In Vivo Pharmacodynamic Target Investigation of Two Bacterial Topoisomerase Inhibitors, ACT-387042 and ACT-292706, in the Neutropenic Murine Thigh Model against Streptococcus pneumoniae and Staphylococcus aureus.

ACT-387042 and ACT-292706 are two novel bacterial topoisomerase inhibitors with broad-spectrum activity against Gram-positive and -negative bacteria, including methicillin-resistant Staphylococcus aureus and penicillin- and fluoroquinolone-resistant Streptococcus pneumoniae We used the neutropenic murine thigh infection model to characterize the pharmacokinetics (PK)/pharmacodynamics (PD) of these investigational compounds against a group of 10 S. aureus and S. pneumoniae isolates with phenotypic resistance to beta-lactams and fluoroquinolones. The in vitro activities of the two compounds were very similar (MIC range, 0.03 to 0.125 mg/liter). Plasma pharmacokinetics were determined for each compound by using four escalating doses administered by the subcutaneous route. In treatment studies, mice had 10(7.4) to 10(8) CFU/thigh at the start of therapy with ACT-387042 and 10(6.7) to 10(8.3) CFU/thigh at the start of therapy with ACT-292706. A dose-response relationship was observed with all isolates over the dose range. Maximal kill approached 3 to 4 log10 CFU/thigh compared to the burden at the start of therapy for the highest doses examined. There was a strong relationship between the PK/PD index AUC/MIC ratio (area under the concentration-time curve over 24 h in the steady state divided by the MIC) and therapeutic efficacy in the model (R(2), 0.63 to 0.82). The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-387042 against S. aureus and S. pneumoniae were 43 and 10, respectively. The 24-h free-drug AUC/MIC ratios associated with net stasis for ACT-292706 against S. aureus and S. pneumoniae were 69 and 25, respectively. The stasis PD targets were significantly lower for S. pneumoniae (P < 0.05) for both compounds. The 1-log-kill AUC/MIC ratio targets were ∼2- to 4-fold higher than stasis targets. Methicillin, penicillin, or ciprofloxacin resistance did not alter the magnitude of the AUC/MIC ratio required for efficacy. These results should be helpful in the design of clinical trials for topoisomerase inhibitors.

Are ducks contributing to the endemicity of highly pathogenic H5N1 influenza virus in Asia?

Wild waterfowl are the natural reservoir of all influenza A viruses, and these viruses are usually nonpathogenic in these birds. However, since late 2002, H5N1 outbreaks in Asia have resulted in mortality among waterfowl in recreational parks, domestic flocks, and wild migratory birds. The evolutionary stasis between influenza virus and its natural host may have been disrupted, prompting us to ask whether waterfowl are resistant to H5N1 influenza virus disease and whether they can still act as a reservoir for these viruses. To better understand the biology of H5N1 viruses in ducks and attempt to answer this question, we inoculated juvenile mallards with 23 different H5N1 influenza viruses isolated in Asia between 2003 and 2004. All virus isolates replicated efficiently in inoculated ducks, and 22 were transmitted to susceptible contacts. Viruses replicated to higher levels in the trachea than in the cloaca of both inoculated and contact birds, suggesting that the digestive tract is not the main site of H5N1 influenza virus replication in ducks and that the fecal-oral route may no longer be the main transmission path. The virus isolates' pathogenicities varied from completely nonpathogenic to highly lethal and were positively correlated with tracheal virus titers. Nevertheless, the eight virus isolates that were nonpathogenic in ducks replicated and transmitted efficiently to naïve contacts, suggesting that highly pathogenic H5N1 viruses causing minimal signs of disease in ducks can propagate silently and efficiently among domestic and wild ducks in Asia and that they represent a serious threat to human and veterinary public health.

Role of domestic ducks in the propagation and biological evolution of highly pathogenic H5N1 influenza viruses in Asia.

Wild waterfowl, including ducks, are natural hosts of influenza A viruses. These viruses rarely caused disease in ducks until 2002, when some H5N1 strains became highly pathogenic. Here we show that these H5N1 viruses are reverting to nonpathogenicity in ducks. Ducks experimentally infected with viruses isolated between 2003 and 2004 shed virus for an extended time (up to 17 days), during which variant viruses with low pathogenicity were selected. These results suggest that the duck has become the "Trojan horse" of Asian H5N1 influenza viruses. The ducks that are unaffected by infection with these viruses continue to circulate these viruses, presenting a pandemic threat.

Decontamination with vaporized hydrogen peroxide is effective against Mycobacterium tuberculosis.

AIMS: To determine the efficacy of room fumigation with vaporized hydrogen peroxide (VHP) in decontamination of viable Mycobacterium tuberculosis. METHODS AND RESULTS: About 8 x 10(4)-2.3 x 10(6) CFU of M. tuberculosis H37Rv and M. tuberculosis Beijing were dried in 10-microl drops in tissue culture plates, placed in steam-permeable Tyvek pouches and distributed on laboratory surfaces. The room was exposed to VHP delivered by air conditioning. Different exposure conditions were tested. Exposure to VHP resulted in sterilization of the bacterial samples in three different test runs. CONCLUSIONS: VHP treatment is an effective means of reducing and eliminating room contaminations of M. tuberculosis. SIGNIFICANCE AND IMPACT OF THE STUDY: Fumigation with VHP represents an alternative to formaldehyde fumigation, particularly for decontamination of animal rooms in tuberculosis research laboratories.

A systems interpretation for observations of bird V-formations.

Birds in V formations are frequently observed and two main hypotheses have emerged to explain this particular geometry: (i) it offers aerodynamic advantages and (ii) it is used to improve visual communication. Both explanations require a bird to track its predecessor. However, most V-formations observed in nature are small and the distribution of wing-tip spacings has a large variation. This suggests that tracking the lateral position of the preceding bird is a difficult task. Control theorists, when trying to control platoons of vehicles, also noted that predecessor following is difficult. In this paper, we apply a result from systems theory to explain the observations of bird V-formations. The strength of this result is that it does not rely on the details of the bird flight model. Thus we claim that formation flight is inherently difficult for birds.

Limited mycobacterial infection of the liver as a consequence of its microanatomical structure causing restriction of mycobacterial growth to professional phagocytes.

Among sites of extrapulmonary growth of Mycobacterium tuberculosis, the liver is the least infected. Our data suggest that this is due to the complete restriction of mycobacterial growth to liver macrophages. Unlike in organs more persistently seeded by M. tuberculosis, in the liver the bacteria do not infect cell types other than professional phagocytes.

Highly functionalized dimeric tetraethynylethenes and expanded radialenes: strong evidence for macrocyclic cross-conjugation.

A selection of dimeric tetraethynylethenes (TEEs) and perethynylated expanded radialenes, containing different donor/acceptor substitution patterns, have been prepared and fully characterized. The first X-ray crystal structure of an expanded [6]radialene, with twelve peripheral 3,5-di(tert-butyl)phenyl substituents, is presented. This macrocycle, the all-carbon core of which is isomeric with fullerene C60, adopts a non-planar, "chair-like" conformation. Also a TEE dimer, carrying N,N-dimethylaniline donor substituents, has been subjected to an X-ray crystallographic analysis. The electronic properties were studied by UV/Vis spectroscopy and electrochemistry, providing fundamental insight into mechanisms of pi-electron delocalization in the acyclic and macrocyclic chromophores. Donor or donor-acceptor-substituted dimeric TEE derivatives show very strong absorptions extending over the entire UV/Vis region; their longest wavelength absorption bands have high charge-transfer character. Macrocyclic cross-conjugation in the expanded radialenes becomes increasingly efficient with increasing donor-acceptor polarization. A dual, strongly solvent-polarity-dependent fluorescence was observed for a tetrakis(N,N-dimethylaniline)-substituted dimeric TEE; this interesting emission behavior is explained by the twisted intramolecular charge-transfer (TICT) state model. Donor-substituted expanded radialenes display huge resonance-enhanced third-order nonlinear optical coefficients.

Tetrathiafulvalene-acetylene scaffolding: new pi-electron systems for advanced materials.

Novel extended tetrathiafulvalenes (TTFs) with hexa-2,4-diyne-1,6-diylidene spacers between the two 1,3-dithiole rings and laterally appended alkynyl moieties for one- and two-dimensional scaffolding were synthesised and investigated for their electronic properties.

A novel tracking technique for the continuous precise measurement of tumour positions in conformal radiotherapy.

Changing tumour positions induced by organ motion can impede the full exploitation of the strengths of conformal radiotherapy. The unnecessary irradiation of healthy tissue surrounding the target volume can be the consequence. To overcome this, one should measure tumour positions directly and continuously with high resolution in space and time. We have developed a novel tracking technique which will allow this. The method can also be used to survey and monitor the patient positioning. The proper functioning of our method has been technically demonstrated at PSI with the help of phantom irradiation with protons. Implementation into the clinical environment is now beginning.

Additive effect of neutralizing antibody and antiviral drug treatment in preventing virus escape and persistence.

Poorly cytopathic or noncytopathic viruses can escape immune surveillance and establish a chronic infection. Here we exploited the strategy of combining antiviral drug treatment with the induction of a neutralizing antibody response to avoid the appearance of neutralization-resistant virus variants. Despite the fact that H25 immunoglobulin transgenic mice infected with lymphocytic choriomeningitis virus mounted an early neutralizing antibody response, the virus escaped from neutralization and persisted. After ribavirin treatment of H25 transgenic mice, the appearance of neutralization-resistant virus was prevented and virus was cleared. Thus, the combination of virus-neutralizing antibodies and chemotherapy efficiently controlled the infection, whereas each defense line alone did not. Similar additive effects may be unexpectedly efficient and beneficial in humans after infections with persistent viruses such as hepatitis C virus and hepatitis B virus and possibly human immunodeficiency virus.

Sperm number and condition affect the number of basal cells and their expression of macrophage antigen in the murine epididymis.

Unilateral ligation of the mid-corpus epididymis, the proximal vas deferens and imposition of an abdominal temperature for 6 days as well as bilateral castration for 3, 6 or 14 days, resulted in a change in epithelial composition of the adult murine epididymis with regard to the number and antigen expression of basal cells. There were fewer basal cells per tubule cross-section with fewer expressing F4/80 antigen when spermatozoa were absent from the proximal lumen following short-term castration. Conversely, more basal cells with more of them demonstrating macrophage antigen expression were evident when more or damaged spermatozoa were in the proximal lumen after corpus ligation and exposure to abdominal temperature or in the cauda after long-term withdrawal of androgen support. By contrast, ligation of the vas deferens did not lead to tubule distension, and hence sperm accumulation, and did not alter the basal cell population in the cauda epididymis. The data suggest that epididymal basal cells respond in number and macrophage antigen expression to the presence of sperm autoantigens in the lumen with little dependence on circulating androgens. These changes may represent responses to minimise the interaction of sperm autoantigens with the immune system and the risk of immunological infertility.

Rapid neutrophil response controls fast-replicating intracellular bacteria but not slow-replicating Mycobacterium tuberculosis.

Being one of the first cells to invade the site of infection, neutrophils play an important role in the control of various bacterial and viral infections. In the present work, the contribution of neutrophils to the control of infection with different intracellular bacteria was investigated. Mice were treated with the neutrophil-depleting monoclonal antibody RB6-8C5, and the time course of infection in treated and untreated mice was compared by using intracellular bacterial species and strains varying in virulence and replication rate. The results indicate that neutrophils are crucial for the control of fast-replicating intracellular bacteria, whereas early neutrophil effector mechanisms are dispensable for the control of the slow-replicating Mycobacterium tuberculosis.

Regional variation in macrophage antigen expression by murine epididymal basal cells and their regulation by testicular factors.

Factors controlling the appearance in the epididymis of basal cells and their expression of macrophage antigens were examined by ligating the efferent ducts to prevent the entry of spermatozoa in adult and juvenile mice. Fixation and antigen retrieval techniques were developed to preserve tissue morphology and expression of two macrophage antigens in paraffin-embedded epididymal tissue. A combination of periodate-lysine-phosphate fixation, low-temperature embedding and enzyme predigestion of sections permitted immunohistochemical detection of the mature macrophage antigen Mac-1, whereas the panmacrophage marker F4/80 required fixation in neutral-buffered formalin. Epididymal basal cells were immunostained for F4/80 and quantified with avidin-biotin-peroxidase. In the adult mouse, the total number of basal cells per millimeter length of tubule cross section perimeter and the percentage expressing the F4/80-antigen were significantly higher in the initial segment and caput region than in all other epididymal regions. In the initial segment, immunostained basal cells surrounded the tubule in a network, and some extended towards the lumen. Ligation of the efferent ducts to prevent inflow of testicular secretions significantly reduced the number of basal cells per cross section in the initial segment of the adult and juvenile; the percentage of basal cells expressing macrophage antigens in the initial segment and the caput epididymidis was also reduced. Since basal cells still appeared in the ligated postpubertal epididymis, it is concluded that testicular exocrine secretions entering the epididymal lumen around puberty are not the major influence on basal cell appearance in the murine epididymis, but they may modulate their expression of macrophage antigens.

Autoimmune intestinal pathology induced by hsp60-specific CD8 T cells.

Due to their ubiquitous distribution and high degree of structural similarity, heat shock proteins (hsp) are potential target antigens in autoimmune diseases. Here, we describe induction of intestinal inflammation following transfer of hsp60-reactive CD8 T cells into mice. Inflammatory reactions were MHC class I dependent and developed primarily in the small intestine. IFN gamma and TNF alpha, as well as gut-derived hsp60, were elevated at sites of T cell infiltration. Intestinal lesions were drastically reduced in mice lacking receptors for TNF alpha. Pathology also developed in germ-free mice, indicating recognition of host-derived hsp60 by CD8 T cells. This report demonstrates that CD8 T cells with defined antigen specificity cause intestinal inflammation, emphasizing a link between infection and autoimmune disease.

Application of a peptide bank from porcine brain in isolation of regulatory peptides.

Over the past years, the introduction of biological assay systems, random peptide sequencing and orphan receptor screening has led to the isolation and identification of new regulatory peptides with potential clinical impact. We have developed a method for separating peptides into about 300 fractions from large amounts of porcine brain tissue. The preparation of this peptide bank consists of three steps including ultrafiltration followed by cation-exchange separation and reversed-phase chromatography. These fractions represent the peptide bank with desalted and lyophilized peptides from brain tissue. Molecular masses of the peptides in the fractions are determined by matrix-assisted laser desorption ionization MS and a mass data bank is subsequently generated. For systematic analysis of the peptides, a subsequent two-step purification procedure is followed by Edman sequencing resulting in the identification of different peptides. A survival assay with a neuronal cell line revealing the stimulatory and inhibitory activities is applied as a model to test the 300 fractions. This primary screen indicates that the biological activities of the extracted peptides are easily characterized and, moreover, can be related to the biochemical entities. We conclude that the established peptide bank is an efficient and useful tool for the isolation of regulatory brain peptides applying different purification strategies.

In vivo selection of neutralization-resistant virus variants but no evidence of B cell tolerance in lymphocytic choriomeningitis virus carrier mice expressing a transgenic virus-neutralizing antibody.

B cell tolerance is maintained by active deletion and functional anergy of self-reactive B cells depending on the time, amount, and site of the self-antigen expression. To study B cell tolerance toward a transplacentally transmitted viral Ag, we crossed transgenic mice expressing the mu heavy and the kappa light chain of the lymphocytic choriomeningitis virus (LCMV)-neutralizing mAb KL25 (HL25-transgenic mice) with persistently infected LCMV carrier mice. Although HL25-transgenic LCMV carrier mice exhibited the same high virus titers as nontransgenic LCMV carrier mice, no evidence for B cell tolerance was found. In contrast, enhanced LCMV-neutralizing Ab titers were measured that, however, did not clear the virus. Instead, LCMV isolates from different tissues turned out to be neutralization resistant Ab escape variants expressing different substitutions of amino acid Asn119 of the LCMV-glycoprotein 1 that displays the neutralizing B cell epitope. Virus variants with the same mutations were also selected in vitro in the presence of the transgenic mAb KL25 confirming that substitutions of Asn119 have been selected by LCMV-neutralizing Abs. Thus, despite abundant expression of viral neo-self-antigen in HL25-transgenic LCMV carrier mice, transgenic B cells expressing LCMV-neutralizing Abs were rather stimulated than tolerized and neutralization resistant Ab escape variants were selected in vivo.

The appearance of basal cells in the developing murine epididymis and their temporal expression of macrophage antigens.

This work demonstrates similarities between epididymal basal cells and macrophages in the mouse. Light microscopic studies of the postnatal development of the murine epididymis showed that basal cells were not present before days 12, 14 and 16 in the cauda, caput and corpus epididymis, respectively. An increase in cell number per unit length of tubule perimeter was demonstrated in all segments between days 20 and 27, when testicular fluid and spermatozoa start entering the epididymis. In the adult, there were more basal cells per unit perimeter in the cauda than caput or corpus epididymis. Conspicuous and consistent expression by basal cells of antigens detected by antibodies against tissue-fixed macrophages (F4/80) and mature macrophages (Mac-1) occurred only after they became established within the epithelium. Basal cells in the cauda epididymis did not display either antigen in the adult, although they persisted in the caput region. Such developmental patterns are compatible with the hypothesis that basal cells play a role in immune defence against sperm autoantigens.

Enhanced virus clearance by early inducible lymphocytic choriomeningitis virus-neutralizing antibodies in immunoglobulin-transgenic mice.

Following infection of mice with lymphocytic choriomeningitis virus (LCMV), virus-neutralizing antibodies appear late, after 30 to 60 days. Such neutralizing antibodies play an important role in protection against reinfection. To analyze whether a neutralizing antibody response which developed earlier could contribute to LCMV clearance during the acute phase of infection, we generated transgenic mice expressing LCMV-neutralizing antibodies. Transgenic mice expressing the immunoglobulin mu heavy chain of the LCMV-neutralizing monoclonal antibody KL25 (H25 transgenic mice) mounted LCMV-neutralizing immunoglobulin M (IgM) serum titers within 8 days after infection. This early inducible LCMV-neutralizing antibody response significantly improved the host's capacity to clear the infection and did not cause an enhancement of disease after intracerebral (i.c.) LCMV infection. In contrast, mice which had been passively administered LCMV-neutralizing antibodies and transgenic mice exhibiting spontaneous LCMV-neutralizing IgM serum titers (HL25 transgenic mice expressing the immunoglobulin mu heavy and the kappa light chain) showed an enhancement of disease after i.c. LCMV infection. Thus, early-inducible LCMV-neutralizing antibodies can contribute to viral clearance in the acute phase of the infection and do not cause antibody-dependent enhancement of disease.