Characterization and review of MTHFD1 deficiency: four new patients, cellular delineation and response to folic and folinic acid treatment.

In the folate cycle MTHFD1, encoded by MTHFD1, is a trifunctional enzyme containing 5,10-methylenetetrahydrofolate dehydrogenase, 5,10-methenyltetrahydrofolate cyclohydrolase and 10-formyltetrahydrofolate synthetase activity. To date, only one patient with MTHFD1 deficiency, presenting with hyperhomocysteinemia, megaloblastic anaemia, hemolytic uremic syndrome (HUS) and severe combined immunodeficiency, has been identified (Watkins et al J Med Genet 48:590-2, 2011). We now describe four additional patients from two different families. The second patient presented with hyperhomocysteinemia, megaloblastic anaemia, HUS, microangiopathy and retinopathy; all except the retinopathy resolved after treatment with hydroxocobalamin, betaine and folinic acid. The third patient developed megaloblastic anaemia, infection, autoimmune disease and moderate liver fibrosis but not hyperhomocysteinemia, and was successfully treated with a regime that included and was eventually reduced to folic acid. The other two, elder siblings of the third patient, died at 9 weeks of age with megaloblastic anaemia, infection and severe acidosis and had MTFHD1 deficiency diagnosed retrospectively. We identified a missense mutation (c.806C > T, p.Thr296Ile) and a splice site mutation (c.1674G > A) leading to exon skipping in the second patient, while the other three harboured a missense mutation (c.146C > T, p.Ser49Phe) and a premature stop mutation (c.673G > T, p.Glu225*), all of which were novel. Patient fibroblast studies revealed severely reduced methionine formation from [(14)C]-formate, which did not increase in cobalamin supplemented culture medium but was responsive to folic and folinic acid. These additional cases increase the clinical spectrum of this intriguing defect, provide in vitro evidence of disturbed methionine synthesis and substantiate the effectiveness of folic or folinic acid treatment.

Vertically stacked quantum dot pairs fabricated by nanohole filling.

Strain-free, vertically coupled GaAs quantum dots (QDs) with an ultra-low density below 1 × 10(7) cm(-2) are fabricated by filling of self-assembled nanoholes with a GaAs/AlGaAs/GaAs layer sequence. The sizes of the two QDs, forming a QD pair (QDP), as well as the AlGaAs tunnel-barrier between the dots are tuned independently. We present atomic force microscopy studies of the QDP formation steps. We have performed photoluminescence studies of single QDPs with varied dot size and tunnel-barrier thickness. The data indicate non-resonant tunnelling between the dots. Furthermore, we apply the quantum confined Stark effect to tune the photoluminescence energy by up to 25 meV.

Regulatory demands on data quality for the environmental risk assessment of pharmaceuticals.

The evaluation of the quality of data and their use within the review of environmental risk assessment of human as well as veterinary pharmaceuticals is described from a regulatory point of view. A definition and differentiation in three categories for the reliability of data are given. Existing criteria relating to international testing standards for categorising reliability are adopted for their use within the environmental risk assessment of pharmaceuticals. A systematic documentation of evaluating reliability for literature data as well as for experimental studies (effect and environmental fate studies) is proposed. The data quality criteria are defined in order to increase the transparency of the evaluation process in Germany and thus the quality of the environmental risk assessment of pharmaceuticals.

Prophylactic ibuprofen versus placebo in very premature infants: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Patent ductus arteriosus is a common complication of prematurity that frequently requires surgical or medical treatment. The benefit of prophylactic treatment by indometacin, a cyclo-oxygenase inhibitor, remains uncertain compared with curative treatment. This benefit could be improved with ibuprofen, another cyclo-oxygenase inhibitor with fewer adverse effects than indometacin on renal, mesenteric, and cerebral perfusion. We aimed to compare prophylactic and curative ibuprofen in the treatment of this abnormality in very premature infants. METHODS: We did a randomised controlled trial in infants younger than 28 weeks of gestation, who were randomly assigned to receive either three doses of ibuprofen or placebo within 6 h of birth. After day 3, symptomatic patent ductus arteriosus was treated first by open curative ibuprofen, then back-up indometacin, surgery, or both. The primary endpoint was need for surgical ligation. Analysis was per protocol. FINDINGS: The study was stopped prematurely after 135 enrollments because of three cases of severe pulmonary hypertension in the prophylactic group. 65 infants received prophylactic ibuprofen, and 66 received placebo. Prophylaxis reduced the need for surgical ligation from six (9%) to zero (p=0.03), and decreased the rate of severe intraventricular haemorrhage from 15 (23%) to seven (11%) (p=0.10). However, survival was not improved (47 [71%] placebo vs 47 [72%] treatment, p=1.00), because of high frequency of adverse respiratory, renal, and digestive events. INTERPRETATION: In premature infants, prophylactic ibuprofen reduces the need for surgical ligation of patent ductus arteriosus, but does not reduce mortality or morbidity. Therefore, it should not be preferred to early curative ibuprofen.

Use of immobilized synthetic peptides for the identification of contact sites between human interleukin-6 and its receptor.

Synthetic peptides immobilized on cellulose membranes proved to be a powerful tool for the identification of sites in the cytokine IL-6 involved in receptor binding. Similarly, a region in the extracellular part of the IL-6 receptor which is important for interaction with its ligand was identified.

Different epitopes are required for gp130 activation by interleukin-6, oncostatin M and leukemia inhibitory factor.

Gp130 is the common signal transducing receptor subunit of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor and cardiotrophin-1. IL-6 and IL-11 induce gp130 homodimerization whereas the others lead to the formation of heterodimers with LIFR or OSMR. Binding epitopes for IL-6 and IL-11 are located in the immunoglobulin-like domain and the cytokine binding module (CBM). Here we show that a gp130 mutant lacking domain 1, although unresponsive to IL-6 and IL-11, can still activate signal transducer and activator of transcription (STAT) transcription factors in response to LIF or OSM. Moreover, point mutations in the CBM of gp130 (F191E and V252D) that severely impair signal transduction in response to IL-6 and IL-11 differentially interfere with gp130 activation in response to LIF and OSM. Thus, epitopes involved in gp130 homodimerization are distinct from those leading to the formation of gp130/LIFR or gp130/OSMR heterodimers. These findings may serve as the base for rational design of gp130 antagonists that specifically interfere with bioactivity of distinct IL-6-type cytokines.

Inhibition of cytosolic phospholipase A(2) attenuates activation of mitogen-activated protein kinases in human monocytic cells.

Eicosanoids and platelet-activating factor generated upon activation of cytosolic phospholipase A(2) enhance activity of transcription factors and synthesis of proinflammatory cytokines. Here, we show that selective inhibitors and antisense oligonucleotides against this enzyme suppressed expression of the interleukin-1beta gene at the level of transcription and promoter activation in human monocytic cell lines. This inhibitory effect was due to failure of activation of mitogen-activated protein kinases (MAPK) through phosphorylation by upstream mitogen-activated protein kinase kinases (MKK). Consequently, phosphorylation and degradation of inhibitor-kappaBalpha (I-kappaBalpha) and subsequent cytoplasmic mobilization, DNA-binding and the transactivating potential of nuclear factor-kappaB (NF-kB), nuclear factor-interleukin-6 (NF-IL6), activation protein-1 (AP-1) and signal-transducer-and-activator-of-transcription-1 (STAT-1) were impaired. It is concluded, that lipid mediators promote activation of MAPKs, which in turn lead to phosphorylation and liberation of active transcription factors. Since inhibition of cytosolic phospholipase A(2) ameliorates inflammation in vivo, this potency may reside in interference with the MAPK pathway.

Feasibility of surgery for patent ductus arteriosus of premature babies in a neonatal intensive care unit.

Twenty-two premature neonates underwent surgical haemoclip closure of patent ductus arteriosus in a neonatal intensive care unit (NICU). Surgery was performed in the NICU in order to prevent hypothermia and interruption of care, and to avoid accidental vascular access removal and extubation. The results suggest that morbidity can be limited by performing the surgery in an NICU, and by switching from conventional to high-frequency mechanical ventilation in case of severe respiratory failure.

Reporting and evaluation criteria as means towards a transparent use of ecotoxicity data for environmental risk assessment of pharmaceuticals.

Ecotoxicity data with high reliability and relevance are needed to guarantee the scientific quality of environmental risk assessments of pharmaceuticals. The main advantages of a more structured approach to data evaluation include increased transparency and predictability of the risk assessment process, and the possibility to use non-standard data. In this collaboration, between the research project MistraPharma and the German Federal Environment Agency, a new set of reporting and evaluation criteria is presented and discussed. The new criteria are based on the approaches in the literature and the OECD reporting requirements, and have been further developed to include both reliability and relevance of test data. Intended users are risk assessors and researchers performing ecotoxicological experiments, but the criteria can also be used for education purposes and in the peer-review process for scientific papers. This approach intends to bridge the gap between the regulator and the scientist's needs and way of work.

Development of liver disease despite mannose treatment in two patients with CDG-Ib.

We report here the 6- and 2-year follow-up of two patients diagnosed at 2 months of age with CDG-Ib who were treated with mannose, with digestive symptoms, liver involvement and hyperinsulinemic hypoglycaemia. Both developed liver fibrosis while general condition improved and other symptoms disappeared.

Further psychometric evaluation of the Spanish language health-promoting lifestyle profile.

The reliability and validity of the Spanish language version of the Health-Promoting Lifestyle Profile (Spanish-HPLP) was evaluated for a predominantly Central American sample. A convenience sample (N = 106) completed the demographic sheet and Spanish-HPLP. Alpha coefficients were 0.94 for the total scale and ranged from 0.64 to 0.89 for the subscales. Significant Pearson correlations were found between Spanish-HPLP scores and the variables of age, education, income, length of residence in the United States, and perceived health status. Significant differences based on sex and marital status were noted. The Spanish-HPLP was found to be reliable for this sample. Although content and construct validity were supported, the instrument is in need of further convergent, or criterion-related validation.

Temperature dependent pH-instability of some alpha-L-arabinofuranosidases.

In this paper are described alpha-L-arabinofuranosidases, partially purified from some moulds, with very high lability of their activity to weakly alkaline or neutral pH-values. This pH-lability strongly depends on temperature. The form of the inactivation curves suggests involvement of charge changes in this irreversible process. These enzymes may be useful for optimization of the tumor therapy concept described by Graffi (5).

Pulmonary hypertension after ibuprofen prophylaxis in very preterm infants.

We report three cases of severe hypoxaemia after ibuprofen administration during a randomised controlled trial of prophylactic treatment of patent ductus arteriosus with ibuprofen in premature infants born at less than 28 weeks of gestation. Echocardiography showed severely decreased pulmonary blood flow. Hypoxaemia resolved quickly on inhaled nitric oxide therapy. We suggest that investigators involved in similar trials pay close attention to pulmonary pressure if hypoxaemia occurs after prophylactic administration of ibuprofen.

[Experiments on the modification of the distribution of the activity of various alpha-L-arabinofuranosidases following i.v. application in tumor-bearing mice]. / Versuche zur Beeinflussung der Verteilung der Aktivität verschiedener alpha-L-Arabinofuranosidasen nach i.v. Applikation in tumortragende Mäuse.

Graffi et al. have proposed the use of exogenous enzymes for selective cleavage of inactive transport forms of cancerostatic substances in tumor tissue. Enzymes appropriate for this purpose should, if possible, show also a certain enrichment in the tumor tissue. In studies on the pH-labile alpha-L-arabinofuranosidase from G. myabena in tumor-bearing mice, under defined conditions in the tumors there could be demonstrated a higher concentration of the active form of the enzyme than in most of the normal tissues. However, the enzyme activity is eliminated from the organism in a relatively short time through excretion and inactivation. The application of glucose led to a strong increase of activity of the enzyme injected, both in the tumors and in the normal tissues. It has been shown in the present investigations that this increase in the enzyme activity can be curtailed more strongly in normal tissue by alkali application than in the tumors. In this way, a distribution of the enzyme activity favorable for therapy experiments is obtained. Only in the kidney and urine has a higher activity of the applied enzyme been measured than in the tumors. In the second part of this work it has been attempted to achieve accumulation of activity of the pH-stable alpha-L-arabinofuranosidase from A. niger through application of angiotensin, but no positive results have been reached under the various experimental conditions used.

[Pharmacokinetic studies of the activity of a pH-labile alpha-L-arabinofuranosidase following i.v. injection into tumor-bearing mice]. / Pharmakokinetische Untersuchungen der Aktivität einer pH-labilen alpha-L-Arabinofuranosidase nach i.v.-Injektion in tumortragende Mäuse.

In order to increase the selectivity of tumor chemotherapy, Graffi et al. have proposed the application of xenogenic enzymes, which are able to split transport forms of carcinostatics under the pH-conditions in the tumor more vigorously than in the normal tissues. This paper describes the distribution within the body and elimination of the activity of the pH-labile alpha-L-arabinofuranosidase from G. myabena compared with the pH-stable enzyme from A. niger, using tumor bearing mice. In vitro, the pH labile arabinosidase was irreversibly inactivated within a few minutes at pH 7.4 and 37 degrees C; however at pH 6.5 it remained active even after several hours. After injection, this enzyme activity was eliminated from the organism by excretion and inactivation within a few hours. Hereby a relatively favourable distribution of the enzyme activity for therapeutic application was reached after 60 minutes. At this time higher activity than in the tumor was measured only in the kidney. The application of glucose led to a strong increase of the enzyme activity in both tumor and normal tissues. This effect was also seen in tumor free mice. In further experiments it will be tried to find out conditions which reduce the glucose induced acidosis. The activity and distribution of the pH-stabile enzyme from A. niger were not influenced by glucose application.

Studies on the interleukin-6-type cytokine signal transducer gp130 reveal a novel mechanism of receptor activation by monoclonal antibodies.

The transmembrane glycoprotein gp130 belongs to the family of hematopoietic cytokine receptors. It represents the common signal transducing receptor component of the so called interleukin-6-type cytokines. For several cytokine receptors including gp130 it has been shown that receptor activation cannot only be achieved by the natural ligand but also by single monoclonal antibodies raised against the receptor ectodomain. These findings have been interpreted in a way that dimerization of cytokine receptors is sufficient for receptor activation. Here we show that the recently described gp130-activating antibody B-S12 actually consists of two different monoclonal antibodies. By subcloning of B-S12 the monoclonal antibodies B-S12-A5 and B-S12-G7 were obtained. The individual antibodies are biologically inactive, in combination they exert B-S12-like activity on hepatoma cells. On Ba/F3 cells stably transfected with gp130 a combination of B-S12-G7 with another monoclonal gp130 antibody, B-P8, is required to stimulate proliferation. Using gp130 deletion mutants we show that all three antibodies map to domains 2 and 3 of gp130 which constitute the cytokine binding module. The individual antibodies inhibit activation of the signal transducer by interleukin-6 and interfere with binding of interleukin-6 to gp130. Interestingly, the combination of B-S12-G7 and a Fab fragment of B-P8 retains biological activity. We conclude from our data that (i) the monoclonal antibodies activate gp130 by mimicking the natural ligand and (ii) enforcement of gp130 dimerization is not sufficient for receptor activation but additional conformational requirements have to be fulfilled.

Rapid and efficient inactivation of IL-6 gingipains, lysine- and arginine-specific proteinases from Porphyromonas gingivalis.

Deregulation of the cytokine network is an important adaptation of pathogenic bacteria to modulate and evade a host immune response. Here we describe that IL-6 is rapidly and efficiently cleaved and inactivated by the arginine- and lysine-specific proteinases from Porphyromonas gingivalis, referred to as RGP-A, RGP-B, and KGP. One of the primary cleavage sites for RGPs has been mapped between R18 and Q19 within the N-terminal region of the IL-6 polypeptide chain; however, both KGP and RGPs cleave IL-6 within the C-terminal region of the polypeptide chain. After these initial proteolytic cleavages, IL-6 is further degraded by each of the enzymes tested. Although KGP is the most potent IL-6-degrading proteinase, the initial C-terminal cleavage of IL-6 mediated by all gingipains is already sufficient to inactivate this cytokine. Our data are consistent with the observation that in periodontitis the IL-6 concentration is lowest in the gingival tissue adjacent to bacterial plaque, whereas significantly elevated concentrations of this cytokine are detected around the infected area. Degradation of IL-6 by gingipains may, therefore, represent an additional mechanism which influences the balance between pro- and anti-inflammatory reactions at distal versus proximal sites from the periodontal plaque.

Angiographic assessment and control of potential operative hemorrhage with pathologic fractures secondary to metastasis.

Two cases are reported, which demonstrate the contribution of angiography to the operative management of neoplastic, pathologic fractures. The degree of fracture site neovascularity was assessed by pre-operative arteriography. Selective trans-catheter gelatin-foam embolization was then employed to markedly reduce or obliterate the arterial supply to lesions shown to be richly vascular. Open reduction and internal fixation was subsequently accomplished with minimal operative hemorrhage.

[Distribution studies of alpha-L-arabinofuranosidase already unstable at pH 7.0 (37 degrees C) in tumor-bearing mice in connection with its possible use to enhance the selectivity of the chemotherapy of malignant tumors]. / Untersuchungen zur Verteilung einer bereits bei pH 7.0 (37 Grad C) instabilen alpha-L-Arabinofuranosidase in tumortragenden Mäusen im Zusammenhang mit ihrer möglichen Anwendung zur Erhöhung der Selektivität der Chemotherapie maligner Tumoren.

Graffi et al. (1-3) had proposed the use of exogenous enzymes to toxify inactive transport forms of cancerostatic substances. For this purpose, the pH difference between normal tissues and the tumor was to be exploited, which can be essentially increased by the application of glucose and inorganic phosphate (5-7). Earlier studies using alpha-L-arabinofuranosidase obtained from Aspergillus niger have shown that the selectivity of tumor chemotherapy can be increased in this way (4). The alpha-L-arabinofuranosidases known to date are stabile in a wide pH range (9). However, in some moulds we found pH-labile enzymes of this kind that become irreversibly inactivated in the weakly alkaline or neutral pH range (10, 11). Studies on the distribution of the activity of a pH-labile alpha-L-arabinofuranosidase from Glomerella myabana in tumor-bearing mice have shown that this enzyme is rapidly eliminated from the organism, in contrast to the pH-stable alpha-L-arabinofuranosidase from A. niger. Apart from its excretion via kidney and liver, of importance is the inactivation of the enzyme in the normal tissues. The additional application of glucose strongly increased the activity of this enzyme both in the tumor and in normal tissues (12). By injecting alkaline solutions, stronger inactivation in normal tissues than in the tumor was achieved (13). In the present paper, distribution of an alpha-L-arabinofuranosidase from Fusarium species I 50 (11), inactive already at pH 7.0 (37 degrees C), was studied in tumor-bearing mice. The activity of this enzyme could be enriched under various conditions in the tumor, and especially favorable proved to be the additional application of a combination of glucose and inorganic phosphate. Under these conditions, a higher activity than in the tumor was demonstrable only in the kidney, which can possibly be eliminated in larger experimental animals by diuretics or an appropriate alkaline administration. The investigations have shown that the pH-labile alpha-L-arabinofuranosidases, especially those of Fusarium sp., due to their pharmacokinetic behavior are better suited for use in our therapy concept than the hitherto employed enzyme from A. niger. More recently, Tietze (16) has proposed a similar therapy concept, in which also the glucose-increased pH difference between tumor and normal tissue using tumor-own enzymes, exogenous enzymes as well as transport forms of cancerostatic agents spontaneously hydrolysing under weakly acidic pH conditions is to be exploited.