The C-terminal region of the major outer sheath protein of Treponema denticola inhibits neutrophil chemotaxis.

Treponema denticola is an oral spirochete strongly associated with severe periodontal disease. A prominent virulence factor, the major outer sheath protein (Msp), disorients neutrophil chemotaxis by altering the cellular phosphoinositide balance, leading to impairment of downstream chemotactic events including actin rearrangement, Rac1 activation, and Akt activation in response to chemoattractant stimulation. The specific regions of Msp responsible for interactions with neutrophils remain unknown. In this study, we investigated the inhibitory effect of truncated Msp regions on neutrophil chemotaxis and associated signaling pathways. Murine neutrophils were treated with recombinant protein truncations followed by assessment of chemotaxis and associated signal pathway activation. Chemotaxis assays indicate sequences within the C-terminal region; particularly the first 130 amino acids, have the strongest inhibitory effect on neutrophil chemotaxis. Neutrophils incubated with the C-terminal region protein also demonstrated the greatest inhibition of Rac1 activation, increased phosphoinositide phosphatase activity, and decreased Akt activation; orchestrating impairment of chemotaxis. Furthermore, incubation with antibodies specific to only the C-terminal region blocked the Msp-induced inhibition of chemotaxis and denaturing the protein restored Rac1 activation. Msp from the strain OTK, with numerous amino acid substitutions throughout the polypeptide, including the C-terminal region compared with strain 35405, showed increased ability to impair neutrophil chemotaxis. Collectively, these results indicate that the C-terminal region of Msp is the most potent region to modulate neutrophil chemotactic signaling and that specific sequences and structures are likely to be required. Knowledge of how spirochetes dampen the neutrophil response is limited and Msp may represent a novel therapeutic target for periodontal disease.

Long-term findings from COMFORT-II, a phase 3 study of ruxolitinib vs best available therapy for myelofibrosis.

Ruxolitinib is a Janus kinase (JAK) (JAK1/JAK2) inhibitor that has demonstrated superiority over placebo and best available therapy (BAT) in the Controlled Myelofibrosis Study with Oral JAK Inhibitor Treatment (COMFORT) studies. COMFORT-II was a randomized (2:1), open-label phase 3 study in patients with myelofibrosis; patients randomized to BAT could crossover to ruxolitinib upon protocol-defined disease progression or after the primary end point, confounding long-term comparisons. At week 48, 28% (41/146) of patients randomized to ruxolitinib achieved ⩾35% decrease in spleen volume (primary end point) compared with no patients on BAT (P<0.001). Among the 78 patients (53.4%) in the ruxolitinib arm who achieved ⩾35% reductions in spleen volume at any time, the probability of maintaining response was 0.48 (95% confidence interval (CI), 0.35-0.60) at 5 years (median, 3.2 years). Median overall survival was not reached in the ruxolitinib arm and was 4.1 years in the BAT arm. There was a 33% reduction in risk of death with ruxolitinib compared with BAT by intent-to-treat analysis (hazard ratio (HR)=0.67; 95% CI, 0.44-1.02; P=0.06); the crossover-corrected HR was 0.44 (95% CI, 0.18-1.04; P=0.06). There was no unexpected increased incidence of adverse events with longer exposure. This final analysis showed that spleen volume reductions with ruxolitinib were maintained with continued therapy and may be associated with survival benefits.

Long-term self-renewal of postnatal muscle-derived stem cells.

The ability to undergo self-renewal is a defining characteristic of stem cells. Self-replenishing activity sustains tissue homeostasis and regeneration. In addition, stem cell therapy strategies require a heightened understanding of the basis of the self-renewal process to enable researchers and clinicians to obtain sufficient numbers of undifferentiated stem cells for cell and gene therapy. Here, we used postnatal muscle-derived stem cells to test the basic biological assumption of unlimited stem cell replication. Muscle-derived stem cells (MDSCs) expanded for 300 population doublings (PDs) showed no indication of replicative senescence. MDSCs preserved their phenotype (ScaI+/CD34+/desmin(low)) for 200 PDs and were capable of serial transplantation into the skeletal muscle of mdx mice, which model Duchenne muscular dystrophy. MDSCs expanded to this level exhibited high skeletal muscle regeneration comparable with that exhibited by minimally expanded cells. Expansion beyond 200 PDs resulted in lower muscle regeneration, loss of CD34 expression, loss of myogenic activity, and increased growth on soft agar, suggestive of inevitable cell aging attributable to expansion and possible transformation of the MDSCs. Although these results raise questions as to whether cellular transformations derive from cell culturing or provide evidence of cancer stem cells, they establish the remarkable long-term self-renewal and regeneration capacity of postnatal MDSCs.

Mobilisation of cadmium by meso- and racemic-2,3-dimercaptosuccinic acid (DMSA) in rats.

A higher efficiency of cadmium binding with racemic than with meso-2,3-dimercaptosuccinic acid (rac-DMSA; meso-DMSA) was found in an in vitro speciation model by Fang et al. (1996). This finding has not yet been tested in vivo. This paper presents results on mobilisation of cadmium by meso- and rac-DMSA in rats. Cadmium chloride was administered as the radioactive isotope 109Cd intraperitoneally to all animals. One group was an untreated control and two groups were treated with meso- and rac-DMSA, respectively. Treatment with chelators was applied twice, immediately after 109Cd and 24 hr afterwards intraperitoneally at the dose of 1 mmol/kg, each. Six days later radioactivity was measured in the liver and kidneys. Whole-body counting was carried out on days 1, 2, 3 and 6 of the experiment. At the end of the experiment, both treatments caused a decrease in 109Cd whole-body retention with rac-DMSA being more efficient (decrease from 83% in control to 74% and 64% in groups treated with meso- and rac-DMSA, respectively). The same reduction of 109Cd was obtained by both chelators in the liver (from 57% to about 47%). In the kidney only rac-DMSA produced significant reduction of 109Cd (from 5.3% to 3.5%). In conclusion, these results show modest reduction of cadmium in the body by two isoforms of DMSA with rac-DMSA being slightly more efficient than meso-DMSA.

Chelation therapy in aluminum-loaded rats: influence of age.

The influence of age at which aluminum (Al) exposure was initiated on the efficacy of chelation therapy in mobilizing Al was investigated in two groups of male rats exposed to this element at two different stages of the life cycle. Young (21 days old) and old (18 months) rats were exposed to 0 and 50 mg Al/kg/day administered as Al nitrate in drinking water for a preliminary period of 14 days followed by a period of 100 days, in which Al-exposed animals received 100 mg Al/kg/day. At the end of the period of exposure, Al-loaded rats in each age group were given one of the following treatments: s.c. deferoxamine (DFO), oral 1,2-dimethyl-3-hydroxypyrid-4-one (L1) and 1-(p-methylbenzyl)-2-ethyl-3-hydroxypyrid-4-one (MeBzEM) at doses of 0.89 mmol/kg/day for 5 consecutive days. Another group of Al-exposed rats received a concurrent administration of s.c. DFO and oral L1 both at 0.45 mmol/kg/day. During chelation therapy urines were collected daily. Control groups included rats exposed and unexposed to Al. Oral administration of L1 was the most effective treatment in enhancing urinary Al excretion in both age groups of Al-loaded rats. This beneficial effect was similar for old and young animals. Concurrent administration of DFO and L1 had no advantages over the use of either single agent, while MeBzEM was not effective in mobilizing Al from Al-exposed rats.

Estrogen receptor immunoreactivity in the adult primate brain: neuronal distribution and association with p75, trkA, and choline acetyltransferase.

The neuroactive steroid hormone, estrogen, has been implicated in both the prevention and treatment of Alzheimer's disease. Interactions between estrogen and neurotrophic systems may partially explain the beneficial effects of estrogen therapy. Previous studies have identified estrogen binding sites colocalized with neurotrophin-related proteins and mRNA within the rodent brain. Extending these studies to a model more relevant to human systems, we have mapped the distribution of estrogen receptor alpha (ER-alpha)-immunoreactive neurons in adult nonhuman primate brains. In addition, we used double-label immunohistochemistry to examine colocalization of ER-alpha with the low- and high-affinity neurotrophin receptors, p75 and trkA, and with the cholinergic marker choline acetyltransferase. Large numbers of ER-alpha-immunoreactive cells were detected in several amygdaloid and hypothalamic nuclei. ER-alpha-labeled cells were also found in the lateral septum, nucleus of the stria terminals, subfornical organ, and periaqueductal gray. Only rare, scattered ER-alpha-immunoreactive cells were noted in the cholinergic basal forebrain. In contrast to rodents, no cells exhibited ER-alpha and p75 or ER-alpha and trkA double-labeling. However, ER-labeled neurons in the amygdala, a region containing putative nerve growth factor-producing cells and exhibiting a role in memory, were densely and specifically invested with cholinergic terminals projecting from the basal forebrain. Estrogen-labeled neurons were also present in the lateral septal nucleus, a system that receives hippocampal inputs and projects to the neurotrophin-sensitive medial septum. Thus, interactions between neurotrophin-sensitive neurons and ER-bearing neurons exist in the primate brain, providing a potential paracrine basis for estrogen-state modulation of vulnerability to Alzheimer's disease.

Comparative aluminum mobilizing actions of deferoxamine and four 3-hydroxypyrid-4-ones in aluminum-loaded rats.

The efficacy of the Al chelating drugs deferoxamine (DFO) and the hydoxypyridones (HPs): 1,2-dimethyl-3-hydroxypyrid-4-one (L1), 1-[3-hydroxy-2-methyl-4-oxopyridyl]-2-ethanesulfonic acid (L6), 1-benzyl-(4-carboxylic acid)-3-hydroxy-2-methyl-4-oxopyridine (Bzcal) and 1-(p-methylbenzyl)-2-ethyl-3-hydroxypyrid-4-one (MeBzEM) in increasing Al excretion and reducing tissue Al accumulation has been compared in adult male rats which had previously received Al nitrate nonahydrate i.p. at 0.16 mmol/kg per day for 2 months. At the end of this period, DFO was injected s.c. and the HPs were given by gavage at 0.89 mmol/kg per day for five consecutive days. Total urines were collected 24 h after each chelator administration. Following chelation treatment animals were killed and samples of brain, bone, liver, kidney, and spleen were collected. DFO administration increased to about 4 x the cumulative urinary Al elimination for 5 days, while the excretion of Al into urine caused by Bzcal, L1, and MeBzEM administration was about twice that of the control group. On the other hand, treatment with Bzcal, DFO, and MeBzEM for 5 days significantly reduced the Al levels in bone by 31, 33, and 29%, and the Al concentrations in brain by 46, 69, and 71%, respectively. These results suggest that oral administrations of MeBzEM and Bzcal can be potential alternatives to parenteral administration of DFO in Al removal.

Hereditary hemochromatosis.

Genetic (hereditary) hemochromatosis is probably the most common autosomal recessive disorder found in white Americans, of whom about 5/1,000 (0.5 percent) are homozygous for the associated gene. The hemochromatosis gene is probably located close to the HLA-A locus on the short arm of chromosome 6. Homozygous individuals may develop severe and potentially lethal hemochromatosis, especially after age 39. Hereditary hemochromatosis involves an increased rate of iron absorption from the gut with subsequent progressive storage of iron in soft organs of the body. Excess iron storage eventually produces pituitary, pancreatic, cardiac, and liver dysfunction and death may result from cardiac arrhythmias, congestive heart failure, and/or hepatic failure or cancer. Early diagnosis can prevent these excess iron-induced problems. Iron overload owing to HLA-linked hereditary hemochromatosis can be distinguished from other causes of hemochromatosis by liver biopsies and interpretations. Patients at risk for genetic hemochromatosis should be screened, identified, and treated as early as age 20 to prevent or minimize the deadly complications of hemochromatosis. Population screening should include measurements of serum iron concentration, total iron binding capacity (TIBC), percent saturation of transferrin, and serum ferritin concentrations. Family members of hereditary hemochromatosis patients are at increased risk and should be tested. Screening, identification and early treatment (phlebotomies, sometimes in combination with the use of Desferal or other iron-chelating agents) may help prevent or reduce iron-related organ damage and premature deaths. Early diagnosis and treatment will reduce the population of aging individuals with severe, complicated hemochromatosis and dramatically reduce medical costs (billions of U.S. dollars per annum) associated with the management of this disease.

Misoprostol and attempted self-induction of abortion.

PIP: Sporadic reports have documented the use, in countries such as Brazil and Mozambique, of misoprostol to self-induce pregnancy termination (PT). This paper presents the case of a young woman from Mozambique who presented to a UK surgery requesting PT . She stated she was concerned about medication she had taken for epigastric pain in early pregnancy. She reluctantly acknowledged the medication was misoprostol obtained from a friend from Portugal. The patient had taken a total dose of 1000 mcg of misoprostol on the same day. After counseling on the potential adverse effects of misoprostol during pregnancy, specifically miscarriage and fetal abnormalities, the woman accepted but later declined a referral for induced abortion. She gave birth to a healthy infant. There is concern that the access to medical information from unregulated sources through the Internet will increase the potentially dangerous use of misoprostol as an abortifacient.^ieng

Cigarette smoking in a multiethnic population of youth: methods and baseline findings.

BACKGROUND: To expand upon recent research studies that have identified dramatic ethnic differences in adolescent cigarette smoking, this study was designed to characterize smoking among a multiethnic population of adolescents and to identify significant factors that may protect against smoking initiation. METHODS: During the first 2 years, this mixed cross-sectional, longitudinal study recruited and collected baseline data from a volunteer sample of 1,441 Houston-area public school students in the 5th, 8th, or 12th grade. A wide range of new and established predictors of smoking behavior was assessed, and their associations with ever smoking and susceptibility to smoking were assessed within ethnicity (white, N = 537; African-American, N = 454; and Hispanic, N = 297). RESULTS: Consistent with previous studies, white students smoked in substantially higher proportions than African-American students, with Hispanic adolescents in-between. Simultaneously adjusting for other variables, the odds of ever smoking (OR = 0.47, P < 0.01) and susceptibility to smoking (OR = 0.64, P < 0.01) were significantly lower among African-American adolescents when compared with whites; odds ratios for Hispanics and whites did not differ. Across all three ethnicities, the most important predictor of both ever smoking and susceptibility to smoking was the smoking status of the three best friends. Several ethnicity-specific variables also were identified. CONCLUSIONS: In concordance with previous investigations, cigarette smoking prevalence differs by ethnicity, and the factors associated with ever smoking and susceptibility to smoking differ among white, African-American, and Hispanic adolescents. The results of this study may be used to develop theory-based, culturally appropriate smoking intervention programs for adolescents.

Effect of cadmium chelating agents on organ cadmium and trace element levels in mice.

In experiments performed on male mice (CD-1, Charles River), the mobilizing effects of repeated administration of the carbodithioate analogue BLDTC [N-benzyl-4-O-(beta-D-galactopyranosyl)-D-glucamine-N-carbodithioate+ ++] and CaDTPA (calcium trisodium pentetate) on cadmium deposits in the liver, kidneys, brain and testes were compared. The antidotes were injected alternately every 48 h over a period of 16 d (8 doses in total) following a previous loading with 20 doses of CdCl2.2.5 H2O (single doses of 3 mg kg-1 i.p.). The experiments confirmed BLDTC to be one of the most effective cadmium mobilizing agents. The administration of CaDTPA, which is known as a useful antidote in acute cadmium intoxication, increased the mobilizing effect of BLDTC. Cadmium elevated the concentration of zinc in all organs examined and the level of copper in the liver, kidneys and testes. This accumulation of trace elements was only partially corrected by the chelators. The antidotes administered alone exert only a negligible effect on the trace element levels in the organs.

Cadmium-induced apoptosis in the urogenital organs of the male rat and its suppression by chelation.

Cadmium-induced apoptosis is shown to occur, in vivo, in several organs of the male Wistar rat urogenital system, 48 h after cadmium administration i.p. at a dose of 0.03 mmol/kg. Characteristic DNA fragmentation (as measured by an enzyme-linked immunosorbent-assay, ELISA) and histopathologically observed changes characteristic of apoptosis are found in the kidney, prostate, seminal vesicles, testes, and epididymis. TUNEL assay also demonstrates the apoptosis. Such changes are absent from bladder and vas deferens tissue. Timely administration of an appropriate chelating agent capable of reaching intracellular cadmium binding sites can suppress the processes leading to apoptosis. Administration of monoisomyl meso-2,3-dimercaptosuccinate (Mi-ADMS, 0.5 mmol/kg i.p.) to cadmium-treated rats is effective in greatly reducing typical histopathologic signs of apoptosis and the associated chromatin DNA fragmentation as revealed by ELISA when the antagonist is administered 1 h after cadmium. Administration of the chelating agent at law times results in greater degradation of DNA into oligonucleotides and more prominent histopathological evidence of apoptotic changes in the affected organs of the rat urogenital system. There is also a progressive increase in apoptotic changes indicated by TUNEL assay, as the antagonist is administered at progressively greater intervals after cadmium.

Reduction of subacute lethal radiotoxicity of polonium-210 in rats by chelating agents.

The protective effect of N,N'-di(2-hydroxyethyl)ethylene-diamine-N,N'-biscarbodithioate (HOEtTTC) against the subacute lethal radiotoxicity of polonium-210 was investigated in a survival study and by histopathological and haematological examinations of some organs and tissues in Sprague-Dawley rats. This effect was compared with that of N,N'-diethylamine-N-carbodithioate (diethy dithiocarbamate, DDTC). In the survival study, rats injected in intravenously solely with a lethal amount of 210Po (1.45 MBq kg-1 body mass) died within 14-44 days while 90% of rats treated with HOEtTTC survived for 5 months until sacrificed. When treated with DDTC all rats died within 36-93 days. In the histopathological examination, relevant changes resulting from incorporation of 210Po were found in lymph nodes, thymus and humeral bone marrow. After the treatment with HOEtTTC no pathological changes were observed. In the haematological examination, severe reduction in blood and femoral bone marrow (BM) cell counts was revealed in rats injected with 210Po. This reduction was reversed by treatment with HOEtTTC. Treatment with DDTC led only to partial recovery of blood and BM cell count. In conclusion, under the conditions of the experiment only HOEtTTC was fully effective in reducing subacute lethal radiotoxicity of 210Po.

Synthesis and in vitro trypanocidal activity of some novel iron chelating agents.

This report describes the syntheses and in vitro trypanocidal activity of a number of iron (III) chelators against epimastigotes of Trypanosoma cruzi. The compounds examined included a number of lipophilic N-alkyl derivatives of 2-ethyl- and 2-methyl-3-hydroxypyrid-4-ones, N,N'-bis(o-hydroxybenzyl)-(+/-)-trans-1,2-diaminocyclohexane, cyclotetrachromotropylene and four commercially available carboxy derivatives of pyridine, pyrazine, and pyarazole. Benznidazole, the drug clinically used in the treatment of Chagas' disease in humans, served as standard. All compounds were screened in vitro against Trypanosoma cruzi epimastigotes at 50 and 100 micrograms/ml for 72 h of exposure. At 100 micrograms/ml dosage, at least 4 compounds exhibited high epimastigote growth inhibition (65-69%) comparable to benznidazole (72%), whereas 9 compounds showed moderate to fair activity (53-64%) in the in vitro assay. At the lower concentration (50 micrograms/ml), the inhibitory activity of the best of these compounds was reduced significantly (39-48%) compared to the standard drug (59%). The activity of all the carboxylic acids remained in the lower range (4-25%). It is hypothesized that the enhanced activity of some of the compounds is due to their increased lipophilicity which enables them to successfully pass through the cellular membrane of Trypanosoma cruzi epimastigotes. The trypanocidal activities of the most effective compounds were significantly reduced when tested in the presence of added ferric ion.

Selenite suppression of cadmium-induced testicular apoptosis.

The characteristic apoptotic ladder-like patterns of rat testicular DNA on agarose gel electrophoresis which results from treatment with CdCl2 are suppressed by the administration of Na2SeO3. The examination of testicular tissue using an ELISA programmed cell death detection procedure confirmed this selenite suppression of cadmium-induced apoptosis. The administration of the Na2SeO3 at either 0.5, 1, 2 h prior to or 0.5, 1, 2 h after the administration of the CdCl2 appear to be almost equally effective at suppressing the apoptotic response. These results are in accord with previous studies on the Na2SeO3 suppression of cadmium induced necrotic changes in tissues and suggest that Na2SeO3 interferes with both necrosis and apoptosis.

Inhibition of Trypanosoma cruzi epimastigotes in vitro by iron chelating agents.

The relative effectiveness of 20 iron chelating agents in suppressing the growth and multiplication of Trypanosoma cruzi epimastigotes has been examined in vitro. 1,2-Dimethyl-3-hydroxypyrid-4-one (L1) and several of its newly synthesised N-substituted analogs containing hydrophobic substituents were significantly more effective than deferoxamine, even though they possess only two donor sites for iron(III) while deferoxamine has six. Analogs with hydrophilic substituents were uniformly less active than L1 itself. Variations in effectiveness as the polarity of the compound is varied indicate that the ability to cross the cellular membrane is of critical importance in the determination of the in vitro trypanocidal activity of iron(III) chelating agents. A group of four tris(2-aminoethyl)amine based tris-imines were also screened, all of which had poor activity (0-28% inhibition). Among the other iron(III) chelating agents which showed a relatively high level of activity at 50 and 100 micrograms/ml were salicylhydroxamic acid (70 and 73% inhibition) and hydroxyurea (42 and 52% inhibition). N,N'-Di(2-hydroxybenzyl)ethylenediamine-N,N'-diacetic acid and acetohydroxamic acid exhibited only slight activity at 50 and 100 micrograms/ml. The best of these iron(III) chelating agents were as effective against the epimastigote form at both 50 and 100 micrograms/ml (74-82% inhibition) as benznidazole (81% inhibition), the drug currently used in the clinic.

Meso-2,3-dimercaptosuccinic acid mono-N-alkylamides: syntheses and biological activity as novel in vivo cadmium mobilizing agents.

Three meso-2,3-dimercaptosuccinic acid mono-N-alkylamides (meso-RNHCOCH(SH)CH(SH)-COOH, where R = CHMe2, Mi-PDMA; CH2CHMe2, Mi-BDMA; and CH2CH2CHMe2, Mi-ADMA), were prepared via a synthetic route using the sulfhydryl-protected anhydride. 2,2-Dimethyl-1,3-dithiolane-4,5-cis-dicarboxylic acid anhydride was opened up with 1 mol of corresponding amine to give the SH-protected monoamide. Subsequent deblocking of the vicinal dithiol functionality was accomplished by conversion of the dithiolane into the mercury complex followed by reaction with H2S to give the target molecule. The potential utility of these compounds in chronic cadmium intoxication was examined by evaluation of their cadmium mobilizing efficacy in vivo in cadmium-loaded female albino rats using sodium N-benzyl-D-glucamine-N-carbodithioate (BGDTC) as the standard drug. Compared to BGDTC, the new compounds were, except at the highest dosage studied, equally or more effective in decreasing retention of hepatic cadmium, while mostly less effective in decreasing renal cadmium. The greatest reductions were obtained with Mi-BDMS at 4 x 1.5 mmol/kg, where liver and kidney cadmium levels were reduced to 12% and 59% of control levels, while at the same dosage BGDTC induced a reduction to 50% and 13% of control levels. The order of the efficacy of the monoamides as hepatic cadmium mobilizing agents was found to be Mi-PDMA > Mi-BDMA > Mi-ADMA. However, the isopropyl analog, though very effective at reducing hepatic cadmium at a low dosage, was found to be more toxic than the isobutyl and isoamyl monoamides. While the new compounds were shown to be effective cadmium mobilizing agents, the specific compounds examined did not possess optimized structures in terms of the balance between effectiveness and toxicity.

Structure of 5-hydroxy-2-hydroxymethyl-1-methylpyrid-4-one and 13C NMR relaxation studies of its gadolinium(III) complex.

In order to further elucidate the properties and biological behavior of 5-hydroxy-2-hydroxymethyl-1-methylpyrid-4-one (M1), its X-ray structure has been determined, and the ability of its gadolinium complex to enhance the relaxation of 13C nuclei has been examined. X-ray analysis using Mo K alpha radiation shows that M1 crystallizes in the monoclinic space group C2/c with a complex intermolecular array of hydrogen bonding. No water molecules were present within the unit cell. Gd(M1)2NO3 x 3H2O has been prepared and found to be very soluble in water. The effect of low concentrations of Gd(III) on enhancing the 13C relaxation times of M1 was examined. Trace amounts of Gd(NO3)3 x 6H2O resulted in significant decreases in the relaxation time of certain carbon atoms relative to the control measurements, and these data indicate that carbon atoms which bear donor atoms for Gd(III) undergo a significantly greater relaxation than the other carbons. The water solubility and hydrophilic character of this complex suggest that it may prove useful for the determination of metal binding sites on peptides and oligonucleotides.