Regulation of human neutrophil IL-1ß secretion induced by Escherichia coli O157:H7 responsible for hemolytic uremic syndrome.

Shiga-toxin producing Escherichia coli (STEC) infections can cause from bloody diarrhea to Hemolytic Uremic Syndrome. The STEC intestinal infection triggers an inflammatory response that can facilitate the development of a systemic disease. We report here that neutrophils might contribute to this inflammatory response by secreting Interleukin 1 beta (IL-1ß). STEC stimulated neutrophils to release elevated levels of IL-1ß through a mechanism that involved the activation of caspase-1 driven by the NLRP3-inflammasome and neutrophil serine proteases (NSPs). Noteworthy, IL-1ß secretion was higher at lower multiplicities of infection. This secretory profile modulated by the bacteria:neutrophil ratio, was the consequence of a regulatory mechanism that reduced IL-1ß secretion the higher were the levels of activation of both caspase-1 and NSPs, and the production of NADPH oxidase-dependent reactive oxygen species. Finally, we also found that inhibition of NSPs significantly reduced STEC-triggered IL-1ß secretion without modulating the ability of neutrophils to kill the bacteria, suggesting NSPs might represent pharmacological targets to be evaluated to limit the STEC-induced intestinal inflammation.

Autophagy Mediates Interleukin-1ß Secretion in Human Neutrophils.

Interleukin-1ß (IL-1ß), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1ß in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1ß by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1ß secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1ß secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1ß secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1ß secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1ß was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1ß-LC3B in a vesicular compartment peaked before IL-1ß increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1ß and LC3B and then promoted neutrophil IL-1ß secretion. In addition, specific ELISAs indicated that although both IL-1ß and pro-IL-1ß are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1ß secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1ß secretion. Moreover, IL-1ß could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1ß intersect azurophil granules content and that serine proteases also regulate IL-1ß secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1ß secretion in human neutrophils.

Restoring conjunctival tolerance by topical nuclear factor-κB inhibitors reduces preservative-facilitated allergic conjunctivitis in mice.

PURPOSE: To evaluate the role of nuclear factor-κB (NF-κB) activation in eye drop preservative toxicity and the effect of topical NF-κB inhibitors on preservative-facilitated allergic conjunctivitis. METHODS: Balb/c mice were instilled ovalbumin (OVA) combined with benzalkonium chloride (BAK) and/or NF-κB inhibitors in both eyes. After immunization, T-cell responses and antigen-induced ocular inflammation were evaluated. Nuclear factor-κB activation and associated inflammatory changes also were assessed in murine eyes and in an epithelial cell line after BAK exposure. RESULTS: Benzalkonium chloride promoted allergic inflammation and leukocyte infiltration of the conjunctiva. Topical NF-κB inhibitors blocked the disruptive effect of BAK on conjunctival immunological tolerance and ameliorated subsequent ocular allergic reactions. In line with these findings, BAK induced NF-κB activation and the secretion of IL-6 and granulocyte-monocyte colony-stimulating factor in an epithelial cell line and in the conjunctiva of instilled mice. In addition, BAK favored major histocompatibility complex (MHC) II expression in cultured epithelial cells in an NF-κB-dependent fashion after interaction with T cells. CONCLUSIONS: Benzalkonium chloride triggers conjunctival epithelial NF-κB activation, which seems to mediate some of its immune side effects, such as proinflammatory cytokine release and increased MHC II expression. Breakdown of conjunctival tolerance by BAK favors allergic inflammation, and this effect can be prevented in mice by topical NF-κB inhibitors. These results suggest a new pharmacological target for preservative toxicity and highlight the importance of conjunctival tolerance in ocular surface homeostasis.

Agreement between placido topography and Scheimpflug tomography for corneal astigmatism assessment.

PURPOSE: To evaluate inter-device agreement between Placido topography (iTrace; Tracey Technologies, Houston, TX) and Scheimpflug tomography (Pentacam; Oculus Optikgeräte GmbH, Wetzlar, Germany) for measuring corneal power and cylinder and axis of astigmatism. METHODS: Observational case series of 54 eyes from 54 subjects with no ocular disease. Main outcome measures were corneal power, cylinder power, and axis of astigmatism and their agreement was assessed by Bland­Altman analysis. RESULTS: For corneal power and corneal cylinder, 95% limits of agreement (LoA) were considered good (−0.38 to 0.45 diopters [D] and −0.49 to 0.27 D, respectively). In contrast, the 95% LoA for corneal astigmatism axis exceeded the clinically relevant margins (−14.8 to 13.5): 28 eyes (52%) had a greater than 5° difference, 10 eyes (19%) had a greater than 10° difference, and 4 eyes (7%) had a greater than 20° difference between instruments. This absolute difference was significantly correlated with average corneal cylinder (Spearman's r = −0.379, P = .005) but not with average corneal power. In eyes with corneal astigmatism 2 D or greater, the 95% LoA for axis were −8.7° to 6.7°, whereas in those with corneal astigmatism less than 1 D, the 95% LoA for axis were −19.1° to 16.6°. CONCLUSIONS: Placido topography and Scheimpflug tomography show good agreement for corneal power and cylinder, but not for corneal astigmatism axis. These instruments could be used interchangeably only in eyes with corneal astigmatism of 2 D or greater.

Pentacam Scheimpflug tomography findings in topographically normal patients and subclinical keratoconus cases.

PURPOSE: To evaluate Pentacam ectasia detection indices in topographically normal patients and in subclinical keratoconus cases. DESIGN: Prospective, observational case series. METHODS: setting: Institutional. patients: Group 1 comprised 1 eye from 189 patients with unremarkable topography and Groups 2 and 3 included the better and worse eyes, respectively, of 55 keratoconic patients. Group 2 eyes with normal topography (n = 37) were considered subclinical keratoconus cases. observation procedure: Pentacam Scheimpflug tomography. main outcome measures: Eleven Pentacam ectasia detection indices. RESULTS: All Pentacam ectasia indices significantly differed between Groups 1 and 2 and were correlated with keratoconus grade. Only 99 eyes (52%) in Group 1 had normal values for every index, whereas 7 subclinical keratoconus eyes (19%) showed 2 or fewer abnormal indices. Standardized relational thickness and overall deviation indices had 73% and 89% sensitivity for subclinical keratoconus, respectively. Both average and maximum pachymetric progression indices offered 84% sensitivity while maximum relational thickness index showed 78% sensitivity for subclinical keratoconus. Optimized cutoff values for subclinical keratoconus increased the sensitivity of the standardized and maximum relational thickness indices. CONCLUSION: Pentacam Scheimpflug tomography can detect most subclinical keratoconus cases with unremarkable topography, but performance is not as good as reported and varies considerably for each index. The overall deviation, average and maximum pachymetric progression, and maximum relational thickness indices offer the highest sensitivity, which can be improved by using optimized cutoff values. Specificity constitutes an issue for some indices and up to 10% of subclinical keratoconus cases may go undetected by this technology.

Combining ocular response analyzer metrics for corneal biomechanical diagnosis.

PURPOSE: To evaluate corneal biomechanical properties in non-keratoconic myopic eyes and to identify descriptors for improving the specificity of the Ocular Response Analyzer (ORA) (Reichert Ophthalmic Instruments, Depew, NY) testing for subclinical keratoconus detection. METHODS: Observational case series of 52 non-keratoconic non-myopic eyes and 97 non-keratoconic myopic eyes (spherical equivalent < -5 diopters [D]) in dataset 1 and 87 non-keratoconic eyes and 73 eyes with subclinical keratoconus in dataset 2. Examination included corneal topography, tomography, and biomechanical testing with the ORA. Receiver operating characteristic curves and logistic regression were used to identify optimal combinations of biomechanical indices for keratoconus detection. Main outcome measures were corneal thickness-corrected hysteresis (DifCH) and resistance factor (DifCRF), the difference between these two (CH-CRF), and the diagnostic performance of their combinations. RESULTS: Compared to non-keratoconic non-myopic eyes, non-keratoconic myopic eyes with flat corneas (average corneal power < 44.0 D) had reduced DifCH (mean ± standard deviation, 0.11 ± 1.27 vs -0.79 ± 1.50, P < .01) and DifCRF (0.24 ± 1.16 vs -0.70 ± 1.59, P < .01) values, whereas non-keratoconic myopic eyes with steep corneas showed no difference. Keratoconic eyes exhibited lower DifCH and DifCRF values than non-keratoconic myopic eyes. Combinations of DifCH, DifCRF, and CH-CRF had increased specificity (> 80%) for subclinical keratoconus compared to the DifCRF index alone (71%). CONCLUSIONS: In biomechanical keratoconus screening, some non-keratoconic myopic eyes show altered ocular biomechanical properties and are identified as false-positive cases. The low specificity of DifCRF when dealing with these non-keratoconic eyes could be improved by considering additional biomechanical descriptors such as DifCH and CH-CRF, which seem to be indicative of the aforementioned biomechanical profile.

Improved keratoconus detection by ocular response analyzer testing after consideration of corneal thickness as a confounding factor.

PURPOSE: To compare corneal hysteresis (CH) and corneal resistance factor (CRF) between normal eyes and eyes with keratoconus correcting for the effect of central corneal thickness (CCT) and to estimate keratoconus detection sensitivity and specificity of these parameters. METHODS: Observational case series of 102 normal eyes (control group) and 77 eyes with keratoconus (keratoconus group). Examination included corneal topography, tomography, and biomechanical testing with the Ocular Response Analyzer (Reichert Technologies). The confounding effect of CCT was controlled by stratification (20-µm CCT intervals) and linear transformation. Receiver operating characteristic curves were used to identify optimal CH and CRF cutoff points for keratoconus detection. Main outcome measures were CCT, CH, CRF, and diagnostic performance of CH and CRF. RESULTS: Corneal hysteresis and CRF were positively correlated to CCT in both groups. In the control versus keratoconus group, CH was 9.79 ± 1.51 vs 8.49 ± 1.48 (P<.0001) and CRF was 9.55 ± 1.64 vs 7.24 ± 1.43 (P<.0001), respectively. Only CRF remained significantly lower in eyes with keratoconus after CCT stratification in 20-µm intervals, and keratoconus grade influenced CH and CRF. Transformed CH and CRF data confirmed these results. Sensitivity and specificity of CRF cutoff points ranged between 83% and 94% and 69% and 83%, respectively. True positive rate for CRF in eyes with keratoconus with normal topography was 84%. CONCLUSIONS: Corneal resistance factor was better than CH for detecting keratoconic corneas once the effect of CCT on ORA measurements was considered, even for topographically unaffected fellow eyes of patients with keratoconus. The CCT-corrected CRF cutoff values and transformed indices may be of clinical use.

CXCL12-induced chemotaxis is impaired in T cells from patients with ZAP-70-negative chronic lymphocytic leukemia.

BACKGROUND: T cells from patients with chronic lymphocytic leukemia may play an important role in contributing to the onset, sustenance, and exacerbation of the disease by providing survival and proliferative signals to the leukemic clone within lymph nodes and bone marrow. DESIGN AND METHODS: By performing chemotaxis assays towards CXCL12, CCL21 and CCL19, we sought to evaluate the migratory potential of T cells from chronic lymphocytic leukemia patients. We next analyzed the chemokine-induced migration of T cells, dividing the chronic lymphocytic leukemia samples according to their expression of the poor prognostic factors CD38 and ZAP-70 in leukemic cells determined by flow cytometry. RESULTS: We found that T cells from patients with chronic lymphocytic leukemia are less responsive to CXCL12, CCL21 and CCL19 than T cells from healthy adults despite similar CXCR4 and CCR7 expression. Following separation of the patients into two groups according to ZAP-70 expression, we found that T cells from ZAP-70-negative samples showed significantly less migration towards CXCL12 compared to T cells from ZAP-70-positive samples and that this was not due to defective CXCR4 down-regulation, F-actin polymerization or to a lesser expression of ZAP-70, CD3, CD45, CD38 or CXCR7 on these cells. Interestingly, we found that leukemic cells from ZAP-70-negative samples seem to be responsible for the defective CXCR4 migratory response observed in their T cells. CONCLUSIONS: Impaired migration towards CXCL12 may reduce the access of T cells from ZAP-70-negative patients to lymphoid organs, creating a less favorable microenvironment for leukemic cell survival and proliferation.