Biomedical subjects

R L Hunter

Publications and source records attributed to R L Hunter.

At least 55 records · Page 3Linked to original sources

PubMed1995

Induction of protective polyclonal antibodies by immunization with a Plasmodium yoelii circumsporozoite protein multiple antigen peptide vaccine.

Monoclonal Abs against the repeat region of the circumsporozoite protein (CSP) completely protect mice against Plasmodium yoelii (Py), but synthetic peptide and recombinant protein vaccines designed to produce only Abs to the PyCSP repeat region have never been reported to consistently provide protection. This lack of protection in the rodent model system has predicted the poor protection achieved in humans after immunization with synthetic peptide and recombinant protein P. falciparum CSP vaccines and has raised serious questions regarding the capacity for vaccine-induced polyclonal Abs against the CSP to consistently protect humans. We now report immunization studies with a multiple Ag peptide vaccine designed to rely on "universal" T epitopes from tetanus toxin to produce T cell help for induction of protective Abs against the repeat region of the PyCSP. When delivered with a nonionic block co-polymer adjuvant, the vaccine protected 78 to 100% of three inbred strains of mice, and 100% of outbred mice against P. yoelii sporozoite challenge. Protection was associated with Ab titer, and passive transfer of purified IgG from immune mice protected naive recipients. Similar protection was achieved when the peptide was encapsulated in liposomes with lipid A and mixed with aluminum hydroxide. By demonstrating for the first time solid protection against P. yoelii by polyclonal Abs against the CSP, these data provide the rationale for assessment of a similarly constructed and formulated P. falciparum CSP multiple Ag peptide vaccine in humans.

Amino Acid Sequence↗

PubMed1995

Induction of long-lasting immunity to Plasmodium yoelii malaria with whole blood-stage antigens and copolymer adjuvants.

We previously reported that protection of mice from nonlethal Plasmodium yoelii malaria by immunization with whole killed blood-stage parasites was dependent on the adjuvant and that adjuvants influenced both the specificity and isotype of Ab. Additional studies with the most effective formulations were undertaken to better define the protective responses and 100% protection from lethal P. yoelii malaria was produced by three immunizations with Ag in copolymer P1004 and detoxified RaLPS as adjuvants and 83% protection was induced by a single immunization. The protection lasted for 9 mo and was associated with an anamnestic rise in Ab titer of the IgG2a isotype during the challenge infection. Passive immunization with Ab from animals that had been immunized and challenged transferred sterile immunity. Splenectomy reduced, but did not abolish, protection. These data suggest that the effective Ab is directed against labile epitopes on the surface of blood-stage parasites. The vaccines primed animals for production of such Ab, but its synthesis was efficiently induced only by challenge with live organisms.

Adjuvants, Immunologic↗

PubMed1995

Enhancement of antibiotic susceptibility and suppression of Mycobacterium avium complex growth by poloxamer 331.

The resistance of Mycobacterium avium complex (MAC) to antibiotics is thought to be enhanced by its outer glycolipid layer, which protects the organisms from antibiotics and host defense mechanisms. We hypothesized that surfactants which disrupt the lipid barrier might be of therapeutic value. We evaluated the ability of 10 poloxamer surfactants to inhibit the growth of MAC organisms and to potentiate antimycobacterial drug activity in broth culture using a radiometric assay. Very large, small, or hydrophilic poloxamers had little or no effect. However, certain hydrophobic poloxamers, especially P331, retarded the growth of most isolates of MAC and produced a synergistic effect with rifampin. The MIC of rifampin required to inhibit the growth of MAC was reduced from a mean of 14.6 micrograms/ml (range, 4 to > 32 micrograms/ml) to 1.4 micrograms/ml (range, < 1.125 to 4 micrograms/ml) by 1.0 mg of P331 per ml (P < 0.01). Enhancement of antibiotic susceptibility was observed with concentrations of poloxamer as low as 10 micrograms/ml. These studies suggest that P331 might be useful in increasing the effectiveness of antibiotic therapy of MAC infections.

Anti-Bacterial Agents↗

PubMed1995

Activities of poloxamer CRL8131 against Mycobacterium tuberculosis in vitro and in vivo.

A poloxamer surfactant, CRL8131, was evaluated for activity against Mycobacterium tuberculosis (Erdman) by itself and in combination with antibiotics in broth culture, in a macrophage cell line assay, and in testing with mice. In the broth culture, CRL8131 suppressed the growth of M. tuberculosis and produced synergistic effects in combination with isoniazid, rifampin, and streptomycin. It also displayed synergy with isoniazid and rifampin against two drug-resistant isolates. In the macrophage cell line assay, CRL8131 produced a synergistic effect on intracellular killing of M. tuberculosis by isoniazid, rifampin, streptomycin, pyrazinamide, thiacetazone, D-cycloserine, ethionamide, amikacin, clindamycin, and p-aminosalicylic acid. It demonstrated no synergy or antagonism with ethambutol, gentamicin, kanamycin, ciprofloxacin, or nalidixic acid. Finally, with C57BL/6 mice infected with M. tuberculosis, a combination of CRL8131 and either thiacetazone or pyrazinamide produced 100% survival at 40 days whereas the antibiotics produced only 33% survival and CRL8131 produced 0% survival when used as single agents. This improved survival rate was associated with a significant reduction in the number of organisms in the lungs and spleens of infected mice.

Animals↗

PubMed1995

Use of the rhesus monkey as an experimental model to test the degree of efficacy of an anti-sporozoite peptide malaria vaccine candidate combined with copolymer-based adjuvants.

Humoral response against sporozoites is not effective in protecting individuals from getting malaria. Reduction in the infectivity of sporozoites has not been quantified for most anti-sporozoite vaccines tested. Quantification requires animal models providing predictable prepatent periods, e.g., time elapsed between sporozoite inoculation and detection of parasitemia, to be used as an indicator of activity against sporozoites. A delay in prepatent period from vaccinated animals would therefore reflect a protective effect in reducing the number of parasites. We report the vaccination of rhesus monkeys with a synthetic peptide reproducing part of the repeated region of the circumsporozoite protein of Plasmodium cynomolgi. This peptide was conjugated to the carrier protein diphtheria toxoid and injected with four adjuvant formulations that differed only by the type of emulsion or immunomodulator. Because all five control animals had a synchronous prepatent period after challenge with live sporozoites, it was possible to quantify the protective efficacy for each vaccine formulation, even though all monkeys developed parasitemia. Sporozoite elimination correlated with the immunomodulator and the type of emulsion. Such elimination was related neither to antibody titer against the immunizing peptide or the whole sporozoite, nor to antibody isotype induced by the vaccine formulation.

Adjuvants, Immunologic↗

PubMed1994

High-resolution mass spectrometer for protein chemistry.

Matrix-assisted laser desorption/ionization is an accurate and sensitive method for measuring the molecular weights of peptides and proteins. Usually time-of-flight mass spectrometry is used to detect the laser-produced ions, but a new method called Fourier transform mass spectrometry offers greater sensitivity and much higher mass measurement accuracy.

Animals↗

PubMed1994

High-accuracy molecular mass determination for peptides and proteins by Fourier transform mass spectrometry.

A new calibration method has been developed for Fourier transform mass spectrometry (FTMS) that is accurate to better than 0.001% (10 ppm) for peptides and proteins up to 5700 Da. The custom-designed FTMS instrument used for this work has a matrix-assisted laser desorption/ionization (MALDI) source located outside of the magnetic field in a differentially pumped chamber, and ions are injected through the fringing fields of the magnet into the FTMS analyzer cell by a long quadrupole ion guide. The mass spectrometer is calibrated with four model compounds ([Arg8]-vasopressin, melittin, bovine insulin B-chain, and bovine insulin) of known molecular mass. The set of measured ion resonance frequencies (f) for these compounds are fit to a three-term calibration equation of the form f = A(z/m) + B(V) + C(V2) (m/z), where m/z is the mass-to-charge ratio of a calibrant peak, V is the trapping voltage, and A, B, and C are calibration coefficients that depend on the magnetic field strength and the dimensions of the analyzer cell. The same set of calibration coefficients can be used for many weeks because the magnet and the electronics of the FTMS instrument are very stable. This method is useful because unknowns can be run separately without the need to add an internal calibration compound in with the sample.

Fourier Analysis↗

PubMed1994

High-resolution laser desorption mass spectrometry of peptides and small proteins.

Matrix-assisted laser desorption/ionization (MALDI) has been used with an external ion source Fourier-transform mass spectrometer to obtain the highest mass resolution ever, to our knowledge, demonstrated for laser-produced ions (m/delta m = 1,100,000 for [Arg8]vasopressin, 228,000 for melittin, and 90,000 for bovine insulin). The peaks in the isotope cluster for bovine insulin are fully resolved, and the mass measurement accuracy is an order of magnitude better than can be achieved with time-of-flight mass spectrometry. With the method described here, analyte is applied to a sample probe and mixed with a solution containing a matrix material (2,5-dihydroxybenzoic acid) that strongly absorbs ultraviolet light. Upon irradiation with a pulse from an excimer laser (353 nm, 2 mJ), a large number of intact protonated molecular ions are produced. The ions are focused by a 117-cm-long quadrupole ion guide and injected into an ion cyclotron resonance analyzer cell located inside the bore of a 6.5-T superconducting magnet. A pulse of argon buffer gas cools the ions prior to detection. One of the principal advantages of an external ion source Fourier-transform mass spectrometer is that the ion formation and ion detection processes are separated and can be independently optimized.

Animals↗

PubMed1994

Matrix-assisted laser desorption/ionization with an external ion source Fourier-transform mass spectrometer.

Mass spectra of several model oligopeptides (substance P, [Arg8]-vasopressin, tyrothricin, and the B-chain of bovine insulin) have been obtained with a matrix-assisted laser desorption/ionization (MALDI) source and a custom-designed Fourier-transform mass spectrometer (FTMS). The MALDI source is outside the magnetic field in a separately pumped external chamber, and ions are injected through the fringing fields of the magnet and into the FTMS analyzer cell by a long quadrupole mass filter that is operated in the RF-only mode. A large window on the ion source housing makes it easy to point and focus the laser beam onto the sample probe tip. A good quality mass spectrum is achieved for 0.5 pmol of substance P, and the mass resolution for the B-chain of bovine insulin is 19,000.

Animals↗

PubMed1994

Characterization of the trehalose 6,6'-dimycolate surface monolayer by scanning tunneling microscopy.

The toxicity of trehalose 6,6'-dimycolate (TDM), a glycolipid of mycobacteria, requires presentation as a surface monolayer. Our model of the structure of the TDM monolayer was confirmed and extended by scanning tunneling microscopy. It consists of linear arrays with a periodicity of approximately 90 A (9 nm) that clustered in groups of four to form secondary structures with a periodicity of 360 A (36 nm).

Cord Factors↗

PubMed1994

Extravascular coagulation and fibrinolysis in murine lung inflammation induced by the mycobacterial cord factor trehalose-6,6'-dimycolate.

Diffuse pulmonary inflammation in interstitial lung diseases is associated with increased coagulation in the extravascular spaces of the lung. We hypothesized that conditions favoring coagulation over fibrinolysis in the lung are related to inflammation. Pulmonary coagulation and fibrinolysis were studied in two strains of mice susceptible or resistant to the development of lung inflammation in response to the mycobacterial cell wall glycolipid trehalose-6,6'-dimycolate (TDM). Susceptible animals treated with TDM intravenously develop well-organized collections of mononuclear cells in the lung parenchyma referred to as granulomas in this report. More granulomas were found in the susceptible ICR mice than in the resistant A/J mice after intravenous administration of TDM (7 +/- 1 granulomas/mm2 versus 1 +/- 0.3 granulomas/mm2, p = 0.005). Granuloma formation was associated with increased lung procoagulant activity (PCA) measured in bronchoalveolar lavage (BAL) cell lysates from susceptible mice. In contrast, TDM-resistant A/J mice challenged with TDM did not have a significant BAL cell PCA response, but expressed several-fold greater levels of lung BAL fluid plasminogen activator activity (PAA) than ICR mice. To examine the role of coagulation in the TDM pulmonary inflammatory response, susceptible C57Bl/10SnJ mice were anticoagulated by oral administration of warfarin prior to challenge of TDM; these mice developed fewer pulmonary granulomas than TDM-treated mice without warfarin treatment (2.6 +/- 0.5 granulomas/mm2 versus 6.5 +/- 0.8 granulomas/mm2, p < 0.001) but had similar BAL cell PCA and lung inflammatory changes as measured by lung weights and BAL cellularity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Copolymer adjuvants in malaria vaccine development.

Protection against virulent challenge with murine Plasmodium yoelii malaria was induced by immunization with whole killed blood-stage parasites in copolymer P1004 and detoxified lipopolysaccharide as adjuvant. Similar immunization with Freund's complete adjuvant and other water-in-oil emulsions failed to protect. Protection was associated with the production of antibody of the IgG2a isotype against epitopes measured by immunofluorescence. Several formulations that did not protect elicited high antibody titers measured by enzyme-linked immunosorbent assays or titers of other isotypes measured by indirect immunofluorescent assay. The results provide additional evidence that the adjuvants influenced both the isotype and specificity of antibody. The implications of these findings for vaccine development are discussed.

Adjuvants, Immunologic↗

PubMed1994

Mechanisms of action of nonionic block copolymer adjuvants.

Nonionic block copolymer adjuvants typically induce high-titer, long-lasting antibody responses, cell-mediated immunity, CTLs, and modulate the isotype and specificity of antibody. Their primary activity is modulation of hydrophobic adhesive interactions. The copolymers adhere to lipids, promote retention of protein antigen to surfaces, activate complement, and induce expression of class II (IA) on macrophages. They produce a concentrated surface matrix of antigen and activated host mediators that facilitates antigen presentation to cells of the immune system. The copolymer adjuvants act synergistically with multiple MDP and LPS preparations to increase total titers, especially those of the IgG2a and IgG2b isotypes. A surprising discovery was that they influence the specificity of antibody by at least two mechanisms. Saline formulations and oil-in-water (o/w) emulsions induced more antibody against labile, conformationally dependent epitopes on the surface of particles than water-in-oil (w/o) emulsions. Finally, we found that very large copolymers are able to stabilize water-in-oil-in-water (w/o/w) or multiple emulsions that can protect antigen during passage through the upper GI tract. They are therefore attractive vehicles for oral delivery of vaccines.

AIDS Vaccines↗

PubMed1993

Role of adjuvants in the modulation of antibody isotype, specificity, and induction of protection by whole blood-stage Plasmodium yoelii vaccines.

Mice were immunized with whole killed blood stage Plasmodium yoelii parasites in 15 adjuvant formulations then boosted and challenged with parasitized blood. Five of six groups immunized with the Ag in oil-in-water emulsions or formulations without oil were protected. Formulations that induced protection contained saponin, pertussis, copolymer P1004, and detoxified RaLPS. In contrast, none of nine groups of animals immunized with Ag in water-in-oil emulsions were protected. Ineffective adjuvants included CFA and water-in-squalene emulsions with copolymer L141 plus detoxified RaLPS, dimethyldioctadecyl ammonium bromide, and mycobacterial cell wall skeletons. Antibody was measured by ELISA against disrupted parasites and by indirect fluorescent antibody (immunofluorescence) using intact parasites. Protection was associated with antibody of the IgG2a isotype detected by immunofluorescence but not with other isotypes detected by immunofluorescence or any type antibody detected by ELISA. The water-in-oil adjuvants induced high titers by ELISA but low titers by immunofluorescence. These results, together with Western blot analyses, suggested that adjuvant vehicles control the specificity of antibody and that this, in turn, is essential for induction of protective immune responses in this model.

Adjuvants, Immunologic↗

PubMed1993

Development of a trehalose 6,6'-dimycolate model which explains cord formation by Mycobacterium tuberculosis.

Trehalose 6,6'-dimycolate (TDM) is a glycolipid of mycobacteria that displays an unusual toxicity and has been reported to be a virulence factor for Mycobacterium tuberculosis. Lack of understanding of the toxicity has impeded acceptance of TDM as a virulence factor. We previously reported that the toxicity of TDM depends on its presentation as a surface monolayer consisting of 30% trehalose and 70% exposed mycolic acid moieties. This paper further investigates the structure of the TDM monolayer. It began with the observation that beads coated with TDM, but not with closely related analogs, aggregate to form organized structures resembling the cords of virulent mycobacteria. This implied that the TDM molecules in the monolayer were arranged in an organized structure. This structure was investigated by real-time kinetic microscopy, scanning electron microscopy, and gross observations of the adhesion patterns of TDM-coated beads. In each of these models, the structures induced by TDM differed from those of analogs or other amphiphiles studied. These observations were used to construct a model of the structure of TDM monolayer which envisions linear arrays of TDM molecules arranged in a circumferential pattern on beads with discontinuities only at the two poles.

Cord Factors↗

PubMed1993

Elevated D dimer in the lungs and blood of patients with sarcoidosis.

The hypothesis of this study was that D dimer, a specific degradation product of cross-linked fibrin, would be increased in the bronchoalveolar lavage (BAL) fluids of patients with sarcoidosis and that it would be related to other BAL parameters of disease activity. Eight of 10 sarcoidosis patients but none of 18 healthy volunteers had detectable BAL D dimer by enzyme immunoassay. Autoradiography revealed the presence of fibrinogen and D dimer in the BAL fluids from sarcoidosis patients. Bronchoalveolar lavage D dimer levels in sarcoidosis patients correlated with total BAL cells per milliliter, lymphocytes per milliliter, and total protein level, but not macrophages per milliliter. The D dimer in the BAL fluids from sarcoidosis patients did not correlate with D dimer in the blood. Our findings indicate that BAL D dimer parallels directly the lymphocytic alveolitis that characterizes pulmonary sarcoidosis.

Adult↗

PubMed1993

Determination of regional myocardial blood flow using fluorescent microspheres.

Determination of blood flow in tissues at risk for infarction is necessary for in vivo studies of the pathologic effects of vascular occlusion. Such blood flow measurements are traditionally carried out using radioactive microspheres. These are expensive and may pose a significant hazard to laboratory personnel. In addition, disposal of radioactive wastes, especially radioactive carcasses, is an increasingly expensive and inconvenient process. To avoid these problems, we have developed a modified procedure using fluorescent microspheres. To measure blood flow, spheres are injected intravenously and a blood sample is taken. Following cardiectomy, appropriate segments of left ventricular myocardium are digested with proteases to release the spheres, which are then purified through sucrose gradients. The spheres may be counted manually using an epifluorescence microscope, or by flow cytometry. Comparison of this method with the traditional radioactive microsphere procedure reveals similar results for the two methods. Fluorescent microspheres should provide a useful and accurate alternative method for measuring blood flow in studies of vascular disease.

Animals↗

PubMed1992

A comparison of commercially available adjuvants for use in research.

We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.

Adjuvants, Immunologic↗