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A multi-faceted antimicrobial stewardship programme contributed to a 17.8% reduction in antibiotic consumption for our English NHS Trust. This dramatic achievement could be partially attributed to an empirical antibiotic guideline change, introduction of procalcitonin testing to guide in antibiotic decisions in SARS-CoV-2 inpatients and use of electronic antibiotic stewardship strategies. In this article, we describe the multifaceted, step-by-step antibiotic stewardship approach that weathered the SARS-CoV-2 pandemic and led to this dramatic improvement. Also included for completeness are interventions that did not pass the plan, do, study, act (PDSA) cycle and were therefore discontinued., (© The Author(s) 2023. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Human mesenchymal stem cells (hMSCs) are effective in treating disorders resulting from an inflammatory or heightened immune response. The hMSCs derived from induced pluripotent stem cells (ihMSCs) share the characteristics of tissue derived hMSCs but lack challenges associated with limited tissue sources and donor variation. To meet the expected future demand for ihMSCs, there is a need to develop scalable methods for their production at clinical yields while retaining immunomodulatory efficacy. Herein, we describe a platform for the scalable expansion and rapid harvest of ihMSCs with robust immunomodulatory activity using degradable gelatin methacryloyl (GelMA) microcarriers. GelMA microcarriers were rapidly and reproducibly fabricated using a custom microfluidic step emulsification device at relatively low cost. Using vertical wheel bioreactors, 8.8 to 16.3-fold expansion of ihMSCs was achieved over 8 days. Complete recovery by 5-minute digestion of the microcarriers with standard cell dissociation reagents resulted in >95% viability. The ihMSCs matched or exceeded immunomodulatory potential in vitro when compared with ihMSCs expanded on monolayers. This is the first description of a robust, scalable, and cost-effective method for generation of immunomodulatory ihMSCs, representing a significant contribution to their translational potential., (© 2021 The Authors. STEM CELLS TRANSLATIONAL MEDICINE published by Wiley Periodicals LLC on behalf of AlphaMed Press.)

The ongoing evolution of SARS-CoV-2 into more easily transmissible and infectious variants has sparked concern over the continued effectiveness of existing therapeutic antibodies and vaccines. Hence, together with increased genomic surveillance, methods to rapidly develop and assess effective interventions are critically needed. Here we report the discovery of SARS-CoV-2 neutralizing antibodies isolated from COVID-19 patients using a high-throughput platform. Antibodies were identified from unpaired donor B-cell and serum repertoires using yeast surface display, proteomics, and public light chain screening. Cryo-EM and functional characterization of the antibodies identified N3-1, an antibody that binds avidly (K d,app = 68 pM) to the receptor binding domain (RBD) of the spike protein and robustly neutralizes the virus in vitro . This antibody likely binds all three RBDs of the trimeric spike protein with a single IgG. Importantly, N3-1 equivalently binds spike proteins from emerging SARS-CoV-2 variants of concern, neutralizes UK variant B.1.1.7, and binds SARS-CoV spike with nanomolar affinity. Taken together, the strategies described herein will prove broadly applicable in interrogating adaptive immunity and developing rapid response biological countermeasures to emerging pathogens., Competing Interests: Competing Interest Statement JL, ADE, EMM, GG, and DRB declare competing financial interests in the form of provisional and granted patent applications relevant to Ig-Seq. JG, CH, ECG, AH, DRB, EMM, JSM, GCI, ADE, GG, and JDG have filed provisional applications for the discovery of neutralizing antibodies. JG, DRB, ECG, AH, and JDG have filed applications for additional methods relevant to this work.

Plants represent a safe and cost-effective platform for producing high-value proteins with pharmaceutical properties; however, the ability to accumulate these in commercially viable quantities is challenging. Ideal crops to serve as biofactories would include low-input, fast-growing, high-biomass species such as sugarcane. The objective of this study was to develop an efficient expression system to enable large-scale production of high-value recombinant proteins in sugarcane culms. Bovine lysozyme (BvLz) is a potent broad-spectrum antimicrobial enzyme used in the food, cosmetics and agricultural industries. Here, we report a novel strategy to achieve high-level expression of recombinant proteins using a combinatorial stacked promoter system. We demonstrate this by co-expressing BvLz under the control of multiple constitutive and culm-regulated promoters on separate expression vectors and combinatorial plant transformation. BvLz accumulation reached 1.4% of total soluble protein (TSP) (10.0 mg BvLz/kg culm mass) in stacked multiple promoter:BvLz lines, compared to 0.07% of TSP (0.56 mg/kg) in single promoter:BvLz lines. BvLz accumulation was further boosted to 11.5% of TSP (82.5 mg/kg) through event stacking by re-transforming the stacked promoter:BvLz lines with additional BvLz expression vectors. The protein accumulation achieved with the combinatorial promoter stacking expression system was stable in multiple vegetative propagations, demonstrating the feasibility of using sugarcane as a biofactory for producing high-value proteins and bioproducts.

The corn grain biofactory was used to produce Cel7A, an exo-cellulase (cellobiohydrolase I) from Hypocrea jecorina. The enzymatic activity on small molecule substrates was equivalent to its fungal counterpart. The corn grain-derived enzyme is glycosylated and 6 kDa smaller than the native fungal protein, likely due to more sugars added in the glycosylation of the fungal enzyme. Our data suggest that corn seed-derived cellobiohydrolase (CBH) I performs as well as or better than its fungal counterpart in releasing sugars from complex substrates such as pre-treated corn stover or wood. This recombinant protein product can enter and expand current reagent enzyme markets as well as create new markets in textile or pulp processing. The purified protein is now available commercially.

Increased industrial use of sugarcane (Saccharum spp. hybrid) for food and bioenergy has led to considerable improvements in its genetic transformation, which allowed the development of not only pest- and herbicide-resistant lines but also lines expressing high-value bioproducts and biopolymers. However, the economic benefits of using inexpensive transgenic plant systems for the production of industrial proteins could be offset by high downstream processing costs. In this work, transgenic sugarcane expressing recombinant bovine lysozyme (BvLz) was used to evaluate the feasibility of extraction and fractionation of recombinant proteins expressed in sugarcane stalks. Three pH levels (4.5, 6.0 and 7.5) and three salt concentrations (0, 50, and 150 mM NaCl) were tested to determine BvLz and total protein extractability. Two extraction conditions were selected to prepare BvLz extracts for further processing by cross-flow filtration, a suitable method for concentration and conditioning of extracts for direct applications or prior to chromatography. Partial removal of native proteins was achieved using a 100 kDa membrane but 20-30 % of the extracted BvLz was lost. Concentration of clarified extracts using a 3 kDa membrane resulted in twofold purification and 65 % recovery of BvLz. Loading of concentrated sugarcane extract on hydrophobic interaction chromatography (HIC) resulted in 50 % BvLz purity and 69 % recovery of BvLz.

Transgenic Lemna minor has been used successfully to produce several biotherapeutic proteins. For plant-produced mAbs specifically, the cost of protein A capture step is critical as the economic benefits of plant production systems could be erased if the downstream processing ends up being expensive. To avoid potential modification of mAb or fouling of expensive protein A resins, a rapid and efficient removal of phenolics from plant extracts is desirable. We identified major phenolics in Lemna extracts and evaluated their removal by adsorption to PVPP, XAD-4, IRA-402, and Q-Sepharose. Forms of apigenin, ferulic acid, and vitexin comprised ∼ 75% of the total phenolics. Screening of the resins with pure ferulic acid and vitexin indicated that PVPP would not be efficient for phenolics removal. Analysis of the breakthrough fractions of phenolics adsorption to XAD-4, IRA-402, and Q-Sepharose showed differences in adsorption with pH and in the type of phenolics adsorbed. Superior dynamic binding capacities (DBC) were observed at pH 4.5 than at 7.5. To evaluate the cost impact of a phenolics removal step before protein A chromatography, a mAb purification process was simulated using SuperPro Designer 7.0. The economic analysis indicated that addition of a phenolics adsorption step would increase mAb production cost only 20% by using IRA-402 compared to 35% for XAD-4 resin. The cost of the adsorption step is offset by increasing the lifespan of protein A resin and a reduction of overall mAb production cost could be achieved by using a phenolics removal step., (Copyright © 2010 American Institute of Chemical Engineers (AIChE).)

Though seemingly an easy procedure to perform, there is no universally accepted approach to the measurement of waist circumference (WC) in the clinical setting. Measurement of WC can be affected by a myriad of factors including patient movement or position changes, poor positioning of the measuring tape or differences in tension applied to the tape by the clinician. Changes in any one of these factors can reduce the validity and reliability of the measurement. This is of particular importance in clinical trials where changes in WC represent a therapeutic endpoint. Recognition of the need for a more standardized and reliable means to perform WC measurement led to the development of a novel, validated technique. The Height of Iliac Crest (HIC) method uses a standardized technique that increases the reliability of measurement by minimizing some of the weaknesses and variations of previous methods. The purpose of the following study was to validate the accuracy and reproducibly of the HIC method for obtaining WC measurement data. This study demonstrates that accurate and reproducible results can be obtained through the application of the HIC method for measuring waist circumference., (© 2011 American Society of Plastic Surgical Nurses.)

Several pharmaceutical protein products made in transgenic plant hosts are advancing through clinical trials. Plant hosts present a different set of impurities from which the proteins must be purified compared to other expression hosts such as mammalian cells. In this work, phenolic compounds present in extracts of monoclonal antibody (mAb)-expressing Lemna minor were examined. Two different extraction pHs were evaluated to assess the effect of extraction condition on the concentration of mAb and phenolics in the extracts. The extract prepared at pH 4.5 had an enriched level of mAb relative to native protein when compared to a pH 7.5 extract although similar overall mAb was extracted at either pH. Slightly more mAb was recovered from the pH 3 elution of the pH 4.5 extract run on a MabSelect column than was recovered from the pH 7.5 extract. Phenolic levels in extracts were assessed by spectrophotometry, Folin-Ciocalteu assay and by profiling on RP-HPLC. The Folin-Ciocalteu assay results did not agree with those obtained by the other two methods. Therefore phenolic levels were quantified by RP-HPLC comparing the total area of phenolic peaks to those of reference phenolic compounds. The pH 7.5 extract had 22% less phenolics than the pH 4.5 extract. Acidic precipitation of the pH 7.5 extract resulted in further reduction of phenolics originally present in the pH 7.5 extract. The total phenolics present in the extracts were effectively removed by incubation of extracts with a commercially available anion exchange resin, Amberlite IRA-402. We anticipate that early removal of phenolic compounds will prolong the life of more expensive affinity columns used for the purification of potential pharmaceutical proteins and should therefore be considered in process development involving proteins extracted from transgenic plant hosts.

The availability of foods low in sugar content yet high in flavour is critically important to millions of individuals conscious of carbohydrate intake for diabetic or dietetic purposes. Brazzein is a sweet protein occurring naturally in a tropical plant that is impractical to produce economically on a large scale, thus limiting its availability for food products. We report here the use of a maize expression system for the production of this naturally sweet protein. High expression of brazzein was obtained, with accumulation of up to 4% total soluble protein in maize seed. Purified corn brazzein possessed a sweetness intensity of up to 1200 times that of sucrose on a per weight basis. In addition, application tests demonstrated that brazzein-containing maize germ flour could be used directly in food applications, providing product sweetness. These results demonstrate that high-intensity sweet protein engineered into food products can give sweetener attributes useful in the food industry.

The search for inexpensive production systems capable of producing large quantities of recombinant protein has resulted in the development of new technology platforms based on transgenic plants and animals. Over the past decade, these transgenic systems have been used to produce several products and potential therapeutic proteins. Improvements continue to be made, not only in how the proteins are expressed but also in how the end products are obtained. As improvements in expression are realized, cost-saving measures will increasingly focus on downstream processing.

Plant molecular farming is a new and promising industry involving plant biotechnology. In this review, we describe several diverse plant systems that have been developed to produce commercially useful proteins for pharmaceutical and industrial uses. The advantages and disadvantages of each system are discussed. The first plant-derived molecular farming products have reached the marketplace and other products are poised to join them during the next few years. We explain the rationale for using plants as biofactories. We also describe the products currently on the market, and those that appear likely to join them in the near future. Lastly, we discuss the issue of public acceptance of molecular farming products., (Copyright 2004 Springer-Verlag)

Osteoporosis of the hip is associated with hip fracture, a devastating outcome on both an individual and aggregate basis. Height loss is a frequent manifestation of vertebral osteoporosis and is simple to evaluate in the clinical setting. The goal of this study was to determine whether height loss is significantly associated with low bone mineral density at the femur, using a retrospective review of cross-sectional data from 2108 women referred for a bone density scan. Collected data included self-reported maximum adult height, current height, and total hip bone mineral density, along with other demographic and risk factor information. We then investigated the relationship between height loss and osteoporosis using multinomial logistic regression modeling. We found that height loss of 2 in. or more is a highly significant predictor of osteoporosis at the hip. In particular, the odds women had osteoporosis at the hip, as determined by total hip bone mineral density, increased 4.4 times (95% confidence interval, 2.6-7.4) if the women had lost > or = 2 but < 3 in. of height. In addition, women with at least 3 in. of height loss had odds of osteoporosis of the hip that were 9.6 times greater (95% confidence interval, 4.8-19.2) than women with less than an inch of height loss. These odds ratios were adjusted for the confounding variables of age, weight, and maximum adult height. Our findings suggest loss of height may be an important clue in detecting osteoporosis of the hip, implying that evaluation of height loss should be routine in the outpatient setting.

We studied the computed tomographic (CT) appearance and determined Hounsfield units (HU) for normal thyroid tissue in eight cats. Helical CT images (2 mm collimation) were acquired from cranial aspect of the second cervical vertebra (C2) through caudal aspect of the fourth cervical vertebra (C4). Data were acquired before contrast medium administration (n = 7), after delayed contrast medium enhancement (n = 8), and immediately after contrast medium enhancement after a second dose of contrast medium (n = 8). Attenuation of thyroid tissue was compared with surrounding tissues. Before contrast medium enhancement, thyroid tissue was hyperattenuating to the surrounding soft tissues. After delayed contrast medium enhancement, thyroid tissue was hyperattenuating to surrounding soft tissues and isoattenuating to contrast medium-laden blood vessels. Immediately after contrast medium enhancement, thyroid tissue was hyperattenuating to surrounding soft tissues and hypoattenuating to contrast medium-laden blood vessels. The thyroid glands were dorsolateral to the trachea, ovoid, and displayed homogenous contrast medium enhancement. Circular regions of interest were drawn on the right and left thyroid lobes. Densitometric data of thyroid tissue were as follows: precontrast medium enhancement, 123.2 HU (95% CI: 119.4-127.1 HU); delayed contrast medium enhancement, 132.1 HU (95% CI: 127.4-136.8 HU); immediate postcontrast medium enhancement, 168.5 HU (95% CI: 163-173.9 HU). Normal feline thyroid tissue is easily detected using CT without contrast medium enhancement. This information may be useful for CT evaluation of abnormal feline thyroid glands.

Lignolytic enzymes such as laccase have been difficult to over-express in an active form. This paper describes the expression, characterization, and application of a fungal laccase in maize seed. The transgenic seed contains immobilized and extractable laccase. Fifty ppm dry weight of aqueously extractable laccase was obtained, and the remaining solids contained a significant amount of immobilized laccase that was active. Although a portion of the extractable laccase was produced as inactive apoenzyme, laccase activity was recovered by treatment with copper and chloride. In addition to allowing the apoenzyme to regain activity, treatment with copper also provided a partial purification step by precipitating other endogenous corn proteins while leaving >90% of the laccase in solution. The data also demonstrate the application of maize-produced laccase as a polymerization agent. The apparent concentration of laccase in ground, defatted corn germ is approximately 0.20% of dry weight.

Bovine trypsin (EC 3.4.21.4) is an enzyme that is widely used for commercial purposes to digest or process other proteins, including some therapeutic proteins. The biopharmaceutical industry is trying to eliminate animal-derived proteins from manufacturing processes due to the possible contamination of these products by human pathogens. Recombinant trypsin has been produced in a number of systems, including cell culture, bacteria and yeast. To date, these expression systems have not produced trypsin on a scale sufficient to fulfill the need of biopharmaceutical manufacturers where kilogram quantities are often required. The present paper describes commercial-level production of trypsin in transgenic maize (Zea mays) and its physical and functional characterization. This protease, the first enzyme to be produced on a large-scale using transgenic plant technology, is functionally equivalent to native bovine pancreatic trypsin. The availability of this reagent should allow for the replacement of animal-derived trypsin in the processing of pharmaceutical proteins.

Thirty-one ears (16 dogs) with otitis externa originating from bacterial or yeast infections were enrolled in a study to evaluate the in vivo efficacy of an ear cleanser containing 2.5% lactic acid and 0.1% salicylic acid for the treatment of infectious otitis externa. The affected ears were treated with the ear cleanser twice daily for 2 weeks and evaluated after 1 and 2 weeks of treatment. The ear cleanser was effective in resolution of infection in 67.7% of the ears, and clinical signs of infectious otitis externa were significantly reduced within 2 weeks.

The use of recombinant gene technologies by the vaccine industry has revolutionized the way antigens are generated, and has provided safer, more effective means of protecting animals and humans against bacterial and viral pathogens. Viral and bacterial antigens for recombinant subunit vaccines have been produced in a variety of organisms. Transgenic plants are now recognized as legitimate sources for these proteins, especially in the developing area of oral vaccines, because antigens have been shown to be correctly processed in plants into forms that elicit immune responses when fed to animals or humans. Antigens expressed in maize (Zea mays) are particularly attractive since they can be deposited in the natural storage vessel, the corn seed, and can be conveniently delivered to any organism that consumes grain. We have previously demonstrated high level expression of the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis in corn, and have demonstrated that these antigens delivered in the seed elicit protective immune responses. Here we provide additional data to support the potency, efficacy, and stability of recombinant subunit vaccines delivered in maize seed.

The number and types of antibodies expressed in plants has increased steadily since the first reports of this accomplishment in the 1980s, illustrating the versatility of plants as a production system for antibodies. Many recent reviews have detailed the antibody forms that have been derived from plant expression systems. This contribution focuses on the remaining challenges to develop plant-derived therapeutic antibodies into products, and some of the progress that is being made in addressing these challenges.

Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.

One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37 degrees C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained sufficient residual chymase activity to support lysis. Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.

Diphenyl 1-(N-peptidylamino)alkanephosphonate esters are highly reactive, specific, and aqueously stable irreversible inhibitors which can be used to probe the functions of many serine proteases, including the lymphocyte granzymes. We synthesized 16 peptide phosphonates with Ala, Met, Phe, or Val P1 amino acid residues, including two biotinylated derivatives for future functional and biochemical characterization of granzymes. The reactivity of the inhibitors was characterized with human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine chymotrypsin, and the granzymes of natural killer (NK) cells, which include a number of proteolytic activities (Asp-ase, Met-ase, etc.) that cleave peptide substrates with these residues in the P1 position. The reactivity and specificity of the phosphonates depended on the length and sequence of the peptidyl moiety and on the leaving group. Z-Ala-Ala-AlaP(OPh)2 was a good inhibitor of HLE and PPE (k(obsd)/[I] = 38 and 30 M(-1) s(-1), respectively) and had little reactivity with chymotrypsin. Z-Phe-Pro-Phe-P(OPh)2 was a good inhibitor of chymotrypsin (k(obsd)/[I] = 17,000 M(-1) s(-1)) and had little reactivity with the elastases. The leaving group of Z-MetP(OPh-4-Cl)2 made it a more effective chymotrypsin inhibitor than Z-MetP(OPh)2 (k(obsd)/[I] values of 142 and 30 M(-1) s(-1), respectively). With granzymes, the compounds reacted with a fraction of the Met-ase, chymase, and Ser-ase activities and lacked reactivity with Asp-ase and tryptase. Z-MetP(OPh-4-Cl)2 was an excellent inhibitor of Met-ase 1. Bi-Aca-Aca-Phe-Leu-PheP(OPh)2 appears to react specifically with one chymase while leaving other chymases untouched. Perforin-dependent lysis mediated by cytotoxic lymphocyte granules was inactivated by Z-Ala-Ala-AlaP(OPh)2, Z-MetP(OPh-4-Cl)2, Z-Leu-PheP(OPh)2, and Bi-Aca-Aca-Phe-Leu-PheP(OPh)2. The biochemical properties and biological efficacy of these inhibitors make them suitable for cellular and physiological studies of granzyme function.

Serine proteases (granzymes) in killer lymphocytes are required for lymphocyte cytotoxic granules to lyse target cells. Herein we report the development of a 3-step PCR cloning technique to amplify novel granzyme genes and two new rat granzymes are described. Degenerate oligonucleotide primers were designed based on sequence motifs selectively expressed in granzymes. These motifs flank "delta" regions that are unique for each granzyme. Total RNA of RNK-16 cells or activated splenocytes was amplified by reverse transcriptase-PCR to obtain cDNA fragments of several new granzymes. Gene-specific primers based on these delta regions were then used for 3'-RACE to obtain clones with the 3' gene ends. Reverse (antisense) delta-based or active site serine primers were used with a granzyme 5'-UTR primer to obtain clones extending to the 5' ends. Using this technique, two new cDNAs, RNKP-4 and RNKP-7, which encode granzymes of 248 and 241 amino acids, respectively, were cloned from activated lymphocytes. RNKP-4 is likely the rat equivalent of mouse granzyme C. RNKP-7 is most closely related to granzymes F and G. Modeling of the predicted proteins suggests large/polar P1 (Gln/Asn) specificity for RNKP-4 and large/hydrophobic P1 (e.g., Phe) specificity for RNKP-7. These specific protease activities were found in cytotoxic RNK-16 lymphocyte granules indicating that the two new genes may be translated and stored as active granzymes.

Cytotoxic lymphocytes and natural killer cells are able to kill their target cells in minutes. The death of the target cell occurs after the release of cytoplasmic granules from the effector cell. These granules contain the pore-forming protein perforin and serine proteases (granzymes). To date 10 genes encoding lymphocyte granzymes have been discovered; of these only four have been purified and characterized for their substrate specificity. Several are predicted to have a common chymase, like specificity which is found in the granule extracts. Others may need to be enriched as active enzymes before they can be evaluated for substrate hydrolysis. Due to the limitations of detection by substrate hydrolysis, a more sensitive method for the detection of dilute granules was needed. We report the differing reactivities of seven biotin (Bi)-tagged isocoumarin (IC) inhibitors for Asp-ase, chymase, tryptase and Met-ase granzymes. The inhibitors contained different substituents at their no. 3 position: methoxy (OMe), ethoxy (OEt), propoxy (OPr) or 2-phenylethoxy (OEtPh) groups. The OMe group conferred general reactivity, whereas the OEtPh group conferred selective reactivity with chymase granzymes. The inhibitors that contained the longest aminocaproyl (Aca) spacers between the biotin-tag and the isocoumarin ring mediated the most stable granzyme inactivation. These inhibitors were the most effective at blocking lysis of red blood cells by the granule extracts. The inhibitors were used in protein blotting experiments where the biotin was detected with an avidin-enzyme complex. Over 10 granzymes were labelled by the inhibitor Bi-Aca-Aca-IC-OMe. The inhibitors detected granzymes when they were not readily detected by substrate hydrolysis.

Binding of Ag to membrane Ig (mIg) can lead to either activation or desensitization of the B cell. For thymus-independent (TI) Ags the nature and concentration of the Ag determines what type of signal is delivered to the cell. These Ags are capable of directly activating B lymphocytes and are an important model system for the study of mechanisms involved in B cell responses. In this study, we quantified TI Ag binding and B cell receptor involvement as functions of TI Ag structure, concentration, and epitope density. Various epitope densities of two structurally different TI Ags, DNP-polymerized flagellin (pol) and DNP-dextran (dex), were labeled with tetramethylrhodamine isothiocyanate (TRITC) and reacted with DNP-specific murine splenic B lymphocytes and with cells of a cloned DNP-specific cell line. The amount of Ag bound to the cell surface at various doses was measured directly by flow cytometry. For each Ag and dose, FITC-labeled DNP-L-papain was used to quantitate receptor sites not occupied by Ag. Approximately 5% receptor occupancy was observed for immunogenic doses of Ag. Higher Ag concentrations that can induce tolerance caused a substantial increase in the fraction of occupied receptors. This suggests that tolerogenic responses result from an overly restrictive cross-linking of surface receptors. By comparing these data to previously published data on biologic activity of the Ags, we are able to more clearly define those conditions of Ag binding that lead to B cell activation.

Cytotoxic NK and T lymphocytes kill virally infected cells within minutes without causing damage to themselves or bystander cells. One mechanism of killing involves exocytosis of granules containing serine proteases and perforin. Serine protease inhibitors block killing of target cells mediated by the cytotoxic lymphocytes. There are at least five different serine protease activities in cytolytic granules. Ten different serine protease sequences have been identified with the use of cDNA-specific clones. It is not known whether only one or several of these serine proteases are essential for cytolytic activity. In this study we show that an irreversible serine protease inhibitor, biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, selectively inhibits a chymotrypsin-like (chymase) serine protease activity of rat RNK-16 granule extracts. Under the same conditions, only one 30-kDa (reduced) band was detected on protein blots. Furthermore, only one of three chymase peaks separated by hydrophobic interaction chromatography was inhibited. When this granzyme was inhibited, granule-mediated lysis of erythrocytes was diminished. NK cell killing was completely blocked when biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2 was added to cytotoxicity assays at 200 microM with rat splenocytes as effectors. By confocal fluorescence microscopy, we show that this inhibitor localizes to distinct regions within RNK-16 cells and rat NK cells. Inhibitor treatment of intact cells inactivated the chymase activity and reduced lysis found in their dense organelles. Together these data indicate that biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2 inhibits a granule chymase that is essential to cytolytic activity of NK cells.

Three fluorescein- and one Texas Red-labeled derivatives of [1-(N-dipeptidylamino)alkyl]phosphonate diphenyl esters were synthesized and evaluated as inhibitors of serine proteases. The two fluorophores, FITC and TXR, were attached to the peptide phosphonates via an epsilon-aminocaproyl unit that acts as a spacer group and facilitates the binding of the phosphonate inhibitor to the targeted enzymes. These derivatives are potent and specific inhibitors of chymotrypsin, porcine pancreatic elastase (PPE), and human leukocyte elastase (HLE). FTC-Aca-Phe-Leu-PheP(OPh)2 (3) inhibited chymotrypsin very potently (k(obsd)/[I] = 9500 M-1 s-1) and 600-fold better than it did PPE (k(obsd)/[I] = 16 M-1 s-1). FTC-Aca-Ala-Ala-MetP(OPh)2 (1) was a more effective inhibitor of chymotrypsin (k(obsd)/[I] = 190 M-1 s-1) than PPE and HLE (k(obsd)/[I] = 13 and 22 M-1 s-1, respectively). Only HLE and PPE were inhibited by FTC-Aca-Ala-Ala-AlaP(OPh)2 (2) (k(obsd)/[I] = 41 and 22 M-1 s-1, respectively). The specificity of these inhibitors toward the targeted serine proteases depends on the sequence of the tripeptide portion and was not affected by the presence of the fluorescent label. Trypsin, for instance, was not inhibited by any of these compounds. In some cases, the inhibitory potency was increased by the fluorescent label. For example, chymotrypsin was inhibited by the fluorescent compounds, FTC-Aca-Ala-Ala-MetP(OPh)2 (1) and FTC-Aca-Phe-Leu-PheP(OPh)2 (3), more potently than by the nonfluorescent compounds, Boc-Ala-Ala-MetP(OPh)2 (5) and Z-Phe-Leu-PheP(OPh)2 (7).(ABSTRACT TRUNCATED AT 250 WORDS)

The lateral diffusion of the fluorescent lipid analog 3,3'-dioctadecylindocarbocyanine iodide (DiI) was measured in the membranes of murine B lymphocytes treated with the B cell mitogen lipopolysaccharide (LPS). The mobility of DiI, as measured by fluorescence photobleaching recovery (FPR) techniques, was temperature-dependent with a value of 6.1.10(-9) cm2 s-1 at 37 degrees C. Untreated cells exhibited this diffusion coefficient over 72 h in culture. In contrast, DiI mobility decreased to 2.0.10(-9) cm2 s-1 at 37 degrees C in membranes of LPS-stimulated lymphocytes 24 h following LPS exposure. Interestingly, this decreased lipid lateral diffusion was not accompanied by any change in surface immunoglobulin lateral diffusion which remained essentially unchanged at 3.6-4.3.10(-11) cm2 s-1 over 72 h. To determine whether LPS effects on lipid lateral diffusion were due to insertion of LPS into the cell plasma membrane, we examined TRITC-LPS diffusion in B lymphocytes from LPS-responsive Balb/c and C3Heb/FeJ mice and from hypo-responsive C3H/HeJ mice. DiI and TRITC-LPS mobility decreased more than 50% in LPS-stimulated Balb/c and C3Heb/FeJ cells by 72 h. On C3H/HeJ lymphocytes, there was no change in DiI or TRITC-LPS lateral diffusion throughout the incubation period. These data indicate that B lymphocyte membrane composition is altered in LPS-activated lymphoblasts and that the decreased lateral diffusion of lipid probes does not result from membrane perturbation by LPS insertion into the lipid bilayer. Further, similarities between TRITC-LPS and DiI lateral diffusion suggest that most LPS molecules interact non-specifically with B cell membranes, presumably by acyl chain insertion of the lipid A moiety.

A subfamily of serine proteases is uniquely expressed by cytotoxic natural killer lymphocytes and T cells. Protease cleavage of different natural substrates is now implicated in the cytotoxic mechanisms of target cell membrane pore formation, DNA fragmentation and cytostasis.