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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.

The limbus, the vascularized junction between the cornea and conjunctiva, is thought to function as a barrier against corneal neovascularization. However, the exact mechanisms regulating this remain unknown. In this study, the limbal epithelial stem cell (LESC) marker ABCB5 was used to investigate the role of LESCs in corneal neovascularization. In an ABCB5KO model, a mild but significant increase of limbal lymphatic and blood vascular network complexity was observed in developing mice (4 weeks) but not in adult mice. Conversely, when using a cornea suture model, the WT animals exhibited a mild but significant increase in the number of lymphatic vessel sprouts compared to the ABCB5KO, suggesting a contextual anti-lymphangiogenic effect of ABCB5 on the limbal vasculature during development, but a pro-lymphangiogenic effect under inflammatory challenge in adulthood. In addition, conditioned media from ABCB5-positive cultured human limbal epithelial cells (ABCB5+) stimulated human blood and lymphatic endothelial cell proliferation and migration. Finally, a proteomic analysis demonstrated ABCB5+ cells have a pro(lymph)angiogenic as well as an anti-inflammatory profile. These data suggest a novel dual, context-dependent role of ABCB5+ LESCs, inhibiting developmental but promoting inflammatory (lymph)angiogenesis in adulthood and exerting anti-inflammatory effects. These findings are of high clinical relevance in relation to LESC therapy against blindness.

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

Recessive dystrophic epidermolysis bullosa (RDEB) is a rare, incurable blistering skin disease caused by biallelic mutations in type VII collagen (C7). Advancements in treatment of RDEB have come from harnessing the immunomodulatory potential of mesenchymal stem cells (MSCs). Although human bone marrow-derived MSC (BM-MSC) trials in RDEB demonstrate improvement in clinical severity, the mechanisms of MSC migration to and persistence in injured skin and their contributions to wound healing are not completely understood. A unique subset of MSCs expressing ATP-binding cassette subfamily member 5 (ABCB5) resides in the reticular dermis and exhibits similar immunomodulatory characteristics to BM-MSCs. Our work aimed to test the hypothesis that skin-derived ABCB5+ dermal MSCs (DSCs) possess superior skin homing ability compared to BM-MSCs in immunodeficient NOD-scid IL2rgammanull (NSG) mice. Compared to BM-MSCs, peripherally injected ABCB5+ DSCs demonstrated superior homing and engraftment of wounds. Furthermore, ABCB5+ DSCs vs BM-MSCs cocultured with macrophages induced less anti-inflammatory interleukin-1 receptor antagonist (IL-1RA) production. RNA sequencing of ABCB5+ DSCs compared to BM-MSCs showed unique expression of major histocompatibility complex class II and Homeobox (Hox) genes, specifically HOXA3. Critical to inducing migration of endothelial and epithelial cells for wound repair, increased expression of HOXA3 may explain superior skin homing properties of ABCB5+ DSCs. Further discernment of the immunomodulatory mechanisms among MSC populations could have broader regenerative medicine implications beyond RDEB treatment.

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 × 105 autologous ABCB5+ MSCs per cm2 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.

A significant number of chronic venous ulcers (CVUs) fail to heal despite guideline-conform standards of care. Skin-derived ABCB5+ mesenchymal stem cells can dampen the sustained IL-1β‒driven inflammation present in chronic wounds. On the basis of their wound healing‒facilitating effects in a mouse CVU model and an autologous first-in-human study, ABCB5+ mesenchymal stem cells have emerged as a potential candidate for cell-based advanced therapy of nonhealing CVUs. In this interventional, multicenter, single-arm, phase I/IIa clinical trial, subjects whose CVUs had emerged as standard therapy resistant received one or two topical applications of 1 × 106 allogeneic ABCB5+ mesenchymal stem cells per cm2 wound area, in addition to standard treatment. 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+ mesenchymal stem cells 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.

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.

Although liver transplantation is a potential effective cure for patients with end-stage liver diseases, this strategy has several drawbacks including high cost, long waiting list, and limited availability of liver organs. Therefore, stem cell-based therapy is presented as an alternative option, which showed promising results in animal models of acute and chronic liver injuries. ABCB5+ cells isolated from skin dermis represent an easy accessible and expandable source of homogenous stem cell populations. In addition, ABCB5+ cells showed already promising results in the treatment of corneal and skin injury. To date, the effect of these cells on liver injury is still unknown. In the current study, sixteen weeks old Mdr2KO mice were i.v. injected with 500,000 ABCB5+ cells using different experimental setups. The effects of cellular therapy on inflammation, fibrosis, apoptosis, and proliferation were analyzed in the collected liver tissues. Toxicity of ABCB5+ cells was additionally investigated in mice with partial liver resection. In vitro, the fibrosis- and inflammatory-modulating effects of supernatant from ABCB5+ cells were examined in the human hepatic stellate cell line (LX-2). Cell injections into fibrotic Mdr2KO mice as well as into mice upon partial liver resection have no signs of toxicity with regard to cell transformation, cellular damage, fibrosis or inflammation as compared to controls. We next investigated the effects of ABCB5+ cells on established biliary liver fibrosis in the Mdr2KO mice. ABCB5+ cells to some extent influenced the shape of the liver inflammatory response and significantly reduced the amount of collagen deposition, as estimated from quantification of sirius red staining. Furthermore, reduced apoptosis and enhanced death compensatory proliferation resulted from ABCB5+ cell transformation. The stem cells secreted several trophic factors that activated TGF-β family signaling in cultured LX-2 hepatic stellate cells (HSCs), therewith shaping cell fate to an αSMAhigh, Vimentinlow phenotype. Taken together, ABCB5+ cells can represent a safe and feasible strategy to support liver regeneration and to reduce liver fibrosis in chronic liver diseases.

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 × 107 ABCB5+ MSCs/animal and intravenous application of 2 × 106 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 × 105 to 1 × 107 cells/animal and three biweekly intravenous injections of 2 × 106 ABCB5+ MSCs/animal, respectively, to immunocompromised mice were nontoxic and revealed no tumorigenic potential; and (iii) intramuscular injection of 5 × 106 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.

Severe angiopathy is a major driver for diabetes associated secondary complications. Knowledge on underlying mechanisms essential for advanced therapies to attenuate these pathologies is limited. Injection of ABCB5+ stromal precursors (SPs) at the edge of non-healing diabetic wounds in a murine db/db model, closely mirroring human type II diabetes, profoundly accelerates wound closure. Strikingly, enhanced angiogenesis was substantially enforced by the release of the ribonuclease angiogenin from ABCB5+ SPs. This compensates for the profoundly reduced angiogenin expression in non-treated murine chronic diabetic wounds. Silencing of angiogenin in ABCB5+ SPs prior to injection significantly reduced angiogenesis and delayed wound closure in diabetic db/db mice implying an unprecedented key role for angiogenin in tissue regeneration in diabetes. These data hold significant promise for further refining SPs-based therapies of non-healing diabetic foot ulcers and other pathologies with impaired angiogenesis.

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 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.

The continuously decreasing willingness for liver donation aggravates treatment of end-stage liver diseases requiring organ transplantation as the only curative strategy. Cell therapy approaches using human hepatocytes or stem cell-derived hepatocyte-like cells may be a therapeutic option out of this dilemma. ABCB5-positive mesenchymal stromal cells from human skin featured promising potential to treat immune-mediated diseases. Since most of chronic liver diseases involve exaggerating immune mechanisms, it was the aim to demonstrate in this study, whether ABCB5+ stem cells may serve as a resource to generate hepatocytic cells for application in liver cell transplantation. Using an established single-step protocol, which had been successfully applied to differentiate mesenchymal stromal cells into the hepatocytic lineage, ABCB5+ skin-derived stem cells did not gain significant characteristics of hepatocytes. Yet, upon culture in hepatocytic differentiation medium, ABCB5+ stem cells secreted immunomodulatory and anti-fibrotic factors as well as proteins, which may prompt hepatic morphogenesis besides others. Hepatic transplantation of ABCB5+ stem cells, which had been prior cultured in hepatocytic differentiation medium, did not cause any obvious deterioration of liver architecture suggesting their safe application. Thus, human ABCB5+ skin-derived stem cells secreted putative hepatotropic factors after culture in hepatocytic differentiation medium.

Summary ATP binding cassette subfamily B member 5 (ABCB5) has been identified as a tumour-initiating cell marker and is expressed in various malignancies, including melanoma. Moreover, treatment with anti-ABCB5 monoclonal antibodies has been shown to inhibit tumour growth in xenotransplantation models. Therefore, ABCB5 represents a potential target for cancer immunotherapy. However, cellular immune responses against ABCB5 in humans have not been described so far. Here, we investigated whether ABCB5-reactive T cells are present in human melanoma patients and tested the applicability of ABCB5-derived peptides for experimental induction of human T cell responses. Peripheral blood mononuclear cells (PBMNC) isolated from blood samples of melanoma patients (n = 40) were stimulated with ABCB5 peptides, followed by intracellular cytokine staining (ICS) for interferon (IFN)-γ and tumour necrosis factor (TNF)-α. To evaluate immunogenicity of ABCB5 peptides in naive healthy donors, CD8 T cells were co-cultured with ABCB5 antigen-loaded autologous dendritic cells (DC). ABCB5 reactivity in expanded T cells was assessed similarly by ICS. ABCB5-reactive CD8+ T cells were detected ex vivo in 19 of 29 patients, melanoma antigen recognised by T cells (MART-1)-reactive CD8+ T cells in six of 21 patients. In this small, heterogeneous cohort, reactivity against ABCB5 was significantly higher than against MART-1. It occurred significantly more often and independently of clinical characteristics. Reactivity against ABCB5 could be induced in 14 of 16 healthy donors in vitro by repeated stimulation with peptide-loaded autologous DC. As ABCB5-reactive CD8 T cells can be found in the peripheral blood of melanoma patients and an ABCB5-specific response can be induced in vitro in naive donors, ABCB5 could be a new target for immunotherapies in melanoma.

Recessive dystrophic epidermolysis bullosa (RDEB) is a debilitating and ultimately lethal blistering disease caused by mutations to the Col7a1−/− gene. Development of novel cell therapies for the treatment of RDEB would be fostered by having immunodeficient mouse models able to accept human cell grafts; however, immunodeficient models of many genodermatoses such as RDEB are lacking. To overcome this limitation, we combined the clustered regularly interspaced short palindromic repeats and associated nuclease (CRISPR/Cas9) system with microinjection into NOD/SCID IL2rγcnull (NSG) embryos to rapidly develop an immunodeficient Col7a1−/− mouse model of RDEB. Through dose optimization, we achieve F0 biallelic knockout efficiencies exceeding 80%, allowing us to quickly generate large numbers of RDEB NSG mice for experimental use. Using this strategy, we clearly demonstrate important strain-specific differences in RDEB pathology that could underlie discordant results observed between independent studies and establish the utility of this system in proof-of-concept human cellular transplantation experiments. Importantly, we uncover the ability of a recently identified skin resident immunomodulatory dermal mesenchymal stem cell marked by ABCB5 to reduce RDEB pathology and dramatically extend the lifespan of RDEB NSG mice via reduced skin infiltration of inflammatory myeloid derivatives.

Mesenchymal stem cells (MSCs) are crucial for tissue homeostasis and regeneration. Though of prime interest, their potentially protective role on neutrophil-induced tissue damage, associated with high morbidity and mortality, has not been explored in sufficient detail. Here we report the therapeutic skill of MSCs to suppress unrestrained neutrophil activation and to attenuate severe tissue damage in a murine immune-complex mediated vasculitis model of unbalanced neutrophil activation. MSC-mediated neutrophil suppression was due to intercellular adhesion molecule 1-dependent engulfment of neutrophils by MSCs, decreasing overall neutrophil numbers. Similar to MSCs in their endogenous niche of murine and human vasculitis, therapeutically injected MSCs via upregulation of the extracellular superoxide dismutase (SOD3), reduced superoxide anion concentrations and consequently prevented neutrophil death, neutrophil extracellular trap formation and spillage of matrix degrading neutrophil elastase, gelatinase and myeloperoxidase. SOD3-silenced MSCs did not exert tissue protective effects. Thus, MSCs hold substantial therapeutic promise to counteract tissue damage in conditions with unrestrained neutrophil activation.

In this study, we report the beneficial effects of a newly identified dermal cell subpopulation expressing the ATP-binding cassette subfamily B member 5 (ABCB5) for the therapy of nonhealing wounds. Local administration of dermal ABCB5+-derived mesenchymal stem cells (MSCs) attenuated macrophage-dominated inflammation and thereby accelerated healing of full-thickness excisional wounds in the iron-overload mouse model mimicking the nonhealing state of human venous leg ulcers. The observed beneficial effects were due to interleukin-1 receptor antagonist (IL-1RA) secreted by ABCB5+-derived MSCs, which dampened inflammation and shifted the prevalence of unrestrained proinflammatory M1 macrophages toward repair promoting anti-inflammatory M2 macrophages at the wound site. The beneficial anti-inflammatory effect of IL-1RA released from ABCB5+-derived MSCs on human wound macrophages was conserved in humanized NOD-scid IL2rγnull mice. In conclusion, human dermal ABCB5+ cells represent a novel, easily accessible, and marker-enriched source of MSCs, which holds substantial promise to successfully treat chronic nonhealing wounds in humans. Stem Cells 2019;37:1057–1074

The continuously decreasing willingness for liver donation aggravates treatment of end-stage liver diseases requiring organ transplantation as the only curative strategy. Cell therapy approaches using human hepatocytes or stem cell-derived hepatocyte-like cells may be a therapeutic option out of this dilemma. ABCB5-positive mesenchymal stromal cells from human skin featured promising potential to treat immune-mediated diseases. Since most of chronic liver diseases involve exaggerating immune mechanisms, it was the aim to demonstrate in this study, whether ABCB5

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Cell-based strategies represent a new frontier in the treatment of immune-mediated disorders. However, the paucity of markers for isolation of molecularly defined immunomodulatory cell populations poses a barrier to this field. Here, we show that ATP-binding cassette member B5 (ABCB5) identifies dermal immunoregulatory cells (DIRCs) capable of exerting therapeutic immunoregulatory functions through engagement of programmed cell death 1 (PD-1). Purified Abcb5\(^+\) DIRCs suppressed T cell proliferation, evaded immune rejection, homed to recipient immune tissues, and induced Tregs in vivo. In fully major-histocompatibility-complex-mismatched cardiac allotransplantation models, allogeneic DIRCs significantly prolonged allograft survival. Blockade of DIRC-expressed PD-1 reversed the inhibitory effects of DIRCs on T cell activation, inhibited DIRC-dependent Treg induction, and attenuated DIRC-induced prolongation of cardiac allograft survival, indicating that DIRC immunoregulatory function is mediated, at least in part, through PD-1. Our results identify ABCB5\(^+\) DIRCs as a distinct immunoregulatory cell population and suggest promising roles of this expandable cell subset in cellular immunotherapy.

UVB irradiation has been linked to pathogenesis of pterygium, a conjunctival tumor growing onto transparent cornea, the windscreen of the eye. Due to corneal anatomy, ambient UVB irradiation is amplified at the stem cell-containing nasal limbus. The aim of this study was to analyse the effect of a UV-blocking contact lens (UVBCL, senofilcon A, Class 1 UV blocker) on limbal epithelial cells and fibroblasts under UVB irradiation compared to a non-UVB-blocking contact lens. UVBCL prevented UVB-induced DNA damage (as assessed by cyclobutane pyrimidine dimer immunostaining) as well as a decrease in proliferation and scratch wound closure rate of both limbal epithelial and fibroblast cells. Similarly, UVBCL protected limbal epithelial cells from UVB-induced loss of their phenotype in terms of colony forming efficiency and stem cell marker expression (ABCB5, P63α, integrin β1) compared to controls. Moreover, with UVBCL pro-inflammatory cytokines such as TNFα and MCP1 remained unchanged. These data demonstrate the significance of UV-protection in preserving the limbal niche in response to at least short-term UVB. Our data support the use of UVBCL in protecting limbal niche cells, especially after limbal stem cell transplantation and in patients after pterygium surgery, to help prevent recurrences.