A Metalloproteinase Cocktail from the Venom of Protobothrops flavoviridis Cleaves Amyloid Beta Peptides at the α-Cleavage Site.

A disintegrin and metalloproteinase (ADAM) family proteins are a major class of membrane-anchored multidomain proteinases that are responsible for the shedding of cell surface protein ectodomains, including amyloid precursor protein (APP). Human ADAM 9, 10, and 17 proteolyze APPs and produce non-amyloid-genic p3 peptides, instead of neurotoxic amyloid-ß peptides (Aßs; Aß40 and Aß42), which form fibrils and accumulate in the brain of patients with Alzheimer's disease (AD). The ADAM family is closely related to snake venom metalloproteinases (SVMPs), which are derived from ancestral ADAMs but act as soluble proteinases. To test the therapeutic potential of SVMPs, we purified SVMPs from Protobothrops flavoviridis venom using metal ion affinity and pooled into a cocktail. Thus, 9 out of 11 SVMPs in the P. flavoviridis genome were identified in the cocktail. SVMPs inhibited Aß secretion when added to human cell culture medium without affecting APP proteolysis. SVMPs degraded synthetic Aß40 and Aß42 peptides at the same cleavage site (α-site of APP) as ADAM9, 10, and 17. SVMPs did not degrade Aß fibrils but interfered with their formation, assessed using thioflavin-T. Thus, SVMPs have therapeutic potential for AD as an Aß-degrading protease, and the finding adds to the discovery of bioactive peptides from venoms as novel therapeutics.

A review of the fate of inhaled α-quartz in the lungs of rats.

The distribution of dust particles within the lungs and their excretion are highly associated with their pulmonary toxicity. Literature was reviewed to discern pulmonary translocation pathways for inhaled α-quartz compared to those for inhaled TiO2. Accordingly, it was hypothesized α-quartz particles in the alveoli were phagocytized by alveolar macrophages but silica-containing macrophages remained in the alveoli for longer time in contrast to the rapid elimination from the alveoli seen for TiO2-containing macrophages. In addition, it was presumed that free silica particles are translocated in the interstitium, possibly through the cytoplasm of Type I epithelial cells, as observed with TiO2. Free silica particles are presumed to be phagocytized by interstitial macrophages soon after the particles penetrate the interstitium; these dust cells are then translocated to the ciliated airway regions in the lumen through bronchus-associated lymphoid tissue (BALT). The pulmonary retention half-time of dust particles in rats exposed to α-quartz is several times longer than that of rats exposed to TiO2, as long as the lung dust burden is ≈ 3 mg. The reduced pulmonary particle clearance ability in rats exposed to α-quartz aerosol is presumably attributed to the long-term retention of dust cells both in the alveoli and in the interstitium; this retention may be caused by the reduced chemotactic abilities of α-quartz-containing dust cells. However, the accumulation of α-quartz-containing dust cells in the lungs is not associated with the occurrence of pulmonary inflammation.

A mechanistic review of particle overload by titanium dioxide.

Chronic exposure to titanium dioxide (TiO2) induces slight but significant pulmonary inflammation in experimental animals, and among potential mechanisms, particle overload is likely. Although mechanisms of particle overload are poorly understood, excess accumulation of dust particles in dust containing macrophages (dust cells) can impair their mobility, resulting in reduced clearance ability. Accordingly, retention half-times of inhaled TiO2 increase linearly with lung burden in rodents, and mathematical (Michaelis-Menten-like) models for pulmonary clearance rates of TiO2 as a function of lung burden have suggested an alternative mechanism for particle overload, involving excess accumulation of macrophages in the translocation pathway due to the narrow exit to the ciliated airway region, and leading to reduced pulmonary TiO2 clearance rates. This mechanism is consistent with observations showing that TiO2 retention half-times in the lungs of rats and mice show no change from the initial value until the lung burden exceeds around mass burden of 3 mg or surface area burden of 1000 cm2. In addition, excess accumulation of macrophages is consistent with several particle overload-associated pathological changes, including accelerated neutrophilic inflammation, elevation of lymph node burden, and epithelial cell hyperplasia. Because excessive alveolar accumulation of macrophages may accelerate interstitialization of macrophages (including dust cells) and subsequent migration into lymph nodes, alveolar macrophages and dust cells likely migrate into interstitial spaces and escape to the luminal side of the ciliated airway region.

A mechanistic review of silica-induced inhalation toxicity.

Crystalline forms of silica have been proposed as positive control material for the toxicity test of inhaled particulate/fibrous matter, although mechanism of silica-induced inhalation toxicity has not yet been established. Inhalation exposure of α-quartz to rodents induces severe lung inflammation and fibrosis only after a certain period of latency, despite strong surface reactivity. The delayed occurrence of inhalation toxicity by α-quartz may be largely attributed to the sequestration of α-quartz particles by alternatively activated (M2) macrophages that express abundant levels of scavenger receptors but are relatively insensitive to inflammatory stimuli. When exposure to α-quartz continues, lung dust burden reaches a particle overload level, at which M2 macrophages cannot accommodate further quartz particles. Free quartz particles distributed in the interstitium interact with another subtype of macrophages, classically activated/inflammatory (M1) macrophages, which secrete various inflammatory cytokines, but silica-laden M1 macrophages initiate granuloma formation, which sequesters silica particles, too. Furthermore, the ability of M2 macrophages to clear foreign matter, particularly bacterial endotoxins [lipopolysaccharides (LPS)], may decrease due to α-quartz cytotoxicity. When LPS concentration in the lung reaches a certain level, LPS primes M1 macrophages to prepare for interleukin-1ß production in response to α-quartz and also stimulates M1 macrophages and plasmacytoid dendritic cells (pDCs) to produce tumor necrosis factor (TNF)-α and interferon (IFN)-ß, respectively. Besides, IFN-ß may enhance TNF-α production in LPS-stimulated M1 macrophages. The elevated levels of inflammatory cytokines produce progressive lung inflammation and fibrosis.

Interstitial duplication of 2q32.1-q33.3 in a patient with epilepsy, developmental delay, and autistic behavior.

Duplications of the 2q33 region are rare; to date, only 13 patients have been reported to have this chromosomal abnormality. The reported duplications are of varying size, and the patients shared developmental delay and minor dysmorphic findings. In this study, we identified a duplication of 2q32.1-q33.3 in a patient with psychomotor developmental delay, epilepsy, and autistic behavior. The duplicated region of this patient was reciprocal to the 2q32-q33 deletion syndrome. Chromosomal microarray testing confirmed the 19.5 Mb of duplication that includes over 100 genes, some of which could have functional relevance to the neurological features of this patient. The SATB homeobox 2 gene (SATB2)-the primary gene responsible for the 2q32-q33 deletion syndrome-may be one of them, because of its expression in the cortical projection neurons of the developing brain. The duplication of the potassium channel tetramerisation domain-containing 18 gene (KCTD18) and the ADAM metallopeptidase domain 23 gene (ADAM23) may also contribute to the phenotype. FISH analysis confirmed a tandem configuration of the duplicated segments. This result is in agreement with our previous study, in which we observed that duplicated segments as interstitial duplications are generally inserted in the tandem configuration.

Estimating health risk from exposure to 1,4-dioxane in Japan.

Exposure to 1,4-dioxane from the atmosphere around high-emission plants and from consumer products used in daily life that contain the substance may have adverse health effects; however, its emission into the atmosphere is not regulated. In this study, the health risk posed by 1,4-dioxane is assessed to investigate whether measures should be undertaken to reduce exposure to 1,4-dioxane. The notion of the margin of exposure (MOE), given by the ratio of no observed adverse effect level (NOAEL) to actual or projected exposure level, is used to assess risk. In exposure assessment, two types of exposure channel are considered: (a) the use of consumer products that contain 1,4-dioxane and (b) the inhalation of air around high-emission plants. To estimate exposure via channel (a), we measured the concentration of 1,4-dioxane in consumer products and estimated the interindividual variability of exposure by Monte Carlo simulation that reflects the measured data. To estimate exposure via channel (b), we employed a local-level atmospheric dispersion model to estimate the concentration of 1,4-dioxane immediately around high-emission plants. For hazard assessment, we derived the inhalatory and oral NOAELs for liver adenomas and carcinomas and the uncertainty factor. The results suggest that measures are not needed to reduce exposure to 1,4-dioxane from consumer products. As for inhalation exposure around high-emission plants, some residents may be exposed to health risks if certain conservative analytical conditions are assumed. Even in this case, we conclude that it is not necessary for Plant A to stop the use of 1,4-dioxane immediately and that medium- to long-term emission reduction measures should be sufficient.

Research strategies for safety evaluation of nanomaterials, part VIII: International efforts to develop risk-based safety evaluations for nanomaterials.

The use of nanotechnology in consumer and industrial applications will likely have a profound impact on a number of products from a variety of industrial sectors. Nanomaterials exhibit unique physical/chemical properties and impart enhancements to engineered materials, including better magnetic properties, improved electrical activity, and increased optical properties. The United States, Europe, and Japan have each initiated comprehensive programs to promote and expand the utility of nanotechnology for commercial applications. An important component of these programs is the development of reliable risk and safety evaluations for these materials to ensure their safety for human health and the environment. The scope of each of these programs includes efforts to assess the hazards posed by nanomaterials in realistic exposure conditions.

Impact of cytidine deaminase activity on intrinsic resistance to cytarabine in carcinoma cells.

Cytarabine (araC) is a highly active antimetabolite against hematological malignancy while the agent shows limited activity against carcinomas. In this study, we focused on cellular transport and catalysis of the nucleoside in order to elucidate the mechanism of intrinsic resistance to araC in carcinomas. Activities of two metabolizing enzymes for araC, deoxycytidine kinase (DCK) and cytidine deaminase (CDA), and cellular transport of the agent were examined in 9 carcinoma cell lines. These variables in carcinoma lines were compared with those in 14 araC-sensitive leukemia lines and one leukemia line with acquired resistance. The mean IC50 in 9 carcinoma lines was 3 x 10(3)-fold higher than that in 14 leukemia lines (4.6 x 10(3) vs. 1.3 microM, p<0.01). A cell line with acquired resistance (U937R), which was established from U937 monocytoid leukemia cells, showed more than 10(3)-fold higher IC50 than the parent cells (1.6 x 10(3) vs. 1.3 microM). The resistance in carcinomas was associated with higher CDA activity and lower influx when compared to araC sensitive leukemias. Especially, these two types of malignant cell lines were clearly distinguished by CDA activity. The acquired resistance in U937R cells was followed by increase in cytidine deaminase (CDA) activity, decrease in DCK activity and decrease in influx of the drug. In conclusion, carcinomas are intrinsically resistant to cytarabine through high CDA activity and low cellular transport, but not low DCK activity. This finding suggests that treatment of carcinoma with deoxycytidine analogues should conquer the high CDA activity.

Fever as a risk factor causing delayed elimination of methotrexate in pediatric patients receiving high doses of cancer chemotherapy.

PURPOSE: Delayed elimination of methotrexate (MTX) has been reported to be caused by a number of factors. In order to identify these causes, we retrospectively investigated the risk factors for delayed elimination in pediatric patients who received high doses of MTX. SUBJECTS AND METHODS: The study included 69 courses of therapy involving 22 patients who received more than 1000 mg/m(2) of MTX. Plasma MTX concentrations 48 h (C48) and 72 h (C72) after infusion were used as indices of MTX elimination. RESULTS: Neither C48 nor C72 was directly proportional to the dose of MTX infused. Both C48 and C72 were significantly higher in patients who developed fever of above 38 degrees C within a 10-day period (i.e., from 7 days before to 3 days after infusion) than in patients with no fever. Subgroup analysis revealed that C72 was significantly higher in patients who either developed fever and received antipyretics or developed fever but did not receive antipyretics than in patients who did not develop fever and did not receive antipyretics. Changes in the serum creatinine concentrations before and after MTX infusion revealed that renal function was significantly decreased in patients who developed fever compared to patients without fever. CONCLUSIONS: The present results suggest that the development of fever is one of the main risk factors for the delayed elimination of MTX.

Recent advances in the treatment of infant acute myeloid leukemia.

Infant acute myeloid leukemia (AML) of less than 12 months old is generally characterized by a high incidence of acute monoblastic or myelomonoblastic leukemia with hyperleukocytosis and extramedullary involvement. Most of the leukemic cells have 11q23 translocations, which lead to the MLL gene rearrangements. The MLL gene rearrangements occur at a high frequency in monoblastic subtype, hyperleukocytosis or young age in infant AML. Compared with acute lymphoblastic leukemia, however, it remains unknown whether prenatal origin exists in the pathogenesis of infant AML. Recently, the treatment outcome of infant AML has been clarified by two study groups, which confirmed the effect of intensive chemotherapy including repeated cycles of cytarabine and anthracyclines for infant AML. Presence of the MLL gene rearrangements, gender, age and white blood cell count showed no influence on the outcome of infant AML. The allogeneic hematopoietic stem cell transplantation (HSCT) remains the treatment of choice for infant AML when a matched related donor is available. Monitoring of minimal residual disease by real-time PCR is a useful technique to predict the outcome or efficacy of the treatment in infant AML. Although intensive chemotherapy and/or allogeneic HSCT have cured most AML infants, some still relapse and ultimately die. A need remains for future development by exploiting the unusual biologic properties of leukemic progenitor cells expressing the abnormal MLL gene product.

Risk-directed treatment of infant acute lymphoblastic leukaemia based on early assessment of MLL gene status: results of the Japan Infant Leukaemia Study (MLL96).

We studied the effectiveness of risk-directed therapy for infants younger than 13 months of age with acute lymphoblastic leukaemia (ALL). Fifty-five infants were assigned to different treatment programs (from December 1995 to December 1998) on the basis of their MLL gene status at diagnosis. Forty-two cases (76.3%) had a rearranged MLL gene (MLL+) and were treated with remission induction therapy followed by sequential intensive chemotherapy, including multiple genotoxic agents (MLL9601 protocol). Haematopoietic stem cell transplantation (HSCT) was attempted if suitable donors were available. Thirteen infants (23.7%) were classified as MLL- and treated for 2.5 years with intensive chemotherapy for high-risk B-ALL (MLL9602 protocol). Complete remission was induced in 38 of the 42 infants (90.5%) with MLL+ ALL and in all 13 patients (100%) with MLL- disease. In the MLL+ subgroup, the estimated event-free survival (EFS) rate at 3 years post diagnosis was 34.0% +/- 7.5%, compared with 92.3% +/- 7.4% in the MLL- subgroup (overall comparison, P = 0.001 by log-rank analysis). Both age less than 6 months (hazard ratio = 6.87, 95% CI = 0.91-52.3; P = 0.013) and central nervous system (CNS) involvement at diagnosis (hazard ratio = 2.92 95% CI = 1.29-6.63; P = 0.015) were significant independent predictors of an inferior outcome. These findings indicate a strategic advantage in classifying infant ALL as either MLL+ or MLL- early in the clinical course and selecting therapy accordingly. Standard chemotherapy for high-risk B-lineage ALL appeared adequate for MLL- cases. Novel therapeutic initiatives are warranted for infants with MLL+ disease, particularly those with initial CNS leukaemic involvement or age less than 6 months, or both.

Increased Deoxycytidine Kinase mRNA Level After Treatment with Interleukin-3.

Pretreatment of IL-3 to Kasumi-1 human acute myeloid leukemia cells enhanced 1-B-D-arabinofuranosyl cytosine (ara-C) cytotoxicity 1.2. to 1.4-fold (median 1.3). To clarify the mechanism of interleukin-3 (IL-3) on ara-C cytotoxicity, we investigated the level of deoxycytidine kinase mRNA with the competitive polymerase chain reaction method and enzyme activities, the incorporation of [(3)H] ara-C into DNA and intracellular ara-cytidine triphosphate (CTP) levels with high-performance liquid chromatography and analyzed cell cycles. The level of deoxycytidine kinase mRNA showed a fourfold increase (88.3 plus minus 4.33 amol &mgr;g of total RNA) at 3 days after treatment with IL-3 compared to control (20.3 plus minus 4.33 amol &mgr;g). After IL-3 treatment, ara-C incorporation into the DNA was increased to 1.33 to 1.83-fold (median, 1.73-fold). The G0/G1 late-phase and S-phase percentages of cells were increased from 28.99 to 78.73% in the IL-3 treatment group as compared to control. These results indicate that IL-3 pretreatment increases the level of deoxycytidine kinase mRNA and ara-C incorporation into the DNA and also increases ratios of G0/G1 late-phase and S-phase subsequent to an enhancement of ara-C cytotoxicity against leukemia cells.