Autoimmunity Against Surfactant Protein B Is Associated with Pneumonitis During Checkpoint Blockade.

RATIONALE: Immune checkpoint inhibitor-related pneumonitis is a serious autoimmune event affecting up to 20% of patients with non-small cell lung cancer, yet the factors underpinning its development in some patients and not others are poorly understood. OBJECTIVES: To investigate the role of autoantibodies and autoreactive T cells against surfactant-related proteins in the development of pneumonitis. METHODS: The study cohort consisted of non-small cell lung cancer patients who gave blood samples before and during immune checkpoint inhibitor treatment. Serum was used for proteomics analyses and to detect autoantibodies present during pneumonitis. T cell stimulation assays and single-cell RNA sequencing were performed to investigate the specificity and functionality of peripheral autoreactive T cells. The findings were confirmed in a validation cohort comprising patients with non-small cell lung cancer and patients with melanoma. MEASUREMENTS AND MAIN RESULTS: Across both cohorts, patients who developed pneumonitis had higher pre-treatment levels of immunoglobulin G autoantibodies targeting surfactant protein-B. At the onset of pneumonitis, these patients also exhibited higher frequencies of CD4+ interferon-gamma-positive surfactant protein B-specific T cells, and expanding T cell clonotypes recognizing this protein, accompanied by a pro-inflammatory serum proteomic profile. CONCLUSIONS: Our data suggest that the co-occurrence of surfactant protein-B-specific immunoglobulin G autoantibodies and CD4+ T cells is associated with the development of pneumonitis during ICI therapy. Pre-treatment levels of these antibodies may represent a potential biomarker for elevated risk of developing pneumonitis and on-treatment levels may provide a diagnostic aid. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Neuromuscular adaptations after osseointegration of a bone-anchored prosthesis in a unilateral transfemoral amputee - a case study.

PURPOSE: Many individuals with a lower limb amputation experience problems with the fitting of the socket of their prosthesis, leading to dissatisfaction or device rejection. Osseointegration (OI)- the implantation of a shaft directly interfacing with the remaining bone- is an alternative for these patients. In this observational study, we investigated how bone anchoring influences neuromuscular parameters during balance control in a patient with a unilateral transfemoral amputation. MATERIAL AND METHODS: Center of pressure (CoP) and electromyography (EMG) signals from muscles controlling the hip and the ankle of the intact leg were recorded during quiet standing six months before and one and a half years after this patient underwent an OI surgery. Results were compared to a control group of nine able-bodied individuals. RESULTS: Muscle co-activation and EMG intensity decreased after bone anchoring, approaching the levels of able-bodied individuals. Muscle co-activation controlling the ankle decreased in the high-frequency range, and the EMG intensity spectrum decreased in the lower-frequency range for all muscles when vision was allowed. With eyes closed, the ankle extensor muscle showed an increased EMG intensity in the high-frequency range post-surgery. CoP length increased in the mediolateral direction of the amputated leg. CONCLUSIONS: These findings point to shifts in the patient's neuromuscular profile towards the one of able-bodied individuals.

Long-term outcome of patients with vaccine-induced immune thrombotic thrombocytopenia and cerebral venous sinus thrombosis.

We present the long-term outcomes of 44 patients who developed cerebral venous sinus thrombosis after vaccination with the adenoviral vector ChAdOx1 nCoV-19 COVID-19 vaccine. Assessment of the Extended Glasgow Outcome Scale was performed within 3-6 months after the initial hospital admissions. Patient outcomes ranged from good recovery (13 patients, 29.6%) to moderate disability (11 patients, 25.0%) and severe disability or vegetative state (6 patients, 13.6%). Fatal outcomes were reported in 14 patients (31.8%).

Complex karyotype with cryptic FUS gene rearrangement and deletion of NR3C1 and VPREB1 genes in childhood B-cell acute lymphoblastic leukemia: A case report.

B-cell acute lymphoblastic leukemia (B-ALL) is a hematopoietic malignancy characterized by overproduction of immature B-lymphoblasts. B-ALL is the most common pediatric tumor and remains the leading cause of mortality in children and adolescents. Molecular and cytogenetic analyses of B-ALL revealed recurrent genetic and structural genomic alterations which are routinely applied for diagnosis, prognosis and choice of treatment regimen. The present case report describes a 4-year-old female diagnosed with B-ALL. GTG-banding at low resolution revealed an abnormal clone with 46,XX,?t(X;19)(q13;q13.3),der(9) besides normal cells. Molecular cytogenetics demonstrated a balanced translocation between chromosomes 16 and 19, and an unbalanced translocation involving chromosomes 5 and 9. A locus-specific probe additionally identified that the FUS gene in 16p11.2 was split and its 5' region was translocated to subband 19q13.33, whereas the 3' region of the FUS gene remained on the derivative chromosome 16. Overall, this complex karyotype included four different chromosomes and five break events. Further analyses, including array-comparative genomic hybridization, additionally revealed biallelic deletion of the tumor suppressor genes CDKN2A/B, and deletion of the NR3C1 and VPREB1 genes. The patient passed away under treatment due to sepsis.

Detection and Correlation of Single and Concomitant TP53, PTEN, and CDKN2A Alterations in Gliomas.

Gliomas are the most frequent primary tumors of central nervous system and represent a heterogeneous group of tumors that originates from the glial cells. TP53, PTEN, and CDKN2A are important tumor suppressor genes that encode proteins involved in sustaining cellular homeostasis by different signaling pathways. Though genetic alterations in these genes play a significant role in tumorigenesis, few studies are available regarding the incidence and relation of concomitant TP53, PTEN, and CDKN2A alterations in gliomas. The purpose of this study was to evaluate the occurrence of mutation and deletion in these genes, through single-strand conformational polymorphism, array-comparative genomic hybridization, and fluorescence in situ hybridization techniques, in 69 gliomas samples. Molecular results demonstrated a significant higher prevalence of TP53, PTEN, and CDKN2A alterations in astrocytoma than other tumor subtypes, and heterozygous deletion was the most frequent event. In addition, a significant association was observed between TP53 and CDKN2A alterations (p = 0.0424), which tend to coexist in low grade astrocytomas (5/46 cases (10.9%)), suggesting that they are early events in development of these tumors, and PTEN and CDKN2A deletions (p = 0.0022), which occurred concomitantly in 9/50 (18%) patients, with CDKN2A changes preceding PTEN deletions, present preferably in high-grade gliomas.

An exploration of the links between parasites, trophic ecology, morphology, and immunogenetics in the Lake Tanganyika cichlid radiation.

Differences in habitat and diet between species are often associated with morphological differences. Habitat and trophic adaptation have therefore been proposed as important drivers of speciation and adaptive radiation. Importantly, habitat and diet shifts likely impose changes in exposure to different parasites and infection risk. As strong selective agents influencing survival and mate choice, parasites might play an important role in host diversification. We explore this possibility for the adaptive radiation of Lake Tanganyika (LT) cichlids. We first compare metazoan macroparasites infection levels between cichlid tribes. We then describe the cichlids' genetic diversity at the major histocompatibility complex (MHC), which plays a key role in vertebrate immunity. Finally, we evaluate to what extent trophic ecology and morphology explain variation in infection levels and MHC, accounting for phylogenetic relationships. We show that different cichlid tribes in LT feature partially non-overlapping parasite communities and partially non-overlapping MHC diversity. While morphology explained 15% of the variation in mean parasite abundance, trophic ecology accounted for 16% and 22% of the MHC variation at the nucleotide and at the amino acid level, respectively. Parasitism and immunogenetic adaptation may thus add additional dimensions to the LT cichlid radiation.

Ribosome biogenesis factor Tsr3 is the aminocarboxypropyl transferase responsible for 18S rRNA hypermodification in yeast and humans.

The chemically most complex modification in eukaryotic rRNA is the conserved hypermodified nucleotide N1-methyl-N3-aminocarboxypropyl-pseudouridine (m(1)acp(3)Ψ) located next to the P-site tRNA on the small subunit 18S rRNA. While S-adenosylmethionine was identified as the source of the aminocarboxypropyl (acp) group more than 40 years ago the enzyme catalyzing the acp transfer remained elusive. Here we identify the cytoplasmic ribosome biogenesis protein Tsr3 as the responsible enzyme in yeast and human cells. In functionally impaired Tsr3-mutants, a reduced level of acp modification directly correlates with increased 20S pre-rRNA accumulation. The crystal structure of archaeal Tsr3 homologs revealed the same fold as in SPOUT-class RNA-methyltransferases but a distinct SAM binding mode. This unique SAM binding mode explains why Tsr3 transfers the acp and not the methyl group of SAM to its substrate. Structurally, Tsr3 therefore represents a novel class of acp transferase enzymes.

A novel IGH@ gene rearrangement associated with CDKN2A/B deletion in young adult B-cell acute lymphoblastic leukemia.

Acquired copy number changes are common in acute leukemia. They are reported as recurrent amplifications or deletions (del), and may be indicative of involvement of oncogenes or tumor suppressor genes in acquired disease, as well as serving as potential biomarkers for prognosis or as targets for molecular therapy. The present study reported a gain of copy number of 14q13 to 14q32, leading to immunoglobulin heavy chain locus splitting in a young adult female. To the best of our knowledge, this rearrangement has not been previously reported in B-cell acute lymphoblastic leukemia (ALL). Low resolution banding cytogenetics performed at the time of diagnosis revealed a normal karyotype. However, retrospective application of fluorescence in situ hybridization (FISH) banding and locus-specific FISH probes, as well as multiplex ligation-dependent probe amplification and high resolution array-comparative genomic hybridization, revealed previously hidden aberrations. Overall, a karyotype of 46, XX, del(9) (p21.3 p21.3),derivative(14) (pter-> q32.33:: q32.33-> q13 ::q32.33-> qter) was determined. The patient was treated according to the Polish Adult Leukemia Group protocol and achieved complete remission. The results of the present study indicate that a favorable prognosis is associated with these aberrations when the aforementioned treatment is administered.

High rates of submicroscopic aberrations in karyotypically normal acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is not a single uniform disease. It consists of several subgroups with different cytogenetic and molecular genetic aberrations, clinical presentations and outcomes. Banding cytogenetics plays a pivotal role in the detection of recurrent chromosomal rearrangements and is the starting point of genetic analysis in ALL, still. Nowadays, molecular (cyto)genetic tools provide substantially to identify previously non-detectable, so-called cryptic chromosomal aberrations in ALL. However, ALL according to banding cytogenetics with normal karyotype - in short cytogenetically normal ALL (CN-ALL) - represent up to ~50 % of all new diagnosed ALL cases. The overall goal of this study was to identify and characterize the rate of cryptic alterations in CN-ALL and to rule out if one single routine approach may be sufficient to detect most of the cryptic alterations present. RESULTS: Sixty-one ALL patients with CN-ALL were introduced in this study. All of them underwent high resolution fluorescence in situ hybridization (FISH) analysis. Also DNA could be extracted from 34 ALL samples. These DNA-samples were studied using a commercially available MLPA (multiplex ligation-dependent probe amplification) probe set directed against 37 loci in hematological malignancies and/or array-comparative genomic hybridization (aCGH). Chromosomal aberrations were detected in 21 of 61 samples (~34 %) applying FISH approaches: structural abnormalities were present in 15 cases and even numerical ones were identified in 6 cases. Applying molecular approaches copy number alterations (CNAs) were detected in 27/34 samples. Overall, 126 CNAs were identified and only 34 of them were detectable by MLPA (~27 %). Loss of CNs was identified in ~80 % while gain of CNs was present in ~20 % of the 126 CNAs. A maximum of 13 aberrations was detected per case; however, only one aberration per case was found in 8 of all in detail studied 34 cases. Of special interest among the detected CNAs are the following new findings: del(15)(q26.1q26.1) including CHD2 gene was found in 20 % of the studied ALL cases, dup(18)(q21.2q21.2) with the DCC gene was present in 9 % of the cases, and the CDK6 gene in 7q21.2 was deleted in 12 % of the here in detail studied ALL cases. CONCLUSIONS: In conclusion, high resolution molecular cytogenetic tools and molecular approaches like MLPA and aCGH need to be combined in a cost-efficient way, to identify disease and progression causing alterations in ALL, as majority of them are cryptic in banding cytogenetic analyses.

A cryptic three-way translocation t(10;19;11)(p12.31;q13.31;q23.3) with a derivative Y-chromosome in an infant with acute myeloblastic leukemia (M5b).

Acute myeloid leukemia (AML) is a heterogeneous disease characterized by the malignant transformation of hematopoietic precursors to a pathogenic cell clone. Chromosomal band 11q23 harboring MLL (=mixed lineage leukemia) gene is known to be involved in rearrangements with variety of genes as activating partners of MLL in different AML subtypes. Overall, an unfavorable prognosis is associated with MLL abnormalities. Here we investigated an 11-month-old male presenting with hyperleukocytosis being diagnosed with AML subtype FAB-M5b. In banding cytogenetics a der(19)t(19;?)(q13.3;?) and del(Y)(q11.23) were found as sole aberrations. Molecular cytogenetics revealed that the MLL gene was disrupted and even partially lost due to a t(10;19;11)(p12.31;q13.31;q23.3), an MLL/MLLT10 fusion appeared, and the der(Y) was an asymmetric inverted duplication with breakpoints in Yp11.2 and Yq11.23. The patient got hematopoietic stem cell transplantation from his haploidentical mother. Still three months afterwards 15% of blasts were detected in bone marrow and later the patient was lost during follow-up. The present case highlights the necessity to exclude MLL rearrangements, even when there seems to be no actual hint from banding cytogenetics.

Novel Cryptic Rearrangements in Adult B-Cell Precursor Acute Lymphoblastic Leukemia Involving the MLL Gene.

MLL (mixed-lineage-leukemia) gene rearrangements are typical for acute leukemia and are associated with an aggressive course of disease, with a worse outcome than comparable case, and thus require intensified treatment. Here we describe a 69-year-old female with adult B cell precursor acute lymphoblastic leukemia (BCP-ALL) with hyperleukocytosis and immunophenotype CD10- and CD19+ with cryptic MLL rearrangements. G-banding at the time of diagnosis showed a normal karyotype: 46,XX. Molecular cytogenetics using multitude multicolor banding (mMCB) revealed a complex rearrangement of the two copies of chromosome 11. However, a locus-specific probe additionally identified that the MLL gene at 11q23.3 was disrupted, and that the 5' region was inserted into the chromosomal sub-band 4q21; thus the aberration involved three chromosomes and five break events. Unfortunately, the patient died six months after the initial diagnosis from serious infections and severe complications. Overall, the present findings confirm that, by far not all MLL aberrations are seen by routine chromosome banding techniques and that fluorescence in situ hybridization (FISH) should be regarded as standard tool to access MLL rearrangements in patients with BCP-ALL.

The Bowen-Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA.

The Nep1 (Emg1) SPOUT-class methyltransferase is an essential ribosome assembly factor and the human Bowen-Conradi syndrome (BCS) is caused by a specific Nep1(D86G) mutation. We recently showed in vitro that Methanocaldococcus jannaschii Nep1 is a sequence-specific pseudouridine-N1-methyltransferase. Here, we show that in yeast the in vivo target site for Nep1-catalyzed methylation is located within loop 35 of the 18S rRNA that contains the unique hypermodification of U1191 to 1-methyl-3-(3-amino-3-carboxypropyl)-pseudouri-dine (m1acp3Ψ). Specific (14)C-methionine labelling of 18S rRNA in yeast mutants showed that Nep1 is not required for acp-modification but suggested a function in Ψ1191 methylation. ESI MS analysis of acp-modified Ψ-nucleosides in a Δnep1-mutant showed that Nep1 catalyzes the Ψ1191 methylation in vivo. Remarkably, the restored growth of a nep1-1(ts) mutant upon addition of S-adenosylmethionine was even observed after preventing U1191 methylation in a Δsnr35 mutant. This strongly suggests a dual Nep1 function, as Ψ1191-methyltransferase and ribosome assembly factor. Interestingly, the Nep1 methyltransferase activity is not affected upon introduction of the BCS mutation. Instead, the mutated protein shows enhanced dimerization propensity and increased affinity for its RNA-target in vitro. Furthermore, the BCS mutation prevents nucleolar accumulation of Nep1, which could be the reason for reduced growth in yeast and the Bowen-Conradi syndrome.

Backbone resonance assignments of the 48 kDa dimeric putative 18S rRNA-methyltransferase Nep1 from Methanocaldococcus jannaschii.

Nep1 from Methanocaldococcus jannaschii is a 48 kDa dimeric protein belonging to the SPOUT-class of S-adenosylmethionine dependent RNA-methyltransferases and acting as a ribosome assembly factor. Mutations in the human homolog are the cause of Bowen-Conradi syndrome. We report here 1H, 15N and 13C chemical shift assignments for the backbone of the protein in its apo state.

The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site.

Ribosome biogenesis in eukaryotes requires the participation of a large number of ribosome assembly factors. The highly conserved eukaryotic nucleolar protein Nep1 has an essential but unknown function in 18S rRNA processing and ribosome biogenesis. In Saccharomyces cerevisiae the malfunction of a temperature-sensitive Nep1 protein (nep1-1(ts)) was suppressed by the addition of S-adenosylmethionine (SAM). This suggests the participation of Nep1 in a methyltransferase reaction during ribosome biogenesis. In addition, yeast Nep1 binds to a 6-nt RNA-binding motif also found in 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes. Here, we present the X-ray structure of the Nep1 homolog from the archaebacterium Methanocaldococcus jannaschii in its free form (2.2 A resolution) and bound to the S-adenosylmethionine analog S-adenosylhomocysteine (SAH, 2.15 A resolution) and the antibiotic and general methyltransferase inhibitor sinefungin (2.25 A resolution). The structure reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases but contains a novel extension of this common core fold. SAH and sinefungin bind to Nep1 at a preformed binding site that is topologically equivalent to the cofactor-binding site in other SPOUT-class methyltransferases. Therefore, our structures together with previous genetic data suggest that Nep1 is a genuine rRNA methyltransferase.

HMGA2 expression in a canine model of prostate cancer.

Prostate cancer is the most prevalent cancer in western countries, being the third leading cause of male cancer death. To check its possible significance as a prognostic marker, allowing a better prognosis of the tumor, we analyzed the high-mobility group protein-A2 gene (HMGA2) expression level because HMGA2 overexpression has been shown to correlate with the malignant potential of various neoplasias. Aside from man, the dog is the only mammalian species that shows spontaneously occurring prostate carcinoma with striking similarities to prostate cancer growth and progression in man, making it an adequate animal model for this neoplasia. We used real-time quantitative reverse-transcription polymerase chain reaction for HMGA2 expression analyses in a subset of canine prostate tissue samples. Our investigations reveal that HMGA2 expression levels in all carcinomas were higher than those of any of the nonmalignant tissues. Thus, canine prostate cancer represents a spontaneously occurring model to test therapeutic effects resulting from reduced expression of HMGA2.

HMGA2 overexpression in non-small cell lung cancer.

Lung cancer is still the leading cause of death from cancer worldwide primarily because of the fact that most lung cancers are diagnosed at advanced stages. Overexpression of the high mobility group protein HMGA2 has been observed in a variety of malignant tumors and often correlates with poor prognosis. Herein, HMGA2 expression levels were analyzed in matching cancerous and non-cancerous lung samples of 17 patients with adenocarcinoma (AC) and 17 patients with squamous cell carcinoma (SCC) with real-time quantitative RT-PCR (qRT-PCR). Transcript levels were compared to results obtained by immunohistochemistry (IHC). HMGA2 expression was detectable by qRT-PCR in all samples tested and varied from 5422 to 16 991 545 copies per 250 ng total RNA in the carcinoma samples and from 289 to 525 947 copies in the non-cancerous tissue samples. In 33/34 non-small cell lung cancer (NSCLC) samples tested, an overexpression of HMGA2 was revealed with statistically highly significant differences between non-neoplastic and tumor samples for both AC (P < 0.0001) as well as for SCC (P < 0.0001). Expression varies strongly and is increased up to 911-fold for AC and up to 2504-fold for SCC, respectively, with statistically significant higher increase in SCC (P < 0.05). The results presented herein indicate that HMGA2 overexpression is a common event in NSCLC and could serve as molecular marker for lung cancer.

Angiogenetic signaling through hypoxia: HMGB1: an angiogenetic switch molecule.

The initiation of angiogenesis, called the angiogenetic switch, is a crucial early step in tumor progression and propagation, ensuring an adequate oxygen supply. The rapid growth of tumors is accompanied by a reduced microvessel density, resulting in chronic hypoxia that often leads to necrotic areas within the tumor. These hypoxic and necrotic regions exhibit increased expression of angiogenetic growth factors, eg, vascular endothelial growth factor, and may also attract macrophages, which are known to produce a number of potent angiogenetic cytokines and growth factors. A group of molecules that may act as mediators of angiogenesis are the so-called high-mobility group proteins. Recent studies showed that HMGB1, known as an architectural chromatin-binding protein, can be extracellularly released by passive diffusion from necrotic cells and activated macrophages. To examine the angiogenetic effects of HMGB1 on endothelial cells an in vitro spheroid model was used. The results of the endothelial-sprouting assay clearly show that exogenous HMGB1 induced endothelial cell migration and sprouting in vitro in a dose-dependent manner. Thus, this is the first report showing strong evidence for HMGB1-induced sprouting of endothelial cells.

Expression pattern of the HMGB1 gene in sarcomas of the dog.

BACKGROUND: The human high mobility group protein B1 (HMGB1) has attracted considerable interest among oncologists because it sensitises cancer cells to the anticancer drug cisplatin by shielding cisplatin-DNA adducts from nucleotide excision repair. MATERIALS AND METHODS: Since cisplatin is the cornerstone of adjuvant systemic therapy for osteosarcomas, in both humans and dogs, the expression pattern of the HMGB1 gene in seven canine sarcomas was investigated by Northern blot analysis and semi-quantitative RT-PCR. RESULTS: A strong intertumoural variation of HMGB1 expression was detected by Northern blot analysis and confirmed by the semi-quantitative RT-PCR established herein. CONCLUSION: The observed variations of HMGB1 expression in canine sarcomas emphasises the role of HMGB1 as a potential marker of clinical interest as its expression level may predict the clinical outcome of therapies based on cisplatin. The semi-quantitative RT-PCR established allows a quick and convenient determination of the HMGB1 expression level as necessary for clinical applications.