Zygomatic bone shape in intentional cranial deformations: a model for the study of the interactions between skull growth and facial morphology.

Intentional cranial deformations (ICD) were obtained by exerting external mechanical constraints on the skull vault during the first years of life to permanently modify head shape. The repercussions of ICD on the face are not well described in the midfacial region. Here we assessed the shape of the zygomatic bone in different types of ICDs. We considered 14 non-deformed skulls, 19 skulls with antero-posterior deformation, nine skulls with circumferential deformation and seven skulls with Toulouse deformation. The shape of the zygomatic bone was assessed using a statistical shape model after mesh registration. Euclidian distances between mean models and Mahalanobis distances after canonical variate analysis were computed. Classification accuracy was computed using a cross-validation approach. Different ICDs cause specific zygomatic shape modifications corresponding to different degrees of retrusion but the shape of the zygomatic bone alone is not a sufficient parameter for classifying populations into ICD groups defined by deformation types. We illustrate the fact that external mechanical constraints on the skull vault influence midfacial growth. ICDs are a model for the study of the influence of epigenetic factors on craniofacial growth and can help to understand the facial effects of congenital skull malformations such as single or multi-suture synostoses, or of external orthopedic devices such as helmets used to correct deformational plagiocephaly.

Virulence and antimicrobial resistance determinants of verotoxigenic Escherichia coli (VTEC) and of multidrug-resistant E. coli from foods of animal origin illegally imported to the EU by flight passengers.

The aim of this study was to reveal phenotype/genotype characteristics of verotoxigenic Escherichia coli (VTEC) and multidrug resistant E. coli in food products of animal origin confiscated as illegal import at Austrian, German and Slovenian airports. VTEC isolates were obtained by using ISO guidelines 16654:2001 for O157 VTEC or ISO/ TS13136:2012 for non-O157 VTEC, with additional use of the RIDASCREEN® Verotoxin immunoassay. The testing of 1526 samples resulted in 15 VTEC isolates (1.0%) primarily isolated from hard cheese from Turkey and Balkan countries. Genotyping for virulence by using a miniaturized microarray identified a wide range of virulence determinants. One VTEC isolate (O26:H46) possessing intimin (eae) and all other essential genes of Locus of Enterocyte Effacement (LEE) was designated as enterohemorrhagic E. coli (EHEC). None of the other VTEC strains belonged to serogroups O157, O145, O111, O104 or O103. VTEC strains harbored either stx(1) (variants stx1(a) or stx(1c)) or st(x2) (variants stx(2a), stx(2b), stx(2a/d) or stx(2c/d)) genes. Pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) demonstrated high genetic diversity and identified three new sequence types (STs): 4505, 4506 and 4507. Food samples collected from the Vienna airport were also tested for E. coli quantities using the ISO 16649:2001, and for detection of multidrug resistant phenotypes and genotypes. The resulting 113 commensal E. coli isolates were first tested in a pre-screening against 6 selected antimicrobials to demonstrate multidrug resistance. The resulting 14 multidrug resistant (MDR) E. coli isolates, representing 0.9% of the samples, were subjected to further resistance phenotyping and to microarray analyses targeting genetic markers of antimicrobial resistance and virulence. Genotyping revealed various combinations of resistance determinants as well as the presence of class 1, class 2 integrons. The isolates harbored 6 to 11 antibiotic resistance genes as well as 1 to 14 virulence genes. In this panel of 14 MDR E. coli two strains proved to carry CTX-M type ESBLs, and one single isolate was identified as enteropathogenic E. coli (EPEC). In general, isolates carrying a high number of resistance determinants had lower number of virulence genes and vice versa. In conclusion, this first pilot study on the prevalence of VTEC and of MDR/ESBL E. coli in illegally imported food products of animal origin suggests that these strains could represent reservoirs for dissemination of potentially new types of pathogenic and MDR E. coli in Europe.

Direct Electrochemical Addressing of Immobilized Alcohol Dehydrogenase for the Heterogeneous Bioelectrocatalytic Reduction of Butyraldehyde to Butanol.

Modified electrodes using immobilized alcohol dehydrogenase enzymes for the efficient electroreduction of butyraldehyde to butanol are presented as an important step for the utilization of CO2-reduction products. Alcohol dehydrogenase was immobilized, embedded in an alginate-silicate hybrid gel, on a carbon felt (CF) electrode. The application of this enzyme to the reduction of an aldehyde to an alcohol with the aid of the coenzyme nicotinamide adenine dinucleotide (NADH), in analogy to the final step in the natural reduction cascade of CO2 to alcohol, has been already reported. However, the use of such enzymatic reductions is limited because of the necessity of providing expensive NADH as a sacrificial electron and proton donor. Immobilization of such dehydrogenase enzymes on electrodes and direct pumping of electrons into the biocatalysts offers an easy and efficient way for the biochemical recycling of CO2 to valuable chemicals or alternative synthetic fuels. We report the direct electrochemical addressing of immobilized alcohol dehydrogenase for the reduction of butyraldehyde to butanol without consumption of NADH. The selective reduction of butyraldehyde to butanol occurs at room temperature, ambient pressure and neutral pH. Production of butanol was detected by using liquid-injection gas chromatography and was estimated to occur with Faradaic efficiencies of around 40 %.

Management of opportunistic infections in acquired immunodeficiency syndrome. I. Treatment.

A case report of a patient infected with human immunodeficiency virus (HIV) is described. The patient presents with a multitude of medical complaints that are of acute or subacute onset. The medical examination of these complaints is described and includes algorithms for the diagnosis and treatment of the most common HIV-related opportunistic infections, including Pneumocystis carinii pneumonia, toxoplasmosis, Mycobacterium avium complex, cytomegalovirus infection, and cryptococcal meningitis.

Transcription of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene occurs before induction of the BCR2 (Cp) EBNA gene promoter during the initial stages of infection in B cells.

The purpose of this study was to gain insights into the regulation of Epstein-Barr virus (EBV) gene transcription during the establishment of viral latency in B cells. During the early stages of EBV infection in B lymphocytes, transcription of six viral nuclear antigens (EBNAs) is initiated from an early promoter (Wp). This is followed by a switch of promoter usage to an upstream promoter, Cp, whose activity is autoregulated by both EBNA1 and EBNA2. Previously it was demonstrated that infection of primary B cells with EBNA2-negative (EBNA2-) EBNA4-mutant (EBNA4mut) virus resulted only in the expression of mutant EBNA4 protein and failure to express the other EBNA gene products (C. Rooney H. G. Howe, S. H. Speck, and G. Miller, J. Virol. 63:1531-1539, 1989). We extended this research to demonstrate that Wp-to-Cp switching did not occur upon infection of primary B cells with an EBNA2- EBNA4mut virus (M. Woisetschlaeger, X. W. Jin, C. N. Yandara, L. A. Furmanski, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 88:3942-3946, 1991). Further characterization of this phenomenon led to the identification of an EBNA2-dependent enhancer upstream of Cp. On the basis of these data, a model was proposed in which initial transcription from Wp gives rise to the expression of EBNA2 and EBNA4, and then transcription is upregulated from Cp via the EBNA2- dependent enhancer (Woisetschlaeger et al., as noted above). Implicit in this model is that transcription of the EBNA1 and EBNA3a to -3c genes is dependent on the switch from Wp to Cp, since primary cells infected with EBNA2- EBNA4mut virus fail to switch and also fail to express these viral antigens. Here we critically evaluate this model and demonstrate, in contrast to the predictions of the model, that transcription of both the EBNA1 and EBNA2 genes precedes activation of Cp. Furthermore, the level of EBNA1 gene transcription was strongly reduced in primary B cells infected with EBNA2- EBNA4mut virus compared with that of cells infected with wild-type virus. Switching to Cp, as well as EBNA1 gene transcription, was observed upon infection of EBV-negative Burkitt's lymphoma (BL) cell lines with EBNA2- EBNA4mut virus, thus establishing a correlation between early EBNA1 gene transcription and upregulation of transcription initiation from Cp. However, in EBV-negative BL cell lines infected with EBNA2- EBNA4mut virus, transcription of the EBNA1 gene at early time points postinfection initiated from Qp, the EBNA1 gene promoter active in group I BL cells (B. C. Schaefer, J. L. Strominger, and S. H. Speck, Proc. Natl. Acad. Sci. USA 92:10565-10569, 1995), rather than from Wp. The data support a model in which EBNA1 plays an important role in the cascade of events leading to successful switching from Wp to Cp and subsequent immortalization of the infected B cell.

The 'monocytic' cell line I937 displays typical B cell characteristics.

The monocytic cell line I937 was derived from U937 by sorting for cells with high expression of MHC class II molecules. Further analysis of these class II molecules revealed the presence of the HLA-DR3 subtype suggesting that the cell line was a potential candidate for testing antigen presentation to T cells restricted by HLA-DR3. We found that the T cell clones CFTS4:2.80 and CFTS4:2.6 with the required restriction element responded to the house dust mite antigen DPT presented by I937 but not U937, whereas CFTS4:3.1, which is not HLA-DR restricted, did not respond to either cell line. Subsequent analysis of surface markers on I937, however, indicated that the cell line is of B cell origin. In contrast to the parental cell line U937, I937 was tested negative for CD4, CD31 and CD64 but expressed CD19, CD21 and CD40. Although neither surface nor cytoplasmic Ig molecules were detected in either I937 or U937, Southern blot analysis revealed IgH gene rearrangement in I937. In addition, a fragment specific for Epstein-Barr virus nuclear antigen (EBNA2) was amplified in I937 by PCR technique. Therefore, we conclude that I937 is an EBV-transformed B cell line, presumably derived from the same donor and not as reported originally as a subline of U937, which expresses high MHC class II levels.

Repression and derepression of conjugation of plasmid R1 by wild-type and mutated finP antisense RNA.

The finP gene of plasmid R1 is located between the genes traM and traJ, partially overlapping the first few nucleotides of the latter. It codes for a repressor of the conjugation system. The product of this gene is a small RNA of 72 nucleotides and, because it is transcribed from the opposite DNA strand, it is complementary to the 5' non-translated sequences, the ribosome-binding site, and the first two codons of traJ mRNA. The finP transcript is present in much higher concentrations in R1 than in R1-19 containing cells, the latter being a derepressed mutant of the former. A synthetic finP gene expressed from a synthetic lambda PL promoter markedly reduced the conjugation frequency of pDB12, a multicopy derivative of R1-19. Mutagenesis of finP showed that only finP loop II mutants have lost the ability to repress conjugation of R1-19 in trans. They are also the only ones which derepress conjugal DNA transfer of R1, probably by competing for the finO product, a molecule needed as corepressor for maximal activity. Mutations interrupting potential open reading frames of finP do not abolish finP repressor activity. Hence finP acts as an antisense RNA blocking the expression of the traJ gene by interacting with traJ mRNA through loop II.

Using fault analysis methods to improve bioreactor safety.

A simplified fault analysis algorithm has been described and applied to the analysis of containment loss in a model bioreactor system. Using approximate data for component failure rates, the relative merits of three proposed operating regimes were evaluated by means of a computer program that implements the algorithm described. With the data given, operator error is shown to be the dominant cause of possible failure in the proposed system. In view of these results, the simplified algorithm appears useful for the comparative evaluation of various design proposals in simple systems.

Role of macrophage lipids in regulating tumoricidal activity. II. Internal genetic and external physiologic regulatory factors controlling macrophage tumor cytotoxicity also control characteristic lipid changes associated with tumoricidal cells.

Peritoneal macrophages (M phi) from C3H/HeN mice became cytotoxic for 1023 tumor cells after incubation with lymphokine (LK) for 8-12 hr and lost tumoricidal activity by 22 hr in the continued presence of LK; bacterial endotoxin (LPS) was ineffective in inducing tumoricidal activity. M phi from C3H/HeJ mice were not activated for tumor cytotoxicity after treatment with LK or LPS. C3H/HeN M phi acquisition of tumoricidal activity was accompanied by unique changes in M phi lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acids (UFA) increased two- to threefold when the cells showed maximal tumoricidal activity and returned to control levels when the M phi lost tumoricidal activity. LPS treatment of C3H/HeN M phi and LK or LPS treatment of C3H/HeJ M phi did not cause characteristic M phi lipid alterations. To determine at what stage during M phi activation the correlative CHOL and UFA compositional changes were occurring, C3H/HeN M phi were primed with LPS or low concentrations of LK and triggered with LPS or Lk; M phi lipid and fatty acid composition was monitored at each stage. LK was shown to be able to prime and trigger whereas LPS could only trigger LK-primed M phi for tumor cytotoxicity. In all cases, the increase in M phi CHOL and UFA content occurred at the triggering step for tumor cytotoxicity rather than at the priming step. These data suggest that there is a correlation between the effects of endogenous and exogenous factors that control expression of M phi tumoricidal activity and their effects on M phi CHOL and UFA content; the establishment of these changes in M phi lipid composition occurs at a time when the cells are triggered for tumor cytotoxicity.

Role of membrane lipids in the immunological killing of tumor cells: II. Effector cell lipids.

Peritoneal macrophages (M phi) from mice become cytotoxic after incubation in lymphokine (LK)-rich supernatants of antigen-stimulated spleen cell cultures. Tumoricidal activity is evident with M phi treated with LK for 4 hr, becomes maximal after 8-12 hr incubation and decreases to control levels by 24-36 hr. To gain insight into LK-induced functional changes, the lipid composition of M phi cultured with LK for 0-36 hr was analyzed by high pressure liquid chromatography. LK induced marked changes in M phi lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acids increased 2- to 3-fold after 8 hr when the cells showed maximal tumoricidal activity. Cellular lipid and fatty acid content returned to control levels by 24 hr when the M phi had lost tumoricidal activity. These changes were not observed with equal numbers of M phi cultured in control supernatants. To analyze further the role of CHOL and unsaturated fatty acids in M phi tumor cytotoxicity, M phi were enriched in CHOL or linolenic acid (18:3) and tested for their ability to kill 1023 tumor cells. Within 1 hr of culture, M phi showed a 3- to 4-fold increase in CHOL or 18:3 content. 18:3-enriched cells were markedly tumoricidal, whereas controls cultured in delipidized medium alone or enriched with saturated fatty acids were cytotoxic. CHOL-enriched M phi were not tumoricidal; indeed, these cells were inhibited in their killing after treatment with LK compared to M phi cultured in delipidized medium with LK alone. These results suggest that UFA aids, whereas CHOL negates, expression of M phi tumor cytotoxicity.

Role of membrane lipids in the immunological killing of tumor cells: I. Target cell lipids.

The metabolic and physical properties of tumor cells that are associated with their ability to resist or escape from immune attack have been investigated. The susceptibility of P815 murine mastocytoma cells to immune killing can be modulated. Culturing the cells with adriamycin or with hydrocortisone increases or decreases, respectively, the sensitivity of the cells to killing by antibody (Ab) plus complement (C); in addition, culturing the cells with mitomycin C or hydrocortisone increases or decreases, respectively, the sensitivity of the cells to killing by cytotoxic T lymphocytes (CTL). The susceptibility of the cells to Ab-C killing correlates with the ability of the cells to synthesize complex cellular lipids, but not DNA, RNA, protein, or carbohydrate. Further, tumor cells rendered sensitive to Ab-C killing by adriamycin are decreased in total lipid content and in their cholesterol/phospholipid mole ratio; hydrocortisone-treated resistant cells showed the opposite effects. The ability of tumor cells to resist CTL killing did not correlate with their total cellular lipid synthesis, but did correlate with the synthesis and composition of specific cellular phospholipids. In addition, tumor cells increased in sensitivity to Ab-C killing exhibited an increase in cell surface membrane fluidity, whereas cells increased in susceptibility to CTL attack showed an increase in their net negative cell surface charge density. These data show certain unique chemical and physical properties of tumor cells to be of fundamental importance for their ability to resist either humoral or cell-mediated immunologic attack; modulation of one or another of these cellular properties results in a change in the cells' susceptibility to immune killing by antibody plus C or by cytotoxic T lymphocytes.

Efficacy of chemotherapeutic treatment of guinea pig hepatoma reflected in host's serum lipid composition.

A correlation was found between the highly polar phospholipid (HPPL) content of serum lipids and the growth and metastasis of the line-10 hepatoma in strain 2 guinea pigs. Animals whose tumors were treated by intralesional injection of decarbazine (DTIC) or saline showed progressive tumor growth and metastasis resulting in death within 120 days of tumor implantation. The percent HPPl in the serum lipids of these animals rose to 40% by 80 days and remained elevated above 20-25% until death. Animals whose tumors were treated by intralesional injection of adriamycin were either cured of their tumors (67%) or showed little or no tumor growth. The percent HPPL in the serum lipids of these animals remained between 3 and 12% at all times. Adriamycin or DTIC injected into non-tumor-bearing animals resulted in an HPPL content of serum lipids that was not significantly different from that of control, saline-inoculated animals.

Role of macrophage lipids in regulating tumoricidal activity.

Peritoneal macrophages (M phi) from mice became cytotoxic after incubation with lymphokine (LK); tumoricidal activity was evident with M phi treated with LK for 4 hr, became maximal after 8-12 hr of incubation, and decreased to control levels by 24-36 hr. LK induced marked changes in M phi lipid composition: cellular content of cholesterol (CHOL) and polyunsaturated fatty acid (UFA) content of cellular lipids (especially 18:3) increased two- to threefold after 8 hr when the cells showed maximal tumoricidal activity. Cellular lipid and fatty-acid content returned to control levels by 24 hr when the M phi had lost tumoricidal activity. These changes were not observed with equal numbers of M phi cultured in control supernatants. To analyze the role of CHOL and UFA in M phi tumor cytotoxicity, casein-induced peritoneal M phi were enriched in CHOL or linolenic acid (18:3) and then tested for their ability to kill 1023 tumor cells. The 18:3-enriched cells were markedly tumoricidal, whereas controls cultured in delipidized medium alone or enriched with saturated fatty acid (18:0) were not cytotoxic. CHOL-enriched M phi were not tumoricidal; indeed, these cells were inhibited in their killing after treatment with LK compared to M phi cultured in delipidized medium with LK alone. The effects of 18:3 and CHOL enrichment of the M phi on their metabolic status, inflammatory function, and tumor cell-binding capacity were tested. The 18:3-enriched M phi were depressed in their ability to synthesize protein and in phagocytic activity compared to controls; these cells showed a transient increase in superoxide release. M phi cultured with 18:3 for 48 hr were also cytotoxic for P815 tumor cells, but did not show an enhanced capacity for P815 binding compared to controls. CHOL-enriched M phi were similar to control cells in their protein synthesizing and phagocytic activities; these cells also showed an early transient increase in superoxide release. CHOL-enriched M phi were not cytotoxic for P815 cells, but bound the tumor cells more readily than did the 18:3-enriched M phi. The data suggest that endogenous levels of 18:3 and CHOL can regulate M phi tumor cytotoxicity, but not through regulation of M phi protein synthesis, oxidative metabolism, or augmented capacity for tumor target binding.

Cell growth-dependent variation in the sensitivity of human and mouse tumor cells to complement-mediated killing.

Cells removed from asynchronous cultures during lag, log, and stationary phases of growth were found to differ in their sensitivity to antibody/complement-mediated killing. The human lymphoblastoid line, Raji, was relatively more susceptible to killing by human anti-HLA antibody plus rabbit complement during the lag and log phases of growth, while the human lymphoid cell line, PY, was relatively more susceptible to rabbit antilymphocyte serum or human anti-HLA plus rabbit complement during the log and late-log phases of growth. The mouse mastocytoma cell line, P815, was relatively resistant to killing by rabbit anti-P815 plus guinea pig complement during the middle log phase of growth. The variation in sensitivity of the three cell lines was dependent upon the concentration of antibody used to sensitize the cells but not due to differences in antigen expression, antigen density, or net synthesis of DNA, RNA, protein, complex carbohydrate, or lipid-containing macromolecules. These data suggest that the variability in susceptibility of the cells for complement-mediated killing is due to changes in physiological and/or physicochemical properties of the cells which either affect the ability of the cells to repair complement-mediated damage or nullify the activity of cell-bound complement.