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In August 2008, 30% of tomato (Solanum lycopersicum) plants in plots in Lubbock County, Texas showed yellowing, lateral stem dieback, upward leaf curling, enlargement of stems, adventitious roots, and swollen nodes. Yellowing in leaves was similar to that seen with zebra chip disease (ZC) of potato that was confirmed in a potato field 112 km away in July 2008 and was associated with a ‘Candidatus Liberibacter’ species (1), similar to findings earlier in 2008 in New Zealand and California (2,3). Tissue from four symptomatic plants of cv. Spitfire and two of cv. Celebrity were collected and DNA was extracted from midribs and petioles with a FastDNA Spin Kit (Qbiogene, Inc., Carlsbad, CA,). PCR amplification was done with 16S rRNA gene primers OA2 and OI2c, which are specific for “Ca. Liberibacter solanacearum” from potato and tomato and amplify a 1.1-kb fragment of the 16S rRNA gene of this new species (1,3). Amplicons of 1.1 kb were obtained from all samples and these were sequenced in both orientations (McLab, San Francisco, CA). Sequences of the 16S rRNA gene were identical for both Spitfire and Celebrity and were submitted to the NCBI as GenBank Accession Nos. FJ939136 and FJ939137, respectively. On the basis of a BLAST search, sequence alignments revealed 99.9% identity with a new species of ‘Ca. Liberibacter’ from potato (EU884128 and EU884129) in Texas (1); 99.7% identity with the new species “Ca. Liberibacter solanacearum” described from potato and tomato (3) in New Zealand (EU849020 and EU834130, respectively) and from the potato psyllid Bactericera cockerelli in California (2) (EU812559, EU812556); 97% identity with ‘Ca L. asiaticus’ from citrus in Malaysia (EU224393) and 94% identity with both ‘Ca. L. africanus’ and ‘Ca. L. americanus’ from citrus (EU921620 and AY742824, respectively). A neighbor-joining cladogram constructed using the 16S rRNA gene fragments delineated four clusters corresponding to each species, and these sequences clustered with “Ca. L. solanacearum”. A second PCR analysis was conducted with the CL514F/CL514R primer pair, which amplifies a sequence from the rplJ and rplL ribosomal protein genes of “Ca. L. solanacearum”. The resulting 669-bp products were 100% identical to a sequence reported from tomato in Mexico (FJ498807). This sequence was submitted to NCBI (GU169328). ZC, a disease causing losses to the potato industry, is associated with a ‘Candidatus Liberibacter’ species (1–3) and was reported in Central America and Mexico in the 1990s, in Texas in 2000, and more recently in other states in the United States (4). In 2008, a “Ca. Liberibacter solanacearum” was detected on Capsicum annuum, S. betaceum, and Physalis peruviana in New Zealand (3). Several studies have shown that the potato psyllid, B. cockerelli, is a potential vector for this pathogen (2,4). To our knowledge, this is the first report of “Ca. Liberibacter solanacearum” in field tomatoes showing ZC-like foliar disease symptoms in the United States. References: (1). J. A. Abad et al. Plant Dis. 93:108, 2009 (2) A. K. Hansen et al. Appl. Environ. Microbiol. 74:5862, 2008. (3) L. W. Liefting et al. Plant Dis. 93:208, 2009. (4) G. A. Secor et al. Plant Dis. 93:574, 2009.

The limited use of distiller’s grains (DG) in the food industry result out of negative effects on texture and flavour. To investigate the odour, aroma volatiles in the common cereal based food system bread were analyzed. Therefore aroma volatiles in bread containing 0–20% DG were identified with Gas Chromatography-Olfactometry/Mass Spectrometry. Likewise, sensory properties were evaluated. As a result, 42 odour active compounds were identified in DDG bread. Phenylacetic acid and dimethyltrisulphide are transferred from DG to bread crust and crumb, but not interfering bread aroma. After comparison of highest flavour dilution (FD) factors, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (FD = 128) and 2-ethyl-3,5-dimethylpyrazine (FD = 128) were revealed in control bread crust, whereas 3-hydroxy-4,5-dimethyl-2-(5H)-furanone (FD = 512) and 4-hydroxy-23,5-dimethyl-3-(2H)-furanone (FD = 512) were revealed in 20% DG bread crust. Regarding bread crumb, 3-methylthiopropanal and 2-phenylethanol provided highest FD factors (FD = 32) in the control, whereas in 20% DG bread crumb 3-methylbutanoic acid, 2-ethyl-3,5-dimethylpyrazine and 3-hydroxy-4,5-dimethyl-2-(5H)-furanone provided FD factors ≥ 32 as well. Principal component analysis (PCA) of bread samples correlated to sensory attributes and important aroma volatiles revealed differences in odorant perception to the presence of 3-hydroxy-4,5-dimethyl-2(5H)-furanone and phenylacetic acid, with simultaneous absence of 2AP.

Invasive zygomycosis in immunocompromised patients results in a high mortality rate, and early identification is crucial to optimize therapy and to reduce morbidity. However, diagnosing specific species of zygomycetes fungi possess challenge in the clinical laboratories. A need for a rapid and sensitive diagnostic tool for early recognition of a zygomycetes fungus in clinical samples to the species level will lead to prompt and accurate therapy and the PathoChip provides one such platform. We utilized a pathogen array technology referred to as PathoChip, comprised of oligonucleotide probes that can detect all the sequenced viruses as well as known pathogenic bacteria, fungi and parasites and family-specific conserved probes, thus providing a means for detecting previously uncharacterized members of a family. We rapidly identified a zygomycetous fungus, Rhizomucor pusillus, an otherwise challenge for the clinical laboratories, predominantly in a patient with acute myelogenous leukemia. This report highlights the value of PathoChip as a diagnostic tool to identify micro-organisms to the species level, especially for those difficult to identify in most clinical laboratories. It will also help clinicians to obtain a critical snapshot of the infection profile of a patient to plan treatment strategies.

Summary Objectives To investigate the risk factors and outcomes associated with Candida krusei bloodstream. Methods We performed a case control study of patients with C. krusei bloodstream infection at the University of Pennsylvania from 1982 to 2010. Controls were without candidemia, and matched to cases on duration of hospitalization and underlying disease. Results We enrolled 34 cases and 114 matched controls. Most subjects (62%) had hematologic malignancies. In the multivariate model, including a priori the duration of fluconazole use (OR 1.06; 95% CI 1.00, 1.11) and days of neutropenia (OR 1.01; 95% CI 0.98, 1.13), risk factors associated with C. krusei bloodstream infection were splenectomy (OR 11.66; 95% CI 1.04, 130.64), and exposure to antimicrobials with anaerobic activity (OR 5.74; 95% CI 1.76, 18.67). Outcomes of infected patients were poor. Only 32% of case patients survived to hospital discharge, compared to 89% of controls. For 48% death was attributed to C. krusei infection. Conclusions C. krusei bloodstream infection occurs most commonly in neutropenic patients with hematologic malignancy. The association with prior fluconazole exposure is less marked than previously described. Splenectomy and the receipt of antimicrobials with anaerobic activity are significant risk factors. The outcome of infected patients remains poor, despite appropriate antifungal therapy.

Ethanol fuel can be produced renewably from numerous plant and waste materials, but harnessing the energy of lignocellulosic feedstocks has been particularly challenging in the development of this alternative fuel as a substitute for petroleum-based fuels. Consolidated bioprocessing has the potential to make the conversion of biomass to fuel an economical process by combining enzyme production, polysaccharide hydrolysis, and sugar fermentation into a single unit operation. This consolidation of steps takes advantage of the synergistic nature of enzyme systems but requires the use of one or a few organisms capable of producing highly efficient cellulolytic enzymes and fermenting most of the resulting sugars to ethanol with minimal byproduct formation while tolerating high levels of ethanol. In this review, conventional ethanol production, consolidated bioprocessing, and simultaneous saccharification and fermentation are described and compared. Several wild-type and genetically engineered microorganisms, including strains of Clostridium thermocellum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escherichia coli, Flammulina velutipes, and Zymomonas mobilis, among others, are highlighted for their potential in consolidated bioprocessing. This review examines the favorable and undesirable qualities of these microorganisms and their enzyme systems, process engineering considerations for particular organisms, characteristics of cellulosomes, enzyme engineering strategies, progress in commercial development, and the impact of these topics on current and future research.

Recent reports describe increasing incidence of non-Aspergillus mold infections in hematopoietic cell transplant (HCT) and solid organ transplant (SOT) recipients. To investigate the epidemiology of infections with Mucorales, Fusarium spp., and Scedosporium spp. molds, we analyzed data from the Transplant-Associated Infection Surveillance Network, 23 transplant centers that conducted prospective surveillance for invasive fungal infections during 2001–2006. We identified 169 infections (105 Mucorales, 37 Fusarium spp., and 27 Scedosporium spp.) in 169 patients; 124 (73.4%) were in HCT recipients, and 45 (26.6%) were in SOT recipients. The crude 90-day mortality rate was 56.6%. The 12-month mucormycosis cumulative incidence was 0.29% for HCT and 0.07% for SOT. Mucormycosis incidence among HCT recipients varied widely, from 0.08% to 0.69%, with higher incidence in cohorts receiving transplants during 2003 and 2004. Non-Aspergillus mold infections continue to be associated with high mortality rates. The incidence of mucormycosis in HCT recipients increased substantially during the surveillance period.

HISTORY AND CLINICAL FINDINGS: A 65-year-old diabetic (requiring insulin during the last year) was admitted as an emergency because of a septic temperature rising to 40 degrees C with rigor, tachycardia (up to 120/min) and dyspnoea. On examination there was local reddening and swelling of the skin over the right thenar eminence and along the lower arm. Two days before admission a bad scratch had been inflicted on his right hand by a cat. He had first noticed the reddening and swelling 10 hours after the incident; 1 1/2 days after the scratch and 9-10 hours before hospitalization the first bouts of fever had occurred. EXAMINATIONS: The chest radiogram showed interstitial pneumonia. The clinical findings, the laboratory tests (white cell count 21 750/microliters, platelets 140,000/microliters, C-reactive protein 35 mg/l and positive blood cultures pointed to early septicaemia. The germ was identified as Pasteurella multocida two days after blood had been taken for culturing. HbA1c was 11.38%. TREATMENT AND COURSE: From the time of hospitalization the patient had been treated with ceftriaxon, 2 g daily intravenously, and also with erythromycin because atypical pneumonia had been the suspected diagnosis at first and acute chlamydia infection had at first not been excluded. The patient's general condition quickly improved and the fever started to go down a few hours after onset of treatment. Blood cultures became negative after the first administration of antibiotics. He was discharged in a good state on optimal insulin dosage. CONCLUSION: Pasteurella multocida is present in a high percentage of domestic animals and can be the cause of systemic infections in immunocompromised patients (e. g. poorly controlled diabetes mellitus).

Transplant recipients are at a high risk for developing invasive fungal infections. The agents of phaeohyphomycosis are environmental molds found worldwide, and they cause a broad spectrum of disease including skin and subcutaneous lesions, pneumonia, central nervous system disease, fungemia, and disseminated disease. Using data from the Transplant Associated Infection Surveillance Network (TRANSNET), we evaluated patients with proven and probable phaeohyphomycosis. Centers collected data on demographics, co-morbid conditions, clinical features, treatment, and three-month mortality. Fifty-six patients with phaeohyphomycosis were identified from 15 centers, comprising 26 stem cell transplant (SCT) and 30 solid organ transplant (SOT) recipients. Median time to diagnosis post-transplant was 358 days (SCT 100 days; SOT 685 days; P =

A randomized, blinded, multicenter trial was conducted to compare fluconazole (800 mg per day) plus placebo with fluconazole plus amphotericin B (AmB) deoxycholate (0.7 mg/kg per day, with the placebo/AmB component given only for the first 5-6 days) as therapy for candidemia due to species other than Candida krusei in adults without neutropenia. A total of 219 patients met criteria for a modified intent-to-treat analysis. The groups were similar except that those who were treated with fluconazole plus placebo had a higher mean (+/- standard error) Acute Physiology and Chronic Health Evaluation II score (16.8+/-0.6 vs. 15.0+/-0.7; P=.039). Success rates on study day 30 by Kaplan-Meier time-to-failure analysis were 57% for fluconazole plus placebo and 69% for fluconazole plus AmB (P=.08). Overall success rates were 56% (60 of 107 patients) and 69% (77 of 112 patients; P=.043), respectively; the bloodstream infection failed to clear in 17% and 6% of subjects, respectively (P=.02). In nonneutropenic subjects, the combination of fluconazole plus AmB was not antagonistic compared with fluconazole alone, and the combination trended toward improved success and more-rapid clearance from the bloodstream.

BACKGROUND With the availability of ICSI, men with severe oligozoospermia (

A microfluidic device was designed with two parallel laminar flow channels where non-motile spermatozoa and debris would flow along their initial streamlines and exit one outlet, whereas motile spermatozoa had an opportunity to swim into a parallel stream and exit a separate outlet. Motile sperm samples were prepared with density gradient separation (n = 5). Sperm motility was assessed the following day after exposing aliquots to polydimethylsiloxane (PDMS) used to construct the device. There was no difference in sperm motility when compared with unexposed aliquots (P > 0.05). Unprocessed semen samples (n = 10) were placed in wider channels and sperm motility and strict morphology were assessed from sorted outlets. Sperm motility increased from 44 +/- 4.5% to 98 +/- 0.4% (P < 0.05) and morphology increased from 10 +/- 1.05% to 22 +/- 3.3% (P < 0.05) following processing. Finally, density gradient prepared samples (n = 6) containing 5 x 10(6) motile spermatozoa/ml and 50 x 10(6) round immature germ cells/ml were sorted and assessed in a similar fashion. The ratio of motile spermatozoa to round immature germ cells in the wide inlet (1:10) was significantly improved in the thin outlet (33:1) (P < 0.05). This microfluidic device provides a novel method for isolating motile, morphologically normal spermatozoa from semen samples without centrifugation. This technology may prove useful in isolating motile spermatozoa from oligozoospermic samples, even with high amounts of non-motile gamete and/or non-gamete cell contamination. A movie sequence showing streaming and sorting of spermatozoa may be purchased for viewing on the internet at www.rbmonline.com/Article/847 (free to web subscribers).

Human infection with Mycoplasma species other than M. pneumoniae are infrequent, but may be encountered in patients with immunodeficiencies. We report a patient with combined variable immunodeficiency that developed multiple abscesses and destructive bone disease caused by M. orale, an organism generally considered to be non-pathogenic. Molecular laboratory methods, 16S rRNA sequence analysis, were used to detect the organism directly in the surgical specimen and confirmed following isolating of the organism. This case demonstrates the importance of molecular technology in the diagnosis of difficult infectious disease problems.

Invasive Fusarium infections occur in immunosuppressed patients, especially those with haematological malignancies. We conducted a descriptive analysis of data from patients with invasive fusariosis identified in the Prospective Antifungal Therapy Alliance registry, which collected data on invasive fungal infections in the United States and Canada from 2004 to 2008. In this series of 65 patients with proven (83.1%) and probable (16.9%) invasive fusariosis, the most common underlying condition was haematological malignancy, in which neutropenia and corticosteroid usage frequently occurred. Seven patients with invasive Fusarium infections had cross-reactive galactomannan assay results. The survival rate for all patients at 90 days was 44%, which was an improvement compared with historical data. Disseminated disease occurred frequently (35.4%), and patients with and without disseminated disease had survival rates of 33% and 50%, respectively. Posaconazole and voriconazole were the most frequently employed therapies and may be linked to the improved survival rate observed in this patient series. In summary, patients with invasive Fusarium infections continue to have high fatality rates, especially those with disseminated disease. Fusarium infections should be strongly considered in the absence of Aspergillus isolation in patients at high risk of mould infections with positive galactomannan assay test results.

WR 238605 is an 8-aminoquinoline developed for the radical cure of Plasmodium vivax. Forty-four P. vivax-infected patients were randomly assigned to 1 of 4 treatment regimens: 3 groups received a blood schizonticidal dose of chloroquine followed by WR 238605: group A (n=15) received 300 mg daily for 7 days; group B (n=11), 500 mg daily for 3 days, repeated 1 week after the initial dose; group C (n=9), 1 dose of 500 mg. A fourth group (D; n=9) received chloroquine only. Among patients who completed 2-6 months of follow-up (n=23), there was 1 relapse in group B (day 120) and 1 in group C (day 112). Among patients treated with chloroquine only, there were 4 relapses (days 40, 43, 49, and 84). WR 238605 was safe, well tolerated, and effective in preventing P. vivax relapse.

The effect of combining promethazine with chloroquine was examined against Plasmodium falciparum in vitro in the Aotus-P. falciparum model and in bioassays from volunteers given promethazine. The combination of chloroquine plus promethazine (1 x 10(-6) M) reversed chloroquine resistance in standard P. falciparum clones and patient parasite isolates from Nigeria. The combination reduced the 50% inhibitory concentrations (IC50s) for chloroquine against resistant parasites by 32-92%. Coadministration of promethazine with chloroquine also demonstrated a dose-dependent effect in Aotus monkeys infected with chloroquine-resistant P. falciparum. Monkeys were given a chloroquine dose (20 mg/kg of body weight for seven days), which normally has no effect on parasitemia, plus 10, 20, 40, or 80 mg of promethazine/kg of body weight. In one monkey, parasitemia was suppressed at the lowest promethazine dose, but re-treatment with 20 mg/kg resulted in clearance of parasitemia. Initial treatment with chloroquine and 20 or 40 mg/kg of promethazine cleared parasitemia in some animals followed by recrudescence. Re-treatment at higher doses cured one monkey and resulted in initial clearance and delayed recrudescence 28 or 63 days after treatment in two monkeys. Recrudescent parasitemia in the two monkeys was low (10 parasites/microl of blood) and subsequently cleared without re-treatment. An in vitro bioassay model was developed to examine the effects of clinically achievable doses of promethazine on parasites susceptibilities in vitro. Plasma samples taken at hourly intervals from patients given a single oral dose of 25 mg of promethazine decreased the IC50 values for chloroquine by 20-58% with the most significant reductions occurring in plasma obtained from volunteers 3-4 hr after ingestion. Plasma obtained from two volunteers 6 hr after ingestion of the drug demonstrated no effect on chloroquine susceptibility, suggesting that study of the pharmacokinetic disposition and potential interaction is warranted to optimize the dose regimen in patients for antimalarial efficacy. Historic use of this drug combination for treatment or prevention of chloroquine-associated pruritus or as an antiemetic suggest that the combination is safe and effective when used at standard dosages. The results from this study demonstrate that promethazine is a potent modulator of chloroquine resistance. Clinical evaluation of therapeutic regimens is required to validate clinical efficacy of this promising combination for treatment of uncomplicated chloroquine-resistant malaria.

This study was conducted to determine the pharmacokinetics and pharmacodynamics of pyridostigmine given as 30 mg of pyridostigmine bromide every 8 hours in healthy subjects. Plasma pyridostigmine concentration and red blood cell acetylcholinesterase activity were measured in blood samples collected during a 3-week period. Population analysis was performed using standard pharmacokinetic and pharmacodynamic models with the nonlinear mixed-effect modeling software (NONMEM). The pharmacokinetic model that best fit the pyridostigmine plasma levels was a two-compartment open model with first-order absorption, a lag time, and first-order elimination from the central compartment. The pharmacodynamic model that best fit red blood cell acetylcholinesterase activity was an inhibitory E max model with an effect compartment linked to the central compartment. The results showed that the pharmacokinetics of pyridostigmine bromide are both gender and weight dependent. The pharmacodynamic effect does not lag significantly from the plasma concentration and returns to near normal within 8 hours. With the present dosage regimen of 30 mg every 8 hours, 30% of individuals may not have red blood cell acetylcholinesterase inhibition >10% at the time of the trough.

The correlation of P. falciparum lactate dehydrogenase (pLDH) activities and patent infections was evaluated for monitoring therapeutic responses and drug resistance in 70 patients with microscopically confirmed P. falciparum malaria in Nigeria. Each patient was treated with standard dosages of artemether (53 patients), chloroquine (7 patients), sulfadoxine-pyrimethamine (6 patients), or halofantrine (4 patients). Response of infection to treatment was monitored by microscopic examination of thick and thin blood smears, clinical symptoms, and levels of pLDH activities in blood products. pLDH activity was determined using an antibody capture technique and 3-acetyl pyridine adenine dinucleotide developed to enhance sensitivity of the enzyme detection. All patients treated with artemether were cured while 5 patients treated with chloroquine, 1 treated with sulfadoxine-pyrimethamine, and 2 treated with halofantrine suffered recrudescent infections after treatment. pLDH activity was detected in blood products obtained from patients with patent or recrudescent infections determined by microscopy and clinical symptoms. Levels of pLDH activities in whole blood and packed cells from the patients correlated with qualitative detection of parasites in blood smears and in patients with high gametocyte counts. Gametocyte counts in the patients after treatment ranged from 40 gametocytes/microliter of blood to 4923 gametocytes/microliter of blood. There is a consistent relationship between patent infection and pLDH activities that could easily be determined in whole blood and packed cells from the patients. Further development of the procedure will enhance its valuable application in clinical management of drug-resistant malaria in the endemic areas.

In orthodontics, removable acrylic appliances are preferably produced from methylmetacrylates. However, in the adjacent oral mucosal tissue, cell damage may be caused by the evaporation of residual monomer. The aim of this study was to modify the classic agar overlay assay in order to apply histochemical methods by comparing conventionally used transformed mouse fibroblasts (L-929) with primary human gingival fibroblasts to further elucidate the term toxicity. While proliferation was assessed via the incorporation of the base analogon bromdesoxyuridine, differentiation was investigated by detection of the fibroblast-specific intermediate filament vimentin. After the monomers had taken effect, reduced proliferation extending over the inhibition area was observed by indirect immunofluorescence, independent of cell type. In contrast to this observation, a few cells within the inhibition area which could not be clearly detected by neutral red staining still exhibited mitotic activity. Detection of the differentiation-specific intermediate filament vimentin was comparable with the degree of neutral red fading visible in the classic agar overlay assay. The study showed that the inhibition areas with primary gingival fibroblasts were smaller (approximately 1/3) after monomer action compared with conventionally applied transformed fibroblasts. The results indicate that the modified assay is comparable with the classic method. Evaluation of neutral red staining with respect to toxic material influence can moreover be supplemented by histochemical studies of typical cell properties.

Homozygous transgenic mice from line A4 have an early-onset progressive neuromuscular disorder characterized by paralysis of the rear limbs, muscle atrophy, and lethality by 4 weeks of age. The transgene insertion site was mapped to distal chromosome 15 close to the locus motor endplate disease (med). The sequence of mouse DNA flanking the insertion site junctions was determined. A small (

WR 243251 is a dihydroacridinedione that was evaluated for antimalarial blood schizonticidal activity in vitro and in vivo. The in vitro doses calculated to kill 50% of organisms were 11 nM for a chloroquine-susceptible, mefloquine-resistant standard strain and 25 nM for a chloroquine- and pyrimethamine-resistant standard strain. The total dose needed to cure 100% of mice infected with a drug-susceptible strain of Plasmodium berghei was 12 to 20 mg/kg of body weight for both oral and subcutaneous administration. The regimen needed to cure 100% of Aotus monkeys infected with Plasmodium falciparum was 8 mg/kg/day for 3 days (chloroquine-susceptible strain) and 16 mg/kg/day for 3 days (chloroquine-resistant strain). The 100% curative doses for Aotus monkeys did not increase for parasites previously exposed to subcurative doses. The absolute value of the curative doses of WR 243251 was comparable to or lower than the values for clinical antimalarial agents. The high absolute activity, comparability of activities against susceptible and resistant parasites, and inability to induce resistance by exposure to subcurative doses suggest that WR 243251 has strong potential as a blood schizonticidal agent.

New treatments for malaria are urgently needed in areas such as Thailand where highly drug-resistant strains of Plasmodium falciparum are prevalent. Mefloquine is rapidly losing efficacy and conventional doses of halofantrine are ineffective. We therefore used pharmacokinetic simulation to design an extended-dose halofantrine regimen and tested it in 26 soldiers stationed along the Thai-Cambodian border. Halofantrine was given after meals as three doses of 500 mg each at 4-hr intervals on the first day, followed by 500 mg a day for six days (total dose 4.5 g). Twenty-six soldiers treated with quinine-tetracycline for seven days (Q(7)T(7)) served as controls. There were no significant differences in efficacy between halofantrine and Q(7)T(7) (P > 0.1) as assessed by cure rate (92% versus 85%), mean parasite clearance time (82 hr versus 81 hr), or mean fever clearance time (93 hr versus 99 hr). Halofantrine was better tolerated than Q(7)T(7). The side effects score was lower (2 versus 11; P < 0.001), there were less days on which side effects occurred (2.0 days versus 5.5 days; P < 0.001), and fewer patients had adverse effects on every treatment day (4% versus 42%; P < 0.01). High-dose halofantrine is as effective and better tolerated than quinine-tetracycline for multidrug-resistant falciparum malaria.

T cells can recognize tumor cells specifically by their TCR and the transfer of TCR-engineered T cells is a promising novel tool in anticancer therapies. We isolated and characterized four allorestricted TCRs with specificity for the HER2/neu-derived peptide 369 (HER2369) demonstrating high peptide specificity. PBMCs transduced with especially one TCR, HER2-1, mediated specific tumor reactivity after TCR optimization suggesting that this TCR represents a potential candidate for targeting HER2 by TCR-transduced effector cells. Another TCR showed high-peptide specificity without tumor reactivity. However, the TCRα-chain of this TCR specifically recognized HER2369 not only in combination with the original β-chain but also with four other β-chains of the same variable family deriving from TCRs with diverse specificities. Pairing with one β-chain derived from another HER2369-specific TCR potentiated the chimeric TCRs in regard to functional avidity, CD8 independency, and tumor reactivity. Although the frequency of such TCR single chains with dominant peptide recognition is currently unknown, they may represent interesting tools for TCR optimization resulting in enhanced functionality when paired to novel partner chains. However, undirected mispairing with novel partner chains may also result in enhanced cross-reactivity and self-reactivity. These results may have an important impact on the further design of strategies for adoptive transfer using TCR-transduced T cells.

The ability of neutrophils to generate free radicals is a crucial component of host defense (Babior, B. M. (1978) N. Engl. J. Med. 298, 659-668, 721-725. Neutrophil oxidants, however, can cause significant host tissue destruction (Weiss, S. J. (1989) N. Engl. J. Med. 320, 365-376), and the regulation of free radical production is not well understood. We have previously shown that recombinant antichymotrypsin (rACT), a serine protease inhibitor, inhibits superoxide production in intact neutrophils (Kilpatrick, L., Johnson, J. L., Nickbarg, E. B., Wang, Z., Clifford, T. F., Banach, M., Cooperman, B. S., Douglas, S. D., and Rubin, H. (1991) J. Immunol. 146, 2388-2393). Using a cell-free NADPH oxidase preparation, we now demonstrate that rACT alone has no effect on superoxide production and that antichymotrypsin-chymotrypsin (rACT.CT) complexes are required to inhibit superoxide, suggesting that neutrophil chymotrypsin-like proteases produce conformational changes in ACT, allowing it to become active in regulating superoxide production. Additionally, we have identified NADPH oxidase itself as the target for rACT.CT and have demonstrated that rACT.CT interferes specifically with activation of the NADPH oxidase without changing the Km for NADPH or the rate constant describing the rate-limiting step in activation. These observations suggest an important role for antichymotrypsin in the regulation of NADPH-oxidase activation, which is a prerequisite for neutrophil superoxide production, and predict possible therapeutic uses for rACT in conditions where unregulated neutrophil-free radical production has been implicated in the mechanism of tissue destruction.

Reactivation of CMV can cause severe disease after allogeneic hemopoietic stem cell transplantation. Adoptive T cell therapy was successfully used for patients who had received transplants from CMV-positive donors. However, patients with transplants from CMV-negative donors are at highest risk, and an adoptive therapy is missing because CMV-specific T cells are not available from such donors. To address this problem, we used retroviral transfer of CMV-specific TCR genes. We generated CMV-specific T cell clones of several HLA restrictions recognizing the endogenously processed Ag pp65. The genes of four TCRs were cloned and transferred to primary T cells from CMV-negative donors. These CMV-TCR-transgenic T cells displayed a broad spectrum of important effector functions (secretion of IFN-γ and IL-2, cytotoxicity, proliferation) in response to endogenously processed pp65 and could be enriched and expanded by strictly Ag-specific stimulation. Expansion of engineered T cells was accompanied by an increase in specific effector functions, indicating that the transferred specificity is stable and fully functional. Hence, we expect these CMV-TCR-transgenic T cells to be effective in controlling acute CMV disease and establishing an antiviral memory.

The formin protein formin-like 1 (FMNL1) is highly restrictedly expressed in hematopoietic lineage-derived cells and has been previously identified as a tumor-associated antigen. However, function and regulation of FMNL1 are not well defined. We have identified a novel splice variant (FMNL1 gamma) containing an intron retention at the C terminus affecting the diaphanous autoinhibitory domain (DAD). FMNL1 gamma is specifically located at the cell membrane and cortex in diverse cell lines. Similar localization of FMNL1 was observed for a mutant lacking the DAD domain (FMNL1 Delta DAD), indicating that deregulation of autoinhibition is effective in FMNL1 gamma. Expression of both FMNL1 gamma and FMNL1 Delta DAD induces polarized nonapoptotic blebbing that is dependent on N-terminal myristoylation of FMNL1 but independent of Src and ROCK activity. Thus, our results describe N-myristoylation as a regulative mechanism of FMNL1 responsible for membrane trafficking potentially involved in a diversity of polarized processes of hematopoietic lineage-derived cells.

High threshold L-type Ca2+ channels of skeletal muscle are thought to consist of a complex of alpha 1, alpha 2 delta, beta, and gamma subunits. Expression of the cloned alpha 1 subunit from skeletal and cardiac muscle has established that this protein is the dihydropyridine-sensitive ion-conducting subunit. However, the kinetics of the skeletal muscle alpha 1 alone expressed in mouse L-cells were abnormally slow and were accelerated to within the normal range by coexpression with the skeletal muscle beta subunit. The kinetics of cardiac muscle alpha 1 were also slowed but to a lesser extent and were not altered by coexpression with skeletal muscle alpha 2. We show here that coexpression of the skeletal muscle beta subunit with the cardiac alpha 1 subunit in Xenopus laevis oocytes produced: 1) an increase in the peak voltage-sensitive current, 2) a shift of the peak current-voltage relationship to more hyperpolarized potentials, and 3) an increase in the rate of activation. Coexpression of the skeletal muscle gamma subunit did not have a significant effect on currents elicited by alpha 1. However, when gamma was coexpressed with beta and alpha 1, both peak currents and rates of activation at more negative potentials were increased. These results indicate that rather than simply amplifying expression of alpha 1, heterologous skeletal muscle beta and gamma subunits can modulate the biophysical properties of cardiac alpha 1.

Summary The concentration of RCMV in the systemically infectible host Pisum sativum L. was found to decrease significantly after application of 5-azadihydrouracil and alkane monosulfonate. The number of local lesions of the same virus on leaves of the hypersensitive host Phaseolus vulgaris ‘Esto’ was reduced insignificantly by 5-azadihydrouracil and highly significantly by alkane monosulfonate. In Cucumis sativus L. the concentration of CGMMV was found to decrease following the uptake of 5-azadihydrouracil, alkane monosulfonate and cyanoguanidine via the leaves and also the roots. The yield-stabilizing effect of granulated 5-azadihydrouracil in CGMMV infected cucumber plants was demonstrated. Granulated alkane monosulfonate yielded results of minor importance.

Cell-based immunotherapy in settings of allogeneic stem cell transplantation or donor leukocyte infusion has curative potential, especially in hematologic malignancies. However, this approach is severely restricted due to graft-versus-host disease (GvHD). This limitation may be overcome if target antigens are molecularly defined and effector cells are specifically selected. We chose formin-related protein in leukocytes 1 (FMNL1) as a target antigen after intensive investigation of its expression profile at the mRNA and protein levels. Here, we confirm restricted expression in peripheral blood mononuclear cells (PBMCs) from healthy donors but also observe overexpression in different leukemias and aberrant expression in transformed cell lines derived from solid tumors. We isolated allorestricted T-cell clones expressing a single defined TCR recognizing a particular HLA-A2–presented peptide derived from FMNL1. This T-cell clone showed potent antitumor activity against lymphoma and renal cell carcinoma cell lines, Epstein-Barr virus (EBV)–transformed B cells, and primary tumor samples derived from patients with chronic lymphocytic leukemia (CLL), whereas nontransformed cells with the exception of activated B cells were only marginally recognized. Allorestricted TCRs with specificity for naturally presented FMNL1-derived epitopes may represent promising reagents for the development of adoptive therapies in lymphoma and other malignant diseases.

The aim of this study was to determine if, following vasectomy, epididymal obstruction resulted in changes in vasal stump fluid using a rat vasectomy model. One hundred and twenty-two mature male rats underwent bilateral surgical vasectomy and subsequent unilateral epididymal obstruction. Animals were randomly assigned to one of the five cohorts, which determined the time to kill and vasal fluid assessment. Numbers of whole sperm and sperm heads were compared between the obstructed and non-obstructed sides. Parametric analysis of microscopic vasal fluid findings was performed using a paired t-test. Whole sperm and sperm heads were detected bilaterally among the initial five cohorts. On the obstructed epididymis side, percentage of whole sperm dropped from 36.9% to less than 1% and sperm heads increased from 63.2 to 99.7% at 12 weeks post-obstruction (p < 0.05 at each time interval). On the unobstructed side, percentage of whole sperm rose from 66.3 to 89.5% and sperm heads dropped from 33.7 to 10.5% (p < 0.05 at each time interval). At 12 weeks, the difference between the obstructed and non-obstructed sides for both percentage and quantity of whole sperm and heads was significant with a p value of

In vitro fertilization using small amounts of cryopreserved sperm would benefit from efficient microscale methods to sort "healthy" motile sperm from nonmotile ones. This paper describes a microfluidic system with embedded reservoirs that sorts sperm based on the ability of motile sperm to move out of their initial streamlines in a laminar fluid stream. The device has horizontally oriented sample inlet and outlet reservoirs with different relative heights that serve the dual purpose of also being a pumping system that pumps fluids by hydrostatic pressure and surface tension. Sperm sorting is necessary for cryopreservation and intracytoplasmic sperm injection of sperm from men with low viable sperm counts. Since they have few viable sperm, it is difficult to obtain the sperm in an efficient manner using current procedures.

Background : Magnetic Resonance Imaging (MRI) allows a detailed “in vivo” macroscopic study of the human brain; previously, it has been demonstrated that Fluid Attenuated Inversion Recovery (FLAIR) sequence shows higher signal intensity of cortices belonging to limbic structures. Purpose : To measure and compare signal intensities (SI) of cytoarchitectonically different cortical regions. Methods : In 22 adult subjects, without psychiatric or neurological diseases, FLAIR sequence was performed in coronal slices, perpendicular to the main hippocampal axis. Signal intensity was measured, with a region-of-interest (ROI) function, in 12 different cortical regions. We compared these values and grouped the cortices into five groups: (1) limbic cortices, (2) paralimbic agranular cortices, (3) paralimbic granular cortices, (4) parietal-type neopallium, (5) frontal-type neopallium. A t -test for comparison of paired samples was performed, considering p ≤0.05 as statistically significant. Results : We found statistically significant differences amongst the different groups, with the exception of groups 1 and 2, which did not show differences between them. No statistically significant differences were found among cortices belonging to the same group. Conclusion : Structural characteristics of the cerebral cortex cause changes in its signal intensity. Magnetic resonance imaging (FLAIR sequence) allows discrimination of different cytoarchitectonic areas of the human cerebral cortex.

Three new diarylheptanoids, (4Z,6E)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)hepta-4,6-dien-3-one, letestuianin A (1), (4Z,6E)-5-hydroxy-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-4,6-dien-3-one, letestuianin B (2), and 1,7-bis(4-hydroxyphenyl)heptan-3,5-dione, letestuianin C (3), as well as the known (4Z,6E)-5-hydroxy-1,7-bis(4-hydroxyphenyl)hepta-4,6-dien-3-one (5) were isolated from Aframomum letestuianum. The known flavonoids 3-acetoxy-5,7,4'-trihydroxyflavanone, 3-acetoxy-7-methoxy-5,4'-dihydroxyflavanone, 7-methoxy-3,5,4'-trihydroxyflavone, and 3,3',4',5,7-pentahydroxyflavan were also obtained from this plant. Their structures were determined using a combination of 1D and 2D NMR techniques. The four diarylheptanoids were tested for growth inhibitory activity in vitro versus bloodstream forms of African trypanosomes. IC(50) values in the range of 1-3 microg/mL were found for compounds 3 and 5.

Inherited resistance to activated protein C (APCR) was identified as a major risk factor for venous thromboembolism. It is caused by a point mutation at nt 1691 G→A in the factor V gene resulting in the replacement of Arg 506 by Gln (FV Leiden mutation; Bertina et al. 1995). The prevalence in our Northeastern German population is about 7%. Among patients with DVT in 30% a FV Leiden mutation was evident. However, in approximately 5–10% of thrombosis patients with an APC resistance no Factor V Leiden mutation can be observed. Other factors like an elevated FVIII activity, antiphospholipid antibodies or pregnancy may contribute to a modulated APCR in some of those cases but are not evident in all.

This article explores whether organotypic cultures of immortalized gingival keratinocytes constitute a suitable model for assessing the epithelial cell compatibility of two groups of dental resins, each of them representing one group used in orthodontics and temporo-mandibular disorders (TMD) therapy under conditions more closely resembling the actual tissue situation. The resins were tested with the agar diffusion assay (ADA) in conventional monolayer and organotypic cultures. Compared to the control exhibiting a neutral red destaining index of 3, the index of 4 obtained after exposure of monolayers to one soft permanent resin (Durabase TM ) indicated the presence of a non-lytic but physiologically active substance. In contrast, the adaptation of the ADA to organotypic cultures revealed no apparent lesions at the epithelial surface by performing scanning electron microscopy, while histoarchitecture indicated the development of stratified surface epithelia. This was substantiated by undamaged cells in the uppermost cell layers and by the preservation of cell-to-cell contacts. Furthermore, indirect immunofluorescence for Ki-67 and the cytokeratins ck14 and ck4 revealed that cell proliferation and epithelial structure were maintained, while differentiation was enhanced, possibly increasing epithelial resistance. The results obtained from the organotypic cultures suggest that (i) cell-affecting effects of materials visible in monolayer cultures may not be seen in epithelia resembling that in vivo and that (ii) enhanced differentiation may be associated with increased stability of the epithelial cells. Thus, organotypic cultures of gingival cells constitute a tissue model allowing short-term tissue compatibility studies of dental materials and rendering a potential candidate also for long-term studies.

Following the addition of 0.2 mg 2,4-dioxohexahydro-l,3,5-triazine (DHT, formulation CKB 1130) to 1 1 Gamborg's medium for organogenesis, the two viruses strawberry mottle virus and strawberry crinkle virus could not be detected in 74.4, 63.6 or 65.1 %, respectively, of the plants regenerated from 0.8 mm apices of the varieties Redgauntlet, Vesper and Pokahontas after their inoculation onto the indicator strain of Fragaria vesca EMK. In contrast, control explants which were not exposed to DHT proved to be 100 % vims infected.

The aim of this study was to examine whether differences in placental angiotensinase A (glutamyl aminopeptidase, EC 3.4.11.7) activities occurred in hypertensive complications of pregnancy compared with uncomplicated pregnancies. Biochemical and semiquantitative histochemical methods were used and compared for their applicability. Angiotensinase A activity was detected using L-alpha glutamyl-4-methoxy-2-naphthylamide (alpha-Glu-MNA) as substrate and Fast Blue B salt for simultaneous azo-coupling in cryostat sections of placental tissue samples from 32 patients with pre-eclampsia, 11 patients with pregnancy-induced hypertension and 44 participants with uncomplicated pregnancies. The graduated intensity of reaction product in the villous trophoblast and in fetal blood vessels was evaluated semiquantitatively in a double-blind study by light microscopy (semiquantitative score method). Score levels were related to relative frequencies of hypertensive disorders (proportional odds model) and correlated to the severity of gestational hypertension (Spearman's rank correlation). After detection of enzyme activity, the same tissue samples were homogenized and used for kinetic fluorometric measurements under the same substrate and buffer conditions as in enzyme histochemistry. Enhanced villous trophoblastic angiotensinase A activity was significantly associated with an increased frequency of pre-eclampsia in pregnant women (cumulative odds ratio x 0(1) 6.37; P < 0.001) and showed significant correlations with the severity of gestational hypertensive disorders, represented by systolic (r = 0.31; P < 0.05) and diastolic (r = 0.34; P < 0.05 blood pressure and by concomitant proteinuria (r = 044; P < 0.01). Histochemical evaluation of fetal blood vessels and biochemical measurements revealed no statistically significant results. In conclusion this study demonstrates for the first time that increased villous trophoblastic angiotensinase A activity indicates an increased likelihood of the presence of pre-eclampsia and the severity of hypertensive disorders in pregnancy.

A retrospective study of patients who received chloramphenicol for the treatment of serious vancomycin-resistant enterococcal infections between 1 January 1993 and 31 August 1993 was conducted at the University of Pennsylvania Medical Center (Philadelphia). Antimicrobial susceptibilities as well as the clinical course of infection, adverse events, and response to therapy of 16 patients were reviewed. Forty-seven percent of enterococcal isolates were susceptible only to chloramphenicol, tetracycline, and nitrofurantoin. Types of infection included bacteremias (n = 7), abscesses (n = 7), and others (n = 5). Of 14 patients for whom a clinical response could be ascertained, eight (57%) showed improvement after treatment. Of 11 patients for whom a microbiological response could be ascertained, eight (73%) had sterile cultures after treatment. No lasting adverse effect related to the drug occurred. In-hospital mortality was 56%, but only one death could be directly attributed to vancomycin-resistant enterococcal infection. Chloramphenicol appears to be a useful and well-tolerated agent for the treatment of serious vancomycin-resistant enterococcal infections.

We compared the efficacy of azithromycin to the clinical antimalarial doxycycline in Plasmodium berghei-infected mice and in P. falciparum-infected Aotus monkeys. When mice were administered drug orally twice a day for three days, the minimum total dose of azithromycin that cured all mice was 768 mg/kg. Doxycycline at a dose of 1,536 mg/kg cured no mice. The efficacy of fast-acting blood schizonticides (quinine, halofantrine, artemisinin) against P. berghei was augmented by azithromycin. In monkey experiments in which there were two animals per experimental group, azithromycin (100 mg/kg/day for seven days) eliminated parasitemia; azithromycin (30 mg/kg/day) initially cleared 99.8–100% of the parasites with recrudescence in the one completely cleared case. Doxycycline (30 mg/kg/day) cleared 100% of the parasites with recrudescence in both cleared cases. Since azithromycin can be clinically administered at a somewhat higher daily dosage than doxycycline, the data suggest that it may be possible to replace drugs of the tetracycline class with azithromycin in combination with fast-acting blood schizonticides for the treatment of P. falciparum infection.

A patient with a history of alcohol abuse and pancreatitis presented with a pleural effusion resulting from a fistula between the pancreatic duct and left pleural space. Two weeks into her hospitalization, fever and persistent bloodstream infection with Erysipelothrix rhusiopathiae and Candida albicans developed. The patient had no history of exposure to animals. To our knowledge this is the first report of an E. rhusiopathiae infection presenting during hospitalization. This case suggests the possibility of a carrier state of infection and illustrates that a high index of suspicion is necessary for identification of unusual pathogens in hospitalized patients.

The autosomal recessive mutation mnd2 results in early onset motor neuron disease with rapidly progressive paralysis, severe muscle wasting, regression of thymus and spleen, and death before 40 days of age. mnd2 has been mapped to mouse chromosome 6 with the gene order: centromere-Tcrb-Ly-2-Sftp-3-D6Mit4-mnd2-D6Mit6, D6Mit9-D6Rck132-Raf-1, D6Mit11-D6Mit12-D6Mit14. mnd2 is located within a conserved linkage group with homologs on human chromosome 2p12-p13. Spinal motor neurons of homozygous affected animals are swollen and stain weakly, and electromyography revealed spontaneous activity characteristic of muscle denervation. Myelin staining was normal throughout the neuraxis. The clinical observations are consistent with a primary abnormality of lower motor neuron function. This new animal model will be of value for identification of a genetic defect responsible for motor neuron disease and for evaluation of new therapies. 36 refs., 7 figs., 2 tabs.