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Background We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting. Methods Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout—a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams. Results Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes ( n  = 754) with some of the most common ones being COL2A1 ( n  = 12; skeletal dysplasia), SCN1A ( n  = 8; epilepsy), and TNFRSF13B ( n  = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange. Conclusions Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.

Invasive urinary bladder cancer shows high recurrence rates after cystectomy even with apparent complete downstaging at cystectomy. Exosomes are nano-sized vesicles important in cell-cell communication, which have been hypothesized to contribute to cancer dissemination and recurrence. The aim of this study was to investigate if pro-carcinogenic exosomes could be detected in urine from histologically downstaged bladder cancer patients. 13 Patients were included in this study. Paired ureter and urine samples from nine patients underwent mass spectrometry, while samples from the remaining patients were used for exosome characterization. At cystectomy, exosomes were isolated from bladder and ureter urine, whereafter quantitative proteome profiling was performed. Urinary exosomes clustered based on whether they came from the bladder, with tumour contact, or the ureters, without tumour contact, even though all came from completely downstaged patients. Proteins overexpressed in exosomes derived from bladder urine contained several oncogenes and were mainly associated with tumour metabolism pathways. Although patients were histologically tumour-free at cystectomy, the bladder urine contained exosomes with a carcinogenic metabolic profile. This suggests a continuous release of exosomes from the bladder, which may promote recurrence at distant sites through metabolic rewiring, even after apparent complete downstaging. These exosomes, coming from either undetected cancer cells or partly transformed cells, are likely to increase the risk of metastasis and encourages cystectomy even in completely downstaged patients.

Recurrence in oral squamous cell carcinoma (OSCC) significantly reduces overall survival. Improved understanding of the host’s immune status in head and neck cancer may facilitate identification of patients at higher risk of recurrence and improve patients’ selection for ongoing clinical trials assessing the effectiveness of immune checkpoint inhibitors (CPI). We aimed to investigate Sentinel Node-derived T-cells and their impact on survival. We enrolled prospectively 28 OSCC patients treated at Karolinska University Hospital, Stockholm, Sweden with primary tumour excision and elective neck dissection. On top of the standard treatment, the enrolled patients underwent sentinel node procedure. T cells derived from Sentinel nodes, non-sentinel nodes, primary tumour and PBMC were analyzed in flow cytometry. Patients who developed recurrence proved to have significantly lower level of CD4+ CD69+ in their sentinel node (31.38 ± 6.019% vs. 43.44 ± 15.33%, p = 0.0103) and significantly higher level of CD8+ CD HLA-DR+ (38.95 ± 9.479% vs. 24.58 ± 11.36%, p = 0.0116) compared to disease-free individuals. Survival analysis of studied population revealed that patients with low proportion of CD4+ CD69+ had significantly decreased disease-free survival (DFS) of 19.7 months (95% CI 12.6–26.9) compared with 42.6 months (95% CI 40.1–45.1) in those with high CD4+ CD69+ proportion in their Sentinel Nodes (log-rank test, p = 0.033). Our findings demonstrate that characterization of T-cell activation in Sentinel Node serves as a complementary prognostic marker. Flow cytometry of Sentinel Node may be useful in both patients’ surveillance and selection for ongoing CPI clinical trials in head and neck cancer.

A re-examination of former concepts is required to meet today’s medical challenges in allergic rhinitis. Previously, neutrophils have been treated as a relatively homogenous cell population found in the nose both when the patient is suffering at the height of the allergic season as well as when the patient report no symptoms. However, new data indicates that neutrophils can be divided into different subsets with diverse roles in inflammation. We showed increased levels of neutrophils in peripheral blood, nasal biopsies and nasal lavage fluid (NAL) from allergic patients during the pollen season compared to healthy controls. A closer examination revealed that the activated subset of neutrophils, CD16 high CD62L dim , outweighed the normal form CD16 high CD62L high in nasal tissue among these patients. This skewed distribution was not seen in controls. The normal subset prevailed in peripheral blood from patients as well as controls, whereas CD16 high CD62L dim and CD16 dim CD62L dim subsets, the latter considered “end state” neutrophils before apoptosis, were elevated in NAL. Functional in vitro experiments revealed that activated neutrophils exhibit a T cell priming capacity and an ability to enhance eosinophil migration. Activated neutrophils may thus contribute to allergic inflammation seen in allergic rhinitis by priming T cells and attracting eosinophils.

Background Hemodialysis (HD) patients have an increased risk of acquiring infections due to many health care contacts and may, in addition, have a suboptimal response to vaccination and a high mortality from Covid-19 infection. Methods In 50 HD patients (mean age 69.4 years, 62% men) administration of SARS-CoV-2BNT162b2 mRNA vaccine began in Dec 2020 and the immune response was evaluated 7–15 weeks after the last dose. Levels of Covid-19 (SARS-CoV-2) IgG antibody against the nucleocapsid antigen (anti-N) and the Spike antigen (anti-S) and T-cell reactivity testing against the Spike protein using ELISPOT technology were evaluated. Results Out of 50 patients, anti-S IgG antibodies indicating a vaccine effect or previous Covid-19 infection, were detected in 37 (74%), 5 (10%) had a borderline response and 8 (16%) were negative after two doses of vaccine. T-cell responses were detected in 29 (58%). Of the 37 patients with anti-S antibodies, 25 (68%) had a measurable T-cell response. 2 (40%) out of 5 patients with borderline anti-S and 2 (25%) without anti-S had a concomitant T-cell response. Twenty-seven (54%) had both an antibody and T-cell response. IgG antibodies to anti-N indicating a previous Covid-19 disease were detected in 7 (14%) patients. Conclusions Most HD patients develop a B- and/or T-cell response after vaccination against Covid-19 but approx. 20% had a limited immunological response. T-cell reactivity against Covid-19 was only present in a few of the anti-S antibody negative patients.

Background An increase of regulatory T cells, defined as CD25 high - and/or FOXP3 + -expressing CD4 + T cells, within tumors has been reported in several studies. Tregs promote tumor growth by modulating the antitumor immune response, mainly through inhibition of T-cell-mediated tumor cell killing: this has been suggested to be dependent on IL-10 and/or TGF-β. In stomach cancer, the mechanisms behind the accumulation of Tregs in tumor tissue has not been fully elucidated, and neither has Treg gene expression in situ. Materials and methods Stomach tissue from gastric cancer patients undergoing gastric resection was analyzed using flow cytometry and cell sorting, followed by RT-PCR. Results We observed that stomach CD4 + FOXP3 + T cells proliferated to a higher degree than CD4 + FOXP3 − T cells, which may contribute to Treg accumulation in the mucosa. By analyzing DNA methylation, we demonstrated that both proliferating and nonproliferating FOXP3 + T cells exhibited complete demethylation of the FOXP3 gene, indicating a stable FOXP3 expression in both cell populations. Furthermore, analysis of T-cell populations isolated directly from the tumor and tumor-free mucosa demonstrated that CD4 + CD25 high T cells have a higher IL-10/IFN-γ gene expression ratio but express lower levels of TGF-β than CD4 + CD25 low/− T cells. Conclusion We demonstrate strong proliferation among regulatory CD4 + FOXP3 + CD25 high T cells in the gastric cancer mucosa. These local Treg express a suppressive cytokine profile characterized by high IL-10 and low TGF-β and IFN-γ production.

Hypoxic ischemia (HI) is an important cause of neonatal brain injury and subsequent inflammation affects neurological outcome. In this study we performed investigations of systemic and local activation states of inflammatory cells from innate and adaptive immunity at different time points after neonatal HI brain injury in mice. We developed a multiplex flow cytometry based method combined with immunohistochemistry to investigate cellular immune responses in the brain 24 h to 7 months after HI brain injury. In addition, functional studies of ex vivo splenocytes after cerebral hypoxic ischemia were performed. Both central and peripheral activation of CD11b(+) and CD11c(+) antigen presenting cells were seen with expression of the costimulatory molecule CD86 and MHC-II, indicating active antigen presentation in the damaged hemisphere and in the spleen. After one week, naïve CD45rb(+) T-lymphocytes were demonstrated in the damaged brain hemisphere. In a second phase after three months, pronounced activation of CD45rb(-) T-lymphocytes expressing CD69 and CD25 was seen in the damaged hemisphere. Brain homogenate induced proliferation in splenocytes after HI but not in controls. Our findings demonstrate activation of both local and systemic immune responses months after hypoxic ischemic neonatal brain injury. The long term immune activation observed is of general importance for future studies of the inflammatory response after brain injury as most previous studies have focused on the first few weeks after damage, while the effects of the late inflammation phase may be missed. Furthermore, the self-reactive component raises the question if there is a correlation with development of autoimmune brain disease later in life.

Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs. Human CD4(+)CD25(hi) Tregs displayed a demethylated FOXP3 promoter (1.4%+/-0.95% SEM methylated) in contrast to CD4(+)CD25(lo) T cells which were partially methylated (27.9%+/-7.1%). Furthermore, stimulated CD4(+)CD25(lo) T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-beta and/or IL-10 does not induce any additional change in methylation level. The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3(+) activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.

In this randomized blinded study, we investigated caffeine 5 mg/kg treatment given directly after neonatal brain hypoxia ischemia. Brain morphology, behavior and key brain infiltrating immune populations were examined. Caffeine treatment significantly improves outcome when compared to phosphate buffered saline. Flow cytometric analysis of immune responses revealed no persistent immunological alterations. Given its safety caffeine emerges as a candidate for neuroprotective intervention after neonatal brain injury.

Background: The hemodialysis (HD) population has been a vulnerable group during the coronavirus disease 2019 (COVID-19) pandemic. Advanced chronic kidney disease with uremia is associated with weaker immune response to infections and an attenuated response to vaccines. The aim of this study was to study the humoral and cellular response to the second and third doses of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS‑CoV‑2) BNT162b2 mRNA vaccine in HD patients and to follow the response over time. Methods: The patients received their first two vaccine doses from 28 December 2020 within a 4-week interval and the third dose in September 2021 and were followed-up for humoral and cellular immune response at 1) 7–15 weeks and 2) 6–8 months after dose two (no t-cell reactivity measured), and 3) 3 weeks and 4) 3 months after dose three. Fifty patients were initially enrolled, and 40 patients were followed during the entire study. Levels of COVID-19 (SARS-CoV-2) IgG antibody against the Spike antigen (anti-S) and T-cell reactivity testing against the Spike protein using Enzyme-Linked ImmunoSpot (ELISPOT) technology were evaluated. Results: IgG antibodies to anti-S were detected in 35 (88%) of the 40 patients 7–15 weeks after vaccine dose two, 31 (78%) were positive, and 4 (10%) borderline. The median anti-S titer was 606 Abbott Units/milliliter (AU/mL) (interquartile range [IQR] 134–1,712). Three months after the third dose, anti-S was detected in 38 (95%) of 40 patients (P < 0.01 compared to after dose two), and the median anti-S titer was 9,910 AU/mL (IQR 2,325–26,975). Cellular reactivity was detected in 22 (55%), 34 (85%), and 28 (71%) of the 40 patients, and the median T-cell response was 9.5 (IQR 3.5–80), 51.5 (14.8–132), and 19.5 (8.8–54.2) units, respectively, for 6–8 months after dose two, 3 weeks, and 3 months after dose three. Conclusions: Our data show that a third dose of SARS‑CoV‑2 BNT162b2 mRNA vaccine gives a robust and improved immunological response in HD patients, but a few patients did not develop any anti-S response during the entire study, indicating the importance to monitor the vaccine response since those who do not respond could now be given monoclonal antibodies if they contract a COVID-19 infection or in the future antivirals.