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Background: The ability of mesenchymal stromal cells (MSCs) to facilitate regenerative responses in inflamed and injured tissues, coupled with preclinical data suggesting potential to restore defective collagen VII at the dermo-epidermal junction, has raised the hope that MSCs may provide an effective disease-modifying therapy for patients suffering from recessive dystrophic epidermolysis bullosa (RDEB)., Methods: We present a descriptive analysis of the clinical research on systemic MSC administration to RDEB patients available in PubMed, including six early-phase studies and one case report, involving 59 patients who received 1-3 intravenous infusions of MSCs from various sources., Results: Based on 133 MSC infusions, a total of 44 mostly mild adverse events were reported as definitely, possibly or likely related to the study treatment, only two of which led to treatment discontinuation. Improvements were seen in skin manifestations, disease activity, pain, pruritus and quality of life, with considerable heterogeneity in reported outcome variables and measurement tools between studies, and large inter-patient variability within studies., Conclusions: Although the current evidence base is limited, reflecting the typical challenges of clinical research in rare diseases, the reported results suggest potential treatment benefits for patients and provide a rationale for continuing to pursue this therapeutic approach.

Background: Given the high level of product complexity and limited regulatory guidance, designing and implementing appropriate potency assays is often the most challenging part of establishing a quality control testing matrix for a cell-based medicinal product. Among the most elusive tasks are the selection of suitable read-out parameters, the development of assay designs that most closely model the pathophysiological conditions, and the validation of the methods. Here we describe these challenges and how they were addressed in developing an assay that measures the anti-inflammatory potency of mesenchymal stromal cells (MSCs) in an M1 macrophage-dominated inflammatory environment., Methods: An in vitro inflammation model was established by coculturing skin-derived ABCB5 + MSCs with THP-1 monocyte-derived M1-polarized macrophages. Readout was the amount of interleukin 1 receptor antagonist (IL-1RA) secreted by the MSCs in the coculture, measured by an enzyme-linked immunosorbent assay., Results: IL-1RA was quantified with guideline-concordant selectivity, accuracy and precision over a relevant concentration range. Consistent induction of the macrophage markers CD36 and CD80 indicated successful macrophage differentiation and M1 polarization of THP-1 cells, which was functionally confirmed by release of proinflammatory tumor necrosis factor α. Testing a wide range of MSC/macrophage ratios revealed the optimal ratio for near-maximal stimulation of MSCs to secrete IL-1RA, providing absolute maximum levels per individual MSC that can be used for future comparison with clinical efficacy. Batch release testing of 71 consecutively manufactured MSC batches showed a low overall failure rate and a high comparability between donors., Conclusions: We describe the systematic development and validation of a therapeutically relevant, straightforward, robust and reproducible potency assay to measure the immunomodulatory capacity of MSCs in M1 macrophage-driven inflammation. The insights into the challenges and how they were addressed may also be helpful to developers of potency assays related to other cellular functions and clinical indications., Competing Interests: Declaration of Competing of interest MHF is inventor or coinventor of U.S. and international patents assigned to Brigham and Women's Hospital and/or Boston Children's Hospital, licensed to RHEACELL GmbH & Co. KG. MHF and KS-K serve as scientific advisors to RHEACELL. MHF holds stock in RHEACELL. SS, LN, EN-R, AN, SH, LV and JE are employees of RHEACELL. CG is CEO, and MAK is COO of RHEACELL., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Quantitative polymerase chain reaction (qPCR) has emerged as an important bioanalytical method for assessing the pharmacokinetics of human-cell-based medicinal products after xenotransplantation into immunodeficient mice. A particular challenge in bioanalytical qPCR studies is that the different tissues of the host organism can affect amplification efficiency and amplicon detection to varying degrees, and ignoring these matrix effects can easily cause a significant underestimation of the true number of target cells in a sample. Here, we describe the development and drug regulatory-compliant validation of a TaqMan ® qPCR assay for the quantification of mesenchymal stromal cells in the range of 125 to 20,000 cells/200 µL lysate via the amplification of a human-specific, highly repetitive α-satellite DNA sequence of the chromosome 17 centromere region HSSATA17. An assessment of matrix effects in 14 different mouse tissues and blood revealed a wide range of spike recovery rates across the different tissue types, from 11 to 174%. Based on these observations, we propose performing systematic spike-and-recovery experiments during assay validation and correcting for the effects of the different tissue matrices on cell quantification in subsequent bioanalytical studies by multiplying the back-calculated cell number by tissue-specific factors derived from the inverse of the validated percent recovery rate.

Background and Aims: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary, rare, devastating and life-threatening skin fragility disorder with a high unmet medical need. In a recent international, single-arm clinical trial, treatment of 16 patients (aged 6-36 years) with three intravenous infusions of 2 × 10 6  immunomodulatory ABCB5 + dermal mesenchymal stromal cells (MSCs)/kg on days 0, 17 and 35 reduced disease activity, itch and pain. A post-hoc analysis was undertaken to assess the potential effects of treatment with ABCB5 + MSCs on the overall skin wound healing in patients suffering from RDEB., Methods: Documentary photographs of the affected body regions taken on days 0, 17, 35 and at 12 weeks were evaluated regarding proportion, temporal course and durability of wound closure as well as development of new wounds., Results: Of 168 baseline wounds in 14 patients, 109 (64.9%) wounds had closed at week 12, of which 63.3% (69 wounds) had closed already by day 35 or day 17. Conversely, 74.2% of the baseline wounds that had closed by day 17 or day 35 remained closed until week 12. First-closure ratio within 12 weeks was 75.6%. The median rate of newly developing wounds decreased significantly (P = 0.001) by 79.3%., Conclusions: Comparison of the findings with published data from placebo arms and vehicle-treated wounds in controlled clinical trials suggests potential capability of ABCB5 + MSCs to facilitate wound closure, prolongate wound recurrence and decelerate formation of new wounds in RDEB. Beyond suggesting therapeutic efficacy for ABCB5 + MSCs, the analysis might stimulate researchers who develop therapies for RDEB and other skin fragility disorders to not only assess closure of preselected target wounds but pay attention to the patients' dynamic and diverse overall wound presentation as well as to the durability of achieved wound closure and the development of new wounds., Trial Registration: Clinicaltrials.gov NCT03529877; EudraCT 2018-001009-98., Competing Interests: Declaration of Competing Interest NYF and MHF are inventors or co-inventors of U.S. and international patents assigned to Brigham and Women's Hospital, Boston Children's Hospital, and/or the VA Boston Healthcare System, Boston, Massachusetts, USA, licensed to TICEBA GmbH, Heidelberg, Germany, and RHEACELL GmbH & Co. KG, Heidelberg, Germany. MHF serves as scientific advisor and holds stock in TICEBA GmbH and RHEACELL GmbH & Co. KG. DK and JT serve as scientific advisors to RHEACELL. KD, CD and SF are employees of RHEACELL. ENR is employee of TICEBA. CG is CEO, and MAK is CSO of TICEBA and RHEACELL. JAM declares that he has no conflicts of interest., (Copyright © 2023 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Recessive dystrophic epidermolysis (RDEB) is a rare, inherited, and currently incurable skin blistering disorder characterized by cyclically recurring wounds coexisting with chronic non-healing wounds. In a recent clinical trial, three intravenous infusions of skin-derived ABCB5 + mesenchymal stromal cells (MSCs) to 14 patients with RDEB improved the healing of wounds that were present at baseline. Since in RDEB even minor mechanical forces perpetually provoke the development of new or recurrent wounds, a post-hoc analysis of patient photographs was performed to specifically assess the effects of ABCB5 + MSCs on new or recurrent wounds by evaluating 174 wounds that occurred after baseline. During 12 weeks of systemic treatment with ABCB5 + MSCs, the number of newly occurring wounds declined. When compared to the previously reported healing responses of the wounds present at baseline, the newly occurring wounds healed faster, and a greater portion of healed wounds remained stably closed. These data suggest a previously undescribed skin-stabilizing effect of treatment with ABCB5 + MSCs and support repeated dosing of ABCB5 + MSCs in RDEB to continuously slow the wound development and accelerate the healing of new or recurrent wounds before they become infected or progress to a chronic, difficult-to-heal stage.

The ATP-binding cassette superfamily member ABCB5 identifies a subset of skin-resident mesenchymal stem cells (MSCs) that exhibit potent immunomodulatory and wound healing-promoting capacities along with superior homing ability. The ABCB5 + MSCs can be easily accessed from discarded skin samples, expanded, and delivered as a highly homogenous medicinal product with standardized potency. A range of preclinical studies has suggested therapeutic efficacy of ABCB5 + MSCs in a variety of currently uncurable skin and non-skin inflammatory diseases, which has been substantiated thus far by distinct clinical trials in chronic skin wounds or recessive dystrophic epidermolysis bullosa. Therefore, skin-derived ABCB5 + MSCs have the potential to provide a breakthrough at the forefront of MSC-based therapies striving to fulfill current unmet medical needs. The most recent milestones in this regard are the approval of a phase III pivotal trial of ABCB5 + MSCs for treatment of recessive dystrophic and junctional epidermolysis bullosa by the US Food and Drug Administration, and national market access of ABCB5 + MSCs (AMESANAR ® ) for therapy-refractory chronic venous ulcers under the national hospital exemption pathway in Germany.

Background: While rapid healing of diabetic foot ulcers (DFUs) is highly desirable to avoid infections, amputations and life-threatening complications, DFUs often respond poorly to standard treatment. GMP-manufactured skin-derived ABCB5 + mesenchymal stem cells (MSCs) might provide a new adjunctive DFU treatment, based on their remarkable skin wound homing and engraftment potential, their ability to adaptively respond to inflammatory signals, and their wound healing-promoting efficacy in mouse wound models and human chronic venous ulcers., Methods: The angiogenic potential of ABCB5 + MSCs was characterized with respect to angiogenic factor expression at the mRNA and protein level, in vitro endothelial trans-differentiation and tube formation potential, and perfusion-restoring capacity in a mouse hindlimb ischemia model. Finally, the efficacy and safety of ABCB5 + MSCs for topical adjunctive treatment of chronic, standard therapy-refractory, neuropathic plantar DFUs were assessed in an open-label single-arm clinical trial., Results: Hypoxic incubation of ABCB5 + MSCs led to posttranslational stabilization of the hypoxia-inducible transcription factor 1α (HIF-1α) and upregulation of HIF-1α mRNA levels. HIF-1α pathway activation was accompanied by upregulation of vascular endothelial growth factor (VEGF) transcription and increase in VEGF protein secretion. Upon culture in growth factor-supplemented medium, ABCB5 + MSCs expressed the endothelial-lineage marker CD31, and after seeding on gel matrix, ABCB5 + MSCs demonstrated formation of capillary-like structures comparable with human umbilical vein endothelial cells. Intramuscularly injected ABCB5 + MSCs to mice with surgically induced hindlimb ischemia accelerated perfusion recovery as measured by laser Doppler blood perfusion imaging and enhanced capillary proliferation and vascularization in the ischemic muscles. Adjunctive topical application of ABCB5 + MSCs onto therapy-refractory DFUs elicited median wound surface area reductions from baseline of 59% (full analysis set, n = 23), 64% (per-protocol set, n = 20) and 67% (subgroup of responders, n = 17) at week 12, while no treatment-related adverse events were observed., Conclusions: The present observations identify GMP-manufactured ABCB5 + dermal MSCs as a potential, safe candidate for adjunctive therapy of otherwise incurable DFUs and justify the conduct of a larger, randomized controlled trial to validate the clinical efficacy., Trial Registration: ClinicalTrials.gov, NCT03267784, Registered 30 August 2017, https://clinicaltrials.gov/ct2/show/NCT03267784., (© 2022. The Author(s).)

A significant number of chronic venous ulcers (CVUs) fail to heal despite of guideline-conform standard of care. Skin-derived ABCB5 + mesenchymal stem cells (MSCs) can dampen the sustained IL-1β-driven inflammation present in chronic wounds. Based on their wound healing-facilitating effects in a mouse CVU model and an autologous first-in-human study, ABCB5 + MSCs have emerged as a potential candidate for cell-based advanced therapy of non-healing CVUs. In the present interventional, multicenter, single-arm, phase I/IIa clinical trial, subjects whose CVU had emerged as standard therapy-resistant received one or two topical applications of 1×10 6 allogeneic ABCB5 + MSCs/cm 2 wound area in addition to standard treatment. Out of 83 treatment-emergent adverse events, only three were judged related to the cell product; they were mild or moderate and recovered without sequelae. Wound size markedly decreased from baseline to week 12, resulting in a median wound size reduction of 76% (full analysis set, N=31), 78% (per-protocol set, N=27) and 87% (subset of responders; n=21). In conclusion, the study treatment was well tolerated and safe. The treatment elicited a profound wound size reduction within 12 weeks, identifying ABCB5 + MSCs as a potential candidate for adjunctive therapy of otherwise incurable CVUs. These results justify the conduct of a larger, randomized, controlled trial to confirm clinical efficacy., Competing Interests: CONFLICT OF INTEREST NYF and MHF are inventors or co-inventors of US and international patents assigned to Brigham and Women’s Hospital and/or Boston Children’s Hospital, Boston, MA, USA, licensed to TICEBA GmbH, Heidelberg, Germany, and RHEACELL GmbH & Co. KG, Heidelberg, Germany. MHF and KSK serve as scientific advisors to TICEBA and RHEACELL and participate in corporate-sponsored research collaborations with RHEACELL. KD, AKD, KK and HS are employees of RHEACELL. ENR, JE, SB and SS are employees of TICEBA. CG is CEO, and MAK is CSO of RHEACELL and TICEBA. The remaining authors state no conflict of interest.

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a rare, devastating, and life-threatening inherited skin fragility disorder that comes about due to a lack of functional type VII collagen, for which no effective therapy exists. ABCB5+ dermal mesenchymal stem cells (ABCB5+ MSCs) possess immunomodulatory, inflammation-dampening, and tissue-healing capacities. In a Col7a1-/- mouse model of RDEB, treatment with ABCB5+ MSCs markedly extended the animals' lifespans.METHODSIn this international, multicentric, single-arm, phase I/IIa clinical trial, 16 patients (aged 4-36 years) enrolled into 4 age cohorts received 3 i.v. infusions of 2 × 106 ABCB5+ MSCs/kg on days 0, 17, and 35. Patients were followed up for 12 weeks regarding efficacy and 12 months regarding safety.RESULTSAt 12 weeks, statistically significant median (IQR) reductions in the Epidermolysis Bullosa Disease Activity and Scarring Index activity (EBDASI activity) score of 13.0% (2.9%-30%; P = 0.049) and the Instrument for Scoring Clinical Outcome of Research for Epidermolysis Bullosa clinician (iscorEB‑c) score of 18.2% (1.9%-39.8%; P = 0.037) were observed. Reductions in itch and pain numerical rating scale scores were greatest on day 35, amounting to 37.5% (0.0%-42.9%; P = 0.033) and 25.0% (-8.4% to 46.4%; P = 0.168), respectively. Three adverse events were considered related to the cell product: 1 mild lymphadenopathy and 2 hypersensitivity reactions. The latter 2 were serious but resolved without sequelae shortly after withdrawal of treatment.CONCLUSIONThis trial demonstrates good tolerability, manageable safety, and potential efficacy of i.v. ABCB5+ MSCs as a readily available disease-modifying therapy for RDEB and provides a rationale for further clinical evaluation.TRIAL REGISTRATIONClinicaltrials.gov NCT03529877; EudraCT 2018-001009-98.FUNDINGThe trial was sponsored by RHEACELL GmbH & Co. KG. Contributions by NYF and MHF to this work were supported by the NIH/National Eye Institute (NEI) grants RO1EY025794 and R24EY028767.

Background: While therapeutic success of the limbal tissue or cell transplantation to treat severe cases of limbal stem cell (LSC) deficiency (LSCD) strongly depends on the percentage of LSCs within the transplanted cells, prospective LSC enrichment has been hampered by the intranuclear localization of the previously reported LSC marker p63. The recent identification of the ATP-binding cassette transporter ABCB5 as a plasma membrane-spanning marker of LSCs that are capable of restoring the cornea and the development of an antibody directed against an extracellular loop of the ABCB5 molecule stimulated us to develop a novel treatment strategy based on the utilization of in vitro expanded allogeneic ABCB5 + LSCs derived from human cadaveric limbal tissue., Methods: We developed and validated a Good Manufacturing Practice- and European Pharmacopeia-conform production and quality-control process, by which ABCB5 + LSCs are derived from human corneal rims, expanded ex vivo, isolated as homogenous cell population, and manufactured as an advanced-therapy medicinal product (ATMP). This product was tested in a preclinical study program investigating the cells' engraftment potential, biodistribution behavior, and safety., Results: ABCB5 + LSCs were reliably expanded and manufactured as an ATMP that contains comparably high percentages of cells expressing transcription factors critical for LSC stemness maintenance (p63) and corneal epithelial differentiation (PAX6). Preclinical studies confirmed local engraftment potential of the cells and gave no signals of toxicity and tumorgenicity. These findings were sufficient for the product to be approved by the German Paul Ehrlich Institute and the U.S. Food & Drug Administration to be tested in an international multicenter phase I/IIa clinical trial (NCT03549299) to evaluate the safety and therapeutic efficacy in patients with LSCD., Conclusion: Building upon these data in conjunction with the previously shown cornea-restoring capacity of human ABCB5 + LSCs in animal models of LSCD, we provide an advanced allogeneic LSC-based treatment strategy that shows promise for replenishment of the patient's LSC pool, recreation of a functional barrier against invading conjunctival cells and restoration of a transparent, avascular cornea.

Background Aim: Mesenchymal stromal cells (MSCs) hold promise for the treatment of tissue damage and injury. However, MSCs comprise multiple subpopulations with diverse properties, which could explain inconsistent therapeutic outcomes seen among therapeutic attempts. Recently, the adenosine triphosphate-binding cassette transporter ABCB5 has been shown to identify a novel dermal immunomodulatory MSC subpopulation., Methods: The authors have established a validated Good Manufacturing Practice (GMP)-compliant expansion and manufacturing process by which ABCB5 + MSCs can be isolated from skin tissue and processed to generate a highly functional homogeneous cell population manufactured as an advanced therapy medicinal product (ATMP). This product has been approved by the German competent regulatory authority to be tested in a clinical trial to treat therapy-resistant chronic venous ulcers., Results: As of now, 12 wounds in nine patients have been treated with 5 × 10 5  autologous ABCB5 + MSCs per cm 2 wound area, eliciting a median wound size reduction of 63% (range, 32-100%) at 12 weeks and early relief of pain., Conclusions: The authors describe here their GMP- and European Pharmacopoeia-compliant production and quality control process, report on a pre-clinical dose selection study and present the first in-human results. Together, these data substantiate the idea that ABCB5 + MSCs manufactured as ATMPs could deliver a clinically relevant wound closure strategy for patients with chronic therapy-resistant wounds., (Copyright © 2020 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Background: Human dermal mesenchymal stromal cells (MSCs) expressing the ATP-binding cassette (ABC) efflux transporter ABCB5 represent an easily accessible MSC population that, based on preclinical and first-in-human data, holds significant promise to treat a broad spectrum of conditions associated not only with skin-related but also systemic inflammatory and/or degenerative processes., Methods: We have developed a validated Good Manufacturing Practice-compliant expansion and manufacturing process by which ABCB5 + MSCs derived from surgical discard skin tissues are processed to an advanced-therapy medicinal product (ATMP) for clinical use. Enrichment for ABCB5 + MSCs is achieved in a three-step process involving plastic adherence selection, expansion in a highly efficient MSC-selecting medium, and immunomagnetic isolation of the ABCB5 + cells from the mixed culture., Results: Product Quality Review data covering 324 cell expansions, 728 ABCB5 + MSC isolations, 66 ABCB5 + MSC batches, and 85 final drug products reveal high process robustness and reproducible, reliable quality of the manufactured cell therapy product., Conclusion: We have successfully established an expansion and manufacturing process that enables the generation of homogenous ABCB5 + MSC populations of proven biological activity manufactured as a standardized, donor-independent, highly pure, and highly functional off-the-shelf available ATMP, which is currently tested in multiple clinical trials.

Background Aims: Human dermal ABCB5-expressing mesenchymal stromal cells (ABCB5 + MSCs) represent a promising candidate for stem cell-based therapy of various currently uncurable diseases in several fields of regenerative medicine. We have developed and validated a method to isolate, from human skin samples, and expand ABCB5 + MSCs that meet the guideline criteria of the International Society for Cellular Therapy. We are able to process these cells into a Good Manufacturing Practice-conforming, MSC-based advanced-therapy medicinal product., Methods: To support the development of ABCB5 + MSCs for potential therapeutic topical, intramuscular and intravenous administration, we have tested our product in a series of Good Laboratory Practice-compliant nonclinical in-vivo studies addressing all relevant aspects of biosafety, including potential long-term persistence and proliferation, distribution to nontarget tissues, differentiation into undesired cell types, ectopic tissue formation, tumor formation and local tissue reaction., Results: (i) Subcutaneous application of 1 × 10 7 ABCB5 + MSCs/animal and intravenous application of 2 × 10 6 ABCB5 + MSCs/animal, respectively, to immunocompromised mice did not result in safety-relevant biodistribution, persistence or proliferation of the cells; (ii) three monthly subcutaneous injections of ABCB5 + MSCs at doses ranging from 1 × 10 5 to 1 × 10 7 cells/animal and three biweekly intravenous injections of 2 × 10 6 ABCB5 + MSCs/animal, respectively, to immunocompromised mice were nontoxic and revealed no tumorigenic potential; and (iii) intramuscular injection of 5 × 10 6 ABCB5 + MSCs/animal to immunocompromised mice was locally well tolerated., Discussion: The present preclinical in vivo data demonstrate the local and systemic safety and tolerability of a novel advanced-therapy medicinal product based on human skin-derived ABCB5 + MSCs., (Copyright © 2019 International Society for Cell and Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Although the molecular machinery and mechanism of cell secretion in acinar cells of the exocrine pancreas is well documented and clear, only recently has the pharmacophysiology of pancreatic exocrine secretion come to light. Therefore, we focus in this article on the current understanding of the pharmacophysiology of pancreatic exocrine secretion. The pancreatic secretory response to ingestion of a meal is mediated via a complex interplay of neural, humoral and paracrine mediators. A major role in the control of the intestinal phase of pancreatic secretion is attributed to vago-vagal enteropancreatic reflexes. In the scheme of this control mechanism, afferents originating in the duodenal mucosa, and efferents mediating central input on the pancreatic ganglia, activate intrapancreatic postganglionic neurons. Experiments utilizing specific receptor antagonists demonstrate the involvement of both muscarinic M1 and M3 receptors expressed in pancreatic acinar cells. Cholecystokinin (CCK), originally implicated in the humoral secretion of pancreatic enzymes, through a direct action on acinar CCK receptors, is also essential to the enteropancreatic reflex mechanism. CCK stimulation of the exocrine pancreatic secretion through excitation of sensory afferents of the enteropancreatic reflexes, is a paracrine mode of CCK action, and is probably the only one in humans and the predominant one in rats. In dogs, however, CCK acts on the pancreas via both the humoral and a paracrine route. More recent experiments suggest further possible sites of CCK action. Additionally, at the brain stem, vago-vagal enteropancreatic reflexes may be modulated by input from higher brain centres, particularly the hypothalamic-cholinergic system in the tonic stimulation of preganglionic neurons of the dorsal motor nucleus of the vagus projecting into the pancreas.

Objectives: To investigate whether unintended muscle activity induced by motor overflow can result in the unintentional discharge of a firearm., Background: In the past few years an increasing number of people have been injured, sometimes fatally, as a result of police weapons being discharged involuntarily., Methods: Two experiments were performed using a pistol fitted with sensors to register the pressure exerted on the hand stock and the trigger. In Study 1, 34 police officers reacted to a set case using the prepared pistol. In Study 2, 25 participants performed 13 tasks requiring the use of different limbs., Results: In Study 1, about 20% of participating officers made contact with the trigger, thus violating police regulations to keep the trigger finger outside the trigger guard at all times until a conscious decision to fire has been made. Study 2 showed that motor activity in different limbs leads to a significant increase in grip force exerted on a firearm (significant results for 12 of 13 trials, Bonferroni-corrected p < .004). Furthermore, pressure on the trigger resulting from involuntary muscle contractions was, in certain cases, sufficient to unintentionally discharge a weapon., Conclusion: Unintentional discharges attributable to involuntary muscle activity can be a real danger for police officers when carrying out their duties., Application: Results may help improve police training with the aim of reducing the danger of involuntary discharges in real-life situations.

The observation that in dogs electrical stimulation of the vagus nerve elicited a strong secretory activity of the pancreas, prompted I. P. Pavlov in 1888 to conclude that the pancreatic secretory response to nutrients is mediated by enteropancreatic reflexes involving the vagus nerves. It took, however, more than 90 years until by studying the latency of pancreatic amylase response to exogenous and endogenous stimuli for the first time experimental evidence was provided for the actual existence of cholinergic vago-vagal enteropancreatic reflexes. Follow-up studies, based on stepwise extrinsic denervation of the pancreas, ruled out possible splanchnic pathways for enteropancreatic reflexes. In more recent years, experiments utilizing specific antagonists demonstrated a physiological role for both cholinergic M1 and cholecystokinin (CCK) receptors within the enteropancreatic reflex. At least a significant portion of the cholinergic fibres of the enteropancreatic reflex end on muscarinic receptors of the subtype M1. CCK, the most important hormone stimulating pancreatic enzyme secretion, appears to act at least in part on CCK receptors located on vagal afferent nerves, which in turn elicit a vago-vagal reflex, implying that CCK exerts its effect on the pancreas at least in part through vago-vagal reflexes. Furthermore, pharmacological blockade of CCK receptors totally abolished the early pancreatic amylase response to intestinal nutrients, suggesting that the activation of (probably vagal) CCK receptors is essential to run the enteropancreatic reflex.

The present report gives an overview of the experimental, pharmacological and molecular investigations that have been undertaken during the past two decades to characterize and identify the muscarinic receptor subtype(s) involved in the cholinergic control of pancreatic exocrine secretion in humans and different animal species. The results published in the literature clearly indicate that both M1 and M3 receptors contribute to the regulation of pancreatic enzyme secretion, although contradictory conclusions have been drawn from secretory studies using specific M1 and M3 receptor antagonists in vivo and in vitro. Binding studies using specific M1 and M3 receptor antagonists have supported the existence of both M1 and M3 receptors on pancreatic acinar cells, which was confirmed by the demonstration of specific mRNA for both receptor subtypes in rat pancreatic acinar cells. In addition, experimental evidence exists that nonacinar (possibly presynaptic) M1 receptors also contribute to the control of pancreatic enzyme secretion. The role of the different muscarinic receptor subtypes in the control of pancreatic fluid and bicarbonate output, however, still needs to be clarified. Future research should cover the evaluation of the relative contribution of the different receptor subtypes to the regulation of pancreatic exocrine function, the localization of the receptors involved as well as possible species differences., (Copyright 2003 S. Karger AG, Basel and IAP)

Efferent vagal impulses act on the exocrine pancreas via pancreatic ganglia, where the impulses are modulated and modified, and terminate via postganglionic fibers at the acinar cells. Acinar muscarinic receptors of the subtype M1 play an important role for the mediation of the stimulatory vagal influences on pancreatic exocrine secretion. In dogs, a potentiative interaction exists between the two most important mediators of the pancreatic exocrine response to intraduodenal stimuli, efferent vagal impulses and CCK. In contrast to humans and rats, in which all action of CCK on pancreatic enzyme output is vagally mediated, CCK acts in dogs in part as a classical humoral factor independent of the cholinergic system. Although several peptides found in pancreatic nerve cell bodies or fibers can stimulate or inhibit pancreatic exocrine secretion, their physiological importance in the neural control of the exocrine pancreas needs to be further evaluated.

Background: Intraileal carbohydrates and lipids affect the pancreatic exocrine secretion, but the effect of intraileal amino acids and the role of the extrinsic nerves of the ileum as mediators of the pancreatic bicarbonate and enzyme output are unknown., Methods: Four dogs underwent total extrinsic denervation of the entire ileum. Thomas-like cannulas were placed into the stomach, duodenum (to collect pure pancreatic juice), and at the jejuno-ileal junction. Eight neurally intact control dogs received only the three fistulas. After recovery, in both sets of dogs, dose-response studies of the pancreatic secretory response to intraileal infusion with graded loads of tryptophan (0.12-10.0 mmol/h) were performed, given against an intravenous (iv) background of secretin (20.5 pmol/kg/h) and cerulein (29.6 pmol/kg/h). On separate days, control experiments with intraileal infusion of 0.15 M NaCl were performed., Results: In both sets of dogs, iv secretin plus cerulein significantly (p < 0.05) increased pancreatic bicarbonate and protein output above basal. Intraileal tryptophan caused a dose-dependent decrease in the pancreatic bicarbonate and protein response to secretin plus cerulein. In the dogs with denervated ileum, this inhibition was significantly stronger than in the intact animals. In both sets of dogs, the 225-min integrated bicarbonate (IBR) and protein response (IPR) to all loads of tryptophan were significantly lower than in control experiments. Both IBR and IPR were significantly lower in the denervated as compared with the intact animals., Conclusions: 1) Extrinsic denervation of the entire ileum is a valuable preparation to study the role of nerves in the control of pancreatic exocrine secretion; 2) both in the intact and denervated animals the amino acid tryptophan induces an "ileal brake" of the hormonally stimulated pancreatic bicarbonate and protein output; 3) the extrinsic nerves of the ileum are probably not the dominant mediators of the inhibitory action of intraileal tryptophan but rather counteract this effect.

In six conscious dogs with gastric and duodenal cannulas, secretin (164 pmol. kg(-1). h(-1) iv) was given to provide a flow of pancreatic juice of approximately 1 drop/s. Amylase activity was measured in each drop before and after rapid intravenous injection of caerulein (7.4 pmol/kg) or intraduodenal injection of L-tryptophan (1 mmol), sodium oleate (3 mmol), and HCl (3 mmol). All experiments were repeated in the presence of the M1 receptor antagonist telenzepine (81 nmol. kg(-1). h(-) iv) and the cholecystokinin (CCK) receptor antagonist L-364718 (0.1 mg/kg iv). Latency of amylase response (time between injection of stimulant and sustained increase in amylase activity greater than mean + 3 SD of prestimulatory activity) to tryptophan (17 +/- 7 s; n = 6) and oleate (16 +/- 5 s) was significantly (P < 0.05) shorter than to caerulein (28 +/- 4 s) and HCl (120 +/- 47 s). Telenzepine significantly increased the latency of amylase response to tryptophan and oleate by >10-fold but not the latency to caerulein or HCl. L-364718 abolished the amylase response to all stimulants. These findings indicate that the early amylase response to intraduodenal tryptophan and oleate is mediated by a neural enteropancreatic reflex ending on M1 receptors rather than by hormone release. However, the activation of (possibly vagal) CCK receptors is essential to run the reflex. The early amylase response to intraduodenal HCl is probably mediated by the release of CCK into the blood circulation.

Whereas oral or intraduodenal ethanol causes a moderate stimulation of pancreatic bicarbonate and enzyme output, intravenous ethanol inhibits basal and hormonally stimulated pancreatic exocrine secretion in humans, dogs, cats, pigs, rabbits, and rats. This inhibition could be mediated by inhibitory cholinergic mechanisms or be the result of a direct cellular effect of ethanol. In vitro investigations have specified several signaling molecules that may be involved in the action of ethanol on stimulus-secretion coupling in the exocrine pancreas, including cyclic adenosine monophosphate, intracellular calcium, and cholecystokinin and somatostatin receptors. In difference to pure ethanol solutions and distilled spirits, beer strongly stimulates pancreatic enzyme output, probably by nonalcoholic fermentation products. During chronic alcoholism, the ethanol-induced inhibition is replaced by an enhanced enzyme output that causes intraductal protein precipitation. In vitro investigations suggest that this increase is reversible after alcohol withdrawal. The occurrence of protein precipitates is considered to be a crucial step in the development of chronic alcoholic pancreatitis in humans. Other ethanol-induced secretory alterations that may contribute to the development of alcoholic pancreatitis are a decreased secretion of trypsin inhibitor, an increased cholinergic tone, and changes in the concentration of lithostathine.

Conclusions: In dogs, 1. Activation of cholecystokinin-receptors is needed for an adequate pancreatic bicarbonate response to secretin; 2. Cholinergic nerve fibers ending on M1-receptors are probably of little or no importance for the bicarbonate response to secretin in the given dose; 3. The bicarbonate response to tryptophan, given with a secretin background, is controlled by cholinergic M1-fibers and by cholecystokinin; 4. M1-fibers mainly mediate the bicarbonate response to low loads of tryptophan, whereas cholecystokinin controls the response to low and high loads of tryptophan; and 5. Both mediators interact in a synergistic manner., Methods: In six conscious dogs with chronic gastric and duodenal fistulas, we compared the action of the M1-receptor antagonist telenzepine (20.25-81.0 nmol/kg/h), the cholecystokinin-receptor antagonist L-364,718 (0.025-0.1 mg/kg/h), and combinations of both on the pancreatic bicarbonate response to graded loads of intraduodenal tryptophan (0.37-10.0 mmol/h), given against a background of secretin (20.5 pmol/kg/h)., Results: Secretin significantly (p < 0.05) stimulated the pancreatic bicarbonate output above basal levels. All doses of L-374,718, but not telenzepine, significantly decreased the bicarbonate response to secretin by up to 64%. Additional administration of telenzepine together with L-364,718 had no further inhibitory effect on the secretin-stimulated bicarbonate output as compared to L-364,718 given alone. All loads of tryptophan significantly increased the bicarbonate output over that seen with secretin alone (= incremental bicarbonate response to tryptophan). Telenzepine significantly decreased the incremental bicarbonate response to the two lower loads (0.37-1.1 mmol/h) of tryptophan (by 82-124%); L-364,718 decreased the incremental bicarbonate response to all loads of tryptophan (by 50-118%). The incremental bicarbonate output, as well as the 180-min integrated bicarbonate response to all loads of tryptophan, were abolished by all combinations of both antagonists.

This report presents a review of the historic and current methods for performing pancreatic exocrine studies in intact animals. Special emphasis is given to the various surgical procedures--pancreatic fistulas, duodenal pouches, and duodenal fistulas--and practice of collecting pancreatic secretion in dogs. Procedures in other animal species--rat, cat, pig, rabbit, cattle, sheep, and horse--also are specified. The advantages and disadvantages, as well as the indications and limitations of the distinct methods, are discussed.

In six conscious dogs we compared the action of the M1-receptor antagonist telenzepine (20.25-81.0 nmol.kg-1.h-1), the cholecystokinin (CCK) antagonist L-364,718 (0.025-0.1 mg.kg-1.h-1), and combinations of both on the pancreatic secretory response to intraduodenal tryptophan, given against a secretin background before and after truncal vagotomy. Before vagotomy, the higher doses of telenzepine and of L-364,718 significantly (P < 0.05) decreased the protein response to tryptophan by up to 97%. After vagotomy, all doses of L-364,718 abolished the protein response, whereas telenzepine had no further effect. Before and after vagotomy, all combinations abolished the protein response. The plasma CCK-like immunoreactivity basally, during secretin, and in response to tryptophan was not altered by vagotomy, telenzepine, and/or L-364,718. These findings indicate that in dogs 1) potentiation exists between M1 receptors and CCK for stimulation of the pancreatic enzyme response to intraduodenal tryptophan, 2) the cholinergic fibers of the enteropancreatic reflex activated by tryptophan run within the vagus nerves and end at least in part on M1 receptors, 3) CCK acts in part independently of the vagal nerves, and 4) the CCK release by intestinal tryptophan is not influenced by vagotomy, telenzepine, and/or L-364,718.

In two sets of dogs with gastric, duodenal, and jejunal fistulas, we studied the effect of atropine (14 nmol/ kg/h) on the pancreatic secretory response to intrajejunal tryptophan (0.12-10.0 mmol/h; given against a secretin background) before (n = 7) and after extrinsic denervation of the jejunoileum (orthotopical autotransplantation; n = 6). Plasma levels of cholecystokinin were determined by radioimmunoassay. The incremental bicarbonate response to tryptophan was not significantly different between the two sets of dogs. Atropine had no effect on the incremental bicarbonate response to tryptophan. In both sets of dogs, intrajejunal tryptophan caused a dose-dependent increase in pancreatic protein output, which was reduced by atropine. The tryptophan-stimulated levels of plasma cholecystokinin were not significantly altered by denervation and or atropine. We conclude that in dogs (1) intrajejunal tryptophan stimulates pancreatic bicarbonate and protein secretion via release of hormones, (2) extrinsic denervation of the jejunoileum does not significantly alter the incremental bicarbonate and protein responses to intrajejunal tryptophan, (3) the cholinergic intrinsic nerves of the jejunoileum and the hormone cholecystokinin are probably involved in control of the pancreatic protein response to tryptophan, and (4) the release of cholecystokinin by intrajejunal tryptophan does not depend on the extrinsic and intrinsic cholinergic nerves of the jejunoileum.

Xenin is a 25 amino acid peptide detected in the gastric mucosa of various mammals. It has since been found in low concentrations in other tissues. Xenin plasma concentrations increase after meals. The present study reports some gastroenteropancreatic effects of this peptide in the dog. Intravenous infusion of 64 pmol/kg min synthetic xenin significantly inhibited pentagastrin-stimulated gastric acid secretion and stimulated exocrine pancreatic secretion of volume and protein. Further, intravenous infusion of xenin in a dose of 1.0 pmol/kg min stimulated jejunal motility in the anaesthetized dog. An intravenous infusion of 32 pmol/kg min xenin raised plasma concentrations of pancreatic polypeptide, vasoactive intestinal polypeptide, insulin and glucagon. The present experiments therefore indicate manifold bioactive properties of intravenously infused xenin in the dog, with jejunal motility the most sensitive target. Conclusions as to the physiological role of xenin cannot be drawn from the present experiments. The release of other hormonal peptides indicates a complex action of xenin.

The present report gives a review about the localization, release and gastrointestinal actions of peptide YY in different animal species and in humans. Possible mechanisms of action, the physiological and pathophysiological significance of peptide YY and the role of peptide YY 3-36 are discussed. Finally, unanswered questions are specified.

Alteration of pancreatic secretion could be a pathogenetic factor for generation of an acute pancreatitis after administration of angiotensin-converting-enzyme (ACE) inhibitors. Therefore in 6 conscious dogs (weight 10-12 kg) with chronic gastric and duodenal fistulas according to Thomas, we studied single-blind, placebo-controlled and randomized the effect of a long-term (74 d) daily oral administration of 5 mg of the ACE inhibitor enalapril and of additionally daily oral administration of the diuretic furosemide from the 60th day on the secretin- (20.5 pmol/kg/h) and caerulein- (7.4, 14.8, 29.6, 59, and 118 pmol/kg/h) stimulated pancreatic bicarbonate and protein secretion at day 1, 29, 57, 60, and 74 after beginning of enalapril administration. Heart rate was measured daily and during the experiments. Neither enalapril nor enalapril plus furosemide significantly altered heart rate. The hormonally stimulated pancreatic bicarbonate secretion was significantly (p < 0.05) increased at day 57, 60, and 74 by 353%, 397%, and 79%. The hormonally stimulated pancreatic protein secretion was also distinctly increased, although not reaching statistical significance. The present study shows, that a long-term administration of therapeutic doses of enalapril +/-furosemide is capable to enhance the pancreatic bicarbonate secretion. Whether this altered pancreatic secretion can be taken for a primary origin of the observed cases of acute pancreatitis after therapeutic administration of enalapril is uncertain but not refutable.