Unraveling the diversity and functions of tissue-resident plasma cells.

Antibody-secreting plasma cells (PCs) are generated in secondary lymphoid organs but are reported to reside in an emerging range of anatomical sites. Analysis of the transcriptome of different tissue-resident (Tr)PC populations revealed that they each have their own transcriptional signature indicative of functional adaptation to the host tissue environment. In contrast to expectation, all TrPCs were extremely long-lived, regardless of their organ of residence, with longevity influenced by intrinsic factors like the immunoglobulin isotype. Analysis at single-cell resolution revealed that the bone marrow is unique in housing a compendium of PCs generated all over the body that retain aspects of the transcriptional program indicative of their tissue of origin. This study reveals that extreme longevity is an intrinsic property of TrPCs whose transcriptome is imprinted by signals received both at the site of induction and within the tissue of residence.

MacroH2A1.1 as a crossroad between epigenetics, inflammation and metabolism of mesenchymal stromal cells in myelodysplastic syndromes.

Ineffective hematopoiesis is a hallmark of myelodysplastic syndromes (MDS). Hematopoietic alterations in MDS patients strictly correlate with microenvironment dysfunctions, eventually affecting also the mesenchymal stromal cell (MSC) compartment. Stromal cells are indeed epigenetically reprogrammed to cooperate with leukemic cells and propagate the disease as "tumor unit"; therefore, changes in MSC epigenetic profile might contribute to the hematopoietic perturbations typical of MDS. Here, we unveil that the histone variant macroH2A1 (mH2A1) regulates the crosstalk between epigenetics and inflammation in MDS-MSCs, potentially affecting their hematopoietic support ability. We show that the mH2A1 splicing isoform mH2A1.1 accumulates in MDS-MSCs, correlating with the expression of the Toll-like receptor 4 (TLR4), an important pro-tumor activator of MSC phenotype associated to a pro-inflammatory behavior. MH2A1.1-TLR4 axis was further investigated in HS-5 stromal cells after ectopic mH2A1.1 overexpression (mH2A1.1-OE). Proteomic data confirmed the activation of a pro-inflammatory signature associated to TLR4 and nuclear factor kappa B (NFkB) activation. Moreover, mH2A1.1-OE proteomic profile identified several upregulated proteins associated to DNA and histones hypermethylation, including S-adenosylhomocysteine hydrolase, a strong inhibitor of DNA methyltransferase and of the methyl donor S-adenosyl-methionine (SAM). HPLC analysis confirmed higher SAM/SAH ratio along with a metabolic reprogramming. Interestingly, an increased LDHA nuclear localization was detected both in mH2A1.1-OE cells and MDS-MSCs, probably depending on MSC inflammatory phenotype. Finally, coculturing healthy mH2A1.1-OE MSCs with CD34+ cells, we found a significant reduction in the number of CD34+ cells, which was reflected in a decreased number of colony forming units (CFU-Cs). These results suggest a key role of mH2A1.1 in driving the crosstalk between epigenetic signaling, inflammation, and cell metabolism networks in MDS-MSCs.

Rethink analgo-sedation in digestive endoscopy: the role of scientific societies in tracing training path.

OBJECTIVE: The Italian Society of Anesthesia, Analgesia, Reanimation and Intensive Care Medicine (SIAARTI) and the Italian Society of Digestive Endoscopy (SIED) worked together to produce a joint Good Clinical Practice (GCP) on analgo-sedation in digestive endoscopy and launched a survey to support the document. The aim was to identify and describe the actual clinical practice of sedation in Italian digestive endoscopy units and offer material for a wider and more widespread discussion among anesthetists and endoscopists. SUBJECTS AND METHODS: A national survey was planned, in order to support the statements of the GCP. Twelve thousand and five hundred questionnaires were sent to the members of SIAARTI and SIED in June 2020. RESULTS: A total of 662 forms (5.3%) returned completed. Highly complex procedures are performed according to 70% of respondents; daily anesthesiologist's assistance is guaranteed in 26%, for scheduled sessions in 14.5% and as needed in 8%. 69% of respondents declared not to have a dedicated team of anesthesiologists, while just 5% reported an anesthesiologist in charge. A complete monitoring system was assured by 70% of respondents. Dedicated pathways for COVID-19-positive patients were confirmed in <40% of the answers. With regard to moderate/deep sedation, 90% of respondents stated that an anesthetist decides timing and doses. Propofol was exclusively administered by anesthetists according to 94% of answers, and for 6% of respondents the endoscopist is allowed to administer propofol in presence of a dedicated nurse, but with a readily available anesthetist. Only 32.8% of respondents reported institutional training courses on procedural analgo-sedation. CONCLUSIONS: The need to provide patients scheduled for endoscopy procedures with an adequate analgo-sedation is becoming an increasing concern, well-known in almost all countries, but many factors compromise the quality of patient care. Results of a national survey would give strength to the need for a shared GCP in gastrointestinal endoscopy. Training and certification of non-anesthetist professionals should be one of the main ways to center the objective.

Management of surgical diseases of thyroid gland indications of the United Italian Society of Endocrine Surgery (SIUEC).

A task force of the United Italian society of Endocrine Surgery (SIUEC) was commissioned to review the position statement on diagnostic, therapeutic and health­care management protocol in thyroid surgery published in 2016, at the light of new technologies, recent oncological concepts, and tailored approaches. The objective of this publication was to support surgeons with modern rational protocols of treatment that can be shared by health-care professionals, taking into account important clinical, healthcare and therapeutic aspects, as well as potential sequelae and complications. The task force consists of 13 members of the SIUEC highly trained and experienced in thyroid surgery. The main topics concern clinical evaluation and preoperative workup, patient preparation for surgery, surgical treatment, non-surgical options, postoperative management, prevention and management of major complications, outpatient care and follow-up.

Infection prevention in endoscopy practice: comparative evaluation of re-usable vs single-use endoscopic valves.

Re-usable air/water and suction valves used in endoscopes often demonstrate risk of infection. To the authors' knowledge, the safety and efficacy of re-usable and single-use valves have not been compared to date. As such, a laboratory investigation was undertaken to compare the safety and efficacy of re-usable and single-use valves at 11 Italian endoscopy sites. Safety was evaluated by analysing the rinse liquid of reprocessed re-usable valves ready for use, and efficacy was assessed based on the completion of endoscopic procedures without valve malfunction. This study found significantly lower contamination of single-use valves compared with re-usable valves (0 vs 29.1%, respectively; P=0.007) and similar efficacy (97.6 vs 98.8%, respectively; P=ns). Microbiological analysis of the rinse liquid of reprocessed re-usable valves identified various surviving micro-organisms and highlighted their potential pathogenicity. Such data suggest that sterile single-use valves may be safer than re-usable valves, and have comparable performance.

Hemithyroidectomy versus total thyroidectomy in the intermediate-risk differentiated thyroid cancer: the Italian Societies of Endocrine Surgeons and Surgical Oncology Multicentric Study.

The surgical treatment of the intermediate-risk DTC (1-4 cm) remains still controversial. We analyzed the current practice in Italy regarding the surgical management of intermediate-risk unilateral DTC to evaluate risk factors for recurrence and to identify a group of patients to whom propose a total thyroidectomy (TT) vs. hemithyroidectomy (HT). Among 1896 patients operated for thyroid cancer between January 2017 and December 2019, we evaluated 564 (29.7%) patients with unilateral intermediate-risk DTC (1-4 cm) without contralateral nodular lesions on the preoperative exams, chronic autoimmune thyroiditis, familiarity or radiance exposure. Data were collected retrospectively from the clinical register from 16 referral centers. The patients were followed for at least 14 months (median time 29.21 months). In our cohort 499 patients (88.4%) underwent total thyroidectomy whereas 65 patients (11.6%) underwent hemithyroidectomy. 151 (26.8%) patients had a multifocal DTC of whom 57 (10.1%) were bilateral. 21/66 (32.3%) patients were reoperated within 2 months from the first intervention (completion thyroidectomy). Three patients (3/564) developed regional lymph node recurrence 2 years after surgery and required a lymph nodal neck dissection. The single factor related to the risk of reoperation was the histological diameter (HR = 1.05 (1.00-1-09), p = 0.026). Risk stratification is the key to differentiating treatment options and achieving better outcomes. According to the present study, tumor diameter is a strong predictive risk factor to proper choose initial surgical management for intermediate-risk DTC.

Excellent outcomes of transformed lymphomas in the rituximab era without autologous stem cell transplantation: an Australian single-centre experience.

BACKGROUND: Histologic transformation (HT) is an important event with adverse prognosis in the natural history of indolent lymphomas. There are minimal data on HT in the Australian setting. AIMS: To characterise patients with biopsy-proven HT and their outcomes identified at a tertiary Australian Hospital. METHODS: All patients with biopsy-proven HT during a 15-year period (2002-2017) were included. Clinico-pathological data were systematically collected from review of patient records. Survival estimates were assessed using the Kaplan-Meier method and compared using the log-rank test. Associations between variables and clinical outcomes were evaluated using Cox's proportional hazards model. RESULTS: A cohort of 45 patients was identified with a median age of 66 years and the majority (59%) having high-risk disease (Revised-International Prognostic Index score ≥3). R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisolone) induction was used in 69%, with an overall response rate of 82% (complete response (CR), 75%). Sixty-one percent of these induction responders received consolidation, with autologous stem cell transplant (ASCT) performed in only 17% and rituximab maintenance given to 31%. With a median follow up of 47 months (range: 4-136), the 5-year overall survival (OS) was 69% (95% CI: 52%, 81%). Chemotherapy-naivety at HT was associated with a superior rate of CR (84% vs 54%, P = 0.057) and 5-year OS (82% vs 46%, P = 0.012). Rituximab maintenance was associated with a durable progression-free survival in induction responders. CONCLUSIONS: Excellent OS was observed in this modern cohort of patients treated with rituximab-containing induction and low rate of consolidation by ASCT, particularly in those who were chemotherapy-naïve at HT.

Deep profiling of apoptotic pathways with mass cytometry identifies a synergistic drug combination for killing myeloma cells.

Multiple myeloma is an incurable and fatal cancer of immunoglobulin-secreting plasma cells. Most conventional therapies aim to induce apoptosis in myeloma cells but resistance to these drugs often arises and drives relapse. In this study, we sought to identify the best adjunct targets to kill myeloma cells resistant to conventional therapies using deep profiling by mass cytometry (CyTOF). We validated probes to simultaneously detect 26 regulators of cell death, mitosis, cell signaling, and cancer-related pathways at the single-cell level following treatment of myeloma cells with dexamethasone or bortezomib. Time-resolved visualization algorithms and machine learning random forest models (RFMs) delineated putative cell death trajectories and a hierarchy of parameters that specified myeloma cell survival versus apoptosis following treatment. Among these parameters, increased amounts of phosphorylated cAMP response element-binding protein (CREB) and the pro-survival protein, MCL-1, were defining features of cells surviving drug treatment. Importantly, the RFM prediction that the combination of an MCL-1 inhibitor with dexamethasone would elicit potent, synergistic killing of myeloma cells was validated in other cell lines, in vivo preclinical models and primary myeloma samples from patients. Furthermore, CyTOF analysis of patient bone marrow cells clearly identified myeloma cells and their key cell survival features. This study demonstrates the utility of CyTOF profiling at the single-cell level to identify clinically relevant drug combinations and tracking of patient responses for future clinical trials.

IRF4 Activity Is Required in Established Plasma Cells to Regulate Gene Transcription and Mitochondrial Homeostasis.

The transcription factor interferon regulatory factor 4 (IRF4) is critical for the development, maintenance, and function of plasma cells. The mechanism by which IRF4 exerts its action in mature plasma cells has been elusive due to the death of all such cells upon IRF4 loss. While we identify apoptosis as a critical pathway for the death of plasma cells caused by IRF4 loss, we also determine that IRF4 did not regulate the intrinsic apoptotic pathway directly. By using an inducible IRF4 deletion system in the presence of the overexpression of anti-apoptotic BCL2, we identify genes whose expression is coordinated by IRF4 and that in turn specify plasma cell identity and mitochondrial homeostasis.

The role of narrow-band imaging in parathyroid surgery: a preliminary evaluation in five parathyroid adenoma cases.

BACKGROUND: Narrow-band imaging uses selective haemoglobin light absorption to emphasise vascular visualisation and capillary networks. OBJECTIVE: This study aimed to evaluate the application of this technique to parathyroid surgery. METHOD: This preliminary evaluation was carried out on five consecutive patients with single parathyroid adenoma being considered for minimally invasive video-assisted parathyroidectomy. The adenomas were checked for narrow-band imaging vascular patterns. Minimally invasive video-assisted parathyroidectomy was then carried out in accordance with our standard protocol. RESULTS: In four out of the five cases, narrow-band imaging integrated the white endoscopic light and direct vision, but in one case narrow-band imaging allowed distinction between the hidden neoplastic tissue and the surrounding structures thanks to the different vascular patterns. CONCLUSION: Narrow-band imaging was helpful in properly identifying adenoma. It is suggested that this technique be considered as a means for surgeons to improve their confidence in selected surgical treatments and to improve treatment quality.

Five-year risk of CIN3 after short-term HPV-DNA negativity in cytology-negative women: a population-based cohort study.

OBJECTIVE: To assess the 5-year risk of high-grade lesions in women with a transient high-risk HPV infection. DESIGN: Population-based cohort study. SETTING: HPV primary testing within population-based organised cervical cancer screening programmes. POPULATION: Italian women enrolled in seven pilot projects and attending the second round. METHODS: On the basis of the cytology triage performed on HPV-positive women, immediate colposcopy or HPV repeat at 12 months was recommended. Data were collected at the subsequent round 3-4 years after HPV infection clearance. MAIN OUTCOME MEASURES: Rates of HPV infection, CIN2+ and CIN3+ detection at subsequent round after HPV clearance, and relative risks (RR) in comparison with HPV-negative women (with 95% confidence interval). RESULTS: Data on 1230 women (1027 aged 25-64 years and 203 aged 35-64 years) have been analysed. Overall compliance with repeat HPV testing was 84%. In comparison with HPV-negative women, those with a transient HPV infection had higher proportions of HPV positivity (15% versus 3.7%) and of CIN2+ lesions (0.87% versus 0.23%) in round two; most of these (7/10) were CIN2; no cancers were detected, and CIN3 occurred in 3/1230 (0.24%). CONCLUSIONS: HPV-based protocols for cervical cancer screening allow long intervals for HPV-negative women; it is important to monitor the clinical outcome in the women with transient high-risk HPV infection. CIN3 detection is similar to that observed in routine European cytology-based screening programmes (CIN3+: 2.7‰); 5-year intervals may provide reasonable protection but longer intervals are not recommended. TWEETABLE ABSTRACT: A screening interval of 5 years (but no longer) appears safe in women with transient HPV detection.

Genotyping-by-sequencing highlights patterns of genetic structure and domestication in artichoke and cardoon.

Exploiting the biodiversity of crops and their wild relatives is fundamental for maintaining and increasing food security. The species Cynara cardunculus includes three taxa: the globe artichoke, one of the most important Mediterranean vegetables, the leafy cardoon, and the wild cardoon. In this study, genotyping by sequencing (GBS) was successfully applied to reveal thousands of polymorphisms in a C. cardunculus germplasm collection, including 65 globe artichoke, 9 leafy cardoon, and 21 wild cardoon samples. The collection showed a strong population structure at K = 2, separating the globe artichoke from the leafy and wild cardoon. At higher K values, further substructures were observed, in which the wild cardoon was separated from the leafy cardoon, and the latter included the Spanish wild cardoons, while the wild sample from Portugal was admixed. Moreover, subpopulations within the globe artichoke set were highlighted. Structure analysis restricted to the globe artichoke dataset pointed out genetic differentiation between the ˝Catanesi˝ typology and all the other samples (K = 2). At higher values of K, the separation of the ˝Catanesi˝ group still held true, and green headed landraces from Apulia region, Italy (˝Green Apulian˝) formed a distinct subpopulation. ˝Romaneschi˝ artichoke types fell in a variable group with admixed samples, indicating that they should not be considered as a genetically uniform typology. The results of principal component analysis and Neighbor-Joining hierarchical clustering were consistent with structure results, and in addition provided a measure of genetic relationships among individual genotypes. Both analyses attributed the wild material from Spain and Portugal to the cultivated cardoon group, supporting the idea that this might be indeed a feral form of the leafy cardoon. Different reproductive habit and possibly selective pressure led to a slower LD decay in artichoke compared to cardoon. Genotyping by sequencing has proven a reliable methodology to obtain valuable SNPs and assess population genetics in C. cardunculus.

IMiDs prime myeloma cells for daratumumab-mediated cytotoxicity through loss of Ikaros and Aiolos.

Recent studies have demonstrated that the immunomodulatory drugs (IMiDs) lead to the degradation of the transcription factors Ikaros and Aiolos. However, why their loss subsequently leads to multiple myeloma (MM) cell death remains unclear. Using CRISPR-Cas9 genome editing, we have deleted IKZF1/Ikaros and IKZF3/Aiolos in human MM cell lines to gain further insight into their downstream gene regulatory networks. Inactivation of either factor alone recapitulates the cell intrinsic action of the IMiDs, resulting in cell cycle arrest and induction of apoptosis. Furthermore, evaluation of the transcriptional changes resulting from their loss demonstrates striking overlap with lenalidomide treatment. This was not dependent on reduction of the IRF4-MYC "axis," as neither protein was consistently downregulated, despite cell death occurring, and overexpression of either factor failed to rescue for Ikaros loss. Importantly, Ikaros and Aiolos repress the expression of interferon-stimulated genes (ISGs), including CD38, and their loss led to the activation of an interferon-like response, contributing to MM cell death. Ikaros/Aiolos repressed CD38 expression through interaction with the nucleosome remodeling and deacetylase complex in MM. IMiD-induced loss of Ikaros or treatment with interferon resulted in an upregulation of CD38 surface expression on MM cells, priming for daratumumab-induced NK cell-mediated antibody-dependent cellular cytotoxicity. These results give further insight into the mechanism of action of the IMiDs and provide mechanistic rationale for combination with anti-CD38 monoclonal antibodies.

Mining the Plasma Cell Transcriptome for Novel Cell Surface Proteins.

Antibody Secreting Cells (ASCs) are a fundamental component of humoral immunity, however, deregulated or excessive antibody production contributes to the pathology of autoimmune diseases, while transformation of ASCs results in the malignancy Multiple Myeloma (MM). Despite substantial recent improvements in treating these conditions, there is as yet no widely used ASC-specific therapeutic approach, highlighting a critical need to identify novel methods of targeting normal and malignant ASCs. Surface molecules specifically expressed by the target cell population represent ideal candidates for a monoclonal antibody-based therapy. By interrogating the ASC gene signature that we previously defined we identified three surface proteins, Plpp5, Clptm1l and Itm2c, which represent potential targets for novel MM treatments. Plpp5, Clptm1l and Itm2c are highly and selectively expressed by mouse and human ASCs as well as MM cells. To investigate the function of these proteins within the humoral immune system we have generated three novel mouse strains, each carrying a loss-of-function mutation in either Plpp5, Clptm1l or Itm2c. Through analysis of these novel strains, we have shown that Plpp5, Clptm1l and Itm2c are dispensable for the development, maturation and differentiation of B-lymphocytes, and for the production of antibodies by ASCs. As adult mice lacking either protein showed no apparent disease phenotypes, it is likely that targeting these molecules on ASCs will have minimal on-target adverse effects.

Transcriptomic response of durum wheat to nitrogen starvation.

Nitrogen (N) is a key macronutrient representing a limiting factor for plant growth and development and affects productivity in wheat. In this study, durum wheat response to N chronic starvation during grain filling was investigated through a transcriptomic approach in roots, leaves/stems, flag leaf and spikes of cv. Svevo. Nitrogen stress negatively influenced plant height, tillering, flag leaf area, spike and seed traits, and total N content. RNA-seq data revealed 4,626 differentially expressed genes (DEGs). Most transcriptomic changes were observed in roots, with 3,270 DEGs, while 963 were found in leaves/stems, 470 in flag leaf, and 355 in spike tissues. A total of 799 gene ontology (GO) terms were identified, 180 and 619 among the upregulated and downregulated genes, respectively. Among the most addressed GO categories, N compound metabolism, carbon metabolism, and photosynthesis were mostly represented. Interesting DEGs, such as N transporters, genes involved in N assimilation, along with transcription factors, protein kinases and other genes related to stress were highlighted. These results provide valuable information about the transcriptomic response to chronic N stress in durum wheat, which could be useful for future improvement of N use efficiency.