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Competing Interests: Disclosure The authors declare no potential conflicts of interest.

Lymphadenectomy represents a fundamental step in the staging and treatment of non-small cell lung cancer (NSCLC). To date, the extension of lymphadenectomy in early-stage NSCLC is a debated topic due to its possible complications. The detection of sentinel lymph nodes (SLNs) is a strategy that can improve the selection of patients in which a more extended lymphadenectomy is necessary. This pilot study aimed to refine lymph nodal staging in early-stage NSCLC patients who underwent robotic lung resection through the application of innovative intraoperative sentinel lymph node (SLN) identification and the pathological evaluation using one-step nucleic acid amplification (OSNA). Clinical N0 NSCLC patients planning to undergo robotic lung resection were selected. The day before surgery, all patients underwent radionuclide computed tomography (CT)-guided marking of the primary lung lesion and subsequently Single Photon Emission Computed Tomography (SPECT) to identify tracer migration and, consequently, the area with higher radioactivity. On the day of surgery, the lymph nodal radioactivity was detected intraoperatively using a gamma camera. SLN was defined as the lymph node with the highest numerical value of radioactivity. The OSNA amplification, detecting the mRNA of CK19, was used for the detection of nodal metastases in the lymph nodes, including SLN. From March to July 2021, a total of 8 patients (3 female; 5 male), with a mean age of 66 years (range 48-77), were enrolled in the study. No complications relating to the CT-guided marking or preoperative SPECT were found. An average of 5.3 lymph nodal stations were examined (range 2-8). N2 positivity was found in 3 out of 8 patients (37.5%). Consequently, pathological examination of lymph nodes with OSNA resulted in three upstages from the clinical IB stage to pathological IIIA stage. Moreover, in 1 patient (18%) with nodal upstaging, a positive node was intraoperatively identified as SLN. Comparing this protocol to the usual practice, no difference was found in terms of the operating time, conversion rate, and complication rate. Our preliminary experience suggests that sentinel lymph node detection, in association with the accurate pathological staging of cN0 patients achieved using OSNA, is safe and effective in the identification of metastasis, which is usually undetected by standard diagnostic methods.

Purpose: SARS-CoV-2 infection may be limited to the respiratory tract or may spread to multiple organs. Besides disease severity, factors associated with virus spread within the host are elusive. Here, we tried to identify features associated with SARS-CoV-2 spread to endocrine organs., Methods: In a retrospective autoptic cohort of 51 subjects who died because of COVID-19, we analyzed the severity and type of lung pathology, patients' features and the detection of virus in thyroid, testis, adrenal gland, pancreas, anterior pituitary, and the white adipose tissue (WAT)., Results: The SARS-CoV-2 genome was detected in endocrine organs of 30/51 cases. The anterior pituitary and WAT were most frequently positive for virus. While pathological features of lung were not associated with the presence of virus in endocrine organs, obesity (BMI > 30) was significantly associated to virus detection in pancreas (p = 0.01) and thyroid (p = 0.04). WAT infection was detected more frequently in males (p = 0.03)., Conclusion: In subject with obesity dying of COVID-19, the virus frequently spreads to endocrine organs. The findings emphasize the need for optimal treatment of patients with obesity at the very onset of COVID-19. Since post-COVID conditions remain a major issue worldwide, a rigorous follow-up of endocrine function-especially of thyroid and pancreas-is advocated in subjects with obesity., (© 2023. The Author(s).)

Background: Tumors develop within an organism operating in a specific social and physical environment. Cortisol and dehydroepiandrosterone (DHEA), two of the most abundant steroid hormones in humans, are involved in both emotional regulation and the tumor progression. Several studies reported preclinical findings that DHEA can have preventive and therapeutic efficacy in treating major age-associated diseases, including cancer, although the mechanisms of action are not yet defined. The main aim of current study was to investigate the relationship between psychological and physiological emotional regulation and cancer development., Method: This study assessed the quality of life of urogenital cancer male patients using several validated tools, including the Functional Assessment of Cancer Therapy-General and the Profile of Mood States. Saliva samples were collected to monitor peripheral activity of both cortisol and DHEA. It was hypothesized that patients with a better quality of life would have higher levels of the DHEA/cortisol ratios., Results: We found that the quality of life was positively related to DHEA, but not cortisol levels. Negative mood increases were related to lower levels of DHEA. Logistic regression of the predictors of metastases indicated three main independent factors involved: DHEA, age, and cortisol. In other words, the higher the DHEA levels in comparison to cortisol levels, controlling for age, the lower the probability of metastases., Conclusion: Our results appear to support the hypothesis that emotional dysregulation mediated by DHEA/cortisol activity is a key factor in the probability of metastasis in urogenital cancers., (© 2022. The Author(s).)

Pleural mesothelioma (PM) comprises three main subtypes: epithelioid, biphasic and sarcomatoid, which have different impacts on prognosis and treatment definition. However, PM subtyping can be complex given the inter- and intra-tumour morphological heterogeneity. We aim to use immunohistochemistry (IHC) to evaluate five markers (Mesothelin, Claudin-15, Complement Factor B, Plasminogen Activator Inhibitor 1 and p21-activated Kinase 4), whose encoding genes have been previously reported as deregulated among PM subtypes. Immunohistochemical expressions were determined in a case series of 73 PMs, and cut-offs for the epithelioid and non-epithelioid subtypes were selected. Further validation was performed on an independent cohort (30 PMs). For biphasic PM, the percentage of the epithelioid component was assessed, and IHC evaluation was also performed on the individual components separately. Mesothelin and Claudin-15 showed good sensitivity (79% and 84%) and specificity (84% and 73%) for the epithelioid subtype. CFB and PAK4 had inferior performance, with higher sensitivity (89% and 84%) but lower specificity (64% and 36%). In the biphasic group, all markers showed different expression when comparing epithelioid with sarcomatoid areas. Mesothelin, Claudin-15 and CFB can be useful in subtype discrimination. PAI1 and PAK4 can improve component distinction in biphasic PM.

Introduction: Non-small cell lung cancer (NSCLC) is the leading cause of cancer incidence and mortality worldwide. Neoadjuvant chemo-immunotherapy has led to clinical benefits in resectable NSCLC in comparison to chemo-therapy alone. Major pathological response (MPR) and pathological complete response (pCR) have been used as surrogates of neoadjuvant therapy response and clinical outcomes. However, the factors affecting the pathological response are still controversial. Therefore, in this study we retrospectively examined MPR and pCR in two different cohorts of NSCLC patients, 14 treated by chemotherapy and 12 by chemo-immunotherapy in the neoadjuvant setting., Methods: In resected tumor specimens, different histological characteristics were evaluated: necrosis, fibrosis, inflammation, presence of organizing pneumonia, granuloma, cholesterol cleft, and reactive epithelial alterations. In addition, we evaluated how MPR impacts on event-free survival (EFS) and overall survival (OS). In a small group of patients treated by chemo-immunotherapy, a gene expression analysis of the Hippo pathway was performed both in preoperative biopsies and matched post-surgical specimens., Results: We observed a better pathological response in the chemo-immunotherapy treated cohort: 6/12 patients (50.0%) achieved a MPR ≤10% and 1/12 (8.3%) achieved pCR both on primary tumor and on lymph nodes. On the contrary, no patient treated with chemotherapy alone achieved pCR or MPR ≤10%. A higher amount of stroma in the neoplastic bed was observed in patients treated with immuno-chemotherapy. Moreover, patients achieving better MPR (including pCR) had significantly improved overall survival (OS) and event-free survival (EFS). After neoadjuvant chemo-immunotherapy, residual tumors showed a remarkable upregulation of genes consistent with the activation of YAP/TAZ. Also, alternative checkpoint, such as CTLA-4, were enhanced., Discussion: Our findings showed that neoadjuvant chemo-immunotherapy treatment improves MPR and pCR thus resulting in better EFS and OS. Moreover, a combined treatment could induce different morphological and molecular changes in comparison to chemotherapy alone, thus giving new insights in the assessment of pathological response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Alì, Poma, Di Stefano, Zirafa, Lenzini, Martinelli, Romano, Chella, Baldini, Melfi and Fontanini.)

Paragangliomas and pheochromocytomas are rare neuroendocrine tumors, carrying a germ-line mutation in 40% patients. Sclerosis is a rare histological feature in these tumors. We investigated the possible correlations between histological findings, first sclerosis, immunoreactivity for vesicular catecholamine transporters (VMAT1/VMAT2) and patients' genotype in a consecutive series of 57 tumors (30 paragangliomas and 27 pheochromocytomas) from 55 patients. The M-GAPP grading system, sclerosis (0-3 scale) and VMAT1/VMAT2 (0-6 scale) immunoreactivity scores were assessed. Germ-line mutations of Succinate Dehydrogenase genes, RET proto-oncogene and Von Hippel Lindau tumor suppressor gene were searched. A germ-line mutation was found in 25/55 (45.5%) patients, mainly with paraganglioma (N = 14/30, 46,66%). Significant (score ≥ 2) tumor sclerosis was found in 9 (16.1%) tumors, i.e., 7 paragangliomas and 2 pheochromocytomas, most of them (8/9) from patients with a germ-line mutation. M-GAPP score was higher in the mutation status (in 76% of patients involving the SDHx genes, in 12% the RET gene and in the remaining 12% the VHL gene) and in tumors with sclerosis (p < 0.05). Spearman's rank correlation showed a strong correlation of germ-line mutations with M-GAPP (p < 0.0001) and sclerosis (p = 0.0027) scores; a significant correlation was also found between sclerosis and M-GAPP scores (p = 0.029). VMAT1 expression was higher in paragangliomas than in pheochromocytomas (p = 0.0006), the highest scores being more frequent in mutation-bearing patients' tumors (p < 0.01). VMAT2 was highly expressed in all but two negative tumors. Sclerosis and VMAT1 expression were higher in paragangliomas than in pheochromocytomas; tumor sclerosis, M-GAPP and VMAT1 scores were associated to germ-line mutations. Sclerosis might represent a histological marker of tumor susceptibility, prompting to genetic investigations in paragangliomas., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: The Italian Consensus for the Classification and Reporting of Thyroid Cytology (ICCRTC) includes six diagnostic categories (TIR 1/1C, TIR 2, TIR 3A, TIR 3B, TIR 4, and TIR 5), each indicating a different risk of malignancy. The objective of this monocentric retrospective study was to evaluate the distribution of the ICCRTC classes at the authors' institution and assess their cytohistologic correlations., Methods: The authors retrospectively collected 37,371 consecutive cytologic reports of thyroid nodules and described the clinical-pathologic features of the different cytologic categories. The cytologic diagnoses also were compared with histologic outcomes in a subset of patients., Results: The cytologic classes were distributed as follows: nondiagnostic, 15.6%; benign, 66.5%; low-risk indeterminate, 10% (TIR 3A); high-risk indeterminate, 3.5% (TIR 3B); suspicious, 1.7%; and malignant, 2.6%. According to histology, the risk of malignancy was very high in the nondiagnostic category (29.8%), with young male patients more exposed to malignancy, and it was relatively high among benign (7.8%) and indeterminate nodules (32.5% in TIR 3A; 52.1% in TIR 3B), mainly because of the high prevalence of follicular architecture in malignant tumors. On histology, the malignancy rates were 92.4% and 99.3% for the suspicious and malignant categories, respectively; aggressive variants of papillary thyroid carcinoma were mostly diagnosed in these categories., Conclusions: In this series, nondiagnostic nodules showed high prevalence and, surprisingly, high malignancy rates. Malignant tumors with follicular architecture represented a diagnostic pitfall in benign and indeterminate nodules. The suspicious and malignant categories had high specificity for malignancy. Importantly, the ICCRTC had high reliability for identifying preoperatively aggressive histotypes of thyroid carcinoma., (© 2022 The Authors. Cancer Cytopathology published by Wiley Periodicals LLC on behalf of American Cancer Society.)

The expression of voltage-gated potassium Kv1.3 channels is increased in activated microglia, with non-selective blockade reported to attenuate microglial-mediated neuroinflammation. In this study, we evaluated the impact of a potent and selective peptidic blocker of Kv1.3 channels, HsTX1[R14A], on microglial-mediated neuroinflammation in vitro and in vivo. Treatment with both 0.1 and 1 µg/mL lipopolysaccharide (LPS) significantly (p < 0.05) increased Kv1.3 abundance on the surface of BV-2 microglia in association with increased levels of mRNA for tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6). The increased transcription of TNF-α and IL-6 was significantly attenuated (by 24.9 and 20.2%, respectively) by HsTX1[R14A] (100 nM). The concomitant increase in TNF-α and IL-6 release from BV-2 microglia was significantly attenuated by HsTX1[R14A] by 10.7 and 12.6%, respectively. In LPS-treated primary mouse microglia, the levels of TNF-α and nitric oxide were also attenuated by HsTX1[R14A] (26.1 and 20.4%, respectively). In an LPS-induced mouse model of neuroinflammation, both an immediate and delayed subcutaneous dose of HsTX1[R14A] (2 mg/kg) significantly reduced plasma and brain levels of the pro-inflammatory mediators TNF-α, IL-1β and IL-6, with no impact on the anti-inflammatory IL-10. These results demonstrate that HsTX1[R14A] is a promising therapeutic candidate for the treatment of diseases with a neuroinflammatory component., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interests., (Copyright © 2021 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

Objectives: This study is aimed to report a case of SV-related gastritis and the results of a systematic literature review of SV infections., Methods: Following a case presentation, we systematically searched different databases (MEDLINE, PubMed, Scopus, Web of Science, EMBASE, google scholar) for the items "sarcina," "ventriculi," "clostridium" with AND/OR., Results: A total of 55 articles reporting 65 cases of Sarcina Ventriculi were found. Thus, 66 patients, including our case, were reviewed. The median age was 51 years (IQR: 0-87 years). Females accounted for 51% of cases. 68% of patients had one or more comorbidities. SV was isolated in the gastrointestinal tract (88%), respiratory (5%), urine (4%), and bloodstream (3%) systems. Upper endoscopy was performed in 52 patients (79%). Biopsies were obtained in all 52 cases and were normal in 23%. Surgery was warranted in 15 patients (23%), and specific antimicrobial therapy was delivered in 34 (52%) patients. Mortality was 14%. At follow-up, 88% of patients showed complete eradication of the SV infection., Conclusions: Upper gastrointestinal biopsy positive for SV should prompt an evaluation of the clinical conditions, considering the risk of gastric perforation is not negligible. Antibiotic therapy may eradicate the infection and prevent complications. Emergency surgery is required in case of source control., Competing Interests: Declaration of Competing Interest No conflict of interest to declare., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Red Queen dynamic is often brought into play for antagonistic relationships. However, the coevolutionary effects of mutualistic interactions, which predict slower evolution for interacting organisms (Red King), have been investigated to a lesser extent. Lichens are a stable, mutualistic relationship of fungi and cyanobacteria and/or algae, which originated several times independently during the evolution of fungi. Therefore, they represent a suitable system to investigate the coevolutionary effect of mutualism on the fungal genome. We measured substitution rates and selective pressure of about 2000 protein-coding genes (plus the rDNA region) in two different classes of Ascomycota, each consisting of closely related lineages of lichenized and non-lichenized fungi. Our results show that independent lichenized clades are characterized by significantly slower rates for both synonymous and non-synonymous substitutions. We hypothesize that this evolutionary pattern is connected to the lichen life cycle (longer generation time of lichenized fungi) rather than a result of different selection strengths, which is described as the main driver for the Red Kind dynamic. This first empirical evidence of slower evolution in lichens provides an important insight on how biotic cooperative interactions are able to shape the evolution of symbiotic organisms., Competing Interests: The authors have no conflict of interest to declare., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Delta-like protein 3 (DLL3) is a protein of the Notch pathway, and it is a potential therapeutic target for high-grade lung neuroendocrine tumors (NETs), i.e., small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC). However, DLL3 prevalence in lung NETs and its association with clinicopathological characteristics and prognosis remained unclear. We analyzed the immunohistochemical expression of DLL3 and its prognostic role in a consecutive series of 155 surgically resected lung NETs, including typical carcinoid (TC), atypical carcinoid (AC), LCNEC, and SCLC patients. The DLL3 expression was categorized as high (>50% positive tumor cells) or low (<50%). In addition, tumors were categorized by H-score (i.e., percentage of positive cells by staining intensity, ≥150 vs. <150). DLL3 staining was positive in 99/155 (64%) samples, and high DLL3 expression was frequently observed in high-grade tumors. In detail, 46.9% and 75% of SCLC and 48.8% and 53.7% of LCNEC specimens showed a high DLL3 expression by using H-score and percentage of positive tumor cells, respectively. Regarding low-grade NETs, only 4.9% and 12.2% TCs and 19.5% and 24.4% ACs had high DLL3 expression considering H-score and percentage of positive tumor cells, respectively. High DLL3 expression was associated with advanced American Joint Committee on Cancer (AJCC) stage, peripheral location, and chromogranin A expression in high-grade tumors (p < 0.05). In low-grade NETs, high DLL3 expression was associated with female sex, peripheral location, a higher number of mitoses, higher Ki-67 index, presence of necrosis, and pleural infiltration (p < 0.05). No association was observed between high DLL3 expression and overall survival (OS) and disease-free survival (DFS) in high-grade NETs, whereas high DLL3 expression was associated with lower DFS in ACs (p = 0.01). In conclusion, our study demonstrated a high prevalence of DLL3 expression in high-grade lung NET patients and its association with aggressive clinicopathological features. These findings confirm that DLL3 could represent a useful biomarker for target therapy in high-grade tumors. Our results also suggest that the DLL3 expression could identify a subset of AC tumors with more aggressive behavior, thus providing the basis for new therapeutic options in this group of patients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alì, Di Stefano, Poma, Ricci, Proietti, Davini, Lucchi, Melfi and Fontanini.)

The aim of the present study was to evaluate whether prostate cancer (PC) patients can be accurately classified on the bases of tissue expression of gastrin-releasing peptide receptor (GRPR) and prostate-specific membrane antigen (PSMA). This retrospective study included 28 patients with PC. Formalin-fixed paraffin-embedded samples were used for diagnosis. Immunohistochemistry staining techniques were used to evaluate PSMA and GRPR expression (both number of cells expressed and % of area stained). To assess the independent associations among selected variables, a multi-dimensional scaling (MDS) analysis was used. It was found that the PSMA expression was inversely correlated with GRPR expression. Only the number of cells expressing GRPR was significantly related to the Gleason score. Both the percentage of area expressing GRPR and the number of cells expressing PSMA were close to reaching significance at the 0.05 level. MDS provided a map of the overall, independent association confirming that GRPR and PSMA represent inversely correlated measures of the same dimension. In conclusion, our data showed that GRPR expression should be evaluated in prostate biopsy specimens to improve our ability to detect PC with low grades at the earliest phases of development. Considering that GRPRs appear to be directly involved in the mechanisms of tumor proliferation, advancements in nuclear medicine radiotherapy can focus on this receptor to improve the therapeutic approach to PC. Further studies in our laboratory will investigate the molecular mechanisms of activation based on GRPR., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Faviana, Boldrini, Erba, Di Stefano, Manassero, Bartoletti, Galli, Gentile and Bardi.)

Undifferentiated connective tissue disease at risk for systemic sclerosis (UCTD-risk-SSc), otherwise referred to as very early-early SSc, is a condition characterized by Raynaud's phenomenon with serum SSc marker autoantibodies and/or typical capillaroscopic findings and unsatisfying classification criteria for the disease. The aim of the present study was to assess the prevalence of right (RV) or left ventricular (LV) systolic and/or diastolic dysfunction by standard echocardiography and tissue Doppler imaging (TDI). Thirty patients with UCTD-risk-SSc (28 female, mean age 47 ± 13 years, range 21-70) and 30 age- and sex-matched controls underwent cardiac assessment by standard echocardiography and TDI. UCTD-risk-SSc patients and controls did not show any difference at standard echocardiography. Despite results falling within the respective normal ranges, TDI pointed out a mild impairment of LV and RV diastolic (E m 15 ± 4 vs. 19 ± 5, p = 0.0004; E/E m 6.1 ± 1.7 vs. 4.8 ± 1.2, p = 0.001; E t 14 ± 3 vs. 16 ± 2, p = 0.02; E t /A t 0.9 ± 0.4 vs. 1.3 ± 0.3, p = 0.002; E/E t 3.5 ± 1.2 vs. 4.2 ± 0.9, p = 0.02) and systolic function (S m 13 ± 3 vs. 15 ± 2 cm/s, p < 0.0003; S t 14 ± 2 vs. 16 ± 3 cm/s, p < 0.0001) and increased estimated pulmonary artery wedge pressure (9 ± 2 vs. 8 ± 1, p = 0.001) in UCTD-risk-SSc patients as compared to controls. Notably, a statistically significant difference also emerged in the prevalence of TDI detected E'/A' t , (71% of UCTD-risk-SSc patients vs. 19% of controls; p < 0.0001). Our study shows that UCTD-risk-SSc patients show a previously unrecognized, mild biventricular systolic and diastolic dysfunction as compared to controls. The pathophysiologic meaning as well the predictive value of developing overt SSc await to be elucidated.

Expending effort is generally considered to be undesirable. However, both humans and vertebrates will work for a reward they could also get for free. Moreover, cues associated with high-effort rewards are preferred to low-effort associated cues. Many explanations for these counterintuitive findings have been suggested, including cognitive dissonance (self-justification) or a greater contrast in state (e.g., energy or frustration level) before and after an effort-linked reward. Here, we test whether effort expenditure also increases perceived value in ants, using both classical cue-association methods and pheromone deposition, which correlates with perceived value. In 2 separate experimental setups, we show that pheromone deposition is higher toward the reward that requires more effort: 47% more pheromone deposition was performed for rewards reached via a vertical runway (high effort) compared with ones reached via a horizontal runway (low effort), and deposition rates were 28% higher on rough (high effort) versus smooth (low effort) runways. Using traditional cue-association methods, 63% of ants trained on different surface roughness, and 70% of ants trained on different runway elevations, preferred the high-effort related cues on a Y maze. Finally, pheromone deposition to feeders requiring memorization of one path bifurcation was up to 29% higher than to an identical feeder requiring no learning. Our results suggest that effort affects value perception in ants. This effect may stem from a cognitive process, which monitors the change in a generalized hedonic state before and after reward. (PsycINFO Database Record, ((c) 2018 APA, all rights reserved).)

Previous studies of the occurrence of interstitial lung disease (ILD) in undifferentiated connective tissue diseases (UCTD) were conducted in patients admitted to Respiratory Medicine Units. The aim of the present prospective study was to investigate lung involvement in UCTD patients admitted to a Rheumatology Unit. Eighty-one consecutive UCTD patients were enrolled in the study. Each patient underwent history and physical examination, routine laboratory investigations, antinuclear antibody (ANA) profiling, B-mode echocardiography, and lung function study according to previously reported methods. Lung high resolution computed tomography (HRCT) was performed in patients who provided informed consent. Six patients (7.4%) had a history of grade II dyspnea. Three of them had a DLCO ranging from 42 to 55% of the predicted value; and a HRCT-documented ILD with a non-specific interstitial pneumonia (NSIP) pattern. Symptoms in the other three patients were due to cardiac disease. None of the 75 asymptomatic patients, had relevant findings at physical examination, 26/75 had a DLCO <80% (<70% in 10 cases). Of these, 3 of the 30 patients who underwent lung HRCT were affected by NSIP-ILD. Six of the 81 enrolled were affected by ILD, which was symptomatic in three patients. A higher percentage of patients had a reduced DLCO. The latter finding may reflect a preradiographic ILD or a preechocardiographic pulmonary vascular disease.

Background: Several studies indicate that ex vivo cytokine-supported expansion induces defective hematopoietic stem cell engraftment. We investigated the role of alpha4 integrin, alpha5 integrin and CXCR4 in engraftment of unmanipulated and cytokine-treated human cord blood CD34(+) cells., Design and Methods: Uncultured or expanded CD34(+) cells were infused in NOD/SCID-beta(2)microglobulin-null mice. The function of alpha4, and alpha5 integrins and CXCR4 was assessed by incubating cells with specific neutralizing antibodies, prior to transplant. The activation state of alpha4 integrin was further tested by adhesion and migration assays., Results: Neutralization of either alpha4 integrin or CXCR4 abolished engraftment of uncultured CD34(+) cells at 6 week spost-transplant, while alpha5 integrin neutralization had no significant effect. However, after short-term ex vivo culture, blocking alpha4 integrin or CXCR4 did not affect repopulating activity whereas neutralization of alpha5 integrin inhibited engraftment. Using soluble vascular cell adhesion molecule-1 binding assays, we observed that alpha4 integrin affinity in fresh CD34(+) cells was low and susceptible to stimulation while in cultured CD34(+) cells, it was high and insensitive to further activation. In addition, stromal cell-derived factor-1 stimulated migration across vascular cell adhesion molecule-1 in fresh CD34(+) cells but not in cultured CD34(+) cells., Conclusions: Our data show that ex vivo culture of hematopoietic progenitor cells is associated with downregulation of both alpha4 integrin- and CXCR4-mediated engraftment. Further investigations suggest that this is caused by supraphysiological increase of alpha4 integrin affinity, which impairs directional migration across vascular cell adhesion molecule-1 in response to stromal cell-derived factor-1. Such changes may underlie the engraftment defect of cytokine-stimulated CD34(+) cells.

Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.

There is potential interest for combining allogeneic hematopoietic cell transplantation (HCT), and particularly allogeneic HCT with a nonmyeloablative regimen, to the tyrosine kinase inhibitor imatinib (Glivec; Novartis, Basel, Switzerland, http://www.novartis.com) in order to maximize anti-leukemic activity against Philadelphia chromosome-positive leukemias. However, because imatinib inhibits c-kit, the stem cell factor receptor, it could interfere with bone marrow engraftment. In this study, we examined the impact of imatinib on normal progenitor cell function. Imatinib decreased the colony-forming capacity of mobilized peripheral blood human CD133(+) cells but not that of long-term culture-initiating cells. Imatinib also decreased the proliferation of cytokine-stimulated CD133(+) cells but did not induce apoptosis of these cells. Expression of very late antigen (VLA)-4, VLA-5, and CXCR4 of CD133(+) cells was not modified by imatinib, but imatinib decreased the ability of CD133(+) cells to migrate. Finally, imatinib did not decrease engraftment of CD133(+) cells into irradiated nonobese diabetic/severe combined immunodeficient/beta2m(null) mice conditioned with 3 or 1 Gy total body irradiation. In summary, our results suggest that, despite inhibition of hematopoietic progenitor cell growth in vitro, imatinib does not interfere with hematopoietic stem cell engraftment.

Ex vivo expansion of hematopoietic stem/progenitor cells may result in defective engraftment. Human cord blood CD34(+) progenitor cells were synchronized and assayed for adhesion and migration onto fibronectin (Fn) and vascular cell adhesion molecule-1 (VCAM-1) at different stages of a first cell cycle executed ex vivo. During S phase transit, adhesion to Fn was transiently increased while binding to VCAM-1 was reversibly decreased, after which adhesion to both ligands returned to baseline levels with cell cycle completion. Transmigration across Fn and VCAM-1 decreased irreversibly during S phase progression. The function of alpha4 and alpha5 integrins was assessed with specific neutralizing antibodies. In uncultured CD34(+) cells and long-term culture-initiating cells (LTC-ICs), both adhesion and migration on Fn were inhibited by anti-alpha4 but not by anti-alpha5 antibodies. In mitotically activated CD34(+) cells and LTC-ICs, adhesion and migration on Fn were mainly dependent on alpha5 integrin and to a lesser extent on alpha4 integrin. Changes in integrin function were not dependent on parallel modulation of integrin expression. In conclusion, Fn and VCAM-1 binding of progenitor cells fluctuates reversibly during cell cycle transit ex vivo. In addition, our data show that mitogenic activation induces a shift from a dominant alpha4 to a preferential alpha5 integrin-dependent interaction with Fn.

Engraftment of hematopoietic progenitor cells has been shown to decrease during cell cycle transit. We studied cell cycle-associated changes in adhesion and migration of mitotically activated cord blood CD34+ cells. Migration toward medium conditioned by the stromal-derived factor-1-producing cell line MS-5 was studied in bovine serum albumin- and fibronectin (Fn)-coated transwells. Migration was reduced in cycling CD34+ cells and long-term culture-initiating cells (LTC-ICs) compared with their noncycling counterparts across Fn but not across bovine serum albumin. Conversely, Fn binding was higher in cycling CD34+ cells and LTC-ICs compared with noncycling progenitor cells, while adhesion of both subsets to bovine serum albumin was undetectable. The contribution of alpha4 and alpha5 integrins in mediating adhesion and migration of activated CD34+ cells onto Fn was analyzed by neutralization experiments. While alpha4-mediated Fn binding decreased during G(2)/M, alpha5 integrin-mediated adhesion increased during transit from G(0)/G(1) to S and G(2)/M phases. As for migration, the contribution of alpha4 integrin was similar in all phases, whereas alpha5-directed migration was lower in G(2)/M compared with G(0)/G(1) and S phases. Defective migration of cycling CD34+ cells was not due to differences in alpha5 integrin expression. In conclusion, chemotaxis across Fn is less efficient in cycling progenitor cells in correlation with an increased Fn binding capacity. In addition, alpha4 and alpha5 integrin functions are independently modulated during cell cycle transit.

We examined the growth of the right common carotid artery of young rabbits after ligating the left common carotid artery at 3 weeks of age, a procedure that approximately doubled right carotid blood flows. Flow increased from 0.065 +/-0.003 to 0.096+/-0.009 ml/s within 1 h and, at 15 weeks of age, carotid blood flows in experimental animals (0.747+/-0.102 ml/s) were more than double of those of sham-operated control animals (0.334+/-0.053 ml/s). Contralateral carotid ligation resulted in more rapid increases in diameter of the artery with growth in the experimental animals. At 15 weeks of age, the vessel was 15% larger than that of sham-operated controls (2.70+/-0.09 vs. 2.34+/-0.05 mm). This more rapid growth of diameter resulted in shear stresses that were not different from controls despite the higher blood flow rates. Interestingly, however, shear stresses in control arteries fell from 17.4+/-3.4 to 9.19+/-1.16 dyn/cm2 over the experimental period (p < 0.05). Elastin accumulation in the experimental artery was much more rapid than in controls and elastin contents were 49% more than in controls at 15 weeks of age. DNA and collagen contents were not significantly affected by contralateral carotid ligation. Previously, we found that experimental manipulations that decreased flow in the same artery of weanling rabbits substantially affected elastin and DNA accumulation, but had no effect on collagen contents. We conclude that increased blood flow is associated with arterial growth and specifically with accumulation of elastin, a wall constituent that bears much of the wall tension at resting blood pressure, and therefore is a primary determinant of resting vessel dimensions.