Evaluation of a multimodal resin for selective capture of CHO-derived monoclonal antibodies directly from harvested cell culture fluid.

This proof-of-concept study examines the applicability of using multimodal chromatography to selectively capture recombinantly produced monoclonal antibodies (mAb) directly from harvested mammalian cell culture fluid (HCCF) with minimal optimization. Capto MMC is a multimodal resin that contains a ligand with the potential to participate in ionic, hydrophobic, and hydrogen boding interactions with proteins and is coupled to a highly cross-linked agarose bead matrix. Twelve mAb HCCF feedstocks were examined for dynamic binding capacity (DBC) and then two representative feedstocks were selected to develop a systematic approach for elution buffer development.

Efficacy of novel benznidazole solutions during the experimental infection with Trypanosoma cruzi.

Chagas' disease is caused by the protozoan parasite Trypanosoma cruzi. About 8 million people throughout Latin America are infected causing approximately 10,000 deaths annually. Benznidazole, available as unique 100 mg tablets in many of the endemic countries, is currently the drug of choice for the specific treatment of this condition.

TNF-α is involved in the abnormal thymocyte migration during experimental Trypanosoma cruzi infection and favors the export of immature cells.

Previous studies revealed a significant production of inflammatory cytokines together with severe thymic atrophy and thymocyte migratory disturbances during experimental Chagas disease. Migratory activity of thymocytes and mature T cells seem to be finely tuned by cytokines, chemokines and extracellular matrix (ECM) components. Systemic TNF-α is enhanced during infection and appears to be crucial in the response against the parasite.

A high corticosterone/DHEA-s ratio in young rats infected with Trypanosoma cruzi is associated with increased susceptibility.

We have previously established that young male rats are more susceptible to the effects of Trypanosoma cruzi infection than adult rats. To explore underlying age-associated differences in disease outcome, we simultaneously assessed hormone levels and cytokine release throughout the acute infection period in young and adult rats infected with T. cruzi.

Immunoneuroendocrine alterations in patients with progressive forms of chronic Chagas disease.

We studied the features of parallel immunoneuroendocrine responses in patients with different degrees of chronic Chagas myocarditis (indeterminate, mild/moderate or severe). A systemic inflammatory scenario was evident in patients with severe myocarditis compared to healthy subjects. This was paralleled by a disrupted activation of the hypothalamus-pituitary-adrenal axis, characterized by decreased concentrations of dehydroepiandrosterone-sulfate (DHEA-s) and an unbalanced cortisol/DHEA-s ratio, reinforcing the view that severe Chagas disease is devoid of an adequate anti-inflammatory milieu, likely involved in pathology.

Benznidazole treatment attenuates liver NF-κB activity and MAPK in a cecal ligation and puncture model of sepsis.

Recent studies have shown that Benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and nitric oxide (NO) release in activated macrophages by blocking NF-κB through inhibition of IKK in vitro. As so far, little is known about the mechanism by which BZL provokes the inhibition of inflammatory response in sepsis in vivo, we aimed to delineate the possible role of BZL as a modulator in liver inflammation in mice with sepsis induced by cecal ligation and puncture (CLP). Specifically, we analyzed leukocytes, liver production of TNF-α and NO and the intracellular pathways modulated by these mediators, including NF-κB and MAPKs, in the liver of mice 24 h post-CLP.

Benznidazole blocks NF-kappaB activation but not AP-1 through inhibition of IKK.

Previously, we demonstrated that benznidazole (BZL), known for its antiparasitic action on Trypanosoma cruzi, modulates pro-inflammatory cytokines and NO release in macrophages by inhibiting NF-kappaB. We now proceeded to elucidate the molecular mechanisms by which BZL exerts its inhibitory action on NF-kappaB. We demonstrated that the inhibitory effect of BZL is not extended to other macrophage responses, since it did not inhibit other typical hallmarks of macrophage activation such as phagocytosis, MHC-II molecules expression or production of reactive oxygen species (ROS) by NADPH oxidase.

Chagas' disease: an update on immune mechanisms and therapeutic strategies.

* Introduction * Chagas' disease * Chemotherapy * Immune response in experimental T. cruzi infection * Immune response in human beings infected with T. cruzi * Immune response in the treatment of chagasic infection * The need for new therapeutic alternatives for Chagas' disease * Conclusions The final decade of the 20th century was marked by an alarming resurgence in infectious diseases caused by tropical parasites belonging to the kinetoplastid protozoan order.

Tumor necrosis factor alpha induced by Trypanosoma cruzi infection mediates inflammation and cell death in the liver of infected mice.

Trypanosoma cruzi (T. cruzi) infected C57BL/6 mice developed a progressive fatal disease due to an imbalance in the profile of circulating related compounds accompanying infection like tumor necrosis factor alpha (TNFalpha). TNFalpha has been proposed as an important effector molecule in apoptosis.

Short treatment with the tumour necrosis factor-alpha blocker infliximab diminishes chronic chagasic myocarditis in rats without evidence of Trypanosoma cruzi reactivation.

Tumour necrosis factor (TNF)-alpha is crucial for resistance to Trypanosoma cruzi acute infection, but there is scant information on its role during the chronic phase. To address this issue, we analysed whether a short treatment with a TNF-alpha blocker affected the course and characteristics of chronic disease in a rat experimental model of T. cruzi infection.

Novel cytostatic activity of the trypanocidal drug Benznidazole.

We have shown that Benznidazole (BZL), a compound with well documented trypanocidal activity, possesses anti-inflammatory properties and inhibits the nuclear factor kappaB (NF-kappaB). Given the relationship between this transcription factor and cell growth, in this study we address the role of NF-kappaB blockade by BZL in the proliferation of different cell lines. Our studies demonstrate that this compound significantly reduced proliferation of RAW 264.

Beneficial effects of benznidazole during an infectious-based situation of systemic inflammatory response: cecal ligation and puncture.

We have shown that benznidazole (BZL), a drug used to treat Chagas disease, markedly reduced the production of pro-inflammatory cytokines and NO-derived metabolites in experimentally Trypanosoma cruzi-infected rats. Treatment with BZL exerted beneficial effects in a model of inflammation-based pathology like murine experimental endotoxemia. Based on these findings, we wished to ascertain the effect of BZL in a closer situation to sepsis: the cecal ligation and puncture (CLP) model in C57BL/6 mice.

Steroidogenic capacity of Trypanosoma cruzi trypomastigotes.

American Trypanosomiasis is caused by the hemoflagellate Trypanosoma cruzi (T. cruzi) and affects millions of persons causing variable degrees of digestive and heart disturbances. As far as we concerned, T.

Immunization with an engineered mutant trans-sialidase highly protects mice from experimental Trypanosoma cruzi infection: a vaccine candidate.

Chagas' disease is a major tropical disease for which a cure for chronic phase does not exist yet. Trypanosoma cruzi trans-sialidase (TS) seems to be involved in relevant processes such as infectivity, host survival and, very importantly, disease pathogenesis. In this study, we show that mice vaccinated with an engineered enzymatically deficient mutant TS containing the catalytic domain without the immunodominant SAPA (Shed Acute Phase Antigen) repeats, were highly protected against T.

Phenotypic differences on the outcome of the host-parasite relationship: behavior of mice of the CBi stock in natural and experimental infections.

Investigation of defined animal models may help to elucidate the role of the host genetic background in the development and establishment of a parasitic infection. Four lines of mice obtained by disruptive selection for body conformation (CBi+, CBi-, CBi/C and CBi/L) and the unselected control line CBi were examined in their response to different parasites to assess whether these distinct genotypes showed differences in their resistance to natural and experimental parasitosis. Protozoans (Trichomonas muris and Spironucleus muris) and nemathelminths (Syphacia obvelata and Aspiculurus tetraptera) were found naturally parasitizing the mice's intestines.

Improved outcome of Trypanosoma cruzi infection in rats following treatment in early life with suspensions of heat-killed environmental Actinomycetales.

The well-established model of Chagas' disease in "l" rats was used to evaluate the effects of three injections of heat-killed Gordonia bronchialis, Rhodococcus coprophilus or saline on Trypanosoma cruzi parasitaemia and acute and chronic myocarditis, sequelae of the infection. Two vaccinating injections were given prior to challenge with T. cruzi, and the third, immunotherapeutic, injection was given 7 days after challenge.

Deficient control of Trypanosoma cruzi infection in C57BL/6 mice is related to a delayed specific IgG response and increased macrophage production of pro-inflammatory cytokines.

Earlier work in Trypanosoma cruzi-infected C57BL/6 and BALB/c mice revealed an acute disease, of lethal outcome in the former group and lesser severity in BALB/c mice. Fatal course was not accompanied by an increased parasite load, but by a substantial imbalance between pro- and anti-inflammatory cytokine serum levels. To better characterise the mechanisms allowing the host to restrain the infection, we have now studied the specific IgG production and in vitro behaviour of peritoneal macrophages (PMs) when exposed to T.

Blood viscosity changes in experimentally Trypanosoma cruzi-infected rats.

Microcirculatory alterations would explain focal lesions found in Chagas' cardiomyopathy. Trypanosoma cruzi (T. cruzi) infection induces host blood properties modifications and defensive responses capable of producing blood hyperviscosity, an ischemic risk factor able to affect microvascular blood flow.

Benznidazole, a drug used in Chagas' disease, ameliorates LPS-induced inflammatory response in mice.

Benznidazole (BZL) is a drug currently used for treating Chagas' disease. Given our earlier demonstration in which BZL downregulated cytokine and nitric oxide (NO) synthesis by LPS and/or IFN-gamma-stimulated murine macrophages, we have now analysed whether this compound could exert beneficial effects in a model of LPS-induced inflammation in C57BL/6 mice. The lethal model consisted of two LPS intraperitoneal injections, 200 microg each separated by 2 h, with BZL given orally at a dose of 200 mg/kg, 18 and 2 h before the first challenge and 20 and 44 hr following the second one.

Thymocyte depletion during acute Trypanosoma cruzi infection in C57BL/6 mice is partly reverted by lipopolysaccharide pretreatment.

Infection with Trypanosoma cruzi in C57BL/6 mice leads to a progressive fatal disease accompanied by thymocyte depletion, which is not related with a higher parasite burden but with increased serum levels of tumour necrosis factor alpha (TNF- alpha). Because this situation may result from an excessive inflammatory syndrome, mice were now given anti-TNF-alpha mAbs throughout their acute infection, or subjected to a LPS desensitization protocol before parasite challenge. Treatment with anti-TNF-alpha mAbs failed to ameliorate thymocyte depletion but shortened survival time and increased parasite load.

Age-related increase in resistance to acute Trypanosoma cruzi infection in rats is associated with an appropriate antibody response.

Inoculation at weaning with Trypanosoma cruzi in inbred "l" rats resulted in a self-resolving acute infection characterized by marked parasitaemias, whereas challenge to adult rats revealed a mild disease with extremely low parasitaemias. To explore the mechanisms underlying such age-associated differences in disease outcome, we analysed the in vitro replication of T. cruzi, nitric oxide and tumour necrosis factor-alpha (TNF-alpha) production in peritoneal macrophages (PMs), the serum concentrations of the specific immunoglobulins (Igs) IgM and IgG, antibodies exhibiting lytic activity against bloodstream forms of T.

Differential susceptibility to acute Trypanosoma cruzi infection in BALB/c and C57BL/6 mice is not associated with a distinct parasite load but cytokine abnormalities.

Inoculation of Trypanosoma cruzi, Tulahuén strain, into C57BL/6 and BALB/c mice led to an acute infection characterized by marked parasitaemia, myocardial inflammation and thymocyte depletion. While C57BL/6 mice showed a progressive and lethal disease, BALB/c mice partly recovered. To characterize these murine models more effectively, we studied the parasite burden, serum levels of major infection outcome-related cytokines, the in vitro features of T.

Trypanocidal drug benznidazole impairs lipopolysaccharide induction of macrophage nitric oxide synthase gene transcription through inhibition of NF-kappaB activation.

In murine macrophages, inducible NO synthase II (NOSII) gene expression is promoted at a transcriptional level by LPS and/or IFN-gamma with benznidazole (BZL), a trypanocidal drug, acting to down-regulate NOSII gene induction and hence inhibiting NO production. By performing transient transfection experiments, we now report that BZL also inhibited the expression of NOSII gene promoter or multimerized NF-kappaB binding site controlled reporter genes. By contrast, no effect was observed on the expression of a reporter gene under the control of the NOSII promoter-derived IFN regulatory factor element.

Treatment with benznidazole and its immunomodulating effects on Trypanosoma cruzi-infected rats.

Because benznidazole (BZL) was found to downregulate nitric oxide (NO) and cytokine synthesis by murine macrophages, we analyzed the potential immunological repercussions of BZL treatment in Trypanosoma cruzi-infected rats. To evaluate whether the effects of BZL were also observed in the presence of an immunostimulating cytokine, four groups of acutely infected rats were subjected to one of the following 20-day therapeutic schedules: (1) a curative BZL oral regimen, (2) recombinant interferon (IFN-gamma) injections, (3) a suboptimal BZL regimen (25% of curative dose), (4) the latter plus IFN-gamma. All BZL doses markedly reduced NO-derived metabolites either in the circulation or in cultured macrophage supernatants.

Antibody maturation in Trypanosoma cruzi-infected rats.

The study of antibody avidity changes during infection has improved the understanding of the pathologic processes involved in several infectious diseases. In some infections, like toxoplasmosis, this information is being used for diagnostic purposes. Results of the evolution of antibody avidity for different specific antigens in Trypanosome cruzi-infected rats are presented.