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To examine the proximate circumstances of sudden cardiac death (SCD) in the setting of major snowstorms, we reviewed records from the medical examiners' offices of 3 counties in the weeks before, during, and after 2 heavy snowfalls that occurred in the greater metropolitan Detroit area. Of those who experienced SCD due to atherosclerotic cardiovascular disease (n = 271), 36 (33 men, 3 women) were engaged in snow removal, representing the largest number of exertion-related deaths after heavy snowfalls reported to date. [ABSTRACT FROM AUTHOR]

Background & Objective: Background and objective: Perioperative blood transfusion (PBT) during radical urological surgeries has been associated with an increased incidence of complications. The present study analyzes the outcome of perioperative blood transfusion (PBT) and the prognostic implications after radical surgeries on patients with malignant urological tumors., Methods: Our retrospective study included 792 cases of partial or radical nephrectomy /cystectomy/prostatectomy surgeries for kidney/bladder/ prostate carcinoma from 2012 to 2022. Data on preoperative, intraoperative, and pathological parameters were evaluated. PBT was taken as a period of transfusion of allogenic RBC during/preoperative/postoperative surgeries. The effect of PBT on oncological parameters like recurrence-free survival (RFS), overall survival (OS), and cancer-free survival (CSS) was compared using univariate cox regression analysis (Odds ratio, Hazard ratio)., Results: PBT was applied on 124 (20.6%) patients of nephrectomy, 54 (46.5%) patients of cystectomy, and 23 (31%) patients of prostatectomy. The baseline characteristics of the cohort study found symptomatic patients with older age and other co-morbidities to be transfusion-dependent. Also, the patients undergoing radical operations with more blood loss and advanced tumor stage were more likely to receive PBT. PBT was significantly associated with survival outcomes ( P <0.05) in nephrectomy and cystectomy cases but independent of association in prostatectomy cases., Conclusion: The result of this study concludes that in nephrectomy and cystectomy operations, PBT had a significant association with cancer recurrence and mortality; however, in prostatectomy cases, no significant correlation was noted. Thus, proper criteria to prevent the unnecessary use of PBT and more defined parameters for transfusion are needed to improve postoperative survival. Autologous transfusion should be considered more frequently. However, more extensive studies and randomized trials are needed in this area., Competing Interests: The authors declared no conflict of interest.

Immunization based antibody discovery is plagued by the paucity of antigen-specific B cells. Identifying these cells is akin to finding needle in a haystack. Current and emerging technologies while effective, are limited in terms of capturing the antigen-specific repertoire. We report on the bulk purification of antigen-specific B-cells and the benefits it offers to various antibody discovery platforms. Using five different antigens, we show hit rates of 51-88%, compared to about 5% with conventional methods. We also show that this purification is highly efficient with loss of only about 2% antigen specific cells. Furthermore, we compared clones in which cognate chains are preserved with those from display libraries in which chains either from total B cells (TBC) or antigen-specific B cells (AgSC) underwent combinatorial pairing. We found that cognate chain paired clones and combinatorial clones from AgSC library had higher frequency of functional clones and showed greater diversity in sequence and paratope compared to clones from the TBC library. This antigen-specific B-cell selection technique exemplifies a process improvement with reduced cycle time and cost, by removing undesired clones prior to screening and increasing the chance of capturing desirable and rare functional clones in the repertoire., (© 2022. The Author(s).)

Background & Objective: Liver lesions are difficult to diagnose and to differentiate primary from metastatic carcinoma, while Biopsy has its limitations. Cell block technology is easily accessible with high diagnostic accuracy. Our aim is 1) To find the role of cell block technology as an alternative to biopsy in identifying liver lesions; 2) To find the efficacy of cell block along with immunohistochemistry (IHC) and ancillary studies in differentiating primary from metastatic lesions; 3) To identify the site of origin of metastatic lesions. This is a descriptive study undertaken in two tertiary care hospitals over a period of three years., Methods: Retrospective review of adequate samples from fine needle aspirations from liver lesions under radiological coverage, converted into cell block was done. IHC was applied as needed. Usefulness of cell block preparation was evaluated, and the final diagnosis correlated with the biopsy results., Results: Analysis of 323 cases found sensitivity of 98.75% and positive predictive value of 99% for all lesions. Sensitivity for metastatic carcinomas was slightly more than hepatocellular carcinoma. However, accuracy of cell block results for individual metastatic lesions and site of origin was less. IHC and morphological pattern worked as an important adjunct in the final diagnosis. On the other hand, contribution of viral markers as a supplement in the final work up was ambiguous., Conclusion: High precision of validity results of cell block technology in comparison with biopsy highlights its pivotal role in conjunction with supportive tests for diagnosing and differentiating liver lesions as well as identifying primary sites in liver metastasis., Competing Interests: None.

Hybridoma technology has been valuable in the development of therapeutic antibodies. More recently, antigen-specific B-cell selection and display technologies are also gaining importance. A major limitation of these approaches used for antibody discovery is the extensive process of cloning and expression involved in transitioning from antibody identification to validating the function, which compromises the throughput of antibody discovery. In this study, we describe a process to identify and rapidly re-format and express antibodies for functional characterization. We used two different approaches to isolate antibodies to five different targets: 1) flow cytometry to identify antigen-specific single B cells from the spleen of immunized human immunoglobulin transgenic mice; and 2) panning of phage libraries. PCR amplification allowed recovery of paired V H and V L sequences from 79% to 96% of antigen-specific B cells. All cognate V H and V L transcripts were formatted into transcription and translation compatible linear DNA expression cassettes (LEC) encoding whole IgG or Fab. Between 92% and 100% of paired V H and V L transcripts could be converted to LECs, and nearly 100% of them expressed as antibodies when transfected into Expi293F cells. The concentration of IgG in the cell culture supernatants ranged from 0.05 µg/ml to 145.8 µg/ml (mean = 18.4 µg/ml). Antigen-specific binding was displayed by 78-100% of antibodies. High throughput functional screening allowed the rapid identification of several functional antibodies. In summary, we describe a plasmid-free system for cloning and expressing antibodies isolated by different approaches, in any format of choice for deep functional screening that can be applied in any research setting during antibody discovery.

Bispecific molecules are biologically significant, yet their complex structures pose important manufacturing and pharmacokinetic challenges. Nevertheless, owing to similarities with monoclonal antibodies (mAbs), IgG-like bispecifics conceptually align well with conventional expression and manufacturing platforms and often exhibit potentially favorable drug metabolism and pharmacokinetic (DMPK) properties. However, IgG-like bispecifics do not possess target bivalency and current designs often require tedious engineering and purification to ensure appropriate chain pairing. Here, we present a near-native IgG antibody format, the 2xVH, which can create bivalency for each target or epitope and requires no engineering for cognate chain pairing. In this modality, two different variable heavy (VH) domains with distinct binding specificities are grafted onto the first constant heavy (CH1) and constant light (CL) domains, conferring the molecule with dual specificity. To determine the versatility of this format, we characterized the expression, binding, and stability of several previously identified soluble human VH domains. By grafting these domains onto an IgG scaffold, we generated several prototype 2xVH IgG and Fab molecules that display similar properties to mAbs. These molecules avoided the post-expression purification necessary for engineered bispecifics while maintaining a capacity for simultaneous dual binding. Hence, the 2xVH format represents a bivalent, bispecific design that addresses limitations of manufacturing IgG-like bispecifics while promoting biologically-relevant dual target engagement.

Human antibody repertoire data captured through next-generation sequencing (NGS) has enabled deeper insights into B cell immunogenetics and paratope diversity. By analyzing large public NGS datasets, we map the landscape of non-canonical cysteines in human variable heavy-chain domains (V H s) at the repertoire level. We identify remarkable usage of non-canonical cysteines within the heavy-chain complementarity-determining region 3 (CDR-H3) and other CDRs and framework regions. Furthermore, our study reveals the diversity and location of non-canonical cysteines and their associated motifs in human V H s, which are reminiscent of and more complex than those found in other non-human species such as chicken, camel, llama, shark, and cow. These results explain how non-canonical cysteines strategically occur in the human antibodyome to expand its paratope space. This study will guide the design of human antibodies harboring disulfide-stabilized long CDR-H3s to access difficult-to-target epitopes and influence a paradigm shift in developability involving non-canonical cysteines., Competing Interests: Declaration of Interests The authors are employees of Sanofi., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

PD-1 blockade therapy has revolutionized cancer treatments. However, a substantial population of patients is unresponsive. To rescue unresponsive patients, the mechanism of unresponsiveness to PD-1 blockade therapy must be elucidated. Using a 'bilateral tumor model' where responsive and unresponsive tumors were inoculated into different sides of the mouse belly, we demonstrated that unresponsive tumors can be categorized into two groups: with and without systemic immunosuppressive property (SIP). The SIP-positive tumors released uncharacterized, non-proteinaceous small molecules that inhibited mitochondrial activation and T cell proliferation. By contrast, the SIP-negative B16 tumor escaped from immunity by losing MHC class I expression. Unresponsiveness of SIP-positive tumors was partially overcome by improving the mitochondrial function with a mitochondrial activator; this was not successful for B16, which employs immune ignorance. These results demonstrated that the 'bilateral tumor model' was useful for stratifying tumors to investigate the mechanism of unresponsiveness and develop a strategy for proper combination therapy., Competing Interests: AK, KC, PC, TH No competing interests declared, (© 2020, Kumar et al.)

Although PD-1 blockade cancer immunotherapy has shown potential for a wide range of patients with cancer, its efficacy is limited, in part, due to the loss of effector cytotoxic T lymphocytes (CTLs) via terminal differentiation-induced apoptosis. We previously demonstrated that mitochondrial activation, by the agonists of peroxisome proliferator-activated receptor γ (PPARγ) coactivator 1-α (PGC-1α)/transcription factor complexes, had synergistic effects with a PD-1-blocking monoclonal antibody in a mouse tumor model. In the current study, we examined the molecular mechanism of the synergistic effects of bezafibrate, an agonist of PGC-1α/ PPAR complexes, which enhanced the tumoricidal effects of PD-1 blockade. Bezafibrate activated CTL mitochondria and upregulated oxidative phosphorylation as well as glycolysis, resulting in more proliferation of naïve T cells and improved effector function in CTLs. Bezafibrate also increased fatty acid oxidation (FAO) and mitochondrial respiratory capacity, which supports the extra energy demands of cells in emergencies, allowing cell survival. Carnitine palmitoyl transferase 1 (Cpt1), which is needed for FAO, and Bcl2 were both upregulated. Cpt1 and Bcl2 can form a complex to prevent apoptosis of CTLs. Together, these results indicate that bezafibrate increases or maintains the number of functional CTLs by activating mitochondrial and cellular metabolism, leading in turn to enhanced antitumor immunity during PD-1 blockade. Cancer Immunol Res; 6(11); 1375-87. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Programmed death 1 (PD-1) is an immune checkpoint molecule that negatively regulates T-cell immune function through the interaction with its ligand PD-L1. Blockage of this interaction unleashes the immune system to fight cancer. Immunotherapy using PD-1 blockade has led to a paradigm shift in the field of cancer drug discovery, owing to its durable effect against a wide variety of cancers with limited adverse effects. A brief history and development of PD-1 blockade, from the initial discovery of PD-1 to the recent clinical output of this therapy, have been summarized here. Despite its tremendous clinical success rate over other cancer treatments, PD-1 blockade has its own pitfall; a significant fraction of patients remains unresponsive to this therapy. The key to improve the PD-1 blockade therapy is the development of combination therapies. As this approach has garnered worldwide interest, here, we have summarized the recent trends in the development of PD-1 blockade-based combination therapies and the ongoing clinical trials. These include combinations with checkpoint inhibitors, radiation therapy, chemotherapy and several other existing cancer treatments. Importantly, FDA has approved PD-1 blockade agent to be used in combination with either CTLA-4 blockade or chemotherapy. Responsiveness to the PD-1 blockade therapy is affected by tumour and immune system-related factors. The role of the immune system, especially T cells, in determining the responsiveness has been poorly studied compared with those factors related to the tumour side. Energy metabolism has emerged as one of the important regulatory mechanisms for the function and differentiation of T cells. We have documented here the recent results regarding the augmentation of PD-1 blockade efficacy by augmenting mitochondrial energy metabolism of T cell., (© 2017 The Association for the Publication of the Journal of Internal Medicine.)

The human antibody repertoire is increasingly being recognized as a valuable source of therapeutic grade antibodies. However, methods for mining primary antibody-expressing B cells are limited in their ability to rapidly isolate rare and antigen-specific binders. Here we show the encapsulation of two million primary B cells into picoliter-sized droplets, where their cognate V genes are fused in-frame to form a library of scFv cassettes. We used this approach to construct natively paired phage-display libraries from healthy donors and drove selection towards cross-reactive antibodies targeting influenza hemagglutinin. Within 4 weeks we progressed from B cell isolation to a panel of unique monoclonal antibodies, including seven that displayed broad reactivity to different clinically relevant influenza hemagglutinin subtypes. Most isolated antibody sequences were not detected by next-generation sequencing of the paired repertoire, illustrating how this method can isolate extremely rare leads not likely found by existing technologies., Competing Interests: The authors declare to be or have been employed by AstraZeneca and own AstraZeneca stocks.

Background: Dipeptidyl peptidase-4 (DPP-4) may be a suitable biomarker to identify people with severe asthma who have greater activation of the interleukin-13 (IL-13) pathway and may therefore benefit from IL-13-targeted treatments. We report the analytical performance of an Investigational Use Only immunoassay and provide data on the biological range of DPP-4 concentrations., Methods: We assessed assay performance, utilising analyses of precision, linearity and sensitivity; interference from common endogenous assay interferents, and from asthma and anti-diabetic medications, were also assessed. The assay was used to measure the range of serum DPP-4 concentrations in healthy volunteers and subjects with diabetes and severe, uncontrolled asthma., Results: The total precision of DPP-4 concentration measurement (determined using percentage coefficient of variation) was ≤5% over 20 days. Dilution analysis yielded linear results from 30 to 1305 ng/mL; the limit of quantitation was 19.2 ng/mL. No notable endogenous or drug interferences were observed at the expected therapeutic concentration. Median DPP-4 concentrations in healthy volunteers and subjects with asthma or Type 1 diabetes were assessed, with concentrations remaining similar in subjects with diabetes and asthma across different demographics., Conclusion: These analyses indicate that the ARCHITECT DPP-4 Immunoassay is a reliable and robust method for measuring serum DPP-4 concentration.

Introduction: A precise puncture of the renal collecting system is the most essential step for percutaneous nephrolithotomy (PCNL). There are many techniques describing this crucial first step in PCNL including the bull's eye technique, triangulation technique, free-hand technique, and gradual descensus technique. We describe a novel puncture guide to assist accurate percutaneous needle placement during bull's eye technique., Methods: The mini access guide (MAG) stabilizes the initial puncture needle by mounting it on an adjustable multidirectional carrier fixed to the patient's skin, which aids in achieving the "bull's eye" puncture. It also avoids a direct fluoroscopic exposure of the urologist's hand during the puncture. Sixty consecutive patients with solitary renal calculus were randomized to traditional hand versus MAG puncture during bull's eye technique of puncture and the fluoroscopy time was assessed., Results: The median fluoroscopy screening time for traditional free-hand bull's eye and MAG-guided bull's eye puncture (fluoroscopic screening time for puncture) was 55 versus 21 s ( P = 0.001) and the median time to puncture was 80 versus 55 s ( P = 0.052), respectively. Novice residents also learned puncture technique faster with MAG on simulator., Conclusion: The MAG is a simple, portable, cheap, and novel assistant to achieve successful PCNL puncture. It would be of great help for novices to establish access during their learning phase of PCNL. It would also be an asset toward significantly decreasing the radiation dose during PCNL access., Competing Interests: There are no conflicts of interest.

Phage display antibody libraries are a rich resource for discovery of potential therapeutic antibodies. Single-chain variable fragment (scFv) libraries are the most common format due to the efficient display of scFv by phage particles and the ease by which soluble scFv antibodies can be expressed for high-throughput screening. Typically, a cascade of screening and triaging activities are performed, beginning with the assessment of large numbers of E. coli-expressed scFv, and progressing through additional assays with individual reformatting of the most promising scFv to full-length IgG. However, use of high-throughput screening of scFv for the discovery of full-length IgG is not ideal because of the differences between these molecules. Furthermore, the reformatting step represents a bottle neck in the process because each antibody has to be handled individually to preserve the unique VH and VL pairing. These problems could be resolved if populations of scFv could be reformatted to full-length IgG before screening without disrupting the variable region pairing. Here, we describe a novel strategy that allows the reformatting of diverse populations of scFv from phage selections to full-length IgG in a batch format. The reformatting process maintains the diversity and variable region pairing with high fidelity, and the resulted IgG pool enables high-throughput expression of IgG in mammalian cells and cell-based functional screening. The improved process led to the discovery of potent candidates that are comparable or better than those obtained by traditional methods. This strategy should also be readily applicable to Fab-based phage libraries. Our approach, Screening in Product Format (SiPF), represents a substantial improvement in the field of antibody discovery using phage display.

Although immunotherapy by PD-1 blockade has dramatically improved the survival rate of cancer patients, further improvement in efficacy is required to reduce the fraction of less sensitive patients. In mouse models of PD-1 blockade therapy, we found that tumor-reactive cytotoxic T lymphocytes (CTLs) in draining lymph nodes (DLNs) carry increased mitochondrial mass and more reactive oxygen species (ROS). We show that ROS generation by ROS precursors or indirectly by mitochondrial uncouplers synergized the tumoricidal activity of PD-1 blockade by expansion of effector/memory CTLs in DLNs and within the tumor. These CTLs carry not only the activation of mechanistic target of rapamycin (mTOR) and AMP-activated protein kinase (AMPK) but also an increment of their downstream transcription factors such as PPAR-gamma coactivator 1α (PGC-1α) and T-bet. Furthermore, direct activators of mTOR, AMPK, or PGC-1α also synergized the PD-1 blockade therapy whereas none of above-mentioned chemicals alone had any effects on tumor growth. These findings will pave a way to developing novel combinatorial therapies with PD-1 blockade., Competing Interests: The authors declare no conflict of interest.

Background: Periostin is being investigated as a potential biomarker for T-helper-2 (Th2)-driven asthma or eosinophilic inflammation and may help to identify patients more likely to benefit from interleukin-13-targeted treatments. We report the development and analytic performance of the investigational use only ARCHITECT Periostin Immunoassay, a new automated assay developed to detect serum periostin concentrations., Methods: We assessed assay performance in terms of precision, sensitivity, linearity, interference from classical immunoassay interferents and representatives of common asthma medications, specimen handling, and isoform reactivity. The assay was also used to assess the biological variability of serum periostin concentrations in samples from healthy volunteers and from subjects with uncontrolled asthma (the intended use population)., Results: The percentage CVs for 5-day total precision, assessed using two instruments, was <6% across 2 controls and one serum-based panel. Limit of quantitation was 4ng/mL (dilution adjusted concentration), suiting the needs for this application. Dilution analysis yielded linear results and no endogenous sample or drug interferences were observed. All known periostin isoforms expressed in the mature human lung were detected by the assay., Conclusion: Our studies provide support that the ARCHITECT Periostin Immunoassay is a reliable and robust test for measuring serum periostin concentrations., (Copyright © 2016. Published by Elsevier B.V.)

Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool.

The increasing incidence of Klebsiella pneumoniae infections refractory to treatment with current broad-spectrum antibiotic classes warrants the exploration of alternative approaches, such as antibody therapy and/or vaccines, for prevention and treatment. However, the lack of validated targets shared by spectrums of clinical strains poses a significant challenge. We adopted a target-agnostic approach to identify protective antibodies against K. pneumoniae Several monoclonal antibodies were isolated from phage display and hybridoma platforms by functional screening for opsonophagocytic killing activity. We further identified their common target antigen to be MrkA, a major protein in the type III fimbriae complex, and showed that these serotype-independent anti-MrkA antibodies reduced biofilm formation in vitro and conferred protection in multiple murine pneumonia models. Importantly, mice immunized with purified MrkA proteins also showed reduced bacterial burden following K. pneumoniae challenge. Taken together, these results support MrkA as a promising target for K. pneumoniae antibody therapeutics and vaccines., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

Context: Bombay blood group although rare is found to be more prevalent in the Western and Southern states of India, believed to be associated with consanguineous marriage., Aims: To estimate the prevalence of the Bombay blood group (Oh) in the urban population of Puducherry. To find the effect of urbanization on consanguineous marriage and to establish whether consanguinity plays a part in the prevalence of Oh group. To compare Oh group prevalence with that of other neighboring states, where population is not predominantly urban., Settings and Design: This is a descriptive study in a tertiary care hospital in Puducherry, over a period of 6 years., Materials and Methods: All blood samples showing 'O' group were tested with anti-H lectin. Specialized tests like Adsorption Elution Technique, inhibition assay for determination of secretor status were performed on Oh positive cases. Any history of consanguineous marriage was recorded., Statistical Analysis Used: All variables were categorical variable and percentage and proportions were calculated manually., Results: Analysis of the results of 35,497 study subjects showed that the most common group was 'O' group constituting 14,164 (39.90%) of subjects. Only three "Oh" that is, Bombay phenotype (0.008%) were detected. Consanguinity was observed in two cases (66.66%)., Conclusions: This study shows the prevalence of Bombay blood group representing the urban population of Puducherry, to be high (0.008%) and associated with consanguineous marriage (66.66%). Thus, consanguinity is still an important risk factor present, even in an urban population in Southern India.

Release of endogenous damage associated molecular patterns (DAMPs), including members of the S100 family, are associated with infection, cellular stress, tissue damage and cancer. The extracellular functions of this family of calcium binding proteins, particularly S100A8, S100A9 and S100A12, are being delineated. They appear to mediate their functions via receptor for advanced glycation endproducts (RAGE) or TLR4, but there remains considerable uncertainty over the relative physiological roles of these DAMPs and their pattern recognition receptors. In this study, we surveyed the capacity of S100 proteins to induce proinflammatory cytokines and cell migration, and the contribution RAGE and TLR4 to mediate these responses in vitro. Using adenoviral delivery of murine S100A9, we also examined the potential for S100A9 homodimers to trigger lung inflammation in vivo. S100A8, S100A9 and S100A12, but not the S100A8/A9 heterodimer, induced modest levels of TLR4-mediated cytokine production from human PBMC. In contrast, for most S100s including S100A9, RAGE blockade inhibited S100-mediated cell migration of THP1 cells and major leukocyte populations, whereas TLR4-blockade had no effect. Intranasal administration of murine S100A9 adenovirus induced a specific, time-dependent predominately macrophage infiltration that coincided with elevated S100A9 levels and proinflammatory cytokines in the BAL fluid. Inflammatory cytokines were markedly ablated in the TLR4-defective mice, but unexpectedly the loss of TLR4 signaling or RAGE-deficiency did not appreciably impact the S100A9-mediated lung pathology or the inflammatory cell infiltrate in the alveolar space. These data demonstrate that physiological levels of S100A9 homodimers can trigger an inflammatory response in vivo, and despite the capacity of RAGE and TLR4 blockade to inhibit responses in vitro, the response is predominately independent of both these receptors.

Monovalent bispecific IgGs cater to a distinct set of mechanisms of action but are difficult to engineer and manufacture because of complexities associated with correct heavy and light chain pairing. We have created a novel design, "DuetMab," for efficient production of these molecules. The platform uses knobs-into-holes (KIH) technology for heterodimerization of 2 distinct heavy chains and increases the efficiency of cognate heavy and light chain pairing by replacing the native disulfide bond in one of the CH1-CL interfaces with an engineered disulfide bond. Using two pairs of antibodies, cetuximab (anti-EGFR) and trastuzumab (anti-HER2), and anti-CD40 and anti-CD70 antibodies, we demonstrate that DuetMab antibodies can be produced in a highly purified and active form, and show for the first time that monovalent bispecific IgGs can concurrently bind both antigens on the same cell. This last property compensates for the loss of avidity brought about by monovalency and improves selectivity toward the target cell.

Introduction: Lower urinary tract symptoms in men, over age of 50 years is suggestive of benign prostatic enlargement (BPE). Different alpha-blockers have been evaluated for the treatment of benign prostatic hyperplasia for over last 30 years. This study was conducted in a tertiary care institution during the period of year between June 2011 and August 2013 to compare the effect of naftopidil and tamsulosin in reducing the obstructive and irritable symptoms of BPE., Subjects and Methods: A prospective randomized comparative study was carried on 60 patients of BPE by assigning half of them to treatment with tamsulosin and rest with naftopidil. Pre- and post-treatment uroflowmetry (UFM), post-void residue (PVR), International Prostate Symptoms Score (IPSS), were obtained at 15 and 30 days after starting treatment., Results: The age of patients ranged from 51 to 78. At base line there was no statistical difference between UFM parameter, PVR and IPSS in the two groups. UFM and PVR showed significantly better response at both intervals with naftopidil. Comparison of IPSS showed better improvement in Group A both at 15 and 30 days. It was seen that the obstructive symptoms showed a significantly better response with tamsulosin and symptoms of irritability was seen better response with naftopidil., Conclusion: It was seen that during the period of follow-up of 30 days naftopidil had a better effect on UFM, PVR, IPSS compared with tamsulosin. In general, obstructive symptoms showed better improvement in tamsulosin and irritative symptoms showed better improvement in naftopidil.

Bispecific antibodies constitute a valuable class of therapeutics owing to their ability to bind 2 distinct targets. Dual targeting is thought to enhance biological efficacy, limit escape mechanisms, and increase target selectivity via a strong avidity effect mediated by concurrent binding to both antigens on the surface of the same cell. However, factors that regulate the extent of target selectivity are not well understood. We show that dual targeting alone is not sufficient to promote efficient target selectivity, and report the substantial roles played by the affinity of the individual arms, overall avidity and valence. More particularly, various monovalent bispecific IgGs composed of an anti-CD70 moiety paired with variants of the anti-CD4 mAb ibalizumab were tested for preferential binding and selective depletion of CD4(+)/CD70(+) T cells over cells expressing only one of the target antigens that resulted from antibody dependent cell-mediated cytotoxicity. Variants exhibiting reduced CD4 affinity showed a greater degree of target selectivity, while the overall efficacy of the bispecific molecule was not affected.

Introduction: The correction of fossa navicularis strictures poses a distinct reconstructive challenge as it requires attention to cosmesis, in addition to urethral patency. Different graft and flap based repairs have been described with variable success rates. However, the ideal management remains unclear. The feasibility and efficacy of a single stage ventral onlay buccal mucosa graft urethroplasty (VOBMGU) for navicular fossa strictures (NFS) was evaluated in the present study., Subjects and Methods: All patients with NFS attending urology out-patient department from March, 2009 onward accepting VOBMGU were evaluated prospectively. Patients with minimum 1 year of follow-up were included for analysis. The technique involves opening the diseased stenosed meatus ventrally up to the corona. The diseased mucosa is excised leaving a midline strip of native urethral mucosa on the dorsal side. The buccal mucosal graft (BMG) is fixed on either side of this strip over a 24 Fr. silicone catheter. The glans wings are apposed in midline taking anchoring bites on the mucosal graft ventrally. Post-operatively patients were reviewed at 1, 3, 6 and 12 months and annually thereafter. Cosmetic acceptance and splaying of the urinary stream was assessed with individual questionnaires., Results: A total of six patients underwent VOBMGU. Average flow rate at 3 months post-operatively was 12 ml/s. The end result was cosmetically highly acceptable. There was no fistula in any of the cases. With a median follow-up of 37 months, only one patient had a recurrence of stricture in a proximal site., Conclusions: VOBMGU is a viable technique for reconstruction of NFS with promising short term results. However, long-term follow-up is necessary.

Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE-DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo.

Monoclonal antibodies targeting the extracellular region of the human IgE heavy chain membrane-tethering domain have been proposed for treating allergies caused by hyperproliferative monoclonal expansion of IgE-producing B cells. Antibodies against this target are expected to deplete membrane IgE (mIgE) displaying B cells and leave B cells of other immunoglobulin isotypes intact. Because of alternative splicing, the mIgE heavy chain has two isoforms that differ in their membrane-proximal segment. In the long isoform, the CH4 domain is followed by a 67-amino acid-long extracellular portion. Out of these 67 amino acids, the first 52 amino acids following the CH4 domain constitute the CɛmX segment while the rest of the 15 amino acids immediately adjacent to the membrane constitute the ɛ-migis. In the short isoform the CɛmX segment is absent and the CH4 domain is followed only by the 15-amino acid-long ɛ-migis segment. Using antibodies derived from a phage display library, we investigated: (1) ɛ-migis and (2) the junction of CɛmX and ɛ-migis (CɛmX.migis), as potential therapeutic antibody targets. Our results indicate that antibodies obtained from our phage library that target ɛ-migis bind to a variety of human cells irrespective of mIgE expression, possibly due to homology between ɛ-migis and a region of phosphoinositide-binding protein (ARAP3). In contrast, antibodies specific for the CɛmX.migis junctional region, bound specifically to transfected and primary B cells expressing human mIgE and elicited antibody-dependent cellular cytotoxicity and reduction in IgE production. These antibodies did not bind secreted IgE or the mIgE isoform in which CɛmX is absent. These results suggest that CɛmX.migis junctional region is a promising antibody target and the human antibodies we describe warrant further evaluation., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Resistance to VEGF inhibitors is emerging as a major clinical problem. Notch signaling has been implicated in tumor angiogenesis. Therefore, to investigate mechanisms of resistance to angiogenesis inhibitors, we transduced human glioblastoma cells with retroviruses encoding Notch delta-like ligand 4 (DLL4), grew them as tumor xenografts and then treated the murine hosts with the VEGF-A inhibitor bevacizumab. We found that DLL4-mediated tumor resistance to bevacizumab in vivo. The large vessels induced by DLL4-Notch signaling increased tumor blood supply and were insensitive to bevacizumab. However, blockade of Notch signaling by dibenzazepine, a γ-secretase inhibitor, disrupted the large vessels and abolished the tumor resistance. Multiple molecular mechanisms of resistance were shown, including decreased levels of hypoxia-induced VEGF and increased levels of the VEGF receptor VEGFR1 in the tumor stroma, decreased levels of VEGFR2 in large blood vessels, and reduced levels of VEGFR3 overall. DLL4-expressing tumors were also resistant to a VEGFR targeting multikinase inhibitor. We also observed activation of other pathways of tumor resistance driven by DLL4-Notch signaling, including the FGF2-FGFR and EphB4-EprinB2 pathways, the inhibition of which reversed tumor resistance partially. Taken together, our findings show the importance of classifying mechanisms involved in angiogenesis in tumors, and how combination therapy to block DLL4-Notch signaling may enhance the efficacy of VEGF inhibitors, particularly in DLL4-upregulated tumors, and thus provide a rational base for the development of novel strategies to overcome antiangiogenic resistance in the clinic.

The effect of dopant concentration on the blue upconversion (UPC) emission of Tm(3+) -doped ZrO(2) nanocrystals under different excitation wavelengths in the red region is reported. The UPC emissions are due to the f-f electronic transitions from excited states (1)G(4) and (1)D(2) of Tm(3+). We observed a chromatic change in the UPC with tuning the excitation wavelength. The UPC emission bands at 475, 488, and 501 nm are observed under excitation at 649 nm, but bands centered at 454 and 460 nm are observed when the excitation wavelength is tuned to 655 nm. The UPC emission could be tuned from 501 to 454 nm ( approximately 47 nm) by changing the excitation wavelength from 649 to 655 nm ( approximately 6 nm). The pump power dependence of the emission bands at 475, 488, and 501 nm were investigated on excitation intensity at 649 nm, and the emission bands at 454 and 460 nm are investigated on excitation intensity at 655 nm, which confirms that all of these UPC emission lines are a two-photon absorption process.

Therapeutic antibodies represent one of the fastest growing areas of the pharmaceutical industry. There are currently 18 monoclonal antibodies in the market that have been approved by the FDA and over 150 in clinical developments. Driven by innovation and technological developments, scientists have gone beyond the traditional antibody molecules. Antibodies have been engineered in a variety of ways to meet the challenges posed by different biological settings. Described in this review is an abridged account of the different ways antibodies have been tailored to make them efficient drug molecules.

Here, we report the role of crystal structure and crystal size on the photoluminescence properties of Ce3+ ions in Y2SiO5 nanocrystals. The emission at 430 nm (5d1 --> 4f1) and lifetime of the excited state of Ce3+ ion doped Y2SiO5 nanocrystals are found to be sensitive to the crystal structure, crystal size, and dopant concentration. It is found that the overall lifetime tau of 0.5 mol % Ce doped Y2SiO5 nanocrystals are 8.78 and 3.45 ns for 1000 and 1100 degrees C heat-treated samples with the same crystal structure (X1-Y2SiO5 phase), respectively. However, a significant increase in the overall lifetime (35.21 ns) is observed for the 1300 degrees C annealed 0.5 mol % Ce doped Y2SiO5 sample having a different crystal structure (X2-Y2SiO5 phase). We found that the decay kinetic is biexponential. It is explained that the fast component arises due to sequential hole-electron capture on the luminescent ions and the slow component arises from isolated ions. Our analysis suggests that modifications of radiative and nonraditive relaxation mechanisms are due to local symmetry structure of the host lattice and crystal size, respectively.

With the aim of identifying tumor-associated antigens that could be potential markers and/or targets of diagnostic and/or therapeutic approaches, we studied the occurrence of circulating IgG antibodies to human stromelysin-3, collagenase-3, galectin-3 and mesothelin, by Western blot against their purified recombinant forms, in the sera of 50 patients with pharynx/larynx squamous cell carcinoma (PLSCC), as well as in the sera of 50 healthy blood donors. Overall, antibodies to collagenase-3 were detected in 50% of all the cancer patients and 16% of the blood donors examined; this percentage difference was statistically significant (p = 0.00066). With respect to anti-galectin-3 antibodies, the percentages were 32% and 18%, respectively, but they were not statistically different (p = 0.16). Low levels of antibodies to stromelysin-3 and to mesothelin were detected in sera from only two cancer patients. No significant correlations were found in the present study between the presence of antibodies to these proteins and tumor site, clinical and T stages, lymph node involvement, DNA ploidy and histological grade of differentiation of the primary tumors. To our knowledge, this is the first report on the detection of circulating IgG to collagenase-3 in cancer patients. Some of the percentages found here in certain groups of patients are among the highest reported of circulating antibodies to any tumor component studied so far. The monitoring and the use of human antibodies to collagenase-3 could be of diagnostic and therapeutic interest.

To target CD30 on Hodgkin's disease and anaplastic large-cell lymphoma, anti-CD30 single-chain antibodies were obtained by DNA immunization of mice with the complete human CD30 cDNA. Spleens were isolated from mice with high anti-CD30 titer, and the RNA was used for the production of an scFv-displaying phage library. Specific phages were enriched by 3 rounds of panning on soluble CD30 or CD30+ K562 cells. Recombinant immunotoxins (rITs) were made from 3 ELISA-positive scFv phages by fusion to a 38 kDa truncated mutant of Pseudomonas exotoxin (PE38) with or without a KDEL mutant sequence at the C terminus. In vitro cytotoxicity of purified anti-CD30 rITs was measured on CD30-transfected A431 cells. IC50 values ranged from 3 to 7 ng/ml (50-110 pM) for PE38 rITs and 0.1 ng/ml (2 pM) for the PE38-KDEL IT on A431-CD30 cells. The parental A431 cells were resistant, indicating that the cytotoxicity was specific and CD30-mediated. rITs were tested for anti-tumor activity in a nude mouse model. A431-CD30 cells were injected s.c. on day 0; then, mice bearing measurable tumors were treated beginning on day 4 with 3 alternate daily doses i.v. Anti-tumor activity was dose-dependent and not found when irrelevant ITs were administered or when CD30- tumors were treated. Our data show that DNA immunization and antibody phage display may be useful in producing new rITs against hematologic malignancies. Published 2001 Wiley-Liss, Inc.

Mesothelin is a GPI-linked, membrane-associated differentiation antigen that is over-expressed in several forms of human cancers. Intradermal injection into rabbits of plasmid DNA encoding full length mesothelin resulted in antisera with titers as high as 1:100,000. Each immunization consisted of 320 microg of DNA delivered into 4 sites. After the initial three injections antisera titers were moderate (between 10 to 30,000) and fell over the course of about 7 weeks. When the titers had fallen, an injection of a booster dose of DNA resulted in very high titers of antisera. These antisera contained IgGs that could bind to both recombinant mesothelin made in Eschericha coli and to mesothelin present on human cells in Western blots and in immunofluorescence assays. These observations indicate that simple intradermal DNA immunization of rabbits can result in high titers of antibodies that can be used for a variety of purposes.

Background: Generation and cloning of antibodies against cell surface antigens can be simplified by combining DNA immunization which enables generation of antibodies against a protein in its natural configuration without the need for any protein purification step and antibody phage display which due to its immense screening power and physical coupling between the phenotype and genotype of antibodies simplifies the cloning of antibody genes., Objectives: Since DNA immunization is expected to elicit antibodies against a protein in its natural configuration, we wanted to see if it can mimic the antibody response generated by cell immunization., Study Design: A phage display library made from splenic mRNA of a mouse immunized with mesothelin cDNA was panned on mesothelin-positive cells. The single-chain Fvs (scFvs) selected were then analyzed., Results: We obtained several anti-mesothelin scFvs. One of these Fvs is almost identical to the Fv of a monoclonal antibody that was previously obtained from a hybridoma in which the mice were immunized with a mesothelin-positive ovarian cancer cell line. Another Fv was found to be specific for mesothelin present on human cells., Conclusion: Our results indicate that an antibody phage display library made from spleens of DNA-immunized mice is a rapid and efficient alternative to cell immunization for obtaining antibodies against different epitopes of a membrane antigen that is very difficult to purify in a native form.

In vivo affinity maturation of antibodies involves mutation of hot spots in the DNA encoding the variable regions. We have used this information to develop a strategy to improve antibody affinity in vitro using phage display technology. In our experiment with the antimesothelin scFv, SS(scFv), we identified DNA sequences in the variable regions that are naturally prone to hypermutations, selected a few hot spots encoding nonconserved amino acids, and introduced random mutations to make libraries with a size requirement between 10(3) and 10(4) independent clones. Panning of the hot spot libraries yielded several mutants with a 15- to 55-fold increase in affinity compared with a single clone with a fourfold increased affinity from a library in which mutagenesis was done outside the hot spots. The strategy should be generally applicable for the rapid isolation of higher-affinity mutants of Fvs, Fabs, and other recombinant antibodies from antibody phage libraries that are small in size.

The conversion of the anti-mesothelin monoclonal antibody K1 to a single-chain Fv (scFv) that is fused to a truncated form of Pseudomonas exotoxin A (PE) results in a fusion protein (immunotoxin) that is unstable and refolds very inefficiently. We have devised a method that identifies candidate residues in the framework region of K1 Fv that, when mutated, improved the yield and stability of the protein. The method works by initially aligning the framework sequences of K1 VH and VL with those of other scFvs that are stable and give a good yield as immunotoxins. Then we assigned a character to each residue that indicates its state of exposure based on the known crystal structures of Fabs. This identifies residues that are not compatible with their environment in the folded state of the protein. Next we calculated the frequencies of different amino acids for each position of the Fvs based on the available sequence database. This identifies residues that are not commonly present in the conserved positions. If these residues are compatible with their exposure profile they are left unaltered. Otherwise, they are identified as candidate residues for mutation. We identified two such residues in the VH (T82 and A85) and two in the VL (H36 and V60) of K1 that did not seem appropriate for their respective positions. By mutating these residues in K1 into those that occur most commonly in the sequence database or in stable scFvs, we significantly improved the stability and yield of the K1 scFv immunotoxins. By making single and combined mutations we assessed the relative contribution of mutations at these four sites towards the stability and yield of K1 scFv immunotoxins. The method we devised is probably general and can be used to improve other scFvs., (Copyright 1998 Academic Press)

Mesothelin is a differentiation antigen present on the surface of ovarian cancers, mesotheliomas, and several other types of human cancers. Because among normal tissues, mesothelin is present only on mesothelial cells, it represents a good target for antibody-mediated delivery of cytotoxic agents. In the present study mice were immunized with an eukaryotic expression vector coding for mesothelin. When high serum antibody titers were obtained, a phage display library was made from the splenic mRNA of these mice. After three rounds of panning on recombinant mesothelin, a single-chain Fv (scFv)-displaying phage was selected that bound specifically to recombinant mesothelin and mesothelin-positive cells. The scFv was used to construct an immunotoxin by genetically fusing it with a truncated mutant of Pseudomonas exotoxin A. The purified immunotoxin binds mesothelin with high affinity (Kd 11 nm), is stable for over 40 hr at 37 degrees C and is very cytotoxic to cells expressing mesothelin. It also produces regressions of tumors expressing mesothelin. This combination of selective cytotoxicity, high activity, and stability makes the immunotoxin a good candidate for development as a therapeutic agent. This work also shows that DNA immunization can be used to isolate and clone antibodies against epitopes present on human proteins in their native conformation.

Phage display has been used to select single-chain Fvs (scFvs) against mesothelin, a surface antigen present on mesothelial cells as well as mesotheliomas and non-mucinous ovarian cancers. Several attempts to produce anti-mesothelin hybridomas from spleen cells of mice immunized with recombinant mesothelin were unsuccessful. This report describes the isolation of anti-mesothelin scFvs from a phage display library made from the mRNA of the same spleens. Panning on recombinant antigen produced in E. coli or on antigen positive cells was employed. Several scFvs which bind specifically to mesothelin were isolated. Panning on recombinant antigen yielded five different scFvs. Panning on cells selected two different scFvs which also differ from the scFvs selected by recombinant antigen. The heavy chains of the scFvs selected on recombinant antigen are derived from four different heavy chain gene families and the scFvs selected on cells are derived from two of these families. In contrast, the light chains of all of these scFvs are derived from family XI. Moreover, the light chains of the two scFvs selected on cells are very similar to the light chains of two of the scFvs selected by panning on recombinant mesothelin except for a few point mutations. One of these scFvs which have been studied in detail has been shown to bind specifically to mesothelin positive transfected cells.

Phage display of single-chain Fvs (scFvs) is a powerful tool to enrich and isolate specific antibody fragments from large pools (libraries) of Fv coding genes. However, many scFvs and scFv fusion proteins are unstable, not only as soluble proteins but also on the surface of phage. This limits and biases the recovery of specific Fv phage from display libraries to relatively stable scFvs. Also, the peptide linker in scFvs can diminish antigen binding of scFvs and scFv-fusion proteins. Disulfide-stabilized Fvs (dsFv) which have the VH-VL heterodimer stabilized by an interchain disulfide bond connecting framework regions in VH and VL rather than a peptide linker are more stable than scFvs and in some instances show better binding. To analyze whether these advantages can be utilized in a phage display system and to prove the feasibility of dsFv display, we constructed phage for dsFv display of the anti-Tac antibody and a dsFv-phage library. We find that dsFv phage can specifically bind antigen although the titer of dsFv phage in supernatants appears to be reduced compared to scFv phage. But this reduction in titer does not hamper the isolation of dsFv phages from large pools (libraries) as demonstrated by 'panning' of anti-Tac scFv and dsFv phages on living leukemia cells in suspension. In addition, dsFv phage are more stable than scFv phage. Therefore, display of dsFvs on phage is a useful alternative and addition to scFv-phage display.

Staphylococcal protein A (SPA) is ranked as a versatile probe in immunoassays because of its immunoglobulin G (IgG)-binding capability. However, poor binding of SPA to the IgG of some laboratory animals and its inability to bind human IgG3 restricts its universal utility. In the present study, DNA encoding the four IgG-binding domains of SPA (E, D, A and B) or the B domain alone has been fused, in separate phagemid vectors, to the 5' end of gene 111 of the phage M13. Upon infection by helper phage M13KO7, phagemid particles encapsulating single-stranded DNA were produced. Dot immunoblot and Western blot analyses showed the presence of fusion proteins on the M13 surface. Binding of rabbit IgG-horseradish peroxidase (IgG-HRP) complex to the phage particles confirmed that the fusion proteins possessed functional IgG-binding domains. The interaction of these phages with immobilised human IgG and its various subclasses was studied by the phage capture immunoassay where the captured phages were detected by a monoclonal antibody to the major coat protein encoded by gene VIII (gVIII). The phages showed maximal binding to IgG1 kappa, followed by IgG2 kappa, and showed negligible binding to the IgG3 kapa and IgG3 lambda subclasses. The specificity of IgG-binding phages was confirmed in a phage capture and elution assay where the binding of these phages to immobilised human IgG1 kapa weas abolished in the presence of excess of soluble protein A. Moreover, IgG-binding phages could be enriched approx. 1000-fold over non-specific phages in a single round of panning.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of catecholamines from chromaffin cells is mediated by cholinergic and peptidergic neurotransmitters. The cholinergic transmitter acetylcholine activates both nicotinic and muscarinic receptors to trigger catecholamine secretion in rat adrenal medulla. Vasoactive intestinal polypeptide (VIP) has been identified as the peptidergic transmitter in rat adrenal medulla and may also be the non-cholinergic transmitter in bovine adrenal. Pituitary adenylate cyclase activating polypeptide (PACAP), a VIP-like secretin peptide, is also found in the adrenal, and is a potent secretagogue. Thus, PACAP may be another peptidergic transmitter at the adrenal synapse. A most intriguing property of rat chromaffin cells is that stimulation of nicotinic, muscarinic, VIP or PACAP receptors are each able to produce robust catecholamine secretion on their own. This raises the question of whether a single chromaffin cell can respond to each of the above agonists or whether the secretion is due to subpopulations of chromaffin cells. This issue was addressed by using electrochemical techniques to monitor exocytosis from individual chromaffin cells in culture. We demonstrate that acetylcholine, nicotine, muscarine, VIP and PACAP are each able to evoke catecholamine secretion from a single chromaffin cell. Some cells only responded to acetylcholine. Furthermore, each agonist produced a distinct pattern of exocytosis. Muscarine-evoked secretion exhibited a latency of 0.5-2 s, but exocytosis persisted up to 30 s following 500 ms stimulation. Nicotine produced an immediate response which usually ended within 10 s. The secretory pattern following acetylcholine appeared to be the sum of the nicotinic and muscarinic patterns, showing both rapid onset and longer duration.(ABSTRACT TRUNCATED AT 250 WORDS)