ChromaFold predicts the 3D contact map from single-cell chromatin accessibility.

The identification of cell-type-specific 3D chromatin interactions between regulatory elements can help to decipher gene regulation and to interpret the function of disease-associated non-coding variants. However, current chromosome conformation capture (3C) technologies are unable to resolve interactions at this resolution when only small numbers of cells are available as input. We therefore present ChromaFold, a deep learning model that predicts 3D contact maps and regulatory interactions from single-cell ATAC sequencing (scATAC-seq) data alone. ChromaFold uses pseudobulk chromatin accessibility, co-accessibility profiles across metacells, and predicted CTCF motif tracks as input features and employs a lightweight architecture to enable training on standard GPUs. Once trained on paired scATAC-seq and Hi-C data in human cell lines and tissues, ChromaFold can accurately predict both the 3D contact map and peak-level interactions across diverse human and mouse test cell types. In benchmarking against a recent deep learning method that uses bulk ATAC-seq, DNA sequence, and CTCF ChIP-seq to make cell-type-specific predictions, ChromaFold yields superior prediction performance when including CTCF ChIP-seq data as an input and comparable performance without. Finally, fine-tuning ChromaFold on paired scATAC-seq and Hi-C in a complex tissue enables deconvolution of chromatin interactions across cell subpopulations. ChromaFold thus achieves state-of-the-art prediction of 3D contact maps and regulatory interactions using scATAC-seq alone as input data, enabling accurate inference of cell-type-specific interactions in settings where 3C-based assays are infeasible.

Th2 cells inhibit growth of colon and pancreas cancers by promoting anti-tumorigenic responses from macrophages and eosinophils.

BACKGROUND: Immunotherapy of gastrointestinal cancers is challenging; however, several lines of evidence suggest that adoptive transfer of stimulated or modified immune cells support not only protective role of immune cells in tumor microenvironment, but actively participate in the elimination of cancer cells. METHODS: In vivo studies employing cancer cell-derived allograft murine models of gastrointestinal cancers were performed. The effects of T helper (Th) 2 cells on gastrointestinal cancers growth and tumor microenvironment composition using adoptive transfer of Th2 cells, interleukin (IL)-5 treatment, and immunofluorescence, multiplex and real-time PCR were explored. RESULTS: Here, we show that Th2 cells play an essential role in the inhibition of colon and pancreas cancers progression. In murine models of gastrointestinal tumors using adoptive transfer of Th2 cells, we identify that Th2 cells are responsible for generation of apoptotic factors and affect macrophage as well as eosinophil recruitment into tumors where they produce cytotoxic factors. Moreover, we found that Th2 cells lead to IL-5 hypersecretion, which links the anti-tumorigenic function of Th2 cells and eosinophils. Importantly, we noted that recombinant IL-5 administration is also related with inhibition of gastrointestinal tumor growth. Finally, using an in vitro approach, we documented that both Th2 cells and eosinophils are directly responsible for gastrointestinal cancer cell killing. CONCLUSIONS: These data demonstrate the significance of Th2 cells, eosinophils and IL-5 in the inhibition of gastrointestinal tumor growth, and pointed toward tumor microenvironment reprogramming as a Th2 cell-mediated anti-tumorigenic mechanism of action.

An Aged/Autoimmune B-cell Program Defines the Early Transformation of Extranodal Lymphomas.

A third of patients with diffuse large B-cell lymphoma (DLBCL) present with extranodal dissemination, which is associated with inferior clinical outcomes. MYD88L265P is a hallmark extranodal DLBCL mutation that supports lymphoma proliferation. Yet extranodal lymphomagenesis and the role of MYD88L265P in transformation remain mostly unknown. Here, we show that B cells expressing Myd88L252P (MYD88L265P murine equivalent) activate, proliferate, and differentiate with minimal T-cell costimulation. Additionally, Myd88L252P skewed B cells toward memory fate. Unexpectedly, the transcriptional and phenotypic profiles of B cells expressing Myd88L252P, or other extranodal lymphoma founder mutations, resembled those of CD11c+T-BET+ aged/autoimmune memory B cells (AiBC). AiBC-like cells progressively accumulated in animals prone to develop lymphomas, and ablation of T-BET, the AiBC master regulator, stripped mouse and human mutant B cells of their competitive fitness. By identifying a phenotypically defined prospective lymphoma precursor population and its dependencies, our findings pave the way for the early detection of premalignant states and targeted prophylactic interventions in high-risk patients. SIGNIFICANCE: Extranodal lymphomas feature a very poor prognosis. The identification of phenotypically distinguishable prospective precursor cells represents a milestone in the pursuit of earlier diagnosis, patient stratification, and prophylactic interventions. Conceptually, we found that extranodal lymphomas and autoimmune disorders harness overlapping pathogenic trajectories, suggesting these B-cell disorders develop and evolve within a spectrum. See related commentary by Leveille et al. (Blood Cancer Discov 2023;4:8-11). This article is highlighted in the In This Issue feature, p. 1.

Defining biomarkers in oral cancer according to smoking and drinking status.

Introduction: Oral Squamous Cell Carcinomas (OSCC) are mostly related to tobacco consumption eventually associated to alcohol (Smoker/Drinker patients: SD), but 25-30% of the patients have no identified risk factors (Non-Smoker/Non-Drinker patients: NSND). We hypothesized that these patients have distinguishable immune profiles that could be useful for prognosis. Materials and Methods: Cells present in immune tumor microenvironment (TME) and blood from 87 OSCC HPV-negative patients were analyzed using a multiparameter flow cytometry assay, in a prospective case-control study. Cytokine levels in tumor supernatants and blood were determined by a cytometric bead array (CBA) assay. Results: Normal gingiva and blood from healthy donors (HD) were used as controls. A significant increase of granulocytes (p<0.05 for blood), of monocytes-macrophages (p<0.01 for blood) and of CD4+ T cells expressing CD45RO and CCR6 (p<0.001 for blood; p<0.0001 for TME) as well as higher levels of IL-6 (p<0.01 for sera, p<0.05 for tumor supernatant) were observed in SD patients as compared to NSND OSCC patients and HD. High percentages of CD4+ T cells expressing CD45RO and CCR6 cells in tumor tissue (p=0.05) and blood (p=0.05) of SD OSCC patients were also associated with a poorer prognosis while a high percentage of regulatory T cells (Treg) in tumor tissue was associated with a more favorable prognostic factor (p=0.05). Also, a higher percentage of blood CD8+ T lymphocytes among CD45+ cells in NSND patients was associated with a better disease-free survival (p=0.004). Conclusion: Granulocytes, monocytes-macrophages, and CD4+ T cells expressing CD45RO and CCR6 in blood and TME as well as serum IL-6 can therefore distinguish OSCC SD and NSND patients. Quantifying the proportion of CD4+ T cells expressing CD45RO and CCR6 and of Treg in SD patients and CD8+ T cells in NSND patients could help defining the prognostic of OSCC patients.

Granulocyte colony-stimulating factor promotes an aggressive phenotype of colon and breast cancer cells with biochemical changes investigated by single-cell Raman microspectroscopy and machine learning analysis.

Granulocyte colony-stimulating factor (G-CSF) is produced at high levels in several cancers and is directly linked with metastasis in gastrointestinal (GI) cancers. In order to further understand the alteration of molecular compositions and biochemical features triggered by G-CSF treatment at molecular and cell levels, we sought to investigate the long term treatment of G-CSF on colon and breast cancer cells measured by label-free, non-invasive single-cell Raman microspectroscopy. Raman spectrum captures the molecule-specific spectral signatures ("fingerprints") of different biomolecules presented on cells. In this work, mouse breast cancer line 4T1 and mouse colon cancer line CT26 were treated with G-CSF for 7 weeks and subsequently analyzed by machine learning based Raman spectroscopy and gene/cytokine expression. The principal component analysis (PCA) identified the Raman bands that most significantly changed between the control and G-CSF treated cells. Notably, here we proposed the concept of aggressiveness score, which can be derived from the posterior probability of linear discriminant analysis (LDA), for quantitative spectral analysis of tumorigenic cells. The aggressiveness score was effectively applied to analyze and differentiate the overall cell biochemical changes of G-CSF-treated two model cancer cells. All these tumorigenic progressions suggested by Raman analysis were confirmed by pro-tumorigenic cytokine and gene analysis. A high correlation between gene expression data and Raman spectra highlights that the machine learning based non-invasive Raman spectroscopy offers emerging and powerful tools to better understand the regulation mechanism of cytokines in the tumor microenvironment that could lead to the discovery of new targets for cancer therapy.

G-CSF in tumors: Aggressiveness, tumor microenvironment and immune cell regulation.

Granulocyte colony-stimulating factor (G-CSF) is a cytokine most well-known for maturation and mobilization of bone marrow neutrophils. Although it is used therapeutically to treat chemotherapy induced neutropenia, it is also highly expressed in some tumors. Case reports suggest that tumors expressing high levels of G-CSF are aggressive, more difficult to treat, and present with poor prognosis and high mortality rates. Research on this topic suggests that G-CSF has tumor-promoting effects on both tumor cells and the tumor microenvironment. G-CSF has a direct effect on tumor cells to promote tumor stem cell longevity and overall tumor cell proliferation and migration. Additionally, it may promote pro-tumorigenic immune cell phenotypes such as M2 macrophages, myeloid-derived suppressor cells, and regulatory T cells. Overall, the literature suggests a plethora of pro-tumorigenic activity that should be balanced with the therapeutic use. In this review, we present an overview of the multiple complex roles of G-CSF and G-CSFR in tumors and their microenvironment and discuss how clinical advances and strategies may open new therapeutic avenues.

Vacuum Bell: Is It a Useful Innovative Device for Pectus Excavatum Correction?

Pectus Excavatum (PE) or "funnel chest," the most common deformity of the anterior chest wall characterized by sternal depression, can be repaired via either operative or non-invasive techniques. Vacuum Bell (VB) device is the most widespread of the latter one which can be applied either intraoperatively or as monotherapy. The present narrative review examines the efficacy of that innovative method. A thorough search of the literature resulted in 13 English-written articles concerning VB therapy from its first description to February 2019. The studies included patients with mild to moderate PE, mainly evaluated via Haller-Index and/or sternum depth prior to and following treatment. Concerning depth-improvement, 37-90% showed amelioration while 10-40% of them an excellent correction to normal. In 42%, Haller-Index also improved with a median decrease of 0.3 after VB application. A correlation was attempted to be found between the efficacy of VB and factors such as the frequency and duration of VB application, patient age, gender, PE severity and type, and differential pressure of the suction cup. Complications may be frequent yet mild and temporary. Intraoperatively, VB widows Minimally Invasive Repair of Pectus Excavatum (MIRPE) operation a safer procedure with greater results. VB as conservative treatment is an effective and well-tolerated alternative therapeutic option for selected patients with PE who meet specific criteria. It also constitutes a device of significant efficacy, appropriate for intraoperative use during MIRPE procedure.

G-CSF and G-CSFR Induce a Pro-Tumorigenic Macrophage Phenotype to Promote Colon and Pancreas Tumor Growth.

Tumor-associated macrophages (TAMs) in the gastrointestinal tumor microenvironment (TME) are known to polarize into populations exhibiting pro- or anti-tumoral activity in response to stimuli such as growth factors and cytokines. Our previous work has recognized granulocyte colony-stimulating factor (G-CSF) as a cytokine capable of influencing immune cells of the TME exhibiting pro-tumoral activity. Here, we aimed to focus on how G-CSF regulates TAM phenotype and function and the effects on gastrointestinal (GI) tumor progression. Thus, wildtype (WT) and G-CSFR-/- macrophages were examined for cytokine production, gene expression, and transcription factor activity. Adoptive transfer of WT or G-CSFR-/- macrophages into tumor-bearing mice was performed to study their influence in the progression of colon (MC38) and pancreatic (PK5L1940) tumor mouse models. Finally, the difference in cytotoxic potential between WT and G-CSFR-/- macrophages was examined both in vitro and in vivo. Our results indicate that G-CSF promotes increased IL-10 production and decreased IL-12 production, which was reversed in G-CSFR-/- macrophages for a pro-inflammatory phenotype. Furthermore, G-CSFR-/- macrophages were characterized by higher levels of NOS2 expression and NO production, which led to greater tumor related cytotoxicity both in vitro and in vivo. Our results suggest that in the absence of G-CSFR, macrophage-related tumor cytotoxicity was amplified. These findings, along with our previous reports, pinpoint G-CSF /G-CSFR as a prominent target for possible clinical applications that aim to control the TME and the GI tumor progression.

G-CSF and G-CSFR Modulate CD4 and CD8 T Cell Responses to Promote Colon Tumor Growth and Are Potential Therapeutic Targets.

Cytokines are known to shape the tumor microenvironment and although progress has been made in understanding their role in carcinogenesis, much remains to learn regarding their role in tumor growth and progression. We have identified granulocyte colony-stimulating factor (G-CSF) as one such cytokine, showing that G-CSF is linked with metastasis in human gastrointestinal tumors and neutralizing G-CSF in a mouse model of colitis-associated cancer is protective. Here, we set out to identify the role of G-CSF and its receptor, G-CSFR, in CD4+ and CD8+ T cell responses in the tumor microenvironment. MC38 colon cancer cells were injected into WT, G-CSFR-/- mice, or Rag2-/- mice. Flow cytometry, Real Time PCR and Multiplex cytokine array analysis were used for in vitro T cell phenotype analysis. Adoptive transfer of WT or G-CSFR-/- CD4+ of CD8+ T cells were performed. Mouse tumor size, cytokine expression, T cell phenotype, and cytotoxic activity were analyzed. We established that in G-CSFR-/- mice, tumor growth of MC38 colon cancer cells is significantly decreased. T cell phenotype and cytokine production were also altered, as both in vitro and in vivo approaches revealed that the G-CSF/G-CSFR stimulate IL-10-producing, FoxP3-expressing CD4+ and CD8+ T cells, whereas G-CSFR-/- T cells exhibit increased IFNγ and IL-17A production, leading to increased cytotoxic activity in the tumor microenvironment. Furthermore, peritumoral injection of recombinant IFNγ or IL-17A inhibited colon and pancreas tumor growth compared to controls. Taken together, our data reveal an unknown mechanism by which G-CSF, through its receptor G-CSFR, promotes an inhibitory Treg phenotype that limits tumor immune responses and furthermore suggest that targeting this cytokine/receptor axis could represent a novel therapeutic approach for gastrointestinal, and likely other tumors with high expression of these factors.

Host's Endogenous Caveolin-1 Expression is Downregulated in the Lung During Sepsis to Promote Cytoprotection.

The present study focuses on the profile of "endogeneous" caveolin-1 protein in septic lung (CLP model).Caveolin-1, CD25, pP38, pAkt, and 14-3-3b protein expression profiles were studied using flow cytometry and immunohistochemistry 6, 12, 24, 36, and 48 h after sepsis induction. Cell viability was determined by 7-AAD staining and fibrosis by Masson trichrome stain. The effect of protein C zymogen concentrate (PC) on caveolin-1 expression was also investigated given that PC, once dissociated from caveolin-1, elicits a PAR-1-mediated protective signaling by forming a complex with endothelial protein C receptor (EPCR).CLP treatment increased lung inflammation and cell apoptosis. Fibrosis was apparent in vessels and alveoli. Caveolin-1+ cells presented reduced protein expression, especially 12 h post-CLP (P = 0.002). Immunohistochemistry revealed caveolin-1 positive expression mainly in regions with strong inflammatory reaction. Early induction of pP38+ cell population (P = 0.014) and gradual increase of CD25+ cells were also observed. Alternations in 14-3-3b expression related to apoptosis were apparent and accompanied by increased AKT phosphorylation activity late during sepsis progression.After PC administration, cell apoptosis was reduced (P = 0.004) and both the percentile and expression intensity of caveolin-1 positive cells were compromised (P = 0.009 and P = 0.027, respectively). 14-3-3b, CD25, and pP38 protein expression were decreased (P = 0.014, P = 0.004, and P = 0.007, respectively), whereas pAkt expression was induced (P = 0.032).The observed decline of endogenous caveolin-1 protein expression during sepsis implies its involvement in host's cytoprotective reaction either directly, by controlling caveolae population to decrease bacterial burden, or indirectly via regulating 14-3-3b-dependent apoptosis and EPCR-PAR-1-dependent protective signaling.

Bacterial Colonization in Hidradenitis Suppurativa/Acne Inversa: A Cross-sectional Study of 50 Patients and Review of the Literature.

It is unclear whether bacterial colonization in hidradenitis suppurativa/acne inversa (HS) comprises a primary cause, triggering factor or secondary phenomenon of the disease pathogenesis. Furthermore, the connection between certain bacterial species, the disease severity and its localization is unknown. Bacterial species were isolated from HS lesions to reveal a potential correlation with localization and disease severity. Ninety swab tests were prospectively obtained from 90 HS lesions of 50 consecutive patients. The material was cultured under aerobic and anaerobic conditions. The identified species were statistically correlated with Hurley stage and localization of the lesions. The most prevalent isolates were reported. Hurley stage significantly correlated with disease localization. Particular bacterial species were associated with "extended" disease and Hurley III stage with the detection of both aerobic and anaerobic bacteria and with a higher number of species. The presence of bacterial species is dependent on the local milieu, which correlates with the localization of the disease, its clinical manifestations and its extension.

Exacerbation of Darier Disease under Interferon-α-2a Therapy with Clinical Signs of Lichen Nitidus.

Darier disease/dyskeratosis follicularis is a genodermatosis characterized by brown, oily keratotic papules and plaques in the seborrheic areas of the face and chest. Responsible for the disease are mutations in the ATP2A2 gene, encoding SERCA2, a calcium pump of the sarco-/endoplasmic reticulum. Mechanical trauma, heat, humidity, ultraviolet B radiation, oral corticosteroids and lithium are known trigger factors of the disorder. We report on a 48-year-old German woman with a flare-up of Darier disease under interferon-α-2a (IFNα-2a) therapy with clinical signs of lichen nitidus. Due to the fulminant course of the eruption, we suspected IFNα as a possible trigger. To our knowledge there are no reports regarding exacerbation of Darier disease during IFNα therapy. Possible pathogenetic mechanisms are being discussed.

Hidradenitis suppurativa/Acne inversa: an endocrine skin disorder?

Hidradenitis suppurativa / acne inversa (HS) is a chronic inflammatory, debilitating skin disorder with a largely unknown etiology. However, many observations such as the typical onset of the disease after puberty, the female predominance, the pre-menstrual flare ups and the improvement during pregnancy suggest a contribution of endocrinological factors to the emergence of the disease. In addition, the reported efficacy of anti-androgen treatment on HS indicates a possible involvement of androgens in the pathogenesis. Furthermore, the common comorbidity with metabolic syndrome points to possible interactions between endocrinological and metabolic alterations in the development of HS. Taking into account the endocrine functions of the human skin, several studies investigated the effects of hormones and the behavior of the pilosebaceous unit in the skin of patients with HS. In this review we describe the current view on the hormonal dysregulation and metabolic syndrome and their role in HS.

High Copy Numbers of ß-Defensin Cluster on 8p23.1, Confer Genetic Susceptibility, and Modulate the Physical Course of Hidradenitis Suppurativa/Acne Inversa.

Hidradenitis suppurativa/acne inversa (HS) has a multifactorial pathogenesis, with many patients reporting positive family history. Nine ß-defensin genes (among them DEFB4 and DEFB103, encoding for proinflammatory mediators human ß-defensin-2 and human ß-defensin-3, respectively) exist as a cluster (DEFB) affected by copy number (CN). We hypothesized that CNs are greater in patients with HS and that they are linked to genetic susceptibility. CNs of DEFB were studied in two independent patient cohorts: 163 patients from Greece and 98 from Germany. CNs were greater in patients than control subjects in both studied cohorts. Carriage of more than six CNs was associated with a 7.53 odds ratio for HS in the Greek cohort and a 5.76 odds ratio for HS in the German cohort. The common odds ratio after meta-analysis was 6.72 (P < 0.0001). However, presence of fewer than six copies was linked with disease onset at an earlier age (P = 0.048), less frequent presentation of permanent purulence of the affected skin lesions (P = 0.036), and fewer skin localizations (P = 0.042). A robust genetic trait for susceptibility to HS is provided, and this is confirmed in two independent cohorts. Susceptibility arises from carriage of more than six DEFB copies, which interferes directly with the HS phenotype.

EXTENDED CYTOPROTECTIVE EFFECT OF AUTOPHAGY IN THE LATE STAGES OF SEPSIS AND FLUCTUATIONS IN SIGNAL TRANSDUCTION PATHWAYS IN A RAT EXPERIMENTAL MODEL OF KIDNEY INJURY.

The impact of a potential autophagy (LC3a/b) deregulation in hyper and in hypo stages during sepsis-induced kidney injury and the temporal profile of phosphorylated extracellular signal-related kinase, P38 (pP38), Akt (pAKT), and 13-3-3ß protein were investigated in the current study, using a rat cecal ligation and puncture (CLP) model, by means of flow cytometry and immunohistochemistry. Cell viability was assessed by protein C zymogen concentrate (PC), 7-aminoactinomycin D (7-AAD) staining and inflammation by S100 protein immunostaining. The impact of reduced kidney inflammation in autophagy was assessed by PC administration, an anti-inflammatory and cytoprotective substance. Sepsis induction increased LC3a/b expression, which presented two peaks at 6 and 36 h after CLP, both in the percentage of positive cells (P = 0.024, P = 0.025, respectively) and in fluorescence intensity. At 6 h when inflammation was already apparent, LC3a/b increase was escorted by phosphorylated extracellular signal-related kinase stimulation and high cell viability (65%), designating autophagy as a cytoprotective mechanism against microbial infection. The phosphorylation of P38 was delayed to 12 h after CLP, when autophagy was reduced. pAkt and 14-3-3ß expression was stimulated between 6 and 36 h after CLP, although a slight inhibition of pAkt within each cell was detected (lower MnIX value). During the second peak, inflammation was intensified, necrosis was significantly increased with LC3a/b+/7-AAD + cells to present a 1.5-fold increase. Protein C zymogen concentrate administration declined autophagy at 6 and 36 h after CLP and reduced necrosis, whereas double positive LC3a/b and 7-AAD cells were increased by 1.68 and 2.78-fold, respectively. These data open new prospectives in sepsis treatment, since they further support that autophagy represents a cytoprotective mechanism triggered by stress conditions, rather than an alternative cell death pathway.

Endocrinologic Aspects of Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disorder of unknown etiology. The role of hormones in HS remains unclear, but the observation of premenstrual flares, female predominance, and improvement during pregnancy suggest a hormonal/metabolic background. The reported positive effects of antiandrogen therapy supports a possible role of androgens. The predominant onset of the disease years after puberty may indicate a metabolic disorder. Obesity contributes significantly to HS pathogenesis; diabetes, dyslipidemia, the metabolic syndrome, and polycystic ovarian syndrome are among the commonest comorbidities. More studies are required to clarify a potential hormonal dysregulation in HS.

Multiple HPV-Induced Squamous Cell Carcinomas on the Fingers of a Patient with Systemic Lupus Erythematosus: A Case and Review.

Human papilloma virus (HPV) infection is documented to be involved in the development of epithelial malignancies, mostly in cervical cancer. Systemic lupus erythematosus (SLE) patients have an increased prevalence of such an infection. We report the case of a 55-year-old female SLE patient who developed multiple in situ squamous cell carcinomas on her fingers, after chronic HPV infection. HPV-33 DNA was isolated from the lesions. The purpose of this case presentation is to raise awareness about HPV-induced malignancies for this high-risk group and propose an early HPV vaccination to efficiently prevent such comorbidities.

Bacteriology of hidradenitis suppurativa/acne inversa: A review.

Given that follicular papules and pustules, as well as nodules and abscesses, are the clinical hallmarks of hidradenitis suppurativa (HS), an infectious, bacterial pathway has been suspected in the pathogenesis of this chronic, inflammatory condition. Elucidating the behavior and role of bacterial species in HS and their interaction with cutaneous innate immunity will provide more insight into the pathophysiology of this condition. This review of prospective investigations suggests a synergistic relationship between impaired innate immunity and microbial factors in the etiology of HS.