Human tumor-specific CD4+ cytotoxic T lymphocytes (CTL) were generated against duodenum papilloma cell line TGBC18TKB from HLA type-matched peripheral blood mononuclear cells. Concanavalin A (Con A) immobilized on carrier beads stimulated growth of the CTL in a long-term culture without repeated antigen stimulation, while soluble Con A induced death of the CTL. The CTL exhibited the target-specific cytotoxicity in a more potent manner than those before the long-term culture in the presence of the immobilized Con A. Enhanced expression of the adhesion molecule, CD11b, was observed on the CTL. These results suggest that immobilized Con A will be useful for continuous growth stimulation and large scale expansion of CTL without tumor antigen.

Guanosine at position 26 in eukaryotic tRNAs is usually modified to N2 , N2 -dimethylguanosine (m22G26). In Saccharomyces cerevisiae , this reaction is catalysed by the TRM1 encoded tRNA (m22G26)dimethyltransferase. As a prerequisite for future studies, the yeast TRM1 gene was expressed in Escherichia coli and the His-tagged Trm1 protein (rTrm1p) was extensively purified. rTrm1p catalysed both the mono- and dimethylation of G26 in vivo in Escherichia coli tRNA and in vitro in yeast trm1 mutant tRNA. The TRM1 gene from two independent wild-type yeast strains differed at 14 base positions causing two amino acid exchanges . Exchange of the original Ser467 for Leu caused a complete loss of enzyme activity in vitro against trm1 yeast tRNA. Comparatively short N- or C-terminal deletions from the 570 amino acid long Trm1 polypeptide decreased or eliminated the enzyme activity, as did some point mutations within these regions. This indicated that the protein is not a two domain peptide with the enzyme activity localised to one of the domains, but rather that both ends of the polypeptide seem to interact to influence the conformation of those parts that make up the RNA-binding site and/or the active site of the enzyme.

By two PCR-based diagnostic methods, Plasmodium malariae infections have been rediscovered at two foci in the Sichuan province of China, a region where no cases of P. malariae have been officially reported for the last 2 decades. In addition, a variant form of P. malariae which has a deletion of 19 bp and seven substitutions of base pairs in the target sequence of the small-subunit (SSU) rRNA gene was detected with high frequency. Alignment analysis of Plasmodium sp. SSU rRNA gene sequences revealed that the 5′ region of the variant sequence is identical to that of P. vivax or P. knowlesi and its 3′ region is identical to that of P. malariae. The same sequence variations were also found in P. malariae isolates collected along the Thai-Myanmar border, suggesting a wide distribution of this variant form from southern China to Southeast Asia.

Studies were undertaken to examine hepatocyte CD14 expression during endotoxemia. Our results show that lipopolysaccharide (LPS) treatment in vivo caused a marked upregulation in CD14 mRNA and protein levels in rat hepatocytes. Detectable increases in mRNA were seen as early as 1.5 h after LPS treatment; these increases peaked at 20-fold by 3 h and returned to baseline levels by 24 h. In situ hybridization localized the CD14 mRNA expression to hepatocytes both in vitro and in vivo. Increases in hepatic CD14 protein levels were detectable by 3 h and peaked at 12 h. Hepatocytes from LPS-treated animals expressed greater amounts of cell-associated CD14 protein, and more of the soluble CD14 was released by hepatocytes from LPS-treated rats in vitro. The increases in hepatocyte CD14 expression during endotoxemia occurred in parallel to increases of CD14 levels in plasma. To provide molecular identification of the hepatocyte CD14, we cloned the rat liver CD14 cDNA. The longest clone consists of a 1,591-bp insert containing a 1,116-bp open reading frame. The deduced amino acid sequence is 372 amino acids long, has 81.8 and 62.8% homology to the amino acid sequences of mouse and human CD14, respectively, and is identical to the rat macrophage CD14. The expressed CD14 protein from this clone was functional, as indicated by NF-κB activation in response to LPS and fluorescein isothiocyanate-LPS binding in CHO cells stably transfected with rat CD14. A nuclear run-on assay showed that CD14 transcription rates were significantly increased in hepatocytes from LPS-treated animals, indicating that the upregulation in CD14 mRNA levels observed in rat hepatocytes after LPS treatment is dependent, in part, on increased transcription. In vitro and in vivo experiments indicated that interleukin-1β and/or tumor necrosis factor α participate in the upregulation of CD14 mRNA levels in hepatocytes. Our data indicate that hepatocytes express CD14 and that hepatocyte CD14 mRNA and protein levels increase rapidly during endotoxemia. Our observations also support the idea that soluble CD14 is an acute-phase protein and that hepatocytes could be a source for soluble CD14 production.

The Epstein-Barr virus (EBV) lytic switch gene, BZLF1, is tightly regulated in latently infected B cells. The BZLF1 gene promoter (Zp) contains several cis elements that have been previously shown to respond to inducers of the viral lytic cycle. These include four copies of an element referred to as the ZI domains and an element that contains a consensus CRE/AP-1 motif (ZII domain). In addition, Zp is autoregulated through two sites that bind the BZLF1 gene product Zta. The ZI domains have been shown to bind the ubiquitous cellular transcription factors Sp1 and Sp3 and/or the myocyte enhancer factor 2D (Liu et al., EMBO J. 16:143–153, 1997; Liu et al., Virology 228:9–16, 1997). Here we present a functional analysis of the ZII domain and show: (i) ATF-1 and ATF-2 appear to be the predominant cellular factors that bind to the CRE/AP-1 motif present in the ZII domain; and (ii) the region immediately upstream of the CRE/AP-1 motif contains a potent negative cis element, mutation of which results in a >10-fold increase in Zp activity. The negative cis element (ZIIR) in the ZII domain decreases both basal and induced Zp activity and thus is likely to play an important role in regulating reactivation of EBV. In addition, analysis of heterologous promoter constructs indicates that the function of ZIIR is context sensitive. Attempts to demonstrate a cellular factor binding to ZIIR have been unsuccessful, leaving unresolved the mechanism by which repression is mediated.

DNA vaccination is an effective means of eliciting both humoral and cellular immunity, including cytotoxic T lymphocytes (CTL). Using an influenza virus model, we previously demonstrated that injection of DNA encoding influenza virus nucleoprotein (NP) induced major histocompatibility complex class I-restricted CTL and cross-strain protection from lethal virus challenge in mice (J. B. Ulmer et al., Science 259:1745–1749, 1993). In the present study, we have characterized in more detail the cellular immune responses induced by NP DNA, which included robust lymphoproliferation and Th1-type cytokine secretion (high levels of gamma interferon and interleukin-2 [IL-2], with little IL-4 or IL-10) in response to antigen-specific restimulation of splenocytes in vitro. These responses were mediated by CD4+ T cells, as shown by in vitro depletion of T-cell subsets. Taken together, these results indicate that immunization with NP DNA primes both cytolytic CD8+ T cells and cytokine-secreting CD4+ T cells. Further, we demonstrate by adoptive transfer and in vivo depletion of T-cell subsets that both of these types of T cells act as effectors in protective immunity against influenza virus challenge conferred by NP DNA.

We conducted steady-state pharmacokinetic studies with high-dose fluconazole with rabbits and human volunteers. We then derived mathematical equations that predict the doses of fluconazole that should be given to rabbits to produce 24-h area under the concentration-time curve values and maximum concentrations in serum that are similar to those measured for humans given 800 to 2,000 mg of fluconazole per day. These equations provide a rational basis for designing future efficacy studies with rabbits and in evaluating the strength with which results of previously conducted studies using rabbit infection models can be extrapolated to the clinic.

We have used a recently described animal model to characterize the ocular pharmacokinetics of sparfloxacin in vitreous humor of uninfected albino rabbits following systemic administration and direct intraocular injection. The relationships of lipophilicity, protein binding, and molecular weight to the penetration and elimination of sparfloxacin were compared to those of ciprofloxacin, fleroxacin, and ofloxacin. To determine whether elimination was active, elimination rates following direct injection with and without probenecid or heat-killed bacteria were compared. Sparfloxacin concentrations were measured in the serum and vitreous humor by a biological assay. Protein binding and lipophilicity were determined, respectively, by ultrafiltration and oil-water partitioning. Pharmacokinetic parameters were characterized with RSTRIP, an iterative, nonlinear, weighted, least-squares-regression program. The relationship between each independent variable and mean quinolone concentration or elimination rate in the vitreous humor was determined by multiple linear regression. The mean concentration of sparfloxacin in the vitreous humor was 59.4% ± 12.2% of that in serum. Penetration of sparfloxacin, ciprofloxacin, fleroxacin, and ofloxacin into, and elimination from, the vitreous humor correlated with lipophilicity (r2 > 0.999). The linear-regression equation describing this relationship was not improved by including the inverse of the square root of the molecular weight and/or the degree of protein binding. Elimination rates for each quinolone were decreased by the intraocular administration of probenecid. Heat-killed Staphylococcus epidermidis decreased the rate of elimination of fleroxacin. Penetration of sparfloxacin into the noninflamed vitreous humor was greater than that of any quinolone previously examined. There was an excellent correlation between lipophilicity and vitreous entry or elimination for sparfloxacin as well as ciprofloxacin, fleroxacin, and ofloxacin. There are two modes of quinolone translocation into and out of the vitreous humor: diffusion into the eye and both diffusion and carrier-mediated elimination out of the vitreous humor.

In this study we defined the pharmacodynamic parameter that optimizes outcome in deep-seated Candida albicans infections treated with fluconazole. Using a murine model of systemic candidiasis, we conducted single-dose dose-ranging studies with fluconazole to determine the dosage of this drug that resulted in a 50% reduction in fungal densities (50% effective dose [ED50]) in kidneys versus the fungal densities in the kidneys of untreated controls. We found that the ED50 of fluconazole given intraperitoneally was 4.56 mg/kg of body weight/day (95% confidence interval, 3.60 to 5.53 mg/kg/day), and the dose-response relationship was best described by an inhibitory sigmoid maximal effect (Emax) curve. To define the pharmacodynamics of fluconazole, we gave dosages lower than, approximating, and higher than the ED50 of fluconazole (range, 3.5 to 5.5 mg/kg/day, equivalent to the ED16 to the ED75) to various groups of infected animals using three dose-fractionation schedules. For each total dose of fluconazole examined, the dose-fractionation schedules optimized the ratio of the area under the concentration-time curve (AUC) to the MIC (the AUC/MIC ratio), the ratio of the maximum concentration of drug in serum (Cmax) to the MIC, and the time that the drug remained above the MIC for the infecting C. albicans isolate. Similar reductions in fungal densities in kidneys were seen between groups that received the same total dose of fluconazole in one, two, or four equally divided doses. Thus, dose-fractionation studies demonstrated that the pharmacodynamic parameter of fluconazole that best predicted outcome was the AUC/MIC ratio.

A set of new water-soluble organic peroxides has been synthesized and evaluated for in vitro antibacterial activity as part of an effort to develop new antibacterial agents for the treatment of acne vulgaris. The water solubility of these new dialkyl peroxides and peroxyesters was achieved by incorporating either a quaternary ammonium group or a polyethylene glycol moiety. These peroxides are effective against both gram-positive and gram-negative bacteria and have a prolonged activity compared to that of benzoyl peroxide and other peroxide-type antiseptic agents. Among them 4-[[(tert-butylperoxy)carbonyl]benzyl]triethylammonium chloride and [10-(tert-butylperoxy)decyl]trimethylammonium bromide have the broadest antimicrobial spectrums. We have shown that the oxidizing properties of the dioxy group of these compounds are responsible for their antibacterial activities.

Although structurally very similar, the aspartate transcarbamoylases (ATCase) of Serratia marcescens and Escherichia coli have distinct allosteric regulatory patterns. It has been reported that a S. marcescens chimera, SM : rS5′ec, in which five divergent residues (r93 to r97) of the regulatory polypeptide were replaced with their Escherichia coli counterparts, possessed E. coli-like regulatory characteristics. T he reverse chimera EC : rS5′sm, in which the same five residues of E. coli have been replaced with their S. marcescens counterpart, lost both heterotrophic and homotropic responses. These results indicate that the r93-r97 region is critical in defining the ATCase allosteric character. Molecular modeling of the regulatory polypeptides has suggested that the replacement of the S5′ β-strand resulted in disruption of the allosteric-zinc interface. However, the structure-function relationship could be indirect, and the disruption of the interface could influence allostery by altering the global energy of the enzyme. Studies of the temperature-sensitivity of the CTP response demonstrate that it is possible to convert CTP inhibition of the SM : rS5′ec chimera at high temperature to activation below 10 °C. Nonetheless, the temperature response of the native S. marcescens ATCase suggests a strong entropic effect that counteracts the CTP activation. Therefore, it is suggested that the entropy component of the coupling free energy plays a significant role in the determination of both the nature and magnitude of the allosteric effect in ATCase.

The design and synthesis of analogues of diadenosine 5′,5′″-P1,P3-triphosphate that are resistant to pyrophosphate hydrolysis is described in relation to their role in signaling and tumorigenesis involving the Fhit protein, the human fragile histidine triad protein, which is a novel Ap3A binding/cleaving protein.

STUDY OBJECTIVE: This study examined the impact on children's respiratory health of a government air quality intervention that restricted the sulphur content of fuels to 0.5% from July 1990 onwards. DESIGN/SETTING/PARTICIPANTS: This study examined the changes, one and two years after the introduction of the intervention, in airway hyperreactivity of non-asthmatic and non-wheezing, primary 4, 5, and 6, school children aged 9-12 years living in a polluted district compared with those in a less polluted district. Bronchial hyperreactivity (BHR)(a 20% decrease in FEV1 provoked by a cumulative dose of histamine less than 7.8 mumol) and bronchial reactivity slope (BR slope) (percentage change in logarithmic scale in FEV1 per unit dose of histamine) were used to estimate responses to a histamine challenge. The between districts differences after the intervention were studied to assess the effectiveness of the intervention. MAIN RESULTS: In cohorts, comparing measurements made before the intervention and one year afterwards, both BHR and BR slope declined from 29% to 16% (p = 0.026) and from 48 to 39 (p = 0.075) respectively in the polluted district; and from 21% to 10% (p = 0.001) and 42 to 36 (p > 0.100) in the less polluted district. Comparing measurements made in 1991 (one year after intervention) with those in 1992 (two years after intervention), only the polluted district showed a significant decline from 28% to 12% (p = 0.016) and from 46 to 35 (p = 0.014), for BHR and BR slope respectively, with a greater decline in both responses (p = 0.018 and 0.073) than in the less polluted district. CONCLUSION: Bronchial hyperresponsiveness tests can be used to support the evaluation of an air quality intervention. The demonstrated reduction in bronchial hyperresponsiveness is an indication of the effectiveness of the intervention.

 

Background—Tumour necrosis factor (TNF) α and TNF-β are soluble ligands binding to TNF receptors with similar activities; soluble TNF receptors neutralise TNF activity by acting as inhibitors. Little is known about the cytokine/soluble receptor role in inflammatory bowel disease (IBD). 
Aims—To test the hypothesis that an imbalance in secretion between TNF and TNF inhibitors plays a role in gut inflammation in patients with IBD. 
Methods—The secretion of TNF-α, TNF-β, and soluble TNF receptors was compared in the culture supernatants of colonic biopsy specimens and isolated lamina propria mononuclear cells from patients with active colonic IBD. 
Results—Spontaneous secretion of TNF-α in involved IBD mucosa was higher than in normal control and self limited colitis mucosa. Secretion of TNF-β was higher in patients with Crohn's disease than in those with ulcerative colitis. Soluble TNF receptor in IBD mucosa inhibited TNF activity. Type 2 soluble receptor release from IBD mucosa was increased in active inflammation; release from lamina propria cells was not increased. Mucosal TNF-α production correlated with severity of disease. 
Conclusions—Results showed enhanced secretion of TNF-α but failure to release enhanced amounts of soluble TNF receptor in lamina propria mononuclear cells of patients with IBD. An imbalance in secretion between TNF and TNF inhibitor may be implicated in the pathogenesis of IBD. 



Keywords: Crohn's disease; inflammation; mucosal immunology; soluble TNF receptor; tumour necrosis factor; ulcerative colitis

OBJECTIVES—To review the outcome measures commonly used in phase III treatment trials of relapsing-remitting multiple sclerosis and to introduce a method of data analysis which is clinically appropriate for the often reversible disability in this type of multiple sclerosis.
METHODS—The conventional end point measures for disability change are inadequate and potentially misleading. Those using the disability difference between study entry and completion do not take into account serial data or disease fluctuations. Rigid definitions of "disease progression" based on two measurements of change in disability several months apart, do not assess worsening after the defined "end point", nor the significant proportion of erroneous "treatment failures" which result from subsequent recovery from relapses that outlast the end point. Assessing attacks merely by counting their frequency ignores the variation in magnitude and duration. These problems can be largely circumvented by integrating the area under a disability-time curve (AUC), a technique which utilises all serial measurements at scheduled visits and during relapses to summarise the total neurological dysfunction experienced by an individual patient on any particular clinical scale during a study period.
CONCLUSIONS—The "summary measure" statistic AUC incorporates both transient and progressive disability into an overall estimate of the dysfunction that was experienced by a patient during a period of time. It is statistically more powerful and clinically more meaningful than conventional methods of assessing disability changes, particularly for trials which are too short to expect to disclose major treatment effects on irreversible disability in patients with a fluctuating disease.



OBJECTIVES—The metabolic changes in the brain of symptomatic subjects affected with Machado-Joseph disease have been previously documented using PET with fluorine-18-fluorodeoxyglucose (FDG). The aim of this study was to evaluate these changes in asymptomatic Machado-Joseph disease gene carriers.
METHODS—Seven asymptomatic MachadoJoseph disease gene carriers, identified using a molecular test, and 10 normal control subjects were recruited for PET studies using FDG. Regional uptake ratios of FDG were calculated from the radioactivity of the cerebellar hemispheres, brainstem, and the temporal, parietal and occipital cortices, divided by the activity in the thalamus.
RESULTS—In comparison with data obtained from normal control subjects, there was significantly decreased FDG utilisation in the cerebellar hemispheres, brainstem, and occipital cortex, and increased FDG metabolism in the parietal and temporal cortices of asymptomatic Machado-Joseph disease gene carriers, suggesting preclinical disease activity. Discriminant analysis of regional FDG uptake correctly classified genetic status (Machado-Joseph disease mutation carriers v mutation negative subjects) in 25 of 25 subjects (100% sensitivity and 100% specificity), and clinical status (asymptomatic mutation carriers v symptomatic patients) in 14 of 15 subjects (100% sensitivity and 85.7% specificity).
CONCLUSION— Subclinical changes of FDG consumption, as measured by non-invasive PET, can act as an objective marker of preclinical disease activity in Machado-Joseph disease.



The prevalence of respiratory symptoms and illnesses among 2225 schoolchildren in Hong Kong was studied by questionnaires administered independently to them and their parents. The agreement was generally poor for respiratory symptoms. The disparity shows the need for cross validation of clinical information in history taking.



BACKGROUND—Macular corneal dystrophy (MCD) is an inherited autosomal recessive disorder that has been subdivided into two primary immunophenotypes, MCD types I and II. The MCD type I gene has been localised previously to chromosome 16q22 and suggestive evidence provided that MCD type II gene is also linked to this region. Here an unusual family is reported where both MCD types I and II are found in a single sibship.
METHODS—Immunoreactivity to an anti-keratan sulphate monoclonal antibody (5-D-4) was evaluated in patients' serum and in corneal tissue obtained at keratoplasty. Chromosomal haplotypes were constructed using microsatellite repeat markers spanning the region of the MCD type I locus.
RESULTS—Immunological studies demonstrated that two of the affected siblings have MCD type II while one has MCD type I. Haplotype analysis suggests that all three affected sibs inherited one identical parental haplotype. However, the two MCD types differ in their alternative chromosome with both MCD type II children sharing an identical haplotype, different from their MCD type I sibling.
CONCLUSION—The findings in this study support the hypothesis that the genes for MCD types I and II co-localise to the same region of chromosome 16 and are likely to be due to allelic manifestations of the same abnormal gene.

 Keywords: macular corneal dystrophy; immunophenotype; alleles; chromosome 16

Aims—To examine the effects of oestradiol and testosterone on the early carcinogenic changes expressed in rat liver from the diethylnitrosamine (DEN), 2-acetylaminofluorene (AAF), partial hepatectomy (PH) model of hepatocarcinogenesis. 
Methods—Preneoplastic liver lesions were evaluated using immunohistochemical analysis of glutathione-S-transferase placental form (GST-P) expression; oestrogen and androgen receptor levels were measured by radioimmunoassay.
Results—Oestradiol administration to non-castrated DEN-AAF-PH treated males resulted in a decrease in the area of GST-P positive foci, while testosterone increased the serum oestradiol level and reduced the area. In males, castration alone and castration with oestradiol replacement significantly reduced the GST-P positive area, and increased the hepatic oestrogen receptor level. In DEN-AAF-PH treated females, castration with testosterone replacement was associated with a significant increase in the GST-P positive area and the hepatic androgen receptor level. 
Conclusion—These findings suggest that exogenous and endogenous oestradiol can suppress chemical hepatocarcinogenesis. It appears that oestrogen receptors may be involved in the inhibition of malignant transformation of preneoplastic liver cells, while androgens and androgen receptors are involved in hepatocarcinogenesis. 



Keywords: hormone receptor; testosterone; diethylnitrosamine; glutathione-S-transferase placental form; antioxidant

Levels of neutral genetic diversity within and between populations were compared between outcrossing (self-incompatible) and inbreeding populations in the annual plant genus Leavenworthia. Two taxonomically independent comparisons are possible, since self-incompatibility has been lost twice in the group of species studied. Within inbred populations of L.uniflora and L.crassa, no DNA sequence variants were seen among the alleles sampled, but high diversity was seen in alleles from populations of the outcrosser L. stylosa, and in self-incompatible L. crassa populations. Diversity between populations was seen in all species. Although total diversity values were lower in the sets of inbreeding populations, between-population values were as high or higher, than those in the outcrossing taxa. Possible reasons for these diversity patterns are discussed. As the effect of inbreeding appears to be a greater than twofold reduction in diversity, we argue that some process such as selection for advantageous mutations, or against deleterious mutations, or bottlenecks occurring predominantly in the inbreeders, appears necessary to account for the findings. If selection for advantageous mutations is responsible, it appears that it must be some form of local adaptive selection, rather than substitution of alleles that are advantageous throughout the species. This is consistent with the finding of high between-population diversity in the inbreeding taxa.

Purpose:Our purpose was to modify a fixation method using Tween-20 and HCl (TH) and to compare it with a protocol using methanol and acetic acid (MA) for the improvement of preimplantation genetic diagnosis by fluorescence in situ hybridization (FISH).

Methods:Single blastomeres were allocated to either the TH or the MA procedure. The two methods were compared to evaluate efficiency of fixation and the intensities of FISH signals.

Results:With the TH method, 123 (93.9%) of 131 blastomeres were fixed, while only 95 (78.5%) of 121 were fixed with MA. Average scores for the intensity of FISH signals were significantly stronger for TH than for MA (P < 0.05). There was also a significant difference in signal intensity scores between the two methods for type-3 nuclei.

Conclusions:Our results indicate that not only are fewer nuclei lost during fixation but also stronger FISH signals can be obtained with the TH method. Thus, modified TH can improve the overall efficiency of preimplantation genetic diagnosis.

Purpose:Our purpose was to analyze potential interactions between the embryo and the maternal endometrial interface in vivo by analyzing immunolocalization of insulin-like growth factor–binding proteins (IGFBPs) -1, -2, and -3 in implantation sites of the mouse.

Methods:Six-week-old B6D2F1female mice underwent superovulation followed by mating and sacrifice at timed intervals. Formalin-fixed paraffin-embedded tissue was used for avidin–biotin immunocytochemical localization of IGFBPs utilizing standard methodology.

Results:Immunostaining at 1.5 days post coitum revealed light staining in the epithelial glandular cells and faint staining in decidual stroma for both IGFBP-1 and IGFBP-2. At 7.5–10.5 days post coitum, there was moderate–dense immunostaining in the decidualized stromal cells at the implantation site for all three IGFBPs, whereas light immunostaining was seen in nonimplantation site decidua.

Conclusions:Compartmentalization of immunostaining for IGFBP-1, -2, and -3 within decidualized stroma suggests that these proteins may be regulated by trophoblastic and/or embryonic signals.