Functional flexibility of intestinal IgA - broadening the fine line

Intestinal bacteria outnumber our own human cells in conditions of both health and disease. It has long been recognized that secretory antibody, particularly IgA, is produced in response to these microbes and hypothesized that this must play an important role in defining the relationship between a host and its intestinal microbes. However, the exact role of IgA and the mechanisms by which IgA can act are only beginning to be understood. In this review we attempt to unravel the complex interaction between so-called "natural," "primitive" (T-cell-independent), and "classical" IgA responses, the nature of the intestinal microbiota/intestinal pathogens and the highly flexible dynamic homeostasis of the mucosal immune system. Such an analysis reveals that low-affinity IgA is sufficient to protect the host from excess mucosal immune activation induced by harmless commensal microbes. However, affinity-maturation of "classical" IgA is essential to provide protection from more invasive commensal species such as segmented filamentous bacteria and from true pathogens such as Salmonellatyphimurium. Thus a correlation is revealed between "sophistication" of the IgA response and aggressiveness of the challenge. A second emerging theme is that more-invasive species take advantage of host inflammatory mechanisms to more successfully compete with the resident microbiota. In many cases, the function of IgA may be to limit such inflammatory responses, either directly by coagulating or inhibiting virulence of bacteria before they can interact with the host or by modulating immune signaling induced by host recognition. Therefore IgA appears to provide an added layer of robustness in the intestinal ecosystem, promoting "commensal-like" behavior of its residents.

The habitat, double life, citizenship, and forgetfulness of IgA

Immunoglobulin A (IgA) is the main secretory immunoglobulin of mucous membranes and is powerfully induced by the presence of commensal microbes in the intestine. B cells undergo class switch recombination to IgA in the mucosa-associated lymphoid tissues, particularly mesenteric lymph nodes (MLNs) and Peyer's patches, through both T-dependent and T-independent pathways. IgA B cells primed in the mucosa traffic from the intestinal lymphoid structures, initially through the lymphatics and then join the bloodstream, to home back to the intestinal mucosa as IgA-secreting plasma cells. Once induced, anti-bacterial IgA can be extremely long-lived but is replaced if there is induction of additional IgA specificities by other microbes. The mucosal immune system is anatomically separated from the systemic immune system by the MLNs, which act as a firewall to prevent penetration of live intestinal bacteria to systemic sites. Dendritic cells sample intestinal bacteria and induce B cells to switch to IgA. In contrast, intestinal macrophages are adept at killing extracellular bacteria and are able to clear bacteria that have crossed the mucus and epithelial barriers. There is both a continuum between innate and adaptive immune mechanisms and compartmentalization of the mucosal immune system from systemic immunity that function to preserve host microbial mutualism.

The neurovascular unit as a selective barrier to polymorphonuclear granulocyte (PMN) infiltration into the brain after ischemic injury

The migration of polymorphonuclear granulocytes (PMN) into the brain parenchyma and release of their abundant proteases are considered the main causes of neuronal cell death and reperfusion injury following ischemia. Yet, therapies targeting PMN egress have been largely ineffective. To address this discrepancy we investigated the temporo-spatial localization of PMNs early after transient ischemia in a murine transient middle cerebral artery occlusion (tMCAO) model and human stroke specimens. Using specific markers that distinguish PMN (Ly6G) from monocytes/macrophages (Ly6C) and that define the cellular and basement membrane boundaries of the neurovascular unit (NVU), histology and confocal microscopy revealed that virtually no PMNs entered the infarcted CNS parenchyma. Regardless of tMCAO duration, PMNs were mainly restricted to luminal surfaces or perivascular spaces of cerebral vessels. Vascular PMN accumulation showed no spatial correlation with increased vessel permeability, enhanced expression of endothelial cell adhesion molecules, platelet aggregation or release of neutrophil extracellular traps. Live cell imaging studies confirmed that oxygen and glucose deprivation followed by reoxygenation fail to induce PMN migration across a brain endothelial monolayer under flow conditions in vitro. The absence of PMN infiltration in infarcted brain tissues was corroborated in 25 human stroke specimens collected at early time points after infarction. Our observations identify the NVU rather than the brain parenchyma as the site of PMN action after CNS ischemia and suggest reappraisal of targets for therapies to reduce reperfusion injury after stroke.

Age, microbiota, and T cells shape diverse individual IgA repertoires in the intestine

Intestinal immunoglobulin A (IgA) ensures host defense and symbiosis with our commensal microbiota. Yet previous studies hint at a surprisingly low diversity of intestinal IgA, and it is unknown to what extent the diverse Ig arsenal generated by somatic recombination and diversification is actually used. In this study, we analyze more than one million mouse IgA sequences to describe the shaping of the intestinal IgA repertoire, its determinants, and stability over time. We show that expanded and infrequent clones combine to form highly diverse polyclonal IgA repertoires with very little overlap between individual mice. Selective homing allows expanded clones to evenly seed the small but not large intestine. Repertoire diversity increases during aging in a dual process. On the one hand, microbiota-, T cell-, and transcription factor RORγt-dependent but Peyer's patch-independent somatic mutations drive the diversification of expanded clones, and on the other hand, new clones are introduced into the repertoire of aged mice. An individual's IgA repertoire is stable and recalled after plasma cell depletion, which is indicative of functional memory. These data provide a conceptual framework to understand the dynamic changes in the IgA repertoires to match environmental and intrinsic stimuli.

Intestinal bacterial translocation in rats with cirrhosis is related to compromised Paneth cell antimicrobial host defense

Liver cirrhosis is associated with bacterial translocation (BT) and endotoxemia. Most translocating bacteria belong to the common intestinal microbiota, suggesting a breakdown of intestinal barrier function. We hypothesized that diminished mucosal antimicrobial host defense could predispose to BT. Two rodent models of portal hypertension with increased BT were used, CCl(4)-induced ascitic cirrhosis and 2-day portal vein-ligated (PVL) animals. BT was assessed by standard microbiological techniques on mesenteric lymph nodes. Total RNA was isolated systematically throughout the intestinal tract, and expression of Paneth cell α-cryptdins and β-defensins was determined by real-time quantitative polymerase chain reaction (qPCR). To determine functional consequences, mucosal antimicrobial activity was assessed with a fluorescence-activated cell sorting assay. BT was detectable in 40% of rats with cirrhosis. Compared with the group without BT, these animals exhibited diminished intestinal Paneth cell α-cryptdin 5 and 7 expression. In contrast, PVL was associated with BT in all animals but did not affect antimicrobial peptides. The decrease in Paneth cell antimicrobials was most pronounced in the ileum and the coecum. Other antimicrobials showed no changes or even an induction in the case of BT at different sites. Antimicrobial activity toward different commensal strains was reduced, especially in the distal ileum and the cecum in experimental cirrhosis with BT (excluding PVL). Conclusion: Compromised Paneth cell antimicrobial host defense seems to predispose to BT in experimental cirrhosis. Understanding this liver-gut axis including the underlying mechanisms could help us to find new treatment avenues.

[Guidance of interventions in subintimal recanalization and fenestration of dissection membranes using a novel dual-lumen intravascular ultrasound catheter]

PURPOSE: To evaluate the feasibility and effectiveness of IVUS-guided puncture for gaining controlled target lumen reentry in subintimal recanalization of chronic iliac/femoral artery occlusions and in fenestration of aortic dissections. MATERIALS AND METHODS: Between 5/2004 and 12/2005 12 consecutive patients (7 male, 5 female; mean age 64.6 +/- 12.0 years) with chronic critical limb ischemia and ischemic complications of aortic dissection were treated using the Pioneer catheter. This 6.2-F dual-lumen catheter combines a 20-MHz IVUS transducer with a pre-shaped extendable, hollow 24-gauge nitinol needle. This coaxial needle allows real-time IVUS-guided puncture of the target lumen and after successful reentry a 0.014" guidewire may be advanced through the needle into the target lumen. 7 patients were treated for aortic dissection and 5 patients (with failed previous attempts at subintimal recanalization) for chronic arterial occlusion. Patients with aortic dissection (5 type A dissections, 2 type B dissections) had developed renal ischemia (n = 2), renal and mesenteric ischemia (n = 2), or low extremity ischemia (n = 3). Patients with chronic arterial occlusions (2 common iliac artery occlusions, 3 superficial femoral artery occlusions) experienced ischemic rest pain (n = 4), and a non-healing foot ulcer (n = 1). RESULTS: The technical success rate using the Pioneer catheter was 100%. The recanalization/fenestration time was 37 +/- 12 min. Procedure-related complications did not occur. In 10 cases a significant improvement of clinical symptoms was evident. One patient with aortic dissection and ischemic paraplegia required subsequent surgical intervention. One patient had persistent ischemic rest pain despite successful recanalization of a superficial femoral artery occlusion. CONCLUSION: The Pioneer catheter is a reliable device which may be helpful for achieving target lumen reentry in subintimal recanalization of chronic occlusions and in fenestration of aortic dissections.

Evaluation of gastrointestinal permeability and mucosal absorptive capacity in dogs with chronic enteropathy

OBJECTIVE: To assess intestinal mucosal function by measuring permeability and absorptive capacity in dogs with chronic enteropathy (CE) before and after treatment and to determine whether those variables were correlated with clinical disease activity or histologic scoring of intestinal biopsy specimens. ANIMALS: 29 dogs with CE. PROCEDURE: Dogs were designated as having dietresponsive CE or CE requiring glucorticoid treatment. Severity of clinical signs was assessed by calculating the canine inflammatory bowel disease activity index (CIBDAI). Histologic severity of intestinal infiltration was assessed before and after 4 weeks of treatment in the diet-responsive group and before and after 10 weeks of treatment in the glucocorticoid group. Gastrointestinal permeability and mucosal absorptive capacity were assessed by use of intragastric administration of a solution containing lactulose, rhamnose, xylose, 3-O-methylglucose, and sucrose. Urine was collected 6 hours after administration of the sugar solution to determine urinary lactulose-to-rhamnose (L:R), xylose-to-methylglucose (X:M), and sucrose-to-methylglucose (S:M) ratios. RESULTS: Median CIBDAI scores decreased significantly in both groups of dogs after treatment. However, the median histologic grade of intestinal biopsy specimens did not change with treatment in either group. There were no significant differences in L:R, X:M, or S:M ratios after treatment in either group and no significant correlations between L:R, X:M, or S:M ratios and CIBDAI or histologic scores. CONCLUSIONS AND CLINICAL RELEVANCE: Results of tests for intestinal permeability and mucosal absorptive capacity were not useful indicators of clinical disease activity as assessed by the CIBDAI or the sever ity of infiltration as indicated by histologic evaluation.

Adaptations of intestinal macrophages to an antigen-rich environment

Intestinal macrophages, preferentially located in the subepithelial lamina propria, represent in humans the largest pool of tissue macrophages. To comply with their main task, i.e. the efficient removal of microbes and particulate matter that might have gained access to the mucosa from the intestinal lumen while maintaining local tissue homeostasis, several phenotypic and functional adaptations evolved. Most notably, microbe-associated molecular pattern (MAMP) receptors, including the lipopolysaccharide receptors CD14 and TLR4, but also the Fc receptors for IgA and IgG are absent on most intestinal Mø. Here we review recent findings on the phenotypic and functional adaptations of intestinal Mø and their implications for the pathogenesis of inflammatory bowel diseases.

Two alpha subunits and one beta subunit of meprin zinc-endopeptidases are differentially expressed in the zebrafish Danio rerio

Meprins are members of the astacin family of metalloproteases expressed in epithelial tissues, intestinal leukocytes and certain cancer cells. In mammals, there are two homologous subunits, which form complex glycosylated disulfide-bonded homo- and heterooligomers. Both human meprin alpha and meprin beta cleave several basement membrane components, suggesting a role in epithelial differentiation and cell migration. There is also evidence that meprin beta is involved in immune defence owing to its capability of activating interleukin-1beta and the diminished mobility of intestinal leukocytes in meprin beta-knockout mice. Here we show for the first time by reverse transcription PCR, immunoblotting and immunofluorescence analyses that meprins are expressed not only in mammals, but also in the zebrafish Danio rerio. In contrast to the human, mouse and rat enzymes, zebrafish meprins are encoded by three genes, corresponding to two homologous alpha subunits and one beta subunit. Observations at both the mRNA and protein level indicate a broad distribution of meprins in zebrafish. However, there are strikingly different expression patterns of the three subunits, which is consistent with meprin expression in mammals. Hence, D. rerio appears to be a suitable model to gain insight into the basic physiological functions of meprin metalloproteases.

The alpha and beta subunits of the metalloprotease meprin are expressed in separate layers of human epidermis, revealing different functions in keratinocyte proliferation and differentiation

The zinc endopeptidase meprin (EC 3.4.24.18) is expressed in brush border membranes of intestine and kidney tubules, intestinal leukocytes, and certain cancer cells, suggesting a role in epithelial differentiation and cell migration. Here we show by RT-PCR and immunoblotting that meprin is also expressed in human skin. As visualized by immunohistochemistry, the two meprin subunits are localized in separate cell layers of the human epidermis. Meprin alpha is expressed in the stratum basale, whereas meprin beta is found in cells of the stratum granulosum just beneath the stratum corneum. In hyperproliferative epidermis such as in psoriasis vulgaris, meprin alpha showed a marked shift of expression from the basal to the uppermost layers of the epidermis. The expression patterns suggest distinct functions for the two subunits in skin. This assumption is supported by diverse effects of recombinant meprin alpha and beta on human adult low-calcium high-temperature keratinocytes. Here, beta induced a dramatic change in cell morphology and reduced the cell number, indicating a function in terminal differentiation, whereas meprin alpha did not affect cell viability, and may play a role in basal keratinocyte proliferation.

Effects of percutaneous coronary interventions in silent ischemia after myocardial infarction: the SWISSI II randomized controlled trial

CONTEXT: The effect of a percutaneous coronary intervention (PCI) on the long-term prognosis of patients with silent ischemia after a myocardial infarction (MI) is not known. OBJECTIVE: To determine whether PCI compared with drug therapy improves long-term outcome of asymptomatic patients with silent ischemia after an MI. DESIGN, SETTING, AND PARTICIPANTS: Randomized, unblinded, controlled trial (Swiss Interventional Study on Silent Ischemia Type II [SWISSI II]) conducted from May 2, 1991, to February 25, 1997, at 3 public hospitals in Switzerland of 201 patients with a recent MI, silent myocardial ischemia verified by stress imaging, and 1- or 2-vessel coronary artery disease. Follow-up ended on May 23, 2006. INTERVENTIONS: Percutaneous coronary intervention aimed at full revascularization (n = 96) or intensive anti-ischemic drug therapy (n = 105). All patients received 100 mg/d of aspirin and a statin. MAIN OUTCOME MEASURES: Survival free of major adverse cardiac events defined as cardiac death, nonfatal MI, and/or symptom-driven revascularization. Secondary measures included exercise-induced ischemia and resting left ventricular ejection fraction during follow-up. RESULTS: During a mean (SD) follow-up of 10.2 (2.6) years, 27 major adverse cardiac events occurred in the PCI group and 67 events occurred in the anti-ischemic drug therapy group (adjusted hazard ratio, 0.33; 95% confidence interval, 0.20-0.55; P<.001), which corresponds to an absolute event reduction of 6.3% per year (95% confidence interval, 3.7%-8.9%; P<.001). Patients in the PCI group had lower rates of ischemia (11.6% vs 28.9% in patients in the drug therapy group at final follow-up; P = .03) despite fewer drugs. Left ventricular ejection fraction remained preserved in PCI patients (mean [SD] of 53.9% [9.9%] at baseline to 55.6% [8.1%] at final follow-up) and decreased significantly (P<.001) in drug therapy patients (mean [SD] of 59.7% [11.8%] at baseline to 48.8% [7.9%] at final follow-up). CONCLUSION: Among patients with recent MI, silent myocardial ischemia verified by stress imaging, and 1- or 2-vessel coronary artery disease, PCI compared with anti-ischemic drug therapy reduced the long-term risk of major cardiac events. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00387231.

Drug-eluting stent and coronary thrombosis: biological mechanisms and clinical implications

Although rare, stent thrombosis remains a severe complication after stent implantation owing to its high morbidity and mortality. Since the introduction of drug-eluting stents (DES), most interventional centers have noted stent thrombosis up to 3 years after implantation, a complication rarely seen with bare-metal stents. Some data from large registries and meta-analyses of randomized trials indicate a higher risk for DES thrombosis, whereas others suggest an absence of such a risk. Several factors are associated with an increased risk of stent thrombosis, including the procedure itself (stent malapposition and/or underexpansion, number of implanted stents, stent length, persistent slow coronary blood flow, and dissections), patient and lesion characteristics, stent design, and premature cessation of antiplatelet drugs. Drugs released from DES exert distinct biological effects, such as activation of signal transduction pathways and inhibition of cell proliferation. As a result, although primarily aimed at preventing vascular smooth muscle cell proliferation and migration (ie, key factors in the development of restenosis), they also impair reendothelialization, which leads to delayed arterial healing, and induce tissue factor expression, which results in a prothrombogenic environment. In the same way, polymers used to load these drugs have been associated with DES thrombosis. Finally, DES impair endothelial function of the coronary artery distal to the stent, which potentially promotes the risk of ischemia and coronary occlusion. Although several reports raise the possibility of a substantially higher risk of stent thrombosis in DES, evidence remains inconclusive; as a consequence, both large-scale and long-term clinical trials, as well as further mechanistic studies, are needed. The present review focuses on the pathophysiological mechanisms and pathological findings of stent thrombosis in DES.

Nitric oxide synthase in critically ischaemic muscle and alterations in isoform expression during revascularization surgery

BACKGROUND: Dysfunction of the nitric oxide pathway is implicated in peripheral arterial disease. Nitric oxide synthase (NOS) isoforms and NOS activity were studied in muscle from patients with critical leg ischaemia (CLI). Alterations in NOS during revascularization surgery were also assessed. METHODS: Muscle biopsies were taken from patients with CLI undergoing amputation and also from patients undergoing femorodistal bypass at the start of surgery, after arterial clamping and following reperfusion. The presence of NOS within muscle sections was confirmed using reduced nicotinamide adenine dinucleotide phosphate diaphorase histochemistry. NOS isoform distribution was studied by immunohistochemistry. NOS mRNA and protein levels were measured using real-time reverse transcriptase-polymerase chain reaction and western blotting. NOS activity was assessed with the citrulline assay. RESULTS: All three NOS isoforms were found in muscle, associated with muscle fibres and microvessels. NOS I and III protein expression was increased in CLI (P = 0.041). During revascularization, further ischaemia and reperfusion led to a rise in NOS III protein levels (P = 0.008). NOS activity was unchanged. CONCLUSION: Alterations in NOS I and III occurred in muscle from patients with CLI and further changes occurred during bypass surgery.

Multiple pathways of neuroprotection against oxidative stress and excitotoxic injury in immature primary hippocampal neurons

In the immature brain hydrogen peroxide accumulates after excitotoxic hypoxia-ischemia and is neurotoxic. Immature hippocampal neurons were exposed to N-methyl-D-aspartate (NMDA), a glutamate agonist, and hydrogen peroxide (H(2)O(2)) and the effects of free radical scavenging and transition metal chelation on neurotoxicity were studied. alpha-Phenyl-N-tert.-butylnitrone (PBN), a known superoxide scavenger, attenuated both H(2)O(2) and NMDA mediated toxicity. Treatment with desferrioxamine (DFX), an iron chelator, at the time of exposure to H(2)O(2) was ineffective, but pretreatment was protective. DFX also protected against NMDA toxicity. TPEN, a metal chelator with higher affinities for a broad spectrum of transition metal ions, also protected against H(2)O(2) toxicity but was ineffective against NMDA induced toxicity. These data suggest that during exposure to free radical and glutamate agonists, the presence of iron and other free metal ions contribute to neuronal cell death. In the immature nervous system this neuronal injury can be attenuated by free radical scavengers and metal chelators.

Measurement of nitric oxide and brain tissue oxygen tension in patients after severe subarachnoid hemorrhage

OBJECTIVE: Nitric oxide (NO), one of the most powerful endogenous vasodilators, is thought to play a major role in the development of delayed vasospasm in patients with subarachnoid hemorrhage (SAH). However, the role of the production of cerebral NO in patients with SAH is not known. In other SAH studies, NO metabolites such as nitrite and nitrate have been demonstrated to be decreased in cerebrospinal fluid and in plasma. METHODS: In this study, a microdialysis probe was used, along with a multiparameter sensor, to measure NO metabolites, brain tissue oxygen tension, brain tissue carbon dioxide tension, and pH in the cortex of patients with severe SAH who were at risk for developing secondary brain damage and vasospasm. NO metabolites, glucose, and lactate were analyzed in the dialysates to determine the time course of NO metabolite changes and to test the interrelationship between the analytes and clinical variables. RESULTS: Brain tissue oxygen tension was strongly correlated to dialysate nitrate and nitrite (r2 = 0.326; P < 0.001); however, no correlation was noted between brain tissue oxygen tension and NO metabolites in cerebrospinal fluid (r2 = 0.018; P = 0.734). No significant correlation between NO production, brain tissue carbon dioxide tension, and dialysate glucose and lactate was observed. CONCLUSION: Cerebral ischemia and compromised substrate delivery are often responsible for high morbidity rates and poor outcomes after SAH. The relationship between brain tissue oxygen and cerebral NO metabolites that we demonstrate suggests that substrate delivery and NO are linked in the pathophysiology of vasospasm after SAH.