (p = 0.0006); the number of vacuoles per cell was 0.16 (p = 0.0022) and the percentage of red cells with 1, 2, 3 and 4 vacuoles was 6.51%, 1.73%, 0.4% and 0.2%, respectively (p = 0.0246, 0.0010, < 0.0001 and 0.0002, respectively). CONCLUSIONS: Splenic function of patients with a history of splenic trauma receiving conservative treatment is normal, independently of the degree of the lesion, thereby reinforcing the use of this therapeutic approach to avoid the development of postsplenectomy sepsis. In the patients treated with splenectomy, with or without splenosis, splenic function was absent or very altered being partially conserved in cases treated with splenectomy plus autotransplantation.

Valoración de la función esplénica mediante gammagrafía dinámica y estudio de «pits» de la membrana eritrocitaria y de vacuolas submembranarias en los pacientes con traumatismo leve y grave de bazo tratados de forma conservadora o mediante esplenectomía / Evaluation of splenic function by dynamic gammagraphy, study of pitted erythrocytes and submembranous vacuoles in patients with slight and severe splenic trauma receiving conservative treatment or splenectomy

Fundamento y objetivo: Cuantificar la función esplénica de los pacientes controlados en el Servicio de Cirugía General y Digestiva del Hospital Universitari Germans Trias i Pujol (HUGTiP) desde 1985 hasta 2003 tras haber sufrido diferentes grados de lesión traumática del bazo según la clasificación de la American Association for the Surgery of Trauma (AAST) del año 1994 y relacionarla con el tratamiento recibido (no operatorio, esplenectomía total con o sin esplenosis y esplenectomía más autotrasplante), con la finalidad de detectar disfunciones esplénicas que predispongan al desarrollo de la sepsis tras la esplenectomía. Pacientes y método: Se ha realizado a 43 pacientes un estudio isotópico con gammagrafía esplénica «dinámica», estudio de «pits» de la membrana eritrocitaria (óptica de Nomarsky) y de vacuolas submembranarias (microscopia electrónica de transmisión). Resultados: El grupo de no operados presenta una función normal de fagocitosis y filtración, con una mediana de velocidad de captación esplénica de 3,46 Kcts/s2 (intervalo, 0,8-6,98). El porcentaje mediano de «pits» de membrana fue del 2% (intervalo, 0-8,8%); el número de «pits» por hematíes, de 0,03 (intervalo, 0-0,12), y el porcentaje de hematíes con 1, 2, 3 y 4 «pits», del 1,6, el 0,4, el 0 y el 0%, respectivamente. El porcentaje mediano de hematíes con vacuolas submembranarias fue el 2,55% (intervalo, 0-5,6%); el número de vacuolas por hematíe, de 0,03 (intervalo, 0-0,06), y el porcentaje de hematíes con 1, 2, 3 y 4 vacuolas, del 2, el 0,2, el 0 y el 0%, respectivamente. En el grupo operado, la velocidad mediana de captación esplénica fue de 0,08 Kcts/s2 (intervalo, 0-1,75; p < 0,0001); el porcentaje mediano de «pits» de membrana, del 38% (intervalo, 0,2-64; p < 0,0001); el número mediano de «pits» por hematíe, de 0,86 (intervalo, 0-1,8; p < 0,0001), y el porcentaje de hematíes con 1, 2, 3 y 4 «pits», del 16,39, el 7,2, el 3,59 y el 2,52%, respectivamente (p < 0,0001). La mediana del porcentaje de hematíes con vacuolas submembranarias fue del 11,2% (intervalo, 1,8-31,9; p = 0,0006); el número mediano de vacuolas por hematíe, de 0,16 (p = 0,0022), y el porcentaje de hematíes con 1, 2, 3 y 4 vacuolas, del 6,51, el 1,73, el 0,4 y el 0,2%, respectivamente (p = 0,0246; p = 0,0010; p < 0,0001, y p = 0,0002, respectivamente). Conclusiones: La función esplénica de los pacientes con antecedentes de traumatismo esplénico tratados de forma conservadora es normal, independientemente del grado de la lesión. Ello refuerza la conveniencia de aplicar de entrada esta actitud a todos los protocolos para intentar evitar la sepsis tras la esplenectomía. En los casos tratados con esplenectomía, con o sin esplenosis, la función esplénica está ausente o muy alterada, mientras que en los casos tratados con esplenectomía y autotrasplante está parcialmente conservada

Background and objective: The splenic function of patients followed by the Department of General and Digestive Surgery in the Hospital Universitari Germans Trias i Pujol (HUGTiP) from 1985 to 2003 for different degrees of splenic trauma according to the classification of the American Association for the Surgery of Trauma (AAST) 1994 was quantified and related to the treatment received (non surgical, total splenectomy with or without splenosis and splenectomy plus autotransplantation) to detect splenic dysfunction predisposing the development of postsplenectomy sepsis (PSS). Patients and method: 43 patients underwent an isotopic study with dynamic splenic gammagraphy and pitted erythrocytes (Normarsky optics) and submembranous vacuoles (transmission electron microscopy) were evaluated. Results: The non surgical group presented normal phagocytic and filtration function with the median speed of splenic enhancement being 3.46 Kcts/s2 (interval: 0.8-6.98). The percentage of pitted erythrocytes was 2% (0-8.8), the number of pits per erythrocyte was 0.03 (0-0.12) and the percentage of erythrocytes with 1,2,3 and 4 pits was 1.6%, 0.4%, 0% and 0%, respectively. The percentage of red cells with submembranous vacuoles was 2.55% (0-5.6), the number of vacuoles per red cell was 0.03 (0-0.06) and the percentage of red cells with 1,2,3 and 4 vacuoles was 2%, 0.2%, 0% and 0%, respectively. In the operated group, the splenic enhancement speed was 0.08 Kcts/s2 (0-1.75) (p < 0.0001). The percentage of pitted erythrocytes was 38% (0.2-64) (p < 0.0001), the number of pits per erythrocyte was 0.86 (0-1.8) (p < 0.0001) and the percentage of erythrocytes with 1,2,3 and 4 pits was 16.39%, 7.2%, 3.59% and 2.52%, respectively (p < 0.0001). The percentage of red cells with submembranous vacuoles was 11.2% (1.8-31.9) (p = 0.0006); the number of vacuoles per cell was 0.16 (p = 0.0022) and the percentage of red cells with 1,2,3 and 4 vacuoles was 6.51%, 1.73%, 0.4% and 0.2%, respectively (p = 0.0246, 0.0010, < 0.0001 and 0.0002, respectively). Conclusions: Splenic function of patients with a history of splenic trauma receiving conservative treatment is normal, independently of the degree of the lesion, thereby reinforcing the use of this therapeutic approach to avoid the development of postsplenectomy sepsis. In the patients treated with splenectomy, with or without splenosis, splenic function was absent or very altered being partially conserved in cases treated with splenectomy plus autotransplantation