Host genetic variants associated with COVID-19 reconsidered in a Slovak cohort.

We present the results of an association study involving hospitalized coronavirus disease 2019 (COVID-19) patients with a clinical background during the 3rd pandemic wave of COVID-19 in Slovakia. Seventeen single nucleotide variants (SNVs) in the eleven most relevant genes, according to the COVID-19 Host Genetics Initiative, were investigated. Our study confirms the validity of the influence of LZTFL1 and 2'-5'-oligoadenylate synthetase (OAS)1/OAS3 genetic variants on the severity of COVID-19. For two LZTFL1 SNVs in complete linkage disequilibrium, rs17713054 and rs73064425, the odds ratios of baseline allelic associations and logistic regressions (LR) adjusted for age and sex ranged in the four tested designs from 2.04 to 2.41 and from 2.05 to 3.98, respectively. The OAS1/OAS3 haplotype 'gttg' carrying a functional allele G of splice-acceptor variant rs10774671 manifested its protective function in the Delta pandemic wave. Significant baseline allelic associations of two DPP9 variants in all tested designs and two IFNAR2 variants in the Omicron pandemic wave were not confirmed by adjusted LR. Nevertheless, adjusted LR showed significant associations of NOTCH4 rs3131294 and TYK2 rs2304256 variants with severity of COVID-19. Hospitalized patients' reported comorbidities were not correlated with genetic variants, except for obesity, smoking (IFNAR2), and hypertension (NOTCH4). The results of our study suggest that host genetic variations have an impact on the severity and duration of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Considering the differences in allelic associations between pandemic waves, they support the hypothesis that every new SARS-CoV-2 variant may modify the host immune response by reconfiguring involved pathways.

Diagnostic tools for fecal incontinence: Scoring systems are the crucial first step.

The main aim of this editorial is to comment on the recent article published by Garg et al in the World Journal of Gastroenterology 2023; 29: 4593-4603. This original research presents a new scoring system for fecal incontinence. Fecal incontinence is a chronic disease with a severe impact on the quality of life of the patients. Substantial social stigmatization often leads to significant underreporting of the condition even during visits to a specialist and could lead to further mismanagement or non-existent management of the disease. An important fact is that patients are often unable to describe their condition when not asked precisely defined questions. This problem is partially resolved by scoring questionnaires. Several scoring systems are commonly used; however, each of them has their shortcomings. For example, the absence of different kinds of leakage besides flatus and stool could further lead to underscoring the incontinence severity. Therefore, there has long been a call for a more precise scoring system. The correct identification of the presence and severity of fecal incontinence is paramount for further diagnostic approach and for choosing the appropriate therapy option. This editorial describes fecal incontinence, its effect on quality of life in general and further evaluates the diagnostic approach with a particular focus on symptom scoring systems and their implications for clinical practice.

Esophageal Infusion of Menthol Does Not Affect Esophageal Motility in Patients with Gastroesophageal Reflux Disease.

Menthol is thought to trigger gastroesophageal reflux disease (GERD) symptoms by influencing esophageal peristalsis and lower esophageal sphincter (LES) function. We evaluated the effect of esophageal menthol infusion on esophageal motility and the LES in healthy volunteers and in patients with GERD. High resolution manometry (HRM) catheter with attached thin tube for menthol infusion was placed transnasally. Protocol which included baseline recording, 16 water swallows (5 ml, 10 ml, and 15 ml) and the multiple rapid swallows was performed before and after esophageal infusion of menthol (3 mM, 20 min, 8 ml/min). We evaluated the effect of this infusion on the HRM parameters of esophageal peristalsis (distal contractile integral, distal latency, contractile front velocity) and the lower esophageal sphincter (LES) barrier function (integrated relaxation pressure and the inspiratory augmentation of the LES). Simultaneously we evaluated the quality and intensity of the symptoms during the menthol infusion. Esophageal infusion of menthol did not appreciably affect HRM measurements characterizing esophageal peristalsis and LES pressure in healthy subjects (N = 13) or GERD patients (N = 11). The magnitude of the distal contractile integral (5 ml) was changed neither in the healthy volunteers' group, (735 ± 127 vs. 814 ± 117 mmHg, p = 0.5), nor in the GERD patients (295 ± 78 vs. 338 ± 96 mmHg, p = 0.99). In healthy volunteers menthol did not change the inspiratory augmentation of the LES (8.67 ± 1.09 vs. 7.69 ± 0.96 mmHg, p = 0.15) and neither did for GERD patients (8.8 ± 1.18 vs. 8.22 ± 0.91 mmHg, p = 0.43). We observed no significant difference in any HRM parameter following menthol infusion, except for distal latency in 10 ml swallows. By contrast, menthol infusion induced significantly more intense discomfort in GERD patient than in healthy volunteers. Our results suggest no significant temporal effect of menthol on the esophageal motility or LES function, neither in healthy volunteers, nor in GERD. Arguably, other mechanisms are responsible for menthol-related heartburn.

Increased Sensitivity of Cough Reflex is Not the Mechanism of Cough Attributed to Laryngopharyngeal Reflux.

OBJECTIVES: In laryngopharyngeal reflux (LPR) patients acid reaches laryngopharyngeal area and stimulates/sensitizes respiratory nerve terminals mediating cough. We addressed several hypothesis: if stimulation of respiratory nerves is responsible for coughing then acidic LPR should correlate with coughing and proton pump inhibitor (PPI) treatment should reduce both LPR and coughing. If sensitization of respiratory nerves is responsible for coughing then cough sensitivity should correlate with coughing and PPI should reduce both coughing and cough sensitivity. STUDY DESIGN/METHODS: In this prospective single center study, patients with positive reflux symptom index (RSI > 13) and/or reflux finding score (RFS > 7) and ≥1 LPR episode/24 hours were enrolled. We evaluated LPR by dual channel 24-hour pH/impedance. We determined number of LPR events with pH drop at levels 6.0, 5.5, 5.0, 4.5, and 4.0. Cough reflex sensitivity was determined as lowest capsaicin concentration causing at least 2/5 coughs (C2/C5) by single breath capsaicin inhalation challenge. For statistical analysis C2/C5 values were -log transformed. Troublesome coughing was evaluated on the scale 0-5. RESULTS: We enrolled 27 LPR patients. The number of LPR events with pH 6.0, 5.5, 5.0, 4.5, and 4.0 was 14[8-23],4[2-6],1[1-3],1[0-2] and 0[0-1], respectively. There was no correlation between number of LPR episodes at any pH level and coughing (Pearson range -0.34 to 0.21, P = NS). There was no correlation between cough reflex sensitivity C2/C5 and coughing (R = -0.29 to 0.34, P = NS). Of patients that completed PPI treatment, 11 had RSI normalized (18.36 ± 2.75 vs. 7 ± 1.35, P < 0.01). There was no change in cough reflex sensitivity in PPI-responders. C2 threshold was 1.41 ± 0.19 vs. 1.2 ± 0.19 (P = 0.11) before and after PPI. CONCLUSIONS: No correlation between cough sensitivity and coughing and no change in cough sensitivity despite improvement of coughing by PPI argue that an increased cough reflex sensitivity is not mechanism of cough in LPR. We identified no simple relationship between LPR and coughing suggesting that this relationship is more complex.

Changes in the Urine Metabolomic Profile in Patients Recovering from Severe COVID-19.

Metabolomics is a relatively new research area that focuses mostly on the profiling of selected molecules and metabolites within the organism. A SARS-CoV-2 infection itself can lead to major disturbances in the metabolite profile of the infected individuals. The aim of this study was to analyze metabolomic changes in the urine of patients during the acute phase of COVID-19 and approximately one month after infection in the recovery period. We discuss the observed changes in relation to the alterations resulting from changes in the blood plasma metabolome, as described in our previous study. The metabolome analysis was performed using NMR spectroscopy from the urine of patients and controls. The urine samples were collected at three timepoints, namely upon hospital admission, during hospitalization, and after discharge from the hospital. The acute COVID-19 phase induced massive alterations in the metabolic composition of urine was linked with various changes taking place in the organism. Discriminatory analyses showed the feasibility of successful discrimination of COVID-19 patients from healthy controls based on urinary metabolite levels, with the highest significance assigned to citrate, Hippurate, and pyruvate. Our results show that the metabolomic changes persist one month after the acute phase and that the organism is not fully recovered.

Acute-on-chronic liver failure in patients with severe acute respiratory syndrome coronavirus 2 infection.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has had a significant impact on the lives of millions of people, especially those with other concomitant diseases, such as chronic liver diseases. To date, seven coronaviruses have been identified to infect humans. The main site of pathological action of these viruses is lung tissue. However, a substantial number of studies have proven that SARS-CoV-2 shows affinity towards several organs, including the gastrointestinal tract and the liver. The current state of evidence points to several proposed mechanisms of liver injury in patients with COVID-19 and their combination. Liver impairment is considered to be the result of the direct effect of the virus on the hepatic tissue cells, a systemic reaction consisting of inflammation, hypoxia and cytokine storm, drug-induced liver injury, with the possible contribution of a perturbed gut-liver axis. Reactivation of chronic hepatic disease could be another factor for liver impairment in patients with SARS-CoV-2 infection. Acute-on-chronic liver failure (ACLF) is a relatively new syndrome that occurs in 10%-30% of all hospitalized patients with chronic liver disease. It is crucial to recognize high-risk patients due to the increased morbidity and mortality in these cases. Several published studies have reported virus infection as a trigger factor for ACLF. However, to date, there are few relevant studies describing the presence of ACLF in patients with acute SARS-CoV-2 infection. In this minireview we summarize the current state of knowledge regarding the relation between ACLF and acute SARS-CoV-2 infection.

Gastrointestinal sequalae months after severe acute respiratory syndrome corona virus 2 infection: a prospective, observational study.

INTRODUCTION: Post-coronavirus disease (post-COVID) symptoms arise mostly from impaired function of respiratory tract although in many patients, the dysfunction of gastrointestinal tract and liver among other organ systems may persist. METHODS: Primary data collection was based on a short gastrointestinal symptom questionnaire at the initial screening. A brief telephone survey within the patient and control group was performed 5-8 months after the initial screening. R ver. 4.0.5 and imbalanced RandomForest (RF) machine-learning algorithm were used for data explorations and analyses. RESULTS: A total of 590 patients were included in the study. The general presence of gastrointestinal symptoms 208.2 days (153-230 days) after the initial acute severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) infection was 19% in patients with moderate-to-serious course of the disease and 7.3% in patients with mild course compared with 3.0% in SARS-CoV-2 negative controls (P < 0.001). Diarrhea and abdominal pain are the most prevalent post-COVID gastrointestinal symptoms. RF machine-learning algorithm identified acute diarrhea and antibiotics administration as the strongest predictors for gastrointestinal sequelae with area under curve of 0.68. Variable importance for acute diarrhea is 0.066 and 0.058 for antibiotics administration. CONCLUSION: The presence of gastrointestinal sequelae 7 months after the initial SARS-CoV-2 infection is significantly higher in patients with moderate-to-severe course of the acute COVID-19 compared with asymptomatic patients or those with mild course of the disease. The most prevalent post-COVID gastrointestinal symptoms are diarrhea and abdominal pain. The strongest predictors for persistence of these symptoms are antibiotics administration and acute diarrhea during the initial infection.

Persistence of Metabolomic Changes in Patients during Post-COVID Phase: A Prospective, Observational Study.

Several relatively recently published studies have shown changes in plasma metabolites in various viral diseases such as Zika, Dengue, RSV or SARS-CoV-1. The aim of this study was to analyze the metabolome profile of patients during acute COVID-19 approximately one month after the acute infection and to compare these results with healthy (SARS-CoV-2-negative) controls. The metabolome analysis was performed by NMR spectroscopy from the peripheral blood of patients and controls. The blood samples were collected on 3 different occasions (at admission, during hospitalization and on control visit after discharge from the hospital). When comparing sample groups (based on the date of acquisition) to controls, there is an indicative shift in metabolomics features based on the time passed after the first sample was taken towards controls. Based on the random forest algorithm, there is a strong discriminatory predictive value between controls and different sample groups (AUC equals 1 for controls versus samples taken at admission, Mathew correlation coefficient equals 1). Significant metabolomic changes persist in patients more than a month after acute SARS-CoV-2 infection. The random forest algorithm shows very strong discrimination (almost ideal) when comparing metabolite levels of patients in two various stages of disease and during the recovery period compared to SARS-CoV-2-negative controls.

A machine learning approach for identification of gastrointestinal predictors for the risk of COVID-19 related hospitalization.

Background and aim: COVID-19 can be presented with various gastrointestinal symptoms. Shortly after the pandemic outbreak, several machine learning algorithms were implemented to assess new diagnostic and therapeutic methods for this disease. The aim of this study is to assess gastrointestinal and liver-related predictive factors for SARS-CoV-2 associated risk of hospitalization. Methods: Data collection was based on a questionnaire from the COVID-19 outpatient test center and from the emergency department at the University Hospital in combination with the data from internal hospital information system and from a mobile application used for telemedicine follow-up of patients. For statistical analysis SARS-CoV-2 negative patients were considered as controls in three different SARS-CoV-2 positive patient groups (divided based on severity of the disease). The data were visualized and analyzed in R version 4.0.5. The Chi-squared or Fisher test was applied to test the null hypothesis of independence between the factors followed, where appropriate, by the multiple comparisons with the Benjamini Hochberg adjustment. The null hypothesis of the equality of the population medians of a continuous variable was tested by the Kruskal Wallis test, followed by the Dunn multiple comparisons test. In order to assess predictive power of the gastrointestinal parameters and other measured variables for predicting an outcome of the patient group the Random Forest machine learning algorithm was trained on the data. The predictive ability was quantified by the ROC curve, constructed from the Out-of-Bag data. Matthews correlation coefficient was used as a one-number summary of the quality of binary classification. The importance of the predictors was measured using the Variable Importance. A 2D representation of the data was obtained by means of Principal Component Analysis for mixed type of data. Findings with the p-value below 0.05 were considered statistically significant. Results: A total of 710 patients were enrolled in the study. The presence of diarrhea and nausea was significantly higher in the emergency department group than in the COVID-19 outpatient test center. Among liver enzymes only aspartate transaminase (AST) has been significantly elevated in the hospitalized group compared to patients discharged home. Based on the Random Forest algorithm, AST has been identified as the most important predictor followed by age or diabetes mellitus. Diarrhea and bloating have also predictive importance, although much lower than AST. Conclusion: SARS-CoV-2 positivity is connected with isolated AST elevation and the level is linked with the severity of the disease. Furthermore, using the machine learning Random Forest algorithm, we have identified the elevated AST as the most important predictor for COVID-19 related hospitalizations.

The infusion of menthol into the esophagus evokes cold sensations in healthy subjects but induces heartburn in patients with gastroesophageal reflux disease (GERD).

Recent studies in animal models have reported that some afferent fibers innervating the esophagus express the cold receptor TRPM8. In the somatosensory system the stimulation of TRPM8 leads to cold sensations and in certain circumstances alleviates pain. It is therefore hypothesized in this paper that the esophageal infusion of the TRPM8 activator menthol evokes cold sensations from the esophagus and alleviates heartburn in humans. The esophageal infusion of menthol (3 mM, 20 min) evoked cold sensations in 11 of 12 healthy subjects. In striking contrast, the esophageal infusion of menthol evoked heartburn in 10 of 10 patients with gastroesophageal reflux disease (GERD). In healthy subjects the cold sensation evoked by menthol was perceived only as a minor discomfort as evaluated by the visual analog scale (VAS score 1.9 ± 0.3 on the scale 1-10). However, in patients with GERD the menthol-induced heartburn was perceived as painful (VAS score 5.6 ± 0.6, P < 0.01 compared to healthy subjects). It is concluded that the sensations evoked by esophageal infusion of menthol change from relatively nonpainful cold sensations in healthy subjects to painful heartburn sensations in patients with GERD. These qualitative and quantitative changes indicate substantial alterations in afferent signaling mediating sensations from the esophagus in patients with GERD.

Effects of ginger constituent 6-shogaol on gastroesophageal vagal afferent C-fibers.

BACKGROUND: Ginger has been used as an herbal medicine worldwide to relieve nausea/vomiting and gastrointestinal discomfort, but the cellular and molecular mechanisms of its neuronal action remain unclear. The present study aimed to determine the effects of ginger constituent 6-shogaol on gastroesophageal vagal nodose C-fibers. METHODS: Extracellular single-unit recording and two-photon nodose neuron imaging were performed, respectively, in ex vivo gastroesophageal-vagal preparations from wild type and Pirt-GCaMP6 transgenic mice. The action potential discharge or calcium influx evoked by mechanical distension and chemical perfusions applied to the gastroesophageal vagal afferent nerve endings were recorded, respectively, at their intact neuronal cell soma in vagal nodose ganglia. The effects of 6-shogaol on nodose C-fiber neurons were then compared and determined. KEY RESULTS: Gastroesophageal application of 6-shogaol-elicited intensive calcium influxes in nodose neurons and evoked robust action potential discharges in most studied nodose C-fibers. Such activation effects were followed by a desensitized response to the second application of 6-shogaol. However, action potential discharges evoked by esophageal mechanical distension, after 6-shogaol perfusion, did not significantly change. Pretreatment with TRPA1 selective blocker HC-030031 inhibited 6-shogaol-induced action potential discharges in gastric and esophageal nodose C-fiber neurons, suggesting that TRPA1 played a role in mediating 6-shogaol-induced activation response. CONCLUSION AND INFERENCES: This study provides evidence that ginger constituent 6-shogaol directly activates vagal afferent C-fiber peripheral gastrointestinal endings. This activation leads to desensitization to subsequent application of 6-shogaol but not subsequent esophageal mechanical distension. Further investigation is required to establish a possible contribution in its anti-emetic effects.

Idiopathic Pan-Colonic and Small-Intestine Varices.

Idiopathic colonic varices represent a rare source of gastrointestinal haemorrhage with a presumed incidence around 0.0007%. Herein, we present a case of idiopathic colonic and small-intestine varices. According to our knowledge, this case report is the first description of both pan-colonic and small-intestine idiopathic varices of this extent. A young male patient without any previous notable medical history was admitted to the hospital because of massive enterorrhagia with haemodynamic instability. Colonoscopy revealed massive pan-colonic varices. After stabilization, numerous diagnostic procedures were performed in order to investigate the aetiology of pan-colonic varices without any explanation of the patient's condition. In addition, capsule endoscopy revealed varices through the whole length of the small intestine. The final diagnosis was idiopathic varices of the colon and small intestine. Because of the rapid clinical stabilization, the single incident of haemorrhage and the extension of the disease, a conservative approach was chosen (venotonics and ß-blockers). During the 12-month follow-up period, the patient reported no gastrointestinal haemorrhage.

The combination of ACE inhibition plus sympathetic denervation is superior to ACE inhibitor monotherapy in the rat renal ablation model.

BACKGROUND: The blood pressure-independent renoprotective actions of the blockade of the renin-angiotensin and the sympathetic nervous system are well documented, but monotherapies fail to completely abrogate progression. We investigated whether combined inhibition of the two systems provides additive renoprotection. METHODS: After subtotal nephrectomy (SNX) or sham operation, rats underwent resection of dorsal roots, i.e. rhizotomy or sham rhizotomy. Subsequently, they received tap water or quinapril in drinking water for 16 weeks (n = 18/group). Albuminuria, blood pressure and kidneys were assessed (morphometry, immunohistochemistry). RESULTS: At the end of the study telemetric blood pressure in SNX was 118 +/- 16 mm Hg, in SNX + rhizotomy 110 +/- 10 mm Hg, in SNX + quinapril 103 +/- 9 mm Hg and in SNX + quinapril + rhizotomy 95 +/- 7 mm Hg. Albuminuria in the respective groups was 169 +/- 75, 86 +/- 45, 15 +/- 23 and 5 +/- 4 mg/24 h. The glomerulosclerosis index was 1.40 +/- 0.6, 0.80 +/- 0.23, 0.37 +/- 0.16 and 0.31 +/- 0.15 (p < 0.001). Only combined intervention caused significant reduction of the glomerular volume and podocyte hypertrophy. The lowest indices for nitrotyrosine, NOS-1 (nNOS), TGF-beta and interstitial collagen were seen with combined interventions (p < 0.05). CONCLUSION: In angiotensin-converting enzyme inhibitor-treated SNX animals, abrogation of sympathetic overactivity provides additional renoprotection and less nitro-oxidative stress of podocytes than single interventions. The added benefits were partially blood pressure independent.

Primer: histopathology of polyomavirus-associated nephropathy in renal allografts.

The BK polyomavirus exhibits tropism for the renal tubular epithelium, where it establishes latent infection. Vigorous immunosuppression of renal allograft recipients can lead to reactivation of the infection and the development of polyomavirus-associated nephropathy (PVAN). Clinically, gradually decreasing renal function, viremia and viruria are observed several months after transplantation; allograft failure occurs in 1-10% of patients. Definitive diagnosis requires an allograft biopsy. Histologically, viral replication results in tubular epithelial cell enlargement, karyomegaly and nuclear inclusion bodies. The cytopathic changes are often associated with lysis of tubular epithelial cells, denudation of the basement membrane and an interstitial inflammatory response. The involvement is multifocal; distal nephron segments are more severely affected than proximal segments. Changes observed during light microscopy are suggestive but not pathognomonic for PVAN, and the diagnosis must be confirmed by adjunct studies. Adjunct studies consist of immunohistochemistry on paraffin sections using an antibody to the SV40 large T antigen, or electron microscopy of infected tubular epithelial cells (virions 40 nm in diameter). PVAN manifests in three histologic patterns: pattern A, viral cytopathic changes with no or only minimal inflammation; pattern B, cytopathic and cytolytic lesions with interstitial inflammation; or pattern C, predominantly interstitial fibrosis and tubular atrophy, with variable cytopathic and inflammatory changes. These patterns correlate with clinical outcomes.

Primer: Histopathology of calcineurin-inhibitor toxicity in renal allografts.

Calcineurin inhibitors (ciclosporin and tacrolimus) can cause acute and chronic nephrotoxicity. The serum levels of these drugs do not correlate well with the extent of renal damage caused, and the clinical manifestation is nonspecific. Renal biopsy is a reliable tool with which to diagnose calcineurin-inhibitor-induced nephrotoxicity. Ciclosporin and tacrolimus produce identical lesions, which are focal in nature and can be overlooked, necessitating the evaluation of serial tissue sections. Acute toxicity is characterized histologically by necrosis and early hyalinosis of individual smooth muscle cells in the afferent arterioles, and/or isometric vacuolation of the proximal straight tubules; thrombotic microangiopathy is a rare manifestation. In chronic toxicity, the damaged media smooth muscle cells in afferent arterioles are replaced by beaded medial hyaline deposits that bulge into the adventitia; the interstitium displays striped fibrosis and tubular atrophy. As maintenance doses of calcineurin inhibitors in renal transplant recipients have been lowered during the past decade, the incidence of acute toxicity has decreased markedly. Chronic toxicity, however, is still prevalent, and causes chronic allograft damage.

Ultrastructural analysis of the Fisher to Lewis rat model of chronic allograft nephropathy.

Chronic allograft nephropathy (CAN) is the leading cause of graft loss following kidney transplantation. One factor contributing to CAN is chronic alloimmune injury. However, the involvement of alloantigen-dependent and -independent factors in CAN is unclear. The pathomechanism of CAN has been extensively studied by utilizing the Fischer-to-Lewis (F344-to-LEW) rat model. Transplant capillaropathy (circumferential multiplication of the peritubular capillary basement membrane) and transplant glomerulopathy (reduplication of the glomerular basement membrane) have recently been validated clinicopathologically as ultrastructural indicators of chronic alloimmune injury. To investigate the presence of these markers, F344-to-LEW kidneys were examined by electron and light microscopy 32, 40 and 52 weeks after implantation. F344 rats with or without 30-min ischemia of the left kidney following right nephrectomy served as controls. All transplanted rats displayed marked proteinuria. On electron microscopy, transplant capillaropathy, transplant glomerulopathy, and T-cell cytotoxicity (indicator of ongoing cellular rejection) were absent. On light microscopy, the arteries were devoid of intimal fibrosis. Focal-segmental glomerulopathy resembling hyperfiltration injury was encountered, with mild interstitial infiltration, fibrosis, and tubular atrophy. The proteinuria and kidney pathology were more severe in transplanted than in ischemic or uninephrectomized rats. Because chronic-active rejection could not be detected between weeks 32 and 52, we propose that the alloantigen-dependent initial graft injury subsides, but induces the late events: glomerular hyperfiltration, proteinuria, and glomerulosclerosis. Accordingly, the model - in the late phase - is suitable to investigate alloantigen-independent factors of CAN and lacks markers of alloantigen-dependent processes.