Unnecessary Abdominal Surgeries in Attacks of Hereditary Angioedema with Normal C1 Inhibitor.

Hereditary angioedema (HAE) is an autosomal dominant disease mostly due to the deficiency of C1 inhibitor (C1-INH). HAE with normal C1-INH was first described in 2000 and associated with mutations in the coagulation factor XII in 2006. Both diseases are associated with high bradykinin production, resulting in increased vascular permeability. Gastrointestinal edema due to HAE can be misdiagnosed as acute abdomen and unnecessary surgical procedures may be performed. The present study evaluates the prevalence of surgical procedures and/or acute abdomen in HAE patients with the coagulation factor XII mutation. It is a retrospective study where patients were diagnosed with recurrent angioedema without urticaria, normal C1-INH levels, and positive family history of angioedema. All patients were evaluated for the known mutations located at exon 9 of the F12 gene. Medical records were evaluated and questionnaires were applied to 52 patients with normal C1-INH levels (age range 13-76 years; 47/52, 90.38% women; 5/52, 9.61% men). F12 mutation was present in 32/52 patients (61.5%). Acute abdominal pain was diagnosed in 16/52 (30.76%) patients, appendicitis in 9/16 (56.2%), and undetermined diagnosis in 7/16 (43.7%). Among patients diagnosed with acute abdominal pain, 13/16 (81.2%) underwent surgery and 3/16 (18.7%) improved without surgical intervention. We conclude that many HAE patients with coagulation factor XII mutation were misdiagnosed with acute abdomen and subjected to unnecessary invasive procedures. It is critical to disseminate information about this rare mutation in patients with otherwise normal C1-INH activity, in order to speed up diagnosis and avoid misconduct.

Correlation between GLA variants and alpha-Galactosidase A profile in dried blood spot: an observational study in Brazilian patients.

BACKGROUND: Fabry disease is a rare X-linked inherited disorder caused by deficiency of α-Galactosidase A. Hundreds of mutations and non-coding haplotypes in the GLA gene have been described; however, many are variants of unknown significance, prompting doubts about the diagnosis and treatment. The α-Galactosidase A enzymatic activity in dried blood spot (DBS) samples are widely used for screening purposes; however, even when values below the normal are found, new tests are required to confirm the diagnosis. Here we describe an analysis of GLA variants and their correlation with DBS α-Galactosidase A enzymatic activity in a large Brazilian population with Fabry disease symptoms. RESULTS: We analyzed GLA variants by DNA sequencing of 803 male patients with suspected Fabry disease or belonging to high-risk populations; in 179 individuals, 58 different exonic variants were detected. From these, 50 are variants described as pathogenic and eight described as variants of unknown significance. The other individuals presented complex non-coding haplotypes or had no variants. Interestingly, the enzymatic activity in DBS was different among pathogenic variants and the other genotypes, including variants of unknown significance; the first presented mean of 12% of residual activity, while the others presented levels above 70% of the activity found in healthy controls. CONCLUSION: The activity of α-Galactosidase A in DBS was markedly reduced in males with known pathogenic variants when compared with subjects presenting variants of unknown significance, non-coding haplotypes, or without variants, indicating a possible non-pathogenic potential of these latter genotypes. These findings bring a better understanding about the biochemical results of α-Galactosidase A in DBS samples, as well as the possible non-pathogenic potential of non-coding haplotypes and variants of unknown significance in GLA gene. These results certainly will help clinicians to decide about the treatment of patients carrying variants in the gene causing this rare but life-threatening disease.

Pathogenicity Reclasssification of RPE65 Missense Variants Related to Leber Congenital Amaurosis and Early-Onset Retinal Dystrophy.

A challenge in molecular diagnosis and genetic counseling is the interpretation of variants of uncertain significance. Proper pathogenicity classification of new variants is important for the conclusion of molecular diagnosis and the medical management of patient treatments. The purpose of this study was to reclassify two RPE65 missense variants, c.247T>C (p.Phe83Leu) and c.560G>A (p.Gly187Glu), found in Brazilian families. To achieve this aim, we reviewed the sequencing data of a 224-gene retinopathy panel from 556 patients (513 families) with inherited retinal dystrophies. Five patients with p.Phe83Leu and seven with p.Gly187Glu were selected and their families investigated. To comprehend the pathogenicity of these variants, we evaluated them based on the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) classification guidelines. Initially, these RPE65 variants met only three pathogenic criteria: (i) absence or low frequency in the population, (ii) several missense pathogenic RPE65 variants, and (iii) 15 out of 16 lines of computational evidence supporting them as damaging, which together allowed the variants to be classified as uncertain significance. Two other pieces of evidence were accepted after further analysis of these Brazilian families: (i) p.Phe83Leu and p.Gly187Glu segregate with childhood retinal dystrophy within families, and (ii) their prevalence in Leber congenital amaurosis (LCA)/early-onset retinal dystrophy (EORD) patients can be considered higher than in other inherited retinal dystrophy patients. Therefore, these variants can now be classified as likely pathogenic according to ACMG/AMP classification guidelines.

Association of Daily Dietary Intake and Inflammation Induced by Marathon Race.

Daily food intake is crucial to maintain health and determine endogenous fuel to practice endurance exercise. We investigated the association between quantity of macronutrient and micronutrient daily intake and inflammation induced by long-distance exercise. Methods. Forty-four Brazilian male amateurs' marathon finishers from 30 to 55 years old participated in this study. Blood samples were collected 1 day before, immediately after, and 1 day and 3 days after São Paulo International Marathon. The serum levels of IL-6, IL-1ß, IL-10, IL-8, IL-12p70, and TNF-α were measured to evaluate inflammation. Dietary intake was determined using a prospective method of three food records in the week before marathon race. Results. Marathon race promoted an elevation on IL-6, IL-8, IL-1-ß, and IL-10 immediately after the race. The energy intake (EI), carbohydrate, fiber, folic acid, vitamin E, vitamin D, calcium, magnesium, and potassium intakes was below recommended. Immediately after the marathon race, we observed a negative correlation between IL-8 and daily EI, carbohydrate, fiber, fat, iron, calcium, potassium, and sodium intakes, and higher levels of IL-8 on runners with <3 g/kg/day of carbohydrate intake compared to runners with >5 g/kg/day. We demonstrated a positive correlation between daily carbohydrate intake and IL-10 and a negative correlation between TNF-α and % of energy intake recommended, carbohydrate and fiber intakes. Finally, runners with adequate EI had lower levels of IL-1ß and TNF-α compared with low EI immediately after the race. Conclusion. Nutrition strategies to promote balanced diet in amateur runners seem to be as important as immunonutrition sports market. Daily food intake, mainly EI, electrolyte and carbohydrate intakes, may modulate exacerbated inflammation after endurance exercise.

Thimet Oligopeptidase (EC 3.4.24.15) Key Functions Suggested by Knockout Mice Phenotype Characterization.

Thimet oligopeptidase (THOP1) is thought to be involved in neuropeptide metabolism, antigen presentation, neurodegeneration, and cancer. Herein, the generation of THOP1 C57BL/6 knockout mice (THOP1-/-) is described showing that they are viable, have estrus cycle, fertility, and a number of puppies per litter similar to C57BL/6 wild type mice (WT). In specific brain regions, THOP1-/- exhibit altered mRNA expression of proteasome beta5, serotonin 5HT2a receptor and dopamine D2 receptor, but not of neurolysin (NLN). Peptidomic analysis identifies differences in intracellular peptide ratios between THOP1-/- and WT mice, which may affect normal cellular functioning. In an experimental model of multiple sclerosis THOP1-/- mice present worse clinical behavior scores compared to WT mice, corroborating its possible involvement in neurodegenerative diseases. THOP1-/- mice also exhibit better survival and improved behavior in a sepsis model, but also a greater peripheral pain sensitivity measured in the hot plate test after bradykinin administration in the paw. THOP1-/- mice show depressive-like behavior, as well as attention and memory retention deficits. Altogether, these results reveal a role of THOP1 on specific behaviors, immune-stimulated neurodegeneration, and infection-induced inflammation.

Leucurogin and melanoma therapy.

Leucurogin is an ECD disintegrin-like protein, cloned from Bothrops leucurus venom gland. This new protein, encompassing the disintegrin region of a PIII metalloproteinase, is produced by recombinant technology and its biological and functional activity was partially characterized in this study. Biological activity was characterized in vitro using human fibroblasts. Functional activity of leucurogin was analysed in vitro and in vivo with murine B16F10 Nex-2 and human melanoma BLM cells. The results show that leucurogin inhibits cellular processes dependent on collagen type I. In a competition assay with collagen, leucurogin inhibits, in a dose-dependent manner, the adhesion of fibroblast to collagen. At 10 µM leucurogin reduces adhesion (40%) and migration (70%) of hFb and inhibits migration (32%) and proliferation (65%) of BLM cells. At 2.5 µM leucurogin inhibits 80% cell proliferation of B16F10 Nex-2 melanoma cells. At 4.8 µM leucurogin inhibits, in vitro, the vascular structures formation by endothelial cells by 66%. Leucurogin, injected intraperitoneally, i.p. (5 µg/animal, two-month old C57/Bl6 male mice) on alternate days for 15 days, inhibits lung metastasis of B16F10 Nex-2 cells by 70-75%. In the treatment of human melanoma, grafted intradermally in the nude mice flank, leucurogin (7.5 µg/kg in alternate days during 17 days) inhibits tumor growth by more than 40%. Leucurogin can be considered a promising agent for melanoma treatment.

Kinin-B2 Receptor Activity in Skeletal Muscle Regeneration and Myoblast Differentiation.

The bioactive peptide bradykinin obtained from cleavage of precursor kininogens activates the kinin-B2 receptor functioning in induction of inflammation and vasodilatation. In addition, bradykinin participates in kidney and cardiovascular development and neuronal and muscle differentiation. Here we show that kinin-B2 receptors are expressed throughout differentiation of murine C2C12 myoblasts into myotubes. An autocrine loop between receptor activation and bradykinin secretion is suggested, since bradykinin secretion is significantly reduced in the presence of the kinin-B2 receptor antagonist HOE-140 during differentiation. Expression of skeletal muscle markers and regenerative capacity were decreased after pharmacological inhibition or genetic ablation of the B2 receptor, while its antagonism increased the number of myoblasts in culture. In summary, the present work reveals to date no functions described for the B2 receptor in muscle regeneration due to the control of proliferation and differentiation of muscle precursor cells.

Effect of the bradykinin 1 receptor antagonist SSR240612 after oral administration in Mycobacterium tuberculosis-infected mice.

The role, if any, played by the kinin system in tuberculosis infection models, either in vivo or in vitro, was investigated. The effects of Mycobacterium tuberculosis infection on C57BL/6 wild type, B1R-/-, B2R-/- and double B1R/B2R knockout mice were evaluated. Immunohistochemistry analysis was carried out to assess B1R and B2R expression in spleens and lungs of M. tuberculosis-infected mice. In addition, in vitro experiments with M. tuberculosis-infected macrophages were performed. The in vivo effects of HOE-140 and SSR240612 on the mice model of infection were also evaluated. Infected B2R-/- mice exhibited increased splenomegaly, whereas decreased spleen weight in infected double B1R/B2R knockout mice was observed. The bacterial load, determined as colony-forming units, did not differ in the spleens and lungs of the studied mouse strains. Importantly, immunohistochemical analysis revealed that B1R was upregulated in both spleens and lungs of infected mice. M. tuberculosis-infected macrophages incubated with SSR240612, alone or in combination with des-Arg9-BK, for four days, displayed a marked inhibitory effect on CFU counts. However, the pre-incubation of the selective B1R (des-Arg9-BK and SSR240612) and B2R (BK and HOE-140) agonists and antagonists, respectively, did not significantly affect the bacterial loads. A statistically significant reduction in the CFU of M. tuberculosis in lungs and spleens of animals treated with SSR240612, but not with HOE-140, was observed. Further efforts should be pursued to clarify whether or not SSR240612 might be considered an option for the treatment of tuberculosis.

Brazilian Guidelines for Hereditary Angioedema Management - 2017 Update Part 1: Definition, Classification and Diagnosis

Hereditary angioedema is an autosomal dominant disease characterized by recurrent angioedema attacks with the involvement of multiple organs. The disease is unknown to many health professionals and is therefore underdiagnosed. Patients who are not adequately diagnosed and treated have an estimated mortality rate ranging from 25% to 40% due to asphyxiation by laryngeal angioedema. Intestinal angioedema is another important and incapacitating presentation that may be the main or only manifestation during an attack. In this article, a group of experts from the "Associação Brasileira de Alergia e Imunologia (ASBAI)" and the "Grupo de Estudos Brasileiro em Angioedema Hereditário (GEBRAEH)" has updated the Brazilian guidelines for the diagnosis and treatment of hereditary angioedema.

The Challenge of Diagnosis and Indication for Treatment in Fabry Disease

Abstract Fabry disease, caused by deficient alpha-galactosidase A lysosomal enzyme activity, remains challenging to health-care professionals. Laboratory diagnosis in males is carried out by determination of alpha-galactosidase A activity; for females, enzymatic activity determination fails to detect the disease in about two-thirds of the patients, and only the identification of a pathogenic mutation in the GLA gene allows for a definite diagnosis. The hurdle to be overcome in this field is to determine whether a mutation that has never been described determines a ''classic'' or ''nonclassic'' phenotype, because this will have an impact on the decision-making for treatment initiation. Besides the enzymatic determination and GLA gene mutation determination, researchers are still searching for a good biomarker, and it seems that plasma lyso-Gb3 is a useful tool that correlates to the degree of substrate storage in organs. The ideal time for treatment initiation for children and nonclassic phenotype remains unclear.

Expression and inactivation of osteopontin-degrading PHEX enzyme in squamous cell carcinoma.

Proteolytic enzymes mediate the activation or inactivation of many physiologic and pathologic processes. The PHEX gene (Phosphate-regulating gene with homologies to endopeptidase on the X chromosome) encodes a metallopeptidase, which is mutated in patients with a prevalent form (1:20,000) of inherited rickets-X-linked hypophosphatemia (XLH). XLH shows growth retardation, hypophosphatemia, osteomalacia, and defective renal phosphate reabsorption and metabolism of vitamin D. Most PHEX studies have focused on bone, and recently we identified osteopontin (OPN) as the first protein substrate for PHEX, demonstrating in the murine model of XLH (Hyp mice) an increase in OPN that contributes to the osteomalacia. Besides its role in bone mineralization, OPN is expressed in many tissues, and therein has different functions. In tumor biology, OPN is known to be associated with metastasis. Here, we extend our PHEX-OPN studies to investigate PHEX expression in a squamous cell carcinoma (SCC) cell line and its possible involvement in modulating OPN function. Real-time PCR showed PHEX-OPN co-expression in SCC cells, with sequencing of the 22 exons showing no mutation of the PHEX gene. Although recombinant PHEX hydrolyze SCC-OPN fragments, unlike in bone cells, SCC-PHEX protein was not predominantly at the plasma membrane. Enzymatic activity assays, FACs and immunoblotting analyses demonstrated that membrane PHEX is degraded by cysteine proteases and the decreased PHEX activity could contribute to inappropriate OPN regulation. These results highlight for the first time PHEX in tumor biology.

Host kinin B1 receptor plays a protective role against melanoma progression.

Melanoma is a very aggressive tumor that arises from melanocytes. Late stage and widely spread diseases do not respond to standard therapeutic approaches. The kallikrein-kinin system (KKS) participates in biological processes such as vasodilatation, pain and inflammatory response. However, the role of KKS in tumor formation and progression is not completely understood. The role of the host kinin B1 receptor in melanoma development was evaluated using a syngeneic melanoma model. Primary tumors and metastasis were respectively induced by injecting B16F10 melanoma cells, which are derived from C57BL/6 mice, subcutaneously or in the tail vein in wild type C57BL/6 and B1 receptor knockout mice (B1(-/-)). Tumors developed in B1(-/-) mice presented unfavorable prognostic factors such as increased incidence of ulceration, higher levels of IL-10, higher activation of proliferative pathways such as ERK1/2 and Akt, and increased mitotic index. Furthermore, in the metastasis model, B1(-/-) mice developed larger metastatic colonies in the lung and lower CD8(+)immune effector cells when compared with WT animals. Altogether, our results provide evidences that B1(-/-) animals developed primary tumors with multiple features associated with poor prognosis and unfavorable metastatic onset, indicating that the B1 receptor may contribute to improve the host response against melanoma progression.

Kinin B1 and B2 receptor deficiency protects against obesity induced by a high-fat diet and improves glucose tolerance in mice.

The kallikrein-kinin system is well known for its role in pain and inflammation, and has been shown recently by our group to have a role also in the regulation of energy expenditure. We have demonstrated that B1 receptor knockout (B1KO) mice are resistant to obesity induced by a high-fat diet (HFD) and that B1 receptor expression in adipocytes regulates glucose tolerance and predisposition to obesity. However, it is also known that in the absence of B1 receptor, the B2 receptor is overexpressed and can take over the function of its B1 counterpart, rendering uncertain the role of each kinin receptor in these metabolic effects. Therefore, we investigated the impact of ablation of each kinin receptor on energy metabolism using double kinin receptor knockout (B1B2KO) mice. Our data show that B1B2KO mice were resistant to HFD-induced obesity, with lower food intake and feed efficiency when compared with wild-type mice. They also had lower blood insulin and leptin levels and higher glucose tolerance after treatment with an HFD. Gene expression for tumor necrosis factor-alpha and C-reactive protein, which are important genes for insulin resistance, was reduced in white adipose tissue, skeletal muscle, and the liver in B1B2KO mice after the HFD. In summary, our data show that disruption of kinin B1 and B2 receptors has a profound impact on metabolic homeostasis in mice, by improving glucose tolerance and preventing HFD-induced obesity. These novel findings could pave the way for development of new pharmacological strategies to treat metabolic disorders such as insulin resistance and obesity.

Kinin-B1 and B2 receptor activity in proliferation and neural phenotype determination of mouse embryonic stem cells.

The kinins bradykinin and des-arg(9) -bradykinin cleaved from kininogen precursors by kallikreins exert their biological actions by stimulating kinin-B2 and B1 receptors, respectively. In vitro models of neural differentiation such as P19 embryonal carcinoma cells and neural progenitor cells have suggested the involvement of B2 receptors in neural differentiation and phenotype determination; however, the involvement of B1 receptors in these processes has not been established. Here, we show that B1 and B2 receptors are differentially expressed in mouse embryonic E14Tg2A stem cells undergoing neural differentiation. Proliferation and differentiation assays, performed in the presence of receptor subtype-selective agonists and antagonists, revealed that B1 receptor activity is required for the proliferation of embryonic and differentiating cells as well as for neuronal maturation at later stages of differentiation, while the B2 receptor acts on neural phenotype choice, promoting neurogenesis over gliogenesis. Besides the elucidation of bradykinin functions in an in vitro model reflecting early embryogenesis and neurogenesis, this study contributes to the understanding of B1 receptor functions in this process.

Kinin B1 Receptor Deletion Affects Bone Healing in Type 1 Diabetic Mice.

The effects of kinin B1 receptor (B1 R) deletion were examined on femur bone regeneration in streptozotocin (STZ)-type 1 diabetes. Diabetes induction in wild-type C57/BL6 (WTC57BL6) mice led to decrease in body weight and hyperglycemia, compared to the non-diabetic group of the same strain. The lack of B1 R did not affect STZ-elicited body weight loss, but partially prevented hyperglycemia. Diabetic mice had a clear delay in bone regeneration, and displayed large areas of loose connective tissue within the defects, with a reduced expression of the mineralization-related protein osteonectin, when compared to the non-diabetic WTC57/BL6. The non-diabetic and diabetic B1 R knockout (B1 RKO) mice had bone regeneration levels and osteonectin expression comparable to that seen in control WTC57/BL6 mice. WTC57/BL6 STZ-diabetic mice also showed a marked reduction of collagen contents, with increased immunolabeling for the apoptosis marker caspase-3, whereas diabetic B1 RKO had collagen levels and caspase-3 activity comparable to those observed in non-diabetic WTC57/BL6 or B1 RKO mice. No significant difference was detected in the number of tartrate-resistant acid phosphatase (TRAP)-stained cells, or in RANK/RANKL/OPG system immunolabeling throughout the experimental groups. Data bring novel evidence on the relevance of kinin B1 R under type 1 diabetes with regards to its role in bone regeneration.

The kinin B1 receptor regulates muscle-specific E3 ligases expression and is involved in skeletal muscle mass control.

Regulation of muscle mass depends on the balance between synthesis and degradation of proteins, which is under the control of different signalling pathways regulated by hormonal, neural and nutritional stimuli. Such stimuli are altered in several pathologies, including COPD (chronic obstructive pulmonary disease), diabetes, AIDS and cancer (cachexia), as well as in some conditions such as immobilization and aging (sarcopenia), leading to muscle atrophy, which represents a significant contribution to patient morbidity. The KKS (kallikrein-kinin system) is composed of the enzymes kallikreins, which generate active peptides called kinins that activate two G-protein-coupled receptors, namely B1 and B2, which are expressed in a variety of tissues. The local modulation of the KKS may account for its participation in different diseases, such as those of the cardiovascular, renal and central nervous systems, cancer and many inflammatory processes, including pain. Owing to such pleiotropic actions of the KKS by local modulatory events and the probable fine-tuning of associated signalling cascades involved in skeletal muscle catabolic disorders [for example, NF-κB (nuclear factor κB) and PI3K (phosphoinositide 3-kinase)/Akt pathways], we hypothesized that KKS might contribute to the modulation of intracellular responses in atrophying skeletal muscle. Our results show that kinin B1 receptor activation induced a decrease in the diameter of C2C12 myotubes, activation of NF-κB, a decrease in Akt phosphorylation levels, and an increase in the mRNA levels of the ubiquitin E3 ligases atrogin-1 and MuRF-1 (muscle RING-finger protein-1). In vivo, we observed an increase in kinin B1 receptor mRNA levels in an androgen-sensitive model of muscle atrophy. In the same model, inhibition of the kinin B1 receptor with a selective antagonist resulted in an impairment of atrogin-1 and MuRF-1 expression and IκB (inhibitor of NF-κB) phosphorylation. Moreover, knockout of the kinin B1 receptor in mice led to an impairment in MuRF-1 mRNA expression after induction of LA (levator ani) muscle atrophy. In conclusion, using pharmacological and gene-ablation tools, we have obtained evidence that the kinin B1 receptor plays a significant role in the regulation of skeletal muscle proteolysis in the LA muscle atrophy model.

Kinin B1 receptor deficiency attenuates cisplatin-induced acute kidney injury by modulating immune cell migration.

UNLABELLED: Cisplatin is a chemotherapeutic agent that causes severe renal dysfunction. The kinin B1 receptor has been associated with the migration of immune cells to injured tissue as well as with renal inflammation. To examine the role of the kinin B1 receptor in cisplatin-induced acute kidney injury, we used kinin B1 receptor knockout mice and treatment with a receptor antagonist before and after cisplatin administration. Cisplatin injection caused exacerbation of renal macrophage and neutrophil migration, higher levels of serum creatinine and blood urea, upregulation of B1 receptor mRNA and an increase in pro-inflammatory cytokines expression. B1 receptor knockout mice exhibited a reduction in serum creatinine and blood urea levels, diminished apoptosis, and decreased cisplatin-induced upregulation of inflammatory components. Moreover, treatment with the B1 receptor antagonist prior to cisplatin administration normalized serum creatinine, blood urea levels, protected from acute tubular necrosis, apoptosis-related genes, and prevented upregulation of pro-inflammatory cytokines. Thus, we propose that kinins have an important role in cisplatin-induced acute kidney injury by impairing immune cells migration to renal tissue during cisplatin nephrotoxicity. KEY MESSAGE: Kinin B1 receptor is upregulated after cisplatin exposure. Kinin B1 receptor deficiency diminishes the nephrotoxicity caused by cisplatin. Kinin B1 receptor deficiency ameliorates the inflammatory response. Kinin B1 receptor deficiency diminishes apoptosis caused by cisplatin. Kinin B1 receptor antagonism ameliorates renal function after cisplatin injection.

Activation of the kinin B1 receptor attenuates melanoma tumor growth and metastasis.

Melanoma is a very aggressive tumor that does not respond well to standard therapeutic approaches, such as radio- and chemotherapies. Furthermore, acquiring the ability to metastasize in melanoma and many other tumor types is directly related to incurable disease. The B1 kinin receptor participates in a variety of cancer-related pathophysiological events, such as inflammation and angiogenesis. Therefore, we investigated whether this G protein-coupled receptor plays a role in tumor progression. We used a murine melanoma cell line that expresses the kinin B1 receptor and does not express the kinin B2 receptor to investigate the precise contribution of activation of the B1 receptor in tumor progression and correlated events using various in vitro and in vivo approaches. Activation of the kinin B1 receptor in the absence of B2 receptor inhibits cell migration in vitro and decreases tumor formation in vivo. Moreover, tumors formed from cells stimulated with B1-specific agonist showed several features of decreased aggressiveness, such as smaller size and infiltration of inflammatory cells within the tumor area, higher levels of pro-inflammatory cytokines implicated in the host anti-tumor immune response, lower number of cells undergoing mitosis, a poorer vascular network, no signs of invasion of surrounding tissues or metastasis and increased animal survival. Our findings reveal that activation of the kinin B1 receptor has a host protective role during murine melanoma tumor progression, suggesting that the B1 receptor could be a new anti-tumor GPCR and provide new opportunities for therapeutic targeting.

Intracellular proteolysis of kininogen by malaria parasites promotes release of active kinins.

BACKGROUND: The malaria burden remains a major public health concern, especially in sub-Saharan Africa. The complex biology of Plasmodium, the apicomplexan parasite responsible for this disease, challenges efforts to develop new strategies to control the disease. Proteolysis is a fundamental process in the metabolism of malaria parasites, but roles for proteases in generating vasoactive peptides have not previously been explored. RESULTS: In the present work, it was demonstrated by mass spectrometry analysis that Plasmodium parasites (Plasmodium chabaudi and Plasmodium falciparum) internalize and process plasma kininogen, thereby releasing vasoactive kinins (Lys-BK, BK and des-Arg9-BK) that may mediate haemodynamic alterations during acute malaria. In addition, it was demonstrated that the P. falciparum cysteine proteases falcipain-2 and falcipain-3 generated kinins after incubation with human kininogen, suggesting that these enzymes have an important role in this process. The biologic activity of peptides released by Plasmodium parasites was observed by measuring ileum contraction and activation of kinin receptors (B1 and B2) in HUVEC cells; the peptides elicited an increase in intracellular calcium, measured by Fluo-3 AM fluorescence. This effect was suppressed by the specific receptor antagonists Des-Arg9[Leu8]-BK and HOE-140. CONCLUSIONS: In previously undescribed means of modulating host physiology, it was demonstrated that malaria parasites can generate active kinins by proteolysis of plasma kininogen.

A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells.

BACKGROUND: Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd(2) [S((-))C(2), N-dmpa](2) (µ-dppe)Cl(2)} named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. METHODS: B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. RESULTS: Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. CONCLUSIONS: The cyclopalladated C7a complex is an effective chemotherapeutic anticancer compound against primary and metastatic murine and human tumors, including cisplatin-resistant cells, inducing apoptotic cell death via the intrinsic pathway.