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This report shows the impact of a pentavalent vaccine that includes Haemophilus influenzae type b (Hib) conjugate vaccine on bacterial meningitis in children younger than 5 years in Ghana. A review of the first 3 years of a pediatric bacterial meningitis surveillance program, started in August 2001 in Accra, Ghana, was undertaken. There was a significant reduction, P = 0.042 and 0.017, in percentage of purulent meningitis in children younger than 1 year, comparing the first year when the vaccine was introduced, to the second and third years, respectively.

Background: Most neonatal deaths in developing countries are caused by infections, birth asphyxia and prematurity. Even though most of these deaths occur at home, newborns admitted to hospital neonatal units have a high risk of contracting fatal multi-drug resistant infections., Objective: To compare the type of bacteria and the pattern of antimicrobial susceptibility of organisms causing neonatal infections in 2001/02 with 1991/92 in the same neonatal unit., Design: We reviewed the hospital records of newborns admitted to the neonatal unit in 2001/02 that had positive blood cultures and compared the findings with similar work done 1991/92., Setting: Neonatal Unit, Korle Bu Teaching Hospital, Ghana., Results: Gram negative organisms (predominantly Enterobacter, Klebsiella and Acinetobacter) remained the predominant cause of neonatal infection. There was a reduction in the proportion of gram negative bacteraemia [70.9% in 1991/92 vs. 54.2% in 2001/02 (p<0.001)] due to the increased prevalence of coagulase negative staphylococcus (31.9% in 2001/02 vs. 0% in 1991/92) as a cause of neonatal bacteraemia ten years later. In 1991/92 as 2001/02 all bacterial isolates showed less than 40% susceptibility to ampicillin. The susceptibility of Klebsiella and Enterobacter to commonly used aminoglycosides and cephalosporins had decreased from over 80% in 1991/92 to less than 35% in 2001/02., Conclusion: Bacterial causes of neonatal infections change over time and antimicrobial resistance is a major cause for concern in neonatal units in resource-poor hospitals. Improving infection control practices and instituting systems to monitor antimicrobial use and resistance will compliment community efforts to reduce neonatal mortality.

Summary Background: The distribution of Shigella serotypes is of epidemiological importance and antimicrobial therapy for shigellosis can prevent potential complications of shigellosis. Studies done fifty years ago in Ghana indicated the predominance of Shigella flexneri., Objectives: To describe the distribution of Shigella serogroups and serotypes and their antibiogram profiles., Study Design: A prospective descriptive study., Setting: The Microbiology Department of the Korle Bu Teaching Hospital., Methods: Consecutive stool specimens from patients with diarrhoea submitted between February 2004 and June 2005 were cultured for Shigella and the isolates typed with commercial anti-sera. The susceptibilities of the isolates were also tested against eleven antimicrobial agents by the disc diffusion method. Minimum inhibitory concentrations (MIC) of isolates to ciprofloxacin were also determined by the E-test., Results: Five hundred and ninety four diarrhoea stool specimens yielded 24 Shigella isolates with the following serogroup distribution: S. flexneri 70.8%, S. dysenteriae 16.7%, S. sonnei 8.3% and S. boydii 4.2%. Approximately 96% of the isolates were multi-drug resistant but all twenty four were susceptible to nalidixic acid and the fluoroquinolones (ofloxacin and ciprofloxacin). The MICs of twenty one of the isolates to ciprofloxacin were

In the present work, we explore the possibility of introducing selectivity to existing chemotherapeutics via the design of non-pro-drug, bi-functional molecules comprising a microtubule-binding agent and a substrate for a disease-associated kinase. The design, synthesis, and in vitro biological evaluation of paclitaxel-thymidine and vinblastine-thymidine bi-functional conjugates are reported here. This work provides the first account of 'kinase-mediated trapping' of cancer therapeutics.

Background: T cells recognize a complex between a specific major histocompatibility complex (MHC) molecule and a particular pathogen-derived epitope. A given epitope will elicit a response only in individuals that express an MHC molecule capable of binding that particular epitope. MHC molecules are extremely polymorphic and over a thousand different human MHC (HLA) alleles are known. A disproportionate amount of MHC polymorphism occurs in positions constituting the peptide-binding region, and as a result, MHC molecules exhibit a widely varying binding specificity. In the design of peptide-based vaccines and diagnostics, the issue of population coverage in relation to MHC polymorphism is further complicated by the fact that different HLA types are expressed at dramatically different frequencies in different ethnicities. Thus, without careful consideration, a vaccine or diagnostic with ethnically biased population coverage could result., Results: To address this issue, an algorithm was developed to calculate, on the basis of HLA genotypic frequencies, the fraction of individuals expected to respond to a given epitope set, diagnostic or vaccine. The population coverage estimates are based on MHC binding and/or T cell restriction data, although the tool can be utilized in a more general fashion. The algorithm was implemented as a web-application available at http://epitope.liai.org:8080/tools/population., Conclusion: We have developed a web-based tool to predict population coverage of T-cell epitope-based diagnostics and vaccines based on MHC binding and/or T cell restriction data. Accordingly, epitope-based vaccines or diagnostics can be designed to maximize population coverage, while minimizing complexity (that is, the number of different epitopes included in the diagnostic or vaccine), and also minimizing the variability of coverage obtained or projected in different ethnic groups.

Background: ONT-093 is an orally bioavailable inhibitor of P-glycoprotein (P-gp). In pre-clinical studies, ONT-093 could inhibit P-gp and reverse multidrug resistance at nM concentrations with no effect on paclitaxel pharmacokinetics. Phase I trials of ONT-093 in normal human volunteers showed no dose-limiting toxicities at serum concentrations associated with biologic activity achieved with doses ranging from 300 to 500 mg., Methods: Phase I pharmacokinetic trial of ONT-093 in doses from 300 to 500 mg administered before and after intravenous paclitaxel doses of 150 to 175 mg/m(2) repeated every 21 days. All patients received paclitaxel alone on cycle 1., Results: 18 patients were enrolled onto 4 dose levels. Toxicity of the combination included neutropenia, arthralgia, myalgia, and peripheral neuropathy. Four of 6 patients receiving 500 mg doses of ONT-093 and paclitaxel at 175 mg/m(2) (dose level 4) had higher-grade neutropenia with cycle 2, with 1 patient experiencing febrile neutropenia. Plasma pharmacokinetic parameters of paclitaxel were unchanged between cycle 1 and 2 for dose levels 1 to 3, but at dose level 4, 45-65% increases in paclitaxel AUC were observed in 4 of the 6 patients. Mean C(max) of ONT-093 was 9 microM (range 5-15 microM) which were 3- to 5-fold higher than in single agent studies of ONT-093., Conclusions: Doses of ONT-093 achieving serum concentrations associated with biological activity were well tolerated in combination with standard doses of paclitaxel. Toxicities of the combination in this schedule were mainly attributable to paclitaxel and dose-limiting toxicity was limited to febrile neutropenia. There was an apparent pharmacokinetic interaction between paclitaxel and ONT-093, possibly related in part to the excipient, Cremophor, present in the paclitaxel formulation.

Objective: To determine the vaginal flora of first time urban Family Planning clients at Korle-Bu Teaching Hospital and to assess its implications for the contraceptive choices made., Design: A cross sectional study., Methods: A standardized questionnaire surveying the sociodemographic characteristics and the choice of Family Planning method was administered to 100 clients at the Korle Bu Teaching Hospital between March and September 2001. High vaginal and endocervical swabs were also taken during the inspection of the vagina and cervix using a sterile bivalve speculum. The specimens were transported in Amies transport medium to the Microbiology laboratory for processing., Results: The age range of the clients was 19-48 years with a modal age of 28 years. Ninety-six percent of them were married while 86% lived in urban slums. Sixty-three percent were sure of their last menstrual period. Potential pathogens were isolated from culture in 56% of the clients. Organisms causing bacterial vaginosis were the most prevalent in their genital tract. The intrauterine contraceptive device (IUCD) was the most common Family Planning method chosen, followed by the Norplant and the Depo-Provera injections. Potential pathogens were isolated from culture in 50% of those who chose the IUCD., Conclusion: Potential pathogens were isolated from culture in 56% of these first-time clients and organisms causing bacterial vaginosis were the most prevalent in the genital tract. It is suggested that Family Planning clients who screened positive for potential pathogens and opt for the IUCD should be considered for prophylactic antibiotics at insertion.

Problem Based Learning (PBL) has been adopted in educational programs in a variety of disciplines, including veterinary medicine. There is a voluminous literature on the subject, but it often remains unclear just what is being done in the name of PBL, and different accounts highlight different, often contradictory, positions on the key features of the approach. Similarly, despite the many claims made for the advantages of PBL, the evidentiary basis of such claims is often questionable. This article provides an introductory overview of what appear to be the key features of the approach and a brief summary of empirical evidence on its effectiveness.

Recognition by CD8(+) T lymphocytes (CTL) of epitopes that are derived from conserved gene products, such as Gag and Pol, is well documented and conceptually supports the development of epitope-based vaccines for use against diverse HIV-1 subtypes. However, many CTL epitopes from highly conserved regions within the HIV-1 genome are highly variable, when assessed by comparison of amino acid sequences. The TCR is somewhat promiscuous with respect to peptide binding, and, as such, CTL can often recognize related epitopes. In these studies, we evaluated CTL recognition of five sets of variant HIV-1 epitopes restricted to HLA-A*0201 and HLA-A*1101 using HLA transgenic mice. We found that numerous different amino acid substitutions can be introduced into epitopes without abrogating their recognition by CTL. Based on our findings, we constructed an algorithm to predict those CTL epitopes capable of inducing responses in the HLA transgenic mice to the greatest numbers of variant epitopes. Similarity of CTL specificity for variant epitopes was demonstrated for humans using PBMC from HIV-1-infected individuals and CTL lines produced in vitro using PBMC from HIV-1-uninfected donors. We believe the ability to predict CTL epitope immunogenicity and recognition patterns of variant epitopes can be useful for designing vaccines against multiple subtypes and circulating recombinant forms of HIV-1.

Experimental carbohydrate-conjugate vaccines composed of the 13 amino acid universal Pan HLA-DR Epitope (PADRE) and Streptococcus pneumoniae capsular polysaccharides from serotypes 14, 6B and 9V were produced. Simple carbodiimide-mediated condensation chemistry was used to conjugate the PADRE synthetic peptide to the three chemically different capsular polysaccharides in a 1:1 molar ratio. The immunogenicity of the PADRE peptide component of the conjugate vaccines was confirmed by the induction of PADRE-specific CD4+ helper T cell (HTL) responses following immunization of C57BL/6 mice. High titer antibody responses specific for polysaccharides of S. pneumoniae serotypes 14, 6B and 9V were induced using Complete Freund's Adjuvant (CFA) and alhydrogel Al(OH)3 formulations. The HTL, or carrier, effect of the PADRE synthetic peptide was only evident using the PADRE-polysaccharide conjugates; simple mixtures of the PADRE peptide and polysaccharides were essentially nonimmunogenic. The functional or potential protective value of the polysaccharide-specific antibodies was measured as a function of opsonophagocytic activity for the 6B serotype. High titers of opsonophagocytic activity were measured in sera from mice immunized with formulations containing both adjuvants. These data demonstrate that the PADRE synthetic peptide can induce the HTL responses needed to support the development of antibodies specific for bacterial carbohydrates used in conjugate vaccines.

Protein-tyrosine phosphatases (PTPs) are considered important therapeutic targets because of their pivotal role as regulators of signal transduction and thus their implication in several human diseases such as diabetes, cancer, and autoimmunity. In particular, PTP1B has been the focus of many academic and industrial laboratories because it was found to be an important negative regulator of insulin and leptin signaling, and hence a potential therapeutic target in diabetes and obesity. As a result, significant progress has been achieved in the design of highly selective and potent PTP1B inhibitors. In contrast, little attention has been given to other potential drug targets within the PTP family. Guided by x-ray crystallography, molecular modeling, and enzyme kinetic analyses with wild type and mutant PTPs, we describe the development of a general, low molecular weight, non-peptide, non-phosphorus PTP inhibitor into an inhibitor that displays more than 100-fold selectivity for PTPbeta over PTP1B. Of note, our structure-based design principles, which are based on extensive bioinformatics analyses of the PTP family, are general in nature. Therefore, we anticipate that this strategy, here applied to PTPbeta, in principle can be used in the design and development of selective inhibitors of many, if not most PTPs.

A highly constrained pseudo-tetrapeptide (OC252-324) further defines a new allosteric binding site located near the center of fructose-1,6-bisphosphatase. In a crystal structure, pairs of inhibitory molecules bind to opposite faces of the enzyme tetramer. Each ligand molecule is in contact with three of four subunits of the tetramer, hydrogen bonding with the side chain of Asp187 and the backbone carbonyl of residue 71, and electrostatically interacting with the backbone carbonyl of residue 51. The ligated complex adopts a quaternary structure between the canonical R- and T-states of fructose-1,6-bisphosphatase, and yet a dynamic loop essential for catalysis (residues 52-72) is in a conformation identical to that of the T-state enzyme. Inhibition by the pseudo-tetrapeptide is cooperative (Hill coefficient of 2), synergistic with both AMP and fructose 2,6-bisphosphate, noncompetitive with respect to Mg2+, and uncompetitive with respect to fructose 1,6-bisphosphate. The ligand dramatically lowers the concentration at which substrate inhibition dominates the kinetics of fructose-1,6-bisphosphatase. Elevated substrate concentrations employed in kinetic screens may have facilitated the discovery of this uncompetitive inhibitor. Moreover, the inhibitor could mimic an unknown natural effector of fructose-1,6-bisphosphatase, as it interacts strongly with a conserved residue of undetermined functional significance.

Epitope-based vaccines designed to induce CTL responses specific for HIV-1 are being developed as a means for addressing vaccine potency and viral heterogeneity. We identified a set of 21 HLA-A2, HLA-A3, and HLA-B7 restricted supertype epitopes from conserved regions of HIV-1 to develop such a vaccine. Based on peptide-binding studies and phenotypic frequencies of HLA-A2, HLA-A3, and HLA-B7 allelic variants, these epitopes are predicted to be immunogenic in greater than 85% of individuals. Immunological recognition of all but one of the vaccine candidate epitopes was demonstrated by IFN-gamma ELISPOT assays in PBMC from HIV-1-infected subjects. The HLA supertypes of the subjects was a very strong predictor of epitope-specific responses, but some subjects responded to epitopes outside of the predicted HLA type. A DNA plasmid vaccine, EP HIV-1090, was designed to express the 21 CTL epitopes as a single Ag and tested for immunogenicity using HLA transgenic mice. Immunization of HLA transgenic mice with this vaccine was sufficient to induce CTL responses to multiple HIV-1 epitopes, comparable in magnitude to those induced by immunization with peptides. The CTL induced by the vaccine recognized target cells pulsed with peptide or cells transfected with HIV-1 env or gag genes. There was no indication of immunodominance, as the vaccine induced CTL responses specific for multiple epitopes in individual mice. These data indicate that the EP HIV-1090 DNA vaccine may be suitable for inducing relevant HIV-1-specific CTL responses in humans.

Degradation of coral reef ecosystems began centuries ago, but there is no global summary of the magnitude of change. We compiled records, extending back thousands of years, of the status and trends of seven major guilds of carnivores, herbivores, and architectural species from 14 regions. Large animals declined before small animals and architectural species, and Atlantic reefs declined before reefs in the Red Sea and Australia, but the trajectories of decline were markedly similar worldwide. All reefs were substantially degraded long before outbreaks of coral disease and bleaching. Regardless of these new threats, reefs will not survive without immediate protection from human exploitation over large spatial scales.

The regulation of lipid metabolism and it's effect on glucose control and diabetes has received intense interest. Hormone-sensitive lipase (HSL) is a vital enzyme in lipid metabolism. A series of novel pyrrolopyrazinediones has been discovered that demonstrate submicromolar activity both in the enzyme assay and in a (14)C-emulsion assay employing cholesteryl oleate as a substrate as a secondary measure of HSL activity. These compounds represent novel inhibitors of the human HSL enzyme.

Background: A multicentre study was carried out in Ghana and southern India to determine the aetiology of suppurative keratitis in two regions located at similar tropical latitudes. Studies of fungal keratitis from the literature were reviewed., Methods: Patients presenting at rural and urban eye units with suspected microbial keratitis were recruited to the study. Corneal ulceration was defined as loss of corneal epithelium with clinical evidence of infection with or without hypopyon. Microscopy and culture were performed on all corneal specimens obtained., Results: 1090 patients were recruited with suspected microbial keratitis between June 1999 and May 2001. Overall the principal causative micro-organisms in both regions were filamentous fungi (42%): Fusarium species and Aspergillus species were the commonest fungal isolates. Pseudomonas species were most frequently isolated from cases of bacterial keratitis in Ghana but in India the commonest bacterial isolates were streptococci., Conclusion: Infections of the cornea due to filamentous fungi are a frequent cause of corneal damage in developing countries in the tropics and are difficult to treat. Microscopy is an essential tool in the diagnosis of these infections. A knowledge of the "local" aetiology within a region is of value in the management of suppurative keratitis in the event that microscopy cannot be performed.

Improvement in the care and treatment of neonates had contributed to their increased survival. Nosocomial infection remains an important problem in intensive care units. Hospital wards had been shown to act as reservoirs of pathogenic microorganisms associated with infection. To assess the prevalence of pathogenic organisms in the environment of the neonatal unit, 92 swabs were randomly collected from cots, incubators and various equipments in the unit and were cultured on Blood agar and MacConkey agar plates. Air contamination was detected by exposing the same types of agar plates for 3 hours in several areas of the unit. After 48 hours incubation, isolates were identified biochemically. There is marked congestion in the unit. Ninety one percent of swabs yielded growth, with coagulase negative Staphylococcus being the predominant organism (44%), followed by Bacillus species (20%), E. coli (12.5%), and Klebsiella (8.5%), Pseudomonas species (3.6%) and moulds (3.6%). Sedimentation plates had colony counts of from 10 - 100 per plate and the majority of the cultures were polymicrobial cultures. The presence of various Gram-negative bacili including known neonatal pathogens (like E. Coli and Pseudomonas) especially on ward equipment and congestion in the ward has the potential to cause nosocomial infection.

Reversible phosphorylation and dephosphorylation of key proteins on tyrosine residues are important parts of intracellular signaling triggered by hormones and other agents. Recent knock-out studies in mice have identified PTP1B as a potential target for the treatment of diabetes and obesity. As a consequence, a number of academic and industrial groups are aggressively pursuing the development of selective PTP1B inhibitors. In addition, other protein-tyrosine phosphatases (PTPs) appear to be critically involved in major diseases such as cancer and autoimmunity. Given the diversity of PTPs and their potential as drug targets in different diseases, we have taken a broad approach to develop active site-directed selective inhibitors of specific members of this family of enzymes. Using a high throughput screening, we have previously identified 2-(oxalylamino)benzoic acid 3a as a relatively weak but classical competitive inhibitor of several PTPs.(4) On the basis of our early studies, indicating that 3a might be used as a starting point for the synthesis of selective PTP inhibitors, we now present our efforts in expansion of this concept and provide here a number of new chemical scaffolds for the development of inhibitors of different members of the PTP family. Although the core structure of these inhibitors is charged, good oral bioavailability has been observed in rat for some compounds. Furthermore, we have observed enhancement of 2-deoxy-glucose accumulation in C2C12 cells with prodrug analogues.

Cellular immune responses mediated by CD8+ cytotoxic T-lymphocytes (CTL) and CD4+ helper T-lymphocytes (HTL) are needed to effectively control and clear many viral pathogens, including HIV-1. Thus, vaccines for HIV-1 capable of inducing CTL and HTL responses are now the focus of multiple academic and industry-based research and development programs. The use of defined, minimal CTL and HTL epitopes in vaccines has several potential advantages. Firstly, it is possible to use epitopes that are conserved thus targeting the majority of viral variants within a given clade or across clades. Secondly, epitopes from multiple viral structural or accessory gene products can be included in vaccines, which supports the induction cellular immune responses with significant breadth. Finally, dominance relationships between epitopes can be altered to increase immune recognition of subdominant epitopes. HTL and CTL epitopes from HIV-1 have recently been identified and characterized in numbers that are large enough to support their use in experimental vaccines. Initial studies with prototype DNA vaccines encoding epitopes indicate the need to include intracellular targeting sequences, to direct the encoded gene products to different cellular compartments, and amino acid spacer sequences between epitopes to optimize the processing, and subsequent presentation, of individual epitopes. Vaccines composed of CTL or HTL epitopes are now being developed for clinical testing.

The idea of using polymeric nanoparticles as drug carriers is receiving an increasing amount of attention both in academia and industry, Nanoparticles have a number of potential applications in protein, drug and vaccine delivery, as well as gene therapy applications. In this article, we focus on this unique drug delivery technology as a method to control the release rate of substances, not only for protein delivery but also for delivering an experimental vaccine immunogen. Nanoparticles were assembled on the basis of ionic interaction between water-soluble polymers so that the resulting particles were stable in physiologic media. Among the typical polymers used to assemble nanoparticles, different polysaccharides, natural amines, and poly-amines were investigated. The entrapped substances tested included a protein and antigens. Polydextran aldehyde was incorporated into the particle core, to enable physiologic cross-linking as a method to control permeability. This resulted in long-term retention of substances that would otherwise rapidly leak out of the nanoparticles. Results of cross-linking experiments clearly demonstrated that the release rate could be substantially reduced, depending on the degree of cross-linking. For vaccine antigen delivery tests, we measured an antibody production after subcutaneous and oral administration. The data indicated that only the cross-linked antigen was immunogenic when the oral route of administration was used. The data presented in this article address primarily the utility of nanoparticulates for oral delivery of vaccine antigen., (Copyright 2002 Wiley Periodicals, Inc.)

DNA-based vaccines were employed to assess protective immunity against herpes simplex virus in experimental infections of hairless (strain SKH1) and BALB/c mice. Mice were vaccinated with plasmids containing the herpes simplex virus type-1 (HSV-1) glycoprotein B (gB) or D (gD) genes under the human cytomegalovirus immediate-early promoter control. Vaccines were injected intramuscularly (i.m.) or intraperitoneally (i.p.) as purified DNA alone or as formulations supplemented with different non-ionic block copolymers. Antibody responses were assessed by immunofluorescence and radio-immunoprecipitation assays. Mice inoculated with either gB or gD plasmid, alone or with non-ionic block copolymers CRL 1029 and CRL 1190, produced high levels of antibodies specific for gB or gD. Three weeks after the last vaccination, mice were challenged with a clinical HSV-1 isolate (ABGK-1) by inoculation of a shaved and subsequently scarified area between the third and fourth lumbar vertebrae. Mice immunised with either gD or gB plasmid alone or mixed with copolymers were protected against lethal HSV-1 challenge when immunisation was performed via the i.m. route. Immunisations given via the i.p. route induced humoral responses in some mice and protected the animals against lethal HSV-1 challenge only when the formulations contained copolymers. The BALB/c mouse model was shown to be as good a model as the hairless mouse model.

Cellular immune responses, specifically those mediated by CD8+ cytotoxic T-lymphocytes and CD4+ helper T-lymphocytes, are needed to control HIV-1 in vivo and to mediate viral clearance. Vaccines capable of inducing cellular immune responses are, therefore, widely considered as necessary for controlling the spread of HIV-1. Numerous different vaccine delivery formats designed to induce cellular immune responses have been evaluated in animal models and clinical trials. These include adjuvant-supplemented peptide- or protein-based formulations, DNA vaccines and viral vectors. While none of the current generation vaccine delivery technologies have proved potent enough for stand-alone use against HIV-1-infection, mixed delivery vaccine formats, vaccination schemes known as heterologous prime-boost, have proved to be significantly potent methods for inducing cellular immune responses. Different forms of heterologous prime-boost vaccines are currently being tested in numerous clinical trials and hold significant promise.

Knowledge of antibiotic resistance in bacteria strains colonizing healthy people is important for several reasons, one of which is that; these organisms form one of the largest reservoirs of resistant genes. Frequency of resistance to eleven different antimicrobial agents was examined in faecal flora of adults with no history of recent antimicrobial treatment. Using the disc diffusion sensitivity test, 106 strains of Escherichia coli were examined, 68% of these were resistant to tetracycline, and 57% were resistant to ampicillin and cotrimoxazole respectively. There was no resistance to cefuroxime but resistance to ceftazidime was 13%. Fifty six out of the eighty eight (64%) isolates, which showed any resistance, were resistant to three or more antimicrobials. The most common resistant pattern was to three drugs tetracycline, ampicillin and cotrimoxazole. Six strains were susceptible to all antibiotics. One strain of Escherichia coli was resistant to eight antimicrobials. Thirty per cent of the Escherichia coli were resistant to gentamicin. This study reveals a high prevalence of resistant bacteria in faecal flora of healthy adults.

Inhibitors of P gLycoprotein (Pgp) may be useful for the enhancement of blood-brain barrier penetration of anti-epileptic drugs (AEDs). Due to polypharmacy and the need for chronic treatment, Pgp inhibitors used in epilepsy should be highly specific and non-toxic. In particular, it may be essential to use compounds that produce minimal inhibition of enzymes involved in metabolism of AEDs and other drugs used by epilepsy patients. OC144-093 is a novel substituted diarylimidazole generated using the OntoBLOCK system, a solid-phase combinatorial chemistry technology, in combination with high-throughput cell-based screening. The compound is an extremely potent inhibitor of Pgp-mediated multidrug resistance (MDR) in cancer with an average FC50 of 32 nM, but does not inhibit multidrug resistance-associated protein (MRP1). OC144-093 is the least non-specifically toxic Pgp inhibitor described to date, with an average cytostatic IC50 of >60 microM in 15 cell types. It is not metabolized by cytochrome P450s CYP3A4, 2C8 or 2C9 enzymes involved in AED metabolism. OC144-093 does not produce a pharmacokinetic (PK) interaction with paclitaxel and has exhibited an excellent PK and safety profile in phase I clinical trials. Our results suggest that OC144-093 may represent an ideal candidate for use in enhancement of AED blood-brain barrier penetration.

Utilizing structure-based design, we have previously demonstrated that it is possible to obtain selective inhibitors of protein-tyrosine phosphatase 1B (PTP1B). A basic nitrogen was introduced into a general PTP inhibitor to form a salt bridge to Asp48 in PTP1B and simultaneously cause repulsion in PTPs containing an asparagine in the equivalent position [Iversen, L. F., et al. (2000) J. Biol. Chem. 275, 10300-10307]. Further, we have recently demonstrated that Gly259 in PTP1B forms the bottom of a gateway that allows easy access to the active site for a broad range of substrates, while bulky residues in the same position in other PTPs cause steric hindrance and reduced substrate recognition capacity [Peters, G. H., et al. (2000) J. Biol. Chem. 275, 18201-18209]. The current study was undertaken to investigate the feasibility of structure-based design, utilizing these differences in accessibility to the active site among various PTPs. We show that a general, low-molecular weight PTP inhibitor can be developed into a highly selective inhibitor for PTP1B and TC-PTP by introducing a substituent, which is designed to address the region around residues 258 and 259. Detailed enzyme kinetic analysis with a set of wild-type and mutant PTPs, X-ray protein crystallography, and molecular modeling studies confirmed that selectivity for PTP1B and TC-PTP was achieved due to steric hindrance imposed by bulky position 259 residues in other PTPs.

The A*0201, A *0202, A*0203, A*0206, and A*6802 binding capacity of single amino acid substitution analogs of known A2-supertype binding peptides and of large nonredundant peptide libraries was measured. The results were utilized to rigorously define the peptide binding specificities of these A2-supertype molecules. Although each molecule was noted to have unique preferences, large overlaps in specificity were found. The presence of L, I, V, M, A, T, and Q residues in position 2, and L, I, V, M, A, and T residues at the C-terminus of peptide ligands were tolerated by all molecules. Likewise, whereas examination of secondary influences on peptide binding revealed allele specific preferences, shared features could also be identified. These shared features were utilized to define an A2-supermotif and were noted to correlate with crossreactivity. Over 70% of the peptides that bound A *0201 with high affinity were found to bind at least two other A2-supertype molecules. Because the A2-supertype molecules studied herein cover the variants most common in different major ethnicities, these findings have important implications for epitope-based approaches to vaccination, immunotherapy, and the monitoring of immune responses.

Single amino acid substitution analogs of the known Mamu A*01 binding peptide gag 181-190 and libraries of naturally occurring sequences of viral or bacterial origin were used to rigorously define the peptide binding motif associated with Mamu A*01 molecules. The presence of S or T in position 2, P in position 3, and hydrophobic or aromatic residues at the C terminus is associated with optimal binding capacity. At each of these positions, additional residues are also tolerated but associated with significant decreases in binding capacity. The presence of at least two preferred and one tolerated residues at the three anchor positions is necessary for good Mamu A*01 binding; optimal ligand size is 8-9 residues. This detailed motif has been used to map potential epitopes from SIVmac239 regulatory proteins and to engineer peptides with increased binding capacity. A total of 13 wild type and 17 analog candidate epitopes were identified. Furthermore, our analysis reveals a significantly lower than expected frequency of epitopes in early regulatory proteins, suggesting a possible evolutionary- and/or immunoselection directed against variants of viral products that contain CTL epitopes.

To distinguish sinusitis from uncomplicated "colds," we examined lactoferrin and eosinophilic cationic protein (ECP) in nasal secretions. Lactoferrin titers were >/=1:400 in 4% of persons with uncomplicated colds and controls but in 79% of persons with sinusitis or purulent sputa. ECP levels were >200 ng/ml in 61% of persons with colds and >3,000 ng/ml in 62% of persons with sinusitis. Nasal lactoferrin helps distinguish sinusitis from colds.

OC144-093 is a novel substituted diarylimidazole (Mr 495) generated using the OntoBLOCK system, a solid-phase combinatorial chemistry technology, in combination with high-throughput cell-based screening. OC144-093 reversed multidrug resistance (MDR) to doxorubicin, paclitaxel, and vinblastine in human lymphoma, breast, ovarian, uterine, and colorectal carcinoma cell lines expressing P-glycoprotein (P-gp) with an average EC50 of 0.032 microM. Inhibition of MDR by OC144-093 was reversible, but the effect persisted for at least 12 h after removal of compound from the culture medium. OC144-093 had no effect on the response to cytotoxic agents by cells in vitro lacking P-gp expression or expressing a multidrug resistance-associated protein (MRP-1). OC144-093 was not cytotoxic by itself against 15 normal, nontransformed, or tumor cell lines, regardless of P-gp status, with an average cytostatic IC50 of >60 microM. OC144-093 blocked the binding of [3H]azidopine to P-gp and inhibited P-gp ATPase activity. The compound was >50% p.o. bioavailable in rodents and dogs and did not alter the plasma pharmacokinetics of i.v.-administered paclitaxel. OC144-093 increased the life span of doxorubicin-treated mice engrafted with MDR P388 leukemia cells by >100% and significantly enhanced the in vivo antitumor activity of paclitaxel in MDR human breast and colon carcinoma xenograft models, without a significant increase in doxorubicin or paclitaxel toxicity. The results demonstrate that OC144-093 is an orally active, potent, and nontoxic inhibitor of P-gp-mediated multidrug resistance that exhibits all of the desired properties for treatment of P-gp-mediated MDR, as well as for prevention of MDR prior to selection and/or induction of refractory disease.

The use of adjuvants is one approach to improve influenza vaccine immunogenicity and efficacy, particularly in aged populations. The response of BALB/c mice to subcutaneously administered formalin-inactivated whole influenza virus vaccine in the presence or absence of a nonionic block copolymer adjuvant CRL1005 was evaluated. In young adult naïve mice, the copolymer adjuvant significantly enhanced virus-specific IgG and hemagglutination-inhibition (HI) antibody responses and augmented the production of IL-2 following vaccination. Influenza vaccine formulated with 2.5 mg CRL1005 significantly enhanced the protective efficacy of the inactivated vaccine in the upper and lower respiratory tract. In mice previously infected with influenza virus or naïve aged mice, inactivated vaccine administered with the copolymer adjuvant substantially enhanced the serum HI antibody response to inactivated influenza vaccine and significantly reduced lung virus titers following subsequent challenge with live virus compared with mice administered vaccine alone. These results suggest that the copolymer adjuvant warrants further investigation as a potential adjuvant for use in human vaccination against influenza.

Anoikis is a form of programmed cell death induced in normal epithelial cells by detachment from the extracellular matrix [1] [2] [3]. In epithelial cells of the intestine and other organs, activated rasinduces resistance to anoikis [3] [4], but the actual molecular effectors directly involved in the apoptotic machinery that execute or block anoikis have not yet been identified. Bak, a pro-apoptotic member of the Bcl-2 family, is downregulated in a high proportion of colorectal tumours [5]. In addition, Bak is an important regulator of apoptosis in normal intestinal epithelial cells [6] [7]. Here, we show that activated rasinduces the downregulation of Bak in rat and human intestinal epithelial cells. This ras-induced downregulation of Bak expression could be suppressed by an inhibitor of phosphatidylinositol (PI) 3-kinase, an enzyme already implicated in ras-induced resistance to anoikis [8]. Ectopic expression of Bak in ras-transformed rat intestinal epithelial IEC-18 cells inhibited ras-induced resistance to anoikis and significantly reduced their tumorigenicity. We conclude, therefore, that the ability of rasto downregulate Bak, and the consequent resistance to anoikis, are essential components of the transforming capacity of this oncogene in intestinal epithelial cells.

Nonionic block copolymers are surfactants synthesized using propylene oxide and ethylene oxide, and they can be designed so that individual copolymers have unique vaccine adjuvant properties. We have designed and produced nonionic block copolymers based on high molecular weight (MW), 9-15 kDA, cores of poly(oxypropylene) (POP) coupled with smaller poly(oxyethylene) (POE) end blocks. Copolymers synthesized with less than 10% (w/w) POE will spontaneously assemble into 300 nm-3 microm micelles or microparticles in aqueous solutions at physiological pH, and when formulated with protein, complex microparticles consisting of both the protein and copolymers are formed. The adjuvant activity of nonionic block copolymers is influenced by both size and POE content; maximal activity is associated with low POE content, 5-10%, and a molecular size of 11-12 kDa. The type of immune response produced is also influenced by the POE content. Copolymers with 10% POE significantly augmented Type 2 helper T-lymphocyte responses whereas copolymers with lower POE contents augmented both Type 1 and Type 2 helper T-lymphocyte responses. This property allows for vaccines to be "customized" by using adjuvant-active nonionic block copolymers that will augment the most appropriate types of immune responses.

Nonionic block copolymers are surfactants synthesized using propylene oxide (PO) and ethylene oxide (EO) which are organized as 'blocks' of polyoxyethylene (POE) and polyoxypropylene (POP). These copolymers can be designed and synthesized using variable amounts of the PO and EO and with differential arrangement of the POP and POE blocks so that individual products have unique physicochemical properties. The copolymers that have been most thoroughly evaluated in vaccine research are linear with the polymer blocks organized as POE-POP-POE. Low molecular weight (MW) copolymers, 3-6 kDa, of this type have been used in oil-based emulsion formulations, whereas high-MW copolymers, >9 kDa, can be used in aqueous formulations. The adjuvant activity of nonionic block copolymers is influenced greatly by the size of the POP core block. As the size of this block is increased so is the adjuvant activity of the copolymer; peak activity is achieved using copolymers with POP cores that are 12-15 kDa. However, adjuvant activity is also affected by the amount of POE with low concentrations, 5-10%, being optimal. The type of immune responses produced is also influenced by the POE content. Copolymers with 10% POE preferentially augment Type 2 helper T-lymphocyte responses which support antibody responses, including mucosal antibody responses. Copolymers with <10% POE augment both Type 1 and Type 2 helper T-lymphocyte responses, which support a broader range of antibody responses and cellular immune responses. This property may allow for vaccines to be 'customized' by using adjuvant-active nonionic block copolymers that will augment the most appropriate types of immune responses.

The regulation of epithelial cell cycle progression by extracellular matrix proteins was investigated in mink lung epithelial cells (Mv1Lu cells) and primary human keratinocytes. Exogenous type V collagen was able to mimic all of the inhibitory effects of type 1 transforming growth factor beta (TGF-beta1). No significant inhibitory effect was observed with collagen types I, III, and IV; laminin; or fibronectin. The type V collagen used was not contaminated with TGF-betas. TGF-beta1 increased the rate of type V collagen protein secretion in Mv1Lu cells, which occurred coincident with DNA synthesis inhibition. Both TGF-beta1 and type V collagen inhibited retinoblastoma protein phosphorylation and the expression of cyclin-dependent kinase (cdk) 4 and cdk2, but not p27Kip expression. Mv1Lu cells constitutively expressing the SV40 T antigen or cdk4 were resistant to the inhibitory effects of both TGF-beta1 and type V collagen. Our results demonstrate that type V collagen is a novel and specific epithelial cell cycle inhibitor and suggest that it may act as an autocrine mediator of the inhibitory effects of TGF-beta1.

The current influenza virus vaccines induce systemic humoral immunity and short lived cellular immunity in young adults. Unfortunately these vaccines are only 50% efficacious in the elderly (> 65 years) and high risk groups of the very young. The use of a vaccine adjuvant to correct this deficit would therefore be very beneficial to these population groups. We have developed high molecular weight synthetic non-ionic block copolymers with adjuvant activity. These copolymers are compatible with, and active in, aqueous, physiological formulations in which they spontaneously assemble into 500-3000 nm particles. By varying both the molecular weight and the proportions of hydrophilic and hydrophobic components of the molecule, we have designed the optimal copolymer adjuvant for use with influenza hemagglutinin. This copolymer, termed CRL-1005, was investigated for its ability to augment the immune response of mice to the commercially-available human influenza vaccine, Fluogen. Co-formulation of CRL-1005 with the vaccine resulted in markedly increased antibody titres measured by both ELISA and the functional haemagglutination inhibition assay, indicating that critical immunogen epitopes were not destroyed. A single dose of copolymer and vaccine produced both long term rising antibody titres (six months) and primed for a potent secondary response. This high molecular weight copolymer is non-toxic and should therefore be well suited for widespread use.

Nonionic block copolymers synthesized from ethylene oxide and propylene oxide were developed specifically for use as surfactants. Because the sizes and relative positions of the hydrophobic polyoxypropylene (POP) and hydrophilic polyoxyethylene (POE) blocks can be altered during synthesis, copolymers with significantly different surfactant characteristics can be produced. Copolymers of this type are currently used as excipients in a wide variety of pharmaceutical products where they act as emulsifying, wetting, thickening, stabilizing, and dispersing agents. Copolymers with unique physicochemical properties have recently been developed through the use of new manufacturing and purification techniques, and these copolymers are being used as drug-active and drug-delivery components. In this review, we summarize the current status of these new copolymers in terms of research and product development. This includes the use of new, high molecular weight copolymers as vaccine adjuvants and as vaccine-delivery vehicles. The use of purified, pharmaceutical-grade copolymers as anti-infectives and as antibiotic-delivery systems for the treatment of established bacterial and viral infections is also reviewed. These novel uses for copolymers are significantly different from the excipient uses common to this type of product and demonstrate the widespread utility of synthetic surfactant polymers.

Treatment of human autoimmune diseases may be enhanced by using adjuvants that can selectively induce immunoregulatory responses. Two versions of a novel nonionic block copolymer adjuvant suitable for human use, Optivax Oil Formulation (OF) and Optivax Aqueous Formulation (AF), were evaluated for induction of immunity to encephalitogenic and regulatory T-cell receptor (TCR) V-gene determinants. In Lewis rats immunized with myelin basic protein (BP), Optivax OF was more efficient than Optivax AF for inducing delayed type hypersensitivity (DTH), T-cell proliferation, antibodies, and even mild clinical signs of experimental autoimmune encephalomyelitis (EAE). Similarly, Optivax OF was more efficient for inducing inflammatory T-cell and antibody responses to immunoregulatory V beta 8.2 proteins and peptides than Optivax AF, which induced a noninflammatory Th2 response. In general, DTH response to the various immunogens was reflected by increased cellularity and mRNA levels for IFN-gamma in draining lymph nodes, whereas LN cell proliferation without DTH was characterized by increased IL-2 mRNA levels but low or absent IFN-gamma message. These data suggest important differential adjuvant effects of Optivax OF versus Optivax AF on the respective induction of Th1 versus Th2 responses that may be useful in the selective treatment of human immune disorders.

The clinical and immunological effects of a vaccine consisting of CTP37, a synthetic peptide corresponding to the COOH-terminal peptide (CTP) of beta-human chorionic gonadotropin (beta-hCG) conjugated to diphtheria toxoid, combined with CRL 1005, a novel synthetic nonionic block copolymer adjuvant, were examined. Twenty-one patients with metastatic, nontrophoblastic cancers received up to four immunizations by i.m. injection of a fixed dose of CTP37 and escalating doses of CRL 1005. Doses of CRL 1005 adjuvant as high as 75 mg were administered with 1 mg of CTP37 without evidence of significant local or systemic toxicity. Immunizations resulted in the production of IgG antibody to beta-hCG. CRL 1005 doses of 3-25 mg appeared to be optimal for antibody induction. Immunizations also resulted in increases in the cellular response of peripheral blood mononuclear cells (PBMCs) to the unconjugated CTP, hCG, and diphtheria toxoid. Responding PBMCs specifically secreted the TH1-associated cytokines IFN-gamma and interleukin (IL)-2 as well as the TH2-associated IL-5 and IL-10. Increased expression of IFN gamma and IL-5 mRNAs by PBMCs 4 h after immunization was also observed. CRL 1005 administered with CTP37 in aqueous solution is well tolerated. The CTP37-CRL 1005 subunit vaccine has the capacity to stimulate potentially beneficial humoral and cellular immune responses in patients with advanced cancer.

High performance liquid chromatography has been coupled simultaneously to high field NMR and MS detectors, giving UV, NMR and mass spectra for each component in a mixture, after on-line separation. This powerful new tool for the structure elucidation of components in mixtures without isolation has been successfully applied to the analysis of the metabolites of paracetamol in human urine.

The antigenic variation associated with Human Immunodeficiency Virus type-1 (HIV-1) envelope proteins could limit their utility in vaccines if the immune responses induced are specific for immunodominant variable epitopes. We evaluated the ability of experimental subunit vaccines, containing recombinant forms of the envelope glycoprotein (rgp120) from two HIV-1 variants, to induce immune responses capable of recognizing unrelated HIV-1 variants. A vaccine formulation based on HIV-1IIIB/LAI rgp120 and supplemented with saponin adjuvant (QS-21) induced neutralizing antibodies specific for the HIV-1IIIB/LAI variant. This antibody response was presumably specific for the variable principle neutralizing determinant (PND) of the third variable region of gp120, the V-3 region. This formulation induced cytotoxic T-lymphocytes (CTL) specific for the dominant V-3 epitope but also to an additional unidentified epitope outside of this region. The CTL specific for this second epitope also recognized gp120 from the HIV-1MN and HIV-1RF variants in a "cross-reactive" manner. A second vaccine formulation based on HIV-1MN rgp120 and QS-21 adjuvant induced neutralizing antibodies that were again variant-specific but also CTL that recognized all three HIV-1 variants in a cross-reactive manner. These data demonstrate that CTL capable of recognizing different HIV-1 variants, which are presumed to be specific for a conserved HIV-1 gp120 epitope, can be induced using subunit vaccines with the appropriate adjuvant while variant-specific antibody responses are produced. These findings support further evaluation of this vaccine format.

Nonionic block copolymers, synthesized from repeating units of oxypropylene and oxyethylene, can be designed so that individual copolymers have unique physical properties with differential levels of adjuvant activity. We have designed high molecular weight block copolymers that spontaneously assemble into 500 nm-3 mum particles when formulated with protein antigens in aqueous solutions at physiological pH. The adjuvant activity of one of these copolymers, termed CRL1005, was compared to selected research adjuvants using ovalbumin (OVA) as the prototype vaccine antigen. Suboptimal doses of OVA were formulated with complete and incomplete. Freund's adjuvant (CFA/IFA), alum Quil-A saponins Ribi Adjuvant System (RAS) or the CRL1005 copolymer and these formulations were used to immunize C57BL/6 mice. The CRL1005 copolymer appeared to be more potent than either Quil-A or alum and comparable to the RAS formulation, based on the numbers of responding mice and the OVA-specific antibody titers. Alum. RAS and Quil-A all augmented the production of IgG1 and IgG2l, similarly whereas only the CFA/IFA boosted IgG2a levels significantly. The effect of adjuvants on relative antibody affinity was more variable with the CRL1005 and CFA/IFA inducing antibodies with the highest affinity scores. This high molecular weight nonionic copolymer is nontoxic in aqueous formulations and should therefore be compatible with a wide variety of protein or polysaccharide vaccine antigens.

High molecular weight nonionic block copolymers consisting of a large hydrophobic core made from repeat oxypropylene units and smaller hydrophilic blocks of oxyethylene repeat units were evaluated as adjuvants in experimental influenza virus vaccine formulations. The goal was to identify a copolymer that would increase the immunogenicity of the commercial Fluogen trivalent influenza virus vaccine. Vaccine experiments done using BALB/c mice provided data that allowed us to identify a copolymer that increased both antibody titers specific for total virus proteins as well as antibodies with hemagglutination inhibition (HAI) activity. This copolymer, termed CRL1005, increased the production of IgG1, IgG2a and IgG2b which suggested it increased the activity of both Type-1 and Type-2 T-helper lymphocytes. The CRL1005 copolymer was tested further in rhesus monkeys with similar results. Levels of antibodies specific for total virus protein preparations were increased as were HAI antibody titers following vaccination with the copolymer-supplemented Fluogen vaccine. Thus, the CRL1005 copolymer adjuvant appears to be compatible for use with the current generation of inactivated viron-based influenza vaccines and useful for increasing the immunogenicity. A more potent influenza virus vaccine could well be more efficacious in the aged segment of our population than current vaccines.

OBJECTIVE: To examine the frequency of community-acquired salmonella bloodstream infection in Ghanaian children and the occurrence of antibiotic resistance in salmonellae. METHODS: The study comprised 472 patients with a blood culture obtained within 48 h of admission to the pediatric department of Korle Bu Teaching Hospital in Accra, Ghana, over a 3-month period. All Salmonella isolates from blood cultures were speciated and antibiotic susceptibility tests were performed. Clinical data of children with salmonella bloodstream infection were compared to those of controls. Two control groups were identified: all children enrolled in the study without salmonella bloodstream infection (group 1), and those with bloodstream infection due to other organisms (group 2). RESULTS: A pathogen was isolated from 111 children (23.5%), and salmonellae were among the most common isolates (n=24; 21.6%). Among Salmonella strains, S. enteritidis (n=14; 59%) predominated over S. typhi (n=6; 25%). Resistance to several antibiotics was only found in S. enteritidis isolates (n=8; 57%). Children with salmonella bloodstream infection presented more often than controls with severe anemia, jaundice, abdominal pain and distension as well as hepatomegaly and splenomegaly. They were also hospitalized for a significantly longer period, but the case-fatality rate was similar. CONCLUSIONS: Salmonella bloodstream infection, especially due to non-typhoidal strains, is a potential health problem for Ghanaian children and may be complicated by resistance to the commonly available antibiotics.

Multidrug resistance (MDR) is a major impediment to the effective treatment of cancer. We have used multicellular tumor spheroids (MTS) as a model to investigate whether MDR can be reversed in a three dimensional structure. MTS are tightly associated aggregates of tumor cells that exhibit many of the properties of solid tumors. A human MDR breast carcinoma cell line was selected by exposure to taxol under monolayer conditions. The sensitive (parental) and drug-resistant phenotypes were retained when the cells were grown as MTS. Thus, the three dimensional conditions in this novel model system did not affect the MDR phenotype. SDZ PSC 833 is an efficient MDR reversing agent as determined under monolayer conditions and is currently being evaluated in clinical trials. Resistance to taxol and doxorubicin of the MDR cells grown as MTS was almost completely reversed by SDZ PSC 833. Our results suggest that SDZ PSC 833 has the potential to reverse the MDR phenotype in solid tumors.

The earlier use of combined liquid chromatography/NMR spectrometry/mass spectrometry (LC/NMR/MS) involved the use of a particle beam interface. This paper describes further developments of this hyphenated technology, in particular the incorporation of an electrospray interface into the LC/NMR/MS system. This improved LC/NMR/MS system was designed for the support of a combinatorial library program. The power of this technique is demonstrated in the successful structural elucidation of each compound in a mixture of commercially available peptides.

An outbreak of nosocomial infection due to multiple-resistant Salmonella Group "G' infection in a neonatal intensive care unit in a temporary ward is reported. It started with five cases of Septicaemia and one case of meningitis over a period of about six weeks. Investigation of the outbreak resulted in isolation of a multiple-resistant Salmonella Group G from the rectal swab of 21 out of 72 babies (29%). Surveillance culture from staff yielded two fully-sensitive salmonella species. Stool culture from mother of colonised babies were all negative. Environmental cultures from the nursery grew multiple-resistant Salmonella Group G from three of four incubator mattresses and also from the radiant warmer. Institution of strict aseptic measures, followed by closure of the ward was able to stop the epidemic.