Development, Validation, and Implementation of an Augmented Multiwell, Multitarget Quantitative PCR for the Analysis of Human Papillomavirus Genotyping through Software Automation, Data Science, and Artificial Intelligence.

The value of human papillomavirus (HPV) testing for cervical cancer screening is well established; however, its use as a primary screening option or as a reflex test after atypical cytology results is now gaining wider acceptance. The importance of full genotyping and viral load determination has been demonstrated to enhance the clinical understanding of the viral infection progression during follow-up or after treatment, thereby providing clinicians with supplementary tools for optimized patient management. We developed a new analysis method for the RIATOL quantitative PCR assay, and validated and implemented it in the laboratory of clinical molecular pathology at Algemeen Medisch Laboratorium (AML), under national accreditation and following the International Organization for Standardization guidelines. This study presents the successful validation of a high-throughput, multitarget HPV analysis method, with enhanced accuracy on both qualitative and quantitative end results. This is achieved by software standardization and automation of PCR curve analysis and interpretation, using data science and artificial intelligence. Moreover, the user-centric functionality of the platform was demonstrated to enhance both staff training and routine analysis workflows, thereby saving time and laboratory personnel resources. Overall, the integration of the FastFinder plugin semi-automatic analysis algorithm with the RIATOL real-time quantitative PCR assay proved to be a remarkable advancement in high-throughput HPV quantification, with demonstrated capability to provide highly accurate clinical-grade results and to reduce manual variability and analysis time.

Characterization of type-specific HPV prevalence in a population of persistent cutaneous warts in Flanders, Belgium.

Cutaneous warts are benign skin lesions caused by the human papillomavirus (HPV). Even though they are considered benign, they can have a considerable impact on the quality of life and cause serious illness in certain immunocompromised populations. Studies have shown that the efficacy of wart treatment is dependent on the causative HPV type. Therefore, in this article, we aim to determine the HPV genotype-specific prevalence in cutaneous warts of a Flemish population as part of the Omnivirol-Salycilic acid randomized controlled trial. Swab samples of cutaneous warts (n = 269) were collected during enrollment. The DNA extraction was performed on the automated NucliSENS® easyMAG® system (bioMérieux). The samples were analyzed with two separate in-house PCR assays capable of detecting the most prevalent cutaneous HPV types (i.e. wart-associated HPV qPCR) as well as the most relevant mucosal types (i.e. RIATOL qPCR assay). In total, the type-specific prevalence of 30 distinct HPV genotypes was determined. The beta-globin gene was used as a cellularity control and for viral load quantification. Data concerning wart persistence, previous treatment, wart type, and other relevant wart and patient characteristics was collected through a baseline questionnaire. The study population consisted mostly of persistent warts considering that 98% (n = 263) of the sampled skin lesions were older than six months and 92% (n = 247) had undergone previous treatment. The most prominent wart type was the mosaic verruca plantaris (42%, n = 113). The most prevalent HPV types were cutaneous HPV types 27 (73%, n = 195), 57 (63%, n = 169), and 2 (42%, n = 113). Only 2% (n = 6) of the lesions was HPV negative. The highest median viral loads were observed with HPV27 and 57 (i.e. 6.29E+04 and 7.47E+01 viral copies per cell respectively). The multivariate analysis found significant associations between wart persistence and certain wart types, the number of warts, and HPV genotypes. Based on these findings, persistent warts are more likely to: (1) be verruca vulgaris, verruca plantaris simple or mosaic, (2) to manifest as multiple warts, (3) and to be negative for HPV type 2 or 4. These characteristics can be useful in the clinical setting for future risk stratification when considering treatment triage and management. Trial registration: NCT05862441, 17/05/2023 (retrospectively registered).

SARS-CoV-2 infection reduces quality of sperm parameters: prospective one year follow-up study in 93 patients.

BACKGROUND: Short- and long-term implications of SARS-CoV-2 on the quality of the sperm and the results of this on fertility remain largely unknown due to lack of longitudinal studies. In this longitudinal observational cohort study, we aimed to analyse the differential effect and the impact of SARS-CoV-2 infection on different semen quality parameters. METHODS: Sperm quality was assessed using the World Health Organization criteria, DNA damage to sperm cells by quantifying the DNA fragmentation index (DFI) and the high-density stainability (HDS), IgA- and IgG-anti-sperm antibodies (ASA) were assessed with light microscopy. FINDINGS: SARS-CoV-2 infection was associated with sperm parameters that were independent of spermatogenic cycle like progressive motility, morphology, DFI and HDS, as well as spermatogenic cycle dependent parameters such as sperm concentration. Detection of IgA- and IgG-ASA allowed classification of patients in three different groups according to its sequence of appearance in sperm during post-COVID-19 follow-up. The maximum progressive motility was lowest during follow-up in patients without ASA (41.9%), intermediate in patients with only IgA-ASA (46.2%) and highest inpatients who had both IgA- and IgG-ASA (54.9%). INTERPRETATION: SARS-CoV-2 infection was associated with changes of all analysed sperm parameters to a different degree which is also observed in their return to normality and is suggestive of individual variations in the patient's immune system performance. Firstly, sperm production is decreased through temporal immune mediated arrest of active meiosis, and secondly immune induced sperm DNA damage prevents fertilization if transferred to the oocyte. Both mechanisms are temporal, and most sperm parameters return to baseline after infection. FUNDING: AML (R20-014), Femicare.

Insulin-like growth factor-1 delays Fas-mediated apoptosis in human neutrophils through the phosphatidylinositol-3 kinase pathway.

Apoptosis of human neutrophils is a crucial mechanism for the resolution of inflammation. We previously showed that insulin-like growth factor-1 (IGF1) delays spontaneous neutrophil apoptosis without influencing the secretion of cytokines by these cells. In the present study, we further addressed the role of IGF1 in regulating neutrophil survival in the presence of other factors present during inflammation, and the mechanism involved in delaying apoptosis. We show that IGF1 delays neutrophil apoptosis triggered by the agonistic anti-Fas antibody CH11 and that the effect of IGF1 is comparable in magnitude to that of the acknowledged anti-apoptotic cytokines interferon-gamma (IFNG) and granulocyte-macrophage colony-stimulating factor (GM-CSF; now known as CSF2). Furthermore, IGF1 exerted additional effects on cell survival in the presence of these cytokines. IGF1 did not affect Fas expression or activation by anti-Fas of caspase-8, but inhibited the depolarization of the mitochondrial membrane. Inhibitor studies indicate that the phosphatidylinositol-3 kinase (PI3K) pathway, but not the MEK-ERK pathway, mediates the effects of IGF1. However, in contrast to CSF2, IGF1 did not induce phosphorylation and translocation to the membrane of AKT, the canonical downstream target of PI3K. We therefore speculate that other downstream targets of PI3K are involved in the delay of neutrophil apoptosis by IGF1, possibly through stabilization of the mitochondrial membrane.

Regulation of interleukin-8 expression in human prostate cancer cells by insulin-like growth factor-I and inflammatory cytokines.

OBJECTIVE: Since serum IGF-I levels are related to risks of prostate cancer and cytokines like interleukin (IL)-6 and IL-8 have been implicated in prostate cancer progression, we investigated the effects of IGF-I on IL-6 and IL-8 expression in the prostate cancer cell. DESIGN: In order to address the regulation by IGF-I of cytokine expression in prostate cancer cells we used cell cultures of established androgen dependent (LNCaP) and androgen-independent cell lines (DU-145 and PC-3). RESULTS: We found that IGF-I stimulates IL-8 mRNA expression and secretion in DU-145 cells, whereas the secretion of IL-6 was hardly affected. IGF-I enhances IL-8 expression in synergy with IL-1beta, but not with tumour necrosis factor (TNF)alpha. Similarly, on IL-8 promoter activity, IGF-I exerted synergistic effects with IL-1beta, but not with TNFalpha. Although IGF-I stimulated the phosphorylation of both Akt (protein kinase B) and extracellular-regulated kinase (ERK), the effect of IGF-I at IL-8 expression was inhibited only by U0126, a pharmacological inhibitor of MAPK/ERK kinase (MEK) and not by inhibition of the upstream activator of Akt, phosphatidylinositol-3 kinase (PI3K). CONCLUSIONS: Our results indicate that IGF-I stimulates IL-8 expression through the MEK-ERK pathway in DU-145 cells, at least in part, by augmentation of transcriptional activity. This finding is in accordance with our observations that IGF-I did not influence cytokine secretion and phosphorylation of ERK in LNCaP or PC-3 cells. It remains to be established whether IL-8 mediates certain effects of IGF-I on prostate cancer cells and whether differential responsiveness of prostate cancer cells to IGF-I relates to certain stages of prostate cancer.

Insulin-like growth factor-I augments interleukin-8 promoter activity through induction of activator protein-1 complex formation.

We previously established that stimulation by IGF-I of interleukin (IL)-8 expression in leukocytes required activation of extracellular-regulated kinase (ERK) and basal activity of c-Jun N-terminal kinase (JNK). In this study, we tested the hypothesis that IGF-I stimulates IL-8 expression at the transcriptional level through induction of Fos/Jun activator protein (AP)-1 complex formation. Inhibition studies using the transcriptional inhibitor actinomycin D and IL-8 promoter activation studies indicate that IGF-I act at the transcriptional level. Using gel shift assays we demonstrate that IGF-I induces the formation of active c-Jun/c-Fos AP-1 complexes. Promoter activation studies using mutated IL-8 promoter constructs show that the AP-1 response element is required for promoter activation by IGF-I whereas CAAT-enhancer binding protein (C/EBP) and nuclear factor of kappa B (NFkappaB) sites were not essential. These results indicate that IGF-I can augment IL-8 expression through activation of AP-1 independent of other inducible transcription factors which have shown to be involved in IL-8 regulation by immune stimuli. This finding is in agreement with our previous observation that IGF-I is able to enhance basal IL-8 production in peripheral blood mononuclear cells (PBMC) in the absence of other stimuli.

Insulin-like growth factor-I stimulates IL-10 production in human T cells.

There is vast body of evidence that the insulin-like growth factor (IGF)-I exerts immunomodulatory effects in vitro and in vivo. In vitro studies indicate that stimulatory effects of IGF-I may be exerted through augmentation of inflammatory cytokine production. To further explore the immunomodulatory effects of IGF-I through regulation of cytokine production, we tested the in vitro effects of IGF-I on the secretion of inflammatory T helper cell type 1 (Th1) and Th2 cytokines by human peripheral blood mononuclear cells (PBMC). To this end, PBMC were stimulated with the T cell mitogen phytohemagglutinin (PHA), and cytokines in the culture media were assessed after 18, 42, 66, and 80 h of culture. We found that IGF-I stimulated the secretion of the Th2 cytokine interleukin (IL)-10 by 40-70% in PHA-stimulated PBMC. In addition, we observed a small stimulatory effect (15%) on the secretion of another Th2 cytokine IL-4. The secretion of IL-2, IL-5, IL-6, interferon-gamma, and the inflammatory cytokines IL-1beta, IL-8, and tumor necrosis factor alpha was not or was hardly affected. IL-10 secretion was also stimulated in purified T cells, and we established that IGF-I also stimulated IL-10 mRNA expression by 100-150%. The monocyte-activating bacterial cell-wall product lipopolysaccharide induced IL-10 production in PBMC, but this was not affected by IGF-I. As IL-10 predominantly exerts anti-inflammatory actions and suppresses Th1-dependent immune responses, our results indicate that IGF-I may exert inhibitory actions on inflammatory and Th1-mediated cellular immune responses through stimulation of IL-10 production in T cells.

IGF-I stimulates IL-8 production in the promyelocytic cell line HL-60 through activation of extracellular signal-regulated protein kinase.

Interleukin (IL)-8 serves as a major chemoattractant for neutrophils and has also been proposed to affect cancer progression. In the present study, we show that IGF-I stimulates IL-8 mRNA expression and IL-8 secretion in the leukemic cell line HL-60. Stimulation of IL-8 expression was completely attenuated by two inhibitors of mitogen-activated protein kinase (MAPK) kinase (MEK), which phosphorylates the MAPKs extracellular-regulated kinase (ERK)1 and ERK2, and by the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125. In contrast, inhibitors of p38 MAPK and phosphatidylinositol-3 kinase (PI3K) did not abrogate the effect of IGF-I. We also show that IGF-I stimulates the activation of ERK1 and ERK2, but we could not detect any effect of IGF-I on the phosphorylation of p38, JNK(p46) or JNK(p54). Collectively, our results suggest that basal JNK activity and activation of the MEK-ERK pathway are required for upregulation of IL-8 by IGF-I in HL-60 cells.

Igf-I inhibits spontaneous apoptosis in human granulocytes.

Granulocytes are key cells in inflammatory processes that are recruited to sites of inflammation by chemoattractants such as IL-8 produced by neutrophils and monocytes. Programmed cell death (apoptosis) of granulocytes and subsequent recognition and phagocytosis by macrophages is a crucial mechanism for resolution of inflammation. Because IGF-I is a potent antiapoptotic factor, we addressed the effects of IGF-I on in vitro apoptosis of human peripheral blood granulocytes. We detected 1390 +/- 467 IGF-I receptors with a dissociation constant of 2.3 +/- 0.9 nM on purified granulocytes. Using microscopical analysis, annexin V binding assays to detect relocation of phosphatidylserine to the cell surface, and DNA fragmentation assays, we showed that IGF-I inhibits spontaneous apoptosis of granulocytes in serum-free culture by 32-45%. IGF-I did not modulate the secretion of IL-6, TNF alpha, and IL-8 by granulocytes, but IL-8 secretion by peripheral blood mononuclear cells was enhanced by 40%. These observations indicate that IGF-I may promote granulocyte functions by increasing granulocyte longevity.