Identification of CDKN3 as a Key Gene that Regulates Neuroblastoma Cell Differentiation.

We conducted a high-content screening (HCS) in neuroblastoma BE(2)-C cells to identify cell cycle regulators that control cell differentiation using a library of siRNAs against cell cycle-regulatory genes. We discovered that knocking down expression of cyclin dependent kinase inhibitor 3 (CDKN3) showed the most potent effect in inducing neurite outgrowth, the morphological cell differentiation marker of neuroblastoma cells. We then demonstrated that CDKN3 knockdown increased expression of neuroblastoma molecular differentiation markers, neuron specific enolase (NSE), ßIII-tubulin and growth associated protein 43 (GAP43). We further showed that CDKN3 knockdown reduced expression of cell proliferation markers Ki67 and proliferating cell nuclear antigen (PCNA), and reduced colony formation of neuroblastoma cells. More importantly, we observed a correlation of high tumor CDKN3 mRNA levels with poor patient survival in the investigation of public neuroblastoma patient datasets. In exploring the mechanisms that regulate CDKN3 expression, we found that multiple strong differentiation-inducing molecules, including miR-506-3p and retinoic acid, down-regulated CDKN3 expression. In addition, we found that N-Myc promoted CDKN3 expression at the transcriptional level by directly binding to the CDKN3 promoter. Furthermore, we found that CDKN3 and two additional differentiation-regulating cell cycle proteins identified in our HCS, CDC6 and CDK4, form an interactive network to promote expression of each other. In summary, we for the first time discovered the function of CDKN3 in regulating neuroblastoma cell differentiation and characterized the transcriptional regulation of CDKN3 expression by N-Myc in neuroblastoma cells. Our findings support that CDKN3 plays a role in modulating neuroblastoma cell differentiation and that overexpression of CDKN3 may contribute to neuroblastoma progression.

Geographical variations in long term colorectal cancer outcomes in England: a contemporary population analysis revealing the north-south divide in colorectal cancer survival.

BACKGROUND: Regional variations in healthcare outcomes in England have been historically reported. This study analyses the variations in long term colorectal cancer survival across different regions in England. METHODS: Relative survival analysis of population data obtained from all cancer registries in England between 2010 and 2014. RESULTS: Totally, 167,501 patients were studied. Regions in the southern England had better outcomes with Southwest and Oxford registries having 63.5 and 62.7% 5 year relative survival. In contrast, Trent and Northwest cancer registries had 58.1% relative survival (p < 0.01). The regions in the north fared below the national average. The survival outcomes reflected socio-economic deprivation status, the best performing regions in the south having low levels of deprivation (5.3 and 6.5% having maximum deprivation in Southwest and Oxford, respectively). The regions with worst long term cancer outcomes had high levels of deprivation with 25% and 17% having high levels of deprivation in Northwest and Trent regions. CONCLUSION: There are significant variations in long term colorectal cancer survival between different regions in England, southern England had better relative survival when compared with the northern regions. Disparities in socio-economic depravation status in different regions may be associated with worse colorectal cancer outcomes.

Attitudes of Australian dermatologists on the use of genetic testing: A cross-sectional survey with a focus on melanoma.

Background: Melanoma genetic testing reportedly increases preventative behaviour without causing psychological harm. Genetic testing for familial melanoma risk is now available, yet little is known about dermatologists' perceptions regarding the utility of testing and genetic testing ordering behaviours. Objectives: To survey Australasian Dermatologists on the perceived utility of genetic testing, current use in practice, as well as their confidence and preferences for the delivery of genomics education. Methods: A 37-item survey, based on previously validated instruments, was sent to accredited members of the Australasian College of Dermatologists in March 2021. Quantitative items were analysed statistically, with one open-ended question analysed qualitatively. Results: The response rate was 56% (256/461), with 60% (153/253) of respondents between 11 and 30 years post-graduation. While 44% (112/252) of respondents agreed, or strongly agreed, that genetic testing was relevant to their practice today, relevance to future practice was reported significantly higher at 84% (212/251) (t = -9.82, p < 0.001). Ninety three percent (235/254) of respondents reported rarely or never ordering genetic testing. Dermatologists who viewed genetic testing as relevant to current practice were more likely to have discussed (p < 0.001) and/or offered testing (p < 0.001). Respondents indicated high confidence in discussing family history of melanoma, but lower confidence in ordering genetic tests and interpreting results. Eighty four percent (207/247) believed that genetic testing could negatively impact life insurance, while only 26% (63/244) were aware of the moratorium on using genetic test results in underwriting in Australia. A minority (22%, 55/254) reported prior continuing education in genetics. Face-to-face courses were the preferred learning modality for upskilling. Conclusion: Australian Dermatologists widely recognise the relevance of genetic testing to future practice, yet few currently order genetic tests. Future educational interventions could focus on how to order appropriate genetic tests and interpret results, as well as potential implications on insurance.

The Function and Regulation of SAPCD2 in Physiological and Oncogenic Processes.

The Suppressor APC Domain Containing 2 (SAPCD2) gene, also known by its aliases p42.3 and c9orf140, encodes a protein with an approximate molecular weight of 42.3 kDa. It was initially recognized as a cell cycle-associated protein involved in mitotic progression. Since the initial discovery of this gene, emerging evidence has suggested that its functions extend beyond that of regulating cell cycle progression to include modulation of planar polarization of cell progenitors and determination of cell fate throughout embryonic development. The underlying mechanisms driving such functions have been partially elucidated. However, the detailed mechanisms of action remain to be further characterized. The expression level of SAPCD2 is high throughout embryogenesis but is generally absent in healthy postnatal tissues, with restored expression in adult tissues being associated with various disease states. The pathological consequences of its aberrant expression have been investigated, most notably in the development of several types of cancers. The role of SAPCD2 in tumorigenesis has been supported by in vitro, in vivo, and retrospective clinical investigations and the mechanisms underlying its oncogenic function have been partially revealed. The potential of SAPCD2 as a diagnostic marker and therapeutic target of cancers have also been explored and have shown great promise. However, many questions pertaining to its oncogenic mechanisms as well as its value as a diagnostic marker and therapeutic target remain to be answered. In addition to its function as an oncogene, an involvement of SAPCD2 in other pathological processes such as inflammation has also been implicated and provides additional directions that warrant future investigation. This article reviews the current understanding of the normal cellular functions of SAPCD2 and the relevance of SAPCD2 in disease development with a primary focus on tumorigenesis. The mechanisms that regulate p43.2 expression, including the potential role of microRNAs in regulating its expression, are also reviewed. To the best of our knowledge, we are the first to comprehensively review the published findings regarding the physiological and pathological functions of this gene.

Young-onset colorectal cancer: Insights from an English population-based study.

AIM: Young colorectal cancer (CRC) patients are reported to have more aggressive disease, an advanced stage at diagnosis and conflicting survival outcomes. The aim of this study was to analyse the demographics, clinicopathological features and prognosis of young CRC at a population-based level in England. METHOD: This is a retrospective review of all CRC patients using data from Public Health England collated from regional cancer registries in England between 2010 and 2014. Those aged 40 years and below were classified as young and those over 40 were classified as older. RESULTS: Overall, 167,501 patients had CRC. Of these, 3757 patients (2.2%) were young. Right-sided cancers were more common in younger patients (48.2% vs. 32.9%, p < 0.001). Favourable histological grade (well or moderately differentiated) was present in 83.1% and 73.5% of young and older patients, respectively. The percentage of young and older patients being diagnosed at an early stage (Stages 1 and 2) was similar at 40.6% vs. 42.9%. The 5-year age- and gender-adjusted relative survival (cancer specific) was significantly better for young patients when compared with older patients diagnosed with CRC. Additionally, overall 5-year survival was better for younger patients (71.6% and 47.2%, p < 0.001 in young and older CRC patients respectively). CONCLUSION: The increased right-sided colon cancer in young CRC patients in England warrants attention. Contrary to previous reports, they do not present at later stage. Young CRC patients have better overall and relative survival than older patients with CRC.

The anti-tumour activity of DNA methylation inhibitor 5-aza-2'-deoxycytidine is enhanced by the common analgesic paracetamol through induction of oxidative stress.

The DNA demethylating agent 5-aza-2'-deoxycytidine (DAC, decitabine) has anti-cancer therapeutic potential, but its clinical efficacy is hindered by DNA damage-related side effects and its use in solid tumours is debated. Here we describe how paracetamol augments the effects of DAC on cancer cell proliferation and differentiation, without enhancing DNA damage. Firstly, DAC specifically upregulates cyclooxygenase-2-prostaglandin E2 pathway, inadvertently providing cancer cells with survival potential, while the addition of paracetamol offsets this effect. Secondly, in the presence of paracetamol, DAC treatment leads to glutathione depletion and finally to accumulation of ROS and/or mitochondrial superoxide, both of which have the potential to restrict tumour growth. The benefits of combined treatment are demonstrated here in head and neck squamous cell carcinoma (HNSCC) and acute myeloid leukaemia cell lines, further corroborated in a HNSCC xenograft mouse model and through mining of publicly available DAC and paracetamol responses. The sensitizing effect of paracetamol supplementation is specific to DAC but not its analogue 5-azacitidine. In summary, the addition of paracetamol could allow for DAC dose reduction, widening its clinical usability and providing a strong rationale for consideration in cancer therapy.

Social Cooperativity of Bacteria during Reversible Surface Attachment in Young Biofilms: a Quantitative Comparison of Pseudomonas aeruginosa PA14 and PAO1.

What are bacteria doing during "reversible attachment," the period of transient surface attachment when they initially engage a surface, besides attaching themselves to the surface? Can an attaching cell help any other cell attach? If so, does it help all cells or employ a more selective strategy to help either nearby cells (spatial neighbors) or its progeny (temporal neighbors)? Using community tracking methods at the single-cell resolution, we suggest answers to these questions based on how reversible attachment progresses during surface sensing for Pseudomonas aeruginosa strains PAO1 and PA14. Although PAO1 and PA14 exhibit similar trends of surface cell population increase, they show unanticipated differences when cells are considered at the lineage level and interpreted using the quantitative framework of an exactly solvable stochastic model. Reversible attachment comprises two regimes of behavior, processive and nonprocessive, corresponding to whether cells of the lineage stay on the surface long enough to divide, or not, before detaching. Stark differences between PAO1 and PA14 in the processive regime of reversible attachment suggest the existence of two surface colonization strategies. PAO1 lineages commit quickly to a surface compared to PA14 lineages, with early c-di-GMP-mediated exopolysaccharide (EPS) production that can facilitate the attachment of neighbors. PA14 lineages modulate their motility via cyclic AMP (cAMP) and retain memory of the surface so that their progeny are primed for improved subsequent surface attachment. Based on the findings of previous studies, we propose that the differences between PAO1 and PA14 are potentially rooted in downstream differences between Wsp-based and Pil-Chp-based surface-sensing systems, respectively.IMPORTANCE The initial pivotal phase of bacterial biofilm formation known as reversible attachment, where cells undergo a period of transient surface attachment, is at once universal and poorly understood. What is more, although we know that reversible attachment culminates ultimately in irreversible attachment, it is not clear how reversible attachment progresses phenotypically, as bacterial surface-sensing circuits fundamentally alter cellular behavior. We analyze diverse observed bacterial behavior one family at a time (defined as a full lineage of cells related to one another by division) using a unifying stochastic model and show that our findings lead to insights on the time evolution of reversible attachment and the social cooperative dimension of surface attachment in PAO1 and PA14 strains.

The PLAGL2/MYCN/miR-506-3p interplay regulates neuroblastoma cell fate and associates with neuroblastoma progression.

BACKGROUND: The oncogene MYCN is critical for tumorigenesis of several types of cancers including neuroblastoma. We previously reported that miR-506-3p repressed MYCN expression in neuroblastoma cells. However, the mechanism underlying such regulation was undetermined since there is no miR-506-3p target site in MYCN 3'UTR. METHODS: By a systematic investigation combining microarray, informatics and luciferase reporter assay, we identified that the transcriptional factor pleiomorphic adenoma gene-like 2 (PLAGL2) is a direct target of miR-506-3p that mediates its regulation on MYCN expression. Using CHIP-PCR and luciferase reporter assay, we validated the transcriptional regulation of MYCN by PLAGL2 and we further demonstrated the transcriptional regulation of PLAGL2 by MYCN. We examined the function of PLAGL2 in regulating neuroblastoma cell fate by cell viability assay, colony formation and Western blotting of differentiation markers. We examined the effect of retinoic acid, the differentiation agent used in neuroblastoma therapy, on miR-506-3p, PLAGL2 and MYCN expressions by quantitative PCR and Western blots. We investigated the clinical relevance of PLAGL2 expression by examining the correlation of tumor PLAGL2 mRNA levels with MYCN mRNA expression and patient survival using public neuroblastoma patient datasets. RESULTS: We found that miR-506-3p directly down-regulated PLAGL2 expression, and we validated a PLAGL2 binding site in the MYCN promoter region responsible for promoting MYCN transcription, thereby establishing a mechanism through which miR-506-3p regulates MYCN expression. Conversely, we discovered that MYCN regulated PLAGL2 transcription through five N-Myc-binding E-boxes in the PLAGL2 promoter region. We further confirmed the reciprocal regulation between endogenous PLAGL2 and MYCN in multiple neuroblastoma cell lines. Moreover, we found that PLAGL2 knockdown induced neuroblastoma cell differentiation and reduced cell proliferation, and combined knockdown of PLAGL2 and MYCN showed a synergistic effect. More strikingly, we found that high tumor PLAGL2 mRNA levels were significantly correlated with high MYCN mRNA levels and poor patient survival in neuroblastoma patients. Furthermore, we found that retinoic acid increased expression of miR-506-3p and repressed expression of MYCN and PLAGL2. CONCLUSIONS: Our findings altogether suggest that the interplay network formed by PLAGL2, MYCN and miR-506-3p is an important mechanism in regulating neuroblastoma cell fate, determining neuroblastoma prognosis, and mediating the therapeutic function of retinoic acid.

Ethanol Decreases Pseudomonas aeruginosa Flagellar Motility through the Regulation of Flagellar Stators.

Pseudomonas aeruginosa frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced P. aeruginosa swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in ΔmotAB or ΔsadC ΔgcbA mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in P. aeruginosa motility, thereby promoting P. aeruginosa colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol.IMPORTANCE Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by Pseudomonas aeruginosa, here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence P. aeruginosa colocalization in the context of infection and in other polymicrobial settings.

Multigenerational memory and adaptive adhesion in early bacterial biofilm communities.

Using multigenerational, single-cell tracking we explore the earliest events of biofilm formation by Pseudomonas aeruginosa During initial stages of surface engagement (≤20 h), the surface cell population of this microbe comprises overwhelmingly cells that attach poorly (∼95% stay <30 s, well below the ∼1-h division time) with little increase in surface population. If we harvest cells previously exposed to a surface and direct them to a virgin surface, we find that these surface-exposed cells and their descendants attach strongly and then rapidly increase the surface cell population. This "adaptive," time-delayed adhesion requires determinants we showed previously are critical for surface sensing: type IV pili (TFP) and cAMP signaling via the Pil-Chp-TFP system. We show that these surface-adapted cells exhibit damped, coupled out-of-phase oscillations of intracellular cAMP levels and associated TFP activity that persist for multiple generations, whereas surface-naïve cells show uncorrelated cAMP and TFP activity. These correlated cAMP-TFP oscillations, which effectively impart intergenerational memory to cells in a lineage, can be understood in terms of a Turing stochastic model based on the Pil-Chp-TFP framework. Importantly, these cAMP-TFP oscillations create a state characterized by a suppression of TFP motility coordinated across entire lineages and lead to a drastic increase in the number of surface-associated cells with near-zero translational motion. The appearance of this surface-adapted state, which can serve to define the historical classification of "irreversibly attached" cells, correlates with family tree architectures that facilitate exponential increases in surface cell populations necessary for biofilm formation.

Limited utility of routine chest X-ray in initial evaluation of neutropenic fever in patients with haematological diseases undergoing chemotherapy.

BACKGROUND: Routine chest X-ray (CXR) is recommended for neutropenic fever (NF) management however its role is relatively understudied in haematology patients. AIM: To investigate the utility of CXR in the diagnosis and management of patients with haematological conditions complicated by NF. METHODS: Retrospective, single-centre analysis of haematology patients admitted with NF between January 2011 and December 2015. Baseline demographics, treatment details and outcomes were collected from electronic patient records. CXR underwent independent radiology review. Primary endpoints were a proportion of NF episodes in which CXR detected a probable chest infection in the absence of respiratory symptoms/signs and/or resulted in a change in antibiotic management. RESULTS: Four hundred and thirty-five episodes were identified; CXR was performed in 75% of patients (65% within 2 days of NF). In 4 of 164 (2.4%) asymptomatic patients, CXR was consistent with infection, in contrast to 19 of 119 (16%) patients with clinical signs of respiratory infection. Only 3 of 283 (1.1%) CXR resulted in a change to antibiotics. CXR consistent with infection was not associated with increased mortality or increased admission length, although there was an association with intensive care unit admission (odds ratios: 7.61, 95% confidence interval: 2.04-28.31). CONCLUSION: In haematology patients with NF, CXR rarely detected chest infection or changed management in patients with no respiratory symptoms or signs. CXR in our institution is no longer part of routine assessment of NF in the absence of these features.

Bacteria, Rev Your Engines: Stator Dynamics Regulate Flagellar Motility.

Many bacteria move through liquids and across surfaces by using flagella-filaments propelled by a membrane-embedded rotary motor. Much is known about the flagellum: its basic structure, the function of its individual motor components, and the regulation of its synthesis. However, we are only beginning to identify the dynamics of flagellar proteins and to understand how the motor structurally adapts to environmental stimuli. In this review, we discuss the external and cellular factors that influence the dynamics of stator complexes (the ion-conducting channels of the flagellar motor). We focus on recent discoveries suggesting that stator dynamics are a means for controlling flagellar function in response to different environments.

A hierarchical cascade of second messengers regulates Pseudomonas aeruginosa surface behaviors.

UNLABELLED: Biofilms are surface-attached multicellular communities. Using single-cell tracking microscopy, we showed that a pilY1 mutant of Pseudomonas aeruginosa is defective in early biofilm formation. We leveraged the observation that PilY1 protein levels increase on a surface to perform a genetic screen to identify mutants altered in surface-grown expression of this protein. Based on our genetic studies, we found that soon after initiating surface growth, cyclic AMP (cAMP) levels increase, dependent on PilJ, a chemoreceptor-like protein of the Pil-Chp complex, and the type IV pilus (TFP). cAMP and its receptor protein Vfr, together with the FimS-AlgR two-component system (TCS), upregulate the expression of PilY1 upon surface growth. FimS and PilJ interact, suggesting a mechanism by which Pil-Chp can regulate FimS function. The subsequent secretion of PilY1 is dependent on the TFP assembly system; thus, PilY1 is not deployed until the pilus is assembled, allowing an ordered signaling cascade. Cell surface-associated PilY1 in turn signals through the TFP alignment complex PilMNOP and the diguanylate cyclase SadC to activate downstream cyclic di-GMP (c-di-GMP) production, thereby repressing swarming motility. Overall, our data support a model whereby P. aeruginosa senses the surface through the Pil-Chp chemotaxis-like complex, TFP, and PilY1 to regulate cAMP and c-di-GMP production, thereby employing a hierarchical regulatory cascade of second messengers to coordinate its program of surface behaviors. IMPORTANCE: Biofilms are surface-attached multicellular communities. Here, we show that a stepwise regulatory circuit, involving ordered signaling via two different second messengers, is required for Pseudomonas aeruginosa to control early events in cell-surface interactions. We propose that our studies have uncovered a multilayered "surface-sensing" system that allows P. aeruginosa to effectively coordinate its surface-associated behaviors. Understanding how cells transition into the biofilm state on a surface may provide new approaches to prevent formation of these communities.

Lymphatic function is impaired following irradiation of a single lymph node.

INTRODUCTION: Lymph nodes are often the target of radiotherapy procedures. Unfortunately, the impact of nodal irradiation on lymphatic function is uncertain. In this study, our aim was to quantify the impact of lymph node irradiation on lymph flow. METHODS AND RESULTS: The popliteal node or the nodal excision site of rabbits was treated with four daily 8 Gy doses of radiation. A FITC-dextran tracer was infused into a prenodal popliteal lymphatic. The area under the tracer blood recovery curve (AUC) indicated lymphatic functionality and the inflow pressure versus flow rate relationship inferred resistance through the system. Fluoroscopic and histological examination provided supporting data. Radiation of intact nodes decreased lymph transport significantly at 1 week, 1 month, and 6 months post-treatment (AUCs of 207.9 ± 79.87, 191.6 ± 62.95, and 250.44 ± 46.45) in comparison to controls (667.32 ± 104.18). Surprisingly, this functional decline was similar to that detected with a combination of node removal and irradiation of the excision site. The pressure-flow relationships in all treatment groups were significantly different from controls. This may be due in part to fibrosis and the thickening of the nodal capsules and trabeculae observed at 1 and 6 months. Fluoroscopy and Evans blue dye studies revealed vigorous new lymphatic vessel growth and occasionally, vessels anastomosed with local veins. CONCLUSIONS: Irradiation of the popliteal lymph node impaired lymph transport and increased the pressure required to maintain flow through the system. New vessel formation and the growth of lymph-venous anastomoses indicated the development of alternative drainage pathways as a compensatory response.

End-stage kidney disease: a survey of recent research to support a palliative approach.

This survey examines the quantity, quality, and accessibility of recent research that contributes to the evidence-based implementation of a palliative approach to end-stage kidney disease (ESKD). An electronic search identified published articles (between September 2009 and August 2011) relevant to adults with ESKD (n = 1628). Few articles (n = 136) referred to key themes in a palliative approach to care: life-limiting illness, holistic care, and unit of care. Most of the relevant empirical articles used designs that did not allow a causal variable to be identified, and evaluations of interventions were rare. The literature was dispersed and often in journals unlikely to be regularly accessed by renal clinicians. Literature supporting the implementation of a full evidence-based palliative approach to ESKD is expanding but remains limited and is difficult to identify and access.

Utility of AAOS OITE scores in predicting ABOS Part I outcomes: AAOS exhibit selection.

BACKGROUND: Residency programs commonly use performance on the Orthopaedic In-Training Examination (OITE) developed by the American Academy of Orthopaedic Surgeons (AAOS) to identify residents who are lagging behind their peers and at risk for failing Part I of the American Board of Orthopaedic Surgery (ABOS) Certifying Examination. This study was designed to investigate the utility of the OITE score as a predictor of ABOS Part I performance. METHOD: Results for 3132 examinees who took Part I of the ABOS examination for the first time from 2002 to 2006 were matched with records from the 1997 to 2006 OITE tests; at least one OITE score was located for 2852 (91%) of the ABOS Part I examinees. After OITE performance was rescaled to place scores from different test years on comparable scales, descriptive statistics and correlations between ABOS and OITE scores were computed, and regression analyses were conducted to predict ABOS results from OITE performance. RESULTS: Substantial increases in the mean OITE score were observed as residents progressed through training. Stronger correlations were observed between OITE and ABOS performance during later years in training, reaching a maximum of 0.53 in years 3 and 4. Logistic regression results indicated that residents with an OITE score below the 10th percentile were much more likely to fail Part I compared with those with an OITE score above the 50th percentile. CONCLUSIONS: OITE performance was a good predictor of the ABOS score and pass-fail outcome; the OITE can be used effectively for early identification of residents at risk for failing the ABOS Part I examination.

Experimental assessment of pro-lymphangiogenic growth factors in the treatment of post-surgical lymphedema following lymphadenectomy.

INTRODUCTION: Lymphedema is a frequent consequence of lymph node excision during breast cancer surgery. Current treatment options are limited mainly to external compression therapies to limit edema development. We investigated previously, post-surgical lymphedema in a sheep model following the removal of a single lymph node and determined that autologous lymph node transplantation has the potential to reduce or prevent edema development. In this report, we examine the potential of lymphangiogenic therapy to restore lymphatic function and reduce post-surgical lymphedema. METHODS: Lymphangiogenic growth factors (vascular endothelial growth factor-C (VEGF-C) and angiopoitein-2 (ANG-2)) were loaded into a gel-based drug delivery system (HAMC; a blend of hyaluronan and methylcellulose). Drug release rates and lymphangiogenic signaling in target endothelial cells were assessed in vitro and vascular permeability biocompatibility tests were examined in vivo. Following, the removal of a single popliteal lymph node, HAMC with the growth factors was injected into the excision site. Six weeks later, lymphatic functionality was assessed by injecting (125)Iodoine radiolabelled bovine serum albumin ((125)I-BSA) into prenodal vessels and measuring its recovery in plasma. Circumferential leg measurements were plotted over time and areas under the curves used to quantify edema formation. RESULTS: The growth factors were released over a two-week period in vitro by diffusion from HAMC, with 50% being released in the first 24 hours. The system induced lymphangiogenic signaling in target endothelial cells, while inducing only a minimal inflammatory response in sheep. Removal of the node significantly reduced lymphatic functionality (Nodectomy 1.9 ± 0.9, HAMC alone 1.7 ± 0.8) compared with intact groups (3.2 ± 0.7). There was no significant difference between the growth factor treatment group (2.3 ± 0.73) and the intact group. An increase in the number of regenerated lymphatic vessels at treatment sites was observed with fluoroscopy. Groups receiving HAMC plus growth factors displayed significantly reduced edema (107.4 ± 51.3) compared with non-treated groups (nodectomy 219.8 ± 118.7, and HAMC alone 162.6 ± 141). CONCLUSIONS: Growth factor therapy has the potential to increase lymphatic function and reduce edema magnitude in an animal model of lymphedema. The application of this concept to lymphedema patients warrants further examination.

Experimental assessment of autologous lymph node transplantation as treatment of postsurgical lymphedema.

BACKGROUND: The authors' objective was to test whether the transplantation of an autologous lymph node into a nodal excision site in sheep would restore lymphatic transport function and reduce the magnitude of postsurgical lymphedema. METHODS: As a measure of lymph transport, iodine-125 human serum albumin was injected into prenodal vessels at 8 and 12 weeks after surgery, and plasma levels of the protein were used to calculate the transport rate of the tracer to blood (percent injected per hour). Edema was quantified from the circumferential measurement of the hind limbs. RESULTS: The transplantation of avascular lymph nodes at 8 (n = 6) and 12 weeks (n = 6) produced lymphatic function levels of 12.3 +/- 0.5 and 12.6 +/- 0.8, respectively. These values were significantly less (p < 0.001) than those measured at similar times in the animals receiving sham surgical procedures (16.6 +/- 0.7, n = 6; and 16.1 +/- 0.7, n = 6, respectively). When vascularized transplants were performed, lymphatic function was similar to the sham controls and significantly greater (p < 0.001) than that of the avascular group (8 weeks, 15.8 +/- 0.9, n = 8; 12 weeks, 15.7 +/- 1.0, n = 10). Lymph transport correlated significantly with the health of the transplanted nodes (scaled with histologic analysis) (p < 0.0001). The vascularized node transplants (n = 18) were associated with the greatest clinical improvement, with the magnitude of edema in these limbs exhibiting significantly lower levels of edema (p = 0.039) than nontreated limbs (n = 18). CONCLUSIONS: The successful reimplantation of a lymph node into a nodal excision site has the potential to restore lymphatic function and facilitate edema resolution. This result has important conceptual implications in the treatment of postsurgical lymphedema.

Lymphedema development and lymphatic function following lymph node excision in sheep.

BACKGROUND: Our objective was to develop an animal model of postsurgical lymphedema that would permit quantitation of edema and lymphatic function after the removal of a single popliteal lymph node in sheep. METHODS: As a measure of lymph transport, (125)I-human serum albumin was injected into prenodal vessels at 8, 12 and 16 weeks after nodal excision, and plasma levels of the protein tracer were used to calculate the transport rate of the tracer to blood (percent injected per hour). Edema was quantified from the circumferential measurement of the hind limbs. RESULTS AND CONCLUSIONS: Following nodal excision, the limbs became progressively more edematous up to 3 days after nodectomy. After this, the swelling decreased but had not resolved even at 16 weeks after surgery. Compared with control limbs (17.2 +/- 0.6; n = 7), lymphatic function was depressed at 8 weeks after surgery (10.6 +/- 1.5; n = 7). At 12 (14.4 +/- 1.0; n = 7) and 16 weeks (13.9 +/- 1.0; n = 6), regeneration of lymphatic vessels at the excision site helped to restore about 80% of lymphatic capacity. These techniques may be helpful in understanding the pathophysiology associated with cancer-related postsurgical lymphedema and may facilitate the development of new strategies to treat or prevent this condition.