Safety and efficacy of the anti-PD1 immunotherapy with nivolumab in trichoblastic carcinomas.

Trichoblastic carcinoma is a rare malignant cutaneous adnexal tumor with a risk of local invasion and distant metastasis. As of today, there is no consensus for the treatment of locally advanced or metastatic trichoblastic carcinoma. "AcSé Nivolumab" is a multi-center Phase II basket clinical trial (NCT03012581) evaluating the safety and efficacy of nivolumab in several cohorts of rare, advanced cancers. Here we report the results of nivolumab in patients with trichoblastic carcinoma. Of the eleven patients enrolled in the study, five patients had been previously treated by sonic hedgehog inhibitors. The primary endpoint 12-week objective response rate was 9.1% (N = 1/11) with 1 partial response. Six patients who progressed under previous lines of treatment showed stable disease at 12 weeks, reflecting a good control of the disease with nivolumab. Furthermore, 54.5% of the patients (N = 6/11) had their disease under control at 6 months. The 1-year overall survival was 80%, and the median progression-free survival was 8.4 months (95%CI, 5.7 to NA). With 2 responders (2 complete responses), the best response rate to nivolumab at any time was 18.2% (95%CI, 2.3-51.8%). No new safety signals were identified, and adverse events observed herein were previously described and well known with nivolumab monotherapy. These results are promising, suggesting that nivolumab might be an option for patients with advanced trichoblastic carcinomas. Further studies on larger cohorts are necessary to confirm these results and define the role of nivolumab in the treatment of trichoblastic carcinomas.

Assessment of two glycated hemoglobin immunoassays.

INTRODUCTION: Glycated hemoglobin (HbA1c) level reflects chronic glycemic status if reliable tests are used, however, in some regions worldwide high performing assays might not be readily available. This study aimed to asses two HbA1c immunoassays, comparing them with high-performance liquid chromatography (HPLC) assay, three methods available in Ecuador. MATERIAL AND METHODS: HbA1c were measured in 114 fresh whole blood-samples by DCA-Vantage point-of-care analyzer, I-Chroma portable fluorescent scanner immunoassay and BioRad Variant II Turbo HPLC. Normal and pathological HbA1c ranges were included. Blood samples with variants of hemoglobin were excluded. HbA1c values were expressed in National Glycohemoglobin Standardization Program percentages and mmol/mol, as mean±standard deviation. RESULTS: HbA1c results by HPLC and DCA-Vantage were similar: 6.3±1.7% (45±18.6mmol/mol) vs. 6.3±1.8% (45±19.7mmol/mol), respectively, P=0.057; while HbA1c values by I-Chroma were lower than HPLC, 5.8±1.9% (40±20.8mmol/mol), P<0.001. The coefficient of variation was below 2% for high and low HbA1c levels, in all methods studied. HbA1c values by HPLC and DCA-Vantage were highly correlated (Spearman's Rank Correlation [SRC]: 0.916), while the correlation among HPLC and I-Chroma was weak (SRC: 0.368). The mean bias between DCA-Vantage and HPLC was -0.02±0.29% (-0.2±3.2mmol/mol), while for I-Chroma and HPLC mean bias was -0.50±1.62% (-5.5±17.7mmol/mol). CONCLUSION: HbA1c immunoassays DCA-Vantage was comparable to HPLC assay, showing good correlation, appropriate precision and low bias, whereas I-Chroma assay was precise but inaccurate. Therefore, DCA-Vantage has better performance than I-Chroma. These findings suggest that is convenient to assess the HbA1c immunoassays commercially available in our country, Ecuador.

Milk yield loss in response to feed restriction is associated with mammary epithelial cell exfoliation in dairy cows.

In dairy cows, feed restriction is known to decrease milk yield by reducing the number of mammary epithelial cells (MEC) in the udder through a shift in the MEC proliferation-apoptosis balance, by reducing the metabolic activity of MEC, or both. The exfoliation of MEC from the mammary epithelium into milk is another process that may participate in regulating the number of MEC during feed restriction. The aim of the present study was to clarify the mechanisms that underlie the milk yield loss induced by feed restriction. Nineteen Holstein dairy cows producing 40.0 ± 0.7 kg/d at 77 ± 5 d in milk were divided into a control group (n = 9) and a feed-restricted group (n = 10). Ad libitum dry matter intake (DMI) was recorded during a pre-experimental period of 2 wk. For 29 d (period 1), cows were fed either 100 (control) or 80% (feed-restricted) of their ad libitum DMI measured during the pre-experimental period. Then, all cows were fed ad libitum for 35 d (period 2). Milk production and DMI were recorded daily. Blood and milk samples were collected once during the pre-experimental period; on d 5, 9, and 27 of period 1; and on d 5, 9, and 30 of period 2. Mammary epithelial cells were purified from milk using an immunomagnetic method to determine the rate of MEC exfoliation. Mammary tissue samples were collected by biopsy at the end of each period to analyze the rates of cell proliferation and apoptosis and the expression of genes involved in synthesizing constituents of milk. Feed restriction decreased milk yield by 3 kg/d but had no effect on rates of proliferation and apoptosis in the mammary tissue or on the expression of genes involved in milk synthesis. The daily MEC exfoliation rate was 65% greater in feed-restricted cows than in control cows. These effects in feed-restricted cows were associated with reduced insulin-like growth factor-1 and cortisol plasma concentrations. When all cows returned to ad libitum feeding, no significant difference on milk yield or MEC exfoliation rate was observed between feed-restricted and control cows, but refeeding increased prolactin release during milking. These results show that the exfoliation process may play a role in regulating the number of MEC in the udders of dairy cows during feed restriction without any carryover effect on their milk production.

Methods for orientation and phase identification of nano-sized embedded secondary phase particles by 4D scanning precession electron diffraction.

The diffraction patterns acquired with transmission electron microscopes gather reflections from all crystallites that overlap in the foil thickness. The superimposition renders automated orientation or phase mapping difficult, in particular when secondary phase particles are embedded in a dominant diffracting matrix. Several numerical approaches specifically developed to overcome this issue for 4D scanning precession electron diffraction data sets are described. They consist either in emphasizing the signature of the particles or in subtracting the matrix information out of the collected set of patterns. The different strategies are applied successively to a steel sample containing precipitates that are in Burgers orientation relationship with the matrix and to an aluminium alloy with randomly oriented Mn-rich particles.

Ultrafine heat-induced structural perturbations of bone mineral at the individual nanocrystal level.

The nanoscale characteristics of the mineral phase in bone tissue such as nanocrystal size, organization, structure and composition have been identified as potential markers of bone quality. However, such characterization remains challenging since it requires combining structural analysis and imaging modalities with nanoscale precision. In this paper, we report the first application of automated crystal orientation mapping using transmission electron microscopy (ACOM-TEM) to the structural analysis of bone mineral at the individual nanocrystal level. By controlling the nanocrystal growth of a cortical bovine bone model artificially heated up to 1000 °C, we highlight the potential of this technique. We thus show that the combination of sample mapping by scanning and the crystallographic information derived from the collected electron diffraction patterns provides a more rigorous analysis of the mineral nanostructure than standard TEM. In particular, we demonstrate that nanocrystal orientation maps yield valuable information for dimensional analysis. Furthermore, we show that ACOM-TEM has sufficient sensitivity to distinguish between phases with close crystal structures and we address unresolved questions regarding the existence of a hexagonal to monoclinic phase transition induced by heating. This first study therefore opens new perspectives in bone characterization at the nanoscale, a daunting challenge in the biomedical and archaeological fields, which could also prove particularly useful to study the mineral characteristics of tissue grown at the interface with biomaterials implants. STATEMENT OF SIGNIFICANCE: In this paper, we propose a new approach to assess the mineral properties of bone at the individual nanocrystal level, a major challenge for decades. We use a modified Transmission Electron Microscopy acquisition mode to perform an Automated Crystal Orientation Mapping (ACOM-TEM) by analyzing electron diffraction patterns. We tune the mineral nanocrystal size by heating a model bovine bone system and show that this method allows precisely assessing the mineral nanocrystal size, orientation and crystallographic phase. ACOM-TEM therefore has sufficient sensitivity to solve problems that couldn't be answered using X-ray diffraction. We thus revisit the fine mechanisms of bone nanocrystal growth upon heating, a process currently used for bone graft manufacturing, also of practical interest for forensic science and archaeology.

Orientation and phase mapping in the transmission electron microscope using precession-assisted diffraction spot recognition: state-of-the-art results.

A recently developed technique based on the transmission electron microscope, which makes use of electron beam precession together with spot diffraction pattern recognition now offers the possibility to acquire reliable orientation/phase maps with a spatial resolution down to 2 nm on a field emission gun transmission electron microscope. The technique may be described as precession-assisted crystal orientation mapping in the transmission electron microscope, precession-assisted crystal orientation mapping technique-transmission electron microscope, also known by its product name, ASTAR, and consists in scanning the precessed electron beam in nanoprobe mode over the specimen area, thus producing a collection of precession electron diffraction spot patterns, to be thereafter indexed automatically through template matching. We present a review on several application examples relative to the characterization of microstructure/microtexture of nanocrystalline metals, ceramics, nanoparticles, minerals and organics. The strengths and limitations of the technique are also discussed using several application examples.

NEMO specifically recognizes K63-linked poly-ubiquitin chains through a new bipartite ubiquitin-binding domain.

An important property of NEMO, the core element of the IKK complex involved in NF-kappaB activation, resides in its ability to specifically recognize poly-ubiquitin chains. A small domain called NOA/UBAN has been suggested to be responsible for this property. We recently demonstrated that the C-terminal Zinc Finger (ZF) of NEMO is also able to bind ubiquitin. We show here by ZF swapping and mutagenesis that this represents its only function. While neither NOA nor ZF shows any preference for K63-linked chains, we demonstrate that together they form a bipartite high-affinity K63-specific ubiquitin-binding domain. A similar domain can be found in two other proteins, Optineurin and ABIN2, and can be freely exchanged with that of NEMO without interfering with its activity. This suggests that the main function of the C-terminal half of NEMO is to specifically bind K63-linked poly-ubiquitin chains. We also demonstrate that the recently described binding of NEMO to linear poly-ubiquitin chains is dependent on the NOA alone and does not require the presence of the ZF.

Acyclic phosphonate nucleotides and human adenylate kinases: impact of a borano group on alpha-P position.

Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The alpha-borano or alpha-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the alpha-phosphate, resulting in substrate bad positioning.

Inhibition of NEMO, the regulatory subunit of the IKK complex, induces apoptosis in high-risk myelodysplastic syndrome and acute myeloid leukemia.

In high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML), blasts constitutively activate the antiapoptotic transcription factor nuclear factor-kappaB (NF-kappaB). Here, we show that this NF-kappaB activation relies on the constitutive activation of the IkappaB kinase (IKK) complex, which is formed by the IKKalpha, IKKbeta and IKKgamma/NF-kappaB essential modulator (NEMO) subunits. A cell-permeable peptide that mimics the leucine zipper subdomain of IKKgamma, thus preventing its oligomerization, inhibited the constitutive NF-kappaB activation and induced apoptotic cell death in a panel of human MDS and AML cell lines (P39, MOLM13, THP1 and MV4-11). Small interfering RNA-mediated knockdown of the p65 NF-kappaB subunit or the three IKK subunits including IKKgamma/NEMO also induced apoptotic cell death in P39 cells. Cell death induced by the IKKgamma/NEMO-antagonistic peptide involved the caspase-independent loss of the mitochondrial transmembrane potential as well as signs of outer mitochondrial membrane permeabilization with the consequent release of cytochrome c, apoptosis-inducing factor and endonuclease G. Primary bone marrow CD34(+) cells from high-risk MDS and AML patients also succumbed to the IKKgamma/NEMO-antagonistic peptide, but not to a mutated control peptide. Altogether, these data indicate that malignant cells in high-risk MDS and AML cells critically depend on IKKgamma/NEMO to survive. Moreover, our data delineate a novel procedure for their therapeutic removal, through inhibition of IKKgamma/NEMO oligomerization.

Natural variation can significantly alter the sensitivity of influenza A (H5N1) viruses to oseltamivir.

Geographic spread of highly pathogenic avian H5N1 influenza viruses may give rise to an influenza pandemic. During the first months of a pandemic, control measures would rely mainly on antiviral drugs, such as the neuraminidase (NA) inhibitors oseltamivir and zanamivir. In this study, we compare the sensitivities to oseltamivir of the NAs of several highly pathogenic H5N1 viruses isolated in Asia from 1997 to 2005. The corresponding 50% inhibitory concentrations were determined using a standard in vitro NA inhibition assay. The K(m) for the substrate and the affinity for the inhibitor (K(i)) of NA were determined for a 1997 and a 2005 virus, using an NA inhibition assay on cells transiently expressing the viral enzyme. Our data show that the sensitivities of the NAs of H5N1 viruses isolated in 2004 and 2005 to oseltamivir are about 10-fold higher than those of earlier H5N1 viruses or currently circulating H1N1 viruses. Three-dimensional modeling of the N1 protein predicted that Glu248Gly and Tyr252His changes could account for increased sensitivity. Our data indicate that genetic variation in the absence of any drug-selective pressure may result in significant variations in sensitivity to anti-NA drugs. Although the clinical relevance of a 10-fold increase in the sensitivity of NA to oseltamivir needs to be investigated further, the possibility that sensitivity to anti-NA drugs could increase (or possibly decrease) significantly, even in the absence of treatment, underscores the need for continuous evaluation of the impact of genetic drift on this parameter, especially for influenza viruses with pandemic potential.

Chagas disease in bone marrow transplantation: an approach to preemptive therapy.

The efficacy of preemptive therapy was evaluated in bone marrow transplantation (BMT) recipients associated with Chagas disease (CD). The criterion to include patients in the protocol was the serological reactivity for CD in recipients and/or donors before transplant. After BMT, the monitoring was performed using the direct Strout method (SM), which detects clinical levels of Trypanosome cruzi parasitemia, and CD conventional serological tests. Monitoring took place during 60 days in ABMT and throughout the immunosuppressive period in allogeneic BMT. Reactivation of CD was diagnosed by detecting T. cruzi parasites in blood or tissues. In primary T. cruzi infection, an additional diagnostic criterion was the serological conversion. A total of 25 CD-BMT patients were included. Two ABMT and four allogeneic BMT recipients showed CD recurrences diagnosed by SM. One patient also showed skin lesions with T. cruzi amastigotes. Benznidazole treatment (Roche Lab), an antiparasitic drug, was prescribed at a dose of 5 mg/kg/day during 4-8 weeks with recovery of patients. Primary T. cruzi infection was not observed. This report proves the relevance of monitoring CD in BMT patients and demonstrates that preemptive therapy was able to abrogate the development of clinical and systemic disease.

Adenosine phosphonoacetic acid is slowly metabolized by NDP kinase.

NDP kinase catalyzes the last step in the phosphorylation of nucleotides. It is also involved in the activation by cellular kinases of nucleoside analogs used in antiviral therapies. Adenosine phosphonoacetic acid, a close analog of ADP already proposed as an inhibitor of ribonucleotide reductase, was found to be a poor substrate for human NDP kinase, as well as a weak inhibitor with an equilibrium dissociation constant of 0.6 mM to be compared to 0.025 mM for ADP. The X-ray structure of a complex of adenosine phosphonoacetic acid and the NDP kinase from Dictyostelium was determined to 2.0 A resolution showing that the analog adopts a binding mode similar to ADP, but that no magnesium ion is present at the active site. As ACP may also interfere with other cellular kinases, its potential as a drug targeting NDP kinase or ribonucleotide reductase is likely to be limited due to strong side effects. The design of new molecules with a narrower specificity and a stronger affinity will benefit from the detailed knowledge of the complex ACP-NDP kinase.

Interactions of tachykinin receptor antagonists with lipopolysaccharide-induced airway inflammation in mice.

1. Several observations suggest that tachykinins are involved in the pathogenesis of bronchopulmonary alterations. We have investigated the effect of antagonists for tachykinin NK1 (SR 140333), NK2 (SR 48968) or NK3 (SR 142801) receptors on inflammatory cell recruitment, tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 release and matrix metalloproteinase (MMP)-9 activity in the bronchoalveolar lavage fluid (BALF) of mice exposed to lipopolysaccharide (LPS; 100 microg/mL aerosol for 30 min). 2. Treatment of mice with a combination of SR 140333 and SR 48968 (10(-6) mol/L, aerosol) significantly reduced the increase in the number of total cells and neutrophils and MMP-9 activity in the BALF of mice 2.5 h after LPS exposure. Treatment with the NK3 antagonist SR 142801 (10(-6) mol/L, aerosol) did not inhibit the influx of neutrophils, but markedly reduced the increase in TNF-alpha and IL-6 levels at 2.5 h and MMP-9 activity at 20 h. 3. These results show that the three tachykinin receptor antagonists may interfere with the development of airway inflammation, namely neutrophilia, TNF-alpha release or MMP-9 activity in the BALF of mice exposed to LPS and suggest that not only NK1 and NK2 receptors, but also NK3 receptors are involved in the modulation of the inflammatory response and airway remodelling.

Antiviral nucleoside analogs phosphorylation by nucleoside diphosphate kinase.

The reaction of NDP kinase was studied in vitro with several antiviral derivatives, using kinetic steady state and presteady state analysis. The enzyme is highly efficient with natural nucleotides but most of the analogs are slow substrates. The catalytic efficiency, also related to the affinity of the analog, is mainly dependent on the presence of a 3'-OH group on the ribose moiety.

A scaled quantum mechanical force field for the sulfuryl halides. I. The symmetric halides SO2X2 (X=F, Cl, Br).

Force fields and vibrational wavenumbers were calculated for the molecules SO2X2 (X=F, Cl, Br) using DFT techniques. The previously available experimental data and assignments for SO2F2 and SO2Cl2 were compared with the theoretical results and revised, and new low temperature infrared and Raman data were obtained for SO2Cl2. These data were subsequently used in the definition of scaled quantum mechanics force fields for such molecules. Adjusted wavenumbers were also predicted for the still unknown SO2Br2. A comparison is made with results published for the VO2X2- anions.

X-ray diffraction from rectangular slits.

It is shown that for micrometre-sized beams the X-ray diffraction from slits is a source of strong parasitic background, even for slits of high quality. In order to illustrate this effect, the coherent diffraction from rectangular slits has been studied in detail. A large number of interference fringes with strong visibility have been observed using a single set of slits made of polished cylinders. For very small apertures, asymmetrical slits generate asymmetrical patterns. This pattern is calculated from the theory of electromagnetic field propagation and compared with experiment in the far-field regime. The use of guard slits to remove Fraunhofer diffraction from the beam-defining slits is treated theoretically. Numerical simulations yield the optimum aperture of the guard slits with respect to the distance to the primary slits. Diffraction theory is shown to be essential to understand how to reduce the background-to-signal ratio in high-resolution experiments.

Addition and correction: the NF-kappa B-like DNA binding activity observed in Dictyostelium nuclear extracts is due to the GBF transcription factor.

We have previously reported that a NF-kappa B transduction pathway was likely to be present in the cellular slime mold Dictyostelium discoideum. This conclusion was based on several observations, including the detection of developmentally regulated DNA binding proteins in Dictyostelium nuclear extracts that bound to bona fide kappa B sequences. We have now performed additional experiments which demonstrate that the protein responsible for this NF-kappa B-like DNA binding activity is the Dictyostelium GBF (G box regulatory element binding factor) transcription factor. This result, along with the fact that no sequence with significant similarity to components of the mammalian NF-kappa B pathway can be found in Dictyostelium genome, now almost entirely sequenced, led us to reconsider our previous conclusion on the occurrence of a NF-kappa B signal transduction pathway in Dictyostelium.

Activation of anti-reverse transcriptase nucleotide analogs by nucleoside diphosphate kinase: improvement by alpha-boranophosphate substitution.

Nucleoside activation by nucleoside diphosphate kinase and inhibition of HIV-1 reverse transcriptase were studied comparatively for a new class of nucleoside analogs with a borano (BH3-) or a thio (SH) group on the alpha-phosphate. Both the alpha-Rp-borano derivatives of AZT and d4T improved phosphorylation by NDP kinase, inhibition of reverse transcription as well as stability of alpha-borano nonophosphate derivatives in terminated viral DNA chain.