Molecular and experimental biology in medicine

WHAT OPTIONS FOR FERTILITY PRESERVATION DO PRE-PUBERTAL BOYS AND GIRLS HAVE (IN CROATIA)?

2022-12-16T10:02:50+01:00

The latest progress in therapies indicated for the treatment of childhood cancers has enabled the improvement of survival rates in affected patients. However, the treatment can have gonadotoxic effects and sometimes results in infertility. Therefore, strategies for fertility preservation are developed for storage of intact reproductive tissue and possible future fertility restoration when the patient is cured of the initial disease. Currently, only the cryopreservation of immature reproductive tissue is well established. In contrast, the approach to restoring fertility using the stored tissue is, for now, in most cases, only theoretical, especially concerning pre-pubertal boys.

On the other hand, fertility restoration in pre-pubertal girls is much more advanced, but further studies are needed to ensure safety regarding malignancies with a high risk of metastases. In order to overcome the risk of malignant contamination, when the cryopreserved reproductive tissue is intended to be applied (whether it is immature testicular or ovarian tissue), a suitable alternative to reproductive tissue autotransplantation is in vitro culture and development of mature gametes. For now, these methods are still in their onset and far from clinical application. Still, their advancement can be expected in the near future, given the rapid development of scientific techniques.

FIRST CYTOGENOMIC CHARACTERIZATION OF MURINE TESTIS TUMOR CELL LINE MLTC-1

2022-12-16T10:02:50+01:00

The cell line MLTC-1 was established in 1982 as a transplantable Leydig cell tumor from a C57BL/6 mouse. The cell line has already been applied in >100 studies: still, the only information about its genetic content is given in the first description: MLTC-1 initially had a polyploid karyotype. Here, a molecular karyotyping and multicolor banding-based molecular cytogenetic study was done to provide the first chromosomal/ (molecular) cytogenetic characterization of MLTC-1. A hexaploid karyotype with 72 to 105 chromosomes was hereby characterized. Besides polyploidy, unbalanced two- and three-way translocations, dicentrics and one neocentric derivative were identified. Also, no Y-chromosome could be detected in this clearly male derived cell line. Overall, a completely unbalanced genome is present in MLTC-1 with ~20 regions being subject to copy number losses or gains. After translating these imbalances into the human genome in a standardized way, a 40% match of imbalances with human Leydig cell tumors was evident; however, about the same rate of concordance was detectable for human spermatocytic seminomas and non-seminomas as well as testicular germ cell tumor. Accordingly, MLTC-1 is better suited as an advanced testicular germ cell tumor model in general, rather than specifically for human Leydig cell tumors.

PREDICTIVE IMMUNOCYTOCHEMISTRY IN NON-SMALL CELL LUNG CANCER CYTOLOGICAL SAMPLES

2022-12-16T10:02:51+01:00

Non-small cell lung cancer (NSCLC) molecular biomarker testing is obligatory for determining therapy. The aim of this study was to compare immunocytochemistry (ICC) results of NSCLC predictive biomarkers between bronchoscopic and non-bronchoscopic type of cytology samples. This study included archive records of 1109 predictive ICC results (ALK, ROS1, and PD-L1). The ICC was done on bronchoscopic, and non-bronchoscopic NSCLC samples prepared as cytological smears and cytospins, using Dako EnVisionTM FLEX detection visualization system. The ALK, ROS1, and PD-L1 distribution between bronchoscopic and non-bronchoscopic samples was analysed. The future perspective of cytology in precision medicine was reconsidered. The obtained positive results of ALK, ROS1, and PD-L1 ICC were in concordance with the previously observed range. There was no statistically significant difference in ALK, ROS1, and PD-L1 ICC distribution between the bronchoscopic and non-bronchoscopic groups of samples (p=0.730). The comparison of PD-L1 expression, and, separately PD-L1 ≥50% expression, between two groups of samples showed no statistically significant difference (p=0.236; p=0.436). Bronchoscopic and non-bronchoscopic samples prepared as cytological smears and cytospins are a suitable, but underutilized resource for ALK, ROS1, and PD-L1 biomarker analysis. The implementation of optimized predictive immunocytochemistry assays to provide rapid and reliable results for limited tumour samples is necessary.

HLA ANTIGEN AND HLA EPLET MISMATCHES - IMPORTANT FACTORS FOR DEVELOPING DONOR-SPECIFIC ANTIBODIES AFTER KIDNEY TRANSPLANTATION

2022-12-16T10:02:51+01:00

Kidney donor-recipient mismatches (MM) in human leukocyte antigen (HLA) system can have the impact on graft survival as de novo formation of donor-specific HLA antibodies (DSA) post-transplant can increase the risk of acute and chronic rejections. Eplets, the smallest functional units of epitopes, are in recent years also being considered in recipient-donor matching. We have retrospectively analysed the relationship between MM at HLA-A, -B, -DRB1 and -DQB1 loci and the development of DSA in 47 kidney transplant recipients that were negative for the presence of HLA antibodies pre-transplant. A total of 19 patients (40%) developed DSA (DSA+) post-transplant, revealing sensitization to HLA class II MM antigens as the most prevalent (84% of DSA+ patients). MM at all HLA loci contributed to the development of HLA locus-specific antibodies, with the prevalence of HLA-DQ sensitization, as 41% of recipients with HLA-DQ MM developed DSA. HLAMatchmaker analysis for HLA-DR/DQ total eplet MM showed statistically significant difference between DSA- (N=33) and DSA+ recipients (N=14) for a total number of eplet MM (300 in DSA- vs 248 in DSA+; P=0.0004). These results emphasize the importance of HLA Class II matching in kidney transplantation.

HLA GAMMA BLOCK MATCHING IN UNRELATED HEMATOPOIETIC STEM CELL TRANSPLANTATION AND THE DEVELOPMENT OF GRAFT VERSUS HOST DISEASE

2022-12-16T10:02:52+01:00

The human leukocyte antigen (HLA) genes routinely typed for hematopoietic stem cell transplantation (HSCT) are HLA-A, -B, -C, -DRB1 and -DQB1. HLA mismatches (MM), which have been associated with many post-transplantation complications, including acute graft-versus-host disease (GvHD), sometimes occur when a fully matched donor is not available. Gamma block (GB) is located in the central HLA region between Beta and Delta blocks and contains many inflammatory and immune regulatory genes, including the C4 gene. C4 was proposed as a marker when predicting haplotype matching due to positive linkage disequilibrium (LD) with its surrounding loci. Our aim was to investigate the association between GB matching in patients and their 9/10 HLA matched unrelated donors (UD) and the occurrence of GvHD. Patients and their UDs were typed using the PCR-SSP kit that detects 25 SNPs within GB. Gamma block mismatch occurred in 25 (75.8%) of the 33 studied patient-UD pairs. There was a significant difference in GvHD occurrence between Gamma type matched (GT-M) and mismatched (GT-MM) patient-UD pairs (p=0.0302). The probability of GvHD occurrence had also shown an increase, although insignificant, along with the number of GT-MM between patient-UD pairs (p=0.0913). These results suggest that GT matching could be useful in reducing the risk of post-HSCT complications.

COMPARISON BETWEEN THE HOMOLOGOUS BNT162b2 AND THE HETEROLOGOUS Gam-COVID-Vac/BNT162b2 VACCINE REGIMEN IN REPUBLIC OF NORTH MACEDONIA

2022-12-16T10:02:52+01:00

The medical and socio-economic consequences that stemmed from the COVID-19 pandemic, forced the healthcare policymakers in Republic of North Macedonia to rely on five different vaccines against the SARS-CoV-2 virus, in order to reach a satisfactory level of herd immunity. It is here were we got the idea to compare the heterologous Gam-COVID-Vac/BNT162b2 regimen to the homologous BNT162b2 regimen, with our main focus being the immunogenicity differences between the two of them. Additionally, we researched the variation in humoral immune response relative to age strata; the reactogenicity differences; and discrepancies in SARS-CoV-2 infection incidence between the two regimens. To achieve this, antibody titers in sera samples from fifty-three (53) healthcare workers, divided in heterologous and homologous group, were analyzed at six different time checkpoints. Our results showed robust immunogenic response after the administration of the booster dose (4. 2-fold increase in antibody titers), followed by a slower-waning humoral immune protection in the heterologous regimen, compared to the homologous BNT162b2 schedule, furthermore confirmed by non-inferiority testing (Geometric Mean Ratio=0,98) at the final checkpoint. That, coupled with the similar reactogenicity (p=0,767) of both regimens, imply that the Gam-COVID-Vac/BNT162b2 combination might be a feasible approach in the effort to contain the COVID-19 pandemic.

EVALUATION OF A MULTIPLEX REAL-TIME PCR ASSAY FOR THE DIAGNOSIS OF SEXUALLY-TRANSMITTED INFECTIONS

2022-12-16T10:02:53+01:00

Diagnostic methods with simultaneous multi-targeting are applied for the accurate identification of sexually transmitted infections (STIs). The aim of this study was to evaluate a molecular real-time PCR-based assay (Vivalytic STI assay) for the diagnosis of STIs in comparison with other routine methods. Fifteen individuals suspected of having an STI were tested with the Vivalytic STI assay. As a routine testing, 8/15 patients were tested with the Abbott RealTime CT/NG PCR Assay, 5 specimens were tested with the Simplexa HSV 1 and 2 Direct Kit. Two samples were not tested with a routine assay. Clinical specimens were of different origin (rectal, vaginal, pharyngeal, urine, cerebrospinal fluid (CSF)). Ten out of 15 specimens were positive according to routine diagnostic assays. Three were positive for C. trachomatis (CT) and 2 for N. gonorrhoeae (NG) in single infection, one showed a coinfection with CT and NG. Two samples each were tested positive for Herpes simplex virus 1 (HSV 1) and Herpes simplex virus 2 (HSV 2). All the pathogens detected by routine molecular methods were also detected with Vivalytic STI except for one invalid sample. The Vivalytic STI assay additionally detected M. hominis and U. urealyticum each in one sample. The assay in the focus of the study showed good agreement with other molecular assays and proved to be a simple and a reliable method for the diagnosis of STIs that could improve the diagnostic methods used at our institution.