Domenico Caccavo

Enteric bacteria, lipopolysaccharides and related cytokines in inflammatory bowel disease: biological and clinical significance.

Ulcerative colitis (UC) and Crohns disease (CD) [inflammatory bowel disease (IBD)] are both characterized by an exaggerated immune response at the gut associated lymphoreticular tissue level. Such an abnormal and dysregulated immune response may be directed against luminal and/or enteric bacterial antigens, as also supported by murine models of inflammatory bowel disease (IBD) caused by organisms such as Citrobacter rodentium and Helicobacter hepaticus. Bacterial endotoxins or lipopolysaccharides (LPS) have been detected in the plasma of IBD patients and an abnormal microflora and/or an increased permeability of the intestinal mucosa have been invoked as cofactors responsible for endotoxemia. At the same time, the evidence that phagocytosis and killing exerted by polymorphonuclear cells and monocytes and the T-cell dependent antibacterial activity are decreased in IBD patients may also explain the origin of LPS in these diseases. In IBD, pro-inflammatory cytokines and chemokines have been detected in elevated amounts in mucosal tissue and/or in peripheral blood, thus suggesting a monocyte/macrophage stimulation by enteric bacteria and/or their constituents (e.g. LPS). On these grounds, in experimental models and in human IBD, anti-cytokine monoclonal antibodies and interleukin receptor antagonists are under investigation for their capacity to neutralize the noxious effects of immune mediators. Finally, the administration of lactobacilli is beneficial in human IBD and, in murine colitis, this treatment leads to a normalization of intestinal flora, reducing the number of colonic mucosal adherent and translocated bacteria.

Treatment of idiopathic mixed cryoglobulinemia with alpha interferon

Seven patients with type II idiopathic mixed cryoglobulinemia were treated with recombinant human leukocyte interferon (alpha interferon). In all of them, a conspicuous reduction of circulating cryoglobulins was noted, together with a definite, remarkable improvement of the clinical pattern. The immunologic parameters (natural killer cell activity and phagocytosis, among others) improved as well; side effects were usually mild and transient. Increases in the cryoglobulin level occurred in a few cases, but they were at least partly sensitive to readministration of alpha interferon treatment. The favorable results obtained in these cases of idiopathic cryoglobulinemia seem to be consistent and prolonged.

The role of the liver in the response to LPS: experimental and clinical findings

The liver plays an important physiological role in lipopolysaccharide (LPS) detoxification and, in particular, hepatocytes are involved in the clearance of endotoxin of intestinal derivation. In experimental shock models, tumor necrosis factor (TNF)-α induces hepatocyte apoptosis and lethal effects are due to secreted TNF-α and not to cell-associated TNF-α. An exaggerated production of TNF-α has been reported in murine viral infections, in which mice become sensitized to low amounts of LPS and both interferon (IFN)-γ and IFN-α/β are involved in the macrophage-induced release of TNF-α. The prominent role of LPS and TNF-α in liver injury is also supported by studies of ethanol-induced hepatic damage. In humans, evidence of LPS-induced hepatic injury has been reported in cirrhosis, autoimmune hepatitis, and primary biliary cirrhosis and a decreased phagocytic activity of the reticulo-endothelial system has been found in these diseases. The origin of endotoxemia in hepatitis C virus (HCV) infected patients seems to be multifactorial and LPS may be of exogenous or endogenous derivation. In endotoxemic HCV-positive patients responsive to a combined treatment with IFN-α/ribavirin (RIB), endotoxemia was no longer detected at the end of the therapeutic regimen. By contrast, 48% of the non-responders to this treatment were still endotoxemic and their monocytes displayed higher intracellular TNF-α and interleukin (IL)-1β levels than responders. Moreover, in responders, an equilibrium between IFN-γ and IL-10 serum levels was attained. In the non-responders, serum levels of IL-10 did not increase following treatment. This may imply that an imbalance between T helper (Th)1 and Th2 derived cytokines could be envisaged in the non-responders.

Neuropsychiatric lupus syndromes: relationship with antiphospholipid antibodies.

The authors assessed the prevalence of neuropsychiatric manifestations occurring in patients with systemic lupus erythematosus (NPSLE), according to the American College of Rheumatology standardized definitions for NPSLE, and evaluated the relationship between NPSLE and antiphospholipid antibodies. Sixty-one consecutive SLE patients were studied. Neuropsychiatric manifestations consistent with the diagnosis of NPSLE occurred in 44 (72%). Patients with NPSLE showed significantly higher levels of anticardiolipin antibodies.

Antimicrobial and immunoregulatory functions of lactoferrin and its potential therapeutic application.

Lactoferrin is an iron-binding glycoprotein present in various secretions (e.g. milk, tears, saliva, pancreatic juice, etc.). It is also stored in specific granules of polymorphonuclear granulocytes from which it is released following activation. Lactoferrin exerts a bactericidal activity by damagingthe outer membrane of Gram-negative bacteria, as well as immunoregulatory functions by decreasing the release of interleukin-1 (IL-1), IL-2 and tumor necrosis factor- (TNF-) and enhancing monocyte and natural killer cell cytotoxicity. Lactoferrin binds with high affinity to lipid A, the toxic moiety of the lipopolysaccharide, or endotoxin from Gram-negative bacteria. Lipopolysaccharide interaction with monocytes/macrophages results in the production and release of TNF- , that plays an important role in inducing septic shock. In this respect, it has recently been demonstrated that lactoferrin inhibits the lipopolysaccharide interaction with CD14 on monocytes/macrophages by competition with the lipopolysaccharide binding protein. Therefore, besides its bactericidal activity, lactoferrin may also act by neutralizing the toxic effects of lipopolysaccharide and this protective role against endotoxin lethal shock has been demonstrated in animal models. Moreover, in vitro and in vivo neutralization of endotoxin by a human lactoferrin-derived peptide was also reported and lactoferrin or lactoferrin-derived peptides could represent useful tools for the treatment of endotoxin-induced septic shock. The recent production and characterization of monoclonal antibodies against different epitopes of human lactoferrin, including monoclonal antibodies selectively neutralizinglactoferrin binding to lipid A, may allow a better elucidation of the consequence of lactoferrin-lipopolysaccharideinteraction.

Phagocytosis, killing, lymphocyte-mediated antibacterial activity, serum autoantibodies, and plasma endotoxins in inflammatory bowel Disease

OBJECTIVE:Alteration of mucosal and systemic immune responses may play an important role in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to evaluate natural immune responses (i.e., phagocytosis, killing, and antibacterial activity), serum autoantibodies (antineutrophil cytoplasmic antibodies [ANCA] and antilactoferrin [LF] antibodies), and plasma endotoxins in patients affected by ulcerative colitis (UC) and Crohns disease (CD).METHODS:Blood samples were obtained from 71 patients with UC, 32 patients with CD, and 32 control subjects. Disease activity was scored using Trueloves criteria in patients with UC and the Crohns Disease Activity Index (CDAI) in patients with CD. Candida albicans served as a target for evaluation of phagocytosis and killing exerted by polymorphonuclear cells (PMN) and monocytes (MØ), whereas Salmonella typhi was used for assessing lymphocyte-mediated antibacterial activity. ANCA were detected by indirect immunofluorescence, whereas anti-LF antibodies were assayed by means of enzyme-linked immunosorbent assay. Plasma endotoxins were measured by Limulus amoebocyte lysate assay.RESULTS:Phagocytosis and killing exerted by PMN and MØ, as well as lymphocyte-mediated antibacterial activity, were significantly reduced (p < 0.0001) in patients affected by UC and CD in comparison with controls, irrespective of either disease activity or treatment. Plasma endotoxins were detected in 12/71 (17%) patients with UC, and in 10/32 (31%) patients with CD. ANCA were present in 42/71 (59%) patients with UC and in 3/32 (9%) patients with CD, whereas anti-LF antibodies were detected in 31 (44%) UC patients and in six (19%) CD patients. No significant differences in phagocytosis and killing exerted by PMN were found between ANCA-positive and ANCA-negative UC patients.CONCLUSIONS:Our data demonstrate an impairment of natural immunity exerted by peripheral blood phagocytes and lymphocytes in patients with UC and CD. ANCA and anti-LF antibodies were present mainly in UC patients but their presence did not affect PMN-mediated phagocytosis and killing. Finally, plasma endotoxins may contribute to the chronic inflammatory status, likely by inducing release of proinflammatory mediators.

Biological and Clinical Significance of Endotoxemia in the Course of Hepatitis C Virus Infection

Endotoxins or lipopolysaccharides (LPS), major components of the cell wall of Gram-negative bacteria, once released from the bacterial outer membrane bind to specific receptors and, in particular, to a membrane-bound receptor, the CD14 (mCD14) and the toll-like receptor 4 present on monocytes/ macrophages. In turn, LPS-activated monocytes/ macrophages release in the host tissue an array of so-called proinflammatory cytokines and, among them, Tumor Necrosis Factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, IL-8 and IL-12 are the major mediators. Before therapy (To) and at the end of 6-month interferon (IFN)-alpha/Ribavirin (RIB) treatment (T6), circulating endotoxin levels were measured in responder and non responder HCV+ patients. At T0, 57% of the non responders were endotoxin-positive and had, on average, 54 pg/ml of plasma LPS while in 50% of the responder patients endotoxin were found with an average of 29 pg/ml. At T6, in responders LPS were no longer detectable, while in 42% of the non responders LPS were found (average levels 45 pg/ml). In terms of serum cytokine concentration, at T6 IFN-gamma levels when compared to those detected at T0 were increased in both endotoxin-positive and endotoxin-negative patients. However, at T6 IL-10 concentration was significantly increased only in the group of endotoxin-negative subjects (responder patients), in comparison to T0 values. The origin of endotoxemia in HCV+ patients seems to be multifactorial, likely depending on impaired phagocytic functions and reduced T-cell mediated antibacterial activity. In these patients, however, one cannot exclude the passage of LPS from the gut flora to the blood stream, owing a condition of altered intestinal permeability. At the same time, a less efficient detoxification of enteric bacterial antigens at the hepatic level should be taken into consideration. Finally, novel therapeutic attempts aimed to neutralize LPS in the host are discussed.

Anti‐cardiolipin antibodies in autoimmune thyroid diseases

OBJECTIVE In recent years anti‐phospholipid antibodies have gained much attention since they are frequently associated with thrombosis, recurrent abortion, and thrombocytopenia. Besides disease‐specific autoantibodies, other autoantibodies reactive with both organ and non‐organ specific autoantigens have been found in patients with autoimmune thyroid diseases. Therefore the objective of this study was to evaluate the presence and significance of anti‐phospholipid antibodies in untreated patients with different forms of autoimmune thyroid diseases.

Portosystemic shunts in a large cohort of patients with liver cirrhosis: detection rate and clinical relevance

BackgroundThis study aimed to determine the detection rate and clinical relevance of portosystemic collaterals.MethodsWe studied 326 cirrhotics. Portosystemic collaterals, portal vein diameter, and splenic area were evaluated by color Doppler sonography; esophageal varices were detected by endoscopy.ResultsOf the cirrhotics, 130 had portosystemic collaterals (39.9% total, left gastric vein 11%, paraumbilical vein 7.4%, splenorenal shunts 13.8%, and combined shunts 7.7%). Cirrhotics without portosystemic collaterals or with a paraumbilical vein had a significantly narrower portal vein diameter than cirrhotics with a left gastric vein (P < 0.001). Cirrhotics with a paraumbilical vein had a significantly smaller splenic area than cirrhotics with a left gastric vein (P < 0.001), splenorenal shunts (P < 0.001), combined shunts (P < 0.001), or without portosystemic collaterals (P < 0.05). A significant association between portosystemic collaterals and Child’s classes or presence and type of esophageal varices was found (P < 0.0001 and P = 0.0004, respectively). The highest prevalence of Child’s class C and large (F-3) esophageal varices was found in cirrhotics with a left gastric vein (41.7% and 36.1%, respectively), whereas esophageal varices were absent in 47.4% of cirrhotics without portosystemic collaterals and in 58.3% of cirrhotics with a paraumbilical vein.ConclusionsThe left gastric vein is associated with some sonographic and clinical markers of disease severity, whereas the absence of portosystemic collaterals or the presence of paraumbilical veins seems to identify cirrhotics with markers predictive of a more favorable clinical course.

Expression of lactoferrin on human granulocytes: analysis with polyclonal and monoclonal antibodies

Lactoferrin (LF), an iron‐binding protein present in specific granules of neutrophils, is expressed on membrane after granulocyte activation. It may represent a target for anti‐neutrophil cytoplasmic antibodies (ANCA) in patients affected by some immunomediated diseases. We recently produced two MoAbs, AGM 2.29 and AGM 10.14, that recognize two spatially distant epitopes of human LF. In this study we perform a cytometric analysis in order to evaluate the expression of LF on the surface of granulocytes obtained from freshly drawn blood or after purification, in both the presence and absence of stimuli. Our results demonstrate that LF is not constitutively expressed on membrane of circulating neutrophils. After priming with phorbol myristate acetate (PMA) or tumour necrosis factor‐alpha (TNF‐α), an increased mean fluorescence intensity (MFI) was obtained on neutrophils stained with polyclonal anti‐LF antibodies and with AGM 2.29. The kinetics of LF expression during activation demonstrated a progressive increase in MFI within 45 min. No increase in MFI was documented when primed granulocytes were stained with MoAb AGM 10.14, thus indicating that the epitope recognized by AGM 10.14 is not exposed at the cell surface. Following membrane permeabilization, performed in order to analyse the binding of anti‐LF MoAbs to cytoplasmic LF, a marked increase in MFI was obtained by staining granulocytes with both anti‐LF MoAbs. Indirect immunofluorescence (IIF) analysis confirmed that AGM 2.29 and AGM 10.14 reacted with human granulocytes, showing a cytoplasmic pattern on formalin–acetone‐fixed neutrophils and a perinuclear one on ethanol‐fixed cells.