Intestinal mononuclear cells primed by systemic interleukin-12 display long-term ability to aggravate colitis in mice.

To address whether the burst of systemic interleukin-12 (IL-12) influences intestinal inflammation elicited by luminal stimuli, we induced IL-12 release by cDNA injection in C57BL/6 mice and simultaneously started dextran sulphate sodium administration. The sequence of the inflammatory response triggered by IL-12 release was characterized by assessing myeloperoxidase activity and histological damage in colon samples on days 1, 3, 5 and 7 after colitis induction. To evaluate the persistence of IL-12 priming, colitis was induced in mice 7 or 60 days after cDNA injection. Under IL-12 influence, the development of acute colitis presented a faster and selective infiltration of inflammatory mononuclear cells in the lamina propria. Recruitment was driven by systemic cytokines rather than luminal antigens. Interestingly, when colitis was triggered 7 or 60 days after the cytokine storm, cells maintained the ability to worsen clinical signs of intestinal inflammation. Together, a systemic IL-12 burst effectively primed intestinal cells that became more prone to develop inflammatory responses. Activation was long-lasting because intestinal cells maintained their inflammatory potential and their ability to aggravate colitis.

Divergent Annexin A1 expression in periphery and gut is associated with systemic immune activation and impaired gut immune response during SIV infection.

HIV-1 disease progression is paradoxically characterized by systemic chronic immune activation and gut mucosal immune dysfunction, which is not fully defined. Annexin A1 (ANXA1), an inflammation modulator, is a potential link between systemic inflammation and gut immune dysfunction during the simian immunodeficiency virus (SIV) infection. Gene expression of ANXA1 and cytokines were assessed in therapy-naïve rhesus macaques during early and chronic stages of SIV infection and compared with SIV-negative controls. ANXA1 expression was suppressed in the gut but systemically increased during early infection. Conversely, ANXA1 expression increased in both compartments during chronic infection. ANXA1 expression in peripheral blood was positively correlated with HLA-DR+CD4+ and CD8+ T-cell frequencies, and negatively associated with the expression of pro-inflammatory cytokines and CCR5. In contrast, the gut mucosa presented an anergic cytokine profile in relation to ANXA1 expression. In vitro stimulations with ANXA1 peptide resulted in decreased inflammatory response in PBMC but increased activation of gut lymphocytes. Our findings suggest that ANXA1 signaling is dysfunctional in SIV infection, and may contribute to chronic inflammation in periphery and with immune dysfunction in the gut mucosa. Thus, ANXA1 signaling may be a novel therapeutic target for the resolution of immune dysfunction in HIV infection.

Role of the protein annexin A1 on the efficacy of anti-TNF treatment in a murine model of acute colitis.

TNF-α is involved in the mechanisms that initiate inflammatory bowel diseases (IBDs). Anti-TNF-α drugs, such as infliximab (IFX), cause non-responsiveness and side effects, indicating the need to investigate alternative therapies for these diseases. The anti-inflammatory protein, annexin A1 (AnxA1), has been associated with the protection of the gastrointestinal mucosa. To further address the role of endogenous AnxA1 on the TNF-α blockade efficacy in a murine model, we assessed colitis induced by Dextran Sulfate Sodium (DSS) in wild-type (WT) and AnxA1(-/-) Balb/c mice treated with IFX. We consistently observed endogenous AnxA1 prevented clinical and physiological manifestations of experimental colitis treated with IFX, additionally the manifestation of the disease was observed earlier in AnxA1(-)(/-) mice. Rectal bleeding, diarrhea, histological score, epithelial damages and collagen degradation caused by DSS were prevented following IFX treatment only in WT mice. IL-6 increased during colitis in WT and AnxA1(-)(/-) mice, decreasing under IFX treatment in WT. The influx of neutrophils and TNF-α secretion were largely elevated in AnxA1(-)(/-) mice when compared to WT mice. In the group WT/DSS+IFX, phagocytes were more susceptible to apoptosis following treatment with IFX. Endogenous expression of AnxA1 increased after DSS and decreased with IFX treatment, demonstrating an attenuated inflammatory response. The data indicate that AnxA1 contributes to the establishment of intestinal homeostasis after blocking of TNF-α was used as a treatment of IBD, constituting a key molecule in the mechanism of action and a potential biomarker of therapeutic efficacy.

Chromosomal disruption and rearrangements during murine sarcoma development converge to stable karyotypic formation kept by telomerase overexpression.

BACKGROUND: Tumor initiation presents a complex and unstable genomic landscape; one of the earliest hallmark events of cancer, and its progression is probably based on selection mechanisms under specific environments that lead to functional tumor cell speciation. We hypothesized that viable tumor phenotypes possess common and highly stable karyotypes and their proliferation is facilitated by an attuned high telomerase activity. Very few investigations have focused on the evolution of common chromosomal rearrangements associated to molecular events that result in functional phenotypes during tumor development. RESULTS: We have used cytogenetic, flow cytometry and cell culture tools to investigate chromosomal rearrangements and clonality during cancer development using the murine sarcoma TG180 model, and also molecular biology techniques to establish a correlation between chromosome instability and telomerase activity, since telomeres are highly affected during cancer evolution. Cytogenetic analysis showed a near-tetraploid karyotype originated by endoreduplication. Chromosomal rearrangements were random events in response to in vitro conditions, but a stable karyotypic equilibrium was achieved during tumor progression in different in vivo conditions, suggesting that a specific microenvironment may stabilize the chromosomal number and architecture. Specific chromosome aberrations (marker chromosomes) and activated regions (rDNAs) were ubiquitous in the karyotype, suggesting that the conservation of these patterns may be advantageous for tumor progression. High telomerase expression was also correlated with the chromosomal rearrangements stabilization. CONCLUSIONS: Our data reinforce the notion that the sarcoma cell evolution converges from a highly unstable karyotype to relatively stable and functional chromosome rearrangements, which are further enabled by telomerase overexpression.

Systemic IL-12 burst expands intestinal T-lymphocyte subsets bearing the α4 ß7 integrin in mice.

The intestinal immune system is complex and displays unique anatomic and functional characteristics. Numerous immune cell subsets are located beneath the epithelial barrier and their activity is highly regulated. Using hydrodynamic shear of IL-12 cDNA to achieve systemic expression of IL-12 in mice, we evaluated the effect of a transient burst of this cytokine on the activation status of T cells from Peyer's patches (PPs), mesenteric lymph nodes (MLNs), and colonic lamina propria (LP). Following systemic IL-12 release, intestinal T lymphocytes became activated, exhibiting a CD44(high) CD62L(-) phenotype. After 5 days of the cytokine burst, the frequency of α4ß7(+) CD4(+) and CD8(+) cells increased, and CD8(+) α4ß7(+) cells mainly expressed T bet, a critical regulator of the Th1 differentiation program. The incremental increase in α4ß7 expression involved the IL-12 receptor-signal transducer and activator of transcription (STAT)-4 axis, and occurred independently of IFN-γ, IL-4, IL-10, and TNF-α signaling. Moreover, IL-12 priming exacerbated the outcome of acute dextran sodium sulphate (DSS)-induced colitis with higher scores of weight loss, blood in stool, and diarrhea and lower hematocrit. Together, our findings demonstrate that systemic polarizing signals could effectively expand the number of effector cells able to home to the LP and contribute to local inflammation.

Lack of TNFRI signaling enhances annexin A1 biological activity in intestinal inflammation.

We evaluated whether the lack of TNF-α signaling increases mucosal levels of annexin A1 (AnxA1); the hypothesis stems from previous findings showing that TNF-α neutralization in Crohn's disease patients up-regulates systemic AnxA1 expression. Biopsies from healthy volunteers and patients under anti-TNF-α therapy with remittent ulcerative colitis (UC) showed higher AnxA1 expression than those with active disease. We also evaluated dextran sulfate sodium (DSS)-acute colitis in TNF-α receptor 1 KO (TNFR1-/-) strain with impaired TNF-α signaling and C57BL/6 (WT) mice. Although both strains developed colitis, TNFR1-/- mice showed early clinical recovery, lower myeloperoxidase (MPO) activity and milder histopathological alterations. Colonic epithelium from control and DSS-treated TNFR1-/- mice showed intense AnxA1 expression and AnxA1+ CD4+ and CD8+ T cells were more frequent in TNFR1-/- animals, suggesting an extra supply of AnxA1. The pan antagonist of AnxA1 receptors exacerbated the colitis outcome in TNFR1-/- mice, supporting the pivotal role of AnxA1 in the early recovery. Our findings demonstrate that the TNF-α signaling reduction favors the expression and biological activity of AnxA1 in inflamed intestinal mucosa.

A novel highly reactive Fab antibody for breast cancer tissue diagnostics and staging also discriminates a subset of good prognostic triple-negative breast cancers.

The discovery of novel markers for breast cancer (BC) has been recently relied on antibody combinatorial libraries and selection through phage display. We constructed a recombinant Fab library, and after selections against BC tissues, the FabC4 clone was thoroughly investigated by immunohistochemistry in 232 patients with long-term follow-up. The FabC4 ligand was determined by mass spectrometry. The FabC4 expression was associated with younger age, lack of progesterone receptor, higher histological grades and non-luminal subtypes, and it also identified a subset of good prognostic triple-negative BCs, possibly targeting a conformational epitope of Cytokeratin-10 (CK10). This new CK10-epitope specific antibody may open new possibilities in diagnostic and therapeutic strategies.

Dysregulation of anti-inflammatory annexin A1 expression in progressive Crohns Disease.

BACKGROUND: Development of inflammatory bowel disease (IBD) involves the interplay of environmental and genetic factors with the host immune system. Mechanisms contributing to immune dysregulation in IBD are not fully defined. Development of novel therapeutic strategies is focused on controlling aberrant immune response in IBD. Current IBD therapy utilizes a combination of immunomodulators and biologics to suppress pro-inflammatory effectors of IBD. However, the role of immunomodulatory factors such as annexin A1 (ANXA1) is not well understood. The goal of this study was to examine the association between ANXA1 and IBD, and the effects of anti-TNF-α, Infliximab (IFX), therapy on ANXA1 expression. METHODS: ANXA1 and TNF-α transcript levels in PBMC were measured by RT PCR. Clinical follow up included the administration of serial ibdQs. ANXA1 expression in the gut mucosa was measured by IHC. Plasma ANXA1 levels were measured by ELISA. RESULTS: We found that the reduction in ANXA1 protein levels in plasma coincided with a decrease in the ANXA1 mRNA expression in peripheral blood of IBD patients. ANXA1 expression is upregulated during IFX therapy in patients with a successful intervention but not in clinical non-responders. The IFX therapy also modified the cellular immune activation in the peripheral blood of IBD patients. Decreased expression of ANXA1 was detected in the colonic mucosa of IBD patients with incomplete resolution of inflammation during continuous therapy, which correlated with increased levels of TNF-α transcripts. Gut mucosal epithelial barrier disruption was evident by increased plasma bacterial 16S levels. CONCLUSION: Loss of ANXA1 expression may support inflammation during IBD and can serve as a biomarker of disease progression. Changes in ANXA1 levels may be predictive of therapeutic efficacy.

Epitope-based vaccines with the Anaplasma marginale MSP1a functional motif induce a balanced humoral and cellular immune response in mice.

Bovine anaplasmosis is a hemoparasitic disease that causes considerable economic loss to the dairy and beef industries. Cattle immunized with the Anaplasma marginale MSP1 outer membrane protein complex presents a protective humoral immune response; however, its efficacy is variable. Immunodominant epitopes seem to be a key-limiting factor for the adaptive immunity. We have successfully demonstrated that critical motifs of the MSP1a functional epitope are essential for antibody recognition of infected animal sera, but its protective immunity is yet to be tested. We have evaluated two synthetic vaccine formulations against A. marginale, using epitope-based approach in mice. Mice infection with bovine anaplasmosis was demonstrated by qPCR analysis of erythrocytes after 15-day exposure. A proof-of-concept was obtained in this murine model, in which peptides conjugated to bovine serum albumin were used for immunization in three 15-day intervals by intraperitoneal injections before challenging with live bacteria. Blood samples were analyzed for the presence of specific IgG2a and IgG1 antibodies, as well as for the rickettsemia analysis. A panel containing the cytokines' transcriptional profile for innate and adaptive immune responses was carried out through qPCR. Immunized BALB/c mice challenged with A. marginale presented stable body weight, reduced number of infected erythrocytes, and no mortality; and among control groups mortality rates ranged from 15% to 29%. Additionally, vaccines have significantly induced higher IgG2a than IgG1 response, followed by increased expression of pro-inflammatory cytokines. This is a successful demonstration of epitope-based vaccines, and protection against anaplasmosis may be associated with elicitation of effector functions of humoral and cellular immune responses in murine model.

The interaction of AGT and NOS3 gene polymorphisms with conventional risk factors increases predisposition to hypertension.

Renin-angiotensin and kallikrein-kinin systems are interconnected, regulating blood pressure homeostasis. We have demonstrated the interactions among polymorphisms of the angiotensinogen (AGT) and endothelial nitric oxide synthase (NOS3) genes and conventional risk factors affecting the hypertension occurrence. Individuals were recruited (n=192) and classified into hypertensive (HG; n=140) and normotensive (NG; n=52) groups. The genotypic distribution of the Met235Thr (AGT) and Glu298Asp (NOS3) polymorphisms demonstrated that both are independent risk factors of hypertension (p=0.02 and p=0.008, respectively). The concomitant presence of these polymorphisms in the HG group was significantly different (p=0.001) from the NG. Both gene polymorphisms presented an additive effect for the unfavourable alleles T and A, respectively, and 95% of the double mutant homozygotes were classified into the HG. Specific interactions among certain conventional factors and the presence of at least one unfavourable allele presented significant odds towards hypertension. Blood pressure homeostasis was affected by genetic polymorphisms conditioned by the T and A alleles of the AGT and NOS3 genes, respectively, which acted independently. However, their interaction with smoking, sedentariness, age and total cholesterol may have increased the predisposition to hypertension, which may explain most of the hypertension cases.

Annexin 1 mimetic peptide protects against renal ischemia/reperfusion injury in rats.

Inflammation is currently recognized as a key mechanism in the pathogenesis of renal ischemia-reperfusion (I/R) injury. The importance of infiltrating neutrophil, lymphocytes, and macrophage in this kind of injury has been assessed with conflicting results. Annexin 1 is a protein with potent neutrophil anti-migratory activity. In order to evaluate the effects of annexin A1 on renal I/R injury, uninephrectomized rats received annexin A1 mimetic peptide Ac2-26 (100 µg) or vehicle before 30 min of renal artery clamping and were compared to sham surgery animals. Annexin A1 mimetic peptide granted a remarkable protection against I/R injury, preventing glomerular filtration rate and urinary osmolality decreases and acute tubular necrosis development. Annexin A1 infusion aborted neutrophil extravasation and attenuated macrophage infiltration but did not prevent tissue lymphocyte traffic. I/R increased annexin A1 expression (assessed by transmission electron microscopy) in renal epithelial cells, which was attenuated by exogenous annexin A1 infusion. Additionally, annexin A1 reduced I/R injury in isolated proximal tubules suspension. Annexin A1 protein afforded striking functional and structural protection against renal I/R. These results point to an important role of annexin A1 in the epithelial cells defense against I/R injury and indicate that neutrophils are key mediators for the development of tissue injury after renal I/R. If these results were confirmed in clinical studies, annexin A1 might emerge as an important tool to protect against I/R injury in renal transplantation and in vascular surgery.

Expression of annexin A1 mRNA in peripheral blood from oral squamous cell carcinoma patients.

Several studies have been suggesting annexin A1 protein as an active player in tumorigenesis of many organs. Nevertheless, its tumor biomarker role has been mainly studied in tissues by immunohistochemistry or cell culture. Hence, in this investigation, the peripheral blood from 27 oral squamous cell carcinoma (OSCC) patients and 25 negative control individuals were examined by quantitative real-time PCR. Down-regulated ANXA1 expression at mRNA level was observed in OSCC samples (p=0.026). Significantly diminished mRNA levels correlated to age, sex and the anatomical site of the tumor lesion were observed. Moreover, the ROC curve analysis revealed the performance of ANXA1 expression as a suitable biomarker for patients with oral cavity cancer, especially those with 60years of age or older and/or women. For the first time, ANXA1 mRNA is revealed as blood-based biomarker, and its adoption for complementary non-invasive diagnosis of OSCC is suggested. These results suggest that, beyond the anti-inflammatory function, annexin A1 may also play a tumor suppressor role in peripheral blood cells, such as leukocytes.

Avaliação da toxicidade aguda e subaguda, em ratos, do extrato etanólico das folhas e do látex de Synadenium umbellatum Pax / Acute and subacute toxicity studies of the latex and of the ethanolic extract of the leaves of Synadenium umbellatum Pax in rats

O uso de plantas medicinais tem sido muito significativo nos últimos anos, sendo incentivado pela Organização Mundial de Saúde (OMS). Synadenium umbellatum Pax, Euphorbiacea (vulgo cola-nota, cancerola, milagrosa) tem o látex usado empiricamente como antitumoral e antiinflamatório. Por existir espécies tóxicas nesta família e visando à segurança no uso de extratos vegetais, tal estudo avaliou a toxicidade pré-clínica do látex e do extrato etanólico das folhas (EEF) de S. umbellatum, por via oral, em ratas Wistar. O estudo seguiu diretrizes do Guideline 423 (toxicidade aguda) e Guideline 407 (toxicidade subaguda) da OECD (Organisation for Economic Cooperation and Development). Na toxicidade aguda do látex e do EEF, não se observou letalidade nem alterações fisiológicas e comportamentais das ratas na dose de 2000 mg/kg, sendo praticamente atóxico. Porém, na análise histopatológica, o látex ocasionou congestão e infiltrado leucocitário nos rins, fígado e pulmões, efeitos não observados com o EEF. Na toxicidade subaguda, doses de 50, 100 e 200 mg/kg de EEF não produziram alterações dose-dependentes significativas nos parâmetros laboratoriais e fisiológicos, nem alterações macroscópicas e histopatológicas nos órgãos das ratas. Contudo, o uso crônico da planta S. umbellatum merece mais estudos.

The use of medicinal plants has been being very significant in the last years, being the use encouraged by WHO. Synadenium umbellatum Pax, Euphorbiacea (popularly known as cola-note, cancerola, miraculous) has the latex used empirically as anti-cancerous and anti-inflammatory. For there being toxic species in this family and aiming at the safety in the use of vegetable extracts, such study evaluated the pre-clinical toxicity of the latex and of the ethanolic extract of the leaves (EEL) of S. umbellatum, administrated by oral route, in Wistar female rats. The study followed OECD's Guidelines for test of acute toxicity (Guideline 423) and for subacute toxicity (Guideline 407). In the acute toxicity of latex and EEL, behavioral and physiological alterations were not observed neither animal's death in the dose level of 2,000 mg/kg. However, the latex caused congestion and leukocytes infiltration of the kidneys, liver and lungs, effects not observed with EEL. In the subacute toxicity, dose levels of 50, 100 and 200 mg/kg of EEL did not produced significant dose-dependent alterations in the lab results and no physiologic, macroscopic and hystopathological alterations. EEL of S. umbellatum is practically poisonless in acute exposure; already the latex can cause hystological damages. The chronic use of S. umbellatum needs more specific studies.

Inclusão do tema plantas medicinais no currículo dos cursos da área de saúde: uma realidade necessária / Including herbal medicine in the health curriculum - a necessary reality / Including herbal medicine in the health curriculum: a necessary reality / Inclusión del tema plantas medicinales en el plan de estudios de los cursos del área de salud - una realidad necesaria

Esta pesquisa, realizada com docentes de universidades da região sul do Rio Grande do Sul e da Campanha, buscou verificar como acontecia a organização do tema plantas medicinais, a importância atribuída, mostrando a necessidade da inclusão do tema no currículo. É uma pesquisa quantitativa realizada com 183 docentes (61%). Verificou-se que o tema não é desenvolvido pela maioria dos docentes; 92,16% consideram importante a difusão do tema; e, 81,70% reconhecem a necessidade da inclusão do tema no currículo; precedida de sua ampla discussão nos meios acadêmicos, pois docentes apontam a necessidade da inclusão desse conteúdo em cursos relacionados à saúde.

This research, carried out with teachers at universities in the south of the State of Rio Grande do Sul and in Campanha, sought to understand how herbal medicine is organized, what status it has, and demonstrated the need to include this issue in the curriculum. It is a quantitative research carried out with 183 teachers (61%). We found that this issue is not discussed by most teachers; 92.16% consider it an important issue and 81.70% recognize the need to include this in the curriculum after wide discussion in academic circles. The teachers state the need to include this content in health courses.

Esta investigación, llevada a cabo con docentes de universidades de la región sur del Estado de Río Grande do Sul y de Campanha, buscaba observar cómo se organizaba el tema de las plantas medicinales, la importancia atribuida y señala la necesidad de incluir dicho tema en el plan de estudios. Se trata de una investigación cuantitativa con 183 docentes (61%); quedó comprobado que la mayoría de los docentes no desarrollan el tema, que 92,16% consideran importante la difusión del tema y que 81,70% reconocen la necesidad de incluirlo en el plan de estudios. Por lo tanto, es importante la discusión del tema en el medio académico pues algunos docentes piensan que debe incluirse en los cursos relacionados con la salud

Visão docente sobre plantas medicinais como um saber e sua utilização como medicamento / The vision of educators on medicinal plants as a knowledge and on it's use as a medication

Considerando o distanciamento do conhecimento e práticas acadêmicas de saúde das práticas populares, este artigo objetiva verificar o posicionamento de docentes de Cursos de Graduação em Enfermagem e Medicina frente ao tema plantas medicinais. A partir de estudos sobre práticas populares de saúde, foi realizada pesquisa descritiva com aplicação do método estatístico, em 2004. Foram realizadas entrevistas com 183 docentes de três Cursos de Enfermagem e dois de Medicina do Rio Grande do Sul. A maioria docente de Faculdades de Rio Grande (55,56%) e de Pelotas (77,78%) considera que o saber científico e o popular se encontram no tema plantas medicinais, e entre os docentes dos cursos de Medicina predominou a concepção saber popular; 79,94% dos entrevistados atribuem valor terapêutico a essas plantas; a maioria as utiliza como recurso terapêutico. Conclui-se que os docentes associam o saber científico e o popular às plantas medicinais, valorizando a influência cultural no desenvolvimento das práticas terapêuticas.