Neural differentiation of canine mesenchymal stem cells/multipotent mesenchymal stromal cells.

BACKGROUND: The ability of adipose tissue-derived multipotent mesenchymal stromal cells/mesenchymal stem cells (ASCs) to differentiate in neural lineages promises progress in the field of regenerative medicine, especially for replacing neuronal tissue damaged by different neurological disorders. Reprogramming of ASCs can be induced by the growth medium with neurogenic inductors and specific growth factors. We investigated the neural differentiation potential of canine ASCs using several growth media (KEM, NIMa, NIMb, NIMc) containing various combinations of neurogenic inductors: B27 supplement, valproic acid, forskolin, N2-supplement, and retinoic acid. Cells were first preconditioned in the pre-differentiation neural induction medium (mitogenically stimulated; STIM1), followed by the induction of neuronal differentiation. RESULTS: After 3, 6, and 9 days of neural induction, elongated neural-like cells with bipolar elongations were observed, and some oval cells with light nuclei appeared. The expression of neuronal markers tubulin beta III (TUBB3), neurofilament H (NF-H), microtubule-associated protein-2 (MAP2), and glial fibrillary acidic protein (GFAP) was observed using immunocytochemistry, which confirmed the differentiation into neurons and glial cells. Flow cytometry analysis showed high GFAP expression (between 70 and 90% of all cells) after cells had been growing three days in the neural induction medium a (NIMa). Around 25% of all cells also expressed adult neuronal markers NF-H and MAP2. After nine days of ASCs differentiation, the expression of all neural markers was reduced. There were no differences between the neural differentiation of ASCs isolated from female or male dogs. CONCLUSIONS: The differentiation repertoire of canine ASCs extends beyond mesodermal lineages. Using a defined neural induction medium, the canine ASCs differentiated into neural lineages and expressed markers of neuronal and glial cells, and also displayed the typical neuronal morphology. Differentiated ASCs can thus be a source of neural cellular lineages for the regenerative therapy of nerve damage and could be useful in the future for therapy or the modelling of neurodegenerative diseases.

Neutrophil CD64 index in cerebrospinal fluid as a marker of bacterial ventriculitis in children with external ventricular drainage.

BACKGROUND: Bacterial ventriculitis is a common complication in children with temporary external ventricular drains (EVD) and the diagnosis is challenging. The present study compared the diagnostic accuracy of novel cerebrospinal fluid (CSF) marker - CD64 expression on neutrophils measured as neutrophil CD64 index (CD64in) to routine laboratory CSF and blood markers for bacterial ventriculitis in children with EVD. METHODS: We conducted a prospective, observational study, enrolling children with EVD. CD64in in CSF together with CSF markers (leukocyte count, percentage of neutrophils, glucose, and proteins) and blood markers (leukocyte and differential count, C-reactive protein (CRP), and procalcitonin (PCT)) were studied at the time of suspected bacterial ventriculitis. CD64in was measured by flow cytometry. Diagnostic accuracy determined by the area under the receiver-operating characteristic (ROC) curves (AUC) was defined for each marker. RESULTS: Thirty-three episodes of clinically suspected ventriculitis in twenty-one children were observed during a 26-month period. Episodes were classified into those with microbiologically proven ventriculitis (13 episodes) and into those with microbiologically negative CSF (20 episodes). CD64in and leukocyte count were the only CSF markers that could differentiate between groups with diagnostic accuracy of 0.875 and 0.694, respectively. Among blood markers only CRP and band neutrophils differentiated between groups with diagnostic accuracy of 0.792 and 0.721, respectively. CONCLUSIONS: CD64in in CSF is a promising diagnostic marker of bacterial ventriculitis in children with EVD as it has higher diagnostic accuracy than routine blood and CSF markers for diagnosing bacterial ventriculitis at the time of clinical suspicion.

Diagnostic accuracy of presepsin (sCD14-ST) for prediction of bacterial infection in cerebrospinal fluid samples from children with suspected bacterial meningitis or ventriculitis.

Children with temporary external ventricular drains (EVD) are prone to nosocomial infections. Diagnosis of bacterial meningitis and ventriculitis in these children is challenging due to frequent blood contamination of cerebrospinal fluid (CSF) and the presence of chemical ventriculitis. The aim of this study was to compare diagnostic accuracy of presepsin (sCD14-ST), a novel biomarker of bacterial infection in CSF, to predict bacterial infection in comparison to the accuracy of established biomarkers like those demonstrated in biochemical analysis of CSF. We conducted a prospective study with 18 children with suspected bacterial meningitis or ventriculitis who had 66 episodes of disease. CSF samples were taken from external ventricular drainage. We measured presepsin in CSF, as well as CSF leukocyte count, glucose, and proteins. CSF was also taken to prove bacterial infection with culture methods or with 16S rRNA gene broad-range PCR (SepsiTest; Molzym, Germany). Infection was clinically confirmed in 57 (86%) episodes of suspected meningitis or ventriculitis. Chemical ventriculitis was diagnosed in 9 (14%) episodes of suspected meningitis or ventriculitis. Diagnostic accuracies presented as area under the curve (AUC) for sCD14-ST, leukocytes, and proteins measured in CSF were 0.877 (95% confidence interval [CI], 0.793 to 0.961), 0.798 (95% CI, 0.677 to 0.920), and 0.857 (95% CI, 0.749 to 0.964), respectively. With CSF culture, we detected bacteria in 17 samples, compared to 37 detected with broad-range PCR. It was found that presepsin was present at a significantly higher level in children with clinically proven ventriculitis than in those without meningitis or ventriculitis. Diagnostic accuracies of presepsin were superior to those of leukocytes or proteins in CSF. Presepsin-guided 16S rRNA gene PCR could be used in everyday clinical practice to improve etiological diagnosis of meningitis and ventriculitis and to prescribe more appropriate antibiotics.

Differences in the antigens of Helicobacter pylori strains influence on the innate immune response in the in vitro experiments.

The immune response to Helicobacter pylori importantly determines the pathogenesis of infection as well as the success of antibiotic eradication of the bacteria. Strains of H. pylori were gathered from 14 patients who failed to eradicate H. pylori infection with antibiotics-therapy resistant strains (TRS)-or from patients who were able to eradicate H. pylori infection-therapy susceptible strains (TSS). The THP-1 cells were stimulated with H. pylori antigens. Cathepsin X expression on THP-1 cells and concentration of cytokines in the supernatant of THP-1 cells were measured with a flow cytometer. TSS H. pylori antigens increased the proportion of cathepsin X positive cells compared to TRS H. pylori antigens. TSS H. pylori antigens induced higher secretion of IL-12 and IL-6 compared to TRS H. pylori antigens (P < 0.001; 0.02). Polymyxin B, a lipid A inhibitor, lowered the secretion of IL-12 and IL-6 in TRS and TSS. We demonstrated a H. pylori strain-dependent cathepsin X and cytokine expression that can be associated with H. pylori resistance to eradication due to lack of effective immune response. Differences in lipid A of H. pylori might have an influence on the insufficient immune response, especially on phagocytosis.

Multi locus sequence typing (MLST) used as a tool to confirm the ability of susceptible Helicobacter pylori strains to gain resistance to clarithromycin during eradication therapy.

BACKGROUND/AIMS: Primary resistance of H. pylori to clarithromycin is the most common reason for eradication failure, followed by mixed susceptible/ resistant H. pylori strain infection. To distinguish between mixed infections and H. pylori switch to resistance phenotype during eradication therapy, we proceeded with multi locus sequence typing (MLST) of H. pylori strains isolated from gastric biopsy samples of patients before and after eradication therapy. METHODOLOGY: We collected H. pylori isolates from gastric biopsies from 133 patients who were never treated for H. pylori. Five patients had eradication failure with the first isolate susceptible and second isolate resistant to clarithromycin. To analyse genotypes of first and second H. pylori isolates, we compared H. pylori strain sequences of 7 housekeeping genes with MLST. RESULTS: Five patients had clarithromycin-sensitive H. pylori before eradication therapy and gained H. pylori-resistant to clarithromycin after eradication therapy. The sensitive and resistant colonies of each of the H. pylori populations, taken from patients before/after antibiotic therapy, had identical sequence types (ST) obtained with MLST. CONCLUSIONS: The factors favouring H. pylori survival and switch to antibiotic-resistance during eradication therapy probably enable milder environmental conditions for H. pylori persistence during therapy. One of such factor is the ineffective destruction of mucosa-adhered H. pylori by immune cells during therapy which may be due to locally induced immune deficit by H. pylori molecules like strain specific H. pylori lipopolysaccharides.

Biomarkers for prediction of CAR T therapy outcomes: current and future perspectives.

Chimeric antigen receptor (CAR) T cell therapy holds enormous potential for the treatment of hematologic malignancies. Despite its benefits, it is still used as a second line of therapy, mainly because of its severe side effects and patient unresponsiveness. Numerous researchers worldwide have attempted to identify effective predictive biomarkers for early prediction of treatment outcomes and adverse effects in CAR T cell therapy, albeit so far only with limited success. This review provides a comprehensive overview of the current state of predictive biomarkers. Although existing predictive metrics correlate to some extent with treatment outcomes, they fail to encapsulate the complexity of the immune system dynamics. The aim of this review is to identify six major groups of predictive biomarkers and propose their use in developing improved and efficient prediction models. These groups include changes in mitochondrial dynamics, endothelial activation, central nervous system impairment, immune system markers, extracellular vesicles, and the inhibitory tumor microenvironment. A comprehensive understanding of the multiple factors that influence therapeutic efficacy has the potential to significantly improve the course of CAR T cell therapy and patient care, thereby making this advanced immunotherapy more appealing and the course of therapy more convenient and favorable for patients.

Low CD8 T cells in neonates and infants prior to surgery, and health-care-associated infections: prospective observational study.

BACKGROUND: Major surgery suppresses the cell-mediated immune response in children and adults. Data on preoperative and postoperative T-cell counts in pediatric surgical patients and their relationship to health-care-associated infection (HAI) are not yet known. METHODS: A prospective observational study was carried out in a level III multidisciplinary neonatal and pediatric intensive care unit. Before and after, and in the first 3 days after surgery, lymphocyte subsets in peripheral blood were measured in 28 neonates and infants on flow cytometry. HAI were classified according to CDC/NHSN criteria. RESULTS: Six out of 28 neonates and infants (21.4%) developed HAI (group I-HAI), while 22 out of 28 (78.6%) remained infection free (group II-non-HAI). In group I with HAI, the preoperative median cytotoxic T-lymphocyte (CD8-T-cell) level was found to be below normal, and remained very low throughout the study period. In addition, the median and interquartile CD8 T-cell range (358 cells/µL; 304-424 cells/µL) were twice as low compared to group II without HAI (822 cells/µL; 522-933 cells/µL; P = 0.013). No differences were found between the two groups with regard to patient demographics and clinical data. CONCLUSION: Neonates and infants who underwent a major surgical procedure and who had a very low preoperative CD8 T-cell level, developed HAI postoperatively.

Inhibition of cathepsin X enzyme influences the immune response of THP-1 cells and dendritic cells infected with Helicobacter pylori.

BACKGROUND: The immune response to Helicobacter pylori importantly determines the outcome of infection as well as the success of eradication therapy. We demonstrate the role of a cysteine protease cathepsin X in the immune response to H. pylori infection. MATERIALS AND METHODS: We analysed how the inhibition of cathepsin X influenced the immune response in experiments when THP-1 cells or dendritic cells isolated from patients were stimulated with 48 strains of H. pylori isolated from gastric biopsy samples of patients which had problems with the eradication of bacteria. RESULTS: The experiments, performed with the help of a flow cytometer, showed that the expression of Toll-like receptors (TLRs), especially TLR-4 molecules, on the membranes of THP-1 cells or dendritic cells was higher when we stimulated cells with H. pylori together with inhibitor of cathepsin X 2F12 compared to THP-1 cells or dendritic cells stimulated with H. pylori only, and also in comparison with negative control samples. We also demonstrated that when we inhibited the action of cathepsin X in THP-1 cells, the concentrations of pro-inflammatory cytokines were lower than when THP-1 cell were stimulated with H. pylori only. CONCLUSIONS: We demonstrated that inhibition of cathepsin X influences the internalization of TLR-2 and TLR-4. TLR-2 and TLR-4 redistribution to intra-cytoplasmic compartments is hampered if cathepsin X is blocked. The beginning of a successful immune response against H. pylori in the case of inhibition of cathepsin X is delayed.

Effects of Anthropogenic Disturbance and Seasonal Variation on Aerobiota in Highly Visited Show Caves in Slovenia.

Aerosols in caves are natural tracers and, together with climatic parameters, provide a detailed insight into atmospheric conditions, responses to climatic changes and anthropogenic influences in caves. Microbiological air monitoring in show caves is becoming increasingly useful to understand changes in cave ecosystems and to implement and review measures for sustainable cave use and tourism development. In 2017 and 2018, air along tourist trails in caves Postojnska jama and Skocjanske jame (Slovenia) was sampled before and after tourist visits. Samples were analysed using culture-dependent methods, flow cytometry, detection of ß-D-glucan and lipopolysaccharide and compared with CO2 and temperature data to measure anthropogenic influences and seasonality on aerobiota. While the presence of tourists significantly increased concentrations of airborne microorganisms (p < 0.05), ß-D-glucan and CO2 did not show such a trend and were more dependent on seasonal changes. Locally, concentrations of cultivable microorganisms above 1000 CFU/m3 were detected, which could have negative effects on the autochthonous microbiota and possibly on human health. A mixture of bacteria typically associated with humans was found in the air and identified with MALDI-TOF MS. Using MALDI-TOF MS, we achieved a 69.6% success rate in identification. Micrococcus luteus, Streptococcus mitis, Staphylococcus epidermidis and Moraxella spp. were recognized as good indicators of cave anthropisation.

Macrophage polarization during Streptococcus agalactiae infection is isolate specific.

Introduction: Streptococcus agalactiae (Group B Streptococcus, GBS), a Gram-positive commensal in healthy adults, remains a major cause of neonatal infections, usually manifesting as sepsis, meningitis, or pneumonia. Intrapartum antibiotic prophylaxis has greatly reduced the incidence of early-onset disease. However, given the lack of effective measures to prevent the risk of late-onset disease and invasive infections in immunocompromised individuals, more studies investigating the GBS-associated pathogenesis and the interplay between bacteria and host immune system are needed. Methods: Here, we examined the impact of 12 previously genotyped GBS isolates belonging to different serotypes and sequence types on the immune response of THP-1 macrophages. Results: Flow cytometry analysis showed isolate-specific differences in phagocytic uptake, ranging from 10% for isolates of serotype Ib, which possess the virulence factor protein ß, to over 70% for isolates of serotype III. Different isolates also induced differential expression of co-stimulatory molecules and scavenger receptors with colonizing isolates inducing higher expression levels of CD80 and CD86 compared to invasive isolates. In addition, real-time measurements of metabolism revealed that macrophages enhanced both glycolysis and mitochondrial respiration after GBS infection, with isolates of serotype III being the most potent activators of glycolysis and glycolytic ATP production. Macrophages also showed differential resistance to GBS-mediated cell cytotoxicity as measured by LDH release and real-time microscopy. The differences were evident both between serotypes and between isolates obtained from different specimens (colonizing or invasive isolates) demonstrating the higher cytotoxicity of vaginal compared with blood isolates. Conclusions: Thus, the data suggest that GBS isolates differ in their potential to become invasive or remain colonizing. In addition, colonizing isolates appear to be more cytotoxic, whereas invasive isolates appear to exploit macrophages to their advantage, avoiding the immune recognition and antibiotics.

Alterations in immunophenotype and metabolic profile of mononuclear cells during follow up in children with multisystem inflammatory syndrome (MIS-C).

Introduction: Although children seem to be less susceptible to COVID-19, some of them develop a rare but serious hyperinflammatory condition called multisystem inflammatory syndrome in children (MIS-C). While several studies describe the clinical conditions of acute MIS-C, the status of convalescent patients in the months after acute MIS-C is still unclear, especially the question of persistence of changes in the specific subpopulations of immune cells in the convalescent phase of the disease. Methods: We therefore analyzed peripheral blood of 14 children with MIS-C at the onset of the disease (acute phase) and 2 to 6 months after disease onset (post-acute convalescent phase) for lymphocyte subsets and antigen-presenting cell (APC) phenotype. The results were compared with six healthy age-matched controls. Results: All major lymphocyte populations (B cells, CD4 + and CD8+ T cells, and NK cells) were decreased in the acute phase and normalized in the convalescent phase. T cell activation was increased in the acute phase, followed by an increased proportion of γ/δ-double-negative T cells (γ/δ DN Ts) in the convalescent phase. B cell differentiation was impaired in the acute phase with a decreased proportion of CD21 expressing, activated/memory, and class-switched memory B cells, which normalized in the convalescent phase. The proportion of plasmacytoid dendritic cells, conventional type 2 dendritic cells, and classical monocytes were decreased, while the proportion of conventional type 1 dendritic cells was increased in the acute phase. Importantly the population of plasmacytoid dendritic cells remained decreased in the convalescent phase, while other APC populations normalized. Immunometabolic analysis of peripheral blood mononuclear cells (PBMCs) in the convalescent MIS-C showed comparable mitochondrial respiration and glycolysis rates to healthy controls. Conclusions: While both immunophenotyping and immunometabolic analyzes showed that immune cells in the convalescent MIS-C phase normalized in many parameters, we found lower percentage of plasmablasts, lower expression of T cell co-receptors (CD3, CD4, and CD8), an increased percentage of γ/δ DN Ts and increased metabolic activity of CD3/CD28-stimulated T cells. Overall, the results suggest that inflammation persists for months after the onset of MIS-C, with significant alterations in some immune system parameters, which may also impair immune defense against viral infections.

Dendritic cell activation and cytokine response in vaccine breakthrough TBE patients after in vitro stimulation with TBEV.

Tick-borne encephalitis (TBE) is a viral infection of the human central nervous system caused by the TBE virus (TBEV). The most effective protective measure against TBE is vaccination. Despite the highly immunogenic vaccine, cases of vaccine breakthroughs (VBTs) occur. One of the first targets of infection is dendritic cells (DC), which represent a fundamental bridge between innate and adaptive immunity through antigen presentation, costimulation, and cytokine production. Therefore, we investigated the activation and maturation of DCs and cytokine production after in vitro TBEV stimulation of peripheral blood mononuclear cells (PBMCs) obtained from VBT and unvaccinated TBE patients. Our results showed that the expression of HLA-DR and CD86 on DCs, was upregulated to a similar extent in both vaccinated and unvaccinated TBE patients but differed in cytokine production after stimulation with TBEV. PBMCs from patients with VBT TBE responded with lower levels of IFN-α and the proinflammatory cytokines IL-12 (p70) and IL-15 after 24- and 48-hour in vitro stimulation with TBEV, possibly facilitating viral replication and influencing the development of cell-mediated immunity. On the other hand, significantly higher levels of IL-6 in addition to an observed trend of higher expression of TNF-α measured after 6 days of in vitro stimulation of PBMC could support disruption of the blood-brain barrier and promote viral and immune cell influx into the CNS, leading to more severe disease in VBT TBE patients.

Microbiota entrapped in recently-formed ice: Paradana Ice Cave, Slovenia.

Paradana is one of the biggest ice caves in Slovenia, with an estimated ice volume of 8,000 m3. Reflecting climatological conditions, the cave ice undergoes repeated freeze-thaw cycles and regular yearly deposition of fresh ice. Three distinct ice block samples, collected from the frozen lake in May 2016, were analysed to obtain data on ice physicochemical properties and the composition of associated microbiota. Isotopic composition of the ice samples (18O, 2H) and a local meteoric water line (LMWL) constructed for monthly precipitation at Postojna were used to estimate the isotopic composition of the water that formed the ice, which had high values of deuterium excess and low concentrations of chloride, sulphate and nitrate. The values of total organic carbon (1.93-3.95 mg/l) within the ice blocks fall within the range of those measured in karst streams. Total cell count in the ice was high and the proportion of cell viability increased along the depth gradient and ranged from 4.67 × 104 to 1.52 × 105 cells/ml and from 51.0 to 85.4%, respectively. Proteobacteria represented the core of the cave-ice microbiome (55.9-79.1%), and probably play an essential role in this ecosystem. Actinobacteria was the second most abundant phylum (12.0-31.4%), followed in abundance by Bacteroidetes (2.8-4.3%). Ice phylotypes recorded amounted to 442 genera, but only 43 genera had abundances greater than 0.5%. Most abundant were Pseudomonas, a well-known ice dweller, and Lysobacter, which previously was not reported in this context. Finally, two xanthophytes, Chloridella glacialis and Ellipsoidion perminimum, known from polar environments, were cultured from the ice. This indicates that the abundance and ecological role of phototrophs in such environments might be greater than previously deduced.

Inflammatory molecules in aqueous humour and on ocular surface and glaucoma surgery outcome.

PURPOSE: To investigate the influence of inflammatory molecules in the aqueous humour and on the ocular surface on the outcome of glaucoma surgery. METHODS: Thirty patients who needed antiglaucomatous surgery were included. The interleukin- (IL-) 8, IL-1beta, IL-6, IL-10, tumour necrosis factor- (TNF-) alpha; and IL-12 were determined from aqueous humour preoperatively and the imprints of conjunctiva were analysed for expression of human leukocyte antigen- (HLA-)-DR after surgery by flow cytometry. The success of trabeculectomy was defined as intraocular pressure less than 21 mmHg without antiglaucoma medication. RESULTS: Eyes with trabeculectomy failure at 3 months showed significantly higher TNF-alpha and IL-6 levels in the aqueous than eyes with successful surgery. Increased expression of HLA-DR on epithelial cells and antigen-presenting cells was not associated with the trabeculectomy outcome. CONCLUSIONS: Higher preoperative levels of TNF-alpha and IL-6 in aqueous humour may contribute to the development of inflammatory milieu and were associated with worse outcome of glaucoma surgery.

Comparison of Lymphocyte Populations in Patients With Dobrava or Puumala orthohantavirus Infection.

Hemorrhagic fever with renal syndrome (HFRS), caused by Dobrava (DOBV) and Puumala (PUUV) orthohantaviruses, is an endemic disease in Slovenia. DOBV is mainly responsible for a more severe disease, whereas PUUV usually causes a milder form. Therefore, the aim of our study was to determine whether any differences in lymphocyte population in patients infected with these two viruses exist. Mononuclear cells from peripheral blood (PBMCs) were isolated from DOBV or PUUV infected patients and different lymphocyte subpopulations were analyzed with flow cytometry. Decreased concentrations of lymphocyte subpopulation were observed in DOBV and in PUUV infected patients compared with a healthy control, which was especially evident in DOBV infected patients. The lower values of T cells are likely due to the extravasation of the activated cells from the circulation to the infected tissue. Higher percentage of NK cells were detected in DOBV infected patients in comparison to PUUV infected patients, which could be associated with a more severe HFRS caused by DOBV. PUUV infected patients had a significantly higher concentration of activated T cell subsets, expressing markers CD25, CD69, and HLA-DR in comparison to DOBV infected patients. Higher activation of T cell subsets in PUUV infected patients could be a contributor to a milder HFRS. Further studies are necessary to elucidate the relation between the protective and the harmful role of activated lymphocytes subsets in HFRS pathogenesis.

Vaccine immune response, autoimmunity and morbidity after neonatal blood exchange transfusion.

INTRODUCTION: A blood exchange transfusion (BET) is most commonly performed to treat severe neonatal haemolytic disease. A distinct form of blood transfusion adverse reaction is transfusion-related immunomodulation. The purpose of our retrospective single-centre case-control cohort study was to investigate whether a blood exchange transfusion in the neonatal period provokes immunomodulation and affects humoral immune response to vaccination, morbidity and occurrence of autoantibodies. METHODS: Study subjects were 74 apparently healthy children, who were born at term as appropriate for gestational age and received four doses of diphtheria and tetanus toxoid vaccine. Forty-one received BET due to neonatal hemolytic disease and no other blood product afterwards, while 33 did not receive any blood products. Analysis of diphtheria, tetanus and autoimmune antibodies was performed and their medical records were analyzed for infectious, allergic, cancerous and autoimmune diseases. RESULTS: A clearly exaggerated immune response to diphtheria (1.016 IU/mL, 95% confidence interval (CI) 0.662-1.369 IU/mL vs. 0.515 IU/mL, 95% CI 0.363 to 0.626 IU/mL, P = 0.011) and slightly exaggerated immune response to tetanus vaccine (1.798 IU/mL, 95% CI 1.180-2.416 IU/mL vs. 1.036 IU/mL, 95% CI 0.398-1.673 IU/mL, P = non-specific) were observed in BET subjects. A propensity towards autoimmunity (25.8% vs. 12.5%, P = non-specific) was observed in BET subjects. However, BET in the neonatal period did not influence the occurrence of bacterial, childhood viral diseases with exception of varicella (43.9% vs. 21.2%, P = 0.040), autoimmune and cancer diseases. CONCLUSION: BET impacted humoral immune response to diphtheria and tetanus vaccine and occurrence of autoimmune antibodies, but did not affect morbidity and the occurrence of autoimmune diseases. These effects could be related to massive antigenic load of BET and an accelerated priming of immune cells and consequent immunomodulation.

Identification and characterization of dendritic cell subtypes in preserved and cultured cadaveric human corneolimbal tissue on amniotic membrane.

PURPOSE: Rejection is the leading cause of failure of limbal allogafts. Resident dendritic cell (DC) maturation plays a critical role in host allosensitization. There are two lineages: myeloid (mDC) and lymphoid (pDC), with different biological properties. The aim was to analyse the distribution of DC subtypes in limbal explant cultures on amniotic membrane (AM), cultivated on either the epithelial or stromal side and to compare the results with directly isolated cells from cadaveric whole corneoscleral tissue divided into specific areas. METHODS: The expression of CD11c (mDC), CD303/CD123 (pDC) and costimulatory molecules CD80, CD86 and activation markers HLA-DR, CD83 was investigated by flow cytometry. Additionally, the corneal epithelium marker CK12 and ABCB5, a new epithelial stem cell marker, were investigated. RESULTS: Cells positive for pDC and mDC markers were found in all examined areas, with a nonsignificant prevalence of pDC. In limbal explant cultures on AM, the percentage of pDC and mDC was similar, with no statistically significant difference between cultures on epithelial or stromal sides of AM. However, with ex vivo limbal explant cultivation on AM, the pDC content declined significantly (p < 0.05) and the ABCB5 marker was likewise statistically significantly reduced. CONCLUSION: This is the first study to characterize the distribution of pDC and mDC subsets in cultured and noncultured human corneolimbal tissue. Additionally, ABCB5 positive cells were identified. These findings might be important for future strategies, allowing preparation of corneolimbal allografts with optimal stem cell content for a longer lasting therapeutic effect.

Dendritic cell profiles in the inflamed colonic mucosa predict the responses to tumor necrosis factor alpha inhibitors in inflammatory bowel disease.

Background Dendritic cells play crucial roles in the control of inflammation and immune tolerance in the gut. We aimed to investigate the effects of tumor necrosis factor alpha (TNFa) inhibitors on intestinal dendritic cells in patients with inflammatory bowel disease and the potential role of intestinal dendritic cells in predicting the response to treatment. Patients and methods Intestinal biopsies were obtained from 30 patients with inflammatory bowel disease before and after treatment with TNFa inhibitors. The proportions of lamina propria dendritic cell phenotypes were analysed using flow cytometry. Disease activity was endoscopically assessed at baseline and after the induction treatment. Results At baseline, the proportion of conventional dendritic cells was higher in the inflamed mucosa (7.8%) compared to the uninflamed mucosa (4.5%) (p = 0.003), and the proportion of CD103+ dendritic cells was lower in the inflamed mucosa (47.1%) versus the uninflamed mucosa (57.3%) (p = 0.03). After 12 weeks of treatment, the proportion of conventional dendritic cells in the inflamed mucosa decreased from 7.8% to 4.5% (p = 0.014), whereas the proportion of CD103+ dendritic cells remained unchanged. Eighteen out of 30 (60%) patients responded to their treatment by week 12. Responders had a significantly higher proportion of conventional dendritic cells (9.16% vs 4.4%, p < 0.01) with higher expression of HLA-DR (median fluorescent intensity [MFI] 12152 vs 8837, p = 0.038) in the inflamed mucosa before treatment compared to nonresponders. Conclusions A proportion of conventional dendritic cells above 7% in the inflamed inflammatory bowel disease mucosa before treatment predicts an endoscopic response to TNFa inhibitors.

Sperm DNA fragmentation and mitochondrial membrane potential combined are better for predicting natural conception than standard sperm parameters.

OBJECTIVE: To evaluate whether DNA fragmentation and/or mitochondrial membrane potential (MMP) predict natural conception better than standard sperm parameters. DESIGN: Prospective cross-sectional study. SETTING: University medical center. PATIENT(S): Eighty-five infertile and 51 fertile men. INTERVENTION(S): Assessment of sperm DNA fragmentation, MMP, and standard semen parameters over a 6- to 12-month observation period. MAIN OUTCOME MEASURE(S): Comparison between the results of DNA fragmentation, MMP, and standard sperm parameters alone or combined and achievement of natural conception. RESULT(S): Twenty-six of the 85 (31%) men from infertile couples conceived naturally. The median values of DNA fragmentation and MMP in the men who conceived within the observation period were similar to those in the fertile controls. Optimal threshold values of DNA fragmentation and MMP were 25% as determined by receiver operating characteristic analysis (area under the curve [AUC], 0.70; 95% confidence interval (CI) 0.58-0.82) and 62.5% (AUC, 0.68, 95% CI 0.56-0.80), respectively. The men in the infertile group with values of DNA fragmentation ≤25% and with MMP values ≥62.5% had significantly higher odds for conception (odds ratio [OR], 5.22; 95% CI 1.82-14.93] and OR, 4.67; 95% CI 1.74-12.5, respectively). Normal semen analysis alone had no predictive value for natural conception (OR, 1.84; 95% CI 0.67-5.07]). Both sperm function tests combined had significant odds for natural conception (OR, 8.24; 95% CI 2.91-23.33]), with a probability of 0.607 (60.7%) for both normal values and 0.158 (15.8%) for abnormal values. CONCLUSION(S): Sperm DNA fragmentation and MMP combined may be superior to standard semen parameters for the prediction of natural conception.

Effect of Cryopreserved Amniotic Membrane Orientation on the Expression of Limbal Mesenchymal and Epithelial Stem Cell Markers in Prolonged Limbal Explant Cultures.

PURPOSE: To evaluate the effect of prolonged limbal explants cultured without any scaffolds or on amniotic membrane (AM) on the viability, proliferation and differentiation potential of putative phenotypically defined cultured limbal mesenchymal (LMSC) and epithelial stem cells (LESC). METHODS: Limbal explants were cultivated on cryopreserved intact AM or plastic plates using medium supplemented with only human serum. AM was positioned with either the epithelial or stromal side up. The outgrowing cells were immunophenotyped for the co-expression of mesenchymal stem cell markers (CD73/CD90/CD105 positive and CD45 negative), proliferation and putative progenitor markers (CXCR4, CD117), epithelial markers and antigen presenting cell markers (CD80, CD83, CD86) by flow cytometry. Immunohistochemistry on limbal cultures cultivated on AM was carried out with antibodies against pan-cytokeratin, p63, Ki67. RESULTS: Morphological and immunostaining analyses revealed two distinct stem cell population types, which could be identified over prolonged culturing time periods. Expression of LMSC markers and CXCR4 was significantly higher (p < 0.05) in cultures cultivated without AM. However, no statistically significant difference was observed in CD117 expression. The cells cultivated on AM retained an epithelial cell structure, which was further confirmed by histology examination. Histology revealed limbal epithelial growth and p63, Ki67 positive cells on both sides of AM. CONCLUSION: Limbal cells cultivated on AM exhibited a lower expression profile of LMSC and CXCR4 markers as limbal cells cultivated on plastic culture plates. However, CD117 expression was similar. Histology confirmed limbal epithelial cell growth on both sides of AM, with no morphological differences, or positivity of cells for p63 and Ki67.