6E11, a highly selective inhibitor of Receptor-Interacting Protein Kinase 1, protects cells against cold hypoxia-reoxygenation injury.

Necroptosis is a programmed cell death pathway that has been shown to be of central pathophysiological relevance in multiple disorders (hepatitis, brain and cardiac ischemia, pancreatitis, viral infection and inflammatory diseases). Necroptosis is driven by two serine threonine kinases, RIPK1 (Receptor Interacting Protein Kinase 1) and RIPK3, and a pseudo-kinase MLKL (Mixed Lineage Kinase domain-Like) associated in a multi-protein complex called necrosome. In order to find new inhibitors for use in human therapy, a chemical library containing highly diverse chemical structures was screened using a cell-based assay. The compound 6E11, a natural product derivative, was characterized as a positive hit. Interestingly, this flavanone compound: inhibits necroptosis induced by death receptors ligands TNF-α (Tumor Necrosis Factor) or TRAIL (TNF-Related Apoptosis-Inducing Ligand); is an extremely selective inhibitor, among kinases, of human RIPK1 enzymatic activity with a nM Kd; has a non-ATP competitive mode of action and a novel putative binding site; is weakly cytotoxic towards human primary blood leukocytes or retinal pigment epithelial cells at effective concentrations; protects human aortic endothelial cells (HAEC) from cold hypoxia/reoxygenation injury more effectively than necrostatin-1 (Nec-1) and Nec-1s. Altogether, these data demonstrate that 6E11 is a novel potent small molecular inhibitor of RIPK1-driven necroptosis.

Cognitive compensation failure in multiple sclerosis.

OBJECTIVES: Compensatory processes involving the recruitment of additional cerebral areas can limit cognitive impairment caused by brain damage as revealed by fMRI. Multiple sclerosis (MS) is characterized by frequent cognitive deficiencies and diffuse brain damage. Understanding the missing or disturbed processes resulting in cognitive compensation failure is a major challenge in MS. METHODS: Fifteen patients with relapsing-remitting (RR) MS and 20 healthy controls underwent an fMRI paradigm based on Go/No-go task with increasing complexity and neuropsychological and morphologic MRI examinations. RESULTS: To perform all the Go/No-go conditions, patients with RRMS exhibited supplementary cerebral recruitment compared to controls. For the most complex condition, patients presented both collapse of additional cerebral recruitment and significant lower cognitive performance compared to controls. In patients, both response times and diffuse tissue damage were correlated with medial frontal activations. Functional connectivity analysis demonstrated strong correlation between dorsolateral prefrontal cortex and medial frontal region activations. CONCLUSIONS: High cognitive demand causes beneficial cerebral recruitment failure, leading to cognitive impairment in patients with RRMS. Functional compensatory mechanisms preserving good cognitive performances operate by a new cerebral strategy involving medial prefrontal regions recruitment, instead of cerebellar regions seen in controls. This new recruitment is diffuse tissue damage-dependent. Missing cerebellar involvement argues for an inability to generate proficient cognitive automation processes in patients, directly leading to recruitment of high-level decision-making areas. Recurrent mobilization of cortical regions could explain the limiting effect of the cognitive load on the cognitive compensatory phenomena in patients with MS.

High pretransplantation soluble CD30 levels: impact in renal transplantation.

In a retrospective study, the impact of the level of pretransplantation soluble CD30 molecule (sCD30) was evaluated on 3 year transplant survival, as well as the number and grade of acute rejection episodes among kidney recipients engrafted between 2000 and 2002. One hundred and ninety sera of 190 patients sampled on the cross-match day were tested for sCD30 concentrations using an enzyme-linked immunosorbent assay (ELISA) kit (Biotest). For the analysis, a sCD30 cutoff level of 100 U/mL was chosen: 87 (46%) recipients had a level >100, and 103 (54%) <100. All cases (5) of immunological graft loss showed a high sCD30 level. The rate of biopsy-proven acute rejection was 26% in the sCD30 >100 group versus 22% in the sCD30 <100 groups. Among the first graft population (n = 157), the rate was 27% for sCD30 >100 versus 20% for the lower level. The difference was more important for grade II acute rejection (Banff criteria): 6/87 (7%) showed high sCD30 versus 2/103 (2%) with sCD30 <100. This analysis became significant for anti-HLA immunization: 11 (13%) recipients developed anti-HLA class II antibodies in the first group (sCD30 >100) versus 1 (1%) in the second group (sCD30 <100; P < .01). A high pretransplantation sCD30 was not a significant risk factor for an acute rejection episode, but it seemed to be more predictive for antibody-mediated acute rejection and immunological graft loss. However, many recipients showed an increased pretransplantation concentration without any rejection episode or graft loss. Consequently, sCD30 pregraft measurements cannot be used as a predictor for acute kidney rejection among our transplant center, nor as an aid to adapt the immunosuppressive regimen.

How to detect cognitive dysfunction at early stages of multiple sclerosis?

Detecting cognitive dysfunction may be clinically important during the early stages of multiple sclerosis (MS). We assessed a self-report questionnaire on cognitive complaints and individual neuropsychological tests to select patients with early relapsing-remitting MS (RRMS) who needed comprehensive cognitive testing. Fifty-seven patients underwent neurological and neuropsychological assessment, including Rao's Brief Repeatable Battery (BRB) and the complete SEP-59 Questionnaire, a French adaptation of the MSQOL-54, which contains four specific questions about self-perception of cognitive functions. Predictive values, specificity, sensitivity and accuracy of five individual neuropsychological tests--Selective Reminding Test, Symbol Digit Modalities Test (SDMT), Similarities Subtest, PASAT and Stroop Test--were calculated to predict cognitive impairment. Only 10.5% of patients did not report any cognitive complaint, while most reported complaints. On the basis of cognitive performances, 59.7% of patients were classified as cognitively impaired, although only one cognitive score was correlated with cognitive complaints. Depressive symptoms and fatigue were associated with more cognitive complaints. Sensitivity of the SDMT to predict cognitive impairment was 74.2%, specificity was 76.9% and accuracy was 75.4%. Since, at this stage, patients' cognitive complaints are already influenced by depression and fatigue and do not accurately reflect cognitive performances, the SDMT may help to select patients for testing with a more complete cognitive battery.

Local immunotherapy of spontaneous feline fibrosarcomas using recombinant poxviruses expressing interleukin 2 (IL2).

We tested the canarypox virus vector ALVAC and the genetically attenuated vaccinia virus vector NYVAC as vehicles for achieving local immunomodulation in domestic animals bearing spontaneous tumours. Following intratumoral administration of ALVAC-, or NYVAC-luciferase in dogs with melanoma, it was demonstrated that viral recombinants remained localized along the needle track, with no virus detectable in the periphery of the tumour. Given these distribution characteristics and their well-documented safety profile, ALVAC- or NYVAC-based recombinants expressing feline or human IL2, respectively, were administered to domestic cats, in order to prevent the recurrence of spontaneous fibrosarcomas. In the absence of immunotherapy, tumour recurrence was observed in 61% of animals within a 12-month follow-up period after treatment with surgery and iridium-based radiotherapy. In contrast, only 39 and 28% of cats receiving either NYVAC-human IL2 or ALVAC-feline IL2, respectively, exhibited tumour recurrences. Based on such results, and in the context of ongoing clinical studies conducted in humans, we discuss the utilization of ALVAC- or NYVAC-based recombinants as viable therapeutic modalities for local immunotherapy or therapeutic vaccination against cancer, both in humans and companion animals.

The role of the phosphorus BI-BII transition in protein-DNA recognition: the NF-kappaB complex.

We examined, by 1H and 31P NMR, the solution structure of a 16 bp non-palindromic DNA fragment (16M2) containing the HIV-1 NF-kappaB-binding site, in which the sequences flanking the kappaB site had been mutated. 31P NMR was particularly useful for obtaining structural information on the phosphodiester backbone conformation. Structural features were then compared with those of the two previously studied DNA fragments corresponding, respectively, to the native kappaB fragment (16N) and a fragment in which mutations have been introduced at the 5' end of the kappaB site (16M1). For the mutated 16M2 duplex, NMR data showed that the BI-BII equilibrium, previously reported for the native fragment (16N) at the kappaB flanking steps, was lost. The role of the BI-BII equilibrium in NF-kappaB recognition by DNA was then investigated by electrophoretic mobility shift assay. We found that the isolated kappaB site has the potential to bind efficiently due to the BI-BII equilibrium of the kappaB flanking sequences.

Therapeutic vaccines against melanoma and colorectal cancer.

Our overall strategy is to develop multivalent recombinant vaccines capable of eliciting broad immune responses in patients with malignant melanoma or colorectal cancer. We report herein results from initial studies conducted in cancer patients to evaluate the effect of intratumoral administration of recombinant canarypox viruses carrying cytokine genes. Our current focus is on the induction of tumor-specific T-cell responses using a prime/boost immunization schedule with a unique vector system derived from the canary pox virus called ALVAC, in which we incorporate genes encoding Tumor Associated Antigens (TAAs) of interest. Clinical studies in colorectal cancer evaluating an ALVAC CEA candidate vaccine have shown that this approach is safe and can induce tumor-specific T cell responses. Additional clinical studies evaluating candidate vaccines against melanoma and colorectal cancer, targeting either the gp100, Mage 1, Mage 3 or p53 molecules are ongoing.

Safety and immunogenicity of ALVAC wild-type human p53 (vCP207) by the intravenous route in rhesus macaques.

p53 is over-expressed in approximately 50% of human cancers, and transfer of cytotoxic T lymphocytes (CTL) against wild-type p53 protects mice against p53-over-expressing tumors, suggesting that p53 might be an attractive target for immunotherapy. Immunization of mice with a recombinant canarypox virus, ALVAC, expressing human wild-type p53 (vCP207) prevented growth of p53-over-expressing tumors. Since intravenous administration induced better immune responses in mice than other routes, we have proposed to use this route in cancer patients. However, because this vector has never been administered intravenously to humans, and because of the possibility of inducing auto-immunity to a self-antigen, we felt it was necessary to first evaluate safety in rhesus macaques. We found that three intravenous administrations of vCP207 at proportional doses up to 10x those proposed for humans produced no abnormalities in hematologic or clinical chemistry parameters. Serologic markers of autoimmunity and inflammation were unaffected, despite the >95% amino acid identity between human and rhesus p53. Pathological examination of numerous tissues yielded findings comparable to those in animals given placebo. Some animals showed anti-p53 antibody responses following vaccination, indicating that tolerance could be broken to some extent. However, with the exception of one animal with a possible delayed type hypersensitivity reaction to p53 protein, we did not see evidence for a cell-mediated response. The safety profile in monkeys with ALVAC-p53 provides encouragement for using such live, modified vectors via the intravenous route for human immunotherapy.

Recombinant viruses as a tool for therapeutic vaccination against human cancers.

Viral vectors can be used to express a variety of genes in vivo, that encode tumor associated antigens, cytokines, or accessory molecules. For vaccination purposes, the ideal viral vector should be safe and enable efficient presentation of expressed antigens to the immune system. It should also exhibit low intrinsic immunogenicity to allow for its re-administration in order to boost relevant specific immune responses. Furthermore, the vector system must meet criteria that enable its industrialization. The characteristics of the most promising viral vectors, including retroviruses, poxviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and alphaviruses, will be reviewed in this communication. Such recombinant viruses have been successfully used in animal models as therapeutic cancer vaccines. Based on these encouraging results, a series of clinical studies, reviewed herein, have been undertaken. Human clinical trials, have as of today, allowed investigators to establish that recombinant viruses can be safely used in cancer patients, and that such recombinants can break immune tolerance against tumor-associated antigens. These promising results are now leading to improved immunization protocols associating recombinant viruses with alternate antigen-presentation platforms (prime-boost regimens), in order to elicit broad tumor-specific immune responses (humoral and cellular) against multiple target antigens.

PKR stimulates NF-kappaB irrespective of its kinase function by interacting with the IkappaB kinase complex.

The interferon (IFN)-induced double-stranded RNA-activated protein kinase PKR mediates inhibition of protein synthesis through phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) and is also involved in the induction of the IFN gene through the activation of the transcription factor NF-kappaB. NF-kappaB is retained in the cytoplasm through binding to its inhibitor IkappaBalpha. The critical step in NF-kappaB activation is the phosphorylation of IkappaBalpha by the IkappaB kinase (IKK) complex. This activity releases NF-kappaB from IkappaBalpha and allows its translocation to the nucleus. Here, we have studied the ability of PKR to activate NF-kappaB in a reporter assay and have shown for the first time that two catalytically inactive PKR mutants, PKR/KR296 and a deletion mutant (PKR/Del42) which lacks the potential eIF2alpha-binding domain, can also activate NF-kappaB. This result indicated that NF-kappaB activation by PKR does not require its kinase activity and that it is independent of the PKR-eIF2alpha relationship. Transfection of either wild-type PKR or catalytically inactive PKR in PKR(0/0) mouse embryo fibroblasts resulted in the activation of the IKK complex. By using a glutathione S-transferase pull-down assay, we showed that PKR interacts with the IKKbeta subunit of the IKK complex. This interaction apparently does not require the integrity of the IKK complex, as it was found to occur with extracts from cells deficient in the NF-kappaB essential modulator, one of the components of the IKK complex. Therefore, our results reveal a novel pathway by which PKR can modulate the NF-kappaB signaling pathway without using its kinase activity.

Infusion of Fc gamma fragments for treatment of children with acute immune thrombocytopenic purpura.

Treatment of acute immune thrombocytopenic purpura (ITP) with intravenous immunoglobulin (IVIG) induces partial or complete responses, shown by transient or persistent increases in platelet count. The clinical benefit could be due to blockade of the Fc gamma receptor (Fc gamma R); platelets sensitised by IgG could not be cleared by cells of the reticuloendothelial system if Fc gamma R on these cells was blocked with IVIG. To find out whether this putative mechanism is correct, we treated twelve children who had acute ITP with intravenous infusions of Fc gamma fragments. Eleven children showed rapid increases in platelet counts to above the critical value of 50 x 10(9)/L, thereby avoiding major haemorrhagic risk. The response was stable in six patients and transient in five. No adverse reactions were observed. In responders who had detectable platelet-associated IgG before treatment (> 1500 IgG per platelet), platelet IgG fell substantially with treatment. Serum soluble CD16 (sCD16 or sFc gamma RIII) concentrations, measured in five children, showed transient or stable increases that correlated with the rise in platelet count. No sCD16 was detected in the Fc gamma preparation used. We conclude that the infusion of Fc gamma fragments is an efficient treatment of acute ITP in children. The efficacy of Fc gamma fragments strengthens the hypothesis that Fc gamma R blockade is the main mechanism of action of IVIG in ITP, although other immunoregulatory mechanisms triggered by the presence of increased sCD16 concentrations in serum could be involved in the clinical benefit observed.

[Necrosis of the columella after prolonged intranasal intubation]. / Nécrose columellaire après intubation prolongée.

A case is reported of necrosis of the columella nasi due to prolonged intubation in a 21-year-old road traffic accident patient. He was intubated by the nasal route because of combined head and facial injuries. The necrosis was discovered during surgery for repair of the bony facial injuries. As the patient was in a poor condition, the lesion was not immediately repaired, and the necrotic area was only removed. Definitive repair was carried out four and a half months later, with closure of the oronasal communication with an intravelar veloplasty. Such maxillofacial complications of prolonged endotracheal intubation are rare. They are related to prolonged vascular compression by the tube. Such accidents must be recognized without any delay by carefully examining patients.

A condition resembling pagetoid reticulosis in a laboratory mouse.

A raised, hairless cutaneous nodule was found incidentally at necropsy of a 24-month-old CD1 mouse. Histologically there was infiltration of the epidermis by a monomorphic population of moderate to large lymphocytoid cells. Many large cells had bizarre convolutions of the nuclear membrane and resembled the so-called Sézary or mycosis cells seen in epidermotropic T-cell proliferative disorders. The pattern of cutaneous involvement and the presence of the large cells with convoluted nuclei is characteristic of pagetoid reticulosis. No previous reports of such cutaneous lymphoid neoplasms in mice were found in an extensive literature search.

Immunocytochemistry of thyroid C-cell complexes in dogs.

Ten thyroid C-cell complexes from five male and five female Beagle dogs, 1 to 2 years old, were studied using single and double immunocytochemical staining with the peroxidase-antiperoxidase technique. The antigens tested included thyroglobulin, calcitonin, calcitonin gene-related peptide, somatostatin, neuron specific enolase, and neurotensin. All C-cell complexes contained four cell types in various proportions: 1) follicular cells staining for thyroglobulin; 2) C-cells staining for calcitonin, calcitonin gene-related peptide, and neuron specific enolase; 3) stellate cells and cuboidal cells in follicle-like structures staining positively for somatostatin; and 4) undifferentiated cells staining negatively for all antigens. No positive immunoreactivity for neurotensin was detected. These findings support the hypothesis that thyroid C-cell complexes are ultimobranchial remnants that can give rise to thyroid follicles and C-cells.

[Anesthetics responsible for anaphylactic shock. A French multicenter study]. / Substances anesthésiques responsables de chocs anaphylactiques. Enquête multicentrique française.

Combined allergological and anaesthetic consultations have been started in the last few years in eight French Teaching Hospitals so as to explore peranaesthetic anaphylactoid shocks. A survey was carried out in these centers in order to collect patients investigated with the same protocol, for the assessment of the incidence of anaphylaxis in France, as well as the involved drugs. Investigations were always carried out at least 6 to 8 weeks after the accident. The tests used to diagnose IgE-dependent anaphylaxis were skin tests (prick and intradermal tests, carried out in all eight centers), the radioimmunological assay of specific anti-quaternary ammonium IgE, together with an inhibition test with thiopentone and propofol (six centers), leukocyte histamine release (five centers) and human basophil degranulation tests (three centers) for those drugs for which no specific antibody assay exists. The collected data involved 1,240 patients, investigated within the last four years. Anaphylaxis was diagnosed in 821 patients (66.2%). Muscle relaxants were responsible in 668 cases (80% of cases of anaphylaxis). Suxamethonium was the main cause (54.3% of shocks due to muscle relaxants), followed by vecuronium (15.3%). General anaesthetics (hypnotics and benzodiazepines) were responsible for 9.2% of all cases of anaphylaxis opioids for 2.6%. There were only three cases of shock due to local anaesthetic agents. Latex and ethylene oxide are becoming increasingly involved. It would therefore seem mandatory to carry out after any anaphylactoid accident an assessment with sensitive and specific tests for anaphylaxis. Diagnosing anaphylaxis means that the involved drug should be used never again in that patient. Because muscle relaxants are by far the most involved drugs, anaesthetists should use them only when really required.