Background: Culturing primary epithelial cells has a major advantage over tumor-derived or immortalized cell lines as long as their functional phenotype and genetic makeup are mainly maintained. The swine model has shown to be helpful and reliable when used as a surrogate model for human diseases. Several porcine cell lines have been established based on a variety of tissues, which have shown to extensively contribute to the current understanding of several pathologies, especially cancer. However, protocols for the isolation and culture of swine gastric epithelial cells that preserve cell phenotype are rather limited. We aimed to develop a new method for establishing a primary epithelial cell culture from the fundic gland region of the pig stomach. Results: Mechanical and enzymatic dissociation of gastric tissue was possible by combining collagenase type I and dispase II, protease inhibitors and antioxidants, which allowed the isolation of epithelial cells from the porcine fundic glands showing cell viability > 90% during the incubation period. Gastric epithelial cells cultured in RPMI 1640, DMEM-HG and DMEM/F12 media did not contribute enough to cell adhesion, cluster formation and cell proliferation. By contrast, William’s E medium supplemented with growth factors supports confluency and proliferation of a pure epithelial cell monolayer after 10 days of incubation at 37 °C, 5% CO2. Mucin-producing cell phenotype of primary isolates was confirmed by PAS staining, MUC1 by immunohistochemistry, as well as the expression of MUC1 and MUC20 genes by RT-PCR and cDNA sequencing. Swine gastric epithelial cells also showed origin-specific markers such as cytokeratin cocktail (AE1/AE3) and cytokeratin 18 (CK-18) using immunohistochemical and immunofluorescence methods, respectively. Conclusions: A new method was successfully established for the isolation of primary gastric epithelial cells from the fundic gland zone through a swine model based on a combination of tissue-specific proteases, protease inhibitors and antioxidants after mechanical cell dissociation. The formulation of William’s E medium with growth factors for epithelial cells contributes to cell adhesion and preserves functional primary cells phenotype, which is confirmed by mucin production and expression of typical epithelial markers over time.

Chronic hepatitis B virus (HBV) infection is an increasing cause of morbidity and mortality in human immunodeficiency virus (HIV)-infected individuals. HIV-positive patients are commonly co-infected with HBV due to shared routes of transmission. Our aim was to determine the risk factors, prevalence, genotypes, and mutations of the Surface S gene of HBV, and occult hepatitis B infection (OBI) among patients infected with HIV in a northeastern Colombian city. A cross-sectional study was conducted with 275 HIV-positive patients attending an outpatient clinic in Bucaramanga, Colombia during 2009–2010. Blood samples were collected and screened for serological markers of HBV (anti-HBs, anti-HBc and HBsAg) through ELISA assay. Regardless of their serological profile, all samples were tested for the HBV S gene by nested-PCR and HBV genotypes were determined by phylogenetic inference. Clinical records were used to examine demographic, clinical, virological, immunological and antiretroviral therapy (ART) variables of HIV infection. Participants were on average 37±11 years old and 65.1% male. The prevalence of HIV-HBV coinfection was 12% (95%CI 8.4–16.4) of which 3.3% had active HBV infection and 8.7% OBI. The prevalence of HIV-HBV coinfection was associated with AIDS stage and ART treatment. Sequence analysis identified genotype F, subgenotype F3 in 93.8% of patients and genotype A in 6.2% of patients. A C149R mutation, which may have resulted from failure in HBsAg detection, was found in one patient with OBI. The present study found a high prevalence of HIV-HBV coinfection with an incidence of OBI 2.6-fold higher compared to active HBV infection. These findings suggest including HBV DNA testing to detect OBI in addition to screening for HBV serological markers in HIV patients.

Hepatitis B virus (HBV) antiviral Resistance-Associated Mutations (RAMs) in human immunodeficiency virus (HIV) coinfected patients undergoing highly active antiretroviral therapy (HAART) are complex and incompletely understood. We aimed to determine the prevalence of HBV coinfection, HBV genotypes, and RAMs in a cohort of people living with HIV (PLWH) in the northeastern region of Colombia. This cross-sectional study was carried out between February 2013 and February 2014. Virological, immunological and HAART data were collected from clinical records. In-house nested PCR and Sanger sequencing of the HBV pol gene were used to identify coinfections, genotypes, RAMs and HBV s antigen (HBsAg) escape mutants. Among 275 PLWH, HBV coinfection was confirmed in 32 patients (11.6%), of whom nine (28.2%) were HBsAg positive (active hepatitis B), and 23 (71.8%) were occult hepatitis B infections (OBI). All HBV sequences (n = 23) belonged to the genotype F3. Among HIV/HBV coinfections, 71.9% had CD4+ T cell counts above 200 cells/mm³ and 37.5% had undetectable HIV viral loads. The RAMs rtL80I, rtL180M, and rtM204V, which confer resistance to Lamivudine/Telbivudine and partially resistant to Entecavir, were found in all HBV isolates. An unknown rt236Y mutation to Tenofovir was also identified. Most patients under HAART received first-generation HBV antiviral therapy with a low genetic barrier to resistance. Antiviral Drug-associated Potential Vaccine-escape Mutations (ADAPVEMs) in the S gene were observed in all isolates ranging from 1–20 amino acid substitutions. However, no vaccine escape mutants were detected. In Conclusion, these findings highlight the importance of HBV molecular screening, antiviral resistance monitoring and new guidelines for PLWH to overcome RAMs and prevent HBV-related liver disease.

The establishment of reliable in vitro oral mucosa models is essential for advancing studies in epithelial barrier function, wound healing, and host–microbe interactions. However, the widespread use of immortalized cell lines such as HaCaT or TR146 limits physiological relevance owing to altered differentiation profiles and genetic drift. In this study, we developed a robust mechanical–enzymatic protocol for isolating and expanding primary human gingival keratinocytes from healthy gingival explants without feeder layers. The resulting cells demonstrated high viability, maintained consistent proliferative capacity across passages, and exhibited characteristic cobblestone morphology. Comprehensive phenotypic validation included immunofluorescence and immunohistochemistry confirming strong expression of epithelial markers CK18, AE1/AE3, and MUC1, with absence of the mesenchymal marker vimentin. Transcriptomic analysis using RT-qPCR corroborated epithelial lineage fidelity, revealing stable MUC1 expression and lack of MUC5AC transcripts, indicative of a nonglandular phenotype. Metabolic competence was supported by WST-1 assays that correlated strongly with manual cell counts, underscoring functional viability. Importantly, AGS and 293T/17 cell lines were processed in parallel as orthogonal controls to confirm assay specificity and lineage discrimination. Under rigorously standardized, within-laboratory conditions, our workflow yielded high interdonor concordance in epithelial identity and growth kinetics across a young-adult cohort ( n = 3), supporting its use as a practical primary-cell platform for downstream applications. Generalizable reproducibility—across age strata, operators, and sites—will require formal, preregistered multicenter validation. By mitigating limitations inherent to immortalized lines, this approach enables more accurate investigations of epithelial biology and strengthens the reliability of in vitro experimental systems relevant to oral regenerative medicine and mucosal immunology.

Introduction: Helicobacter pylori exhibit variations in antibiotic resistance in America and Colombia. Despite the use of various treatment regimens containing amoxicillin in the department of Antioquia, Colombia, as well as in several countries across the continent, the pattern of genotypic and phenotypic susceptibility to this antibiotic remains unknown. Objective: To determine the frequency of H. pylori infection by detecting the ureA gene and amoxicillin susceptibility through beta-lactamases in H. pylori isolates from patients with gastroduodenal diseases in Antioquia-Colombia. Methods: Cross-sectional analytical study with 179 patients attending endoscopy units of seven healthcare institutions. Sociodemographic and risk factor data were collected. Moreover, biopsies from the antrum and body were taken for microbiological isolation and molecular detection of H. pylori. The sociodemographic data were assessed through frequency analysis and bivariate statistics. DNA obtained from the biopsies was amplified by PCR for the ureA gene, and beta-lactamase production was evaluated using the nitrocefin test as well as the amplification of the TEM and SHV genes. Results: It was found that 61 % (109 / 179) of patients tested positive for H. pylori; out of 84 isolates obtained by culture, none tested positive for the nitrocefin test. Significant differences were found in socioeconomic status, healthcare system, and geographical location in patients positive for the ureA gene. Two patients tested positive for molecular detection of beta-lactamases from biopsies; however, SHV and TEM genes were not found in any isolate. Conclusions: The frequency of H. pylori for ureA-gene detection is higher than previous studies and similar to other reports in other Colombian regions; consequently, this will become a good diagnostic test in the country. Finally, none of the H. pylori isolates tested positive for beta-lactamases TEM and SHV.

Purpose Occult hepatitis B infection (OBI) can sustain residual transfusion risk because HBV DNA may persist at very low levels while HBsAg remains undetectable by routine serology. We assessed OBI among anti-HBc–reactive, HBsAg-negative (anti-HBc+/HBsAg−) donors in Bucaramanga, Colombia and characterized small-S variation within genotype F3. Methods In 2021, we analyzed 61,188 donations in three licensed blood banks using routine serology. From anti-HBc+/HBsAg− donations, 219 randomly selected sera underwent nested PCR targeting the small-S/reverse-transcriptase overlap, followed by Sanger sequencing. Genotyping and phylogeny used a curated 960-nt small-S fragment. Associations with OBI were estimated by logistic regression. Results HBsAg reactivity was 0.21% and anti-HBc prevalence was 1.77%. OBI occurred in 16/219 anti-HBc+/HBsAg− donations (7.3%; 95% CI, 4.5–11.5), approximately 130 per 100,000 donations (95% CI, 80–204). Among the 16 OBI cases, 12 were anti-HBs reactive. Sex, age, and anti-HBs were not associated with OBI after adjustment. All sequences clustered within genotype F3. Small-S amino acid variation at positions previously linked to altered HBsAg detectability was observed in 11/16 (68.8%): A166G (5/16) outside “a”; “a” substitutions L127R, K141N, N146Y, C147S; variants at the pre-“a” boundary K122R/T123A; loop-proximal cysteine changes C149S/C149F and S117C; stop codons W156 and L216*; additional MHR changes included V106D, T118S, I150V, P151R, I152S, K160I. Conclusions All OBI cases belonged to genotype F3 and showed small-S variants at positions previously associated with HBsAg detectability, which may contribute to reduced detection rather than classical antigenic escape. Among anti-HBc+/HBsAg− donations, targeted NAT identified donors with detectable HBV DNA within this serologic profile that routine screening alone could not resolve, indicating a residual risk that may be reduced through selective NAT where universal implementation is not feasible.