Radical metastasectomy followed by sorafenib versus observation in patients withclear cell renal cell carcinoma: extended follow -up of efficacy results from the randomized phase II RESORT trial.

Background: The RESORT trial showed no longer relapse free survival (RFS) with sorafenib following radical metastasectomy in metastatic renal cell carcinoma. We present the updated 42-month follow-up data.Methods: The phase II RESORT trial randomized patients to sorafenib or observation within 12 weeks from surgery. RFS was the primary endpoint.Results: We analyzed 68 patients (32 in sorafenib and 36 in the observation arm), randomized between November 2012 and November 2017. Eighty-one percent in the sorafenib arm and 80% in the observation arm had one metastasis . At a median follow-up of 42 months (interquartile range 31-58), in the observation arm the median RFS was 35 months, RFS probability was 57% (95% CI 42-76%) at 24 and 44% (95% CI 30-65%) at 48 months. In the sorafenib arm, median RFS was 21 months, RFS probability was 50% (95% CI 34-71%) at 24 and 32% (95% CI 18-57%) at 48 months (p = 0.342;HR 1.35;95% CI 0.72-2.54). Forty-seven percent and 37.5% of the patients in the two arms, respectively, are disease free. The site of relapses was independent of the previous metastasectomy site.Expert commentary: Sorafenib after metastasectomy did not improve RFS, but surgery in selected patients should be considered in order to potentially improve survival.Clinical trial registration: www.clinicaltrials.gov identifier is NCT0144480.

Dose-finding/phase II trial: bevacizumab, immunotherapy, and chemotherapy (BIC) in metastatic renal cell cancer (mRCC). Antitumor effects and variations of circulating T regulatory cells (Treg).

The aim of this study was to explore the efficacy and toxicities of a combined regimen of bevacizumab plus immunotherapy and chemotherapy (BIC) and the circulating T regulatory cells (Treg) in metastatic renal cell cancer (mRCC). Nephrectomized mRCC patients were enrolled into a multicenter single-arm dose-finding study with five escalated dose levels of chemotherapy with intravenous gemcitabine and 5-fluorouracil associated with fixed intravenous doses of bevacizumab, subcutaneous low doses of interleukin-2, and interferon-α-2a. An expanded cohort (phase II study) was treated at the recommended dose for additional safety and efficacy information according to minimax Simon two-stage design. Blood samples for Treg were collected and evaluated by fluorescence-activated cell sorting (FACS) analysis on cycle 1. Fifty-one patients were entered to receive one of five dose levels. Median age was 58 years (male 67 %, pretreated 49 %): 15 patients were low risk according to Memorial Sloan-Kettering Cancer Center (MSKCC) criteria, while 27 and nine were respectively intermediate- and high-risk patients. More frequent grade 3 and 4 toxicities included nonfebrile neutropenia, thrombocytopenia, and fever. Among patients evaluable for response (49), 29.5 % had partial response and 37 % stable disease. Overall median time to progression and median overall survival were 8.8 and 22.67 months, respectively. We observed a rapid increase in the percentage of Treg after immunotherapy and a reduction after bevacizumab only in patient who obtained a partial response or stable disease. The BIC was feasible, well tolerated, and shown interesting activity. Further studies are needed to explore if Treg could have a role in clinical response in mRCC treated with bevacizumab.

Lead poisoning in cattle in southern Brazil

Em setembro de 2000, três novilhas, provenientes de um pequeno rebanho de bovinos de corte, apresentaram severa depressão, tremores musculares, ato de pressionar a cabeça contra objetos e de ranger de dentes, intensa salivação, cegueira e morte. Envenenamento por chumbo foi diagnosticado com base nos sinais clínicos e em função da presença de grandes concentrações de chumbo nos rins e no fígado de um dos animais afetados. Latas de tinta velhas, presentes no potreiro onde estavam os animais e cujo conteúdo extravasava através de fendas provocadas pela ferrugem, foram identificadas como a fonte de chumbo que causou a toxicose nos animais.

Mechanisms of expression of NADPH oxidase components in human cultured monocytes: role of cytokines and transcriptional regulators involved.

Human blood monocytes lose their capability to produce microbicidal oxidants during culture. We report that this process is associated with decreased gp91phox, p22phox and p47phox expression, release of PU.1 and CP-1 from gp91phox promoter, and PU.1 from p47phox promoter. However, in presence of IFN-gamma or TNF-alpha, the superoxide anion (O(2)(-)) production, the p47phox, gp91phox and p22phox expression, and the binding of PU.1 and CP-1 to DNA are maintained at the high levels observed in blood monocytes. To clarify the role of PU.1 in the expression of NADPH oxidase components, oligonucleotides competing for PU.1-DNA binding were added to cultured monocytes. These oligonucleotides abrogated the maintenance of gp91phox and p22phox expression by IFN-gamma and TNF-alpha, but did not inhibit the effect of these cytokines on p47phox expression and O(2)(-) production. Our results indicate that in monocytes the IFN-gamma- and TNF-alpha-induced expression of gp91phox and p22phox, but not p47phox, requires the binding of PU.1 to gp91phox promoter. However, the preservation of O(2)(-) production by IFN-gamma and TNF-alpha is unrelated to their effect on gp91phox and p22phox expression.

A single-chain antibody fragment is functionally expressed in the cytoplasm of both Escherichia coli and transgenic plants.

Despite the well-known crucial role of intradomain disulfide bridges for immunoglobulin folding and stability, the single-chain variable fragment of the anti-viral antibody F8 is functionally expressed when targeted to the reducing environment of the plant cytoplasm. We show here that this antibody fragment is also functionally expressed in the cytoplasm of Escherichia coli. A gel shift assay revealed that the single-chain variable fragment (scFv) accumulating in the plant and bacterial cytoplasm bears free sulfhydryl groups. Guanidinium chloride denaturation/renaturation studies indicated that refolding occurs even in a reducing environment, producing a functional molecule with the same spectral properties of the native scFv(F8). Taken together, these results suggest that folding and functionality of this antibody fragment are not prevented in a reducing environment. This antibody fragment could therefore represent a suitable framework for engineering recombinant antibodies to be targeted to the cytoplasm.

Nicotinamide-adenine dinucleotide phosphate oxidase assembly and activation in EBV-transformed B lymphoblastoid cell lines of normal and chronic granulomatous disease patients.

This paper deals with the mechanisms of activation of NADPH oxidase investigated using EBV-transformed human B lymphoblastoid cell lines (B cells) from normal subjects and from patients affected by X-linked chronic granulomatous disease (CGD). The results reported are as follows. 1) In normal B cells, the NADPH oxidase components p67phox, p40phox, p22phox, and gp91phox were less expressed than in polymorphonuclear neutrophils. 2) In normal B cells stimulated with PMA, p47phox, p67phox, and p40phox translocated to the membranes as occurs in polymorphonuclear neutrophils. 3) In CGD, B cells expressing p22phox in the absence of gp91phox, p47phox, p67phox, and p40phox did not translocate to the membranes after stimulation with PMA. 4) In PMA-stimulated B cells from an X91+ CGD patient in which p22phox was normally expressed and gp91phox was present but lacked five amino acids, translocation of p47phox to the membranes was unaffected, but p67phox and p40phox were poorly translocated, and the production of O2- was greatly reduced with respect to that by normal B cells. Taken together, these findings indicate that 1) a low expression of some NADPH oxidase components may represent the molecular basis of the low production of O2- in B lymphocytes; 2) the cytosolic components of NADPH oxidase cannot bind to p22phox on the membranes in the absence of gp91phox; 3) p47phox can translocate to the membranes independently of p67phox and p40phox; and 4) gp91phox may have a role in mediating and/or stabilizing the binding of p67phox and p40phox to the membranes of activated cells.

Identification of a double mutation (D160V-K161E) in the p67phox gene of a chronic granulomatous disease patient.

In neutrophils of a chronic granulomatous disease (CGD) patient with a lack of p67phox the mRNA for p67phox was present in normal amount and size. This mRNA was reverse transcribed, and the coding region was analyzed by single-strand conformation polymorphism analysis. Direct DNA sequencing allowed the identification of a A479-to-T and A481-to-G substitution in exon 5 of the p67phox gene resulting in a double nonconservative amino acid change 160Lys-to-Glu and 161Asp-to-Val (D160V-K161E). This defect was found in the genomic DNA of this patient in heterozygous state and does not correspond to those previously found in other cases of CGD lacking the p67phox.

Mechanisms of stimulation of the respiratory burst by TNF in nonadherent neutrophils: its independence of lipidic transmembrane signaling and dependence on protein tyrosine phosphorylation and cytoskeleton.

This study concerns the controversial problem of whether the TNF-alpha (TNF) induces a respiratory burst in human neutrophils in suspension. The results have shown that in these cells TNF induces a classical respiratory burst. In fact, the production of oxygen free radicals 1) is linked to the translocation of NADPH oxidase components from cytosol to the plasma membrane, 2) does not take place in neutrophils from a patient lacking the cytochrome b558, and 3) does not involve other sources such as mitochondrial respiratory chain or xanthine oxidase. Signal transduction studies have demonstrated that this respiratory burst 1) is not accompanied by calcium transients, stimulation of phosphoinositide turnover, and phospholipase D activity (moreover, this burst is associated with the stimulation of the activity of phospholipase A2, but not of sphingomyelinase); 2) is strictly dependent on activation of tyrosine kinases, which is functional to the translocation to the plasma membrane of the cytosolic NADPH oxidase component rac; and 3) is dependent on the integrity of the cytoskeleton because it is completely suppressed by cytochalasin B. The integrity of the cytoskeleton is required for a full translocation of all the NADPH oxidase components and for an optimal activation of tyrosine kinases, but not for phospholipase A2 activation. Taken together, these findings demonstrate that TNF activates the NADPH oxidase through stimulation of tyrosine kinases, whose function is cytoskeleton-dependent, and raise the problem of whether the activation of this respiratory burst involves signals arising from TNF-activated beta2 integrins.

Mechanisms of NADPH oxidase activation: translocation of p40phox, Rac1 and Rac2 from the cytosol to the membranes in human neutrophils lacking p47phox or p67phox.

On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox, p47phox, p40phox, as well as the Ras-related G-proteins Rac1 and Rac2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b, forming a functional complex responsible for the production of oxygen radicals in phagocytes. In this paper we show that (a) in neutrophils from a patient with a form of chronic granulomatous disease (CGD) in which p67phox is absent, p47phox and Rac2, but not p40phox and Rac1 were translocated from the cytosol to the membrane on stimulation with formylmethionyl-leucylphenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA); (b) in neutrophils from a patient with a form of CGD in which p47phox is absent, p67phox, p40phox and Rac1 failed to associate with the membrane on stimulation with fMLP or PMA, whereas Rac2 was translocated as in normal neutrophils. We also show that in neutrophils from a patient lacking p67phox, the amount of cytosolic p40phox was decreased by about 40%. These findings indicate that, on neutrophil stimulation, p67phox mediates the translocation of p40phox and Rac1 from the cytosol to cell membranes and that Rac2 associates with the membranes independently of p47phox and p67phox.

Role of 55- and 75-kDa TNF receptors in the potentiation of Fc-mediated phagocytosis in human neutrophils.

Human neutrophils respond with an increased phagocytosis when exposed to TNF. Two types of TNF receptors have been identified, namely 55 kDa (TR55) and 75 kDa (TR75). We addressed the problem of the role of these receptors in the priming effect of TNF. By using monoclonal antibodies (MoAbs) directed either against TR55 or TR75, we have shown that 1) only TR55 is the signaling receptor for the potentiation of Fc-mediated phagocytosis and upregulation of beta 2-integrin CD11b/CD18; 2) TR75 may control the function of TR55 by regulating the binding of TNF to the signaling receptor.

Mechanisms of NADPH oxidase activation in human neutrophils: p67phox is required for the translocation of rac 1 but not of rac 2 from cytosol to the membranes.

NADPH oxidase is the enzyme complex responsible for the production of oxygen radicals in phagocytes. On neutrophil stimulation, the cytosolic components of NADPH oxidase, p67phox and p47phox, as well as the Ras-related G-protein rac 2, are translocated from the cytosol to cell membranes where they associate with a flavocytochrome b to form a functional complex. Besides rac 2, rac 1 G-protein is also involved in the activation of the NADPH oxidase, but, to date, it has not been documented whether it is also translocated in activated neutrophils. In this paper we show that: (a) in neutrophils stimulated with formylmethionyl-leucylphenylalanine, concanavalin A or phorbol 12-myristate 13-acetate, both rac 1 and rac 2 are translocated from cytosol to the membranes; (b) in neutrophils from a patient with a form of chronic granulomatous disease in which p67phox is absent, rac 2 and p47phox were translocated as in normal neutrophils on stimulation with the above agonists, but rac 1 failed to be translocated from the cytosol to the membranes. This is the first demonstration that, in activated neutrophils, rac 1 is translocated from the cytosol to the membranes and this translocation requires p67phox. These results, coupled with those showing that rac 2 is not translocated in activated neutrophils lacking p47phox [El Benna, Ruedi and Babior (1994) J. Biol. Chem. 269, 6729-6734], may suggest that the assembly of the cytosolic components of NADPH oxidase on the plasma membrane takes place through selective coupling of activated rac 1 and rac 2 with p67phox and p47phox respectively.

Influence of radiotherapy on intestinal microflora in cancer patients.

We investigated in 15 patients with carcinoma of the uterine cervix or endometrium, who were undergoing postoperative radiation therapy, the effects of different fractionated radiation exposures on counts of fecal bacteria, on the growth of Clostridium difficile and Clostridium perfringens enterotoxin production. We observed a generally significant decrease in intestinal microflora after the first radiation exposure, whereas at the end of radiotherapy all bacteria increased and reached basal values except Enterococcus faecium 1, lactobacilli and total anaerobes. In some patients we observed an overgrowth of some Clostridium spp. which were potential pathogens associated with clinical symptoms. We did not observe an influence of multiple radiations on C. perfringens enterotoxin fecal contents. We conclude that patients receiving radiotherapy may benefit from the intake of oral bacteriotherapy, i.e. live beneficial bacteria such as Bacillus subtilis at the beginning of the irradiation exposure.