Selective imaging of active pharmaceutical ingredients in powdered blends with common excipients utilizing two-photon excited ultraviolet-fluorescence and ultraviolet-second order nonlinear optical imaging of chiral crystals.

Second order nonlinear optical imaging of chiral crystals (SONICC) and two-photon excited fluorescence measurements [both autofluorescence and two-photon excited UV-fluorescence (TPE-UVF)] were assessed for the selective detection of APIs relative to common pharmaceutical excipients. Active pharmaceutical ingredients (APIs) compose only a small percentage of most tabulated formulations, yet the API distribution within the tablet can affect drug release and tablet stability. Complementary measurements using either UV-SONICC (266 nm detection) or TPE-UVF were shown to generate signals >50-fold more intense for a model API (griseofulvin) than those produced by common pharmaceutical excipients. The combined product of the measurements produced signals >10(4)-fold greater than the excipients studied. UV-SONICC or TPE-UVF produced greater selectivity than analogous measurements with visible-light detection, attributed to the presence of aromatic moieties within the API exhibiting strong one and two photon absorption at ~266 nm. Complementary SONICC and fluorescence measurements allowed for the sensitive detection of the three-dimensional distribution of tadalafil within a Cialis tablet to a depth of >140 µm.

Single particle nonlinear optical imaging of trace crystallinity in an organic powder.

Microscopic characterization of crystallinity in powders can reveal information lost in ensemble-averaged measurements. Nonlinear optical imaging based on second harmonic generation (SHG) provides rapid and highly selective detection of individual chiral microcrystals, enabling insights into the fundamental mechanism of action for the observed crystallinity loss of an organic powder induced by mechanical grinding. Using griseofulvin as the model compound, the results from second order nonlinear optical imaging of chiral crystals (SONICC) compared favorably with those of powder X-ray diffraction (PXRD) over the linear dynamic range of the PXRD measurements. However, the SHG measurements demonstrated three decade improvements in linear dynamic range. The detection limit of SHG was estimated to be 4 ppm crystallinity in the powder. The rate of crystallinity loss induced by milling followed a first order process with a half-life of 15 ± 1 min. Recrystallization of cryomilled powder is ~40 times faster than that prepared by melt-quenched powder, suggesting that the disordered state obtained by exhaustive cryomilling appears to contain ordered domains that are larger than the critical nucleation size, but below the detection limit of SONICC. The presence of such domains provides a barrier-less nucleation source resulting in rapid crystallization, the kinetics of which depends only on crystal growth.

Interaction of environmental moisture with powdered green tea formulations: effect on catechin chemical stability.

Green tea and tea catechins must be stable in finished products to deliver health benefits; however, they may be adversely affected by tea processing/storage conditions and the presence of other components. The objective of this study was to determine the effects of storage relative humidity (RH) and addition of other ingredients on catechin stability in simulated dry beverage mixtures. Samples of green tea powder alone and mixed with sucrose, citric acid, and/or ascorbic acid were prepared and stored in desiccators at 22 degrees C and 0-85% RH for up to 3 months. Epicatechin, epigallocatechin, epicatechin gallate, and epigallocatechin gallate were determined by high-performance liquid chromatography (HPLC). Formulation and the interaction of formulation and RH significantly promoted catechin degradation ( P < 0.0001). The chemical degradation of total and individual catechins in green tea powder formulations was significantly increased ( P < 0.0001) by exposure to increasing RH, and the degradation was exacerbated at > or = 58% RH by the presence of powdered citric acid and at > or = 75% RH by the presence of ascorbic acid. Catechins degraded the most in formulations containing both acids. Although catechin chemical stability was maintained at < or = 43% RH in all samples stored at 22 degrees C for 3 months, caking was observed in samples at these relative humidities. These results are the first to demonstrate that addition of other dry components to tea powders may affect catechin stability in finished dry blends and highlight the importance of considering the complex interplay between a multicomponent system and its environment for developing stable products.

Fourier transform Raman spectroscopic study of the interaction of water vapor with amorphous polymers.

Water associated with amorphous polymers is known to affect their chemical and physical properties. The purpose of this study was to investigate the nature of water-polymer interactions for some polymers of pharmaceutical interest. Using Raman spectroscopy, polymer-water hydrogen bond interactions were probed for two molecular weight grades of poly(vinylpyrrolidone), namely PVP K90 and PVP K12, and also for poly(vinylacetate) and poly(vinyl pyrrolidone-co-vinyl acetate). Water vapor absorption isotherms were obtained for the polymers, and the effect of the absorbed water on the glass transition temperature was determined. A knowledge of the water content and physical state of the polymer was used to aid interpretation of Raman spectral changes. The strength of the hydrogen bond formed with water was found to depend on the chemistry of the polymer, with the pyrrolidone group interacting more strongly than the acetate group. However, minor differences were also observed between the degree of interaction of water and polymer for PVP K12 and PVP K90 at some water contents. This result is attributed to differences in the structural relaxation changes accompanying plasticization by water for the two molecular weight grades. Using principal components analysis of the spectral data, it was also possible to differentiate between samples in the rubbery state and samples in the glassy state. In conclusion, water sorbed into polymers causes changes in the polymer Raman spectra not only because of hydrogen bonding, but also as a result of the plasticizing effect of water on polymer mobility.

A comparison of alternative polymer excipients and processing methods for making solid dispersions of a poorly water soluble drug.

Solid dispersions were prepared with the extremely poorly water soluble drug, probucol and the water soluble polymers, polyvinyl pyrrolidone (PVP), polyacrylic acid (PAA) or polyethylene oxide (PEO) and blends of these polymers. The solid dispersions were prepared either by the solvent evaporation method, or by compression moulding into films. The materials were characterised by a combination of thermal analysis and FT-Raman spectroscopy. The physical state of the drug was observed to be dependent on the carrier, thus the PVP solid dispersions contained amorphous probucol, whilst the PAA and PEO systems contained the crystalline polymorph II. The method of production was not found to greatly influence the state of the drug in the solid dispersion. The greatest extent of release into solution was observed for the binary blend of drug and PEO, and the blending of polymers was not found to have any advantageous effects in this study.

Evaluation of solid-state forms present in tablets by Raman spectroscopy.

In this study the potential of Fourier transform (FT)-Raman spectroscopy as a method to probe the solid-state form of active substances present in tablets and capsules is explored. Raman spectra were obtained from intact tablets and capsules containing enalapril maleate, prednisolone, form I and form II polymorphs of ranitidine, anhydrous and monohydrate theophylline, and warfarin sodium clathrate. Spectra were also collected from the corresponding drug substances. These studies show that it is possible to detect the active ingredients in the intact dosage form, even where the substance comprises <1% of the total mass of the tablet. Moreover, it is shown that, in some cases, Raman spectroscopy can also be used to investigate the solid-state form of a drug present in the dosage form and even to determine if a mixture of forms are present.

Myeloid specific human CD33 is an inhibitory receptor with differential ITIM function in recruiting the phosphatases SHP-1 and SHP-2.

CD33 is a myeloid specific member of the sialic acid-binding receptor family and is expressed highly on myeloid progenitor cells but at much lower levels in differentiated cells. Human CD33 has two tyrosine residues in its cytoplasmic domain (Y340 and Y358). When phosphorylated, these tyrosines could function as docking sites for the phosphatases, SHP-1 and/or SHP-2, enabling CD33 to function as an inhibitory receptor. Here we demonstrate that CD33 is tyrosine phosphorylated in the presence of the phosphatase inhibitor, pervanadate, and recruits SHP-1 and SHP-2. Co-expression studies suggest that the Src-family kinase Lck is effective at phosphorylating Y340, but not Y358, suggesting that these residues may function in the selective recruitment of adapter molecules and have distinct functions. Further support for overlapping, but nonredundant, roles for Y340 and Y358 comes from peptide-binding studies that revealed the recruitment of both SHP-1 and SHP-2 to Y340 but only SHP-2 to Y358. Analysis using mutants of SHP-1 demonstrated that binding Y340 of CD33 was primarily to the amino Src homology-2 domain of SHP-1. The potential of CD33 to function as an inhibitory receptor was demonstrated by its ability to down-regulate CD64-induced calcium mobilization in U937. The dependence of this inhibition on SHP-1 was demonstrated by blocking CD33-mediated effects with dominant negative SHP-1. This result implies that CD33 is an inhibitory receptor and also that SHP-1 phosphatase has a significant role in mediating CD33 function. Further studies are essential to identify the receptor(s) that CD33 inhibits in vivo and its function in myeloid lineage development. (Blood. 2000;96:483-490)

Three-dimensional sonoelastography: principles and practices.

Sonoelastography is an ultrasound imaging technique where low amplitude, low-frequency shear waves (less than 0.1 mm displacement and less than 1 kHz frequency) are propagated through internal organs, while real-time Doppler techniques are used to image the resulting vibration pattern. When a discrete hard inhomogeneity, such as a tumour, is present within a region of soft tissue, a decrease in the vibration amplitude will occur at its location. This forms the basis for tumour detection using sonoelastography. For three-dimensional (3D) imaging the acquisition of sequential tomographic slices using this technique, combined with image segmentation, enables the reconstruction, quantification and visualization of tumour volumes. Sonoelastography and magnetic resonance images (MRI) of a tissue phantom containing a hard isoechoic inclusion are compared to evaluate the accuracy of this method. The tumour delineation from sonoelastography was found to have good agreement with the tumour from MRI except for a bleeding at one of its ends. Although sonoelastography is still in an experimental phase, the principles behind this imaging modality are explained and some practical aspects of acquiring sonoelastography images are described. Results from a 3D sonoelastography reconstruction of a tissue mimicking phantom and an ex vivo whole prostate specimen are presented.

Pathogen-specific loss of host resistance in mice lacking the IFN-gamma-inducible gene IGTP.

Interferon-gamma (IFN-gamma) is critical for defense against pathogens, but the molecules that mediate its antimicrobial responses are largely unknown. IGTP is the prototype for a family of IFN-gamma-regulated genes that encode 48-kDa GTP-binding proteins that localize to the endoplasmic reticulum. We have generated IGTP-deficient mice and found that, despite normal immune cell development and normal clearance of Listeria monocytogenes and cytomegalovirus infections, the mice displayed a profound loss of host resistance to acute infections of the protozoan parasite Toxoplasma gondii. By contrast, IFN-gamma receptor-deficient mice have increased susceptibility to all three pathogens. Thus, IGTP defines an IFN-gamma-regulated pathway with a specialized role in antimicrobial resistance.

Paired inhibitory and activating receptor signals.

The immunological literature has become inundated with reports regarding paired inhibitory receptors. Paired inhibitory receptor systems are highly conserved families that contain receptors involved in either cellular inhibition or activation. In most cases the paired putative biochemical antagonists are co-expressed on a given cell and thought to bind similar, if not identical, ligands making their biological role difficult to understand. Examples of these systems include immunoglobulin (Ig)-like receptors (Killer Ig Receptors, Immunoglobulin-like Transcripts/Leukocyte Ig-like Receptors/Monocyte Macrophage Ig Receptors, and Paired Ig-like Receptors), and type II lectin-like receptor systems (NKG2 and Ly49). General characteristics of these inhibitory receptors include a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). The ITIM is phosphorylated upon engagement and recruits protein tyrosine phosphatases that dephosphorylate cellular substrates that would otherwise mediate activation. In contrast, the activating receptors of these pairs use charged residues within their transmembrane domains to associate with various signal transduction chains including the gamma chain of the receptor for the Fc portion of IgE, DAP12 or DAP10. Once phosphorylated, these chains direct the signal transduction cascade resulting in cellular activation. Here we review the signaling of several paired systems and present the current models for their signal transduction cascades.

Cloning and characterization of a novel activating Ly49 closely related to Ly49A.

The majority of the known Ly49 family members have been isolated from either C57BL/6 (B6) or BALB/c mice. Interestingly, the anti-Ly49 Ab reactivities observed in 129/J mice are different from those of B6 mice. Furthermore, immunoprecipitation of 129/J NK cell lysates with YE1/32 and YE1/48, Abs specific for the inhibitory Ly49A in B6, resulted in detection of the activation-associated DAP12 molecule. These results indicated a need for a more detailed study of this strain. Therefore, a cloning strategy was devised to isolate Ly49 cDNAs from 129/J mice. An immunoreceptor tyrosine-based inhibitory motif-containing, Ly49D-related clone was discovered that we have named Ly49O, and one immunoreceptor tyrosine-based inhibitory motif-lacking, Ly49A-related clone was discovered that we have named Ly49P. No anti-Ly49 mAb reacted with Ly49O, whereas the molecule encoded by the Ly49P cDNA was found to react with YE1/32 and YE1/48. Ly49P was found to associate with mouse DAP12, and Ab-mediated cross-linking of Ly49P resulted in mouse DAP12 phosphorylation and Ca2+ mobilization, indicating that Ly49P is a competent activation receptor. Ly49P, therefore, represents a novel member of the Ly49 activating receptor subfamily.

Functional association of FcepsilonRIgamma with arginine(632) of paired immunoglobulin-like receptor (PIR)-A3 in murine macrophages.

Paired immunoglobulin-like receptors (PIR) are expressed on B cells and macrophages and include inhibitory and putative activating receptors referred to as PIR-B and PIR-A, respectively. Although PIR-B's inhibitory pathway has been described, it is unknown whether PIR-A receptors can deliver activation signals to macrophages, and if so, through what mechanism. Here we use chimeric receptors to address the mechanisms of PIR-A signaling. Cotransfection of chimeric receptors comprised of the extracellular region of human CD4 and the transmembrane and cytoplasmic domains of murine PIR-A3 showed the ability of PIR-A3 to physically interact with the FcepsilonRIgamma chain in 293T cells. This interaction is dependent on Arg(632) within the PIR-A3 transmembrane domain. We also demonstrate PIR-A3 interaction with the endogenous FcepsilonRIgamma of the ANA-1 macrophage cell line, again in an Arg(632)-dependent manner. Furthermore, we show that crosslinking of these chimeric receptors synergizes with IFN-gamma in the production of nitric oxide. Our data are the first to show the potential of PIR-A3 to deliver activation signals to macrophages and establish its dependence on Arg(632). These findings suggest that further study of the PIR-A receptors should be aggressively pursued toward a complete understanding of the intricate regulation of macrophage biology.

Experiences of ethnic minority primary care physicians with managed care: a national survey.

OBJECTIVES: To determine if ethnic minority physicians experience more barriers in acquiring and maintaining managed care contracts than white physicians, and to determine if the physician's perceptions of his or her ability to provide appropriate care to patients varies with physician ethnicity. STUDY DESIGN: Using a national sample, we identified 4 research areas germane to this topic and analyzed them by physician ethnic group. METHODS: Analysis involved a pre-existing data set from a national survey that employed a random sampling approach to achieve reasonably accurate national population estimates with acceptable margins of error (95% CI = +/- 2). RESULTS: A total of 1032 primary care physicians completed the survey (response rate of 48%). After controlling for confounding variables, we found that Asian physicians have the most difficulty keeping managed care contracts. Type of practice varies with physician ethnicity, and solo practitioners have more problems securing contracts than physicians in other types of practices. Board-certified physicians are more likely to have managed care contracts than those who are not. Latino physicians have significantly fewer managed care patients than primary care physicians who are white, African American, or Asian. The perceptions of the physicians of their ability to deliver appropriate care overall did not vary by ethnicity, but 2 major subcategories of this item did vary by physician ethnicity: quality of care, and limitations to providing care. CONCLUSIONS: Although we did not find overwhelming evidence of discrimination against ethnic minority physicians, differences in rates of termination, type of practice, board certification rates, and managed care affiliation were related to physician ethnicity.

DAP12-mediated signal transduction in natural killer cells. A dominant role for the Syk protein-tyrosine kinase.

The murine Ly49 family contains nine genes in two subgroups: the inhibitory receptors (Ly49A, B, C, E, F, G2, and I) and the noninhibitory receptors (Ly49D and H). Unlike their inhibitory counterparts, Ly49D and H do not contain immunoreceptor tyrosine-based inhibitory motifs but associate with a recently described co-receptor, DAP12, to transmit positive signals to natural killer (NK) cells. DAP12 is also expressed in myeloid cells, but the receptors coupled to it there are unknown. Here we document the signaling pathways of the Ly49D/DAP12 complex in NK cells. We show that ligation of Ly49D results in 1) tyrosine phosphorylation of several substrates, including phospholipase Cgamma1, Cbl, and p44/p42 mitogen-activated protein kinase, and 2) calcium mobilization. Moreover, we demonstrate that although human DAP12 reportedly binds the SH2 domains of both Syk and Zap-70, ligation of Ly49D leads to activation of Syk but not Zap-70. Consistent with this observation, Ly49D/DAP12-mediated calcium mobilization is blocked by dominant negative Syk but not by catalytically inactive Zap-70. These data demonstrate the dependence of DAP12-coupled receptors on Syk and suggest that the outcome of Ly49D/DAP12 engagement will be regulated by Cbl and culminate in the activation of transcription factors.

Particle size dependent molecular rearrangements during the dehydration of trehalose dihydrate in situ FT-Raman spectroscopy.

PURPOSE: (1) To characterise the different phases of trehalose using FT-Raman spectroscopy. (2) To monitor the changes in the structure of trehalose dihydrate on isothermal heating at 80 degrees C. METHODS: Different phases of trehalose were prepared and FT-Raman spectra obtained. Trehalose dihydrate was sieved to < 45 microns and > 425 microns particle size fractions and FT-Raman spectra were obtained at various time intervals during heating at 80 degrees C. RESULTS: During heating at this temperature, the spectra of a < 45 microns particle size fraction showed a loss of peak resolution with time and after 210 minutes resembled the spectrum of amorphous trehalose prepared by lyophilisation, indicating that the material was rendered amorphous by heating. In contrast, spectra obtained from a > 425 micron particle size fraction altered with time and became characteristic of the crystalline anhydrate. The approximate kinetics of this transformation to the anhydrate were monitored by analysis of peak intensity ratios with time. A two state rearrangement was indicated; some functional groups appeared to manoeuvre into the spatial arrangement found in the anhydrate initially before the rest of the ring structure relaxed into this conformation. This may be due to some parts of the molecule being immediately affected by the loss of the water molecules on dehydration prior to the subsequent reorientation of the entire molecule into the anhydrate crystal lattice. CONCLUSIONS: The < 45 micron particle size fraction becomes disordered on dehydration induced by heating at 80 degrees C whilst the > 425 micron particle size fraction crystallises to the anhydrate under the same conditions.

Mixing behavior of colyophilized binary systems.

The purpose of this study was to investigate the factors which govern the mixing of amorphous sucrose with trehalose, poly(vinylpyrrolidone) (PVP), dextran, and poly(vinylpyrrolidone-co-vinyl acetate) (PVP/VA). These materials were chosen as model systems to represent multicomponent freeze-dried pharmaceutical preparations. Mixtures were prepared by colyophilization of the components from aqueous solutions. The glass transition temperatures (Tg) of these mixtures were measured using differential scanning calorimetry (DSC) and were compared to predictions based on simple mixing rules. FT-Raman spectroscopy was used to probe selected mixtures for evidence of molecular interactions between components. Colyophilized mixtures were confirmed to be amorphous by X-ray powder diffraction. The Tg values of the various mixtures generally were lower than values predicted from free volume and thermodynamic models, indicating that mixing is not ideal. The FT-Raman spectra of colyophilized sucrose-PVP and sucrose-PVP/VA mixtures provided evidence for interaction between the components through hydrogen bonding. Hydrogen bonds formed between components in colyophilized sucrose-additive mixtures are formed at the expense of hydrogen bonds within sucrose and in some cases within the additive. A thermodynamic analysis of these mixtures indicates that mixing is endothermic, which is consistent with a net loss in the degree of hydrogen bonding on mixing. There is also a positive excess entropy of mixing which accompanies the net loss in hydrogen bonds. Despite this gain in excess entropy, the excess free energy of mixing is positive, consistent with the observed deviations in Tg from values predicted using models which assume ideal mixing.

The quantitative analysis of crystallinity using FT-Raman spectroscopy.

PURPOSE: To establish if FT-Raman spectroscopy can be used to quantitate the degree of crystallinity in a model compound. METHODS: Mixtures containing different proportions of amorphous and crystalline indomethacin were prepared. Using the peak intensity ratio 1698 cm(-1) (crystalline) to 1680 cm(-1) (amorphous), a correlation curve was prepared. This correlation curve was validated by testing further samples of known composition. Partially crystalline indomethacin was prepared by milling crystalline indomethacin. RESULTS: A linear correlation curve was obtained across the entire range of 0-100% crystallinity. Using this method, it was possible to detect down to either 1% amorphous or crystalline content. The largest errors were found to result from inhomogeneities in the mixing of the calibration and validation samples. The spectra of the mechanically processed samples were similar to the spectra of the calibration samples, and the degree of crystallinity could be estimated in these samples. CONCLUSIONS: FT-Raman spectroscopy is a potentially useful method to complement existing techniques for the quantitative determination of crystallinity.

Characterization of the phase transitions of trehalose dihydrate on heating and subsequent dehydration.

Many pharmaceutical compounds of interest form hydrates. The phase behavior of different particle size fractions of trehalose dihydrate was studied as the sugar was dehydrated by heating. Hot-stage microscopy, X-ray powder diffraction, thermogravimetric analysis, and differential scanning calorimetry were used to characterize the phase changes. Small particles (<45 microm) formed an amorphous phase on dehydration and subsequently liquefied at temperatures above the glass transition temperature of amorphous trehalose. Crystallization to the anhydrate was observed from this supercooled liquid. Large particles (>425 microm) underwent a solid-solid conversion from the dihydrate to the anhydrate at temperatures as low as 80 degrees C. This solid-solid conversion was explained by a catalytic effect of the liberated dihydrate water on the rearrangement of the dehydrated phase to the anhydrate. The large surface area-to-volume ratio of the small particles resulted in dehydration prior to attaining the threshold temperature for rearrangement, explaining why solid-solid conversion was absent for these particles.

Sugar-polymer hydrogen bond interactions in lyophilized amorphous mixtures.

The objective of this work was to investigate hydrogen bonding interactions between a variety of glass-forming sugars and a model polymer, poly(vinylpyrrolidone) (PVP), in binary amorphous solid solutions, produced by lyophilization. The glass transition temperatures of the sugars and sugar-PVP colyophilized mixtures were assessed using differential scanning calorimetry. The hydrogen bonding interactions between each sugar and PVP were monitored using FT-Raman spectroscopy. Sucrose was found to hydrogen bond to a greater extent with PVP at a particular sugar:polymer ratio than the other disaccharides studied including trehalose and the trisaccharide raffinose. Maltodextrins showed a decreased tendency to hydrogen bond with the polymer compared to the lower molecular weight sugars. The extent of hydrogen bonding was found to correlate inversely with the glass transition temperature of the sugar, with the tendency to hydrogen bond decreasing as the Tg increased. The importance of hydrogen bonding interactions to the thermodynamics of mixing in amorphous solids is discussed.

Bryostatin-1 and IFN-gamma synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages.

Bryostatin-1 (Bryo) is a nontumor-promoting protein kinase C modulator that has been shown to have both in vitro and in vivo activity against several murine and human tumors. In this study, we investigated the effects of Bryo on nitric oxide production, measured as accumulated nitrite (NO2-) in culture supernatant, and inducible nitric oxide synthase (iNOS) gene expression in the murine macrophage cell line ANA-1. ANA-1 macrophages did not produce NO2- or iNOS mRNA constitutively, and very little or no NO2- or iNOS mRNA were detectable upon exposure to IFN-gamma. Bryo, although ineffective alone, and IFN-gamma synergized to produce high levels of NO2- and iNOS mRNA. The activity of Bryo was evident at a concentration of 0.1 ng/ml and reached its maximum at 1 ng/ml. The effects of Bryo were time dependent because expression of iNOS mRNA was detectable as early as 6 h and increased through 24 h. Analyses of the molecular mechanisms involved indicate that Bryo and IFN-gamma mainly regulate iNOS gene expression posttranscriptionally through stabilization of iNOS mRNA. Experiments designed to investigate the role of tumor necrosis factor alpha (TNF-alpha) in NO2- production by Bryo- and IFN-gamma-activated macrophages revealed that ANA-1 macrophages expressed low levels of TNF-alpha mRNA constitutively that were not augmented in the presence of IFN-gamma. However, Bryo alone augmented the TNF-alpha mRNA expression, which was only slightly increased with the addition of IFN-gamma. A polyclonal antibody to TNF-alpha was able to completely neutralize TNF-alpha secreted in either medium or Bryo plus IFN-gamma-treated cultures. Neutralizing concentrations of anti-TNF-alpha antibody suppressed the Bryo plus IFN-gamma-induced NO2- production approximately by 50%, suggesting that NO2- produced by Bryo plus IFN-gamma-treated ANA-1 macrophages may involve both TNF-alpha-dependent and TNF-alpha-independent mechanisms. Overall, these findings provide the first evidence that Bryo and IFN-gamma can synergize for the induction of NO2- production as well as iNOS gene expression and show the involvement of posttranscriptional mechanisms in the induction of iNOS mRNA.