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.

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.

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.

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.

Peptidic determinants and structural model of human NDP kinase B (Nm23-H2) bound to single-stranded DNA.

Isoform B of human NDP kinase (NDPK-B) was previously identified as a transcription factor stimulating in vitro and ex vivo the transcription of the c-myc oncogene, which involves this enzyme in carcinogenesis. We have studied the enzymatic properties of NDPK-B in the presence of several single-stranded oligonucleotides. We show that the oligonucleotides are competitive inhibitors of the catalytic activity, indicating that the active site acts as a binding template for the anchorage of the oligonucleotide. Furthermore, the presence of a guanine at the 3'-end of several different aptamers increases its affinity 10-fold. To define the surface of the protein contacting the DNA within the nucleoprotein complex, we used single nanosecond laser pulses as the cross-linking reagent and MALDI-TOF mass spectrometry to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Using 11-mer and 30-mer single-stranded oligonucleotides, the same three different nucleopeptides were identified after irradiation of the complexes, indicating a common binding mode for these two aptamers. Taken together, these results allowed us to propose a structural model of NDPK-B bound to single-stranded DNA.

Mechanism of phosphoryl transfer by nucleoside diphosphate kinase pH dependence and role of the active site Lys16 and Tyr56 residues.

Nucleoside diphosphate (NDP) kinase phosphorylates nucleoside diphosphates with little specificity for the base and the sugar. Although nucleotide analogues used in antiviral therapies are also metabolized to their triphosphate form by NDP kinase, their lack of the 3'-hydroxyl of the ribose, which allows them to be DNA chain terminators, severely impairs the catalytic efficiency of NDP kinase. We have analyzed the kinetics parameters of several mutant NDP kinases modified on residues (Lys16, Tyr56, Asn119) interacting with the gamma-phosphate and/or the 3'-OH of the Mg2+-ATP substrate. We compared the relative contributions of the active-site residues and the substrate 3'-OH for point mutations on Lys16, Tyr56 and Asn119. Analysis of additional data from pH profiles identify the ionization state of these residues in the enzyme active form. X-ray structure of K16A mutant NDP kinase shows no detectable rearrangement of the residues of the active site.

A cross-sectional study of the health effects of work schedules on 3212 hospital workers in France: implications for the new French work schedules policy.

This study was designed to investigate the effects of work schedules on the health of hospital workers at the Assistance Publique-Hôpitaux de Paris (AP-HP). Out of 40 hospitals, 17 volunteered to participate in this study. The Standard Shiftwork Index and a questionnaire concerning physicians' work schedules were used. Ten thousand questionnaires were distributed anonymously to hospital workers between March and April 1999. Professional categories comprised head nurses, nurses, nursing auxiliaries, hospital agents, midwives and full time physicians. Departments included internal and geriatric medicine, general paediatrics, orthopaedic and general surgery, operating and emergency rooms, and anaesthesiology and intensive care units. 3250 questionnaires were returned. Demographics for the respondents were: 79.2% female, average age 38.1 +/- 9.1 years old. Eleven work schedules were identified. One fourth of the personnel had fixed morning work schedules. The highest level of job satisfaction was found in personnel working in paediatrics while dissatisfaction was strongest in the gerontology and, emergency room personnel. General Health Questionnaire (GHQ) scores were high for head nurses, operating room nurses and junior doctors as well as for personnel with rotating and flexible shifts. This study will be used to make recommendations concerning the reduction of working time for French hospital workers.

The mechanism of phosphorylation of anti-HIV D4T by nucleoside diphosphate kinase.

The last step in the intracellular activation of antiviral nucleoside analogs is the addition of the third phosphate by nucleoside diphosphate (NDP) kinase resulting in the synthesis of the viral reverse transcriptase substrates. We have previously shown that dideoxynucleotide analogs and 3'-deoxy-3'-azidothymidine (AZT) as di- or triphosphate are poor substrates for NDP kinase. By use of protein fluorescence, we monitor the phosphotransfer between the enzyme and the nucleotide analog. Here, we have studied the reactivity of D4T (2',3'-dideoxy-2',3'-didehydrothymidine; stavudine) as di- (DP) or triphosphate (TP) at the pre-steady state. The catalytic efficiency of D4T-DP or -TP is increased by a factor of 10 compared with AZT-DP or -TP, respectively. We use an inactive mutant of NDP kinase to monitor the binding of a TP derivative, and show that the affinity for D4T-TP is in the same range as for the natural substrate deoxythymidine triphosphate, but is 30 times higher than for AZT-TP. Our results indicate that D4T should be efficiently phosphorylated after intracellular maturation of a prodrug into D4T-monophosphate.

Morphofunctional plasticity in the adult hypothalamus induces regulation of polysialic acid-neural cell adhesion molecule through changing activity and expression levels of polysialyltransferases.

Polysialic acid-neural cell adhesion molecule (PSA-NCAM) expression in the adult nervous system is restricted to regions retaining a capacity for morphological plasticity. For the female rat hypothalamoneurohypophysial system (HNS), we have previously shown that lactation induces a dramatic decrease in PSA-NCAM, while leaving the level of total NCAM protein unchanged. Here, we wanted to elucidate the molecular mechanisms leading to a downregulation of PSA, thereby stabilizing newly established synapses and neurohemal contacts that accompany the increased activity of oxytocinergic neurons. First, we show that the overall specific activity of polysialyltransferases present in tissue extracts from supraoptic nuclei decreases by approximately 50% during lactation. So far, two polysialyltransferase enzymes, STX and PST, have been characterized for their capacity to transfer PSA onto NCAM in vitro. Using a competitive RT-PCR on RNA extracts from the HNS, we demonstrate furthermore a significant decrease in the expression levels of both STX and PST mRNAs in lactating versus virgin animals. Interestingly, this downregulation of NCAM polysialylation is not correlated with the post-transcriptional regulation of variable alternative spliced exon splicing, in contrast to neural development. The control of polysialylation via a regulation of both enzyme activity and expression underlines the important role of this post-translational modification of NCAM in morphofunctional plasticity in adult brain.

Mycoses in the transplanted patient.

The incidence of invasive fungal infection (IFI) has increased considerably over the past 20 years, and transplant recipients are at especially high risk for fungal infections owing to their overall immunosuppressed condition. Organ transplantation procedures were incorporated as a therapeutic option for many patients who lacked the normal functions of organs such as the heart, liver, kidney, lung, pancreas and small bowel. The prevalence of IFI in solid organ transplant (SOTR) patients ranges from 5 to 50% in kidney and liver transplants, respectively. In bone marrow transplant (BMT) patients, IFI are major causes of morbidity and mortality due to the protracted neutropenic period and graft-versus-host disease. Candida spp. and Aspergillus spp. account for >80% of fungal episodes in both SOTR and BMT. The development of new immunosuppressive agents, new prophylaxis strategies (as pre-emptive therapy) and the improvement in surgical techniques led to increase survival of transplant recipients. In this session, a clear and concise update of the recent advances in the laboratory diagnosis of candidiasis and aspergillosis in this kind of patients was presented. However, we still need to establish more rapid, sensitive and specific methods for IFI diagnosis. Representatives of the 'Subcomision de Infecciones en el Paciente Neutropenico y Transplantado (SIPNYT)' de la Sociedad Argentina de Infectologia (SADI), presented the results of an unusual multicenter study both retrospective and descriptive studies of IFI in SOTR and BMT patients in Argentina. In addition, a study of IFI in 1,861 SOTR patients from four centers and the analysis of IFI in 2,066 BMT patients from all 12 BMT centers from Argentina was presented. From these studies it can be concluded that 'all transplant recipients are not the same' and that they should be stratified according to their different risk degrees in order to determine the best prophylaxis and treatment strategies.

The binding mode of human nucleoside diphosphate kinase B to single-strand DNA.

In this paper, we studied the interaction of the human isoform B of nucleoside diphosphate kinase (NDP kinase B) with the nuclease hypersensitive element (NHE) present in the promoter element of the c-myc oncogene. The DNA-binding properties of NDP kinase B and other NDP kinases are compared and the nucleotide requirement for binding are discussed. Using quantitative methods, we identified the DNA-binding sites on the protein and we proposed a structural model for a complex of one hexameric NDP kinase B with an oligonucleotide.

Single strand DNA specificity analysis of human nucleoside diphosphate kinase B.

Nucleoside diphosphate kinases (NDP kinases) form a family of oligomeric enzymes present in all organisms. Eukaryotic NDP kinases are hexamers composed of identical subunits (approximately 17 kDa). A distinctive property of human NDPK-B encoded by the gene nm23-H2 is its ability to stimulate the gene transcription. This property is independent of its catalytic activity and is possibly related to the role of this protein in cellular events including differentiation and tumor metastasis. In this paper, we report the first characterization of human NDPK-B.DNA complex formation using a filter-binding assay and fluorescence spectroscopy. We analyzed the binding of several oligonucleotides mimicking the promoter region of the c-myc oncogene including variants in sequence, structure, and length of both strands. We show that NDPK-B binds to single-stranded oligonucleotides in a nonsequence specific manner, but that it exhibits a poor binding activity to double-stranded oligonucleotides. This indicates that the specificity of recognition to DNA is a function of the structural conformation of DNA rather than of its specific sequence. Moreover, competition experiments performed with all nucleotides provide evidence for the contribution of the six active sites in the DNA.protein complex formation. We propose a mechanism through which human NDPK-B could stimulate transcription of c-myc or possibly other genes involved in cellular differentiation.

Nucleophilic activation by positioning in phosphoryl transfer catalyzed by nucleoside diphosphate kinase.

The nonenzymatic reaction of ATP with a nucleophile to generate ADP and a phosphorylated product proceeds via a dissociative transition state with little bond formation to the nucleophile. Consideration of the dissociative nature of the nonenzymatic transition state leads to the following question: To what extent can the nucleophile be activated in enzymatic phosphoryl transfer? We have addressed this question for the NDP kinase reaction. A mutant form of the enzyme lacking the nucleophilic histidine (H122G) can be chemically rescued for ATP attack by imidazole or other exogenous small nucleophiles. The ATP reaction is 50-fold faster with the wild-type enzyme, which has an imidazole nucleophile positioned for reaction by a covalent bond, than with H122G, which employs a noncovalently bound imidazole nucleophile [(kcat/KM)ATP]. Further, a 4-fold advantage for imidazole positioned in the nucleophile binding pocket created by the mutation is suggested from comparison of the reaction of H122G and ATP with an imidazole versus a water nucleophile, after correction for the intrinsic reactivities of imidazole and water toward ATP in solution. X-ray structural analysis shows no detectable rearrangement of the residues surrounding His 122 upon mutation to Gly 122. The overall rate effect of approximately 10(2)-fold for the covalent imidazole nucleophile relative to water is therefore attributed to positioning of the nucleophile with respect to the reactive phosphoryl group. This is underscored by the more deleterious effect of replacing ATP with AlphaTauPgammaS in the wild-type reaction than in the imidazole-rescued mutant reaction, as follows. For the wild-type, AlphaTauPgammaS presumably disrupts positioning between nucleophile and substrate, resulting in a large thio effect of 300-fold, whereas precise alignment is already disrupted in the mutant because there is no covalent bond to the nucleophile, resulting in a smaller thio effect of 10-fold. In summary, the results suggest a catalytic role for activation of the nucleophile by positioning in phosphoryl transfer catalyzed by NDP kinase.

Random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit from Dictyostelium discoideum.

The green fluorescent protein (GFP) is currently being used for diverse cellular biology approaches, mainly as a protein tag or to monitor gene expression. Recently it has been shown that GFP can also be used to monitor the activation of second messenger pathways by the use of fluorescence resonance energy transfer (FRET) between two different GFP mutants fused to a Ca2+sensor. We show here that GFP fusions can also be used to obtain information on regions essential for protein function. As FRET requires the two GFPs to be very close, N- or C-terminal fusion proteins will not generally produce FRET between two interacting proteins. In order to increase the probability of FRET, we decided to study the effect of random insertion of two GFP mutants into a protein of interest. We describe here a methodology for random insertion of GFP into the cAMP-dependent protein kinase regulatory subunit using a bacterial expression vector. The selection and analysis of 120 green fluorescent colonies revealed that the insertions were distributed throughout the R coding region. 14 R/GFP fusion proteins were partially purified and characterized for cAMP binding, fluorescence and ability to inhibit PKA catalytic activity. This study reveals that GFP insertion only moderately disturbed the overall folding of the protein or the proper folding of another domain of the protein, as tested by cAMP binding capacity. Furthermore, three R subunits out of 14, which harbour a GFP inserted in the cAMP binding site B, inhibit PKA catalytic subunit in a cAMP-dependent manner. Random insertion of GFP within the R subunit sets the path to develop two-component FRET with the C subunit.

3'-Phosphorylated nucleotides are tight binding inhibitors of nucleoside diphosphate kinase activity.

Nucleoside diphosphate (NDP) kinase catalyzes the phosphorylation of ribo- and deoxyribonucleosides diphosphates into triphosphates. NDP kinase is also involved in malignant tumors and was shown to activate in vitro transcription of the c-myc oncogene by binding to its NHE sequence. The structure of the complex of NDP kinase with bound ADP shows that the nucleotide adopts a different conformation from that observed in other phosphokinases with an internal H bond between the 3'-OH and the beta-O made free by the phosphate transfer. We use intrinsic protein fluorescence to investigate the inhibitory and binding potential of nucleotide analogues phosphorylated in 3'-OH position of the ribose to both wild type and F64W mutant NDP kinase from Dictyostelium discoideum. Due to their 3'-phosphate, 5'-phosphoadenosine 3'-phosphate (PAP) and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) can be regarded as structural analogues of enzyme-bound ADP. The KD of PAPS (10 microM) is three times lower than the KD of ADP. PAPS also acts as a competitive inhibitor toward natural substrates during catalysis, with a KI in agreement with binding data. The crystal structure of the binary complex between Dictyostelium NDP kinase and PAPS was solved at 2.8-A resolution. It shows a new mode of nucleotide binding at the active site with the 3'-phosphate of PAPS located near the catalytic histidine, at the same position as the gamma-phosphate in the transition state. The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.

Functional analysis of the catalytic subunit of Dictyostelium PKA in vivo.

The catalytic subunit of the cAMP-dependent protein kinase (PKA) from Dictyostelium discoideum contains several domains, including an unusually long N-terminal extension preceding a highly conserved catalytic core. We transformed the aggregationless PkaC-null strain with several deletion constructs of both domains. Strains transformed with genes expressing catalytically-inactive polypeptides could not rescue development. Cotransformation with constructs encoding the N-terminal extension and the catalytic core, both unable to rescue development by themselves, yielded transformants able to proceed to late development. A 27-amino acid long hydrophobic region, immediately upstream of the catalytic core, was found indispensable for PKA function. A putative role of this sequence in the acquisition of the active conformation of the protein is discussed.