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Recently, genotypic testing (the identification of viral mutations and their associated drug resistance) has be- come a popular procedure to identify drug resistanc e before advising alternative therapy regimens. Since major drug resistance factors are associated with the frequency of viral mutations, many researchers have explored HIV’s mutation frequency at specified nucl e- otide sequence positions in response to different t ypes of drug therapy. However, only a handful of papers dis- cuss major genetic signatures that lead to positive pa- tients’ responses to a specific type of drug therap y. Using existing clinical drug resistance libraries, we were able to determine the most common mutations in the HIV protease (PR) enzymes associated with the s uc- cess and failure of Protease Inhibitor (PI) HIV/AID S drug regimens. A total of 2,079 patient records sel ected from the Stanford HIV drug resistance database has been considered in identifying genetic sequences associa ted with positive responses to PR-inhibitors drug regim ens. We show that patients who responded positively to P ro- tease Inhibitor therapy have consistently maintaine d specific nucleic acids bases at specific positions of their HIV nucleotide sequences. When virus sequences ob- tained from groups of patients who did not respond well to PI therapy were compared against virus sequences at the same positions from patients who did respond we ll, we noticed that the two patient groups differ at th ese positions.

Motivation: We present a spatial-temporal (ST) human immunodeficiency virus (HIV) simulation model to investigate the spatial distribution of viral load and T-cells during HIV progression. The proposed model uses the Finite Element (FE) method to divide a considered infected region into interconnected subregions each containing viral population and T-cells. HIV T-cells and viral load are traced and counted within and between subregions to estimate their effect upon neighboring regions. The objective is to estimate overall ST changes of HIV progression and to study the ST therapeutic effect upon HIV dynamics in spatial and temporal domains. We introduce sub-regional (spatial) parameters of T-cells and viral load production and elimination to estimate the spatial propagation and interaction of HIV dynamics under the influence of a 3TC D4T Reverse Transcriptase Inhibitors (RTI) drug regimen. Results: In terms of percentage change standard deviation, we show that the average rate per 10 weeks (throughout a 10-year clinical trial) of the ST CD4+ change is 5.35% 1.3 different than that of the CD4+ rate of change in laboratory datasets, and the average rate of change of the ST CD8+ is 4.98% 1.93 different than that of the CD8+ rate of change. The half-life of the ST CD4+ count is 1.68% 3.381 different than the actual half-life of the CD4+ count obtained from laboratory datasets. The distribution of the viral load and T-cells in a considered region tends to cluster during the HIV progression once a threshold of 86–89% viral accumulation is reached. Availability: Executable code and data libraries are available by contacting the corresponding author. Implementation: C++ and Java have been used to simulate the suggested model. A Pentium III or higher platform is recommended. Contact: george.towfic@clarke.edu Supplementary information: Supplementary data are available at Bioinformatics online.

Background Allergen-mediated mast cell activation is a key feature of ocular allergic diseases. Evidence of eosinophil-derived mediators in tears and conjunctival biopsy specimens has been associated with chronic ocular allergic inflammation. Objective To examine the role of conjunctival mast cell mediators in eosinophil adhesion to conjunctival epithelial cells and eosinophil degranulation. Methods Conjunctival cells were obtained by enzymatic digestion of cadaveric conjunctival tissues. Eosinophils were obtained from peripheral blood samples using negative magnetic bead selection. The effect of IgE-activated mast cell supernates on eosinophil degranulation and adherence to epithelial cells was compared with supernates obtained from mast cells pretreated with a degranulation inhibitor (olopatadine). Eosinophil adhesion was measured by eosinophil peroxidase assay, and eosinophil degranulation was measured by eosinophil-derived neurotoxin radioimmunoassay. Results IgE-activated conjunctival mast cell supernates stimulated adhesion of eosinophils to epithelial cells (20.4% ± 6.3% vs 3.1% ± 1.0%; P = .048). Degranulation was not required for this process (no effect of olopatadine). IgE-activated mast cell supernates stimulated eosinophil-derived neurotoxin release (108.89 ± 8.27 ng/10 6 cells vs 79.45 ± 5.21 ng/10 6 cells for controls, P = .02), which was significantly inhibited by pretreatment of mast cells with a degranulation inhibitor (79.22 ± 4.33 ng/10 6 cells vs 61.09 ± 5.39 ng/10 6 cells for olopatadine pretreated and untreated, respectively, P = .02). Conclusions Mediators released from conjunctival mast cells promote eosinophil adhesion to conjunctival epithelial cells and eosinophil degranulation. Degranulation inhibition studies suggest that different mast cell mediators are involved in regulation of these events.

Allergic eye disease is associated with IgE-mediated conjunctival inflammation leading to signs of immediate hypersensitivity, including redness, itching, and tearing. Pathologic studies using conjunctival mast cells demonstrate that these cells, when sensitized with IgE antibody and exposed to environmental allergens, release mediators involved with allergic inflammation. The type, release kinetics, and concentration of these mediators in the conjunctiva have not been completely characterized. The ability to isolate and purify mast cells and epithelial cells from human conjunctival tissue has permitted the study of mediator release and cell-to-cell signaling in this tissue. Our laboratory has developed in vitro and in vivo models to better understand how inflammatory cells are recruited to and infiltrate conjunctival tissues. These models demonstrate that mast-cell activation may supply sufficient cytokine signaling to initiate and direct the well-orchestrated trafficking of eosinophils to the ocular surface, facilitate their adhesion, and cause release of potent mediators of ocular inflammation.

Allergic eye disease is a common clinical problem adversely affecting the quality of life for millions of sufferers. This ocular process is associated with IgE-mediated conjunctival inflammation leading to signs of immediate hypersensitivity including redness, itching, and tearing. Pathologic studies have shown that the conjunctiva contains mast cells that when sensitized with IgE antibody and exposed to environmental allergens can release mediators of allergic inflammation. The type, release kinetics, and concentration of these mediators in the conjunctiva have not been completely characterized. The ability to isolate and purify mast cells and epithelial cells from human conjunctival tissue has permitted the study of mediator release and cell-to-cell signaling in this tissue. Our laboratory has developed in vitro and in vivo models to better understand how inflammatory cells are recruited to and infiltrate conjunctival tissues. These models demonstrate that mast cell activation may supply sufficient cytokine signaling to initiate and direct the well-orchestrated trafficking of eosinophils to the ocular surface, facilitate their adhesion, and cause release of potent mediators of ocular inflammation.

Background Tumor necrosis factor-α (TNFα) release likely plays a crucial role in allergic ocular inflammation via increasing ICAM-1 on epithelial cells and triggering other proinflammatory events. The immediate and prolonged release of TNFα from human conjunctival mast cells in response to allergen challenge is potentially an important target for therapeutic intervention, yet the effect of ocular anti-allergic agents on this process has not been examined. Olopatadine (Patanol) is a clinically effective dual-action ophthalmic anti-allergic agent that has been shown to inhibit mast cell histamine, tryptase, and PGD 2 release in vitro and promote decreased H 1 receptor binding activity in vitro and functional H 1 receptor antagonism in vivo. Objective To investigate the effect of olopatadine on TNFα release from anti-IgE antibody challenged purified human conjunctival mast cells. Methods Human conjunctival mast cells were purified (>95%) from cadaveric tissues using a procedure combining enzymatic digestion and Percoll gradient centrifugation. These cells were incubated with olopatadine for 30 minutes then challenged with anti-IgE antibody for 90 minutes. Supernatants were analyzed for TNFα. Results Purified human conjunctival mast cells responded to anti-IgE antibody challenge with TNFα release in a concentration dependent manner (optimum concentration was 10 μg/mL). Olopatadine pre-incubation resulted in a dose-dependent decrease in anti-IgE antibody mediated TNFα release (IC 50 = 13.1 μM). At a concentration of 3 mM olopatadine reduced TNFα release to the level of unchallenged controls. Conclusion Olopatadine inhibited anti-IgE antibody-mediated release of TNFα from human conjunctival mast cells. This effect could contribute to the long duration of anti-allergic activity reported for the drug.

Over the past several years, a number of cytokines with chemoattractive properties (chemokines) have been identified. These low molecular weight molecules have been shown to be important leukocyte chemical attractants to sites of inflammation and infection. Chemokines act on leukocytes through selective receptors and are now known to function also in leukocyte maturation, trafficking, and homing of these cells. RANTES and eotaxin (among other chemokines) are important chemoattractants for eosinophils. Since eosinophils seem to play a critical role in the production of allergic inflammation, an understanding of the mechanism of action of these chemokines may lead to new therapies for asthma and other allergic processes.

Trimellitic anhydride (TMA) is a small molecular weight chemical used in the paint and plastics industry that can cause asthma-like symptoms in humans. Guinea pigs sensitized intradermally with TMA will respond to antigen challenge with asthma-like symptoms, including an immediate bronchoconstriction and a delayed cellular infiltration into the lung, particularly eosinophil infiltration. Sensitized guinea pigs produce TMA-specific IgG1, which is thought to be important in asthmatic reactions in this animal model; however, they also produce TMA-specific IgG2 antibody. The purpose of the present study was to determine the role of IgG1 and IgG2 in the TMA-induced immediate bronchoconstriction and delayed cellular infiltration in the guinea pig. Guinea pigs were passively sensitized by intratracheal instillation of TMA-specific IgG2, an antibody preparation enriched with TMA-specific IgG1, or a combination of the two. The allergic response was induced by intratracheal instillation of TMA conjugated to guinea pig serum albumin (TMA-GPSA). A significantly greater bronchoconstrictor response was observed in animals sensitized with a combination of the IgG2 and IgG1 preparation compared to those sensitized with IgG2 or the IgG1 preparation alone. Cellular infiltration was quantified 24 h after antigen challenge by differential cell counts of bronchoalveolar lavage (BAL) cells as well as by using eosinophil peroxidase (EPO) and myeloperoxidase (MPO) activity as a measure of the numbers of eosinophils and neutrophils, respectively. In the BAL, passively sensitizing with IgG2 alone resulted in an increase in both TMA-induced MPO and EPO activity. In contrast, in the lung, passively sensitizing with a partially purified preparation of TMA-specific IgG1 alone resulted in a significant increase in TMA-induced EPO activity. Passively sensitizing with IgG2 in conjunction with the IgG1 preparation resulted in an enhanced cellular infiltration and lung injury over that seen with either antibody preparation alone. These data demonstrate an augmentation of IgG1-mediated responses by the addition of IgG2 and suggest a significant role for both subclasses of IgG antibodies in this guinea pig model of TMA-induced occupational asthma.

Eotaxin is the major eosinophil chemoattractant found in bronchoalveolar lavage (BAL) fluid from sensitized guinea pigs after antigen challenge. In this study we have performed immunostaining for eotaxin in airways obtained from challenged animals and examined purified guinea pig lung cells (epithelial cells > 98% purity, mast cells > 90% purity) for eotaxin mRNA and protein. In the airways of antigen (ovalbumin) challenged animals, significant amounts of epithelial cell eotaxin immunostaining were observed. Northern analysis of total RNA obtained from unchallenged, freshly isolated airway epithelial cells contained high levels of eotaxin mRNA. Semi-pure and high purity lung mast cell preparations (challenged or unchallenged) did not express eotaxin mRNA. Western analysis of supernatant fluids obtained from incubated airway epithelial cells demonstrated detectable amounts of eotaxin protein, with the majority of the protein being cell-associated. Thus, airway epithelial cells are identified as a major cellular source of eotaxin in the guinea pig pulmonary system.

Summary Purified pulmonary mast cells were obtained from bovine lung using a combination of enzymatic digestion of tissue, density gradient centrifugation using Percoll, and centrifugal elutriation. In the initial procedure, lung tissue was enzymatically digested with collagenase, hyaluronidase, protease and elastase in three 30 min incubations at 37 °C. Monodispersed cell suspensions contained between 2 and 6 % mast cells. Further purification of these mast cells by Percoll gradients and elutriation consistently yielded mast cells of >90 % purity. These cells were morphologically intact, viable and functional, as determined by histamine release evoked by secretagogue challenge. Incubation of BLMCs with Pasteurella haemolytica A1 culture supernate containing leucotoxin (LCT) alone, resulted in increased histamine release compared to controls. LCT also potentiated calcium ionophore (Cal)-induced histamine release from BLMCs.

Human conjunctiva was enzymatically digested into monodispersed cell preparations using collagenase and hyaluronidase. The preparations were enriched for mast cells using Percoll gradient centrifugation prior to challenge with various secretagogues. Mast cell percentage (9.7 ± 2.9%) and viability (96 ± 3%) were determined using toluidine blue staining and vital dye exclusion, respectively. The mast cell enriched preparations were challenged with calcium ionophore A(23187') compound 48/80, concanavalin A, anti-IgE, morphine and substance P. Mediator release was quantified by radioimmunoassay. The calcium ionophore A(23187), compound 48/80, concanavalin A and anti-IgE stimulated histamine release from the conjunctival mast cell preparations, with EC(30) values of 0.028, 62.5, 24.4 and 1.9 ϵg/ml, respectively. Morphine but not substance P challenge induced significant but low levels of histamine release. Tryptase, sulfidopeptide leukotriene, and prostaglandin D(2) levels in cell supernatants were also increased following stimulation. The profile of mediator release observed with these cells compared to that reported from human lung and human skin mast cells suggest that human conjunctival mast cells may have unique biological responses.

The relative contributions of mucosal/submucosal and detrusor layers to the release of inflammatory mediators were investigated in isolated, ovalbumin (OVA) sensitized guinea pig urinary bladders. Ovalbumin challenge of sensitized mucosa induced release of prostaglandins (PG): PGE2, PGD2 and PGF2 alpha, in that order of magnitude. The total release of PGs was significantly higher from the mucosa/submucosa than from the detrusor/serosa. Under the same conditions, net release of leukotriene (LT) was observed predominantly from the detrusor. The total amount of histamine released from the mucosa was greater than that from the muscle layer. These results indicate differential production and release of inflammatory mediators from the mucosal/submucosal and detrusor smooth muscle layers. These results may have serious implications in disorders, such as interstitial cystitis, involving bladder mucosal damage. The cytoprotective effect of PGE2 is likely to be lost when the mucosa is damaged, and LT release from deeper layers may contribute significantly to symptoms of bladder inflammation.

Mast cells were isolated by enzymatic digestion of synovium obtained from 48 patients with rheumatoid arthritis (RA) and 42 patients with osteoarthritis (OA). A significantly lower percentage of stainable synovial mast cells was obtained by tissue digestion from patients with clinically active RA compared with those with less active disease. The 54 patients treated with nonsteroidal antiinflammatory drugs had a significantly lower percentage of stainable synovial mast cells in cell suspension than did the other 36 patients. When anti-IgE antibody was used as a secretagogue in vitro, significantly greater histamine release was observed from synovial mast cells of RA patients compared with OA patients. Greater histamine release in response to anti-IgE was observed in the RA patients with more clinically active disease and those who were treated with prednisone, compared with RA patients without these features. Synovial mast cells of RA patients treated with a disease-modifying antirheumatic drug had a significantly lower mean histamine content than did cells from patients not receiving such treatment. Our data suggest that there are differences between synovial mast cells from tissues of patients with RA and OA and suggest that synovial mast cells may be activated in clinically active RA. In addition, the data indicate an effect of systemic antirheumatic therapy on mast cells isolated from synovium of patients with arthritis.

This study examined the possibility that there is cross-desensitization between immunologic and nonimmunologic stimuli that evoke contraction and histamine release (HR) in the isolated guinea pig trachea. Compound 48/80 and D-tubocurarine were found to cause homologous and heterologous desensitization for both contraction and HR from superfused trachea. Specific antigen challenge of trachea obtained from animals sensitized with either IgG1 (ovalbumin [OA]) or IgE (oxazalone-human serum albumin [OX-HSA]) also resulted in homologous desensitization for both contraction and HR. However, in experiments with animals sensitized with both IgG1 and IgE antibodies, prechallenge with OA resulted in cross-desensitization to OX-HSA, whereas the reverse sequence was ineffective in eliciting this phenomenon. This may be related to the type of desensitization produced by each antigen (specific versus nonspecific) or to heterogeneity of mast cells in the tissue. Prechallenge of the trachea with compound 48/80 or D-tubocurarine failed to alter subsequent effects of antigen after active sensitization with OA or passive sensitization with either IgG1 or IgE antibodies. Small but statistically significant decreases in tracheal responses to D-tubocurarine were observed after antigen prechallenge to active both IgG1 and IgE antibodies. This is the first study to demonstrate a cross-desensitization between compound 48/80 and D-tubocurarine and the first to examine cross-desensitization with IgG1 and IgE antibodies in the guinea pig trachea. The overall conclusion is that there is no major overlap in the desensitization mechanisms between immunologic and nonimmunologic stimuli in the guinea pig trachea.

Sulfamethoxazole (SMX) is an effective drug for the management of opportunistic infections, but its use is limited by hypersensitivity reactions, particularly in HIV-infected patients. The oxidative metabolite SMX-nitroso (SMX-NO), is thought to be a proximate mediator of SMX hypersensitivity, and can be reduced in vitro by ascorbate or glutathione. Leukocytes from patients with SMX hypersensitivity show enhanced cytotoxicity from SMX metabolites in vitro; this finding has been attributed to a possible "detoxification defect" in some individuals. The purpose of this study was to determine whether variability in endogenous ascorbate or glutathione could be associated with individual differences in SMX-NO cytotoxicity. Thirty HIV-positive patients and 23 healthy control subjects were studied. Both antioxidants were significantly correlated with the reduction of SMX-NO to its hydroxylamine, SMX-HA, by mononuclear leukocytes, and both were linearly depleted during reduction. Controlled ascorbate supplementation in three healthy subjects increased leukocyte ascorbate with no change in glutathione, and significantly enhanced SMX-NO reduction. Ascorbate supplementation also decreased SMX-NO cytotoxicity compared to pre-supplementation values. Rapid reduction of SMX-NO to SMX-HA was associated with enhanced direct cytotoxicity from SMX-NO. When forward oxidation of SMX-HA back to SMX-NO was driven by the superoxide dismutase mimetic, Tempol, SMX-NO cytotoxicity was increased, without enhancement of adduct formation. This suggests that SMX-NO cytotoxicity may be mediated, at least in part, by redox cycling between SMX-HA and SMX-NO. Overall, these data indicate that endogenous ascorbate and glutathione are important for the intracellular reduction of SMX-NO, a proposed mediator of SMX hypersensitivity, and that redox cycling of SMX-HA to SMX-NO may contribute to the cytotoxicity of these metabolites in vitro.

PURPOSE To gain better understanding of conjunctival epithelial cell responses to proinflammatory cytokines, the individual and combined effects of TNFalpha, IL-1beta, and IFNgamma on chemokine release (IL-8, regulated on activation normal T-cell expressed and secreted [RANTES]) and surface receptor expression (intercellular adhesion molecule [ICAM]-1, and HLA-DR, -DP, and -DQ) were examined. METHODS Conjunctival epithelial cells were isolated from cadaveric conjunctival tissues and cultured in 24-well plates until almost confluent. Recombinant cytokines (0.005-50 ng/mL) were added, alone or in various combinations, 24 hours before harvesting of supernates for ELISAs and cells for flow cytometry. RESULTS TNFalpha, IL-1beta, and IFNgamma had distinctive individual and combined effects on the parameters tested. Although TNFalpha and IL-1beta had similar and synergistic effects on increasing expression of ICAM-1, IL-1beta was a more potent upregulator of the release of IL-8 than was TNFalpha. Upregulation of IL-8 was additive when IL-1beta was combined with TNFalpha. Neither TNFalpha nor IL-1beta increased expression of HLA. In contrast, IFNgamma was a potent upregulator of both surface receptors (ICAM-1 and HLA) but IFNgamma alone had no effect on mediator release (IL-8 and RANTES). Release of RANTES required two cytokine signals, with IFNgamma and TNFalpha being the most potent combination. CONCLUSIONS Knowledge of the differential and combined effects of proinflammatory cytokines on conjunctival epithelial cells allows better understanding of ocular inflammation.

Tears play an essential role in maintaining corneal and conjunctival integrity by providing a tightly regulated, optimal extracellular environment critical to its numerous functions, which include anti-microbial defense, wound healing and inflammatory responses such as allergies. Elevated levels of inflammatory cytokines have been reported in tears from various ocular disease states. Characterization of tear cytokines has been limited by the small volume (microliter amounts) attainable. This limitation was addressed with the newly developed Becton Dickinson Cytometric Bead Array (CBA), which combines the principles of the "sandwich" immunoassay with the capability of flow cytometry for simultaneous measurement of the characteristics of multiple particles. This technique allows determination of six human cytokine (IFNgamma, TNFalpha, IL-2, IL-4, IL-5, IL-10) concentrations simultaneously in a single tear sample. Tears were collected from the inferior fornix of non-allergic (n=7) and allergic (n=9) donors. Each tear sample or cytokine standard was incubated with a mixture of capture Ab-bead reagent and detector Ab-phycoerythrin (PE) reagent, and analyzed using flow cytometry. All six cytokines were detectable in both non-allergic and allergic tears. Tears from allergic donors contained significantly less IL-10 (p=0.035), and had significant increases in the ratios of TNFalpha/IFNgamma, IL-5/IFNgamma and IL-5/IL-10 (p=0.0008, 0.0124 and 0.011, respectively). The small volume required (5-10 microl/test) by the Cytometric Bead Array allows measurement of all six cytokines from a single collection of tears. This decreases collection time, minimizing the confounding effect of stimulation on cytokine concentration in tears, as well as allowing calculation of cytokine ratios.

Evaluation of drug resistance by human immunodeficiency virus (HIV) genotyping has proven to be useful for the selection of drug combinations with maximum antiretroviral activity. We compared three genotyping methods for identification of mutations known to confer drug resistance in the reverse transcriptase (RT) and protease genes of HIV type 1 (HIV-1). The HIV-GenotypR method (GenotypR; Specialty Laboratories, Inc., Santa Monica, Calif.) with the ABI 377 DNA sequencer (Applied Biosystems Inc.), the HIV PRT GeneChip assay (GeneChip; Affymetrix, Santa Clara, Calif.), and the HIV-1 RT Line Probe Assay (LiPA; Innogenetics, Alpharetta, Ga.) were used to genotype plasma samples from HIV-infected patients attending the University of Wisconsin Hospitals and Clinics and the Mayo Clinic. At the time of analysis, patients were failing combination therapy ( n = 18) or were treatment naive ( n = 6). Forty codons of the RT and protease genes were analyzed by GenotypR and GeneChip for resistance-associated mutations. LiPA analyzed seven RT codons for mutations. Each sample was genotyped by all three assays, and each assay was subjected to pairwise comparisons. At least 92% of the codons tested (by the three assays) in paired comparisons were concordant. GenotypR and GeneChip demonstrated 96.6% concordance over the 40 codons tested. GenotypR identified slightly more mutations than GeneChip and LiPA; GeneChip identified all primary mutations that corresponded to failing treatment regimens. Each assay identified at least 84% of the mutations identified by the other assays. Mutations that were discordant between the assays mainly comprised secondary mutations and natural polymorphisms. The assays had better concordance for mutations that corresponded to current failing regimens, present in the more predominant viral quasispecies. In the treatment-naive patients, GenotypR, GeneChip, and LiPA mainly identified wild-type virus. Only the LiPA identified K70R, a possible transmitted zidovudine resistance mutation, in the RT gene of a treatment-naive patient. We conclude that although discrepancies in results exist between assays, each assay showed a similar capacity to identify potentially clinically relevant mutations related to patient treatment regimens.

A 31-year-old African-American man complained of a I-month history of a new-onset headache. His headache was constant, most intense behind his eyes, and 10 of 10 in severity. He noticed the headache upon awakening, and it persisted throughout the day. Mild photophobia and light­ headedness were associated with the headache. The patient denied visual changes, hearing changes, nausea, vomiting, vertigo, shortness of breath, chest pain, cough, fevers, or chills. His medical history was remarkable only for hypertension, and he was not taking any medica­ tions. He smoked 1 pack of cigarettes per day, used alcohol occasionally, and used no other drugs. The patient had lived in Oklahoma, Texas, Arkansas, and Illinois, and had traveled to Mexico within the past 5 years. His work history included laboring on cargo boats, clipping chicken wings on a chicken farm, and most recently, installing fuel pumps at gas stations. Pertinent physical findings included a blood pressure of 160/105 mm Hg. His pupils were equal and reactive to light, he had full visual fields, and there was no evidence of papilledema or uveitis on an ophthalmologic examination. He also had bilateral strength reduction (4/5) in the proximal lower extremities, but had normal sensation in his lower extremities. Findings of the remainder of his physical examination were unremarkable. Because of the duration and severity of the head­ aches, a computed tomographic (CT) scan was or­ dered. The scan showed an enhancing soft-tissue mass in the posterior left temporal and occipital lobe (Figure 1). The patient was given dexametha­ sone and mannitol to reduce intracranial pressure. A cerebral angiogram, ordered to establish further the nature of the lesion, showed the lesion to be avascular. A brain biopsy determined that the lesion

This study has compared the abilities of isoproterenol and forskolin to inhibit immunologic- and nonimmunologic-induced histamine release from guinea-pig superfused trachea and enzymatically dispersed tracheal cells. Contraction was also measured in the former preparation. The potency of isoproterenol was similar for inhibition of all parameters associated with immunologic (ovalbumin) challenge in the two preparations. In contrast, forskolin appeared less potent in inhibiting ovalbumin-induced histamine release from dispersed tracheal cells. Histamine release by the nonimmunologic secretagogues d-tubocurarine and compound 48/80 was not altered by either substance. However, inhibition by isoproterenol and forskolin of tracheal contraction was evident when challenge was conducted with d-tubocurarine and compound 48/80. Inhibition of contraction appears to be a result of functional antagonism at the level of the smooth muscle. The superfused trachea is a useful preparation in which to explore the effects of substances that modulate mast cell mediator release.

Guinea pig lung mast cells can be isolated and purified to high purity. This has given us the opportunity to study in greater detail mediator release from these cells. Both immunologic (ovalbumin sensitized) and nonimmunologic (calcium ionophore, CaI) stimuli caused a dose-dependent release of histamine and pLT from monodispersed lung cells and highly purified lung mast cells. Examination of the time release curve for pLT revealed a 5-min lag in the release of this mediator and a peak release at 60 min after challenge with antigen. Verification of pLT release was obtained by use of the 5-lipoxygenase inhibitor A64077 (Zileuton). Pretreatment of lung mast cells with the 5-lipoxygenase inhibitor prevented release of pLT by either antigen or CaI but had no appreciable effect on histamine release (HR). The pulmonary mast cell appears to be an important contributor to pLT release in the guinea pig lung.

We have previously reported differences in mediator release during equivalent levels of antigen (Ag)-induced smooth muscle contraction of guinea pig pulmonary tissues after passive sensitization with IgG1 versus IgE antibodies (Abs). In the present study, we have examined the influence of indomethacin (5 x 10 −6 mol/L) and l-cysteine (3 or 10 mmol/L) on mediator release from superfused trachea taken from guinea pigs passively sensitized with IgG1 or IgE Ab 1 day before in vitro studies. Tissues were challenged with Ag (oxazolone-human serum albumin conjugate), and contractions and superfusate histamine and peptidoleukotrienes were monitored at discrete time intervals thereafter. Superfusate mediator contents were determined by spectrophotofluorimetry (histamine) and RAST (peptidoleukotrienes). The profiles of peptidoleukotrienes were examined with high-pressure liquid chromatography. At equivalent levels of contraction, significantly less histamine and peptidoleukotrienes were found in superfusate samples after sensitization with IgE Abs. None of the drug pretreatments significantly altered Ag-induced histamine release after IgGI or IgE sensitization. Indomethacin resulted in an increase in total measurable peptidoleukotrienes found only after IgGI receptor activation, but it did prolong tracheal contractions with both Abs. l-cysteine, 10 mmol/L, resulted in an increase in total measurable superfusate peptidoleukotriene content under all experimental conditions. The percentage increase in peptidoleukotriene content from that found without drug pretreatment was larger in the case of IgE compared to IgGI sensitization. During early time periods, after Ag challenge, measurable peptidoleukotriene levels in superfusate samples were similar for both Abs in the presence of l-cysteine, 10 mmol/L. These data suggest that there is a differential pattern of peptidoleukotriene metabolism after activation of IgG1 versus IgE receptors in guinea pig trachea.

The proinflammatory cytokine TNFα is believed to play an important role in ocular surface inflammation such as conjunctivitis (allergic, bacterial, and viral) and dry eye disease.1-3 However, the regulation of TNFα on the human conjunctival surface has not been studied. It has been demonstrated in vitro that HCECs respond to recombinant TNFα with proinflammatory responses consistent with those observed in vivo, such as the upregulation of intercellular adhesion molecule (ICAM)-1 expression and increased amounts of the chemokine IL-8. However, regulation of the receptor for TNFα has not been studied in HCECs. In ocular allergic inflammation, conjunctival mast cells are an important source of TNFα. We have previously demonstrated that IgE-activated human conjunctival mast cell supernates upregulate the expression of ICAM-1 on HCECs by a mechanism specifically dependent on TNFα released from the conjunctival mast cell.4 Our work has further suggested that conjunctival mast cell supernates render HCECs more sensitive to TNFα-mediated ICAM-1 upregulation. In these experiments, we demonstrated that TNFα in mast cell supernates could promote ICAM-1 upregulation at log (10−3) lower concentrations than recombinant TNFα alone.4 We hypothesized that IgE-activated conjunctival mast cell supernates upregulate the expression of TNFR1 on HCECs and that the upregulation of TNFR1 expression results in increased sensitivity to TNFα-mediated activation responses (e.g., upregulation of ICAM-1). Our first experiments confirmed the first part of our hypothesis, demonstrating that supernates from IgE-activated conjunctival mast cells upregulate the expression of TNFR1 on primary HCECs. However, because purified conjunctival mast cell supernates require large amounts of cadaveric conjunctival tissues, we sought an alternative mechanism to upregulate TNFR1 expression and to test the biological significance of changes in expression of this receptor. Although TNFR1 expression has not been studied in HCECs, in other types of cells it has been demonstrated that TNFR1 is constitutively expressed and that it can, in some cases, be induced at the transcriptional level by IFNγ.5 TNFR1 is primarily regulated posttranscriptionally by the metalloprotease, TNFα-converting enzyme (TACE; also known as a disintegrin and a metalloprotease [ADAM]-17). TACE-mediated proteolytic cleavage of TNFR1 to its soluble form (sTNFR1) is one important mechanism for the down-regulation of TNFα-mediated inflammatory responses.6 TACE is naturally inhibited in vivo by tissue inhibitor of metalloprotease-3 (TIMP-3) and can be inhibited pharmaceutically by a variety of compounds. For the present study, we examined the mechanisms of TNFR1 expression and shedding in HCECs (primary and IOBA-NHC cell line) using the phorbol ester phorbol myristate acetate (PMA), which activates the protein kinase C pathway (a common pathway of HCEC activation) and stimulates reactive oxygen species (known to activate TACE) and the pharmaceutical TACE inhibitor TNFα protease inhibitor-2 (TAPI-2). With these tools we were able to examine the second part of our hypothesis, which demonstrates how changes in surface expression of TNFR1 affect the threshold of responsiveness to TNFα.

To study the function of mast cells in bladder tissue, guinea pigs were sensitized with ovalbumin by intraperitoneal injections, bladder tissue strips were superfused, and tissue contractile force and histamine release were studied. Upon challenge with ovalbumin, bladder tissue contracted 64 ± 4% (mean ± S.E.M.) of the maximum carbachol contraction and released 14.1 ± 1.6% of the total tissue histamine content. Incubation of sensitized bladder tissue with indomethacin led to an increased force and duration of the contraction while incubation with nordihydroguaiaretic acid combined with pyrilamine reduced histamine release and abolished the contraction. Tissue histamine content was significantly higher in the bladder neck than in the dome, and significantly elevated following sensitization. Histochemical studies of bladder tissue demonstrated mast cell degranulation in antigen challenge experiments. In addition, a group of guinea pigs were sensitized to ovalbumin through bladder instillations. With this model, study of the functional characteristics of bladder mast cells and the acute actions of mast cell products on the bladder microenvironment, should now be feasible. ( J. Urol., 144: 1293–1300, 1990 )

The pathogenesis of the late asthmatic response has been of interest due to the fact that its development has been associated with airway obstruction, airway inflammation, and airway hyperresponsiveness: all features of chronic asthma. In order to study more precisely late responses, a number of animal models have been developed. Previous reports have described alterations in airway mechanics at both 3 to 6 and 17 h after antigen challenge in sensitized guinea pigs. In these experiments, however, animals were challenged through the nose with aerosolized antigen, and airway conductance was measured using whole body plethysmography while the animals breathed through the upper airways. In rats similarly challenged, the predominant immediate change in resistance occurs in the upper airways. The purpose of this study, therefore, was to evaluate the contribution made by both the upper and lower airways to late changes after allergen challenge in guinea pigs. Outbred female Hartley guinea pigs were actively or passively sensitized (IgG1 antibody response) to three different antigens and challenged either by direct tracheal insufflation (sheep gamma globulin [SGG] or oxazolone-human serum albumin [OX-HSA]) or by aerosolization (ovalbumin [OA]). Lung resistance (RL) and dynamic compliance (Cdyn) were measured in anesthetized guinea pigs through a tracheostomy tube, and specific airway conductance (SGaw) was measured in unanesthetized, nose-breathing guinea pigs using a whole body plethysmograph. Late responses were defined as a RL greater than the mean + 2 SD RL or as a decrease in SGaw from baseline greater than 2 SD from the placebo-challenged (bovine serum albumin) group.(ABSTRACT TRUNCATED AT 250 WORDS)

PURPOSE The mechanism by which eosinophils adhere to the ocular surface during allergic inflammation is unknown. This study examined whether the incubation of human conjunctival epithelial cells (HCEs) with tears from allergic subjects promotes eosinophil adhesion, and it examined the effect of treatment with olopatadine on this process. METHODS Allergic subjects (n = 6) and nonallergic subjects (n = 4) were treated in season for 1 week with olopatadine in one eye while the other eye remained untreated. Tears were collected from both eyes with the use of a microcapillary tube. HCEs were acquired by enzymatic digestion of cadaveric conjunctival tissues. Confluent cultures of HCEs were treated with diluted tears for 24 hours before incubation with peripheral blood eosinophils (purified with negative magnetic bead selection). Eosinophil adhesion was measured with an eosinophil peroxidase assay. RESULTS Incubation of HCEs with tears from allergic subjects significantly upregulated eosinophil adhesion compared with eosinophil adhesion to untreated HCEs or with HCEs treated with nonallergic tears and untreated HCEs (P < 0.05). Eosinophil adhesion to HCEs treated with tears from olopatadine-treated allergic subjects was inhibited (P < 0.01) compared with tear-stimulated adhesion observed from untreated eyes. Percentage of inhibition was 43.3% +/- 13.9% (mean +/- SD). Blocking antibodies demonstrated that eosinophil adhesion to HCEs in vitro involved beta2 integrins on eosinophils but not intercellular adhesion molecule-1 on human HCEs. CONCLUSIONS Tears collected from allergic subjects contain bioactivity capable of upregulating eosinophil adhesion to HCEs in vitro. Inhibition of this process by treatment of subjects with olopatadine suggests that some of the cellular targets of this drug may play a role in promoting eosinophil adhesion.

Purpose Increased colonization with Staphylococcus aureus (SA) may play a pro-inflammatory role in atopic keratoconjunctivitis (AKC). We previously demonstrated that human conjunctival epithelial cells are activated by SA peptidoglycan via interaction with Toll-like receptor 2 (TLR2) expressed on human conjunctival epithelial cells in vitro. The purpose of this study was to examine human conjunctival epithelial cells derived from subjects with AKC, seasonal allergic conjunctivitis (SAC), and non-allergic subjects for expression of TLR2. Methods For in vivo studies, human conjunctival epithelial cells were acquired via impression cytology, fixed, and examined for TLR2 expression via flow cytometry. Anti-CD45 was used to gate out contaminating leukocytes. For in vitro studies, human conjunctival epithelial cells and conjunctival mast cells were enzymatically dispersed and purified from cadaveric tissues and cultured on 24-well plates. Conjunctival mast cells were challenged with anti-IgE antibody to obtain activated mast cell supernates. Conjunctival epithelial cells were incubated with the activated mast cell supernates for 24 hours, then harvested using trypsin-EDTA for immunostaining via flow cytometry. Results Staining of conjunctival epithelial cells for TLR2 was positive in 4/5 subjects with AKC, 3/5 subjects with SAC, and 0/3 non-allergic subjects. Intensity of TLR2 staining was significantly greater on conjunctival epithelial cells obtained from AKC subjects compared to conjunctival epithelial cells obtained from non-allergic subjects (p Conclusion While TLR2 may not be constitutively expressed on conjunctival epithelium, ocular allergic inflammation appears to increase TLR2 expression, which is significant in AKC. This increased expression could promote ocular defense against colonizing pathogenic gram-positive bacteria.

Rationale The mechanism by which eosinophils adhere to the ocular surface during allergic inflammation is unknown. This study examined whether tears from allergic subjects upregulate the ability of human conjunctival epithelial cells (HCE) to promote eosinophil adhesion and the effect of the anti-allergic drug olopatadine on this process. Methods Allergic subjects were treated for 48 hrs with olopatadine in one eye. Tears were collected 24 hrs prior to allergen challenge (both eyes) and 30 min and 90 min post-challenge. HCE were pre-incubated with tears (diluted 1:125 in media) or controls (TNFα, IFNγ) for 24 hrs after which peripheral blood eosinophil adhesion to HCE was measured using an eosinophil peroxidase assay. Blocking antibodies to β2 integrins on eosinophils (A-β2), and ICAM-1 on HCE (A-ICAM-1), examined integrin specificity. Results Both pre and post-challenge tears activated HCE resulting in upregulated eosinophil adhesion (equivalent to levels of adhesion to TNFα, IFNγ stimulated HCE). Olopatadine treatment completely inhibited the ability of both pre and post-challenge tears to upregulate eosinophil adhesion (equivalent to levels of adhesion to unstimulated HCE). Blocking antibodies demonstrated that eosinophil adhesion to conjunctival epithelial cells in vitro was mediated by β2 integrins on eosinophils (completely inhibited by A-β2) but not via ICAM-1 on HCE (not inhibited by A-ICAM-1). Conclusions Olopatadine treatment inhibits the pro-inflammatory effect of allergic tears on eosinophil adhesion to HCE in vitro. In vivo, an effect of this magnitude (3–6×102 less eosinophils/cm2) could result in a significant decrease in the numbers of eosinophils maintained on the ocular surface in allergic inflammation.

Objective To characterize the expression and regulation of conjunctival mast cell surface receptors important in allergic inflammation. Methods Mast cells were isolated from human conjunctival tissues of cadavers. Mast cell surface markers were identified using flow cytometry with antibodies to IgE, FcϵRI, c-kit, and intercellular adhesion molecule-1 (ICAM-1). We evaluated the effect of 24-hour tumor necrosis factor α (TNF-α) or interleukin 4 (IL-4) incubation on the expression of mast cell c-kit, ICAM-1, and surface-bound IgE. Results Staining of mast cells ( c-kit and/or tryptase positive) yielded positive results for all of the variables measured. The intensity of mast cell c-kit staining increased with TNF-α incubation, but decreased below that of unstimulated mast cells when incubated with IL-4. Anti–ICAM-1 and anti–IgE staining were increased over that of unstimulated cells when incubated with TNF-α or IL-4. Conclusions In this model, TNF-α up-regulates mast cell surface receptors and cell-bound IgE. Interleukin 4 up-regulates mast cell ICAM-1 and cell-bound IgE, but down-regulates c-kit. Clinical Relevance Conjunctival mast cells play a critical role in the pathogenesis of atopic ocular disease. Characterization of the expression and regulation of mast cell surface receptors is important to the development of potential novel treatments for ocular inflammation.

We used Western blot analysis to examine the occurrence and titer of antibody to cytoskeletal neurofilament protein antigens in patients with neuropsychiatric manifestations of systemic lupus erythematosus (SLE) and in controls. Twenty-two patients with neuropsychiatric SLE (NPSLE) had an increased incidence of antineurofilament antibody (ANFA) compared with 34 patients with SLE without neuropsychiatric symptoms, 78 patients with other disease processes, and 22 healthy controls. ANFA were found to be directed against the 205,000- and 160,000-dalton proteins of the neurofilament triplet. Patients with a diffuse NPSLE clinical presentation had the greatest frequency of serum ANFA (7 of 12, 58%) compared with all other groups examined. Magnetic resonance imaging and serum anticardiolipin antibody testing were also performed in selected patients with NPSLE. Patients with a focal clinical presentation of NPSLE, positive magnetic resonance imaging findings, and negative serum ANFA had significantly elevated levels of anticardiolipin antibody.

Pulmonary mast cells were obtained from guinea pig lung using a combination of enzymatic digestion of tissue, centrifugal elutriation, and density gradient centrifugation on Percoll. In the initial procedure, lung tissue was enzymatically digested with collagenase and elastase in four 30 min incubations. Typically, monodispersed cell suspensions contained 4% mast cells. Further purification of these lung mast cells using elutriation and Percoll gradients consistently yielded mast cells of 40–78% (mean 51%) purity. These cells were morphologically intact, viable and found to be functional as determined by histamine release evoked by antigen and anti-guinea pig IgG 1 antibody.

Neutrophils are prominent in some IgE-mediated allergic reactions and may contribute to the pathophysiology of immediate hypersensitivity. Antigen challenge of fragments of guinea pig lung tissue that were passively sensitized with IgE or IgG antibody evoked the release of neutrophil chemotactic activity (NCA) in parallel with histamine. The NCA released from lung tissue by both IgG- and IgE-dependent stimulation coeluted from a column of Sephacryl S-300 with synthetic leukotriene B4 (LTB4). The NCA in eluates from the Sephacryl S-300 column contained LTB4, as determined by high-performance liquid chromatography and specific radioimmunoassay, in quantities that accounted for the observed chemoattractant activity in the eluates. Furthermore, the NCA of supernatants from antigen-challenged lung fragments was reduced by a mean of 80% after absorption with a monoclonal antibody to LTB4. LTB4 thus constitutes the major functional constituent of NCA released after anaphylactic challenge of IgE- and IgG-sensitized guinea pig lung tissue.

Publisher Summary This chapter discusses the basophil isolation procedures with special emphasis on human and guinea pig techniques. In humans, basophils contain almost all of the blood histamine. Studies of human basophil function have progressed much more rapidly than studies of basophil biochemistry, pharmacology, and physiology. A number of methods have been developed for purifying human basophils, and a more intensive study of this cell has proceeded. Three major procedures have been developed, which take advantage of a positive selection of basophils using affinity chromatography. Two of the methods utilize antibody linked to an insoluble support, and the third utilizes the property of basophil adherence to glass beads. Techniques for the purification of guinea pig basophils utilizing Ficoll density-gradient centrifugation and negative selection using a rabbit antileukocyte antibody and Percoll density gradient centrifugation have been reported. However, the most consistent and reliable technique utilized for guinea pig basophil purification involves both centrifugal elutriation and Percoll density-gradient centrifugation

Ovalbumin-induced histamine release from passively sensitized monodispersed cells (2–4% mast cells) or purified mast cells (50–78%) from guinea-pig lung was inhibited by isoproterenol and sulfonterol and their relative potencies or maximum responses were similar in both cases. The apparent dissociation constant for butoxamine was also independent of mast cell purity. The results indicate that β-adrenoceptors mediating histamine release inhibition from lung are localized to mast cells and act independently of other cell types.

Alphitobius diaperinus is an important beetle in the grain and poultry industries. We evaluated three individuals with work-related symptoms of asthma, rhinitis, conjunctivitis, urticaria, and angioedema on exposure to the insect. Prick skin tests with extracts prepared from the larval, pupal, and adult life stages were positive in all three patients. Specific IgE antibodies to these extracts were demonstrated by RAST or radioimmunoassay. RAST and radioimmunoassay inhibition confirmed the specificity of IgE binding and further demonstrated immunologic cross-reactivity between the three life stages. Peripheral blood leukocytes from two of the individuals demonstrated significant histamine release when they were compared with cells from nonexposed atopic and normal control subjects. The proteins in the extracts of each life stage were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 30 protein bands were detected in each of the extracts; however, the patterns of separation were different for each life stage. After immunoblotting and autoradiography, IgE-binding proteins were recognized by sera from all three individuals in the larval extract at 90 kilodaltons (kd), in the pupal extract at 90, 64, and 38 kd, and in the adult extract at 84 kd. Additionally, several other proteins were identified as being allergenic in some of the patients. We conclude that these three patients developed IgE-mediated sensitivity to A. diaperinus antigens as the result of occupational exposure. To our knowledge, this is the first description of sensitivity to this grain beetle.