Tumour evolution and microenvironment interactions in 2D and 3D space.

To study the spatial interactions among cancer and non-cancer cells1, we here examined a cohort of 131 tumour sections from 78 cases across 6 cancer types by Visium spatial transcriptomics (ST). This was combined with 48 matched single-nucleus RNA sequencing samples and 22 matched co-detection by indexing (CODEX) samples. To describe tumour structures and habitats, we defined 'tumour microregions' as spatially distinct cancer cell clusters separated by stromal components. They varied in size and density among cancer types, with the largest microregions observed in metastatic samples. We further grouped microregions with shared genetic alterations into 'spatial subclones'. Thirty five tumour sections exhibited subclonal structures. Spatial subclones with distinct copy number variations and mutations displayed differential oncogenic activities. We identified increased metabolic activity at the centre and increased antigen presentation along the leading edges of microregions. We also observed variable T cell infiltrations within microregions and macrophages predominantly residing at tumour boundaries. We reconstructed 3D tumour structures by co-registering 48 serial ST sections from 16 samples, which provided insights into the spatial organization and heterogeneity of tumours. Additionally, using an unsupervised deep-learning algorithm and integrating ST and CODEX data, we identified both immune hot and cold neighbourhoods and enhanced immune exhaustion markers surrounding the 3D subclones. These findings contribute to the understanding of spatial tumour evolution through interactions with the local microenvironment in 2D and 3D space, providing valuable insights into tumour biology.

Facial and ocular thermal mapping in black-and-gold howler monkey (Alouatta caraya) by infrared thermography: An ex situ study.

BACKGROUND: This study used infrared thermography (IRT) for mapping the facial and ocular temperatures of howler monkeys, to determine parameters for the diagnosis of febrile processes. There are no published IRT study in this species. METHODS: Were evaluated images of a group of monkeys kept under human care at Sorocaba Zoo (São Paulo, Brazil). The images were recorded during 1 year, in all seasons. Face and eye temperatures were evaluated. RESULTS: There are statistically significant differences in face and eye temperatures. Mean values and standard deviations for facial and ocular temperature were respectively: 33.0°C (2.1) and 36.5°C (1.9) in the summer; 31.5°C (4.5) and 35.3°C (3.6) in the autumn; 30.0°C (4.3) and 35.6°C (3.9) in the winter; 30.8°C (2.9) and 35.5°C (2.1) in the spring. CONCLUSIONS: The IRT was effective to establish a parameter for facial and ocular temperatures of black-and-gold howler monkeys kept under human care.

Retrospective study of the mortality of the Vieira's titi monkey (Plecturocebus vieirai) at the Sorocaba Zoo, Brazil.

BACKGROUND: The Vieira's titi monkey (Plecturocebus vieirai) was recently described and characterized as endemic to Brazil. According to the IUCN red list, this species is classified as critically endangered (CR). At the date of the publication of this manuscript, there are no published data on the health aspects of this species. METHODS: For this study, the necropsy, and histopathological data of the mortality of P. vieirai at Sorocaba Zoo (São Paulo, Brazil) were collected and analyzed. RESULTS: Causes of death diagnosed included disorders of the urinary, gastrointestinal, immune, and circulatory systems. CONCLUSIONS: This study provides information regarding the pathological conditions of P. vieirai and points to urinary and gastrointestinal diseases as the main causes of death in this species at Sorocaba Zoo. These results can help veterinarians who have this species under their care diagnose and deal with it more quickly, increasing the probability of survival.

Periodontal pathogen Aggregatibacter actinomycetemcomitans JP2 correlates with colonic leukocytes decrease and gut microbiome imbalance in mice.

AIM: Evidence suggests that translocation of oral pathogens through the oral-gut axis may induce intestinal dysbiosis. This study aimed to evaluate the impact of a highly leukotoxic Aggregatibacter actinomycetemcomitans (Aa) strain on the gut microbiota, intestinal mucosal integrity and immune system in healthy mice. METHODS: Eight-week-old male C57BL6 mice were divided into control (n = 16) and JP2 groups (n = 19), which received intragastric gavage with PBS and with a suspension of Aa JP2 (HK921), respectively, twice a week for 4 weeks. Colonic lamina propria, fecal material, serum, gingival tissues, and mandibles were obtained for analyses of leukocyte populations, inflammatory mediators, mucosal integrity, alveolar bone loss, and gut microbiota. Differences between groups for these parameters were examined by non-parametric tests. RESULTS: The gut microbial richness and the number of colonic macrophages, neutrophils, and monocytes were significantly lower in Aa JP2-infected mice than in controls (p < .05). In contrast, infected animals showed higher abundance of Clostridiaceae, Lactobacillus taiwanensis, Helicobacter rodentium, higher levels of IL-6 expression in colonic tissues, and higher splenic MPO activity than controls (p < .05). No differences in tight junction expression, serum endotoxin levels, and colonic inflammatory cytokines were observed between groups. Infected animals presented also slightly more alveolar bone loss and gingival IL-6 levels than controls (p < .05). CONCLUSION: Based on this model, intragastric administration of Aa JP2 is associated with changes in the gut ecosystem of healthy hosts, characterized by less live/recruited myeloid cells, enrichment of the gut microbiota with pathobionts and decrease in commensals. Negligible levels of colonic pro-inflammatory cytokines, and no signs of mucosal barrier disruption were related to these changes.

Antimicrobial and anti-biofilm activity of silver nanoparticles biosynthesized with Cystoseira algae extracts.

Antimicrobial resistance is an ever-growing global concern to public health with no clear or immediate solution. Silver nanoparticles (AgNPs) have long been proposed as efficient agents to fight the growing number of antibiotic-resistant strains. However, the synthesis of these particles is often linked to high costs and the use of toxic, hazardous chemicals, with environmental and health impact. In this study, we successfully produced AgNPs by green synthesis with the aid of the extract of two brown algae-Cystoseira baccata (CB) and Cystoseira tamariscifolia (CT)-and characterized their physico-chemical properties. The NPs produced in both cases (Ag@CB and Ag@CT) present similar sizes, with mean diameters of around 22 nm. The antioxidant activity of the extracts and the NPs was evaluated, with the extracts showing important antioxidant activity. The bacteriostatic and bactericidal properties of both Ag@CB and Ag@CT were tested and compared with gold NPs produced in the same algae extracts as previously reported. AgNPs demonstrated the strongest bacteriostatic and bactericidal properties, at concentrations as low as 2.16 µg/mL against Pseudomonas aeruginosa and Escherichia coli. Finally, the capacity of these samples to prevent the formation of biofilms characteristic of infections with a poorer outcome was assessed, obtaining similar results. This work points towards an alternative for the treatment of bacterial infections, even biofilm-inducing, with the possibility of minimizing the risk of drug resistance, albeit the necessary caution implied using metallic NPs.

SURGICAL MANAGEMENT OF UPWARD FIXATION OF THE PATELLA VIA MEDIAL PATELLAR LIGAMENT DESMOTOMY IN A LOWLAND TAPIR (TAPIRUS TERRESTRIS).

Tapirs are endangered terrestrial mammals that inhabit several continents. They have anatomical similarities to horses, sharing a common ancestral lineage. This article reports the case of a 14-yr-old female lowland tapir (Tapirus terrestris) presented for intermittent lameness due to upward fixation of the patella causing extension of the limb in the caudal phase of the stride. Medial patellar desmotomy was performed under general anesthesia, correcting the problem. To date there are no reports of this condition or treatment recommendations in tapirs. An anatomical study including stifle dissection, advanced MRI, and CT was performed in a separate lowland tapir. According to the clinical case and the anatomical findings in the other lowland tapir, upward fixation of the patella may occur in the tapir, although the anatomy varies slightly from that of the horse. Because the lowland tapir does not have parapatellar cartilage or as large of a medial patellar ligament or medial trochlea of the distal femur compared to the horse, more severe disease secondary to complete or persistent upward fixation of the patella may not occur in tapirs. Rather, mild forms of the disease associated with intermittent upward fixation of the patella or delayed patellar release appear more likely in the tapir.

Hybrid treatment for lower limb revascularization in a patient with vascular graft infection: a case report.

Patients with severe arterial obstructive disease and critical limb ischemia associated with vascular graft infection have elevated morbidity and mortality rates and are at high risk of limb loss. We present the case of a 76-year-old male patient with left lower limb critical ischemia and a femoropopliteal vascular graft infection. We used a hybrid treatment approach with an open surgical approach to the inguinal and popliteal regions and used the vascular prosthesis as endovascular access for direct recanalization of the superficial femoral artery, because the long occlusion and extensive calcification had frustrated initial attempts at endovascular treatment. After endovascular recanalization, the infected graft was removed. Used in conjunction with open surgery, advances in endovascular techniques and materials offer new solutions for patients when usual procedures fail.

The Pathogenesis, Molecular Mechanisms, and Therapeutic Potential of the Interferon Pathway in Systemic Lupus Erythematosus and Other Autoimmune Diseases.

Therapeutic success in treating patients with systemic lupus erythematosus (SLE) is limited by the multivariate disease etiology, multi-organ presentation, systemic involvement, and complex immunopathogenesis. Agents targeting B-cell differentiation and survival are not efficacious for all patients, indicating a need to target other inflammatory mediators. One such target is the type I interferon pathway. Type I interferons upregulate interferon gene signatures and mediate critical antiviral responses. Dysregulated type I interferon signaling is detectable in many patients with SLE and other autoimmune diseases, and the extent of this dysregulation is associated with disease severity, making type I interferons therapeutically tangible targets. The recent approval of the type I interferon-blocking antibody, anifrolumab, by the US Food and Drug Administration for the treatment of patients with SLE demonstrates the value of targeting this pathway. Nevertheless, the interferon pathway has pleiotropic biology, with multiple cellular targets and signaling components that are incompletely understood. Deconvoluting the complexity of the type I interferon pathway and its intersection with lupus disease pathology will be valuable for further development of targeted SLE therapeutics. This review summarizes the immune mediators of the interferon pathway, its association with disease pathogenesis, and therapeutic modalities targeting the dysregulated interferon pathway.

In-Block Lateralization as a New Technique for Mobilization of the Inferior Alveolar Nerve: A Technique Case Series.

The edentulous atrophic posterior mandible is often a great challenge for implant rehabilitation. Although a number of treatment options have been proposed, including the use of short implants and surgical grafting techniques, in cases of severe bone atrophy, techniques for mobilization of the inferior alveolar nerve (IAN) have been shown to be efficient, with good results. Four female patients underwent IAN lateralization for prosthetic rehabilitation of the posterior mandible from 2013 to 2019, with 3 years to 5 years and 3 months of follow-up. This case series describes a new technique for mobilization of the IAN, named in-block lateralization, to facilitate access to the IAN and to reduce nerve manipulation. The implant is immediately installed (allowing nerve lateralization in unitary spaces), and the original mandibular anatomy is restored with autogenous bone from the original bed during the same surgical procedure. When well indicated and well performed, this new approach provides better and easier visualization of the IAN and safer manipulation aiming to achieve good results for implant stability and minimal risk of neurosensory disturbances, allowing rehabilitation even in unitary spaces.

Immunohistochemical analysis of growth factors in platelet-rich fibrin membranes.

The use of autogenous materials to promote tissue regeneration has guided the direction of modern dentistry, and platelet-rich fibrin (PRF) is a promising biomaterial for tissue engineering. This in vitro immunohistochemical study aimed to analyze the presence of factors of endothelial growth and cell differentiation in PRF membranes by using the CD31 (endothelial cells) and CD163 (monocytes) markers. Five men and 5 women, aged between 25 and 60 years and without systemic health problems, were enrolled in the study. Blood samples were collected, submitted to a centrifugation protocol, and fixed in 4% formaldehyde, and then immunohistochemical analysis was performed. The histologic analysis of the slides showed that the fibrin clot was formed by a dense fiber network and cells trapped in its structure. One sample was excluded from the markers testing due to poor quality. All 9 of the valid samples were positive for the CD31 and CD163 markers, with reactivity ranging from 5% to 30% and 10% to 40% of cells, respectively. The immunohistochemical analysis showed the presence of CD31 and CD163 in the PRF membranes, indicating the potential for vascular neoformation and the significant presence of monocytes, which play an important role in tissue remodeling via their differentiation into macrophages.

What causes hidradenitis suppurativa ?-15 years after.

The 14 authors of the first review article on hidradenitis suppurativa (HS) pathogenesis published 2008 in EXPERIMENTAL DERMATOLOGY cumulating from the 1st International Hidradenitis Suppurativa Research Symposium held March 30-April 2, 2006 in Dessau, Germany with 33 participants were prophetic when they wrote "Hopefully, this heralds a welcome new tradition: to get to the molecular heart of HS pathogenesis, which can only be achieved by a renaissance of solid basic HS research, as the key to developing more effective HS therapy." (Kurzen et al. What causes hidradenitis suppurativa? Exp Dermatol 2008;17:455). Fifteen years later, there is no doubt that the desired renaissance of solid basic HS research is progressing with rapid steps and that HS has developed deep roots among inflammatory diseases in Dermatology and beyond, recognized as "the only inflammatory skin disease than can be healed". This anniversary article of 43 research-performing authors from all around the globe in the official journal of the European Hidradenitis Suppurativa Foundation e.V. (EHSF e.V.) and the Hidradenitis Suppurativa Foundation, Inc (HSF USA) summarizes the evidence of the intense HS clinical and experimental research during the last 15 years in all aspects of the disease and provides information of the developments to come in the near future.

Intraindividual Neurofilament Dynamics in Serum Mark the Conversion to Sporadic Parkinson's Disease.

BACKGROUND AND OBJECTIVES: With disease-modifying treatment strategies on the horizon, stratification of individual patients at the earliest stages of Parkinson's disease (PD) is key-ideally already at clinical disease onset. Blood levels of neurofilament light chain (NfL) provide an easily accessible fluid biomarker that might allow capturing the conversion from prodromal to manifest PD. METHODS: We assessed longitudinal serum NfL levels in subjects converting from prodromal to manifest sporadic PD (converters), at-risk subjects, and matched controls (72 participants with ≈4 visits), using single-molecule array (Simoa) technique. RESULTS: While NfL levels were not increased at the prodromal stage, subjects converting to the manifest motor stage showed a significant intraindividual acceleration of the age-dependent increase of NfL levels. CONCLUSIONS: The temporal dynamics of intraindividual NfL blood levels might mark the conversion to clinically manifest PD, providing a potential stratification biomarker for individual disease onset in the advent of precision medicine for PD. © 2020 The Authors. Movement Disorders published by Wiley Periodicals, Inc. on behalf of International Parkinson and Movement Disorder Society.

Standardized Photographic Documentation of Hidradenitis Suppurativa/Acne Inversa.

BACKGROUND: Despite existing guidelines and methods for standardized clinical photography in dermatology and plastic surgery, human skin exhibits exquisite site-specific morphologies and functions, and each body region can exhibit an individual pathologic phenotype. The aim of this work was to develop a standardized, representative and reproducible documentation of the multilocular hidradenitis suppurativa/acne inversa (HS) lesions, a disease mostly occurring in skin folds. METHOD: Optimal body positions for medical photography of candidate areas for HS involvement were evaluated. Pictures of volunteers were taken, and indicative scientific graphics were designed. RESULTS: Due to the variability of HS lesions and the fact of their localization in skin folds, a standardized, reproducible photographic documentation of HS candidate skin areas (50 cm from the skin surface) is proposed. The photographic series includes: (1) right armpit, (2) left armpit, (3) right groin, (4) left groin, (5) genital area, (6) anal area and anal fold, (7) right buttock, (8) left buttock, (9) chest area, (10) mons pubis, (11) right submammary area (females), (12) left submammary area (females). The protocol is accompanied with indicative scientific graphics for photography-proper positioning of the relevant body parts for documentation of potentially flat skin areas. In addition, former proposals for technical standardization of photography in dermatology, regarding instrumentation, environmental lighting and background colour, have to be respected. CONCLUSION: Standardized photography of candidate skin areas for HS involvement will allow longitudinal intraindividual and interindividual evaluation of the disease course as well as prospective and retrospective comparative studies.

Long-Wave Medical Infrared Thermography: A Clinical Biomarker of Inflammation in Hidradenitis Suppurativa/Acne Inversa.

BACKGROUND: A more reliable classification of skin inflammation and severity of active disease results from ultrasound sonography and the new hidradenitis suppurativa/acne inversa (HS) classification system IHS4. However, an objective assessment of skin inflammation in a continuous mode is still the ultimate goal. Long-wave medical infrared thermography (MIT) may offer a blood flow and temperature differential assessment in inflammatory conditions. OBJECTIVE: To evaluate the application of MIT in HS. METHODS: Standardized photography of the areas involved or been candidates for HS involvement was performed and MIT pictures were taken simultaneously and superimposed on the photographs of 18 patients (11 female, 7 male, median age 38.75 years [95% confidence interval 28.5 and 51 years], Hurley score I 5.6%, Hurley score II 38.9%, and Hurley score III 55.5%). A modification of the Otsu's method facilitated the automatic lesion segmentation from the background, depicting the inflammation area. Moreover, MIT was administered in real-time mode during radical HS surgery. RESULTS: A 1°C temperature difference from a corresponding symmetric body region was indicative of inflammation. MIT figures detected a gradual increase of skin temperature from 33.0°C in healthy skin on average to 35.0-36.6°C at the center of inflamma tory lesions in the axilla and to 35.4-36.9°C at the center of inflammation in the groin area. Real-time MIT assessment enabled the definition of the margins and depth of the surgical intervention during the procedure. CONCLUSION: MIT is a promising tool for the detection of inflammation severity in HS lesions and can be used as a clinical biomarker in evaluation studies of medical and surgical HS treatment.

Current trends in in silico, in vitro toxicology, and safety biomarkers in early drug development.

The development of new medicines is a long and expensive process. Despite growing efforts in R&D over the last decades, attrition rate due to safety issues (especially cardiac and hepatic toxicity) remains a major challenge for the pharmaceutical industry. This may lead to market withdrawal or late stage halting of a drug development program. Consequently, early detection of toxicity issues is critical to avoid late-stage failures. To this end, development of predictive toxicology assays and models have become a strategic matter for drug makers. An integrated approach confronting knowledge-based data sources with in vitro and in vivo experimental data should be performed. A well-defined balance between in vivo and in vitro assays should guide the safety assessment process and include a rationale taking into account ethical considerations as well as associated resourcing involved with animal use. Innovation in de-risking strategies may support refinement of regulatory testing and contribute to (i) improve drug safety evaluation alleviating assessment of the risk-benefit ratio and (ii) promote the access to safe drugs for patients. In this review, promising innovative approaches aiming at facilitating early detection of toxicity during drug development are described.

Natural variations of copper and sulfur stable isotopes in blood of hepatocellular carcinoma patients.

The widespread hypoxic conditions of the tumor microenvironment can impair the metabolism of bioessential elements such as copper and sulfur, notably by changing their redox state and, as a consequence, their ability to bind specific molecules. Because competing redox state is known to drive isotopic fractionation, we have used here the stable isotope compositions of copper ((65)Cu/(63)Cu) and sulfur ((34)S/(32)S) in the blood of patients with hepatocellular carcinoma (HCC) as a tool to explore the cancer-driven copper and sulfur imbalances. We report that copper is (63)Cu-enriched by ∼0.4‰ and sulfur is (32)S-enriched by ∼1.5‰ in the blood of patients compared with that of control subjects. As expected, HCC patients have more copper in red blood cells and serum compared with control subjects. However, the isotopic signature of this blood extra copper burden is not in favor of a dietary origin but rather suggests a reallocation in the body of copper bound to cysteine-rich proteins such as metallothioneins. The magnitude of the sulfur isotope effect is similar in red blood cells and serum of HCC patients, implying that sulfur fractionation is systemic. The (32)S-enrichment of sulfur in the blood of HCC patients is compatible with the notion that sulfur partly originates from tumor-derived sulfides. The measurement of natural variations of stable isotope compositions, using techniques developed in the field of Earth sciences, can provide new means to detect and quantify cancer metabolic changes and provide insights into underlying mechanisms.

Osteopontin and latent-TGF ß binding-protein 2 as potential diagnostic markers for HBV-related hepatocellular carcinoma.

Chronic Hepatitis B (HB) is the main risk factor for chronic liver disease (CLD) and hepatocellular carcinoma (HCC) in many low-resource countries, where diagnosis is constrained by lack of clinical, histopathological and biomarker resources. We have used proteomics to detect plasma biomarkers that outperform α-Fetoprotein (AFP), the most widely used biomarker for HCC diagnosis in low-resource contexts. Deep-plasma proteome analysis was performed in HCC patients, patients with CLD and in HB-carrier controls from Thailand (South-East Asia) and The Gambia (West-Africa). Mass spectrometry profiling identified latent-transforming growth factor ß binding-protein 2 (LTBP2) and Osteopontin (OPN) as being significantly elevated in HCC versus CLD and controls. These two proteins were further analyzed by ELISA in a total of 684 plasma samples, including 183 HCC, 274 CLD and 227 asymptomatic controls. When combined, LTBP2 and OPN showed an area under the receiver operating curve of 0.85 in distinguishing HCC from CLD in subjects with AFP <20 ng/mL. In a prospective cohort of 115 CLD patients from Korea, increased plasma levels of LTBP2 and/or OPN were detected in plasma collected over 2 years prior to diagnosis in 21 subjects who developed HCC. Thus, the combination of LTBP2 and OPN outperformed AFP for diagnosis and prediction of HCC and may therefore improve biomarker-based detection of HBV-related HCC.

Development of elastin-like recombinamer films with antimicrobial activity.

In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.

Renal Safety Pharmacology in Drug Discovery and Development.

The kidney is a complex excretory organ playing a crucial role in various physiological processes such as fluid and electrolyte balance, control of blood pressure, removal of waste products, and drug disposition. Drug-induced kidney injury (DIKI) remains a significant cause of candidate drug attrition during drug development. However, the incidence of renal toxicities in preclinical studies is low, and the mechanisms by which drugs induce kidney injury are still poorly understood. Although some in vitro investigational tools have been developed, the in vivo assessment of renal function remains the most widely used methodology to identify DIKI. Stand-alone safety pharmacology studies usually include assessment of glomerular and hemodynamic function, coupled with urine and plasma analyses. However, as renal function is not part of the ICH S7A core battery, such studies are not routinely conducted by pharmaceutical companies. The most common approach consists in integrating renal/urinary measurements in repeat-dose toxicity studies. In addition to the standard analyses and histopathological examination of kidneys, novel promising urinary biomarkers have emerged over the last decade, offering greater sensitivity and specificity than traditional renal parameters. Seven of these biomarkers have been qualified by regulatory agencies for use in rat toxicity studies.

Gastrointestinal Safety Pharmacology in Drug Discovery and Development.

Although the basic structure of the gastrointestinal tract (GIT) is similar across species, there are significant differences in the anatomy, physiology, and biochemistry between humans and laboratory animals, which should be taken into account when conducting a gastrointestinal (GI) assessment. Historically, the percentage of cases of drug attrition associated with GI-related adverse effects is small; however, this incidence has increased over the last few years. Drug-related GI effects are very diverse, usually functional in nature, and not limited to a single pharmacological class. The most common GI signs are nausea and vomiting, diarrhea, constipation, and gastric ulceration. Despite being generally not life-threatening, they can greatly affect patient compliance and quality of life. There is therefore a real need for improved and/or more extensive GI screening of candidate drugs in preclinical development, which may help to better predict clinical effects. Models to identify drug effects on GI function cover GI motility, nausea and emesis liability, secretory function (mainly gastric secretion), and absorption aspects. Both in vitro and in vivo assessments are described in this chapter. Drug-induced effects on GI function can be assessed in stand-alone safety pharmacology studies or as endpoints integrated into toxicology studies. In silico approaches are also being developed, such as the gut-on-a-chip model, but await further optimization and validation before routine use in drug development. GI injuries are still in their infancy with regard to biomarkers, probably due to their greater diversity. Nevertheless, several potential blood, stool, and breath biomarkers have been investigated. However, additional validation studies are necessary to assess the relevance of these biomarkers and their predictive value for GI injuries.