Anti-inflammatory pretreatment enables an efficient dendritic cell-based immunotherapy against established tumors

Although animals can be immunized against the growth of some tumor implants, most of the attempts to use immunotherapy to cause the regression of animal and human tumors once they have become established have been disappointing even when strongly immunogenic tumors were used as target. In this paper, we demonstrate that the failure to achieve an efficient immunological treatment against an established strongly immunogenic murine fibrosarcoma was paralleled with the emergence of a state of immunological unresponsiveness (immunological eclipse) against tumor antigens observed when the tumor surpassed the critical size of 500 mm³. In turn, the onset of the immunological eclipse was coincidental with the onset of a systemic inflammatory condition characterized by a high number of circulating and splenic polymorphonucleated neutrophils (PMN) displaying activation and Gr1⁺Mac1⁺ phenotype and an increasing serum concentration of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 cytokines and C-reactive protein (CRP) and serum A amyloid (SAA) phase acute proteins. Treatment of tumor-bearing mice with a single low dose (0.75 mg/kg) of the synthetic corticoid dexamethasone (DX) significantly reduced all the systemic inflammatory parameters and simultaneously reversed the immunological eclipse, as evidenced by the restoration of specific T-cell-dependent concomitant immunity, ability of spleen cells to transfer anti-tumor activity and recovery of T-cell signal transduction molecules. Two other anti-inflammatory treatments by using indomethacin or dimeric TNF-α receptor, also partially reversed the immunological eclipse although the effect was not as striking as that observed with DX. The reversion of the immunological eclipse was not enough on its own to inhibit the primary growing tumor. However, when we used the two-step strategy of inoculating DX to reverse the eclipse and then dendritic cells loaded with tumor antigens (DC) as an immunization booster, a significant inhibition of the growth of both established tumors and remnant tumor cells after excision of large established tumors was observed, despite the fact that the vaccination alone (DC) had no effect or even enhanced tumor growth in certain circumstances. The two-step strategy of tumor immunotherapy that we present is based on the rationale that it is necessary to eliminate or ameliorate the immunological eclipse as a precondition to allow an otherwise ineffective anti-tumor immunological therapy to have a chance to be successful.     

Anti-GM1 antibodies as a model of the immune response to self-glycans

Glycans are a class of molecules with high structural variability, frequently found in the plasma membrane facing the extracellular space. Because of these characteristics, glycans are often considered as recognition molecules involved in cell social functions, and as targets of pathogenic factors. Induction of anti-glycan antibodies is one of the early events in immunological defense against bacteria that colonize the body. Because of this natural infection, antibodies recognizing a variety of bacterial glycans are found in sera of adult humans and animals. The immune response to glycans is restricted by self-tolerance, and no antibodies to self-glycans should exist in normal subjects. However, antibodies recognizing structures closely related to self-glycans do exist, and can lead to production of harmful anti-self antibodies. Normal human sera contain low-affinity anti-GM1 IgM-antibodies. Similar antibodies with higher affinity or different isotype are found in some neuropathy patients. Two hypotheses have been developed to explain the origin of disease-associated anti-GM1 antibodies. According to the "molecular mimicry" hypothesis, similarity between GM1 and Campylobacter jejuni lipopolysaccharide carrying a GM1-like glycan is the cause of Guillain-Barré syndrome associated with anti-GM1 IgG-antibodies. According to the "binding site drift" hypothesis, IgM-antibodies associated with disease originate through changes in the binding site of normally occurring anti-GM1 antibodies. We now present an "integrated" hypothesis, combining the "mimicry" and "drift" concepts, which satisfactorily explains most of the published data on anti-GM1 antibodies.     

Activation of phosphatidylcholine signalling during oxidative stress in synaptic endings

The purpose of the present study was to investigate the involvement of phosphatidylcholine (PC) signalling in synaptic endings incubated under oxidative stress conditions. Synaptosomes purified from adult rats (4 months old) cerebral cortex were exposed to oxidative insult (FeSO₄, 50 μM) or vehicle, and diacylglycerol (DAG) generation and free fatty acid (FFA) release were subsequently evaluated using exogenous [¹⁴C]PC as substrate. DAG formation increased after 5, 30, and 60 min of Fe²⁺-exposure with respect to the control conditions. The contribution of PC-specific phospholipase C (PC-PLC) and phospholipase D (PLD) pathways to DAG generation was evaluated using ethanol in the enzyme assays. Phosphatidylethanol (PEth) production was measured as a marker of PLD activity. In the presence of ethanol (2%) iron significantly stimulated DAG and PEth production at all times assayed. FFA release from PC, however, was inhibited after 5 and 60 min of iron exposure. Similar results were observed in aged animals (28 months old) when compared with adult animals. DAG generation from PC was also evaluated in the presence of the tyrosine kinase inhibitors genistein and herbimycin A. Inhibition of tyrosine kinase activity did not modify the stimulatory effect exerted by iron on PC-PLC and PLD activities. Moreover, the presence of LY294002 (a specific PI3K inhibitor) did not alter DAG production. Our results demonstrate that oxidative stress induced by free iron stimulates the generation of the lipid messenger DAG from PC in synaptic endings in adult and aged rats.     

Interspecific interactions between wild black and gold howlermonkeys (<Emphasis Type="Italic">Alouatta caraya</Emphasis>) and other mammals in northeastern Argentina

We studied the interactions between wild black and gold howler monkeys (Alouatta caraya) and other mammals at three sites with different human disturbance levels and forest structures in northeastern Argentina. The main goal was to evaluate the effects of the study site and type of interaction (agonistic or non-agonistic) on the rate of interaction. In addition, we also described the associations between interspecific interaction rate, species involved, howler monkeys’ activity, and seasonality. We present 50 group-years for 14 groups, collected between 2003 and 2012. We registered a total of 29 interactions (0.22 ± 0.26 interactions/100 h). Most interactions (56.7%) were agonistic (12% of high intensity and 88% of low intensity), 41.9% were neutral, and 1.5% were affiliative. We found that both factors, site and type of interaction, have an effect on the rate of interaction. Interspecific interactions were more frequent at the two sites without human settlement than in the rural site. Interspecific interactions occurred during resting (46.73%), traveling (27.40%), and feeding (25.87%). Interactions occurred throughout the year but were concentrated in August (late winter) and September (beginning of spring). Our results suggest that both site and type of interaction affected the rate of interaction between howler monkeys and other mammals in northeastern Argentina. Agonistic interactions were more frequent that non-agonistic ones, and monkeys reacted agonistically to several mammalian species, but these interactions were mainly of low intensity.     

Synthesis and in vitro antiproliferative activities of (5-aryl-1,2,4-oxadiazole-3-yl) methyl d-ribofuranosides

The emergence of multidrug resistance cell lines is one of the major obstacles in the success of cancer chemotherapeutic treatment. Therefore, it remains a big challenge the development of new and effective drugs to defeat cancer. The presence of nitrogen heterocycles in the architectural design of drugs has led to the discovery of new leading compounds. Herein, we report the synthesis, characterization and in vitro antiproliferative activity against six cancer cell lines of d-ribofuranoside derivatives bearing a 1,2,4-oxadiazolic ring, with the aim of developing new active compounds. Most of these derivatives exhibit significant antiproliferative activities in the micromolar range. Noteworthy, the most potent compound of the series showed better selectivity towards the more resistant colon cancer cell line WiDr.     

Cytoskeletal ultrastructure and lipid composition of I-Z-I fraction in muscle from pre- and post-spawned female hake (Merluccius hubbsi)

Myofibrils from pre- and post-spawned female hake were depleted of thin and thick filaments by KI-treatment and the cytoskeletal ultrastructure of the preparations was investigated by electron microscopy. Irrespective of the gonadal stage of the fish, transmission (TEM) and scanning (SEM) electron micrographs showed an extensive network of filaments connecting Z-structures, which appeared as two circular rings held together. Z-structures of KI-treated myofibrils from post-spawned hake were more compact than those from pre-spawned fish. This ultrastructural difference was absent when KI-treated myofibrils from hake in both gonadal conditions were prepared in the presence of a proteinase inhibitor cocktail (1 mM EDTA+1 mM PMSF+1 mM iodoacetic acid). The total lipid (TL) content of the I-Z-I fraction from fish in pre-spawned condition was about 3.7 mg/g I-Z-I and non-polar lipids (NPL) represent 86.5% of TL. TL and NPL contents of the I-Z-I fraction from post-spawned hake were about 50% and 78.2% lower than those obtained from this fraction in pre-spawning fish. No significant changes (p>0.05) were observed in phospholipids (PL) and acidic phospholipids (AP). No significant differences (p<0.01) were observed among the corresponding lipid fractions of I-Z-I purified from pre- and post-spawning fish in the presence of a protease inhibitor cocktail.     

Sugar uptake and sensitivity to carbon catabolite regulation in Streptomyces peucetius var. caesius

Streptomyces peucetius var. caesius produces a family of secondary metabolites called anthracyclines. Production of these compounds is negatively affected in the presence of glucose, galactose, and lactose, but the greatest effect is observed under conditions of excess glucose. Other carbon sources, such as arabinose or glutamate, show either no effect or stimulate production. Among the carbon sources that negatively affect anthracycline production, glucose is consumed in greater concentrations. We determined glucose and galactose transport in S. peucetius var. caesius and in a mutant of this strain whose anthracycline production is insensitive to carbon catabolite repression (CCR). In the original strain, incorporation of glucose and galactose was stimulated when the microorganism was grown in media containing these sugars, although we also observed basal galactose incorporation. Both the induced and the basal incorporation of galactose were suppressed when the microorganism was grown in the presence of glucose. Furthermore, adding glucose directly during the transport assay also inhibited galactose incorporation. In the mutant strain, we observed a reduction in both glucose (48%) and galactose (81%) incorporation compared to the original. Galactose transport in this mutant showed reduced sensitivity to the negative effect of glucose; however, it was still sensitive to inhibition. The deficient transport of these sugars, as well as CCR sensitivity to glucose in this mutant was corrected when the mutant was transformed with the SCO2127 region of the Streptomyces coelicolor genome. Our results support a role for glucose as the most easily utilized carbon source capable of exerting the greatest repression on anthracycline biosynthesis. In consequence, glucose also prevented the repressive effect of galactose by suppressing its incorporation. This suggests the participation of an integral regulatory system, which is initiated by an increase in incorporation of repressive sugars and their metabolism as a prerequisite for establishing the phenomenon of CCR in S. peucetius var. caesius.     

Novel expression of liver FBPase in Langerhans islets of human and rat pancreas

Several reports have indicated the absence of gluconeogenic enzymes in pancreatic islet cells. In contrast, here we demonstrate that liver fructose-1,6-bisphosphatase (FBPase) is highly expressed both in human and rat pancreas. Interestingly, pancreatic FBPase is active and functional, and is inhibited by AMP and fructose-2,6-bisphosphate (Fru-2,6-P2). These results suggest that FBPase may participate as a component of a metabolic sensing mechanism present in the pancreas. Immunolocalization analysis showed that FBPase is expressed both in human and rat Langerhans islets, specifically in beta cells. In humans, FBPase was also located in the canaliculus and acinar cells. These results indicate that FBPase coupled with phosphofructokinase (PFK) plays a crucial role in the metabolism of pancreatic islet cells. The demonstration of gluconeogenic recycling of trioses as a new metabolic signaling pathway may contribute to our understanding of the differences between the insulin secretagogues trioses, fructose, and glucose in pancreas.