Expression of serum amyloid A, in normal, dysplastic, and neoplastic human colonic mucosa: implication for a role in colonic tumorigenesis.

Serum amyloid A (SAA) is an acute phase reactant, whose level in the blood is elevated in response to trauma, infection, inflammation, and neoplasia. Elevated levels of SAA in the serum of cancer patients were suggested to be of liver origin rather than a tumor cell product. The role of SAA in human malignancies has not been elucidated. We investigated the expression of SAA at various stages of human colon carcinoma progression. Nonradioactive in situ hybridization applied on paraffin tissue sections from 26 colon cancer patients revealed barely detected SAA mRNA expression in normal looking colonic epithelium. Expression was increased gradually as epithelial cells progressed through dysplasia to neoplasia. Deeply invading colon carcinoma cells showed the highest levels of SAA. Expression was also found in colon carcinoma metastases. Cells of lymphoid follicles of the intestinal wall, inflammatory cells, ganglion cells, and endothelial cells, also expressed SAA mRNA. Immunohistochemical staining revealed SAA protein expression that colocalized with SAA mRNA expression. RT-PCR analysis confirmed the expression of the SAA1 and SAA4 genes in colon carcinomas, expression that was barely detectable in normal colon tissues. These findings indicate local and differential expression of SAA in human colon cancer tissues and suggest its role in colonic tumorigenesis.     

Progressive and concordant expression of PKC-eta and iNOS phenotypes in monocytes from patients with rheumatoid arthritis: association with disease severity.

Rheumatoid arthritis (RA) is a relatively common autoimmune disease with strong genetic and environmental determinants. The disease manifests itself as inflammation of the synovia and usually progresses to joint erosion and destruction. The disease can also be considered as a systemic disease because extra-articular manifestations are often observed throughout many organs and tissues of the body. Patients with severe RA have altered peripheral blood monocytes (PBM) that express activation markers. Two such markers, PKC-eta and iNOS, were studied using confocal laser scanning microscopy to determine how these markers are expressed during disease progression. Healthy individuals expressed neither of the two markers, but there was an elevated level of PKC-eta observed as the disease progressed (40% in mild RA and 100% in severe RA patients). Concordant expression of the two markers was observed in only 3% of PBM from mild RA patients, reaching 38% in severe RA patients. No cells expressing iNOS alone were observed in any of the patients studied. These data support the hypothesis linking PKC-eta expression with the regulation and predisposition to the development of the iNOS phenotype in severe RA patients. PKC-eta may therefore be a key regulator in the production of elevated plasma nitric oxide (NO) and corresponding circulating reactive nitrogen intermediates in severe RA and may be a possible target to regulate iNOS induction and NO production by monocytic cells in RA patients and possibly other inflammatory diseases.     

Accumulation of B lymphocytes with a naive,resting phenotype in a subset of hepatitis C patients

Chronic infection with hepatitis C virus (HCV) is associated with disturbances of B lymphocyte activation and function: autoantibody production, mixed cryoglobulinemia, and B cell lymphomas. It has been proposed that these abnormalities reflect chronic antigenic stimulation or aberrant signaling through the B cell coreceptor, the latter mediated by binding of the HCV E2 glycoprotein to CD81. To test this hypothesis, we measured expression of activation and differentiation markers on peripheral blood B cells from patients with chronic HCV infection. Thirty-six HCV patients with and without mixed cryoglobulinemia were compared with 18 healthy control volunteers and 17 sustained virologic responders who had cleared HCV infection. Ten of the 36 HCV patient samples showed increased B cell frequencies; B cell frequency was higher in patients with more severe hepatic fibrosis. However, these samples lacked evidence of Ag-driven activation or proliferation. The expanded cells were low in the activation markers CD25, CD69, CD71, CD80, and CD86. Proliferation of circulating B cells was unchanged in HCV patients. These cells did not express the differentiation marker CD27, suggesting that they were not enriched in memory B cells. Furthermore, the expanded B cells expressed both IgD and IgM, suggesting that they were antigenically naive. Together, these results indicate that B cell expansion in the peripheral blood of HCV patients is not associated with Ag-mediated activation and differentiation. Instead, factors other than antigenic stimulation may promote the accumulation of peripheral blood B cells with a naive phenotype in a subset of HCV patients.     

Pathophysiological aspects of lymphedema of human limbs: I. Lymph protein composition

The knowledge of humoral and cellular factors retained in tissues affected by ineffective lymph transport is necessary to comprehend the pathological mechanisms of the evolving changes in the extremity. In this article, we report our observations on changes in the protein composition of lymph in obstructive lymphedema. We have documented that the protein concentrations in lymph tissue fluid of the extremities remain within normal limits in lymphedema of stages I through III. Normal lymph protein values in lymphedema indicate that the homeostatic mechanism of protein transport remains intact as long as there are no major structural changes in the tissues that imply loss of tissue space compliance. A "high protein" edema means "high protein volume" edema that does not affect the Starling's equilibrium. The levels of cytokines were found to be elevated in lymphedema lymph when compared with controls. There were major differences in cytokine levels among patients, evidently higher than those among the control subjects. The high levels of cytokines might be attributed to their local production by infiltrating immune cells. The serum levels remain low, and the lymph-to-serum ratio was above 1 in all the cases investigated. These findings reflect the intensity of the chronic inflammation that prevails in tissues with lymph stasis, not detectable by measuring the levels of lymph immunoglobulins and complement. Moreover, our studies have documented the presence of apoptotic DNA in the lymph of patients with obstructive lymphedema. There were greater quantities of 400-kb apoptotic and smaller DNA fragments in the lymph from lymphedematous limbs than in the controls. The level of fragmented DNA may be another parameter that reflects cellular changes in tissues with lymph stasis. Taken together, measuring levels of lymph proteins provides insight into the evolving processes in the limb tissues.     

Immune status evaluation of patients with chronic heart failure

Chronic heart failure (CHF) may be considered a state of immune activation and persistent inflammation expressed by increased circulating levels of pro- and anti-inflammatory cytokines. The purpose of the study was to investigate the immune status in patients with CHF compared to normal individuals. We measured serum cytokine levels as well as cytokine production after ex vivo LPS stimulation of whole blood taken from 14 patients with CHF and 14 healthy volunteers. We used 500 pg/ml of LPS for an incubation period of 4h to stimulate 100 microL of whole blood. Patients with CHF had significantly higher levels of TNF-RI, and TNF-RII in serum compared to normal individuals. TNF-alpha, IL-6, and IL-10 did not differ significantly. After LPS stimulation, patients with CHF had significantly higher levels of TNF-alpha and IL-10, and significantly lower IL-6 levels compared to normal individuals. TNF-alpha receptors did not differ significantly. Patients with CHF may be found in a pro- as well as an anti-inflammatory state. They also do not develop endotoxin tolerance in an ex vivo laboratory model using whole blood stimulated with LPS. They may have increased TNF-alpha and IL-10 production after LPS stimulation of whole blood, which may contribute to a worsening of heart function, more severe disease presentation and a worse outcome during infections.     

Spontaneous inflammatory cytokine gene expression in normal human peripheral blood mononuclear cells

Cytokines regulate cellular immune activity and are produced by a variety of cells, especially lymphocytes, monocytes, and macrophages. Measurement of cytokine levels has yielded useful information on the pathological process of different diseases such as AIDS, endotoxic shock, sepsis, asthma, and cancer. It may also be of use in the monitoring of disease progression and/or inflammation. To determine spontaneous cytokine gene expression in whole blood and PBMCs, whole blood was obtained from healthy volunteers and total mRNA was isolated from PBMCs. The kinetics of response were determined by sequential testing of cytokine gene expression by RT-PCR analysis. Our results demonstrated that isolated and incubated PBMCs expressed TNF-alpha and high levels of IL-1beta, IL-6, IL-8, and IL-10. In contrast, WB only expressed the mRNA cytokines of TNF-alpha and IL-8 (p < 0.05). These results suggest that spontaneous myriad mRNA cytokine expression can be avoided with the use of WB incubation and the rapid collection of PBMCs. Furthermore, this method should be employed in all cases where the levels of cytokine gene expression can be evaluated.     

Post-treatment Vascular Leakage and Inflammatory Responses around Brain Cysts in Porcine Neurocysticercosis

Cysticidal treatment of neurocysticercosis, an infection of humans and pig brains with Taenia solium, results in an early inflammatory response directed to cysts causing seizures and focal neurological manifestations. Treatment-induced pericystic inflammation and its association with blood brain barrier (BBB) dysfunction, as determined by Evans blue (EB) extravasation, was studied in infected untreated and anthelmintic-treated pigs. We compared the magnitude and extent of the pericystic inflammation, presence of EB-stained capsules, the level of damage to the parasite, expression of genes for proinflammatory and regulatory cytokines, chemokines, and tissue remodeling by quantitative PCR assays between treated and untreated infected pigs and between EB-stained (blue) and non stained (clear) cysts. Inflammatory scores were higher in pericystic tissues from EB-stained cysts compared to clear cysts from untreated pigs and also from anthelmintic-treated pigs 48 hr and 120 hr after treatment. The degree of inflammation correlated with the severity of cyst wall damage and both increased significantly at 120 hours. Expression levels of the proinflammatory genes for IL-6, IFN-γ, TNF-α were higher in EB-stained cysts compared to clear cysts and unaffected brain tissues, and were generally highest at 120 hr. Additionally, expression of some markers of immunoregulatory activity (IL-10, IL-2Rα) were decreased in EB-stained capsules. An increase in other markers for regulatory T cells (CTLA4, FoxP3) was found, as well as significant increases in expression of two metalloproteases, MMP1 and MMP2 at 48 hr and 120 hr post-treatment. We conclude that the increase in severity of the inflammation caused by treatment is accompanied by both a proinflammatory and a complex regulatory response, largely limited to pericystic tissues with compromised vascular integrity. Because treatment induced inflammation occurs in porcine NCC similar to that in human cases, this model can be used to investigate mechanisms involved in host damaging inflammatory responses and agents or modalities that may control damaging post treatment inflammation.     

The murine antiapoptotic protein A1 is induced in inflammatory macrophages and constitutively expressed in neutrophils.

Myeloid leukocytes are thought to regulate their susceptibility to apoptosis upon migration to a site of inflammation. However, factors that determine survival have not been well characterized in these cells. We have examined the expression of murine A1, an antiapoptotic Bcl-2 relative found in activated myeloid cells, during the course of an acute inflammatory response. Intraperitoneal infection of mice with the virulent RH strain of Toxoplasma gondii led to a 5- to 10-fold increase in A1 mRNA levels in peritoneal cells after several days. Bcl-2 expression was unchanged. The increase in A1 expression depended on the dose of the organism and coincided with a sharp increase in peritoneal cellularity. A1 protein levels were also increased as determined by Western blot analysis and immunohistochemical studies. All neutrophils and approximately half of the macrophages in the inflammatory exudate contained high levels of A1 in cytoplasm. A1 expression did not correlate with intracellular parasitization. Peripheral blood neutrophils from normal mice strongly expressed A1 protein, whereas normal monocytes showed only weak staining. Bax mRNA was induced in parallel with A1 in macrophages. Exudate macrophages and granulocytes that were apoptotic by TUNEL staining occasionally appeared to display A1 throughout the cell nucleus. These studies identify A1 as a potential regulator of apoptosis during acute inflammation.     

Intracellular regulation of Fas-induced apoptosis in human fibroblasts by extracellular factors and cycloheximide

Fibroblasts play an important role in reparative and inflammatory processes by synthesizing extracellular matrix components and releasing growth factors and cytokines. Fibroblast apoptosis has been observed at the termination phase of reparative or fibrotic responses, but its regulation in this context is poorly known. We investigated the susceptibility of human dermal fibroblasts (DF) to Fas-induced apoptosis and its regulation by extracellular factors potentially involved in immune-mediated inflammation and repair. DF expressed all components of the Fas apoptotic pathway: surface Fas, Fas-associated protein with death domain, and caspase-8 proteins. However, Fas activation resulted in caspase-8 activation and apoptosis only in the presence of cycloheximide (CHX). DF constitutively expressed Fas-associated death domain-like IL-1-converting enzyme-like inhibitory protein (FLIP) that was drastically down-regulated by CHX. Exogenous growth factors, cytokines, and adherence to the extracellular matrix shifted the balance of FLIP-caspase-8 proteins and modified the susceptibility of DF to Fas- or Fas-CHX-induced apoptosis. Short-term serum deprivation, suspension culture, and pretreatment with IFN-gamma or TNF-alpha increased, whereas long-term serum-free culture and pretreatment with TGF-beta or IL-10 decreased the apoptotic susceptibility of DF. Surface Fas expression was only modified by TNF-alpha and IFN-gamma, whereas all studied factors modified FLIP-caspase-8 protein expression, consistently with their pro- or antiapoptotic effects. Antisense FLIP oligonucleotides prevented resistance to Fas-induced apoptosis in DF. FLIP-caspase-8 balance seems tightly regulated in fibroblasts by extracellular factors that determine their susceptibility to Fas- or Fas-CHX-induced apoptosis. Th1 and Th regulatory cytokines display opposite effects on fibroblast apoptosis that suggest that their pro- or antifibrotic effects involve direct effects on fibroblast survival.     

Release of cytokines by human nasal epithelial cells and peripheral blood mononuclear cells infected with Mycoplasma pneumoniae

Mycoplasma pneumoniae (Mp) infection is associated with asthma exacerbation in children. We hypothesized that Mp infection may cause airway inflammation by inducing the release of cytokines by respiratory epithelial cells. The levels of chemokines interleukin-8 (IL-8) and released upon activation, normal t cell expressed and secreted (RANTES) released by nasal epithelial cell (NEC) cultures established from asthmatic and nonasthmatic children were measured by ELISA at 4, 24, 48, and 72 hr after cells were inoculated with Mp, and were compared with baseline release of these factors. The presence of MP on apical membranes of NEC after infection was confirmed by transmission electron microscopy, and adherence was shown to be inhibited by erythromycin. Mp infection did not alter NEC release of IL-8 or RANTES at any time point. In contrast, tumor necrosis factor alpha (TNF-alpha) stimulated increased IL-8 at all time points, and respiratory syncytial virus (RSV) infection stimulated RANTES release at 48 and 72 hr by NEC. These results were not significantly different between NEC from asthmatic and nonasthmatic children. As a comparison, peripheral blood mononuclear cells from normal human volunteers were also incubated with Mp and had significantly increased release of IL-2, IL-6, and TNF-alpha. We conclude that Mp, unlike viral pathogens such as RSV, is unlikely to directly stimulate early airway surface cytokine responses via mechanisms involving epithelial cells. We speculate that the chronic presence of mononuclear cells at the airway surface of asthmatics provides a target for Mp-triggered cytokine production.     

Coevolution of markers of innate and adaptive immunity in skin and peripheral blood of patients with erythema migrans

We used multiparameter flow cytometry to characterize leukocyte immunophenotypes and cytokines in skin and peripheral blood of patients with erythema migrans (EM). Dermal leukocytes and cytokines were assessed in fluids aspirated from epidermal suction blisters raised over EM lesions and skin of uninfected controls. Compared with corresponding peripheral blood, EM infiltrates were enriched for T cells, monocytes/macrophages, and dendritic cells (DCs), contained lower proportions of neutrophils, and were virtually devoid of B cells. Enhanced expression of CD14 and HLA-DR by lesional neutrophils and macrophages indicated that these innate effector cells were highly activated. Staining for CD45RO and CD27 revealed that lesional T lymphocytes were predominantly Ag-experienced cells; furthermore, a subset of circulating T cells also appeared to be neosensitized. Lesional DC subsets, CD11c(+) (monocytoid) and CD11c(-) (plasmacytoid), expressed activation/maturation surface markers. Patients with multiple EM lesions had greater symptom scores and higher serum levels of IFN-alpha, TNF-alpha, and IL-2 than patients with solitary EM. IL-6 and IFN-gamma were the predominant cytokines in EM lesions; however, greater levels of both mediators were detected in blister fluids from patients with isolated EM. Circulating monocytes displayed significant increases in surface expression of Toll-like receptor (TLR)1 and TLR2, while CD11c(+) DCs showed increased expression of TLR2 and TLR4; lesional macrophages and CD11c(+) and CD11c(-) DCs exhibited increases in expression of all three TLRs. These results demonstrate that Borrelia burgdorferi triggers innate and adaptive responses during early Lyme disease and emphasize the interdependence of these two arms of the immune response in the efforts of the host to contain spirochetal infection.     

Vdelta1 T lymphocytes expressing a Th1 phenotype are the major gammadelta T cell subset infiltrating the liver of HCV-infected persons

BACKGROUND: Hepatitis C infection induces an acute and chronic liver inflammation that may lead to cirrhosis, liver failure, or hepatocarcinoma. Since the role of alphabeta T lymphocytes in hepatitis C virus (HCV) immunopathology has been analyzed extensively, we investigated the distribution and functional activation of gammadelta T cell subsets in chronically HCV-infected patients. MATERIALS AND METHODS: Blood samples and liver biopsies from 35 patients with compensated chronic HCV infection were compared in terms of T cell subset distribution, expression of activation markers, gammadelta T cell receptor (TCR) repertoire, and pattern of cytokine production. Moreover, we analyzed whether these immunological parameters were associated with other clinical observations (plasma viremia, ALT levels, Ishak index). RESULTS: Differing from peripheral blood distribution, a specific compartmentalization of Vdelta1 T cells (p < 0.001) was observed in the liver of HCV patients. These cells represented a relevant fraction of intrahepatic T lymphocytes (1.8-8.7%) and expressed the memory/effector phenotype (CD62-L- CD45-RO+CD95+). This phenotype was consistent with selective homing upon antigen recognition. Mitogenic stimulation of Vdelta1 + T lymphocytes recruited in the liver revealed the T helper cell type 1 (Th1) pattern of cytokine secretion. Interestingly, the frequency of interferon-gamma (IFN-gamma)-producing Vdelta1 T cells was associated with an higher degree of liver necroinflammation, measured by the Ishak index. Finally, the T-cell repertoire analysis revealed the absence of Vgamma selection in the TCR repertoire of intrahepatic Vdelta1 T cells. CONCLUSIONS: gammadelta T cell distribution in the peripheral blood differs from the Vdelta1 T cell subset because it is policlonally activated and recruited in the liver of chronic HCV-infected patients. During HCV-infection, this T cell subset may release Th1 cytokines and contribute to the necroinflammatory liver disease.     

Characteristics of dengue virus-infected peripheral blood mononuclear cell death that correlates with the severity of illness

The pathogenic mechanism of the severe form of dengue is complicated. Recent reports indicate that apoptotic death of various tissues or organs may be associated with vascular leakage, and ultimately leads to the death of DENV-infected patients. In the present study, we provide additional evidence supporting the detrimental role of apoptosis in DENV infection. A comparison of the rate of apoptosis in PBMCs isolated from patients suffering DF, a mild form of the disease, and the rate in patients with DHF, a life-threatening disease, revealed that PBMCs from DHF patients underwent apoptosis at a significantly higher rate than those suffering from DF alone. This suggests that the severity of natural DENV infection correlates with PBMC apoptosis. In addition, this cell death was induced not only by DENV itself, but also by the apoptotic activities of pro-inflammatory cytokines, such as TNF-α, and IL-1β, that were upregulated in DHF patients. The death of these mononuclear cells that function in an innate immune system may explain the higher viral load in DHF patients than in DF patients. Interestingly, a gene expression profile pattern elucidated that apoptosis occurring during natural DENV infection involved mainly the extrinsic apoptosis pathway, which is mediated via both caspase-dependent and caspase-independent mechanisms. In conclusion, our data highlight the adverse effect of apoptosis induced by DENV and by pro-inflammatory cytokines during natural DENV infection.     

Changes in the Inflammatory Response to Injury and Its Resolution during the Loss of Regenerative Capacity in Developing Xenopus Limbs

Tissue and organ regeneration, unlike development, involves an injury that in postembryonic animals triggers inflammation followed by resolution. How inflammation affects epimorphic regeneration is largely uninvestigated. Here we examine inflammation and its resolution in Xenopus laevis hindlimb regeneration, which declines during larval development. During the first 5 days postamputation, both regeneration-competent stage 53 and regeneration-deficient stage 57 hindlimbs showed very rapid accumulation of leukocytes and cells expressing interleukin-1β and matrix metalloproteinase 9. Expression of genes for factors mediating inflammatory resolution appeared more persistent at stages 55 and 57 than at stage 53, suggesting changes in this process during development. FoxP3, a marker for regulatory T cells, was upregulated by amputation in limbs at all three stages but only persisted at stage 57, when it was also detected before amputation. Expression of genes for cellular reprogramming, such as SALL4, was upregulated in limbs at all 3 stages, but markers of limb patterning, such as Shh, were expressed later and less actively after amputation in regeneration-deficient limbs. Topical application of specific proinflammatory agents to freshly amputated limbs increased interleukin-1β expression locally. With aqueous solutions of the proinflammatory metal beryllium sulfate, this effect persisted through 7 days postamputation and was accompanied by inhibition of regeneration. In BeSO₄-treated limbs expression of markers for both inflammation and resolution, including FoxP3, was prolonged, while genes for cellular reprogramming were relatively unaffected and those for limb patterning failed to be expressed normally. These data imply that in Xenopus hindlimbs postamputation inflammation and its resolution change during development, with little effect on cellular dedifferentiation or reprogramming, but potentially interfering with the expression of genes required for blastema patterning. The results suggest that developmental changes in the larval anuran immune system may be involved in the ontogenetic loss of epimorphic regeneration in this system.     

Gastric mucosal cytokine responses in Helicobacter pylori-infected patients with gastritis and peptic ulcers. Association with inflammatory parameters and bacteria load

Helicobacter pylori is an important pathogen in gastroduodenal inflammation and ulceration. Several mechanisms have been proposed to explain its role. We studied the cytokine production patterns in situ in gastric mucosal biopsies from H. pylori-positive and H. pylori-negative patients with dyspepsia. Immunohistochemistry with monoclonal antibodies was used. The study showed enhanced expression of interleukin (IL) -8, IL-10 and interferon-gamma (IFN-gamma) in H. pylori infection and a significant association was found between these cytokines and the following parameters: bacteria load, chronic inflammation and activity. These parameters were significantly correlated with the cell markers CD19 and CD56. The study indicates a dual effect of H. pylori on the Th1 response, i.e. a stimulation of the response verified by increased IFN-gamma and a feed-back verified by an increase of the counterinflammatory IL-10, which may dampen the inflammatory and cytotoxic effect of the Th1 response. Furthermore, the study confirms the connection between increase of IL-8 and inflammatory activity in gastric mucosa in H. pylori infection.     

Angiogenesis, Kaposi＇s Sarcoma and Kaposi＇s Sarcomaassociated Herpesvirus

Tumor angiogenesis is the uncontrolled growth of blood vessels in tumors,serving to supply nutrients and oxygen,and remove metabolic wastes.Kaposi's sarcoma (KS),a multifocal angioproliferative disorder characterized by spindle cell proliferation,neo-angiogenesis,inflammation,and edema,is associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV).Recent studies indicate that KSHV infection directly promotes angiogenesis and inflammation through an autocrine and paracrine mechanism by inducing pro-angiogenic and pro-inflammatory cytokines.Many of these cytokines are also expressed in KS lesions,implicating a direct role of KSI-IV in the pathogenesis of this malignancy.Several KSHV genes are involved in KSHV-induced angiogenesis.These studies have provided insights into the pathogenesis of KS,and identified potential therapeutic targets for this malignancy.