Hydrogen cyanamide exposure: a case series from Pavia Poison Control Centre.

BACKGROUND: Hydrogen cyanamide is a plant growth regulator introduced in Italy as Dormex in 2000, but recalled from the market in 2008. It's currently not authorized in Europe. Inhalation/dermal contact may cause irritation/caustic burns, ingestion of severe organ damage and concomitant alcohol consumption disulfiram-like reaction due to aldehyde-dehydrogenase inhibition by hydrogen cyanamide. AIMS: To study all exposure cases referred to our centre, evaluating temporal and geographic distribution and analysing clinical manifestations, including the ones after alcohol consumption. METHODS: We retrospectively evaluated all hydrogen cyanamide exposures referred to our Poison Control Centre (January 2007-December 2021). For each case, age, sex, exposure route/year, geographical location, intent of exposure, alcohol co-ingestion, emergency department-admission Poison Severity Score, signs/symptoms and treatment were analysed. RESULTS: Thirty subjects were included. Median case/year was 1 [1; 2]: 79% occurred after market withdrawal, 92% in Sicily. All exposures were unintentional and work related; 41% of patients also co-ingested alcohol. Mean poison severity score at emergency department admission was 1.54, more severe when ingestion occurred. The most common signs/symptoms were flushing, secondary to peripheral vasodilation (41%), hyperaemia/erythema (29%), dyspnoea (25%), nausea (20%), vomiting (12%), oedema (12%), II-III degrees burns (12%) and pharyngodynia (12%). All patients were treated symptomatically and fully recovered. CONCLUSIONS: Hydrogen cyanamide exposure can lead to severe clinical manifestations. Despite its withdrawal from the Italian market, hydrogen cyanamide is still used: through PCC's crucial role in monitoring exposure to agricultural products efforts should be made to contrast illegal trade and increase awareness of its potential toxicity in those countries in which it's still legal.

Optical properties of alkali halide crystals from all-electron hybrid TD-DFT calculations.

We present a study of the electronic and optical properties of a series of alkali halide crystals AX, with A = Li, Na, K, Rb and X = F, Cl, Br based on a recent implementation of hybrid-exchange time-dependent density functional theory (TD-DFT) (TD-B3LYP) in the all-electron Gaussian basis set code CRYSTAL. We examine, in particular, the impact of basis set size and quality on the prediction of the optical gap and exciton binding energy. The formation of bound excitons by photoexcitation is observed in all the studied systems and this is shown to be correlated to specific features of the Hartree-Fock exchange component of the TD-DFT response kernel. All computed optical gaps and exciton binding energies are however markedly below estimated experimental and, where available, 2-particle Green's function (GW-Bethe-Salpeter equation, GW-BSE) values. We attribute this reduced exciton binding to the incorrect asymptotics of the B3LYP exchange correlation ground state functional and of the TD-B3LYP response kernel, which lead to a large underestimation of the Coulomb interaction between the excited electron and hole wavefunctions. Considering LiF as an example, we correlate the asymptotic behaviour of the TD-B3LYP kernel to the fraction of Fock exchange admixed in the ground state functional cHF and show that there exists one value of cHF (∼0.32) that reproduces at least semi-quantitatively the optical gap of this material.

Lifetime and actual prevalence of pain and headache in multiple sclerosis.

The aim of the present study is to assess the actual and lifetime frequency of neuropathic (trigeminal neuralgia, L'Hermitte's sign, dysesthesic pain) and somatic (painful muscle spasms and low back pain) pain and headache (tensive headache and migraine) in a cross-sectional sample of 428 consecutive multiple sclerosis (MS) outpatients followed-up in an Italian University MS center over a 3-month period. The impact of demographic and disease-related variables on pain and headache risk is also studied. A semi-structured questionnaire was administered during a face-to-face interview with MS patients and a multivariate logistic regression model is applied to obtain crude and adjusted risk measures. The mean age of the sample was 38.4 years, and female/male ratio was 1.65. The mean disease duration was 9.6 years and the median Expanded Disability Status Scale was 2.0, with most of the patients (74.8%) being affected by the relapsing-remitting form. Lifetime prevalence at the date of examination of at least one type of neuropathic or somatic pain was 39.8% in MS patients, with 58.5% also including headache, while the actual prevalence was 23.8% and 39.9%, respectively. After multivariate analysis, a progressive course of disease was shown to increase the risk of dysesthesic pain and painful muscle spasms, while greater disability was responsible for a higher risk of back pain. L'Hermitte's sign was more frequent in younger patients, while females had a higher risk of headache. Pain and headache in MS are not negligible symptoms and a neurological examination should not miss the assessment of risk factors for specific types of pain for a more specific and individualized treatment.

Beneficial effects of r-h-CLU on disease severity in different animal models of peripheral neuropathies.

Clusterin is a protein involved in multiple biological events, including neuronal cytoprotection, membrane recycling and regulation of complement-mediated membrane attack after injury. We investigated the effect of recombinant human clusterin in preclinical models of peripheral neuropathies. Daily treatment with clusterin accelerated the recovery of nerve motor evoked potential parameters after sciatic nerve injury. Prophylactic or therapeutic treatment of experimental autoimmune neuritis rats with clusterin also accelerated the rate of recovery from the disease, associated with remyelination of demyelinated nerve fibers. These data demonstrate that clusterin is capable of ameliorating clinical, neurophysiological and pathological signs in models of peripheral neuropathies.

Cortical function abnormalities in migraine: neurophysiological and neuropsychological evidence from reaction times and event-related potentials to the Stroop test.

Cortical hyperexcitability was studied in migraine patients using reaction times (RT's) and Event-Related Potentials (ERP) to the Stroop test. We found a slower RTs in patients if compared to controls, supporting the hypothesis of a mild cortical functions impairment even in interictal periods in this group of patients.

Maturation of dendritic cells is accompanied by rapid transcriptional silencing of class II transactivator (CIITA) expression.

Cell surface expression of major histocompatibility complex class II (MHCII) molecules is increased during the maturation of dendritic cells (DCs). This enhances their ability to present antigen and activate naive CD4(+) T cells. In contrast to increased cell surface MHCII expression, de novo biosynthesis of MHCII mRNA is turned off during DC maturation. We show here that this is due to a remarkably rapid reduction in the synthesis of class II transactivator (CIITA) mRNA and protein. This reduction in CIITA expression occurs in human monocyte-derived DCs and mouse bone marrow-derived DCs, and is triggered by a variety of different maturation stimuli, including lipopolysaccharide, tumor necrosis factor alpha, CD40 ligand, interferon alpha, and infection with Salmonella typhimurium or Sendai virus. It is also observed in vivo in splenic DCs in acute myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalitis. The arrest in CIITA expression is the result of a transcriptional inactivation of the MHC2TA gene. This is mediated by a global repression mechanism implicating histone deacetylation over a large domain spanning the entire MHC2TA regulatory region.

Maturation of the humoral autoimmune response to epitopes of GAD in preclinical childhood type 1 diabetes.

GAD is a major target of autoimmunity in preclinical type 1 diabetes. Here we examine the maturation of the humoral response to GAD epitopes sequentially from birth to diabetes onset or current follow-up in 29 GAD antibody (GADA)+ offspring of parents with diabetes from the BABYDIAB Study. Antibodies were measured against GAD65, GAD67, and GAD65/67 chimeras by radiobinding assay. In 28 of 29 offspring, the first GADAs contained reactivity against epitopes within GAD65 residues 96-444, suggesting that the middle GAD65 region is a primary target of GAD humoral autoimmunity. In 7 of these 28 offspring, initial antibody reactivity was against all epitope regions tested (middle GAD65, COOH-terminal GAD65 residues 445-585, NH2-terminal GAD65 residues 1-95, and GAD67); in 16 offspring, reactivity was to middle and COOH-terminal GAD65 epitopes, and in 5 offspring, reactivity was only to the middle GAD65 epitopes. The single offspring without middle GAD65 reactivity had antibodies to the NH2-terminal epitopes in the absence of all other islet autoimmunity. Subsequent GADA epitope spreading was frequent and seen in 10 of 15 offspring with informative follow-up samples. Spreading was mostly (eight cases) to NH2-terminal GAD65 epitopes. In two offspring, spreading to new epitopes was found when antibody titers to GAD65 and early epitopes were declining, suggesting determinant-specific regulation of the humoral response. None of the GADA reactivities nor any changes in reactivity over time were specifically associated with diabetes onset. The findings suggest that the humoral autoimmune response to GAD found in childhood is dynamic, is initially against epitopes within the middle portion of GAD65, and spreads to epitopes in other regions of GAD65 and GAD67.

Confocal microscopic localization of anti-myelin-associated glycoprotein autoantibodies in a patient with peripheral neuropathy initially lacking a detectable IgM gammopathy.

We report here on a patient with anti-myelin-associated glycoprotein (MAG) neuropathy in whom examination of a sural nerve biopsy by multichannel confocal microscopy showed a partly overlapping distribution of MAG and IgM deposits in myelinated fibers. Our data demonstrate that MAG in Schmidt-Lanterman incisures and paranodal loops, as well as some additional HNK-1-positive components of the basal lamina, are the major targets of the anti-MAG monoclonal IgM autoantibodies in this neuropathy in vivo. Perforation of the basal lamina can allow the penetration and binding of anti-MAG IgM inside myelinated fibers. Our results support and extend the notion that the production of monoclonal anti-MAG IgM may be antigenically driven by MAG molecules and that this process may occur in the immunologically privileged environment of the nerve prior to the appearance of a genuine gammopathy in serum.

Inhibition of Bax channel-forming activity by Bcl-2.

Proteins of the Bcl-2 family are intracellular membrane-associated proteins that regulate programmed cell death (apoptosis) either positively or negatively by as yet unknown mechanisms. Bax, a pro-apoptotic member of the Bcl-2 family, was shown to form channels in lipid membranes. Bax triggered the release of liposome-encapsulated carboxyfluorescein at both neutral and acidic pH. At physiological pH, release could be blocked by Bcl-2. Bcl-2, in contrast, triggered carboxyfluorescein release at acidic pH only. In planar lipid bilayers, Bax formed pH- and voltage-dependent ion-conducting channels. Thus, the pro-apoptotic effects of Bax may be elicited through an intrinsic pore-forming activity that can be antagonized by Bcl-2.

A bacterial signal peptide directs efficient secretion of eukaryotic proteins in the baculovirus expression system.

Escherichia coli remains an organism of choice for the production of recombinant proteins required in large quantities. Whenever possible, secretion is the preferred strategy since it permits easy and efficient purification from the extracellular medium. Our efforts to use E. coli to secrete a human CD23 soluble variant fused to a pair of IgG binding domains via the Staphylococcal protein A signal peptide were unsuccessful. Surprisingly, when the same construct was expressed in the baculovirus system, efficient secretion was observed and cleavage of the signal peptide occurred at the expected site. Varying the genes in the fusions or the tags, or the topology of the gene and the tag, did not affect the high-level secretion and cleavage at the correct site. We envision that fusion of the bacterial signal sequence to eukaryotic recombinant genes will prove to be a tool of value for efficient protein secretion in insect cells using the baculovirus expression system.

Expression and intracellular localization of catechol O-methyltransferase in transfected mammalian cells.

The intracellular localization of soluble and membrane-bound isoforms of rat and human catechol O-methyltransferase (COMT) was studied by expressing the recombinant COMT proteins either separately or together in mammalian cell lines (HeLa and COS-7 cells) and in rat primary neurons. The distribution of soluble and membrane-bound COMT enzyme was visualized by immunocytochemistry. For comparison, the localization of native COMT was studied in rat C6 glioma cells by immunoelectron microscopy. Staining of cells expressing membrane-bound COMT with a COMT-specific antiserum revealed an immunofluorescence signal in intracellular reticular structures and in the nuclear membrane. Double-staining of the cells with antisera against proteins specific for the rough endoplasmic reticulum indicated that they colocalized with membrane-bound COMT, suggesting that it resided in the endoplasmic reticulum. Notably, no COMT-specific fluorescence of plasma membranes was detected. The signal in the endoplasmic reticulum was also evident in the cells expressing both recombinant COMT forms. Intracellular native COMT reaction was detected by immunoelectron microscopy in rat C6 glioma cells and an intense cytoplasmic signal was seen in the primary neurons infected with the recombinant Semliki Forest virus. The cells expressing recombinant soluble COMT revealed intense nuclear staining together with diffuse cytoplasmic immunoreactivity, suggesting that a part of soluble COMT is transported to nuclei. Western blotting from rat liver and brain revealed soluble COMT in the nuclei. Enzyme activity measurements from liver cytoplasmic and nuclear fractions suggested that about 5% of the soluble COMT resided in nuclei. The intracellular localization of both COMT forms implies that COMT acts in the cytoplasm and possibly also in the nuclear compartment, and that the physiological substrates of COMT enzymes may have to be internalized before their methylation by COMT.

Dissecting processing and apoptotic activity of a cysteine protease by mutant analysis.

We have compared the behavior of wild-type mouse NEDD-2, a neural precursor cell-expressed, developmentally down-regulated cysteine protease gene, to various mutant forms of the gene in both apoptotic activity in neuronal cells and proteolytic cleavage in the Semliki Forest virus and rabbit reticulocyte protein expression systems. Our results confirm that NEDD-2 processing and apoptotic activity are linked phenomena. They identify aspartate residues as likely targets for autocatalytic cleavage. They establish that cleavage events only occur at specific sites. Finally, they pinpoint differential effects of individual mutations on the overall proteolytic cleavage patterns, raising interesting questions related to the mechanisms of subunit assembly.

Expression of the 69K movement protein of turnip yellow mosaic virus in insect cells.

The nonstructural 69-kilodalton (K) protein of turnip yellow mosaic virus is necessary for systemic spread of the virus within the plant. To examine the behavior of the 69K protein in vivo, antibodies were raised against the carboxy-terminal region of this protein. The full-length 69K protein was also expressed in insect cells using a recombinant baculovirus. Studies on the posttranslational modifications of the 69K protein in insect cells revealed that the protein is phosphorylated but not glycosylated. Further experiments of subcellular fractionation and indirect immunolocalization in insect cells showed that the 69K protein is localized in the cytoplasm and/or in the plasma membrane.

Treatment of multiple sclerosis with 15 +/- deoxyspergualin. Design of a controlled study with close MRI-monitoring.

The rationale and design of a prospective randomized double blind study of 15 +/- Deoxyspergualin in the treatment of Multiple Sclerosis are presented. The study was started in autumn 1992 and will include more than 200 patients who will be randomized either to DSG low dose or DSG high dose or placebo. Average number of contrast enhancing lesions in monthly cranial MRIs during the first six months of treatment and change in EDSS at the end of the two-year study period will be the main efficacy parameters.

Human granulocyte-macrophage colony-stimulating factor plus phorbol myristate acetate stimulate a promyelocytic cell line to produce an IL-1 inhibitor.

We recently described an IL-1 inhibitor found in urine of febrile patients. It is a 26-kDa glycoprotein that acts by blocking the binding of IL-1 to its receptor. In a search for a cell source for the urinary IL-1 inhibitor, we tested three promyelocytic cell lines, H-161, AML-193, and HL-60, for their ability to produce this protein. Under normal culture conditions none of these cell lines produce detectable IL-1 inhibitory activity. The H-161 cells were treated with differentiation-inducing agents, i.e., sodium butyrate, hemin, retinoic acid, DMSO, vitamin D3, and PMA alone or in combination with IL-1 alpha, IL-2, IL-3, IL-4, IL-5, IL-6, TNF-alpha, IFN-gamma, granulocyte-CSF, macrophage-CSF, granulocyte/macrophage-CSF (GM-CSF), and Con A and tested for the production of IL-1 inhibitor. Production of IL-1 inhibitor was detected in cell supernatant, when H-161 cells were differentiated to adherent macrophage-like cells under the influence of PMA followed by a second signal provided by GM-CSF. Treatment of the other two cell lines, AML-193 and HL-60, with PMA plus GM-CSF also yielded similar IL-1 inhibitor protein. Partial purified H-161-derived IL-1 inhibitor showed specific binding to IL-1R-bearing cells and blocked the binding of IL-1 to its receptor and is thus similar to the urinary-derived molecule. We conclude the GM-CSF provides a signal to adherent macrophage-like cells to become "inhibitory macrophages" and to produce a competitive inhibitor of IL-1.

Production of a 26,000-dalton interleukin 1 inhibitor by human monocytes is regulated by granulocyte-macrophage colony-stimulating factor.

An interleukin 1 (IL 1) inhibitor is secreted into culture medium by a human promyelocytic cell line, H-161, upon stimulation with (PMA) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF). Since the morphological characteristics of this cell line were macrophage-like, human monocytes were tested for their ability to produce similar activity using the same induction conditions. Upon induction of adherent peripheral blood monocytes with rhGM-CSF and/or PMA, an IL 1 antagonistic activity was found in the cell supernatants, as determined by IL 1 receptor binding assay, using the murine EL-4.6.1C10 cell line as the cell target. Most of the inhibition of IL 1 binding induced by PMA or by PMA/rhGM-CSF was shown to be caused by IL 1, since it was neutralized by a mixture of anti-IL 1 alpha/beta antibodies and was active in the murine thymocyte proliferation assay (LAF). The activity induced by GM-CSF alone was not neutralized by anti-IL 1 alpha/beta antibodies and showed no LAF activity. The IL 1 inhibitor activity was induced by rhGM-CSF with a D50 around 40 pg/ml. The activity was produced for more than 3 wk in the presence of GM-CSF; removal of GM-CSF was followed by a rapid decrease of IL 1 antagonistic activity. The specific binding of biosynthetically labeled IL 1 inhibitor to target cells (EL-4.6.1C10) showed a protein of 26 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). This molecule shares biological and physical characteristics with the urinary IL 1 inhibitor and the promyelocytic H-161-derived IL 1 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)