The involvement of T lymphocytes in the pathogenesis of endometriotic tissues overgrowth in women with endometriosis

BACKGROUND: Endometriosis, uncontrolled proliferation of ectopic and eutopic endometriotic tissues, is common in women at reproductive age, and may affect fertility. The role of macrophages in the pathogenesis is well proved, but engagement of T cells in the pathogenesis of endometriosis is a matter of controversy. AIMS: T-cell involvement in the pathogenesis of endometriosis was the objective of our study performed on women aged 24-46 years with diagnosed endometriosis. All the patients that were studied underwent diagnostic laparoscopy. METHODS: We evaluated the distribution of T-lymphocyte subpopulations in peripheral blood (PB), peritoneal fluid (PF) and in endometriotic tissues (ET), as well as cytokines [interleukin (IL)-2, IL-4, IL-10, IL-12, interferon (IFN)-gamma] production by peripheral blood lymphocytes. IFN-gamma, tumor necrosis factor (TNF)-alpha, IL-4 and IL-6 were investigated for their intracellular presence. The experiments were carried out before and after 6 months treatment with the GnRH-Analogous Goserelin (Zeneca Pharmaceuticals). The number of performed investigations is presented. Statistical analysis was performed using Statistica/Win 5.0 software and Student''s t-test, the paired Student t-test and Fisher''s exact test when appropriate. RESULTS: We have compared the lymphocyte subset re-distribution with regard to the American Fertility Society (AFS) stages and scores, but no differences were observed. The significant increase in CD4:CD8 ratio, the decrease in the number of natural killer (NK) cells in PB and the decrease in CD4:CD8 ratio in PF and ET of women with endometriosis was noted. The diminished IFN-gamma secretion by phytohemagglutinim (PHA)-stimulated lymphocytes in vitro derived from women with endometriosis and increased IL-4 production may be responsible for defective immunosurveillance against overgrowth of endometriotic tissues. The diminished NK cells number in PB of women with endometriosis argues for such a hypothesis. The increased deposits of proinflammatory IL-6 and TNF-alpha in the T lymphocytes of women with endometriosis may be related to T-regulatory lymphocyte function and their inability to suppress cell proliferation in endometriosis. GnRH-Analogous Goserelin treatment normalises cytokine production and induces patient recovery. CONCLUSIONS: The significant functional and phenotypic differences between the lymphocytes from healthy women and women with endometriosis were noted. The diminished IFN-gamma production in relation to decreased NK cells number and the increased IL-4 production before the treatment and normalisation after the treatment suggest the involvement of the deregulated T-cell system in the growth stimulation and recruitment of endometriotic cells. The increased CD4:CD8 ratio, IL-6, TNF-alpha deposits and diminished anti-inflammatory IL-10 production by lymphocytes may participate in the pathogenesis of endometriosis, and may secondarily affect the monocyte/macrophage function.     

Intra-Abdominal Pressure Correlates with Extracellular Water Content

Background

Secondary increase in intra-abdominal pressure (IAP) may result from extra-abdominal pathology, such as massive fluid resuscitation, capillary leak or sepsis. All these conditions increase the extravascular water content. The aim of this study was to analyze the relationship between IAP and body water volume.

Material and Methods

Adult patients treated for sepsis or septic shock with acute kidney injury (AKI) and patients undergoing elective pharyngolaryngeal or orthopedic surgery were enrolled. IAP was measured in the urinary bladder. Total body water (TBW), extracellular water content (ECW) and volume excess (VE) were measured by whole body bioimpedance. Among critically ill patients, all parameters were analyzed over three consecutive days, and parameters were evaluated perioperatively in surgical patients.

Results

One hundred twenty patients were studied. Taken together, the correlations between IAP and VE, TBW, and ECW were measured at 408 time points. In all participants, IAP strongly correlated with ECW and VE. In critically ill patients, IAP correlated with ECW and VE. In surgical patients, IAP correlated with ECW and TBW. IAP strongly correlated with ECW and VE in the mixed population. IAP also correlated with VE in critically ill patients. ROC curve analysis showed that ECW and VE might be discriminative parameters of risk for increased IAP.

Conclusion

IAP strongly correlates with ECW.     

Recruitment and abundance of an invasive fish,the common carp,is driven by its propensity to invade and reproduce in basins that experience winter-time hypoxia in interconnected lakes

Although the common carp is globally distributed, it only reaches extreme densities in certain regions. We hypothesized that this phenomenon might be linked to recruitment bottlenecks which carp overcome where environmental conditions create unstable peripheral areas that it can access for spawning and nursery habitat. To test this hypothesis, the abundance, movement and reproductive success of carp was determined in two systems of inter-connected lakes in the North American Midwest whose shallow basins frequently experience winter-hypoxia (‘winterkill’). Radio-tracking demonstrated that while adult carp overwinter in deep lakes that do not winterkill, they aggressively move into winterkill-prone shallow regions in the spring to spawn. The significance of this behavior was demonstrated by ageing analyses which found that carp recruit only in interconnected shallow lakes and then only in years following severe winter hypoxia. Presumably this strategy allows carp to exploit nursery habitat that is relatively free of predators. It likely evolved in response to seasonally variable conditions in the carp’s native habitat in the Ponto-Caspian region. This life history may also explain the carp’s abundance in other unstable regions such as southern Australia and could potentially be exploited to control this damaging invasive.     

Impact of functionalized coligands on the pharmacokinetics of 99mTcIII '4+1' mixed-ligand complexes conjugated to bombesin

Bombesins (BN) containing ^99mTc ‘4 + 1’ complexes may be useful to detect tumors expressing the gastrin-releasing peptide receptor (GRPR). Derivatives of the formula [^99mTc(NS₃R)(L2-BN_st)] were synthesized, in which Tc(III) is coordinated by an isocyanide L2-BN_st bearing the peptide (BN_st = βAla-βAla-Gln-Trp-Ala-Val-Gly-His-Cha-Nle-NH₂) and a tetradentate chelator NS₃R. NS₃R consists of 2,2′,2″-nitrilotriethanethiol (NS₃) bearing a crown ether (NS₃crown), an aliphatic amine (NS₃en) and a tricarboxylic acid (NS₃(COOH)₃). Non-radioactive Re compounds were prepared and analysed by electrospray ionization mass spectrometry. The structural similarity to the ^99mTc conjugates was demonstrated by their identical HPLC elution profiles. The lipophilicity of [^99mTc(NS₃R)(L2-BN_st)] decreased depending on the coligands NS₃crown (log D_O/W, pH = 7.4, 0.98 ± 0.11), NS₃en (− 0.49 ± 0.07) and NS₃(COOH)₃ (− 2.01 ± 0.09). Biodistribution in normal rats was characterized by an increasing kidney uptake and a decreasing uptake into the liver corresponding to the reduced lipophilicity of the conjugates. The pancreatic uptake expressed by the organ/blood ratio of standardized uptake values at 60 min p.i. in rats was 8.6 ± 1.2 for [^99mTc(NS₃en)(L2-BN_st)] and higher compared to the other conjugates. The pancreas/liver ratio of the SUV at 60 min p.i. in rats was highest for [^99mTc(NS₃(COOH)₃)(L2-BN_st)] at 8.4 ± 1.3. [^99mTc(NS₃en)(L2-BN_st)] was further studied in tumor-bearing mice and its pancreas/blood and pancreas/liver ratios were lower, however the pancreas/kidney ratios were higher in mice compared to rats. The activity uptake of [^99mTc(NS₃en)(L2-BN_st)] into the PC-3 tumor xenografts was low (%ID/g: 0.83 ± 0.18 at 60 min; SUV: 0.21 ± 0.05 at 60 min) but specific.     

Entropy versus aromaticity in the conformational dynamics of aromatic rings

Comparison of the results of Car-Parrinello molecular dynamics simulations of isolated benzene, pyrimidine and 1,2,4-triazine molecules reveals that the unusually low population of planar geometry of the benzene ring is caused by entropy effects despite its high aromaticity. The decrease in symmetry of the molecule results in smaller changes in entropy and Gibbs free energy due to out-of-plane deformations of the ring, leading to an increase in the population of planar geometry of the ring. This leads to differences in the topology of potential energy and Gibbs free energy surfaces.

Biochemical and Structural Characterization of SplD Protease from Staphylococcus aureus

Staphylococcus aureus is a dangerous human pathogen. A number of the proteins secreted by this bacterium are implicated in its virulence, but many of the components of its secretome are poorly characterized. Strains of S. aureus can produce up to six homologous extracellular serine proteases grouped in a single spl operon. Although the SplA, SplB, and SplC proteases have been thoroughly characterized, the properties of the other three enzymes have not yet been investigated. Here, we describe the biochemical and structural characteristics of the SplD protease. The active enzyme was produced in an Escherichia coli recombinant system and purified to homogeneity. P1 substrate specificity was determined using a combinatorial library of synthetic peptide substrates showing exclusive preference for threonine, serine, leucine, isoleucine, alanine, and valine. To further determine the specificity of SplD, we used high-throughput synthetic peptide and cell surface protein display methods. The results not only confirmed SplD preference for a P1 residue, but also provided insight into the specificity of individual primed- and non-primed substrate-binding subsites. The analyses revealed a surprisingly narrow specificity of the protease, which recognized five consecutive residues (P4-P3-P2-P1-P1’) with a consensus motif of R-(Y/W)-(P/L)-(T/L/I/V)↓S. To understand the molecular basis of the strict substrate specificity, we crystallized the enzyme in two different conditions, and refined the structures at resolutions of 1.56 Å and 2.1 Å. Molecular modeling and mutagenesis studies allowed us to define a consensus model of substrate binding, and illustrated the molecular mechanism of protease specificity.     

Insights into eukaryotic Rubisco assembly — Crystal structures of RbcX chaperones from Arabidopsis thaliana

Background

Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6 billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures.

Methods

Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX–RbcL was predicted using molecular dynamic simulation.

Results

We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence.

General significance

The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.