To Örebro University

BACKGROUND: Rituximab, a B-cell-depleting therapy widely used for multiple sclerosis (MS), is associated with a progressive decline in immunoglobulin G (IgG) levels, raising concerns regarding infection risk. This study evaluates IgG dynamics and antibiotic use in a real-world MS cohort.

METHODS: A retrospective cohort study was conducted on MS patients treated with Rituximab over a 10-year period. Patients included had at least two infusions and a follow-up of ≥6 months after the last infusion. IgG levels and antibiotic prescriptions were registered longitudinally.

RESULTS: The study included 213 patients (72% female, mean age 40.1 ± 10.6 years). Mean IgG decreased from 11.5 ± 2.4 g/L at baseline to 9.0 ± 1.7 g/L after five years, with an average decline of 0.35 g/L/year (95% CI 0.25-0.44; p < 0.001). Hypogammaglobulinemia (<6.7 g/L) occurred in 7.5% of patients. Cumulative rituximab dose was a significant predictor of lower IgG (p < 0.001). Antibiotic prescriptions were recorded in 62% of patients (mean 2.5 per patient), most commonly for urinary (29%) and respiratory (17%) tract infections. In multivariable analysis, lower IgG levels did not significantly predict the risk of antibiotic prescription.

CONCLUSION: Rituximab treatment resulted in a significant, dose-dependent IgG decline. However, this decline was not associated with an increased risk of antibiotic-treated infections, suggesting a dissociation between total IgG levels and functional immune competence in this cohort. These findings support continued use of rituximab with vigilant monitoring, though dose adjustments may be warranted for patients with rapidly decreasing IgG.

Damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) are key triggers of inflammation in sepsis. However, they have rarely been studied simultaneously. Thus, in the present study of patients with bacteraemic infection, we aimed to study how DAMP dynamics are linked to disease severity and outcome and to compare diagnostic and prognostic properties of a DAMP and a previously analysed PAMP (16S rDNA). In a prospective study of adult patients hospitalized with culture-proven community-onset bacteraemic infection, caused by Streptococcus pneumonia (n = 30), Staphylococcus aureus (n = 27), or Escherichia coli (n = 26), dynamics of a PAMP, i.e. 16S rDNA, have previously been presented. For the present study, blood samples obtained on hospital days 1-2 (when blood culture was positive), 3-4, 7 ± 1, 14 ± 2, and 28 ± 4 were analysed for four different DAMPs, i.e., nuclear DNA (nDNA), mitochondrial DNA (mtDNA), heat shock protein 90 alpha (HSP90α), and extracellular high mobility group box 1 (HMGB1). Sepsis was defined according to the Sepsis-3 criteria. The study outcomes were sepsis at admission and negative outcome, defined as intensive care unit (ICU) admission and/or death within 60 days. Of 83 study patients, sepsis was noted in 41 patients (49%) and a negative outcome was noted in 17 patients (20%). nDNA had areas under the receiver operating characteristic (ROC) curves of 0.78 for sepsis and 0.76 for negative outcome, which were higher than those of the other DAMPs and additional biomarkers (CRP, IL-6, IL-8, and IL-10). The nDNA and positive 16S rDNA results on day 1-2 were correlated with each other (r = 0.68, p < 0.001). Multivariate analyses showed that high day 1-2 concentrations of both nDNA and 16S rDNA were independently associated with sepsis. In addition, high day 1-2 concentration of nDNA was independently associated with negative outcomes. While 16S rDNA dissipated from the circulation within days, nDNA concentrations remained elevated throughout the follow-up period in patients with negative outcome. In conclusion, nDNA outperformed the other DAMPs regarding sepsis detection and outcome prediction. Both nDNA (a DAMP) and 16S rDNA (a PAMP) were independently linked to sepsis; nDNA was also associated with negative outcomes and persisted elevated in such cases. This highlights nDNA as an interesting marker within sepsis pathogenesis and as a promising clinical biomarker, warranting further studies.

Introduction: In the last years B-cell depleting therapies such as rituximab (RTX) have become commonplace in the therapeutic landscape of multiple sclerosis (MS), with this class of drugs showing high efficacy. Nevertheless, B-cell depleting therapies have been associated with a higher risk of bacterial infections compared to other disease modifying therapies.

Objectives/Aims: To report the frequency and type of antibiotic prescriptions during a 10-year period.

Methods: A retrospective cohort study was performed on MS-patients followed at Örebro University Hospital (Örebro, Sweden), having received at least one dose of RTX between 2011 and 2020. The number and types of prescribed antibiotic therapies were registered. Data collection was performed using the Swedish MS-registry and electronic patient files. We registered all prescriptions for antibacterial agents between the first dose of RTX administered and 12 months after the last dose, or until the study cut-off date of December 31, 2020, whichever came first.

Results: A total of 219 patients were included (213 with relapsing-remitting MS; mean age 40 years). The median Expanded Disability Status Scale was 2.0. Median disease duration was 36 months at the start of rituximab, median follow-up time was 43 months, and the median number of rituximab infusions was 6. A total of 415 antibiotic prescriptions were registered in this time period. The mean number of prescriptions per patient and year was 0.5. In the cohort, 115 (47%) of the patients did not receive an antibiotic prescription. The most common type of infection treated was urinary tract infections (UTI), followed by upper respiratory tract ones. The majority (83%) of patients who received antibiotic therapy for UTI were women. There were 16 patients (7%) that required long duration of treatment with antibiotics and 15 (7%) of the study population were hospitalized for infections.

Conclusion: Prescription of antibiotics is frequent among MS-patients treated with RTX, occurring in approximately half of this patient population. UTI is the most common cause of antibiotic treatment.

OBJECTIVE: Increased expression of the amino acid transporter solute Carrier Family 7 Member 5 (SLC7A5) has been observed in neoplastic cells during hypoxic conditions in vitro, indicating an adaptation for cell survival. To further explore this, we evaluated hypoxia-mimetic by CoCl2 as a model for hypoxia in breast cancer cell lines and the effect on SLC257A5 expression. Four different breast cancer cell lines (MCF7, T-47D, BT-474 and ZR-75-1) were exposed to 100 µM CoCl2 for 48 h. Subsequently, cell viability, gene- and protein expression analyses were performed.

RESULTS: The gene expression of VEGF, a marker of hypoxia, was significantly elevated in all four cell lines compared to the control (p < 0.0001), indicating that CoCl2 exposure generates a hypoxic response. Moreover, CoCl2 exposure significantly upregulated SLC7A5 gene expression in T-47D (p < 0.001), BT-474 (p < 0.0001) and ZR-75-1 (p < 0.0001) cells, as compared to vehicle control. Immunofluorescence staining showed increased SLC7A5 protein expression in MCF7, T-47D and BT-474 cells compared to vehicle control. This report suggests that hypoxia-mimetic by CoCl2 can be used as a simple model for inducing hypoxia in breast cancer cell lines and in fact influence SLC7A5 gene and protein expression in vitro.

BACKGROUND: Patients with chronic lymphocytic leukemia (CLL) show an immune dysfunction with increased risk of infections and poor response to vaccination. Streptococcus pneumoniae is a common cause of morbidity and mortality in CLL patients. In a previous randomized clinical trial, we found a superior immune response in CLL patients receiving conjugated pneumococcal vaccine compared to non-conjugated vaccine. The response to revaccination in CLL patients is scarcely studied. In this study, early humoral response to repeated revaccinations with pneumococcal vaccines was evaluated, by determination of B cell subsets and plasmablast dynamics in peripheral blood.

METHOD: CLL patients (n = 14) and immunocompetent controls (n = 31) were revaccinated with a 13-valent pneumococcal conjugate vaccine (PCV13) after previous primary immunization (3-6 years ago) with PCV13 or a 23-valent pneumococcal polysaccharide vaccine (PPSV23). Eight weeks after the first revaccination, all CLL patients received a second revaccination with PCV13 or PPSV23. B cell subsets including plasmablasts were analyzed in peripheral blood by flow cytometry, before and after the first and the second revaccination.

RESULTS: None of the CLL patients, but all controls, had detectable plasmablasts at baseline (p < 0.001). After the first revaccination with PCV13, the plasmablast proportions did not increase in CLL patients (p = 0.13), while increases were seen in controls (p < 0.001). However, after a second revaccination with PCV13 or PPSV23, plasmablasts increased compared to baseline also in CLL patients (p < 0.01). If no response was evident after first revaccination, only a second revaccination with PCV13 increased plasmablasts in contrast to PPSV23 revaccination. Patients with hypogammaglobulinemia and ongoing/previous CLL specific treatment responded poorly, also to a second revaccination.

CONCLUSION: In CLL patients, pneumococcal revaccination induced minor early plasmablast response compared to controls, but the response improved using a strategy of repeated doses with of conjugated T cell dependent pneumococcal vaccine.

BACKGROUND: Basal cell carcinoma (BCC) is the most common type of cancer in fair-skinned individuals worldwide. Altered metabolism is a hallmark of cancer, and a growing body of evidence has shown increased expression of the large neutral amino acid transporter (LAT) small subunit 1 in several types of cancers, including BCC. However, the mechanisms behind changed LAT1 expression in BCC are largely unknown.

OBJECTIVES: To describe the protein expression of LAT1 and its co-localisation with LAT2, and to examine LAT1 in association with BCC tumour biology characteristics such as cell proliferation, apoptosis, and hypoxia.

METHODS: Formalin-fixed and paraffin-embedded tissue samples (n=14) from excised BCCs were stained with immunofluorescence and examined regarding protein-staining patterns.

RESULTS: There was no correlation between expression of LAT1 and LAT2, and the co-localisation was low. The proliferation markers topoisomerase IIα and Ki-67 both showed a significantly higher expression in the BCC tissue than in the normal epidermis (p=0.0063 and p=0.010, respectively). The fraction of LAT1-expressing cells in the BCC was inversely correlated to the fraction of proliferative active tumour cells (p=0.0013). Cleaved caspase-3 was significantly increased in tumour areas with high LAT1 expression (p=0.016).

CONCLUSIONS: The findings of the present study show that LAT1 is not usually expressed by proliferating BCC cells. The morphological localisation suggests that tumour cells use LAT1 in adaption to environmental changes such as starvation and/or hypoxia. These findings could have implications for future development of LAT1-inhibitory BCC treatments.

The amino acid transporter named solute carrier family 7 member 5 (SLC7A5) is suggested to play a part in altered cell metabolism and proliferative signaling and has been reported to be overexpressed in various types of cancer, including breast cancer. Estrogen‑receptor‑positive (ER⁺) breast cancers constitute the most common type of breast malignancies and are often treated with anti‑estrogenic therapies. In this group of patients, endocrine resistance is a challenging problem that could lead to recurrent disease. To overcome this, additional prognostic biomarkers are needed. The present study aimed therefore to determine whether SLC7A5 may be considered as a possible prognostic marker in ER⁺ breast cancer and to investigate its relation with certain cancer‑related genes. We used a local breast cancer cohort (n=154) and immunohistochemistry to analyze the expression of SLC7A5 in association with clinicopathological characteristics and patient outcome. In addition, gene expression analysis was performed on 80 of these tumors. Furthermore, the METABRIC dataset was used for correlation analyses between expression of SLC7A5 and several genes related to breast cancer biology. The results demonstrated that overexpression of SLC7A5 was significantly associated with histopathological grade in patients with breast cancer, and that SLC7A5 mRNA expression was positively correlated with the expression of marker of proliferation Ki‑67 and hypoxia inducible factor 1 subunit alpha. Overexpression of SLC7A5 may therefore play a role in the biology of endocrinologically‑driven disease. However, when further assessing SLC7A5 using the METABRIC dataset, SLC7A5 mRNA expression level was more significantly increased in ER‑ subgroups compared with ER⁺ disease. All breast cancer subtypes included, SLC7A5 mRNA expression was correlated with a higher number of cancer‑related genes than in estrogen receptor positive tumors alone. The present study suggested that SLC7A5 expression may be of importance for breast cancer cell proliferation and survival. In order to further establish the biological and clinical role of SLC7A5 in breast cancer, further investigation using different breast cancer subgroups is required.

Breast cancer patients treated with tamoxifen may experience recurrence due to endocrine resistance, which highlights the need for additional predictive and prognostic biomarkers. The glyco-phosphoprotein osteopontin (OPN), encoded by the SPP1 gene, has previously shown to be associated with poor prognosis in breast cancer. However, studies on the predictive value of OPN are inconclusive. In the present study, we evaluated tissue SPP1 mRNA and OPN protein expression as markers of recurrence in estrogen receptor- positive (ER+) breast cancer tissue. Tamoxifen- treated patients with recurrence or non-recurrence were selected using a matched case-control design. SPP1 mRNA expression was analysed using qPCR (n = 100) and OPN protein by immunohistochemistry (n = 116) using different antibodies. Odds ratios were estimated with conditional logistic regression. The SPP1 expression increased the risk of recurrence with an odds ratio (OR) of 2.50 (95% confidence interval [CI]; 1.30-4.82), after adjustment for tumour grade, HER 2 status and other treatments to OR 3.62 (95% CI; 1.45-9.07). However, OPN protein expression was not associated with risk of recurrence or with SPP1-gene expression, suggesting SPP1 mRNA a stronger prognostic marker candidate compared to tumor tissue OPN protein.

Regional anesthesia may prolong survival following surgery for different types of cancers. The mechanisms behind this are unclear but direct effects on cancer cells by local anesthetics (LA) have been suggested. The aim of this study was to investigate if lidocaine or ropivacaine have a dose-dependent effect on the cell viability and proliferation of a primary and a secondary colon carcinoma cell line in vitro. The colon cancer cell lines SW480 derived from primary tumor and SW620 from a metastatic site in the same patient were exposed to increasing concentrations of lidocaine and ropivacaine (5-1,000 mu M). Cell viability was measured using CellTiter-Blue((R)) and cell proliferation by PKH67 after exposure for up to 72 h. Cell viability was significantly reduced by ropivacaine at the highest concentration (1,000 mu M) after 48 and 72 h in the cell line SW480 and at 72 h in SW620. Exposure to lidocaine did not show any significant reduction in cell viability. Notably, low concentrations of both lidocaine and ropivacaine significantly increased cell viability after 48 and 72 h in SW620. Cell proliferation was significantly reduced by 1,000 mu M lidocaine in SW480 and by 1,000 mu M ropivacaine in SW620. In summary, both lidocaine and ropivacaine showed an anti-proliferative effect in the colon cancer cell lines at high concentrations and after prolonged exposure to LA in vitro. Our findings also indicate that lower concentrations promote cell viability in the metastatic cell line.

Background: The incidence of basal cell carcinoma (BCC) is increasing and the costs for care rising. Therefore, the need for simplified and cost-effective treatment choices is substantial. Aberrant signalling in several pathways, induced by ultraviolet radiation, is of importance in the development of BCC. Alterations in tumour metabolic activity are part of general carcinogenesis; however, these alterations are only partially recognized in skin cancer.

Objectives: To study expression profiles in BCCs compared with individually matched nontumour skin, with a focus on finding differences associated with tumour metabolism.

Materials and methods: Gene expression in biopsies from BCCs (n = 14) compared with biopsies from nontumour gluteal skin was analysed with microarrays (n = 4 + 4) and/or quantitative real-time polymerase chain reaction (qPCR, n = 14 + 14). Protein expression and localization was assessed using immunohistochemistry (IHC) in formalin-fixed and paraffin-embedded BCC samples.

Results: Microarray analysis revealed increased expression of the amino acid transporters SLC7A5, SLC7A7 and SLC7A8 as well as the cytosolic enzyme tryptophan 2,3-dioxygenase (TDO) 2 in BCC. Higher expression of SLC7A5 (P < 0.001), SLC7A8 (P < 0.001) and TDO2 (P = 0.002), but not SLC7A7 (P = 0.50), was confirmed by qPCR, and IHC demonstrated correlating tumour cell protein expression of SLC7A5 and SLC7A8. Protein expression of SLC7A7 was observed in the stratum granulosum, and TDO2 in immune cells.

Conclusions: This study highlights the upregulation of SLC7A5, SLC7A8 and TDO2 in BCC compared with nontumour skin. Our findings imply that amino acid transporters may be further explored as potential targets for future medical treatment.