Short peptides from leucyl-tRNA synthetase rescue disease-causing mitochondrial tRNA point mutations.

Mutations in mitochondrial (mt) genes coding for mt-tRNAs are responsible for a range of syndromes, for which no effective treatment is available. We recently showed that the carboxy-terminal domain (Cterm) of human mt-leucyl tRNA synthetase rescues the pathologic phenotype associated either with the m.3243A>G mutation in mt-tRNA(Leu(UUR)) or with mutations in the mt-tRNA(Ile), both of which are aminoacylated by Class I mt-aminoacyl-tRNA synthetases (mt-aaRSs). Here we show, by using the human transmitochondrial cybrid model, that the Cterm is also able to improve the phenotype caused by the m.8344A>G mutation in mt-tRNA(Lys), aminoacylated by a Class II aaRS. Importantly, we demonstrate that the same rescuing ability is retained by two Cterm-derived short peptides, ß30_31 and ß32_33, which are effective towards both the m.8344A>G and the m.3243A>G mutations. Furthermore, we provide in vitro evidence that these peptides bind with high affinity wild-type and mutant human mt-tRNA(Leu(UUR)) and mt-tRNA(Lys), and stabilize mutant mt-tRNA(Leu(UUR)). In conclusion, we demonstrate that small Cterm-derived peptides can be effective tools to rescue cellular defects caused by mutations in a wide range of mt-tRNAs.

Targeting estrogen receptor ß as preventive therapeutic strategy for Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is a maternally inherited blinding disease characterized by degeneration of retinal ganglion cells (RGCs) and consequent optic nerve atrophy. Peculiar features of LHON are incomplete penetrance and gender bias, with a marked male prevalence. Based on the different hormonal metabolism between genders, we proposed that estrogens play a protective role in females and showed that these hormones ameliorate mitochondrial dysfunction in LHON through the estrogen receptors (ERs). We also showed that ERß localize to the mitochondria of RGCs. Thus, targeting ERß may become a therapeutic strategy for LHON specifically aimed at avoiding or delaying the onset of disease in mutation carriers. Here, we tested the effects of ERß targeting on LHON mitochondrial defective metabolism by treating LHON cybrid cells carrying the m.11778G>A mutation with a combination of natural estrogen-like compounds that bind ERß with high selectivity. We demonstrated that these molecules improve cell viability by reducing apoptosis, inducing mitochondrial biogenesis and strongly reducing the levels of reactive oxygen species in LHON cells. These effects were abolished in cells with ERß knockdown by silencing receptor expression or by using specific receptor antagonists. Our observations support the hypothesis that estrogen-like molecules may be useful in LHON prophylactic therapy. This is particularly important for lifelong disease prevention in unaffected LHON mutation carriers. Current strategies attempting to combat degeneration of RGCs during the acute phase of LHON have not been very effective. Implementing a different and preemptive approach with a low risk profile may be very helpful.

Tracheal and Crico-Tracheal Resection and Anastomosis for Malignancies Involving the Thyroid Gland and the Airway.

OBJECTIVES: To evaluate outcomes in different malignancies involving the thyroid and infiltrating the airway submitted to tracheal (TRA) or crico-tracheal resection and anastomosis (CTRA). METHODS: Retrospective charts review of 27 patients affected by thyroid malignancies involving the airway treated by TRA/CTRA in a single academic institution. Kaplan-Meier curves were used to evaluate the overall (OS) and disease-specific (DSS) survivals and local (LC) and loco-regional control (LRC). Impact on survival of age, comorbidities, previous radiotherapy, types of TRA/CTRA, Shin's stage (II, III, IV), grading (well vs poorly differentiated), and length of airway resected was calculated by the log-rank test. RESULTS: Overall survival and DSS at 3 and 5 years were 82.3% and 71.6%, respectively. Local control and LRC in the entire group were 82.3% at 3 and 5 years. Crico-tracheal resection and anastomosis involving the cricoid arch and plate (type C) and tumor differentiation significantly affected OS and DSS (both P < .001). Type C CTRA and tumor differentiation significantly impacted on LC (P = .002 and P = .009, respectively). CONCLUSIONS: Grading and extension of CTRA to the cricoid plate are the most important factors for oncologic outcomes in thyroid malignancies infiltrating the airway. Except for poorly differentiated tumors, TRA/CTRA allows adequate LC even in advanced stage lesions involving the crico-tracheal junction.

The diagnostic value of narrow band imaging in different oral and oropharyngeal subsites.

The diagnostic value of narrow band imaging (NBI) in the "optical biopsy" of leukoplakias (LP) and erythroplakias (EP) in different oral cavity (OC) and oropharyngeal (OP) subsites is still to be defined. We evaluated 128 unbiopsied and untreated OC/OP LP and EP by conventional oral examination (COE), white light (WL) endoscopy, and NBI and categorized them as "suspicious" or "innocuous". All lesions were treated by excisional biopsy. True positives were those considered as "suspicious" and with histopathology ranging from mild dysplasia to invasive carcinoma. Epithelia were classified as follows: type 1, keratinized thick stratified (gingiva, hard palate, dorsal tongue); type 2a, non-keratinized thin stratified (floor of mouth, vestibule, ventral tongue, soft palate, palatine tonsils, base of tongue); type 2b, non-keratinized, very thick stratified (retromolar trigon, lateral tongue, labial and buccal mucosa). Histopathology revealed 32 % benign lesions, 13 % mild to moderate dysplasias, 15 % severe dysplasias/carcinoma in situ, 16 % microinvasive, and 23 % invasive carcinomas. The false positive rates were 32 % at COE, 27 % at WL, and 15 % at NBI. The false negative rates were 49, 22, and 11 %, respectively. Diagnositic performance was higher for NBI compared to COE (p < 0.001) and to WL (p = 0.004). Comparison of the diagnostic value of NBI among different OC/OP subsites did not show statistically significant difference. NBI as an "optical biopsy" tool significantly reduces the rates of false positives and false negatives in diagnosis of OC/OP cancer compared with COE and WL. No statistically significant difference was noted in its diagnostic value among different OC/OP subsites.

Mouse Sertoli cells sustain de novo generation of regulatory T cells by triggering the notch pathway through soluble JAGGED1.

FOXP3(+) regulatory T cells (Tregs) are central to the maintenance of immunological homeostasis and tolerance. It has long been known that Sertoli cells are endowed with immune suppressive properties; however, the underlying mechanisms as well as the effective nature and role of soluble factors secreted by Sertoli cells have not been fully elucidated as yet. We hypothesized that conditioned medium from primary mouse Sertoli cells (SCCM) may be able and sufficient to induce Tregs. By culturing CD4(+)CD25(-)EGFP(-) T splenocytes purified from FOXP3-EGFP knock-in mice in SCCM, here we show, by flow cytometry and suppression assay, the conversion of peripheral CD4(+)FOXP3(-) T cells into functional CD4(+)FOXP3(+) Tregs. We also demonstrate that the Notch/Jagged1 axis is involved in regulating the de novo generation of Tregs although this process is transforming growth factor-beta1 (TGF-B) dependent. In particular, we identified by Western blot analysis a soluble form of JAGGED1 (JAG1) in SCCM that significantly influences the induction of Tregs, as demonstrated by performing the conversion assay in presence of a JAG1-specific neutralizing antibody. In addition, we show that SCCM modulates the Notch pathway in converted Tregs by triggering the recruitment of the Notch-specific transcription factor CSL/RBP-Jk to the Foxp3 promoter and by inducing the Notch target gene Hey1, as shown by chromatin immunoprecipitation assay and by real time-RT-PCR experiments, respectively. Overall, these results contribute to a better understanding of the molecular mechanisms involved in Sertoli cell-mediated immune tolerance and provide a novel approach to generate ex vivo functional Tregs for therapeutic purpose.

Complications after tracheal and cricotracheal resection and anastomosis for inflammatory and neoplastic stenoses.

OBJECTIVE: This study aimed to evaluate complications and success rates of tracheal resection and anastomosis (TRA) and cricotracheal resection and anastomosis (CTRA) in patients treated in 2 academic institutions. METHODS: Retrospective charts review of 137 patients submitted to TRA/CTRA. Fifty (36.5%) had neoplastic (group A) and 87 (63.5%) benign (group B) stenoses. Using univariate analysis, age, medical comorbidities, previous radiotherapy, type of TRA/CTRA, association with neck dissection and thyroidectomy, length of resected airway, and preoperative tracheotomy were evaluated to identify factors predictive of complications and outcomes. RESULTS: The mean length of resected airway was 2.7 and 3 cm in groups A and B, respectively. Overall decannulation and complication rates for group A were 96% and 36%, and 99% and 46% for group B, respectively. Length of airway resected and presence of preoperative tracheotomy had a statistically significant effect on major surgical complications. Age older than 70 and cardiovascular and pulmonary comorbidities were significantly associated with the incidence of major medical complications. No statistically significant difference was found considering the complication rates of group A versus group B. CONCLUSION: Even though the overall success rate of TRA/CTRA is high, it should always be regarded as a major surgical procedure with a non-negligible incidence of complications.

Scleroderma Mesenchymal Stem Cells display a different phenotype from healthy controls; implications for regenerative medicine.

INTRODUCTION: Vascular involvement is a key feature of Systemic sclerosis (SSc). Although the pericytes/endothelial cells (ECs) cross-talk regulates vessels formation, no evidences about the pericytes contribution to ineffective angiogenesis in SSc are available. Recent findings showed similarities between pericytes and Bone Marrow Mesenchymal Stem Cells (BM-MSCs). Due to difficulties in pericytes isolation, this work explores the possibility to use BM-MSCs as pericytes surrogate, clarifying their role in supporting neo-angiogenesis during SSc. METHODS: To demonstrate their potential to normally differentiate into pericytes, both SSc and healthy controls (HC) BM-MSCs were treated with TGF-ß and PDGF-BB. The expression of pericytes specific markers (α-SMA, NG2, RGS5 and desmin) was assessed by qPCR, western blot, and immunofluorescence; chemioinvasion and capillary morphogenesis were also performed. Cell-sorting of BM-MSCs co-cultured with HC-ECs was used to identify a possible change in contractile proteins genes expression. RESULTS: We showed that BM-MSCs isolated from SSc patients displayed an up-regulation of α-SMA and SM22α genes and a reduced proliferative activity. Moreover during SSc, both TGF-ß and PDGF-BB can specifically modulate BM-MSCs toward pericytes. TGF-ß was found interfering with the PDGF-BB effects. Using BM-MSCs/MVECs co-culture system we observed that SSc BM-MSCs improve ECs tube formation in stressed condition, and BM-MSCs, sorted after co-culture, showed a reduced α-SMA and SM22α gene expression. CONCLUSIONS: BM-MSCs from SSc patients behave as pericytes. They display a more mature and myofibroblast-like phenotype, probably related to microenvironmental cues operating during the disease. After their co-culture with HC-MVECs, SSc BM-MSCs underwent to a phenotypic modulation which re-programs these cells toward a pro-angiogenic behaviour.

Notch3 and canonical NF-kappaB signaling pathways cooperatively regulate Foxp3 transcription.

Notch3 overexpression has been previously shown to positively regulate the generation and function of naturally occurring regulatory T cells and the expression of Foxp3, in cooperation with the pTα/pre-TCR pathway. In this study, we show that Notch3 triggers the trans activation of Foxp3 promoter depending on the T cell developmental stage. Moreover, we discovered a novel CSL/NF-κB overlapping binding site within the Foxp3 promoter, and we demonstrate that the activation of NF-κB, mainly represented by p65-dependent canonical pathway, plays a positive role in Notch3-dependent regulation of Foxp3 transcription. Accordingly, the deletion of protein kinase C, which mediates canonical NF-κB activation, markedly reduces regulatory T cell number and per cell Foxp3 expression in transgenic mice with a constitutive activation of Notch3 signaling. Collectively, our data indicate that the cooperation among Notch3, protein kinase C, and p65/NF-κB subunit modulates Foxp3 expression, adding new insights in the understanding of the molecular mechanisms involved in regulatory T cell homeostasis and function.

Function preservation using transoral laser surgery for T2-T3 glottic cancer: oncologic, vocal, and swallowing outcomes.

Aim of this study was to retrospectively analyze oncologic and functional results of a cohort of T2 and selected T3 glottic tumors treated by transoral laser surgery (TLS). Eighty-nine patients affected by T2 and T3 glottic tumors were treated by TLS from 2005 to 2010 at an academic institution by Type V cordectomies using an "en bloc" or, more frequently, a "piece-meal" technique depending on a number of variables. Kaplan-Meier curves were used to evaluate 5-year overall, disease-specific survivals, local control with laser, and organ preservation rates. Univariate analysis of the impact of different variables was performed. At least 1 year after surgery, we examined: subjective voice evaluation by voice handicap index (VHI), perceptive analysis by GRBAS scale, objective measurements with multidimensional voice program (MDVP), swallowing assessment with the M.D. Anderson Dysphagia Inventory (MDADI), videonasal endoscopic evaluation of swallowing (VEES), and videofluoroscopy (VFS). Fifty-nine patients were pT2 and 30 pT3. The 5-year overall, disease-specific survivals, local control with laser, and organ preservation rates were 92.4, 98.7, 68.5, and 82.1 %, respectively. VHI mean score was 20 (mild dysphonia). Mild and moderate dysphonias were detected by GRBAS in 82 and 18% of patients, respectively. Mean values of Jitter, Shimmer, and noise-to-harmonic ratio by MDVP resulted 7.87%, 24.8%, and 0.37, respectively. Mean value of MDADI was 95.75. Only 2% of patients at VEES and 4% at VFS showed tracheal aspiration. Our results highlight that T2 and selected T3 glottic tumors treated by TLS have favorable oncologic and functional outcomes.

The chemokine Bv8/prokineticin 2 is up-regulated in inflammatory granulocytes and modulates inflammatory pain.

Neutrophil migration into injured tissues is invariably accompanied by pain. Bv8/prokineticin 2 (PK2), a chemokine characterized by a unique structural motif comprising five disulfide bonds, is highly expressed in inflamed tissues associated to infiltrating cells. Here, we demonstrate the fundamental role of granulocyte-derived PK2 (GrPK2) in initiating inflammatory pain and driving peripheral sensitization. In animal models of complete Freund's adjuvant-induced paw inflammation the development and duration of pain temporally correlated with the expression levels of PK2 in the inflamed sites. Such an increase in PK2 mRNA depends mainly on a marked up-regulation of PK2 gene transcription in granulocytes. A substantially lower up-regulation was also detected in macrophages. From a pool of peritoneal granulocytes, elicited in rats by oyster glycogen, we purified the GrPK2 protein, which displayed high affinity for the prokineticin receptors (PKRs) and, when injected into the rat paw, induced hypersensitivity to noxious stimuli as the amphibian prokineticin Bv8 did. Mice lacking PKR1 or PKR2 developed significantly less inflammation-induced hyperalgesia in comparison with WT mice, confirming the involvement of both PKRs in inflammatory pain. The inflammation-induced up-regulation of PK2 was significantly less in pkr1 null mice than in WT and pkr2 null mice, demonstrating a role of PKR1 in setting PK2 levels during inflammation. Pretreatment with a nonpeptide PKR antagonist, which preferentially binds PKR1, dose-dependently reduced and eventually abolished both prokineticin-induced hypernociception and inflammatory hyperalgesia. Inhibiting PK2 formation or antagonizing PKRs may represent another therapeutic approach for controlling inflammatory pain.

Notch-Signaling Deregulation Induces Myeloid-Derived Suppressor Cells in T-Cell Acute Lymphoblastic Leukemia.

Notch receptors deeply influence T-cell development and differentiation, and their dysregulation represents a frequent causative event in "T-cell acute lymphoblastic leukemia" (T-ALL). "Myeloid-derived suppressor cells" (MDSCs) inhibit host immune responses in the tumor environment, favoring cancer progression, as reported in solid and hematologic tumors, with the notable exception of T-ALL. Here, we prove that Notch-signaling deregulation in immature T cells promotes CD11b+Gr-1+ MDSCs in the Notch3-transgenic murine model of T-ALL. Indeed, aberrant T cells from these mice can induce MDSCs in vitro, as well as in immunodeficient hosts. Conversely, anti-Gr1-mediated depletion of MDSCs in T-ALL-bearing mice reduces proliferation and expansion of malignant T cells. Interestingly, the coculture with Notch-dependent T-ALL cell lines, sustains the induction of human CD14+HLA-DRlow/neg MDSCs from healthy-donor PBMCs that are impaired upon exposure to gamma-secretase inhibitors. Notch-independent T-ALL cells do not induce MDSCs, suggesting that Notch-signaling activation is crucial for this process. Finally, in both murine and human models, IL-6 mediates MDSC induction, which is significantly reversed by treatment with neutralizing antibodies. Overall, our results unveil a novel role of Notch-deregulated T cells in modifying the T-ALL environment and represent a strong premise for the clinical assessment of MDSCs in T-ALL patients.

Neonatal Respiratory Distress and Airway Emergency: Report of Two Cases.

We discuss two cases of congenital airway malformations seen in our neonatal intensive care unit (NICU). The aim is to report extremely rare events characterized by immediate respiratory distress after delivery and the impossibility to ventilate and intubate the airway. The first case is a male twin born at 34 weeks by emergency caesarean section. Immediately after delivery, the newborn was cyanotic and showed severe respiratory distress. Bag-valve-mask ventilation did not relieve the respiratory distress but allowed for temporary oxygenation during subsequent unsuccessful oral-tracheal intubation (OTI) attempts. Flexible laryngoscopy revealed complete subglottic obstruction. Postmortem analysis revealed a poly-malformative syndrome, unilateral multicystic renal dysplasia with a complete subglottic diaphragm, and a tracheo-esophageal fistula (TEF). The second case is a male patient that was vaginally born at 35 weeks. Antenatally, an ultrasound (US) arose suspicion for a VACTERL association (vertebral defects, anal atresia, TEF with esophageal atresia and radial or renal dysplasia, plus cardiovascular and limb defects) and a TEF, and thus, fetal magnetic resonance (MRI) was scheduled. Spontaneous labor started shortly thereafter, before imaging could be performed. Respiratory distress, cyanosis, and absence of an audible cry was observed immediately at delivery. Attempts at OTI were unsuccessful, whereas bag-valve-mask ventilation and esophageal intubation allowed for sufficient oxygenation. An emergency tracheostomy was attempted, although no trachea could be found on cervical exploration. Postmortem analysis revealed tracheal agenesis (TA), renal dysplasia, anal atresia, and a single umbilical artery. Clinicians need to be aware of congenital airway malformations and subsequent difficulties upon endotracheal intubation and must plan for multidisciplinary management of the airway at delivery, including emergency esophageal intubation and tracheostomy.

Notch3 and pTalpha/pre-TCR sustain the in vivo function of naturally occurring regulatory T cells.

Dysregulated generation and/or function of naturally occurring 'CD4(+)CD25(+) regulatory T cells' (T(reg)s) play key role in the development of autoimmune diseases, including type 1 diabetes. Recent findings suggest that Notch3 signaling activation promotes thymic generation and peripheral expansion and in vivo function of naturally occurring T(reg)s, thus preventing autoimmune diabetes progression in mouse models. However, the mechanisms underlying these effects have remained elusive, thus far. Here, we show that the expression of pTalpha gene is up-regulated in naturally occurring T(reg)s, at both mRNA and protein levels. Moreover, by using double mutant mice, with T cell-targeted constitutive activation of Notch3 in a pTalpha(-/-) background, we demonstrate that pTalpha deletion significantly counteracts the Notch3-dependent expansion, the increased FoxP3 expression and the enhanced in vitro activity of naturally occurring T(reg)s. Notably, the absence of pTalpha also impairs the Notch3-dependent protection against experimentally induced autoimmune diabetes. Finally, by adoptive cell transfer experiments, we demonstrated that this failure is directly related to the impaired in vivo function of naturally occurring T(reg)s bearing pTalpha deletion. Collectively, our data suggest that pTalpha expression is required for the in vivo function of naturally occurring T(reg)s and that the activation of Notch3 signaling may positively regulate the function of this population, through the pTalpha/pre-T cell receptor pathway.

NF-κB1 Regulates Immune Environment and Outcome of Notch-Dependent T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive pediatric malignancy that arises from the transformation of immature T-cell progenitors and has no definitive cure. Notch signaling governs many steps of T cell development and its dysregulation represents the most common causative event in the pathogenesis of T-ALL. The activation of canonical NF-κB pathway has been described as a critical downstream mediator of Notch oncogenic functions, through the sustaining of tumor cell survival and growth. The potential role of Notch/NF-κB partnership is also emerging in the generation and function of regulatory T cells (Tregs) in the context of cancer. However, little is known about the effects of combined mutations of Notch and NF-κB in regulating immune-environment and progression of T-ALL. To shed light on the topics above we generated double-mutant mice, harboring conventional knock-out mutation of NF-κB1/p50 on the genetic background of a transgenic model of Notch-dependent T-ALL. The immunophenotyping of double-mutant mice demonstrates that NF-κB1 deletion inhibits the progression of T-ALL and strongly modifies immune-environment of the disease. Double-mutant mice display indeed a dramatic reduction of pre-leukemic CD4+CD8+ (DP) T cells and regulatory T cells (Tregs) and, concurrently, the rising of an aggressive myeloproliferative trait with a massive expansion of CD11b+Gr-1+ cells in the periphery, and an accumulation of the granulocyte/monocyte progenitors in the bone-marrow. Interestingly, double-mutant T cells are able to improve the growth of CD11b+Gr-1+ cells in vitro, and, more importantly, the in vivo depletion of T cells in double-mutant mice significantly reduces the expansion of myeloid compartment. Our results strongly suggest that the myeloproliferative trait observed in double-mutant mice may depend on non-cell-autonomous mechanism/s driven by T cells. Moreover, we demonstrate that the reduction of CD4+CD8+ (DP) T cells and Tregs in double-mutant mice relies on a significant enhancement of their apoptotic rate. In conclusion, double-mutant mice may represent a useful model to deepen the knowledge of the consequences on T-ALL immune-environment of modulating Notch/NF-κB relationships in tumor cells. More importantly, information derived from these studies may help in the refinement of multitarget therapies for the disease.

Sialoendoscopy for treatment of juvenile recurrent parotitis: The Brescia experience.

OBJECTIVE: To evaluate the role of sialoendoscopy associated with steroid irrigation for juvenile recurrent parotitis (JRP) at a tertiary referral hospital. METHODS: Clinical records of patients affected by JRP and treated with operative sialoendoscopy between June 2011 and April 2017 were retrospectively reviewed. Data on demographics, number of acute episodes per year before and after surgery, characteristics of the surgical procedure, hospitalization time, and rate of complications were collected. The outcome of the procedure was measured by comparing the number of episodes of parotid swelling before and after salivary endoscopic treatment. RESULTS: Twenty-three patients for a total of 34 operative sialoendoscopies were included in the study. Before the surgical endoscopic procedure, the mean number of parotid swelling was 10 episodes per year. At sialoendoscopy, typical endoscopic findings such as mucous plugs, stenosis of the duct, intraductal debris, and pale ductal appearance were evident. All patients were discharged on the first postoperative day. A significant decrease in the number of swelling episodes per year was observed compared to the preoperative rate (p = .0004). Complete resolution of the disorder was obtained in 35% of patients. CONCLUSIONS: Operative sialoendoscopy with steroid irrigation can be considered a valid therapeutic treatment for JRP. The technique is conservative, effective, safe, and, potentially repeatable. Short hospitalization time, rapid recovery, absence of peri-operative complications, and a high rate of good outcomes are the main advantages of this treatment.

Notch and NF-κB: Coach and Players of Regulatory T-Cell Response in Cancer.

The Notch signaling pathway plays multiple roles in driving T-cell fate decisions, proliferation, and aberrant growth. NF-κB is a cell-context key player interconnected with Notch signaling either in physiological or in pathological conditions. This review focuses on how the multilayered crosstalk between different Notches and NF-κB subunits may converge on Foxp3 gene regulation and orchestrate CD4+ regulatory T (Treg) cell function, particularly in a tumor microenvironment. Notably, Treg cells may play a pivotal role in the inhibition of antitumor immune responses, possibly promoting tumor growth. A future challenge is represented by further dissection of both Notch and NF-κB pathways and consequences of their intersection in tumor-associated Treg biology. This may shed light on the molecular mechanisms regulating Treg cell expansion and migration to peripheral lymphoid organs thought to facilitate tumor development and still to be explored. In so doing, new opportunities for combined and/or more selective therapeutic approaches to improve anticancer immunity may be found.

Laryngeal exposure and margin status in glottic cancer treated by transoral laser microsurgery.

OBJECTIVE: Laryngeal exposure is one of the most limiting factors in transoral laser microsurgery (TLM) for glottic cancer. We evaluated the correlation between the degree of laryngeal exposure, as assessed by an easy previously described scoring tool (Laryngoscore), and histopathologic surgical margin status after TLM. STUDY DESIGN: Prospective evaluation of 147 patients affected by Tis-T2 glottic cancer treated by TLM with curative intent between January 2012 and April 2016. METHODS: All patients were preoperatively assessed and classified as having good (group A including Laryngoscore class 0-I) or suboptimal laryngeal exposure (group B including class II-III). Margins were classified as negative (more than 1 mm margin between healthy tissue and tumor) or positive (one/multiple superficial or deep margins involved by invasive or in situ carcinoma). Patients with multiple superficial or deep margin positivity were scheduled for TLM re-excision, open partial laryngectomy, or postoperative radiotherapy. RESULTS: Twenty-one type I, 54 type II, 19 type III, 7 type IV, 41 type V, and 5 type VI cordectomies (according to the European Laryngological Society classification) were performed with an en-bloc or multi-bloc technique according to the size, site, and exposure of the lesion. Group A included 109 (74%) and group B included 38 (26%) patients. Positive surgical margins were overall observed in 39 (26.5%) cases: 21 (19.2%) in group A versus 18 (47.4%) in group B (P = 0.001). CONCLUSION: Laryngeal exposure is one of the most important factors influencing TLM resection of glottic cancer within safe surgical margins. The importance of its adequate preoperative assessment cannot be overemphasized. LEVEL OF EVIDENCE: 2b. Laryngoscope, 128:1146-1151, 2018.

A non-conserved amino acid variant regulates differential signalling between human and mouse CD28.

CD28 superagonistic antibodies (CD28SAb) can preferentially activate and expand immunosuppressive regulatory T cells (Treg) in mice. However, pre-clinical trials assessing CD28SAbs for the therapy of autoimmune diseases reveal severe systemic inflammatory response syndrome in humans, thereby implying the existence of distinct signalling abilities between human and mouse CD28. Here, we show that a single amino acid variant within the C-terminal proline-rich motif of human and mouse CD28 (P212 in human vs. A210 in mouse) regulates CD28-induced NF-κB activation and pro-inflammatory cytokine gene expression. Moreover, this Y209APP212 sequence in humans is crucial for the association of CD28 with the Nck adaptor protein for actin cytoskeleton reorganisation events necessary for CD28 autonomous signalling. This study thus unveils different outcomes between human and mouse CD28 signalling to underscore the importance of species difference when transferring results from preclinical models to the bedside.

Intrathymic Notch3 and CXCR4 combinatorial interplay facilitates T-cell leukemia propagation.

Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying "pre-leukemic" cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4+CD8+ thymocytes, possibly contributing to "pre-leukemic" cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4+CD8+ cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4+CD8+ cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4+CD8+ cells and such an expression is regulated by Notch3 through ß-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4+CD8+ cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4+CD8+ cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of "pre-leukemic" CD4+CD8+ cells, by a "thymus-autonomous" mechanism.

Stem-like and highly invasive prostate cancer cells expressing CD44v8-10 marker originate from CD44-negative cells.

In human prostate cancer (PCa), the neuroendocrine cells, expressing the prostate cancer stem cell (CSC) marker CD44, may be resistant to androgen ablation and promote tumor recurrence. During the study of heterogeneity of the highly aggressive neuroendocrine PCa cell lines PC3 and DU-145, we isolated and expanded in vitro a minor subpopulation of very small cells lacking CD44 (CD44neg). Unexpectedly, these sorted CD44neg cells rapidly and spontaneously converted to a stable CD44high phenotype specifically expressing the CD44v8-10 isoform which the sorted CD44high subpopulation failed to express. Surprisingly and potentially interesting, in these cells expression of CD44v8-10 was found to be induced in stem cell medium. CD44 variant isoforms are known to be more expressed in CSC and metastatic cells than CD44 standard isoform. In agreement, functional analysis of the two sorted and cultured subpopulations has shown that the CD44v8-10pos PC3 cells, resulting from the conversion of the CD44neg subpopulation, were more invasive in vitro and had a higher clonogenic potential than the sorted CD44high cells, in that they produced mainly holoclones, known to be enriched in stem-like cells. Of interest, the CD44v8-10 is more expressed in human PCa biopsies than in normal gland. The discovery of CD44v8-10pos cells with stem-like and invasive features, derived from a minoritarian CD44neg cell population in PCa, alerts on the high plasticity of stem-like markers and urges for prudency on the approaches to targeting the putative CSC.