Serial measurement of the circulating levels of tumour necrosis factor and its soluble receptors 1 and 2 for monitoring leprosy patients during multidrug treatment

Leprosy is an infectious and contagious spectral disease accompanied by a series of immunological events triggered by the host response to the aetiologic agent, Mycobacterium leprae . The induction and maintenance of the immune/inflammatory response in leprosy are linked to multiple cell interactions and soluble factors, primarily through the action of cytokines. The purpose of the present study was to evaluate the serum levels of tumour necrosis factor (TNF)-α and its soluble receptors (sTNF-R1 and sTNF-R2) in leprosy patients at different stages of multidrug treatment (MDT) in comparison with non-infected individuals and to determine their role as putative biomarkers of the severity of leprosy or the treatment response. ELISA was used to measure the levels of these molecules in 30 healthy controls and 37 leprosy patients at the time of diagnosis and during and after MDT. Our results showed increases in the serum levels of TNF-α and sTNF-R2 in infected individuals in comparison with controls. The levels of TNF-α, but not sTNF-R2, decreased with treatment. The current results corroborate previous reports of elevated serum levels of TNF-α in leprosy and suggest a role for sTNF-R2 in the control of this cytokine during MDT.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.

Detection of Toxoplasma gondii soluble antigen, SAG-1(p30), antibody and immune complex in the cerebrospinal fluid of HIV positive or negative individuals

Active infection by T. gondii was evaluated by immunoassay for soluble SAG-1 (p30), the major surface antigen from T. gondii, specific antibodies and immune complexes in human cerebrospinal fluid (CSF) samples. A total of 263 samples of CSF were collected from hospitalized patients presenting neurological disorders and analyzed for antibodies to HIV. Patients were divided into two groups: HIV positive (n = 96) or HIV negative (n =167). The results of the assays showed that 45% of all samples were positive for soluble SAG-1. Toxoplasma Ag/Ab immune complexes were detected in 19% of the CSF samples and 62% were positive for T. gondii- specific IgG. A combination of these assays in the presence of clinical findings consistent with active Toxoplasma infection may predict the presence of toxoplasmic encephalitis. Moreover, detection of soluble SAG-1 in the CSF of these individuals appears consistent with active infection.

Effect of antibodies to intercellular adhesion molecule type 1 on the protection of distant organs during reperfusion syndrome in rats

We investigated kidney and lung alterations caused by intercellular adhesion molecule type 1 (ICAM-1) blockade after ischemia and reperfusion of hind limb skeletal muscles. Rats were submitted to ligature of the infrarenal aorta for 6 h. The animals were randomized into three groups of 6 rats each: group I, sacrificed after ischemia; group II, reperfusion for 24 h, and group III, reperfusion for 24 h after receiving monoclonal anti-ICAM-1 antibodies. At the end of the experiment, blood samples were collected for creatinine, lactate dehydrogenase, creatine phosphokinase, potassium, pH and leukocyte counts. Samples were taken from the muscles of the hind limbs and from the kidneys and lungs for histological analysis and measurement of the neutrophil infiltrate by myeloperoxidase staining. The groups did not differ significantly with regard to the laboratory tests. There were no major histological alterations in the kidneys. An intense neutrophil infiltrate in the lungs, similar in all groups, was detected. Myeloperoxidase determination showed that after reperfusion there was significantly less retention of polymorphonuclear neutrophils in the muscles (352 ± 70 vs 1451 ± 235 × 10² neutrophils/mg; P<0.01) and in the kidneys (526 ± 89 vs 852 ± 73 × 10² neutrophils/mg; P<0.01) of the animals that received anti-ICAM-1 before perfusion compared to the group that did not. The use of anti-ICAM-1 antibodies in this experimental model minimized neutrophil influx, thus reducing the inflammatory process, in the muscles and kidneys after ischemia and reperfusion of the hind limbs.

Marcadores inflamatórios intracoronarianos após intervenções coronarianas percutâneas

OBJETIVO: Analisar a liberação intracoronariana de marcadores inflamatórios (MI) após intervenções coronarianas percutâneas (ICP) e comparar suas concentrações com relação ao tipo de ICP utilizada (rotablator vs angioplastia por balão). MÉTODOS: Foram randomizados 22 pacientes com média de idade de 60±11,9 anos, 12 do sexo masculino, portadores de síndromes coronarianas estáveis, submetidos ao tratamento eletivo de uma única lesão coronariana utilizando rotablator (N=11) ou pré-dilatação por balão (N=11) para implante de stents. As amostras foram colhidas na raiz da aorta e no seio coronariano, imediatamente antes e 15 minutos após as intervenções. Todas as dosagens foram feitas antes do implante do stent, sendo analisadas as citocinas TNF-a, IL-6 e IL-1 e as moléculas de adesão solúveis ICAM-1, E-selectina e P-selectina, utilizando o método ELISA. RESULTADOS: As concentrações de TNF-a e IL-6 aumentaram após as ICP, passando respectivamente de 9,5±1,5 pg/ml para 9,9±1,8 pg/ml (p=0,017) e de 6,0±2,4 pg/ml para 6,9±3,0 pg/ml (p<0,001). Não houve mudança significativa na expressão de IL-1, ICAM-1 e P-selectina, observando-se diminuição nas concentrações de E-selectina após os procedimentos (52,0±17,5 ng/ml para 49,3±18,7 ng/ml; p=0,009). Não houve diferença significativa entre as concentrações dos MI após as ICP, com relação ao tipo de procedimento utilizado. CONCLUSÃO: No período precoce, pós-intervenções coronarianas percutâneas, observou-se aumento das concentrações intracoronárias de TNF-a e IL-6 e ausência de diferença significativa entre as concentrações dos marcadores inflamatórios liberados na circulação coronariana por rotablator e pela angioplastia com balão.

Pentoxifylline Attenuates Cardiac Remodeling Induced by Tobacco Smoke Exposure

Abstract Background: Tobacco smoke exposure is an important risk factor for cardiac remodeling. Under this condition, inflammation, oxidative stress, energy metabolism abnormalities, apoptosis, and hypertrophy are present. Pentoxifylline has anti‑inflammatory, anti-apoptotic, anti-thrombotic and anti-proliferative properties. Objective: The present study tested the hypothesis that pentoxifylline would attenuate cardiac remodeling induced by smoking. Methods: Wistar rats were distributed in four groups: Control (C), Pentoxifylline (PX), Tobacco Smoke (TS), and PX-TS. After two months, echocardiography, invasive blood pressure measurement, biochemical, and histological studies were performed. The groups were compared by two-way ANOVA with a significance level of 5%. Results: TS increased left atrium diameter and area, which was attenuated by PX. In the isolated heart study, TS lowered the positive derivate (+dp/dt), and this was attenuated by PX. The antioxidants enzyme superoxide dismutase and glutathione peroxidase were decreased in the TS group; PX recovered these activities. TS increased lactate dehydrogenase (LDH) and decreased 3-hydroxyacyl Coenzyme A dehydrogenases (OH-DHA) and citrate synthase (CS). PX attenuated LDH, 3-OH-DHA and CS alterations in TS-PX group. TS increased IL-10, ICAM-1, and caspase-3. PX did not influence these variables. Conclusion: TS induced cardiac remodeling, associated with increased inflammation, oxidative stress, apoptosis, and changed energy metabolism. PX attenuated cardiac remodeling by reducing oxidative stress and improving cardiac bioenergetics, but did not act upon cardiac cytokines and apoptosis.

A study on the pathogenesis of human cerebral malaria and cerebral babesiosis

Cerebral complications are important, but poorly understood pathological features of infections caused by some species of Plasmodium and Babesia. Patients dying from P. falciparum were classified as cerebral or non-cerebral cases according to the cerebral malaria coma scale. Light microscopy revealed that cerebral microvessels of cerebral malaria patients were field with a mixture of parazited and unparazited erythrocytes, with 94% of the vessels showing parasitized red blood cell (PRBC) sequestration. Some degree of PRBC sequestration was also found in non-cerebral malaria patients, but the percentage of microvessls with sequestered PRBC was only 13% Electron microscopy demonstrated knobs on the membrane of PRBC that formed focal junctions with the capillary endothelium. A number of host cell molecules such as CD36, thrombospondim (TSP) and intracellular adhesion molecule I (ICAM-1) may function as endothelial cell surfacereports for P. falciparum-infected erythrocytes. Affinity labeling of CD36 and TSP to the PRBC surface showed these molecules specifically bind to the knobs. Babesia bovis infected erythrocytes procedure projections of the erythrocyte membrane that are similar to knobs. When brain tissue from B. bovis-infected cattle was examined, cerebral capillaries were packed with PRBC. Infected erythrocytes formed focal attachments with cerebral endothelial cells at the site of these knob-like projections. These findings indicate that cerebral pathology caused by B. bovis is similar to human cerebral malaria. A search for cytoadherence proteins in the endothelial cells may lead to a better understanding of the pathogenisis of cerebral babesiosis.

Variation in the cytoadherence characteristics of malaria parasites: is this a true virulence factor?

The sequestration of Plasmodium falciparum-infected erythrocytes to the endothelial cells of brain capillaries is believed to represent one of the determining factors in the pathogenesis of cerebral malaria. In vitro studies of cytoadherence provide an experimental approach to understand the mechanism of sequestration and the respective roles played by parasite and host components in this interaction. This paper critically reviews current studies on cytoadherence, with particular emphasis on the nature of the information provided by such studies and their limitations. The paper also describes how cytoadherence studies using the patient's own monocytes can provide original information on the level of receptor up-regulation in the course of malarial infection.

Plasmodium coatneyi-infected rhesus monkeys: a primate modelfor human cerebral malaria

Although several animal models for human cerebral malaria have been proposed in the past, name have shown pathological findings that are similar to those seen in humans. In order to develop an animal model for human cerebral malaria, we studied the pathology of brains of Plasmodium coatneyi (primate malaria parasite)-infected rhesus monkeys. Our study demonstrated parazitized erythrocyte (PRBC) sequestration and cytoadherence of knobs on PRBC to endothelial cells in cerebral microvessels of these monkeys. This similar to the findings een in human cerebral malaria. Crebral microvessels with sequestred PRBC were shown by immunohistochemistry to possess CD36, TSP and ICAM-1. These proteins were not evident in cerebral microvessels of uninfected control monkeys. Our study indicates, for the first time, that rhesus monkeys infected with P. coatneyi can be used as a primate model to study human cerebral malaria.

Systemic modulation of peripheral eosinophilia (air pouch model) in Schistosoma mansoni infection

Schistosoma mansoni infection induces in their hosts a marked and sustained eosinophilia, which is influenced or modulated by complex mechanisms, that vary according to the phase of infection. To address this phenomenon, we used the air pouch (AP) model in control and infected Swiss webster mice, analyzing the cellular, tissue response and local expression of adhesion molecules [CD18 (beta 2-chain), CD44, ICAM-1 (CD54), L-selectin (CD62L), CD49d (alpha 4-chain), LFA1 (CD11a)]. Infected animals were studied at 3 (pre-oviposition phase), 7 (acute phase), and 14 (chronic phase) weeks after infection (5-6 mice/period of infection). Normal mice were age-matched. Results showed that after egg stimulation, compared with matched controls, the infected mice, at each point of infection, showed a lower eosinophil response in the acute (7 weeks) and chronic phase (14 weeks) of infection. However, when the infected mice were in pre-oviposition phase (3 weeks) their eosinophil response surpassed the control ones. In the AP wall of infected mice, a significant decrease in the expression of ICAM-1 and CD44 in fibroblastic-like cells and a reduction in the number of CD18 and CD11a in migratory cells were observed. The other adhesion molecules were negative or weakly expressed. The results indicated that in the air pouch model, in S. mansoni-infected mice: (1) eosinophil response is strikingly down-regulated, during the acute ovular phase; (2) in the pre-oviposition phase, in contrast, it occurs an up-regulatory modulation of eosinophil response, in which the mechanisms are completely unknown; (3) in the chronic phase of the infection, the down modulation of eosinophil response is less pronounced; 4) Down-regulation of adhesion molecules, specially of ICAM-1 appear to be associated with the lower eosinophil response.

Differential expression of adhesion moleculesshaping the T-cell subset prevalence during the early phase of autoimmune and Trypanosoma cruzi-elicited myocarditis

The participation of cell adhesion molecules (CAMs) in the establishment of autoimmune and infectious myocarditis is an important matter of investigation and may have therapeutic implication. Trypanosoma cruzi infection induces a CD8-mediated myocarditis in patients with severe cardiomyopathy and experimental animals. Previously, we have proposed that this predominance of CD8+ T-cells is, at least in part, consequence of the differential expression of CAMs on circulating CD8+ lymphocytes. In the present study we investigated the participation of CAMs in shaping the phenotypic nature of the autoimmune CD4-mediated myosin-induced and the CD8-mediated T. cruzi-elicited myocarditis. We provide evidence that the prevalence of a certain T-cell subset inside the inflamed heart reflects the differential profile of the adhesion molecules VLA-4, LFA-1, and ICAM-1 displayed on a large proportion of this particular T-cell population in peripheral blood during the early phase of inflammation. Further, the expression of VCAM-1, ligand for VLA-4, and ICAM-1, counter-receptor for LFA-1, was up-regulated on vascular endothelium and paralleled the entrance of inflammatory cells into the cardiac tissue. Thus, this up-regulated expression of receptors-counter-receptors that regulate T-cell transmigration through the vascular endothelium may have an important role in the pathogenesis of the early phase of both autoimmune and infectious myocarditis.

Activated peripheral lymphocytes with increased expression of cell adhesion molecules and cytotoxic markers are associated with dengue fever disease

The immune mechanisms involved in dengue fever and dengue hemorrhagic/dengue shock syndrome are not well understood. The ex vivo activation status of immune cells during the dengue disease in patients was examined. CD4and CD8 T cells were reduced during the acute phase. Interestingly, CD8 T cells co-expressing activation marker HLA-DR, Q, P, and cytolytic granule protein-Tia-1 were significantly higher in dengue patients than in controls. Detection of adhesion molecules indicated that in dengue patients the majority of T cells (CD4 and CD8) express the activation/memory phenotype, characterized as CD44HIGH and lack the expression of the naïve cell marker, CD62L LOW. Also, the levels of T cells co-expressing ICAM-1 (CD54), VLA-4, and LFA-1 (CD11a) were significantly increased. CD8 T lymphocytes expressed predominantly low levels of anti-apoptotic molecule Bcl-2 in the acute phase, possibly leading to the exhibition of a phenotype of activated/effector cells. Circulating levels of IL-18, TGF-b1 and sICAM-1 were significantly elevated in dengue patients. Early activation events occur during acute dengue infection which might contribute to viral clearance. Differences in expression of adhesion molecules among CD4 and CD8 T cells might underlie the selective extravasation of these subsets from blood circulation into lymphoid organs and/or tissues. In addition, activated CD8 T cells would be more susceptible to apoptosis as shown by the alteration in Bcl-2 expression. Cytokines such as IL-18, TGF-b1, and sICAM-1 may be contributing by either stimulating or suppressing the adaptative immune response, during dengue infection, thereby perhaps establishing a relationship with disease severity.

Babesia bovis: expression of adhesion molecules in bovine umbilical endothelial cells stimulated with plasma from infected cattle

Ten male, 12-month-old Jersey with intact spleens, serologically and parasitologically free from Babesia were housed individually in an arthropod-free isolation system from birth and throughout entire experiment. The animals were randomly divided into two groups. Five animals (group A) were intravenously inoculated with 6.6 X10(7) red blood cells parasitized with pathogenic sample of Babesia bovis (passage 7 BboUFV-1), for the subsequent "ex vivo" determination of the expression of adhesion molecules. Five non-inoculated animals (group B) were used as the negative control. The expression of the adhesion molecules ICAM-1, VCAM, PECAM-1 E-selectin and thrombospondin (TSP) was measured in bovine umbilical vein endothelial cells (BUVECs). The endothelial cells stimulated with a pool of plasma from animals infected with the BboUFV-1 7th passage sample had a much more intense immunostaining of ICAM-1, VCAM, PECAM-1 E-selectin and TSP, compared to the cells which did not received the stimulus. The results suggest that proinflammatory cytokines released in the acute phase of babesiosis may be involved in the expression of adhesion molecules thereby implicating them in the pathophysiology of babesiosis caused by B. bovis.

Interferon gamma is a key cytokine in lung phase immunity to schistosomes but what is its precise role?

Vaccination of mice with radiation-attenuated cercariae of Schistosoma mansoni induces a high level of protection against challenge with normal larvae. The immune effector mechanism, which operates in the lungs, is a cell-mediated delayed-type hypersensitivity response and involves the formation of a tight focus of mononuclear cells around embolised larvae. CD4+ T cells with Th1 characteristics are a major component of the infiltrate. They secrete abundant interferon gamma (IFNg) upon antigen stimulation in vitro, whilst in vivo neutralisation of the cytokine results in 90% abrogation of immunity. IFNg can induce a large number of genes and an attempt has been made to identify the ones which are essential components of the effector mechanism. Inducible nitric oxide synthase (iNOS) is such a candidate and nitric oxide (NO) is produced by cultures of airway leucocytes from the lungs of vaccinated mice post-challenge. However, the continued resistance of mice with a disrupted iNOS gene indicates that NO has only a minor role in the protective response. Mice with a disrupted IFNg receptor gene have been used to dissect the role of the cytokine. After vaccination and challenge, CD4+ T cells from the pulmonary interstitium have reduced levels of ICAM-1 and LFA-1 expression, compared to wild-type animals, which coincides with a reduced cohesiveness of foci. However, immunity is not significantly impaired in mice with a disrupted ICAM-1 gene, and focus formation is normal. Similarly, a role has not been found for CD2/CD48 interactions in cell aggregation. Possible IFNg-inducible molecules yet to be fully investigated include other ligand-receptor pairs, chemokines, and tumour necrosis factor a.

Trypanosoma cruzi infection: a continuous invader-host cell cross talk with participation of extracellular matrix and adhesion and chemoattractant molecules

Several lines of evidence have shown that Trypanosoma cruzi interacts with host extracellular matrix (ECM) components producing breakdown products that play an important role in parasite mobilization and infectivity. Parasite-released antigens also modulate ECM expression that could participate in cell-cell and/or cell-parasite interactions. Increased expression of ECM components has been described in the cardiac tissue of chronic chagasic patients and diverse target tissues including heart, thymus, central nervous system and skeletal muscle of experimentally T. cruzi-infected mice. ECM components may adsorb parasite antigens and cytokines that could contribute to the establishment and perpetuation of inflammation. Furthermore, T. cruzi-infected mammalian cells produce cytokines and chemokines that not only participate in the control of parasitism but also contribute to the establishment of chronic inflammatory lesions in several target tissues and most frequently lead to severe myocarditis. T. cruzi-driven cytokines and chemokines may also modulate VCAM-1 and ICAM-1 adhesion molecules on endothelial cells of target tissues and play a key role in cell recruitment, especially of activated VLA-4+LFA-1+CD8+ T lymphocytes, resulting in a predominance of this cell population in the inflamed heart, central nervous system and skeletal muscle. The VLA-4+-invading cells are surrounded by a fine network of fibronectin that could contribute to cell anchorage, activation and effector functions. Since persistent "danger signals" triggered by the parasite and its antigens are required for the establishment of inflammation and ECM alterations, therapeutic interventions that control parasitism and selectively modulate cell migration improve ECM abnormalities, paving the way for the development of new therapeutic strategies improving the prognosis of T. cruzi-infected individuals.