Tungsten carbide nanoparticles show a broad spectrum virucidal activity against enveloped and nonenveloped model viruses using a guideline-standardized in vitro test.

Five tungsten carbide nanoparticle preparations (denoted WC1-WC5) were investigated for broad spectrum virucidal activity against four recommended model viruses. These are modified vaccinia virus Ankara (MVA), human adenovirus type 5 (HAdV-5), poliovirus type 1 (PV-1) and murine norovirus (MNV). All virucidal tests were performed two to five times using the quantitative suspension test, which is a highly standardized test method to evaluate the virucidal efficacy of disinfectants in accordance with the European norm EN 14476+A1 and the German DVV/RKI guidelines. Quantitative detection of viruses was conducted by endpoint titration and quantitative real-time PCR. Results showed that three of the five tested compounds (WC1-WC3) were able to reduce the infectivity of all model viruses by at least four log10 of tissue culture infective dose 50% per ml after 15 min, whereas the other two compounds exhibited only limited efficacy (WC4) or showed cytotoxicity (WC5). Virucidal activity of nanoparticles increased with incubation time and a dose-effect curve showed dependence of virucidal activity with particle concentration. Whereas WC1-WC4 showed little cytotoxicity, WC5 which was doped with copper exhibited a significant cytotoxic effect. These findings propose tungsten carbide nanoparticles to be very promising in terms of new disinfection techniques. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study investigates the virucidal activity of tungsten carbide nanoparticles using the quantitative suspension test in accordance with the European norm EN 14476+A1 and the German DVV/RKI guidelines. Due to highly standardized assay conditions, results of this test are considered very reliable for evaluation of the virucidal activity of disinfectants. Broad-spectrum activity and high efficacy of three different tungsten carbide nanoparticles preparations is concluded.

In vitro cultivation and cryopreservation of duck embryonic hepatocytes.

Hepatitis B-virucidal testing of biocides in quantitative suspension tests using duck hepatitis B virus (DHBV) requires primary duck embryonic hepatocytes for viral propagation. To improve the test system and availability of these cells, commercial culture plates with different growth surfaces were tested for cell cultivation and different approaches for cryopreservation of hepatocyte suspension were examined. After 12 days of culture, the largest amounts of hepatocytes were grown in CellBIND and TTP plates and CellBIND surface showed the lowest tendency of monolayer detachment nearly comparable with collagen 1-coated CELLCOAT plates. For cryopreservation of hepatocyte suspension, the use of growth medium supplemented with fetal calf serum (FCS) and dimethyl sulfoxide (ME(2)SO), FCS supplemented with ME(2)SO or cryosafe-1 as cryoprotective agents provided the highest rates of surviving cells after thawing. The freezing-thawing process did not significantly reduce the susceptibility of hepatocytes to infection with DHBV. In conclusion, plates without collagen 1 such as CellBIND are recommended for cultivation of primary duck embryonic hepatocytes in infectivity experiments of DHBV for virucidal testing of biocides. The use of cryopreserved hepatocytes is possible when freshly isolated cells from the liver of duck embryos are not available.

Validation of biocides against duck hepatitis B virus as a surrogate virus for human hepatitis B virus.

The use of a surrogate virus, namely duck hepatitis B virus (DHBV), has been recommended for testing the virucidal activity of chemical biocides against hepatitis B virus. To date, however, this model has not been recognized as a standard test in European countries, as its laboratory use is associated with considerable difficulties. As previous studies have demonstrated, several alternative procedures may improve the validation of DHBV infection in a cell culture system. Using indirect immunofluorescent antigen staining and the light cycler real-time polymerase chain reaction (PCR) technique, the virucidal activity of peracetic acid (PAA), povidone-iodine (PVP-I) and formaldehyde was tested against DHBV obtained from congenitally infected ducks or prepared from the transfected hepatoma D2 cell line. The results demonstrated that inactivation of DHBV from the D2 cell line was achieved with lower concentrations of the biocides and within shorter exposure time intervals. These lower concentration-exposure time values for DHBV from D2 cells in comparison with DHBV from infected ducks indicated a higher sensitivity of the virus derived from D2 cells. In addition, concentrations of PAA and PVP-I that significantly inactivated DHBV in suspension tests were not able to destroy the viral genome. In conclusion, DHBV from congenitally infected ducks should be used for virucidal testing of chemical biocides against DHBV; DHBV prepared from D2 cells is unsuitable due to its higher sensitivity to biocides. Indirect immunofluorescent staining allows reliable detection of DHBV infectivity, whereas the hepadnavirucidal effect can be evaluated by quantitative PCR.

AsnI: a novel class II restriction endonuclease from Arthrobacter sp., strain N-CM, recognizing 5'-AT/TAAT-3'.

A new class II restriction endonuclease, AsnI, with a novel sequence specificity was isolated from the Gram-positive eubacterium Arthrobacter species, strain N-CM. AsnI recognizes the unambiguously defined palindromic hexanucleotide (Formula: see text) consisting of A- and T-residues. The novel enzyme in the presence of Mg2+ cleaves specifically both strands as indicated by the arrows. The staggered cuts generate 5'-protruding ends with single-stranded 5'-TA-3' dinucleotide extensions. The novel enzyme may be a useful tool for cloning experiments by complementation of the few enzymes such as PstI and PvuI cutting only once in the Ampr-gene of plasmids pBR322 and pBR328.

Electroporation of cell membranes supporting penetration of photodynamic active macromolecular chromophore dextrans.

The aim is to demonstrate that macromolecular chromophore dextrans (Cibacron-dextran) acting as photosensitizers can be transported easily into cancer cells by electroporation of their membranes (short electric pulses on cell suspension between electrodes). There are two possibilities, either:(A)irradiation starts with the electropulse-showed with easily penetrating thiopyronin-yielding nearly 100% dead cells;(B)irradiation starts after a resealing time of membrane pores during which macromolecular photosensitizers can penetrate into cells. In this way, fractions of Cibacron-dextran with molecular weights (Mw) 3300, 10,900 and 500,000 are now able to kill. This combination of bioelectrochemistry and photobiology will be suitable also for other biopolymers, connected with photodynamic active chromophores (e.g. chromopeptides) to transport them through cell walls and membranes into cells and tissues. The human cancer cells U-935 and K-562 (pulsed by 1.15 kV/cm field strength) additionally or synergistically reach high rates of necrotic cells (colored by trypan blue) by this combination.

Shoulder subluxation as a complication of positioning during CT.

Previously unreported, we note the complication of anterior shoulder subluxation that occurred secondary to patient positioning during chest CT examination. Positioning a patient's arms abducted and externally rotated over the head can precipitate anterior shoulder subluxation in patients with anterior shoulder instability.

Reactions to iodinated radiographic contrast agents. How to identify and manage patients at risk.

Systemic adverse reactions to iodinated radiographic contrast material are infrequent but can be dramatic. The number of patients experiencing such reactions can be minimized by selecting the most appropriate diagnostic study for each patient and by identifying patients at increased risk. Pretreatment with corticosteroids and use of low-osmolality contrast materials can minimize the number of severe reactions in high-risk patients.

[Studies using different human cell-culture systems for demonstrating interferon induction under in vitro conditions]. / Untersuchungen mit verschiedenen humanen Zellkultursystemen zum Nachweis einer Interferon-Induktion unter In-vitro-Bedingungen.

Five human cell culture systems (FL-cells, suspensions of tonsillar cells, FL-cells and suspensions of tonsillar cells combined, human fibroblasts, leukocyte suspensions) were tested with the international standard inducer Poly (IC) for the estimation of an in vitro induction of interferon under in vitro conditions. All induced cell systems have produced IFN. It can be concluded that alone the cell culture system represented by fibroblasts and leukocytes allow a reliable prediction concerning the induction of interferon under in vitro conditions.

[Interferon sensitivity of various cell lines]. / Interferonsensitivität verschiedener Zellkulturen.

An economic assay to determine the yield of interferon is necessary for the induction, production and purification of interferon. Using the inhibition of cytopathic effect of vesicular stomatitis virus in a microtitre system, we compared the susceptibility of different cell lines against an internal IFN alpha standard obtained from Sendai virus-induced human leukocytes. Human fibroblasts carrying trisomy G 21 and bovine MDBK cells showed the highest sensitivity followed by normal human fibroblasts. Human RH kidney cells exhibited a susceptibility similar to that of human WISH amnion cells frequently used by others. The human amnion cell line FL and monkey Vero kidney cells, as described here, were unsuitable for the determination of IFN yields.

[Interferon induction under in vitro conditions]. / Methodische Untersuchungen zur Interferon-Induktion unter in vitro-Bedingungen.

The procedures generally used for the in vitro induction of interferon exploit fibroblast or leucocyte culture suspensions. Several factors and conditions influencing the capacity for IFN induction were studied, with the following results. In contrast to previous findings, optimum IFN induction was obtained at 37 degrees C, rather than 30 degrees C, when human fibroblasts were induced with 100 micrograms/ml of Poly (IC). There was a direct correlation between the dose of the inducing agent applied and the amount of IFN produced. Also, the cells were excellently super-inducible with metabolic inhibitors like actinomycin D and cycloheximine. In addition, priming procedures enhanced IFN induction in human fibroblasts. Comparing different cell types with respect to their ability to be induced for IFN production, we found that different cell types show great differences in their inducer sensitivity. Finally, the response of different cell culture systems was highly variable when induced with different compounds.

Inhibition of proliferation of human lymphoma cells U937 by a 50 Hz electromagnetic field.

Weak pulsating electromagnetically induced fields (PEMF) by Helmholtz coils changes cell metabolism, if cells are treated with a certain range of frequency and amplitude. The influence on proliferation of human histiocytic lymphoma cells U937 has been studied applying a sinusoidal 50 Hz field with amplitudes of the flux density B = 0.3 to 4.7 mT for 4 days. No difference between experiment and control was found in the region 0.3 and 2 mT. However, stronger fields (> or =2.5 mT) inhibit cell division. Fields > or =3.5 mT treatment kill > or =80% of the cell number at the beginning (1.5 x 10(5)/ml). This effect may be an electromagnetocally induced cell death as the first step for a non-invasive application on cell proliferation process.

[Sensitivity of different cell lines to interferons: the relative antiviral activity as a function of the interferon subtype]. / Interferonsensitivität verschiedener Zellkulturen: Untersuchungen zur Abhängigkeit der relativen antiviralen Aktivität vom Interferon-Subtyp.

The phenomenon that rHuIFN-alpha1(D) displays an apparently higher antiviral activity when assayed on bovine cells as compared to human cell lines was applied to the elucidation of the nature of recombinant HuIFN prepared in our institute. These investigations were carried out by using a microtitre test, which defines biological activity as the IFN concentration leading to 50% inhibition of the cytopathic effect of vesicular stomatitis virus (VSV). In addition, the ability of IFN to diminish the reproduction of infectious viruses was monitored. The two methods yielded similar results. With bovine cells, antiviral activities of the same order of magnitude were observed, regardless of the interferon types applied, i.e. rHuIFN-alpha 1, rHuIFN-alpha 2 and human leukocyte interferon. On human fibroblasts, however, rHuIFN-alpha 1 had an apparently 45 to 165 times lower activity than the other two interferons. On human WISH cells, the differences in apparent activity between the respective IFNs were even greater, with factors of up to 212 fold being observed. Still more distinctive were the effects on murine L 929 cells where an antiviral effect could be confirmed only for rHuIFN-alpha 1 whereas the other two interferons proved completely inactive.

Hysteresis of the mammalian pCa/tension relation is small and muscle specific.

Small but statistically significant shifts (hysteresis) between the up (increasing tension direction) and down (decreasing tension) pCa/tension relation for several muscle types of the rabbit and rat were found. The largest shift is, at most, 0.093 p units or 60% of that reported for the barnacle muscle. The direction of shift for psoas is opposite to that for the soleus and tibialis.

Silent period induced by cutaneous stimulation.

An electrical stimulus applied to a cutaneous nerve during isometric muscle contraction causes a suppression of EMG activity (silent period) followed by a rebound. The extent of inhibition is related to the stimulus intensity as the silent period is more evident when stimulation is perceived as painful. The silent period is present in different limb and cranial muscles after stimulation of the same cutaneous nerve and in the same muscle after stimulation of distant cutaneous nerves. It also occurs synchronously in antagonist muscles. Within the silent period induced after cutaneous stimulation the maximal inhibition on the opponens pollicis motor neuron pool, as tested by the motor response evoked after transcranial cortical stimulation, occurs between 50 and 70 msec. Using the double stimulus technique to study the recovery cycle, the silent period is present at interstimulus intervals as low as 100 msec, and does not habituate with trains of stimuli at frequencies up to 5 Hz. Our results suggest that motor neuron inhibition from nociceptive stimulation may be mediated by Renshaw cells directly activated by high threshold cutaneous afferents.

Salmonella typhi and pregnancy: a case report.

BACKGROUND: Salmonella typhi may be a cause of significant morbidity and mortality in both the mother and fetus. Febrile illness during pregnancy, especially that associated with hemolysis, is associated with chorioamnionitis, pyelonephritis, or viral syndrome. As such, S. typhi should be considered when a patient presents with a fever and hemolysis. We present a case of S. typhi complicating pregnancy. CASE: A primigravida at 26 weeks gestation presented with persistent spiking temperature and severe hemolysis. Her presenting signs and symptoms included fever, vomiting, cough, earache, jaundice, dark urine, and anemia. After 4 units of blood, her posttransfusion hematocrit was 29%. Hemolysis was evident on a peripheral blood smear. Total bilirubin was 2.5 mg/dl and direct bilirubin was 1.2 mg/dl. Gentamicin and clindamycin were administered empirically. Stool, blood, and urine cultures were obtained. Blood cultures were positive for S. typhi. Antibiotic coverage was changed to gentamicin and ceftriaxone. She defervesced on the 5th day and had no further problems. A healthy male infant was delivered vaginally at term. CONCLUSION: Typhoid fever is a serious infection, and special concern arises when S. typhi complicates pregnancy. The diagnosis of S. typhi should be considered in a gravida presenting with fever with prompt institution of antibiotic therapy. Appropriate cultures are essential for confirming the diagnosis.

[The use of primate tissue homogenates for quality control in the creatine kinase-MB determination with inhibiting antibodies (author's transl)]. / Verwendung von Primaten-Gewebshomogenaten zur Qualitätskontrolle der Bestimmung von Kreatinkinase-MB mit inhibierenden Antikörpern.

The creatine kinase isoenzymes CK-MM, CK-MB and CK-BB from Rhesus monkey heart and brain tissue homogenates were separated chromatographically. Thereafter it could be demonstrated that the activity of the simian subunit CK-M is completely inhibited by anti-inhibitory-CK-M serum. Thus control sera from simian tissue are in principle suited for quality control in an immunological determination of creatine kinase-MB. The intra-assay variance and interassay variance were n = 56, -/x = 29.2 U/1, SD = 3.2 U/1, CV = 11.1% and n= 12, -/x = 166.7 U/1, SD = 5.0 U/1, CV = 3.0% respectively. It is desirable to develop control sera with catalytic concentrations of creatine kinase-MB in a lower range.

Expression of inducible nitric oxide synthase in experimental viral myocarditis.

Nitric oxide (NO) is an important bioactive molecule with regulatory, cytotoxic or cytoprotective properties. In virus-induced myocarditis, NO mediates host defense mechanisms against the infection or causes cardiac dysfunctions. NO is synthesized from L-arginine by the enzyme nitric oxide synthase (NOS). The expression of the inducible form of the nitric oxide synthase (iNOS) is regulated by cytokines, involved in the complex myocardial immune response to enterovirus infections. The present study was undertaken to characterize the role of iNOS and NO in the murine model of viral myocarditis induced by coxsackievirus B3 (CVB3). In response to CVB3 infection we investigated the time course of iNOS induction in correlation with cytokine mRNA expression (TNF-alpha, IL-1 alpha, IFN-gamma, TGF-beta) in the heart of NMRI mice by RT-PCR. Positive PCR signals for viral RNA were found in the acute and chronic stage of disease by seminested PCR, indicating the persistence of viral genome. We found distinct expression of iNOS at all time points (1, 2, 3, 4, 7, 14, 28, 56, 98 days post infection [p.i.]). Higher iNOS mRNA levels were identified between days 4 until 28 p.i. in comparison to day 56 and 98 p.i. using densitometric values. The mRNA of the inflammatory cytokines TNF-alpha, IL-1 alpha, IFN-gamma appeared at days 1, 4, and 7 p.i., peaked at day 7 p.i. and persisted until day 98 p.i. Similar like the iNOS mRNA pattern was the expression profile of TGF-beta. Using in situ hybridization and immunohistochemistry iNOS was localized in infiltrates, vascular endothelial cells, smooth muscle cells, myocytes and throughout the interstitial spaces between myocardial fibers in the heart sections of NMRI mice. Increased levels of NO were measured as total nitrate/nitrite concentration in the sera of mice from day 7 until day 28 p.i.

[Experimental studies of in vitro IFN induction by different polynucleotides in human cell culture systems]. / Experimentelle Untersuchungen zur in vitro-IFN-Induktion durch verschiedene Polynucleotide in humanen Zellkultur-Systemen.

Several polynucleotides were tested for interferon induction in comparison with the standard Poly (IC) in human diploid fibroblasts and in human leukocyte suspensions. We received high IFN-titre following superinduction of the polynucleotides in human fibroblasts. These results show that under superinduction conditions partly 3 times more interferon is induced in comparison with the standard inductor Poly (IC). The tested concentration of 10 micrograms/ml polynucleotides did not result in any IFN yields or only in very low ones in human leukocytes, which were only within the range of detectability.

Congenital skin lesions caused by intrauterine infection with coxsackievirus B3.

BACKGROUND: Serious neonatal coxsackievirus infections transplacentally acquired in late pregnancy involve primarily the central nervous system, heart, liver and rarely the skin. PATIENTS AND METHODS: A boy born with a disseminated papulovesicular, nodular, bullous and necrotic ulcerated rash at 39 weeks gestational age developed pneumonia, carditis and hepatitis during the first days after birth. Molecular biological and serological methods were used for virological diagnosis. RESULTS: Coxsackievirus B3 (CVB3) was found in throat swabs and/or feces of the neonate and his mother. In addition, there was serological evidence of intrauterine infection. CONCLUSION: Intrauterine transmission of CVB3 during late pregnancy may lead to varicella-like congenital skin lesions.

Microcalcifications on postoperative mammograms as an indicator of adequacy of tumor excision.

Mammograms, including microfocus spot magnification views, were obtained before reexcision of the tumor-ectomy site in 43 women with breast carcinoma. These studies were prospectively evaluated by radiologists experienced in breast imaging. All women had mammographically evident microcalcifications associated with their original tumors. In all, tumor was at or near the margin of resection or the status of margins was unknown, necessitating reexcision of the tumorectomy site. Of 29 women with residual microcalifications, 20 had residual tumor. The positive predictive value of residual microcalcifications was 0.69. The positive predictive value was greatest (0.90) in women with ductal carcinoma in situ, when more than five microcalifications were present. Residual microcalifications not due to carcinoma were secondary to sclerosing adenosis, fat necrosis, and foreign body granuloma. Of the 13 cases in which no residual calcifications were left after tumorectomy, residual tumor was found in four. The negative predictive value of the absence of calcifications for total excision of tumor was 0.64 for all tumor types and was 1.0 for the noncomedo subtype of ductal carcinoma in situ.