Biomedical subjects

Stanley L Hem

Publications and source records attributed to Stanley L Hem.

At least 19 recordsLinked to original sources

PubMed2006

Potentiation of the immune response to non-adsorbed antigens by aluminum-containing adjuvants.

The degree of antigen adsorption by aluminum-containing adjuvants is considered an important characteristic of vaccines that is related to immunopotentiation by the adjuvant. This study examined immunopotentiation by aluminum phosphate adjuvant in three model vaccines in which the antigen was not adsorbed in the vaccine formulation nor when mixed in vitro with interstitial fluid. In the first model vaccine, aluminum phosphate adjuvant was pre-treated with 0.5 M KH2PO4 to minimize the adsorption of dephosphorylated alpha casein. The second model vaccine was composed of aluminum phosphate adjuvant and ovalbumin that was dephosphorylated by treatment with potato acid phosphatase. The third model vaccine consisted of aluminum phosphate adjuvant and lysozyme (LYS). In order to prevent adsorption of lysozyme, the aluminum phosphate adjuvant was pre-treated with fibrinogen, a protein present in interstitial fluid that binds strongly to aluminum phosphate adjuvant. Immunopotentiation was evaluated by measuring antibody production in mice. It was found that all three model vaccines induced antibody titers that were statistically higher than induced by a solution of antigen without adjuvant and similar to vaccines in which the antigens were adsorbed by aluminum phosphate adjuvant. Confocal microscopy experiments suggested that the antigens used in these experiments, even though not adsorbed to the aluminum phosphate adjuvant, were trapped in void spaces within the adjuvant aggregates, resulting in uptake of antigen by dendritic cells.

Adjuvants, Immunologic↗

PubMed2006

Aluminum hydroxide adjuvant produced under constant reactant concentration.

Aluminum hydroxide adjuvant, AlO(OH), is used to potentiate the immune response to vaccines by adsorbing the antigen. The structure of aluminum hydroxide adjuvant is unusual as it is crystalline but has a high surface area due to its very small primary particles. The purpose of this study was to investigate the chemical and thermal conditions required to synthesize aluminum hydroxide adjuvant that is stable and exhibits a high protein adsorptive capacity. Aluminum hydroxide adjuvant was precipitated using a procedure in which the concentration of reactants was maintained constant throughout the precipitation. The precipitation variables were: 2.50, 2.75, and 3.00 OH/Al molar ratio; 0.5, 4.0, and 5.0 M NaCl; and 25, 60, and 65 degrees C. High sodium chloride concentration and high temperature facilitated the formation of AlO(OH) rather than crystalline forms of aluminum hydroxide, Al(OH)(3). The AlO(OH) produced was not stable because crystalline forms of aluminum hydroxide formed during aging at room temperature. Aluminum hydroxide adjuvant was stabilized for the study period of 12 weeks at room temperature by either the addition of 3.0 M NaCl after precipitation and washing or hydrothermal treatment at 110 degrees C for 4 h. Stabilization by the addition of sodium chloride required a hypertonic concentration of sodium chloride and was not practical as vaccines for parenteral administration are desired to be isotonic (equivalent to 0.15 M NaCl). Stabilization by hydrothermal treatment produced aluminum hydroxide adjuvant, which exhibited a high protein adsorptive capacity that did not change during the 12-week study period.

Adjuvants, Pharmaceutic↗

PubMed2005

Relationship of adsorption mechanism of antigens by aluminum-containing adjuvants to in vitro elution in interstitial fluid.

The objective of this research was to determine how the mechanism by which antigens adsorb to aluminum-containing adjuvants affects the elution upon exposure to interstitial fluid. Antigens (alpha lactalbumin, bovine serum albumin, lysozyme and myoglobin) that adsorb to aluminum-containing adjuvants principally by electrostatic attraction were found to elute readily in vitro when exposed to interstitial fluid. Phosphorylated antigens (alpha casein, hepatitis B surface antigen and phosphorylated bovine serum albumin) that adsorb to aluminum-containing adjuvants principally by ligand exchange exhibit little if any elution during 12-24 h in vitro exposure to interstitial fluid. Dephosphorylated alpha casein, which contains less than two phosphate groups, was less strongly adsorbed by ligand exchange in comparison to alpha casein, which contains eight phosphate groups. Dephosphorylated alpha casein was completely eluted when exposed to interstitial fluid. The results of this study lead to the generalization that antigens that adsorb to aluminum-containing adjuvants by electrostatic attraction are more likely to elute upon intramuscular or subcutaneous administration than antigens that adsorb by ligand exchange.

Adjuvants, Immunologic↗

PubMed2005

Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro.

An important step in the induction of an immune response to vaccines is the internalization of antigens by antigen presenting cells, such as dendritic cells (DCs). Many current vaccines are formulated with antigens adsorbed to an aluminum-containing adjuvant. Following injection of the vaccine the antigens may either elute or stay adsorbed to the adjuvant surface. Antigens, which elute from the adjuvant surface, are internalized by dendritic cells through macropinocytosis while those that remain adsorbed are internalized with the adjuvant particle by phagocytosis. The relative efficiency of these two routes of internalization was studied. Alpha casein (AC) labeled with a green fluorescent dye was selected as the model antigen. In order to model vaccine antigens that elute from aluminum-containing adjuvants following administration, dendritic cells were incubated with a solution of fluorochrome-labeled alpha casein. To model vaccine antigens that do not elute from aluminum-containing adjuvants following administration, dendritic cells were exposed to fluorochrome-labeled alpha casein adsorbed to aluminum hydroxide adjuvant (AH). Alpha casein has eight phosphate groups and adsorbs to aluminum hydroxide adjuvant through ligand exchange. Alpha casein does not elute from aluminum hydroxide adjuvant upon exposure to cell culture media. The uptake of antigen by dendritic cells was determined at 0.5, 1, 2 and 3h by confocal microscopy and flow cytometry. Dendritic cells internalized both alpha casein in solution and alpha casein adsorbed to aluminum hydroxide adjuvant. However, the mean fluorescence intensity of dendritic cells incubated with adsorbed alpha casein was four times greater than dendritic cells incubated with alpha casein in solution. In addition, the internalization of alpha casein was enhanced when the mean aggregate diameter of the adjuvant in the cell culture media was reduced from 17 microm to 3 microm. It was concluded that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and the aggregate size of the adjuvant was smaller than dendritic cells which are approximately 10 microm in diameter.

Adjuvants, Immunologic↗

PubMed2005

Effect of phosphorylation of ovalbumin on adsorption by aluminum-containing adjuvants and elution upon exposure to interstitial fluid.

The phosphate content of commercial ovalbumin was increased from 1.8 to 3.2 mol PO(4)/mol ovalbumin by conjugation of phosphoserine and reduced to 1.2 or 0.14 mol PO(4)/mol ovalbumin by treatment with potato acid phosphatase. The four ovalbumin samples were completely adsorbed by aluminum hydroxide adjuvant due to electrostatic attraction of the negatively charged ovalbumin and the positively charged aluminum hydroxide adjuvant as well as by ligand exchange of phosphate groups with surface hydroxyl groups. Elution from aluminum hydroxide adjuvant upon exposure to interstitial fluid was inversely related to the degree of phosphorylation of the ovalbumin. The ovalbumin sample containing 3.2 mol PO(4)/mol ovalbumin did not elute while the ovalbumin sample containing 0.14 mol PO(4)/mol ovalbumin eluted completely from aluminum hydroxide adjuvant during exposure to interstitial fluid for 30 min. Adsorption of the four ovalbumin samples by aluminum phosphate adjuvant was directly related to the degree of phosphorylation of ovalbumin. Adsorption was due to ligand exchange as an electrostatic repulsive force operated between the negatively charged ovalbumin samples and the negatively charged aluminum phosphate adjuvant. The potential for ligand exchange decreased as the phosphorylation of ovalbumin decreased. Elution upon exposure to interstitial fluid was inversely related to the degree of phosphorylation and was more extensive than observed for aluminum hydroxide adjuvant. Adsorption of ovalbumin by aluminum-containing adjuvants and elution upon exposure to interstitial fluid can be controlled by the degree of phosphorylation of both ovalbumin and the aluminum-containing adjuvant.

Acid Phosphatase↗

PubMed2004

Structure and adsorption properties of commercial calcium phosphate adjuvant.

Calcium phosphate adjuvant is a commercially available vaccine adjuvant that potentiates the immune response to antigens. Although its name suggests that it is Ca3(PO4)2, X-ray diffraction, FTIR spectroscopy, thermal analysis and the Ca/P molar ratio identify commercial calcium phosphate adjuvant as non-stoichiometric hydroxyapatite, Ca10-x (HPO4)x (PO4)6-x (OH)2-x, where x varies from 0 to 2. The surface charge is pH-dependent (point of zero charge = 5.5). Consequently, commercial calcium phosphate adjuvant exhibits a negative surface charge at physiological pH and electrostatically adsorbs positively charged antigens. The presence of hydroxyls allows calcium phosphate adjuvant to adsorb phosphorylated antigens by ligand exchange with surface hydroxyls.

Adjuvants, Immunologic↗

PubMed2004

Distribution of adsorbed antigen in mono-valent and combination vaccines.

The distribution of alpha-casein, bovine serum albumin (BSA), myoglobin and recombinant protective antigen (rPA) in mono-valent and combination vaccines containing aluminum hydroxide adjuvant was studied by fluorescence microscopy and flow cytometry. Green and red fluorescent probes were conjugated to the antigens. Adsorption isotherms of the fluorescently labeled proteins to aluminum hydroxide adjuvant demonstrated that incorporation of the fluorescent probe did not significantly affect the adsorption. In mono-valent vaccine systems, antigen adsorption occurred within one minute and uniform surface coverage of the adjuvant aggregates was observed within 1h. Content uniformity was achieved through a cycle of de-aggregation and re-aggregation of the aluminum hydroxide adjuvant aggregates caused by mixing. For combination vaccines, two antigens were adsorbed separately to the aluminum hydroxide adjuvant prior to combination. Following combination, cycles of de-aggregation and re-aggregation occurred due to mixing, which led to uniform distribution of both antigens. The results of this study indicate that content uniformity should not be an issue during the production of mono-valent or combination vaccines as long as adequate mixing procedures are followed.

Absorption↗

PubMed2004

Mechanism of adsorption of hepatitis B surface antigen by aluminum hydroxide adjuvant.

Hepatitis B surface antigen (HBsAg) differs from many antigens because of its associated lipid bilayer that is largely composed of phospholipids. In general, phosphate groups adsorb strongly to hydroxylated mineral surfaces by ligand exchange. The purpose of this study was to investigate the mechanism of adsorption of hepatitis B surface antigen to aluminum hydroxide adjuvant with emphasis on the role of phospholipids in this adsorption. The adsorption of HBsAg by aluminum hydroxide adjuvant exhibits a high affinity adsorption isotherm. The Langmuir equation was used to calculate the adsorptive capacity (1.7 microg/microg Al), which is the amount of HBsAg adsorbed at monolayer coverage and the adsorptive coefficient (6.0 ml/microg), which is a measure of the strength of the adsorption force. The relatively high value of the adsorptive coefficient indicates that adsorption is due to a strong attractive force. Ligand exchange between a phosphate of the antigen and a surface hydroxyl of the adjuvant provides the strongest adsorption mechanism. The adsorption capacity of HBsAg was not affected by increased ionic strength indicating that electrostatic attraction is not the predominant adsorption force. Adsorption was also not affected by the addition of ethylene glycol indicating that hydrophobic interactions were not the predominant adsorption force. The strength of the adsorption force was indicated by the resistance of HBsAg to elution when exposed to interstitial fluid. Less than 5% of the HBsAg adsorbed to aluminum hydroxide adjuvant in a model vaccine was eluted during a 12 h in vitro exposure to interstitial fluid at 37 degrees C. Less than 1% of the adsorbed HBsAg in two commercial vaccines was eluted by in vitro exposure to interstitial fluid for 48 h at 37 degrees C. Thus, it was concluded that adsorption of HBsAg by aluminum hydroxide adjuvant is predominantly due to ligand exchange between the phospholipids in HBsAg and surface hydroxyls in aluminum hydroxide adjuvant.

Adjuvants, Immunologic↗

PubMed2004

Effect of microenvironment pH of aluminum hydroxide adjuvant on the chemical stability of adsorbed antigen.

The rate of acid-catalyzed hydrolysis of glucose-1-phosphate (G1P) when adsorbed to aluminum hydroxide adjuvant was significantly slower than the rate of hydrolysis of a solution of G1P at the same pH. It was concluded that the positively charged aluminum hydroxide adjuvant (iep 11.4) electrostatically attracted anions including hydroxyls to form a double layer surrounding the adjuvant particles. Thus, the pH of the microenvironment surrounding the aluminum hydroxide adjuvant was higher than the bulk pH. Adsorbed G1P hydrolyzed at a rate associated with the pH of the microenvironment of the surface of the adjuvant rather than with the pH of the bulk solution. Comparison of the rate constant for the hydrolysis of adsorbed G1P to the pH-stability profile of G1P in solution revealed that adsorbed G1P hydrolyzed at a rate associated with a pH that was approximately two pH units higher than the bulk pH. The results suggest that the chemical stability of antigens that degrade by pH-dependent mechanisms can be optimized by modifying the surface charge of the aluminum-containing adjuvant to produce the pH of maximum stability in the microenvironment of the adjuvant.

Absorption↗

PubMed2003

Water-vapor adsorption and surface area measurement of poorly crystalline boehmite.

Water-vapor adsorption on poorly crystalline boehmite (PCB) was studied using a gravimetric FTIR apparatus that measured FTIR spectra and water adsorption isotherms simultaneously. The intensity of the delta(HOH) band of adsorbed water changed linearly with water content and this linear relationship was used to determine the dry mass of the sample. Adsorption and desorption isotherms of PCB showed a Type IV isotherm. The BET(H2O) surface area of PCB was 514+/-36 m2/g. The mean crystallite dimensions of PCB were estimated to be 4.5 x 2.2 x 10.0 nm (dimensions along the a, b, and c axes, respectively) based on application of the Scherrer equation to powder diffraction data of PCB. A surface area value of 504+/-45 m2/g calculated using the mean crystallite dimensions was in good agreement with the BET(H2O) surface area. This work also demonstrated a method to determine surface areas for materials with minimal perturbation of their surface structure. In addition, the FTIR spectra of PCB were influenced by changes in water content. The delta(AlOH) band at 835 cm(-1) observed under dry conditions was assigned to the non-H-bonded surface OH groups. As the amount of adsorbed water increased, the intensity at 835 cm(-1) decreased and that at 890 and 965 cm(-1) increased. The 890- and 965-cm(-1) bands are assigned to surface OH groups H-bonded with adsorbed water.

Journal Article↗

PubMed2003

Relationship between the degree of antigen adsorption to aluminum hydroxide adjuvant in interstitial fluid and antibody production.

The effect of the degree of adsorption after exposure to interstitial fluid on the immune response in mice to model vaccines containing ovalbumin, alpha casein or dephosphorylated alpha casein adsorbed to aluminum hydroxide adjuvant was studied. Ovalbumin and dephosphorylated alpha casein were adsorbed in the vaccine but were completely eluted when exposed to interstitial fluid for 4 h. The presence of aluminum hydroxide adjuvant in the vaccine produced immunopotentiation compared to a solution of the protein even though the protein desorbed rapidly upon subcutaneous administration. In contrast, alpha casein was completely adsorbed to aluminum hydroxide adjuvant in both the vaccine and upon exposure to interstitial fluid. Immunopotentiation by aluminum hydroxide adjuvant was also observed in this model vaccine compared to a solution of alpha casein. The results indicated that antigen presenting cells can take up desorbed antigen from interstitial fluid as well as antigen adsorbed to aluminum-containing adjuvants.

Adjuvants, Immunologic↗

PubMed2003

Effect of the degree of phosphate substitution in aluminum hydroxide adjuvant on the adsorption of phosphorylated proteins.

Aluminum hydroxide adjuvant was pretreated with six concentrations of potassium dihydrogen phosphate to produce a series of adjuvants with various degrees of phosphate substitution for surface hydroxyl. The adsorption of three phosphorylated proteins (alpha casein, dephosphorylated alpha casein, and ovalbumin) by the phosphate-treated aluminum hydroxide adjuvants was studied. The phosphorylated proteins were adsorbed by ligand exchange of phosphate for hydroxyl even when an electrostatic repulsive force was present. However, the extent (adsorptive capacity) and strength (adsorptive coefficient) of adsorption was inversely related to the degree of phosphate substitution of the aluminum hydroxide adjuvant. Exposure of vaccines containing aluminum hydroxide adjuvant and phosphorylated antigens to phosphate ion in the formulation or during manufacture should be minimized to produce maximum adsorption of the antigen.

Adjuvants, Pharmaceutic↗

PubMed2003

Effect of steam sterilization on the rheology of polymer solutions.

The effect of steam sterilization (dwell@121 degrees C) on the apparent viscosity of solutions of carbomer 940P, guar gum, hydroxyethylcellulose (HEC), and xanthan gum was studied. Guar gum and carbomer 940P could be steam sterilized for 30 min without any change in the apparent viscosity. Steam sterilization of HEC and xanthan gum resulted in a substantial decrease in the apparent viscosity and the desirable shear-thinning rheology. The addition of sodium chloride to either polymer prior to steam sterilization diminished the effect of sterilization on the rheology. The apparent viscosity of xanthan solutions was not significantly affected if the solution was made isotonic with sodium chloride (0.15 M). The addition of sodium chloride to xanthan gum after steam sterilization resulted in full recovery of the initial rheological properties. Carbomers, guar gum, and xanthan (in 0.15 M NaCI) are candidates for use in pharmaceutical solutions/suspensions that are subjected to steam sterilization.

Pharmaceutical Solutions↗

PubMed2003

Using rate of acid neutralization to characterize aluminum phosphate adjuvant.

Five aluminum phosphate adjuvants having P/Al molar ratios ranging from 0.74 to 0.26 were prepared. The adjuvants were characterized by both protein adsorptive capacity and rate of acid neutralization at pH 2.25, 25 degrees C. The protein adsorptive capacity was not a useful parameter to compare the initial properties of the adjuvants, as differences in surface charge of the adjuvants required the use of different proteins. In contrast, the rate of acid neutralization allowed a comparison of the freshly precipitated adjuvants and revealed that the rate of acid neutralization was directly related to the P/Al molar ratio. The protein adsorptive capacity decreased slightly during 39 weeks of aging at room temperature. The changes in the rate of acid neutralization were much greater and indicated that a P/Al molar ratio of at least 0.5 was required to minimize the aging of the adjuvants. Thus, the rate of acid neutralization was found to be the most sensitive parameter to characterize aluminum phosphate adjuvants.

Adjuvants, Immunologic↗

PubMed2002

Elimination of aluminum adjuvants.

In vitro dissolution experiments although perhaps not at typical body concentrations and temperatures demonstrated that the alpha-hydroxycarboxylic acids present in interstitial fluid (citric acid, lactic acid, and malic acid) are capable of dissolving aluminum-containing adjuvants. Amorphous aluminum phosphate adjuvant dissolved more rapidly than crystalline aluminum hydroxide adjuvant. Intramuscular administration in New Zealand White rabbits of aluminum phosphate and aluminum hydroxide adjuvants, which were labelled with 26Al, revealed that 26Al was present in the first blood sample (1 h) for both adjuvants. The area under the blood level curve for 28 days indicated that three times more aluminum was absorbed from aluminum phosphate adjuvant than aluminum hydroxide adjuvant. In vivo studies using 26Al-labelled adjuvants are relatively safe because accelerator mass spectrometry (AMS) can quantify quantities of 26Al as small as 10(-17) g. A similar study in humans would require a whole-body exposure of 0.7 microSv per year compared to the natural background exposure of 3000 microSv per year. The in vitro dissolution and in vivo absorption studies indicate that aluminum-containing adjuvants which are administered intramuscularly are dissolved by alpha-hydroxycarboxylic acids in interstitial fluid, absorbed into the blood, distributed to tissues, and eliminated in the urine.

Adjuvants, Immunologic↗

PubMed2002

The effect of surface charge and partition coefficient on the chemical stability of solutes in O/W emulsions.

Methylparaben (MP) was the model solute used to study the effect of surface charge on the rate of degradation in oil-in-water emulsions. The surface charge was varied by adding small amounts of phosphatidylglycerol (anionic) or stearylamine (cationic) to a standard intravenous lipid emulsion stabilized by egg phospholipid. The rates of hydrolysis at pH 8.0 in the water phase, oil phase, interface, and aqueous micellar phase were determined by application of a four-phase kinetic model. The rate of hydrolysis in the aqueous phase was dependent on the zeta potential. This was attributed to the effect of surface charge on the pH of the microenvironment of the oil drops through the phenomena known as surface acidity. MP in the aqueous phase hydrolyzed at a rate associated with the pH of the microenvironment, not the pH of the bulk. The effect of the partition coefficient of the solute was studied by substituting ethylparaben (EP), propylparaben (PP), and butylparaben (BP) for MP in the emulsions used to study the effect of surface charge. The rate of hydrolysis was inversely related to the partition coefficient. The effect of surface charge on the rate of hydrolysis was evident in the emulsions containing MP and EP. Partitioning had the greatest effect on the emulsions containing PP and BP. In general, the effect of surface charge predominated when the partition coefficient was small. The partition coefficient had a greater effect than surface charge when the partition coefficient was large.

Drug Stability↗

PubMed2002

Measuring the surface area of aluminum hydroxide adjuvant.

The traditional method of determining surface area, nitrogen gas sorption, requires complete drying of the sample prior to analysis. This technique is not suitable for aluminum hydroxide adjuvant because it is composed of submicron, fibrous particles that agglomerate irreversibly upon complete removal of water. In this study, the surface area of a commercial aluminum hydroxide adjuvant was determined by a gravimetric/FTIR method that measures the water adsorption capacity. This technique does not require complete drying of the adjuvant. Five replicate determinations gave a mean surface area of 514 m(2)/g and a 95% confidence interval of 36 m(2)/g for a commercial aluminum hydroxide adjuvant. The X-ray diffraction pattern and the Scherrer equation were used to calculate the dimensions of the primary crystallites. The average calculated dimensions were 4.5 x 2.2 x 10 nm. Based on these dimensions, the mean calculated surface area of the commercial aluminum hydroxide adjuvant was 509 m(2)/g, and the 95% confidential interval was 30 m(2)/g. The close agreement between the two surface area values indicates that either method may be used to determine the surface area of aluminum hydroxide adjuvant. The high surface area, which was determined by two methods, is an important property of aluminum hydroxide adjuvants, and is the basis for the intrinsically high protein adsorption capacity.

Adjuvants, Pharmaceutic↗

PubMed2002

Effect of thermal treatment during the preparation of aluminum hydroxide adjuvant on the protein adsorption capacity during aging.

Six aluminum hydroxide adjuvants, poorly crystalline aluminum oxyhydroxide (AlOOH) were prepared using different thermal treatments of amorphous aluminum hydroxide (Al(OH)3) in an effort to increase the protein adsorption capacity. All of the adjuvants initially exhibited a higher protein adsorption capacity. However, the protein adsorption capacity decreased during aging at room temperature. X-ray and differential centrifugal sedimentation analysis revealed that complete dehydration of amorphous aluminum hydroxide to aluminum oxyhydroxide is required to produce a stable adjuvant. Any residual amorphous aluminum hydroxide will spontaneously transform to crystalline aluminum hydroxide during aging at room temperature. Since crystalline aluminum hydroxide has a small surface area, the protein adsorption capacity of adjuvants containing amorphous aluminum hydroxide decreased by 30-40% when stored for 6 months at room temperature.

Adjuvants, Pharmaceutic↗