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Biomedical subjects

D L Peterson

Publications and source records attributed to D L Peterson.

At least 19 recordsLinked to original sources
PubMed2004

Evaluation of initial base deficit as a prognosticator of outcome in the pediatric trauma population.

Although the utility of the base deficit as an indicator of hypoperfusion and physiologic derangement in adults is well established, its value in the assessment of children is not as clear. The purpose of this study was to evaluate this tool with regard to injury severity, infectious morbidity, and outcome in a pediatric trauma population. A retrospective review of a 6-year period of the database of our level 1 pediatric trauma center was performed. One hundred seventeen severely injured children requiring mechanical ventilation were identified. Initial base deficit, Injury Severity Score, time to correction of this abnormality, ventilator days, infectious morbidity, and mortality were obtained and compared. Of the 117 patients included in this study, 30 patients were identified with an initial BD of less than or equal to -8 mEq/L and were placed into group 1. Group 2 consisted of the remaining 87 patients who presented with a base deficit (BD) of greater than -8 mEq/L. An admission base deficit of -8 mEq/L or less corresponded to a probability of mortality of 23 per cent as opposed to only 6 per cent with a BD greater than -8. Patients in group 1 remained on mechanical ventilation 9.4 +/- 8.1 days, whereas patients in group 2 remained ventilated 6.5 +/- 6.4 days; an increase of nearly 145 per cent. Likewise, the number of infectious complications rose 26 per cent with a worsening initial base deficit from 17 per cent of group 2 patients to 43 per cent of group 1 patients. We conclude that a high initial base deficit in injured children predicts a higher incidence of infectious complications and a less favorable outcome. This readily available laboratory study can identify those children most at risk of potentially preventable complications.

Adolescent↗
PubMed2001

Preclinical evaluation of group B streptococcal polysaccharide conjugate vaccines prepared with a modified diphtheria toxin and a recombinant duck hepatitis B core antigen.

An effective vaccine against group B streptococcal (GBS) disease will undoubtedly include capsular polysaccharides (CPSs) from each of the five serotypes prevalent in the United States individually coupled to immunogenic proteins. This formulation may require the use of two or more different protein carriers. We preclinically examined the potential of two proteins to serve as effective carriers for GBS type III CPS. Recombinant duck hepatitis B core antigen (rdHBcAg), a particulate protein of viral origin, and a newly mutated form of diphtheria toxin (DTm) were covalently and directly coupled to purified type III CPS by reductive amination. Seventy-seven of 79 (97%) newborn pups born to mouse dams actively vaccinated with type III CPS-rdHBcAg conjugate survived GBS type III challenge, whereas none of the pups born to dams that received an uncoupled mixture of type III CPS and rdHBcAg or saline survived. Likewise, 64 (98%) of 65 pups born to dams vaccinated with type III CPS-DTm conjugate survived challenge, in sharp contrast to no survivors among the pups born to dams vaccinated with an uncoupled mixture of type III CPS and DTm. The presence of type III CPS-specific IgG in serum from dams correlated with pup survival in groups that received a conjugate vaccine, and this serum was opsonically active in vitro against GBS type III. In addition, carrier-specific IgG was also measured in serum from vaccinated mice. These data suggest that the rdHBcAg and DTm may be effective carriers for GBS CPSs.

Animals↗
PubMed2001

In vitro assembly of feline immunodeficiency virus capsid protein: biological role of conserved cysteines.

Core assembly, a key step in the retroviral life cycle, is poorly understood. Previous studies have shown that the entire gag region is needed to form the assembled particles. In this report, we have shown that the assembly process is driven by recombinant capsid protein (p26) of feline immunodeficiency virus itself. Proteins are expressed in a bacterial system and soluble forms of wild-type and modified proteins are purified from bacterial extracts and are examined on gel-filtration chromatography fitted to an HPLC system. It has also been shown that changing residue Cys190 (one of the two conserved cysteines of feline immunodeficiency virus which are also conserved for all the immunodeficiency viruses including HIV) to serine by site-directed mutagenesis disrupts the assembly process. In addition, this modification causes considerable thermal instability of the protein while substitutions at nonconserved cysteines do not significantly affect the thermal stability and assembly of the protein. These findings indicate that conserved cysteine residues play a vital role in the capsid protein assembly and, therefore, are critical for virus infectivity.

Amino Acid Sequence↗
PubMed2001

Urea equilibrium unfolding of the major core protein of the retrovirus feline immunodeficiency virus and its tryptophan mutants.

Circular dichroism and fluorescence spectroscopy have been employed to study the urea unfolding mechanism of a recombinant form of the major core protein of feline immunodeficiency virus (FIV-rp24) and its native tryptophan mutants. The equilibrium denaturation curves indicate the existence of two transitions. The first unfolding transition most likely reflects the denaturation of the carboxy-terminal region of FIV-rp24. Consequently, the second transition, where the changes in fluorescence are produced, should reflect the denaturation of the amino-terminal region. If the intermediate observed upon urea denaturation is an on-pathway species, the data described herein can reflect the sequential and independent loss of structure of the two domains that this type of proteins possesses.

Circular Dichroism↗
PubMed2001

ELISPOT analysis of hepatitis C virus protein-specific IFN-gamma-producing peripheral blood lymphocytes in infected humans with and without cirrhosis.

An improved ability to monitor hepatitis C virus (HCV)-specific T cell immunity in infected patients may provide novel information regarding the pathogenesis and prognosis of this infection. We used an ELISPOT assay to analyze a cross-section of HCV-infected humans. HCV-infected patients without cirrhosis, those with cirrhosis, and controls with other liver diseases were tested for recall responses to HCV Core and NS3 proteins. Peripheral blood lymphocytes (PBLs) from HCV-infected patients without cirrhosis responded to NS3 and Core proteins, producing predominantly IFN-gamma, with little IL-4 or IL-5. In contrast, PBLs from HCV-infected patients with cirrhosis responded to NS3, but not to the Core protein, suggesting a selectively altered immune state during cirrhosis. Our data provide support for the notion that HCV-specific IFN-gamma-producing immunity is important in the pathogenesis of progressing HCV-related disease.

Enzyme-Linked Immunosorbent Assay↗
PubMed2001

Cloning, expression, and purification of histidine-tagged preS domains of hepatitis B virus.

The preS domains of the hepatitis B virus are hydrophilic polypeptides that have been implicated, among other functions, in the binding of the virus to hepatocytes and in the induction of virus-neutralizing antibodies. A method of overproducing the preS domains of two different subtypes, adw and ayw, has been developed by adding a 6x His tag at the carboxy-terminal end of the polypeptides. Codons for the 6x His were added in reverse primers used to amplify the corresponding cDNAs. The polymerase chain reaction products were cloned into the expression vectors pET-3d (subtype ayw) and pT7-7 (subtype adw), under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl-beta-d-thiogalactopyranoside, proteins were overexpressed and purified by affinity chromatography on a Ni-nitrilotriacetic acid agarose column. This method yielded 20-40 mg of highly pure and very stable proteins per liter of cell culture. Circular dichroism and fluorescence spectroscopy of isolated preS-his-ayw and preS-his-adw, as well as their ability to bind polymerized human serum albumin, indicate that the 6x His tag does not modify the native-like conformation and, therefore, they may be considered as useful tools to study the function of these viral polypeptide regions.

Amino Acid Sequence↗
PubMed2001

Use of passive ambient ozone (O3) samplers in vegetation effects assessment.

A stochastistic, Weibull probability model was developed and verified to simulate the underlying frequency distributions of hourly ozone (O3) concentrations (exposure dynamics) using the single, weekly mean values obtained from a passive (sodium nitrite absorbent) sampler. The simulation was based on the data derived from a co-located continuous monitor. Although at the moment the model output may be considered as being specific to the elevation and location of the study site, the results were extremely good. This effort for the approximation of the O3 exposure dynamics can be extended to other sites with similar data sets and in developing a generalized understanding of the stochastic O3 exposure-plant response relationships, conferring measurable benefits to the future use of passive O3 samplers, in the absence of continuous monitoring.

Air Pollutants↗
PubMed2001

Characterization of a novel hepatitis B virus mutant: demonstration of mutation-induced hepatitis B virus surface antigen group specific "a" determinant conformation change and its application in diagnostic assays.

We report and characterize a novel mutant of the hepatitis B virus surface antigen (HBsAg). The mutant was isolated from a symptomatic patient who was found to be persistently positive for both HBsAg and anti-hepatitis B surface antibody (anti-HBs) and a long-lasting anti-HBc (core) IgM. Due to the unusual immune serological profile, polymerase chain reaction and sequencing were performed and revealed a genotype D mutant HBV (LBN). Aligned with known HBsAg sequences from GenBank, the LBN variant matched to consensus subtype ayw2 and revealed five mutation positions. Interestingly, none of the mutations was found within the group-specific "a" determinant region (124-147) and, specifically, two of the five mutations, T118K and P120Q, were located only a few amino acids adjacent to the 124-147 region. Using a panel of six monoclonal antibodies and vaccine raised human neutralizing antibodies, the recombinant wild-type HBsAg and the novel variant LBN HBsAg were investigated for their immunological reactivity. Vaccine raised human anti-HBs showed less reactivity to the variant LBN HBsAg than to wild-type HBsAg in enzyme immune assays. Furthermore, our observations demonstrate the influence of adjacent residues on the group-specific "a" determinant structural configuration; this resulted in severe antigenic changes of the immunodominant region as well as in the subtype serology. The importance of the variant LBN lies in the observation that mutations close to the "a" determinant can change the immunodominant region structure and therefore alter the group specific determinant antigenicity even though no mutations are present within this region. Hence, the classical definition of the "a" determinant cluster may need to be extended to cover a broader region to include the requirement of adjacent amino acids to support its conformation. In conclusion, by understanding the HBsAg major immunodominant region structure and by using a combination of antibodies with specificity covering all key mutation locations, maximal anti-HBs-based protection, and highly sensitive diagnostic assays can be achieved.

Adult↗
PubMed2001

Molecular basis for the interaction of the hepatitis B virus core antigen with the surface immunoglobulin receptor on naive B cells.

The nucleocapsid of the hepatitis B virus (HBV) is composed of 180 to 240 copies of the HBV core (HBc) protein. HBc antigen (HBcAg) capsids are extremely immunogenic and can activate naive B cells by cross-linking their surface receptors. The molecular basis for the interaction between HBcAg and naive B cells is not known. The functionality of this activation was evidenced in that low concentrations of HBcAg, but not the nonparticulate homologue HBV envelope antigen (HBeAg), could prime naive B cells to produce anti-HBc in vitro with splenocytes from HBcAg- and HBeAg-specific T-cell receptor transgenic mice. The frequency of these HBcAg-binding B cells was estimated by both hybridoma techniques and flow cytometry (B7-2 induction and direct HBcAg binding) to be approximately 4 to 8% of the B cells in a naive spleen. Cloning and sequence analysis of the immunoglobulin heavy- and light-chain variable (VH and VL) domains of seven primary HBcAg-binding hybridomas revealed that six (86%) were related to the murine and human VH1 germ line gene families and one was related to the murine VH3 family. By using synthetic peptides spanning three VH1 sequences, one VH3 sequence, and one VLkappaV sequence, a linear motif in the framework region 1 (FR1)complementarity-determining region 1 (CDR1) junction of the VH1 sequence was identified that bound HBcAg. Interestingly, the HBcAg-binding motif was present in the VL domain of the HBcAg-binding VH3-encoded antibody. Finally, two monoclonal antibodies containing linear HBcAg-binding motifs blocked HBcAg presentation by purified naive B cells to purified HBcAg-primed CD4(+) T cells. Thus, the ability of HBcAg to bind and activate a high frequency of naive B cells seems to be mediated through a linear motif present in the FR1-CDR1 junction of the heavy or light chain of the B-cell surface receptor.

Amino Acid Sequence↗
PubMed2000

Immune responses to hepatitis C and non-hepatitis C antigens in hepatitis C virus infected and HIV-1 coinfected patients.

OBJECTIVE: To characterize immune phenotype and function in hepatitis C virus (HCV) infection in the presence and absence of HIV-1 infection. DESIGN: Cross-sectional comparison among controls (group A), patients with HCV infection (group B), HCV-HIV-1 coinfected patients (group C), coinfected patients receiving treatment for HIV-1 (group D), and untreated HIV-1 infected patients (group E). METHODS: Flow cytometric analysis for lymphocyte phenotypes, lymphocyte proliferation and cytokine production by ELISPOT. RESULTS: HCV infected patients tended to have an increased percentage of activated (CD38, HLA-DR) CD8 cells (group A, 2+/-1.4%; group B, 6+/-3.9%; P=0.08). Proliferative responses to non-HCV antigens were comparable in group A and group B subjects. A greater proportion of group B patients had stimulation indices (SI) > 3 to the HCV protein NS3 compared to group C and D patients (67%, 0%, and 11% respectively; P < 0.003), but only two patients in group B had SI > or = 5. The SI to NS3 was significantly higher in group B patients [median, 4; interquartile range (IQR), 3-9) than in group C (median, 2; IQR, 1-3; P < 0.04) or group D (median, 1; IQR, 1-4; P < 0.009) patients. Plasma HCV RNA levels correlated directly with alanine aminotransferase levels (p, 0.52; P < 0.05) and inversely with the number of CD4 lymphocytes (rho, -0.55; P < 0.009) and proliferation to NS3 (p, -0.55; P < 0.009). CONCLUSIONS: Lymphocytes of HCV infected patients show weak proliferative responses to HCV antigens while responses to other antigens are preserved. Infection with HIV-1 potentiates this deficiency. Poor CD4 T cell responses to HCV are associated with and may determine the failure to control HCV propagation.

Adult↗
PubMed2000

Selective destabilization of acidic phospholipid bilayers performed by the hepatitis B virus fusion peptide.

A peptide corresponding to the N-terminal region of the S protein of hepatitis B virus (Met-Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu-Val-Leu-Gln) has been previously demonstrated to perform aggregation and destabilization of acidic liposome bilayers and to adopt a highly stable beta-sheet conformation in the presence of phospholipids. The changes in the lipid moiety produced by this peptide have been followed by fluorescence depolarization and electron microscopy. The later was employed to determine the size and shape of the peptide-vesicle complexes, showing the presence of highly aggregated and fused structures only when negatively charged liposomes were employed. 1,6-Diphenyl-1,3,5-hexatriene depolarization measurements showed that the interaction of the peptide with both negatively charged and zwitterionic liposomes was accompanied by a substantial reduction of the transition amplitude without affecting the temperature of the gel-to-liquid crystalline phase transition. These data are indicative of the peptide insertion inside the bilayer of both types of liposomes affecting the acyl chain order, though only the interaction with acidic phospholipids leads to aggregation and fusion. This preferential destabilization of the peptide towards negatively charged phospholipids can be ascribed to the electrostatic interactions between the peptide and the polar head groups, as monitored by 1-(4-(trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene fluorescence depolarization analysis.

Amino Acid Sequence↗
PubMed2000

Spatial distribution of tropospheric ozone in western Washington, USA.

We quantified the distribution of tropospheric ozone in topographically complex western Washington state, USA (total area approximately 6000 km(2)), using passive ozone samplers along nine river drainages to measure ozone exposure from near sea level to high-elevation mountain sites. Weekly average ozone concentrations were higher with increasing distance from the urban core and at higher elevations, increasing a mean of 1.3 ppbv per 100 m elevation gain for all mountain transects. Weekly average ozone concentrations were generally highest in Cascade Mountains drainages east and southeast of Seattle (maximum=55-67 pbv) and in the Columbia River Gorge east of Portland (maximum=59 ppbv), and lowest in the western Olympic Peninsula (maximum=34 ppbv). Higher ozone concentrations in the Cascade Mountains and Columbia River locations downwind of large cities indicate that significant quantities of ozone and ozone precursors are being transported eastward toward rural wildland areas by prevailing westerly winds. In addition, temporal (week to week) variation in ozone distribution is synchronous within and between all drainages sampled, which indicates that there is regional coherence in air pollution detectable with weekly averages. These data provide insight on large-scale spatial variation of ozone distribution in western Washington, and will help regulatory agencies optimize future monitoring networks and identify locations where human health and natural resources could be at risk.

Journal Article↗
PubMed2000

Frequent HHV-6 reactivation in multiple sclerosis (MS) and chronic fatigue syndrome (CFS) patients.

BACKGROUND: HHV-6 is a ubiquitous virus and its infection usually occurs in childhood and then becomes a latent infection. HHV-6 reactivation has been shown to play a role in the pathogenesis of AIDS and several other diseases. OBJECTIVES: To determine what role HHV-6 infection or reactivation plays in the pathogenesis of multiple sclerosis (MS) and chronic fatigue syndrome (CFS). RESULTS: Twenty-one MS and 35 CFS patients were studied and followed clinically. In these patients, we measured HHV-6 IgG and IgM antibody levels and also analyzed their peripheral blood mononuclear cells (PBMCs) for the presence of HHV-6, using a short term culture assay. In both MS and CFS patients, we found higher levels of HHV-6 IgM antibody and elevated levels of IgG antibody when compared to healthy controls. Seventy percent of the MS patients studied contained IgM antibodies for HHV-6 late antigens (capsid), while only 15% of the healthy donors (HD) and 20% of the patients with other neurological disorders (OND) had HHV-6 IgM antibodies. Higher frequency of IgM antibody was also detected in CFS patients (57.1%) compared to HD (16%). Moreover, 54% of CFS patients exhibited antibody to HHV-6 early protein (p41/38) compared to only 8.0% of the HD. Elevated IgG antibody titers were detected in both the MS and the CFS patients. PBMCs from MS, CFS and HD were analyzed in a short term culture assay in order to detect HHV-6 antigen expressing cells and to characterize the viral isolates obtained as either Variant A or B. Fifty-four percent of MS patients contained HHV-6 early and late antigen producing cells and 87% of HHV-6 isolates were Variant B. Isolates from CFS, patients were predominately Variant A (70%) and isolates from HD were predominately Variant B (67%). Moreover, one isolate from OND was also Variant B. Persistent HHV-6 infection was found in two CFS patients over a period of 2.5 years and HHV-6 specific cellular immune responses were detected in PBMCs from ten CFS patients. CONCLUSION: In both MS and CFS patients, we found increased levels of HHV-6 antibody and HHV-6 DNA. A decrease in cellular immune responses was also detected in CFS patients. These data suggest that HHV-6 reactivation plays a role in the pathogenesis of these disorders.

Antibodies, Viral↗
PubMed2000

Characterization of a monoclonal antibody and its single-chain antibody fragment recognizing the nucleoside Triphosphatase/Helicase domain of the hepatitis C virus nonstructural 3 protein.

We have produced a murine monoclonal antibody (MAb), ZX10, recognizing the NTPase/helicase domain of the hepatitis C virus (HCV) nonstructural 3 protein (NS3), from which we designed a single-chain variable fragment (ScFv). The ZX10 MAb recognized a discontinuous epitope of the NTPase/helicase domain, of which the linear sequence GEIPFYGKAIPL at residues 1371 to 1382 constitutes one part. cDNAs from variable regions coding for the heavy and light chains were cloned, sequenced, and assembled into the NS3-ScFv, which was inserted into procaryotic and eucaryotic expression vectors. Escherichia coli-expressed NS3-ScFv inhibited the binding of the ZX10 MAb to NS3, confirming a retained specificity. However, the ability to bind the peptide 1371-1382 had been lost. In vitro-translated NS3-ScFv and HCV NS3/NS4A were coprecipitated by antibodies to HCV NS4A, confirming the in vitro activity of the NS3 ScFv. Thus, we have designed a functional NS3 NTPase/helicase domain-specific ScFv which should be evaluated further with respect to disturbing enzymatic functions of the NS3 protein.

Acid Anhydride Hydrolases↗
PubMed1999

Fusogenic activity of hepadnavirus peptides corresponding to sequences downstream of the putative cleavage site.

Sequence homology between the amino-terminal region of the S protein of hepatitis B Virus (HBV) and known fusion peptides from retroviruses and paramyxoviruses led us to propose that this region might be equally involved in the initial infective steps of hepadnaviruses. In fact, we showed that a synthetic peptide corresponding to the N-terminus region of the S protein of HBV had membrane-interacting properties and was able to induce liposome fusion adopting an extended (beta-sheet) conformation (Rodríguez-Crespo et al., 1996, 1995). We describe herein studies on the interaction of peptides derived from the N-terminal region of the S protein of duck (DHBV: Met-Ser-Gly-Thr-Phe-Gly-Gly-Ile-Leu-Ala-Gly-Leu-Ile-Gly-Leu-Leu) and woodchuck hepatitis B viruses (WHV: Met-Ser-Pro-Ser-Ser-Leu-Leu-Gly-Leu-Leu-Ala-Gly-Leu-Gln-Val-Val) with liposomes. These peptides were able to induce to a different extent aggregation, lipid mixing, and leakage of internal aqueous contents from both neutral and negatively charged phospholipid vesicles in a concentration-dependent and pH-independent manner. Fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene-labeled vesicles indicated that both peptides become inserted into the hydrophobic core of the lipid bilayer. Circular dichroism studies indicated that the DHBV peptide adopts an extended conformation in the presence of lipids, whereas the WHV peptide displays a high content of alpha-helical conformation. Therefore, these results extend our previous findings obtained for human hepatitis B virus to other members of the hepadnavirus family and suggest that this region of the S protein is important in the initial steps of the infective cycle.

Amino Acid Sequence↗
PubMed1999

Model for lentivirus capsid core assembly based on crystal dimers of EIAV p26.

Two crystal forms of recombinant p26 capsid protein (CA) from the equine infectious anemia virus (EIAV) have in common an antiparallel four-helix bundle dimer interface between N-terminal domains (NTDs). The dimer interface provides a lenient scaffold to accommodate the wide sequence variation in these helices within lentivirus CA. Pairs of dimers weakly associate to form exact or approximate D2 symmetry tetramers. In one of the two crystal forms, the tetramers are linked via dimerization of C-terminal domains (CTDs). We propose that the observed NTD and CTD homodimer interactions are involved in the assembly of the lentivirus capsid. The NTD homodimer shape readily suggests a model for the mature capsid core, based on hexagonal packing with dimensions and surface topology resembling described EIAV capsid cores. Combining available data for human immunodeficiency virus and EIAV CA, we also propose an assembly pathway for maturation of the lentivirus capsid core following proteolytic cleavage of the gag polyprotein precursor.

Amino Acid Sequence↗
PubMed1999

Circular dichroism and fluorescence spectroscopic properties of the major core protein of feline immunodeficiency virus and its tryptophan mutants. Assignment of the individual contribution of the aromatic sidechains.

The gene coding for the major capsid protein of feline immunodeficiency virus (FIV) has been cloned into the expression vector pQE60, which allows protein purification by affinity chromatography on a nitrilotriacetic acid/Ni/agarose column. The gene was expressed in Escherichia coli and the resultant soluble protein (FIV-rp24) purified to electrophoretic homogeneity. The amino-acid composition of the recombinant protein is almost identical to that predicted from the DNA sequence. This protein has two tryptophan residues at positions 40 and 126 that have been replaced by phenylalanine by site-directed mutagenesis to obtain two single mutants and a double mutant. Circular dichroism and fluorescence spectroscopy were employed to study the structural features of FIV-rp24 protein and its tryptophan mutants. The analysis of the CD spectra indicated that alpha-helix is the major secondary structural element (48-52%) and that the overall three-dimensional structure is not modified by the mutations. The fluorescence emission spectra showed that both tryptophan residues occupy a highly hydrophobic environment. Moreover, the different tyrosine fluorescence intensities of wild-type and mutant proteins are indicative of the existence of resonance energy transfer processes to nearby tryptophan. The individual contributions of each tryptophan residue to the spectroscopic properties of the wild-type protein were obtained from the spectra of all these proteins. Thermal denaturation studies indicate that the two tryptophan residues do not contribute equally to the stabilization of the three-dimensional structure.

Amino Acid Sequence↗