Total Keratin-18 (M65) as a Potential, Early, Non-Invasive Biomarker of Hepatocyte Injury in Alcohol Intoxicated Adolescents—A Preliminary Study

Background: Underage drinking is associated with health risk behaviors. Serum keratin-18 (CK18) levels are increased in liver diseases and may be biomarkers of outcome. The purpose of this study was to determine if the total CK18 (M65) or caspase-cleaved CK18 (M30) levels were different in adolescents admitted to hospital because of alcohol intoxication and controls with excluded liver diseases. Methods: A prospective study included 57 adolescents after alcohol use and 23 control subjects. The concentrations of M30 and M65 in the serum samples were evaluated using an enzyme-linked immunosorbent assay. Results: The median age was 15 (14–17) years and 49% were male. There were significant differences in M65 levels between the study and control groups (p = 0.03). The concentrations of M30 and M65 were insignificant in adolescents divided into subgroups according to blood alcohol concentrations (BAC). Significant positive correlations were found between BAC and M65 levels (p = 0.038; r = 0.3). In receiver operating characteristic (ROC) analysis M65 (cut-off = 125.966 IU/l, Se = 70.2%, Sp = 43.5%) allowed to differentiate between patients with and without alcohol intoxication (AUC = 0.66, p = 0.03). Conclusion: M65 appears to be a promising non-invasive biomarker of hepatocyte injury during alcohol intoxication in adolescents. Moreover, a higher concentration of M65 may indicate early organ injury before the increase in the activity of liver enzymes, alanine aminotransferase (ALT) and aspartate aminotransferase (AST).

Revealing the Roles of Keratin 8/18-Associated Signaling Proteins Involved in the Development of Hepatocellular Carcinoma

Although hepatocellular carcinoma (HCC) is developed with various etiologies, protection of hepatocytes seems basically essential to prevent the incidence of HCC. Keratin 8 and keratin 18 (K8/K18) are cytoskeletal intermediate filament proteins that are expressed in hepatocytes. They maintain the cell shape and protect cells under stress conditions. Their protective roles in liver damage have been described in studies of mouse models, and K8/K18 mutation frequency in liver patients. Interestingly, K8/K18 bind to signaling proteins such as transcription factors and protein kinases involved in HCC development. Since K8/K18 are abundant cytoskeletal proteins, K8/K18 binding with the signaling factors can alter the availability of the factors. Herein, we discuss the potential roles of K8/K18 in HCC development.

Over-expression of keratin 18 in human endometrial cancer.

Gynecologic cancers including cancers of the endometrium are a clinical problem (1-4). We mined published and public microarray data (5, 6) to discover genes associated with endometrial cancers by comparing transcriptomes of the normal endometrium and endometrial tumors from humans. We identified keratin 18, encoded by KRT18, as among the most differentially expressed genes, transcriptome-wide, in cancers of the endometrium. KRT18 was expressed at significantly higher levels in endometrial tumor tissues as compared to the endometrium. Importantly, in human endometrial cancer, primary tumor expression of KRT18 was correlated with overall survival in white patients with high and low mutational burden. KRT18 may be a molecule of interest in understanding the etiology or progression of human endometrial cancer.

The K18-hACE2 Transgenic Mouse Model Recapitulates Non-Severe and Severe COVID-19 in Response to Infectious Dose of SARS-CoV-2 Virus

A comprehensive analysis and characterization of a SARS-CoV-2 infection model that mimics non-severe and severe COVID-19 in humans is warranted for understating the virus and developing preventive and therapeutic agents. Here, we characterized the K18-hACE2 mouse model expressing human (h)ACE2 in mice, controlled by the human keratin 18 (K18) promoter, in epithelia, including airway epithelial cells where SARS-CoV-2 infections typically start. We found that intranasal inoculation with higher viral doses (2x103 and 2x104 PFU) of SARS-CoV-2 caused lethality of all mice and severe damage of various organs, including lungs, liver, and kidney, while lower doses (2x101 and 2x102 PFU) led to less severe tissue damage and some mice recovered from the infection. In this humanized hACE2 mouse model, SARS-CoV-2 infection damaged multiple tissues, with a dose-dependent effect in most tissues. Similar damage was observed in biopsy samples from COVID-19 patients. Finally, the mice that recovered after infection with a low dose of virus also survived rechallenge with a high dose of virus. Compared to other existing models, the K18-hACE2 model seems to be the most sensitive COVID-19 model reported to date. Our work expands the information available about this model to include analysis of multiple infectious doses and various tissues with comparison to human biopsy samples from COVID-19 patients. In conclusion, the K18-hACE2 mouse model recapitulates both severe and non-severe COVID-19 in humans and can provide insight into disease progression and the efficacy of therapeutics for preventing or treating COVID-19.

Human convalescent plasma protects K18-hACE2 mice against severe respiratory disease

SARS-CoV-2 is the causative agent of COVID-19 and human infections have resulted in a global health emergency. Small animal models that reproduce key elements of SARS-CoV-2 human infections are needed to rigorously screen candidate drugs to mitigate severe disease and prevent the spread of SARS-CoV-2. We and others have reported that transgenic mice expressing the human angiotensin-converting enzyme 2 (hACE2) viral receptor under the control of the Keratin 18 (K18) promoter develop severe and lethal respiratory disease subsequent to SARS-CoV-2 intranasal challenge. Here we report that some infected mice that survive challenge have residual pulmonary damages and persistent brain infection on day 28 post-infection despite the presence of anti-SARS-COV-2 neutralizing antibodies. Because of the hypersensitivity of K18-hACE2 mice to SARS-CoV-2 and the propensity of virus to infect the brain, we sought to determine if anti-infective biologics could protect against disease in this model system. We demonstrate that anti-SARS-CoV-2 human convalescent plasma protects K18-hACE2 against severe disease. All control mice succumbed to disease by day 7; however, all treated mice survived infection without observable signs of disease. In marked contrast to control mice, viral antigen and lesions were reduced or absent from lungs and absent in brains of antibody-treated mice. Our findings support the use of K18-hACE2 mice for protective efficacy studies of anti-SARS-CoV-2 medical countermeasures (MCMs). They also support the use of this system to study SARS-CoV-2 persistence and host recovery.

Caspase-Cleaved Keratin 18 Measurements Identified Ongoing Liver Injury after Bariatric Surgery

Bariatric surgery has emerged as an effective treatment option in morbidly obese patients with non-alcoholic fatty liver disease (NAFLD). However, worsening or new onset of non-alcoholic steatohepatitis (NASH) and fibrosis have been observed. Caspase-cleaved keratin 18 (ccK18) has been established as a marker of hepatocyte apoptosis, a key event in NASH development. Thus, ccK18 measurements might be feasible to monitor bariatric surgery patients. Clinical data and laboratory parameters were collected from 39 patients undergoing laparoscopic Roux-en-Y gastric bypass at six timepoints, prior to surgery until one year after the procedure. ccK18 levels were measured and a high-throughput analysis of serum adipokines and cytokines was carried out. Half of the cohort’s patients (20/39) presented with ccK18 levels indicative of progressed liver disease. 21% had a NAFLD-fibrosis score greater than 0.676, suggesting significant fibrosis. One year after surgery, a mean weight loss of 36.87% was achieved. Six and twelve months after surgery, ccK18 fragments were significantly reduced compared to preoperative levels (p < 0.001). Yet nine patients did not show a decline in ccK18 levels ≥ 10% within one year postoperatively, which was considered a response to treatment. While no significant differences in laboratory parameters or ccK18 could be observed, they presented with a greater expression of leptin and fibrinogen before surgery. Consecutive ccK18 measurements monitored the resolution of NAFLD and identified non-responders to bariatric surgery with ongoing liver injury. Further studies are needed to elicit the pathological mechanisms in non-responders and study the potential of adipokines as prognostic markers.

The Role of Keratin-8 and Keratin-18 Polymorphisms and Protein Levels in the Occurrence and Progression of Vocal Leukoplakia

<b><i>Objective:</i></b> This study aimed to evaluate the association between the single-nucleotide polymorphism (SNP) and tissue protein level of keratin-8/18 and the occurrence and progression of vocal leukoplakia. <b><i>Methods:</i></b> The case-control study enrolled 158 patients with vocal leukoplakia, 326 patients with laryngeal squamous cell carcinoma (LSCC), and 268 healthy controls, which were tested for genotype analysis with keratin-8 and keratin-18 gene polymorphisms using pyrosequencing. The tissue protein expression levels of keratin-8 and keratin-18 were evaluated using immunohistochemistry. <b><i>Results:</i></b> The keratin-8 SNP RS1907671 showed an obvious increased risk for vocal leukoplakia (OR 1.56, <i>p</i> = 0.002), while the other SNPs (RS2035875, RS2035878, RS4300473) were tested as protective factors for vocal leukoplakia and LSCC (OR &#x3c;1, <i>p</i> &#x3c; 0.05). In keratin-18 SNP test, both RS2070876 and RS2638526 polymorphisms demonstrated decreased risks for vocal leukoplakia and LSCC (OR &#x3c;1, <i>p</i> &#x3c; 0.05). The protein levels of keratin-8 and keratin-18 in vocal leukoplakia group were significantly higher than those of the LSCC group (<i>p</i> &#x3c; 0.05). <b><i>Conclusions:</i></b> Keratin-8 and keratin-18 polymorphisms and protein levels are associated with the occurrence and progression of vocal leukoplakia.

The Developmental & Molecular Requirements for Ensuring that Human Pluripotent Stem Cell-Derived Hair Follicle Bulge Stem Cells Have Acquired Competence for Hair Follicle Generation Following Transplantation

When using human induced pluripotent stem cells (hiPSCs) to achieve hair follicle (HF) replacement, we found it best to emulate the earliest fundamental developmental processes of gastrulation, ectodermal lineage commitment, and dermogenesis. Viewing hiPSCs as a model of the epiblast, we exploited insights from mapping the dynamic up- and down-regulation of the developmental molecules that determine HF lineage in order to ascertain the precise differentiation stage and molecular requirements for grafting HF-generating progenitors. To yield an integrin-dependent lineage like the HF in vivo, we show that hiPSC derivatives should co-express, just prior to transplantation, the following combination of markers: integrins α6 and β1 and the glycoprotein CD200 on their surface; and, intracellularly, the epithelial marker keratin 18 and the hair follicle bulge stem cell (HFBSC)-defining molecules transcription factor P63 and the keratins 15 and 19. If the degree of trichogenic responsiveness indicated by the presence of these molecules is not achieved (they peak on Days 11-18 of the protocol), HF generation is not possible. Conversely, if differentiation of the cells is allowed to proceed beyond the transient intermediate progenitor state represented by the HFBSC, and instead cascades to their becoming keratin 14+ keratin 5+ CD200– keratinocytes (Day 25), HF generation is equally impossible. We make the developmental case for transplanting at Day 16-18 of differentiation—the point at which the hiPSCs have lost pluripotency, have attained optimal expression of HFBSC markers, have not yet experienced downregulation of key integrins and surface glycoproteins, have not yet started expressing keratinocyte-associated molecules, and have sufficient proliferative capacity to allow a well-populated graft. This panel of markers may be used for isolating (by cytometry) HF-generating derivatives away from cell types unsuited for this therapy as well as for identifying trichogenic drugs.

Time-Course Changes of Serum Keratin Concentrations after Liver Transplantation: Contrasting Results of Keratin-18 and Keratin-19 Fragments

Objective. Under normal conditions, adult hepatocytes express only keratin-8 (K8) and keratin-18 (K18), whereas cholangiocytes also express K19. In this study, we delineate the pattern of normal time-course changes in serum K19 and K18 levels after liver transplantation. Patients and Methods. Serum levels of the K19 fragment CYFRA 21-1 and the K18 fragments tissue polypeptide specific antigen (TPS) and M30 (a neoepitope that is generated after caspase cleavage during apoptosis) were measured at baseline and at regular intervals (up to 6 months) after liver transplantation in 11 adult patients. Results. There was a gradual decrease in serum K19 concentrations from baseline values after transplantation, following a time-course pattern similar to that of serum bilirubin. In contrast, serum concentrations of K18 fragments increased markedly shortly after transplantation and gradually decreased thereafter, following a time-course pattern similar to that of serum transaminases. The increase in TPS tended to occur earlier than that in M30, suggesting an initial predominance of hepatocyte necrosis followed by a predominance of apoptosis in the first days after transplantation. Five patients presented posttransplant complications (acute rejection in three cases and HCV recurrence in two cases). An early increase in serum K19 concentrations was observed in all cases. An increase in serum concentrations of K18 fragments (M30 and TPS) was observed in the two cases with HCV recurrence and was more variable in the three cases with acute rejection. Conclusions. Serum concentrations of K19 and K18 fragments follow a dissimilar pattern of time-course changes after liver transplantation. The diagnostic value of variations in these normal patterns should be addressed in future studies.