Immunohistochemical evaluation of two antibodies against PD-L1 and prognostic significance of PD-L1 expression in epithelioid peritoneal malignant mesothelioma: A RENAPE study.

BACKGROUND: Epithelioid peritoneal malignant mesothelioma (EPMM) is the most common subtype of this aggressive tumor. We compared two antibodies against PD-L1, a recent theranostic biomarker, and evaluated the prognostic value of PD-L1 expression by mesothelial and immune cells in EPMM. METHODS: Immunohistochemistry was performed on 45 EPMM. Clinical and pathological data were extracted from the RENAPE database. Using E1L3N and SP142 clones, inter-observer agreement, PD-L1 expression by mesothelial and immune cells and inter-antibody agreement were evaluated. The prognostic relevance of PD-L1 expression was evaluated in 39 EPMM by univariate and multivariate analysis of overall survival (OS) and progression-free survival (PFS). RESULTS: Inter-observer agreement on E1L3N immunostaining was moderate for mesothelial and immune cells, and fair for mesothelial and poor for immune cells using SP142. Using E1L3N, 31.1% of mesothelial and 15.6% of immune cells expressed PD-L1, and 22.2% of mesothelial and 26.7% of immune cells using SP142. Inter-antibody agreement was moderate. In most positive cases, 1-5% of tumor cells were positive. Using E1L3N, PD-L1 expression by lymphocytes was associated with better OS and PFS by both univariate and multivariate analysis. Cytoreductive surgery with hyperthermic intraperitoneal chemotherapy predicted better prognosis than other treatments. Solid subtype was an independent prognostic factor for worse OS. CONCLUSION: E1L3N appeared easier to use than SP142 to evaluate PD-L1 expression. A minority of EPMM expressed PD-L1, and only a few cells were positive. PD-L1 expression by immune cells evaluated with E1L3N was an independent prognostic factor in EPMM.

Functional dominance among Hox genes: repression dominates activation in the regulation of Dpp.

Here we investigate the mechanisms by which Hox genes compete for the control of positional identity. Functional dominance is often observed where different Hox genes are co-expressed, and frequently the more posteriorly expressed Hox gene is the one that prevails, a phenomenon known as posterior prevalence. We use dpp674, a visceral mesoderm-specific enhancer of decapentaplegic (dpp), to investigate functional dominance among Hox genes molecularly. We find that posterior prevalence does not adequately describe the regulation of dpp by Hox genes. Instead, we find that abdominal-A (abd-A) dominates over the more posterior Abdominal-B (Abd-B) and the more anterior Ultrabithorax (Ubx). In the context of the dpp674 enhancer, abd-A functions as a repressor whereas Ubx and Abd-B function as activators. Thus, these results suggest that other cases of Hox competition and functional dominance may also be understood in terms of competition for target gene regulation in which repression dominates over activation.

The bx region enhancer, a distant cis-control element of the Drosophila Ubx gene and its regulation by hunchback and other segmentation genes.

The Drosophila homeotic gene Ultrabithorax (Ubx) is regulated by complex mechanisms that specify the spatial domain, the timing and the activity of the gene in individual tissues and in individual cells. In early embryonic development, Ubx expression is controlled by segmentation genes turned on earlier in the developmental hierarchy. Correct Ubx expression depends on multiple regulatory sequences located outside the basal promoter. Here we report that a 500 bp DNA fragment from the bx region of the Ubx unit, approximately 30 kb away from the promoter, contains one of the distant regulatory elements (bx region enhancer, BRE). During early embryogenesis, this enhancer element activates the Ubx promoter in parasegments (PS) 6, 8, 10, and 12 and represses it in the anterior half of the embryo. The repressor of the anterior Ubx expression is the gap gene hunchback (hb). We show that the hb protein binds to the BRE element and that such binding is essential for hb repression in vivo, hb protein also binds to DNA fragments from abx and bxd, two other regulatory regions of the Ubx gene. We conclude that hb represses Ubx expression directly by binding to BRE and probably other Ubx regulatory elements. In addition, the BRE pattern requires input from other segmentation genes, among them tailless and fushi tarazu but not Krüppel and knirps.

Molecular mechanisms of pattern formation by the BRE enhancer of the Ubx gene.

The core activity of the Ubx gene enhancer BRE (bx region enhancer) is encoded within a 500 bp module. bx DNA outside this active module increases the level of expression, expands the expression into ventro-lateral ectoderm and partially stabilizes the late expression pattern. The products of the gap genes hb and tll and of the pair-rule gene ftz bind to the 500 bp BRE module and control directly its initial pattern of expression. ftz enhances expression in even-numbered parasegments within the correct spatial domain whose boundaries are set by hb and tll. In addition, en and twi products activate the enhancer, probably directly. en broadens the parasegmental stripe while twi cooperates with ftz to enhance expression in the mesoderm. Binding sites for the five regulators are closely clustered, often overlapping extensively with one another. In vitro, hb blocks the binding of ftz and can also displace ftz protein pre-bound to an overlapping site, suggesting that competitive binding and/or interference by hb sets the initial boundaries of the domain of expression. Our results also suggest that this interaction is short-range and the long distance interactions among different enhancers may depend on each enhancer's ability to complex with the promoter.

Antagonism between steady light and phosphodiesterase inhibitors on the kinetics of rod photoresponses.

Treatment of toad rods with phosphodiesterase inhibitors (3-isobutyl-1-methylxanthine, caffeine, theophylline, papaverine, and RO 20-1724) modifies the properties of the intracellular voltage responses to dim flashes of light. 3-Isobutyl-1-methylxanthine at 1-20 microM causes an increase in flash sensitivity and a slowing down of the kinetics of the photoresponses. When the drug concentration is greater than 20 microM, rods also show supralinear behavior, whereby doubling the intensity of a dim flash may increase the response by greater than 2-fold. Sensitivity, kinetics, and supralinear behavior can be restored to normal by steady background illumination while still in the presence of 3-isobutyl-1-methylxanthine. However, the intensity of the steady light needed to restore the sensitivity to control levels is not sufficient to accelerate the kinetics back to control values. The antagonism between the effects of 3-isobutyl-1-methylxanthine and the effects of background illumination is explained by assuming that: (i) the length of time to peak voltage responses to dim flashes of light is inversely proportional to the rate of a chemical reaction; (ii) the rate of this reaction is controlled by an enzyme that is inhibited competitively by 3-isobutyl-1-methylxanthine with a Ki of 3 x 10(-6) M; and (iii) the concentration of a cofactor of this reaction increases proportionally with the intensity of the background illumination.

Effects of changing external potassium and chloride concentrations on the photoresponses of Bufo bufo rods.

1. Intracellular responses to light were recorded from Bufo bufo rods in different ionic media. 2. The exposure of the retina to high external [K+] depolarized the rod and modified the time course of the photoresponse. The prominent initial transient of rod responses to bright flashes was drastically reduced in 5 mM-external [K+] and completely disappeared in 26 mM. In high external [K+] the kinetics of responses to dim flashes were considerably slower than in control conditions. 3. When external [Cl-] was changed from 120.6 to 10.6 mM the resting membrane potential decreased and the size of photoresponses increased. Changes in the kinetics similar to those described in high external [K+] were also observed. 4. In many cases exposure of the retina to low external [Cl-] induced oscillations of the resting membrane potential that sometimes became sustained. This instability of the membrane completely disappeared upon restoring to normal conditions. 5. The present results may be explained by assuming the existence of a voltage- and time-dependent conductance active near the dark level of membrane potential. This hypothesis can be represented by an equivalent electrical circuit that includes an inductance (Detwiler, Hodgkin & McNaughton, 1980).

Direct regulation of decapentaplegic by Ultrabithorax and its role in Drosophila midgut morphogenesis.

Drosophila homeotic genes encode transcription factors thought to control segmental identity by regulating expression of largely unknown target genes. The formation of the second midgut constriction requires the Ultrabithorax (Ubx) and abdominal-A (abd-A) homeotic genes and decapentaplegic (dpp), a gene encoding a member of the TGF beta family of proteins. We identified a 674 bp enhancer of dpp controlling its expression in the second constriction domain of the visceral mesoderm (parasegment 7). Normal enhancer function requires positive regulation by Ubx and negative regulation by abd-A. This enhancer contains UBX- and ABD-A-binding sites defined in vitro. By generating complementary alterations of the binding sites and the binding specificity of UBX, we show that Ubx directly regulates dpp expression. These regulatory interactions are relevant to normal development, because a transgene made with this enhancer driving a dpp transcription unit rescues the second midgut constriction and larval lethality phenotypes of dpps mutations.

Mechanisms of generation of signals in vertebrate photoreceptors.

The electrical responses of rods are analyzed in different ionic environments. It is shown that the dark level of the membrane potential is predominantly determined by a sodium current, while the peak of responses to bright light is controlled by the concentration of external potassium. The sag from the peak to the plateau of photoresponses seems to be generated by different ionic mechanism. The effects produced by substituting the external calcium with EGTA are also analyzed. It is suggested that calcium plays a role in different mechanisms of generation of electrical responses.

The effect of phosphodiesterase inhibitors on the electrical activity of toad rods.

The membrane potential of toad rods was recorded during addition of small amounts of phosphodiesterase inhibitors to the extracellular medium. Separate application of 3-isobutyl-1-methylxanthine (IBMX), caffeine, theophylline, papaverine and RO 20-1724 slowed down the time course of rod photo-response to dim flashes of light. These changes were associated with a two to six-fold increase in the amplitude of photoresponse. The effects on kinetics may be described simply by an expansion of the photoresponse time scale. When the drug concentration was raised above a certain level, the rods showed supralinear behaviour whereby doubling of the intensity of a dim flash could increase the response more than two-fold. Under similar conditions rods also showed light sensitization whereby responses to dim flashes were enhanced in the presence of dim backgrounds. Taking the drug concentration that induced a two-fold increase in the time-to-peak, IBMX was found the most effective compound, followed by papaverine, RO 20-1724, theophylline and caffeine with relative effectivities 1, 1/2, 1/7, 1/40 and 1/100. Sensitivity, kinetics and supralinear behaviour may be restored to normal by steady background illumination while still in the presence of IBMX. However the intensity of the steady light, needed to restore the sensitivity to control levels, is not sufficient to accelerate the kinetics back to control values. In the presence of 50 microM-IBMX a dim steady background of light enhanced the response to dim flashes. When the intensity of the light background was increased rods were desensitized and the supralinear behaviour disappeared. The antagonism between the effects of IBMX and the effects of background illumination on the kinetics of photoresponse suggests that phosphodiesterase activity controls the time course of light response in vertebrate rods.

Direct regulation of the muscle-identity gene apterous by a Hox protein in the somatic mesoderm.

Hox genes control segment identity in the mesoderm as well as in other tissues. Most evidence indicates that Hox genes act cell-autonomously in muscle development, although this remains a controversial issue. We show that apterous expression in the somatic mesoderm is under direct Hox control. We have identified a small enhancer element of apterous (apME680) that regulates reporter gene expression in the LT1-4 muscle progenitors. We show that the product of the Hox gene Antennapedia is present in the somatic mesoderm of the second and third thoracic segments. Through complementary alterations in the Antennapedia protein and in its binding sites on apME680, we show that Antennapedia positively regulates apterous in a direct manner, demonstrating unambiguously its cell-autonomous role in muscle development. Finally, we determine that LT1-4 muscles contain more nuclei in the thorax than in the abdomen and we propose that one of the segmental differences under Hox control is the number of myoblasts allocated to the formation of specific muscles in different segments.

The giant gene of Drosophila encodes a b-ZIP DNA-binding protein that regulates the expression of other segmentation gap genes.

The sequence of a cDNA from the giant gene of Drosophila shows that its product has a basic domain followed by a leucine zipper motif. Both features contain characteristic conserved elements of the b-ZIP family of DNA-binding proteins. Expression of the gene in bacteria or by in vitro translation yields a protein that migrates considerably faster than the protein extracted from Drosophila embryos. Treatment with phosphatase shows that this difference is due to multiple phosphorylation of the giant protein in the embryo. Ectopic expression of the protein in precellular blastoderm embryos produces abnormal phenotypes with a pattern of segment loss closely resembling that of Krüppel mutant embryos. Immunological staining shows that giant, ectopically expressed from the hsp70 promoter, represses the expression of both the Krüppel and knirps segmentation gap genes. The analysis of the interactions between Krüppel, knirps and giant reveals a network of negative regulation. We show that the apparent positive regulation of knirps by Krüppel is in fact mediated by a negative effect of Krüppel on giant and a negative effect of giant on knirps. giant protein made in bacteria or in embryos binds in vitro to the Krüppel regulatory elements CD1 and CD2 and recognizes a sequence resembling the binding sites of other b-ZIP proteins.

Voltage gain of signal transfer from retinal rods to bipolar cells in the tiger salamander.

1. Intracellular recordings of the voltage responses of rods and both functional classes of bipolar cell were made in the isolated, perfused retina of the tiger salamander, Ambystoma tigrinum. 2. Brief, dim flashes of 519 nm light delivered to the receptive-field centres were used to measure the flash sensitivities of twenty-one on-centre bipolar cells and thirty-six off-centre cells. In each experiment the flash sensitivity of a rod was also measured using diffuse illumination of the same duration and wave-length. 3. The mean flash sensitivity of the rods (fifty-nine cells) was 4.47 mV photon-1 micron 2 flash. The mean flash sensitivity of the off-centre bipolar cells was 35.4 mV photon-1 micron 2 flash (thirty-six cells). The mean flash sensitivity of the on-centre bipolar cells was 12.5 mV photon-1 micron 2 flash. 4. The ratio of the flash sensitivity of the bipolar cell to that of a rod recorded in the same retina defined the gain of voltage transfer from rod to bipolar cell. For signal transfer to on-centre bipolar cells the mean value of the voltage gain was 5.05 +/- 1.34 (S.E. of mean). For signal transfer to the off-centre bipolar cells, the mean value of the gain was 10.4 +/- 1.29. 5. The on-centre cell gain in the salamander was smaller by a factor of 27 than that of the on-centre cells in the dogfish retina (Ashmore & Falk, 1980 a), while the off-centre cell gain was comparable in the two species. Possible reasons for the large difference between the voltage gains of on-centre cells in the dogfish and salamander are considered.

Fate of a volatile chlorinated solvent in indoor aquatic microcosms: sublethal and static exposure to [14C]dichloromethane. Groupe pour l'Etude du Devenir de Xénobiotiques dans l'Environnement (GEDEXE).

The goal of this work was to study the fate of dichloromethane in indoor aquatic microcosms after a sublethal and static exposure to simulate the accidental contamination of a lenitic ecosystem such as littoral lake zone or a pond. This kind of ecosystem is characterized by high productive capacity and rich biocoenose, and are usually first affected by acute or chronic pollution. Microcosms containing immersed bryophytes (Fontinalis antipyretica), macrophytes (Lemna minor, Groenlendia densa, Elodea canadensis), molluscs (Physa fontinalis), crustaceans (Daphnia magna), and unicellular green algae (Scenedesmus subspicatus) were contaminated with sublethal concentrations of dichloromethane or [14C]dichloromethane. The initial mean concentration was 9.9 +/- 3.7 microM. The mean concentration exposure for organisms was 4.5 +/- 1.5 microM. The fate of 14C radioactivity was monitored by measuring the radioactivity of the sediment, water, macro- and microorganism, and atmospheric compartments. Radioactivity in the water disappeared quickly from the microcosms, most likely as [14C]dichloromethane (t1/2 = 5.31 +/- 0.41 days). At the end of the experiments, radioactivity was mainly located in the atmosphere, with traces remaining in the biomass. Under static conditions, the bioaccumulation of 14C radioactivity from the radiolabeled dichloromethane was negligible.

Ionic movements through light-sensitive channels of toad rods.

Electrical photoresponses of rods in the isolated toad retina were recorded during ionic manipulations of the Na+-free extracellular medium. In the presence of a concentration of external Ca2+ above 10(-5) M, voltage photoresponses were observed only in the presence of external Na+ or Li+. When external Ca2+ was reduced below 10(-6) M, voltage photoresponses of normal polarity could be detected even in the absence of Na+ or Li+, but in the presence of external Mg2+. In the presence of normal extracellular Ca2+ hyperpolarizing photoresponses were observed even in the absence of Na+ or Li+, provided small amounts of phosphodiesterase inhibitors (IBMX, RO 20-1724, papaverine, caffeine, theophylline) were added to the perfusate. Responses obtained in low-Na+ IBMX solutions required the presence of millimolar amounts of a variety of divalent cations, among which Mn2+ and Ba2+ were the most effective. When the concentration of both external Ca2+ and Mg2+ was reduced to micromolar amounts, depolarizing photoresponses were observed. In these conditions measurements with radioactive tracers showed a light-modulated efflux of 42K+ or 86Rb+. The light-modulated 42K+ or 86Rb+ efflux was halved by 2 X 6 mM-external K+ and was completely blocked when K+ was raised above 10 mM. These results show that ionic movements through light-sensitive channels are controlled by Ca2+ and Mg2+ and possibly also be the intracellular level of cyclic nucleotides. Moreover, the movement of ions through the light-sensitive channel, does not obey the independence principle.

The sodium current underlying the responses of toad rods to light.

1. Intracellular responses were recorded from single rods in the retina of the toads Bufo bufo and Bufo marinus during exposure to solutions in which sodium was replaced by equimolar amounts of choline. 2. Upon moderate reduction (80 and 50 mM) of the external sodium the size of responses to bright flashes decreased as a consequence of both an increase in the resting potential and a decrease of the membrane potential at the peak, while the level of the plateau remained fairly constant. 3. Upon reduction of the external sodium to 22 mM or less, rods hyperpolarized to about the plateau level and failed to respond to illumination. Under these circumstances, membrane depolarization induced by an increased external potassium did not restore the cell responsiveness. Addition of 2-5 mM caesium hyperpolarized the membrane and partially restored the photoresponse. 4. Complete replacements of external sodium with potassium depolarized the rod by 40 +/- 10 mV, and no voltage responses to light could be detected. 5. In the presence of caesium, a nearly complete blockage of the photoresponses was obtained when the external sodium was 5 mM or less. Further reductions of the external sodium did not invert the photoresponses. Application of caesium when the external sodium was nominally zero induced a transient hyperpolarization followed by a slow decay. 6. During exposure to steady illumination, the dependence of the plateau level on the external sodium slowly increased. 7. These results indicate that the ionic current which is directly modulated by the light depends primarily on the external sodium. They suggest also that the current associated with the voltage- and time-dependent process responsible for the sag from peak to plateau of the response to a bright flash of light may have multiple components.

Rod photoresponses in the absence of external sodium in retinae treated with phosphodiesterase inhibitors.

Exposure of isolated toad retinae to phosphodiesterase inhibitors, induced changes in the ionic permeability of rod cells. Under similar conditions intracellularly recorded light responses were observed also in the absence of external Na+. Hyperpolarizing photoresponses in Na+-free media required the presence of divalent cations among which Mg2+, Mn2+ and Ba2+ were the most effective.

Constitutive expression of a complement-like protein in toll and JAK gain-of-function mutants of Drosophila.

We show that Drosophila expresses four genes encoding proteins with significant similarities with the thiolester-containing proteins of the complement C3/alpha(2)-macroglobulin superfamily. The genes are transcribed at a low level during all stages of development, and their expression is markedly up-regulated after an immune challenge. For one of these genes, which is predominantly expressed in the larval fat body, we observe a constitutive expression in gain-of-function mutants of the Janus kinase (JAK) hop and a reduced inducibility in loss-of-function hop mutants. We also observe a constitutive expression in gain-of-function Toll mutants. We discuss the possible roles of these novel complement-like proteins in the Drosophila host defense.

The DNA binding specificity of Ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein.

The Ultrabithorax (Ubx) and Antennapedia (Antp) genes of Drosophila encode homeodomain proteins that have very similar DNA binding specificities in vitro but specify the development of different segmental patterns in vivo. We describe cooperative interactions between Ubx protein and a divergent homeodomain protein, extradenticle (exd), that selectively increases the affinity of Ubx, but not Antp, for a particular DNA target. We also provide evidence that Ubx and exd bind to neighboring sites on this DNA and interact directly to stabilize the DNA-bound form of Ubx. Thus, the ability of different homeotic genes to specify distinct segmental patterns may depend on cooperative interactions with proteins such as exd that selectively modulate their otherwise similar DNA binding specificities.

Tissue-specific inducible expression of antimicrobial peptide genes in Drosophila surface epithelia.

The production of antimicrobial peptides is an important aspect of host defense in multicellular organisms. In Drosophila, seven antimicrobial peptides with different spectra of activities are synthesized by the fat body during the immune response and secreted into the hemolymph. Using GFP reporter transgenes, we show here that all seven Drosophila antimicrobial peptides can be induced in surface epithelia in a tissue-specific manner. The imd gene plays a critical role in the activation of this local response to infection. In particular, drosomycin expression, which is regulated by the Toll pathway during the systemic response, is regulated by imd in the respiratory tract, thus demonstrating the existence of distinct regulatory mechanisms for local and systemic induction of antimicrobial peptide genes in Drosophila.

Identification of genes that modify ataxin-1-induced neurodegeneration.

A growing number of human neurodegenerative diseases result from the expansion of a glutamine repeat in the protein that causes the disease. Spinocerebellar ataxia type 1 (SCA1) is one such disease-caused by expansion of a polyglutamine tract in the protein ataxin-1. To elucidate the genetic pathways and molecular mechanisms underlying neuronal degeneration in this group of diseases, we have created a model system for SCA1 by expressing the full-length human SCA1 gene in Drosophila. Here we show that high levels of wild-type ataxin-1 can cause degenerative phenotypes similar to those caused by the expanded protein. We conducted genetic screens to identify genes that modify SCA1-induced neurodegeneration. Several modifiers highlight the role of protein folding and protein clearance in the development of SCA1. Furthermore, new mechanisms of polyglutamine pathogenesis were revealed by the discovery of modifiers that are involved in RNA processing, transcriptional regulation and cellular detoxification. These findings may be relevant to the treatment of polyglutamine diseases and, perhaps, to other neurodegenerative diseases, such as Alzheimer's and Parkinson's disease.