Infections in lung and heart transplant recipients : Studies on the impact of bronchoscopy in the diagnosis of respiratory infections and detection of viral infections in blood and bronchoalveolar lavage fluid

Infection is a major cause of mortality and morbidity after thoracic organ transplantation. The aim of the present study was to evaluate the infectious complications after lung and heart transplantation, with a special emphasis on the usefulness of bronchoscopy and the demonstration of cytomegalovirus (CMV), human herpes virus (HHV)-6, and HHV-7. We reviewed all the consecutive bronchoscopies performed on heart transplant recipients (HTRs) from May 1988 to December 2001 (n = 44) and lung transplant recipients (LTRs) from February 1994 to November 2002 (n = 472). To compare different assays in the detection of CMV, a total of 21 thoracic organ transplant recipients were prospectively monitored by CMV pp65-antigenemia, DNAemia (PCR), and mRNAemia (NASBA) tests. The antigenemia test was the reference assay for therapeutic intervention. In addition to CMV antigenemia, 22 LTRs were monitored for HHV-6 and HHV-7 antigenemia. The diagnostic yield of the clinically indicated bronchoscopies was 41 % in the HTRs and 61 % in the LTRs. The utility of the bronchoscopy was highest from one to six months after transplantation. In contrast, the findings from the surveillance bronchoscopies performed on LTRs led to a change in the previous treatment in only 6 % of the cases. Pneumocystis carinii and CMV were the most commonly detected pathogens. Furthermore, 15 (65 %) of the P. carinii infections in the LTRs were detected during chemoprophylaxis. None of the complications of the bronchoscopies were fatal. Antigenemia, DNAemia, and mRNAemia were present in 98 %, 72 %, and 43 % of the CMV infections, respectively. The optimal DNAemia cut-off levels (sensitivity/specificity) were 400 (75.9/92.7 %), 850 (91.3/91.3 %), and 1250 (100/91.5 %) copies/ml for the antigenemia of 2, 5, and 10 pp65-positive leukocytes/50 000 leukocytes, respectively. The sensitivities of the NASBA were 25.9, 43.5, and 56.3 % in detecting the same cut-off levels. CMV DNAemia was detected in 93 % and mRNAemia in 61 % of the CMV antigenemias requiring antiviral therapy. HHV-6, HHV-7, and CMV antigenemia was detected in 20 (91 %), 11 (50 %), and 12 (55 %) of the 22 LTRs (median 16, 31, and 165 days), respectively. HHV-6 appeared in 15 (79 %), HHV-7 in seven (37 %), and CMV in one (7 %) of these patients during ganciclovir or valganciclovir prophylaxis. One case of pneumonitis and another of encephalitis were associated with HHV-6. In conclusion, bronchoscopy is a safe and useful diagnostic tool in LTRs and HTRs with a suspected respiratory infection, but the role of surveillance bronchoscopy in LTRs remains controversial. The PCR assay acts comparably with the antigenemia test in guiding the pre-emptive therapy against CMV when threshold levels of over 5 pp65-antigen positive leukocytes are used. In contrast, the low sensitivity of NASBA limits its usefulness. HHV-6 and HHV-7 activation is common after lung transplantation despite ganciclovir or valganciclovir prophylaxis, but clinical manifestations are infrequently linked to them.

Part I: Enzyme replacement versus neurotrophic factor viral vector-mediated gene therapy of Parkinson’s disease, Part II: The effects of orally dosed tolcapone and entacapone on dopamine metabolism in the dorsal striatum and nucleus accumbens in rats

Part I: Parkinson’s disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.

Activation and persistence of implicit causality information in spoken language comprehension

A visual world eye-tracking study investigated the activation and persistence of implicit causality information in spoken language comprehension. We showed that people infer the implicit causality of verbs as soon as they encounter such verbs in discourse, as is predicted by proponents of the immediate focusing account (Greene & McKoon, 1995; Koornneef & Van Berkum, 2006; Van Berkum, Koornneef, Otten, & Nieuwland, 2007). Interestingly, we observed activation of implicit causality information even before people encountered the causal conjunction. However, while implicit causality information was persistent as the discourse unfolded, it did not have a privileged role as a focusing cue immediately at the ambiguous pronoun when people were resolving its antecedent. Instead, our study indicated that implicit causality does not affect all referents to the same extent, rather it interacts with other cues in the discourse, especially when one of the referents is already prominently in focus.

Characterization of viral proteins ofOryctes baculovirus and comparison between two geographical isolates

BacilliformOryctes baculovirus particles have been visualized in electron micrographs of midgut sections from virus infectedOryctes rhinoceros beetles. Morphologically the Indian isolate (Oryctes baculovirus, KI) resembled the previously reportedOryctes baculovirus, isolate PV505. The constituent proteins of baculovirus KI have been analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by Western blots using polyclonal antibodies raised against the complete viral particles, as probes. A total of forty eight viral proteins have been identified. Fourteen viral proteins were located on the viral envelope. Among the proteins constituting the nucleocapsid, three were located internally within the capsid. A 23.5 kDa protein was tightly associated with viral DNA in the nucleocapsid core. Two envelope and seven capsid proteins stained positive for glycosylation. Comparison between the viral proteins of KI and PV505 revealed differences in SDS-PAGE profiles and glycosylation patterns. Immunoblotting of KI and PV505 proteins with anti KI antiserum demonstrated antigenic differences between the two viral isolates.

Performance Persistence of International Stock Funds

People saving in mutual funds often look at historical performance before they decide which funds to invest in. The implicit assumption made is that superior performance is likely to be repeated in the future. The findings presented in this study, which investigates funds sold on the Swedish market, support such an approach provided that the time horizons are limited to one year. International stock funds that have performed strongly one year are likely to outperform their peers also the following years. But if the historical and future time horizons are extended to two or three years, the positive relationship between past and future performance vanishes in most cases. Persistence tests focusing on the aggregated performance of fund companies were also carried out. These tests produced results rather similar to those on the individual fund level.

Mother-Infant Antibodies to Pneumococcal Polysaccharides and Proteins : Transfer and Persistence of Maternal Antibodies and Development of Vaccine-Induced and Naturally Acquired Antibodies in Infants

Symptomless nasopharyngeal carriage of Streptococcus pneumoniae (pneumococcus) is very common in young children. Occasionally the carriage proceeds into mild mucosal diseases, such as sinusitis or acute otitis media, or into serious life-threatening diseases, such as pneumonia, sepsis or meningitis. Each year, up to one million children less than five years of age worldwide die of invasive pneumococcal diseases (IPD). Especially in the low-income countries IPD is a leading health problem in infants; 75% of all IPD cases occur before one year of age. This stresses the need of increased protection against pneumococcus in infancy. Anti-pneumococcal antibodies form an important component in the defence against pneumococcal infection. Maternal immunisation and early infant immunisation are two possible ways by which potentially protective antibody concentrations against pneumococci could be achieved in early infancy. The aim of this thesis is to increase the knowledge of antibody mediated protection against pneumococcal disease in infants and young children. We investigated the transfer of maternal anti-pneumococcal antibodies from Filipino mothers to their infants, the persistence of the transferred antibodies in the infants, the immunogenicity of the 23-valent pneumococcal polysaccharide vaccine (PPV) in infants and the response of the children to a second dose of PPV at three years of age. We also investigated the development of antibodies to pneumococcal protein antigens in relation to culture-confirmed pneumococcal carriage in infants. Serum samples were collected from the mothers, the umbilical cords and from the infants at young age as well as at three years of age. The samples were used to determine the antibody concentrations to pneumococcal serotypes 1, 5, 6B, 14, 18C and 19F, as well as to the pneumococcal proteins PspA, PsaA, Ply, PspC, PhtD, PhtDC and LytC by the enzyme immunoassay. The findings of the present study confirm previously obtained results and add to the global knowledge of responses to PPV in young children. Immunising pregnant women with PPV provides the infants with increased concentrations of pneumococcal polysaccharide antibodies. Of the six serotypes examined, serotypes 1 and 5 were immunogenic already in infants. At three years of age, the children responded well to the second dose of PPV suggesting that maternal and early infant immunisations might not induce hyporesponsiveness to polysaccharide antigens after subsequent immunisations. The anti-protein antibody findings provide useful information for the development of pneumococcal protein vaccines. All six proteins studied were immunogenic in infancy and the development of anti-protein antibodies started early in life in relation to pneumococcal carriage.

Characterization of the genome of Oryctes baculovirus, a viral biocide of the insect pest Oryctes rhinoceros

Oryctes baculovirus is a viral biocide exploited for the control of the insect pest Oryctes rhinoceros. We have recently established a physical map of the genome of the Indian isolate of Oryctes baculovirus (OBV-KI). Here we examine the genomic relatedness between OBV-KI and OBV-PV505, the type isolate (originally from the Philippines), by DNA reassociation kinetics and by the use of restriction endonucleases. On the basis of differences in restriction-enzyme profiles between the two genomes, and previously reported differences in protein profiles and antigenic makeup, we propose the taxonomic status of a variant of Oryctes baculovirus for the Indian isolate.

Interaction of Sesbania mosaic virus movement protein with the coat protein - implications for viral spread

Sesbania mosaic virus (SeMV) is a single-stranded positive-sense RNA plant virus belonging to the genus Sobemovirus. The movement protein (MP) encoded by SeMV ORF1 showed no significant sequence similarity with MPs of other genera, but showed 32% identity with the MP of Southern bean mosaic virus within the Sobemovirus genus. With a view to understanding the mechanism of cell-to-cell movement in sobemoviruses, the SeMV MP gene was cloned, over-expressed in Escherichia coli and purified. Interaction of the recombinant MP with the native virus (NV) was investigated by ELISA and pull-down assays. It was observed that SeMV MP interacted with NV in a concentration- and pH-dependent manner. Analysis of N- and C-terminal deletion mutants of the MP showed that SeMV MP interacts with the NV through the N- terminal 49 amino acid segment. Yeast two-hybrid assays confirmed the in vitro observations, and suggested that SeMV might belong to the class of viruses that require MP and NV/coat protein for cell-to-cell movement.

Persistence Problem in Two-Dimensional Fluid Turbulence

We present a natural framework for studying the persistence problem in two-dimensional fluid turbulence by using the Okubo-Weiss parameter Lambda to distinguish between vortical and extensional regions. We then use a direct numerical simulation of the two-dimensional, incompressible Navier-Stokes equation with Ekman friction to study probability distribution functions (PDFs) of the persistence times of vortical and extensional regions by employing both Eulerian and Lagrangian measurements. We find that, in the Eulerian case, the persistence-time PDFs have exponential tails; by contrast, this PDF for Lagrangian particles, in vortical regions, has a power-law tail with an exponent theta = 2.9 +/- 0.2.

Dynamics of zeolite cage and its effect on the diffusion properties of sorbate: persistence of diffusion anomaly in NaA zeolite

Recent computer simulations on zeolites Y and A have found that the diffusion coefficient and the rate of intercage diffusion exhibit, apart from a linear dependence on the reciprocal of the square of the sorbate diameter, an anomalous peak as sorbate diameter approaches the window diameter. Here we report molecular dynamics simulations of zeolite NaA incorporating framework flexibility as a function of sorbate diameter in order to verify the existence of anomalous diffusion. Results suggest persistence of anomalous diffusion or ring effect. This suggests that the anomalous behavior is a general effect characteristic of zeolites Y and A. The barrier for diffusion across the eight-ring window is seen to be negative and is found to decrease with sorbate size. The effect of sorbate on the cage motion has also been investigated. Results suggest that the window expands during intercage migration only if the sorbate size is comparable to the window diameter. Flexible cage simulations yield a higher value for the diffusion coefficient and also the rate of intercage diffusion. This increase has been shown to be due to an increase in the intercage diffusions via the centralized diffusion mode rather than the surface-mediated mode. It is shown that this increase arises from an increase in the single particle density distribution in the region near the cage center.

Japanese encephalitis virus infection of mouse cell lines: ability to prime mice for generation of virus specific cytotoxic T lymphocytes and differences in CTL recognisable viral determinants

Ten different mouse cell lines were examined for Japanese encephalitis virus (JEV) infection in vitro and then tested for their ability to generate virus specific cytotoxic T lymphocytes (CTL). Among all cell lines examined, Neuro La (a neuroblastoma) was readily infected with JEV as examined by immunofluorescence and viral replication. Among other cells, P388D1, RAW 264.7 (Macrophage origin), Sp2/0 (B-cell Hybridoma), YAC-1 (T-cell lymphoma), and L929 (Fibroblast) were semipermissive to JEV infection. The cytopathic effects caused by progressive JEV infection varied from cell line to cell line. In the case of YAC-1 cells long-term viral antigen expression was observed without significant alterations in cell viability. Intermediate degrees of cytopathicity are seen in RAW 264.7 and L929 cells while infection of PS, Neuro 2a, P388D1 and Sp2/0 caused major viability losses. All infected cell lines were able to prime adult BALB/c (H-2(d)) mice for the generation of secondary JEV specific CTL. In contrast to YAC-1, the permissive neuroblastoma cell line Neuro 2a (H-2K(k)D(d)) was found to be least efficient in its ability to stimulate anti-viral CTL generation. Cold target competition studies demonstrated that both Neuro 2a and YAC-1 (H-2K(k)D(d)) cells expressed similar viral determinants that are recognised by CTL, suggesting that the reason for the lower ability of Neuro 2a to stimulate anti-viral CTL was not due to lack of viral CTL determinants. These findings demonstrate that a variety of mouse cell lines can be infected with Japanese encephalitis virus, and that these infected cells could be utilised to generate virus specific CTL in BALB/c mice.

Non-viral ex vivo hepatic gene transfer by in situ lipofection of liver and intraperitoneal transplantation of hepatocytes

Perfusion of liver with plasmid DNA-lipofectin complexes via the portal vein results in efficient accumulation of the vector in hepatocytes. Such hepatocytes, when administered intraperitoneally into a hepatectomized rat, repopulate the liver and express the transgene efficiently. This procedure obviates the need for large-scale hepatocyte culture for ex vivo gene transfer. Further, intraperitoneal transplantation is a simple and cost-effective strategy of introducing genetically modified hepatocytes into liver. Thus, in situ lipofection of liver and intraperitoneal transfer of hepatocytes can be developed into a novel method of non-viral ex vivo gene transfer technique that has applications in the treatment of metabolic disorders of liver and hepatic gene therapy.