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Personal lifestyle and working conditions are closely linked for long haul truck drivers. We compare lifestyle related diseases in long haul drivers with other drivers of goods and the working population at large. Standardized hospital treatment ratios (SHR) for lifestyle related diseases were compared for long haul truck drivers and other truck drivers to the working population at large. The follow up group comprised of 2,175 long haul drivers and 15,060 other truck drivers. An increased risk was found for lifestyle related diseases among truck drivers except for alcohol related diseases. We identified a strong association between hospital treatment for obesity and working as a driver and an association between diabetes and working as a driver. No major differences in lifestyle related diseases were found in long haul drivers compared to other truck drivers with the exception of a significant lower risk for alcohol-related diseases and a possibly higher risk for lung cancer in long haul drivers. It is concluded that diseases related to excess caloric intake or lack of exercise may be a problem for truck drivers. This risk is preventable and of importance both in occupational medicine as in public health. [ABSTRACT FROM AUTHOR]

Background: We studied musculoskeletal diseases (MSD) and injuries among fishermen and seamen with focus on low back disorders, carpal tunnel syndrome (CTS), rotator cuff syndrome and arthrosis.Methods: Cohorts of all male Danish seamen (officers and non-officers) and fishermen employed 1994 and 1999 with at least six months employment history were linked to the Occupational Hospitalisation Register. We calculated standardised incidence ratios (SIR) for the two time periods, using rates for the entire Danish workforce as a reference.Results: Among fishermen, we found high SIRs for knee arthrosis, thoraco-lumbar disc disorders, injuries and statistically significant SIRs above 200 were seen for both rotator cuff syndrome and CTS. The SIR was augmented for injuries and reduced for hip arthrosis between the two time periods. The SIRs for injuries and CTS were high for non-officers. A sub-analysis revealed that the highest risk for CTS was found among male non-officers working as deck crew, SIR 233 (95% CI: 166-317) based on 40 cases. Among officers, the SIRs for injuries and MSDs were low. The number of employed Danish fishermen declined with 25% 1994-1999 to 3470. Short-term employments were common. None of the SIRs increased with increasing length of employment.Conclusion: Both fishermen and non-officers have high SIRs for injuries and fishermen also for MSD. Only the SIR for injuries among fishermen was augmented between 1994 and 1999. Our findings suggest an association between the incidence of rotator cuff syndrome and CTS and work within fishery. Long-term cumulative effects of employment were not shown for any of the disease outcomes. Other conditions may play a role. [ABSTRACT FROM AUTHOR]

Aims: In this proof-of-concept study, we propose a new method for automated digital quantification of PRAME (PReferentially expressed Antigen of MElanoma) as a diagnostic aid to distinguish between benign and malignant melanocytic lesions. The proposed method utilizes immunohistochemical virtual double nuclear staining for PRAME and SOX10 to precisely identify the melanocytic cells of interest, which is combined with digital image analyse to quantify a PRAME-index., Methods: Our study included 10 compound nevi, 3 halo nevi, and 10 melanomas. Tissue slides were stained with PRAME, scanned, the cover glass removed, stained with SOX10, scanned again, and finally analysed digitally. The digitally quantified PRAME-index was compared with a manual qualitative assessment by a dermatopathologist using the standard PRAME-scoring system., Results: The digitally quantified PRAME-index showed a sensitivity of 70 % and a specificity of 100 % for separating melanomas from benign lesions. The manual qualitative PRAME-score showed a sensitivity of 60 % and a specificity of 100 %. Comparing the two methods using ROC-analyses, our digital quantitative method (AUC: 0.931, 95 % CI: 0.834;1.00, SD: 0.050) remains on par with the manual qualitative method (AUC: 0.877, 95 % CI: 0.725;1.00, SD: 0.078)., Conclusion: We found our novel digital quantitative method was at least as precise at classifying lesions as benign or malignant as the current manual qualitative assessment. Our method has the advantages of being operator-independent, objective, and replicable. Furthermore, our method can easily be implemented in an already digitalized pathology department. Given the small cohort size, more studies are to be done to validate our findings., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mette Bak Brogard reports financial support was provided by Harboefonden. Mette Bak Brogard reports financial support was provided by Torben and Alice Frimodts Fund. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Aims: Pathologists often use immunohistochemical staining of the proliferation marker Ki67 in their diagnostic assessment of melanocytic lesions. However, the interpretation of Ki67 can be challenging. We propose a new workflow to improve the diagnostic utility of the Ki67-index. In this workflow, Ki67 is combined with the melanocytic tumour-cell marker SOX10 in a Ki67/SOX10 double nuclear stain. The Ki67-index is then quantified automatically using digital image analysis (DIA). The aim of this study was to optimise and test three different multiplexing methods for Ki67/SOX10 double nuclear staining., Methods: Multiplex immunofluorescence (mIF), multiplex immunohistochemistry (mIHC), and multiplexed immunohistochemical consecutive staining on single slide (MICSSS) were optimised for Ki67/SOX10 double nuclear staining. DIA applications were designed for automated quantification of the Ki67-index. The methods were tested on a pilot case-control cohort of benign and malignant melanocytic lesions (n = 23)., Results: Using the Ki67/SOX10 double nuclear stain, malignant melanocytic lesions could be completely distinguished from benign lesions by the Ki67-index. The Ki67-index cut-offs were 1.8% (mIF) and 1.5% (mIHC and MICSSS). The AUC of the automatically quantified Ki67-index based on double nuclear staining was 1.0 (95% CI: 1.0;1.0), whereas the AUC of conventional Ki67 single-stains was 0.87 (95% CI: 0.71;1.00)., Conclusions: The novel Ki67/SOX10 double nuclear stain highly improved the diagnostic precision of Ki67 interpretation. Both mIHC and mIF were useful methods for Ki67/SOX10 double nuclear staining, whereas the MICSSS method had challenges in the current setting. The Ki67/SOX10 double nuclear stain shows potential as a valuable diagnostic aid for melanocytic lesions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Mette Bak Brogaard reports financial support was provided by Harboe Foundation. Patricia Switten Nielsen reports financial support was provided by Toyota-Fonden, Denmark. Patricia Switten Nielsen reports financial support was provided by Dagmar Marshall Fund. Patricia Switten Nielsen reports financial support was provided by Health Research Foundation of Central Denmark Region. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Background: Systemic inflammation, diagnostically ascribed by measuring serum levels of the acute phase reactant C-reactive protein (CRP), has consistently been correlated with poor outcomes across cancer types. CRP exists in two structurally and functionally distinct isoforms, circulating pentameric CRP (pCRP) and the highly pro-inflammatory monomeric isoform (mCRP). The aim of this pilot study was to map the pattern of mCRP distribution in a previously immunologically well-defined colon cancer (CC) cohort and explore possible functional roles of mCRP within the tumor microenvironment (TME)., Methods: Formalin-fixed, paraffin-embedded (FFPE) tissue samples from 43 stage II and III CC patients, including 20 patients with serum CRP 0-1 mg/L and 23 patients with serum CRP >30 mg/L were immunohistochemically (IHC) stained with a conformation-specific mCRP antibody and selected immune and stromal markers. A digital analysis algorithm was developed for evaluating mCRP distribution within the primary tumors and adjacent normal colon mucosa., Results: mCRP was abundantly present within tumors from patients with high serum CRP (>30 mg/L) diagnostically interpreted as being systemically inflamed, whereas patients with CRP 0-1 mg/L exhibited only modest mCRP positivity (median mCRP per area 5.07‰ (95%CI:1.32-6.85) vs. 0.02‰ (95%CI:0.01-0.04), p<0.001). Similarly, tissue-expressed mCRP correlated strongly with circulating pCRP (Spearman correlation 0.81, p<0.001). Importantly, mCRP was detected exclusively within tumors, whereas adjacent normal colon mucosa showed no mCRP expression. Double IHC staining revealed colocalization of mCRP with endothelial cells and neutrophils. Intriguingly, some tumor cells also colocalized with mCRP, suggesting a direct interaction or mCRP expression by the tumor itself., Conclusion: Our data show that the pro-inflammatory mCRP isoform is expressed in the TME of CC, primarily in patients with high systemic pCRP values. This strengthens the hypothesis that CRP might not only be an inflammatory marker but also an active mediator within tumors., 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 Køstner, Fuglestad, Georgsen, Nielsen, Christensen, Zibrandtsen, Parner, Rajab, Potempa, Steiniche and Kersten.)

Deep learning for the analysis of H&E stains requires a large annotated training set. This may form a labor-intensive task involving highly skilled pathologists. We aimed to optimize and evaluate computer-assisted annotation based on digital dual stains of the same tissue section. H&E stains of primary and metastatic melanoma ( N = 77) were digitized, re-stained with SOX10, and re-scanned. Because images were aligned, annotations of SOX10 image analysis were directly transferred to H&E stains of the training set. Based on 1,221,367 annotated nuclei, a convolutional neural network for calculating tumor burden (CNN TB ) was developed. For primary melanomas, precision of annotation was 100% (95%CI, 99% to 100%) for tumor cells and 99% (95%CI, 98% to 100%) for normal cells. Due to low or missing tumor-cell SOX10 positivity, precision for normal cells was markedly reduced in lymph-node and organ metastases compared with primary melanomas ( p < 0.001). Compared with stereological counts within skin lesions, mean difference in tumor burden was 6% (95%CI, -1% to 13%, p = 0.10) for CNN TB and 16% (95%CI, 4% to 28%, p = 0.02) for pathologists. Conclusively, the technique produced a large annotated H&E training set with high quality within a reasonable timeframe for primary melanomas and subcutaneous metastases. For these lesion types, the training set generated a high-performing CNN TB , which was superior to the routine assessments of pathologists.

Spinal cord injury (SCI) initiates detrimental cellular and molecular events that lead to acute and delayed neuroinflammation. Understanding the role of the inflammatory response in SCI requires insight into the temporal and cellular synthesis of inflammatory mediators. We subjected C57BL/6J mice to SCI and investigated inflammatory reactions. We examined activation, recruitment, and polarization of microglia and infiltrating immune cells, focusing specifically on tumor necrosis factor (TNF) and its receptors TNFR1 and TNFR2. In the acute phase, TNF expression increased in glial cells and neuron-like cells, followed by infiltrating immune cells. TNFR1 and TNFR2 levels increased in the delayed phase and were found preferentially on neurons and glial cells, respectively. The acute phase was dominated by the infiltration of granulocytes and macrophages. Microglial/macrophage expression of Arg1 increased from 1-7 days after SCI, followed by an increase in Itgam , Cx3cr1 , and P2ry12 , which remained elevated throughout the study. By 21 and 28 days after SCI, the lesion core was populated by galectin-3 + , CD68 + , and CD11b + microglia/macrophages, surrounded by a glial scar consisting of GFAP + astrocytes. Findings were verified in postmortem tissue from individuals with SCI. Our findings support the consensus that future neuroprotective immunotherapies should aim to selectively neutralize detrimental immune signaling while sustaining pro-regenerative processes.

Air pollution is a global challenge, and especially urban areas are particularly affected by acute episodes. Traditional approaches used to mitigate air pollution primarily consider the technical aspects of the problem but not the role of citizen behaviour and day-to-day practices. ClairCity, a Horizon 2020 funded project, created an impact assessment framework considering the role of citizen behaviour to create future scenarios, aiming to improve urban environments and the wellbeing and health of its inhabitants. This framework was applied to six pilot cases: Bristol, Amsterdam, Ljubljana, Sosnowiec, Aveiro Region and Liguria Region, considering three-time horizons: 2025, 2035 and 2050. The scenarios approach includes the Business As Usual (BAU) scenario and a Final Unified Policy Scenarios (FUPS) established by citizens, decision-makers, local planners and stakeholders based on data collected through a citizen and stakeholder co-creation process. Therefore, this paper aims to present the ClairCity outcomes, analysing the quantified impacts of selected measures in terms of emissions, air quality, population exposure, and health. Each case study has established a particular set of measures with different levels of ambition, therefore different levels of success were achieved towards the control and mitigation of their specific air pollution problems. The transport sector was the most addressed by the measures showing substantial improvements for NO 2 , already with the BAU scenarios, and overall, even better results when applying the citizen-led FUPS scenarios. In some cases, due to a lack of ambition for the residential and commercial sector, the results were not sufficient to fulfil the WHO guidelines. Overall, it was found in all cities that the co-created scenarios would lead to environmental improvements in terms of air quality and citizens' health compared to the baseline year of 2015. However, in some cases, the health impacts were lower than air quality due to the implementation of the measures not affecting the most densely populated areas. Benefits from the FUPS comparing to the BAU scenario were found to be highest in Amsterdam and Bristol, with further NO 2 and PM10 emission reductions around 10%-16% by 2025 and 19%-28% by 2050, compared to BAU., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Systemic inflammation measured by the acute-phase protein CRP associates with poor outcome across cancer types. In contrast, local tumor-associated inflammation, primarily evaluated by T-lymphocytes, correlates with favorable prognosis. Yet, little is known whether these two responses are related or opposing processes and why elevated CRP in relation to cancer is detrimental for clinical outcome. As proof of concept, we developed a platform combining multiplexed IHC and digital imaging, enabling a virtual readout of both lymphoid and myeloid immune markers and their spatial patterns in the primary tumors of resected stage II and III colon cancer (CC) patients with and without accompanying systemic inflammation. Twenty-one patients with elevated CRP (>30 mg/l) and 15 patients with low CRP (<10 mg/l) were included in the analyses. Whole slides from the primary tumors were stained for markers of adaptive (CD8+, CD4+, foxp3 regulatory T cells, CD20+ B cells) and innate (CD68+ macrophages, CD66b+ neutrophils) immunity and the immune checkpoint molecule PD-L1. Associations between individual immune markers, preoperative CRP values, mismatch repair status (MMR), and risk of recurrence or death were assessed. Unsupervised hierarchical clustering was used to explore whether distinct immune phenotypes were present. Tumors from systemically inflamed patients (CRP >30 mg/l) displayed significantly more myeloid features in terms of higher densities of CD66b+neutrophils (p = 0.001) and CD68+macrophages (p = 0.04) and less lymphoid features (lower CD8 T cell, p = 0.03, and foxp3 regulatory T cell densities, p = 0.03) regardless of MMR status. Additionally, systemically inflamed patients harbored lower mean distances between neutrophils and tumor cells within the TME. Intriguingly, microsatellite instable (MSI) tumor status correlated with systemic inflammation. However, using a combinatorial approach, we found that regardless of an adaptive composite score (compounded CD4+ and CD8+ T cells), a high innate score (CD66b+ neutrophils and CD68+ macrophages) associated significantly with elevated CRP. In conclusion, tumor-associated systemic inflammation correlated with a myeloid-dominated TME in a small cohort of resectable CC patients. Our data highlight the importance of a comprehensive immune classification of tumors including players of innate immunity and support a role for CRP as an informative biomarker of the immune response taking place at the tumor site., 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 Køstner, Nielsen, Georgsen, Parner, Nielsen, Kersten and Steiniche.)

Background: Angiogenesis is prominent in metastatic renal cell carcinoma (mRCC). We compared two angiogenesis assessment methods: dynamic contrast-enhanced computed tomography (DCE-CT)-derived blood volume (BV) and blood flow (BF) and core biopsy microvessel density (MVD)., Methods: As planned in DaRenCa Study-1 study, DCE-CT and core biopsy were performed from the same tumour/metastasis at baseline. MVD was assessed by CD34 immunostaining in tumour (CD34-index T ) or tumour including necrosis (CD34-index TN ). BV and BF were assessed using the DCE-CT software. Overall survival (OS) and progression-free survival (PFS) were assessed by Kaplan-Meier analysis. Spearman coefficient (rho) tested the correlation between MVD and BV, BF, or CT density (HU)., Results: At baseline, 25 patients had analysable scans and tissue. BV deconv , BV Patlak , and BF deconv > median were associated with favourable OS (43.2 versus 14.6 months, p = 0.002; 31.6 versus 20.2 months, p = 0.015; and 31.6 versus 24.5 months, p = 0.019). CD34-index T and CD34-index TN did not correlate with age (p = 0.543), sex (p = 0.225), treatment (p = 0.848), International mRCC Database Consortium category (p = 0.152), synchronous versus metachronous metastatic disease (p = 0.378), or tumour volume (p = 0.848). CD34-index T or CD34-index TN > median was not associated with PFS (p = 0.441 and p = 0.854, respectively) or OS (p = 0.987 and p =0.528, respectively). CD34-index T or CD34-index TN was not correlated with BV, BF, or HU (rho 0.20-0.26)., Conclusions: Differently from MVD, DCE-CT-derived BV and BF had prognostic impact and may better reflect angiogenesis in mRCC., Trial Registration: NCT01274273., (© 2021. The Author(s).)

Spinal cord injury (SCI) is a devastating condition consisting of an instant primary mechanical injury followed by a secondary injury that progresses for weeks to months. The cytokine tumor necrosis factor (TNF) plays an important role in the pathophysiology of SCI. We investigated the effect of myeloid TNF ablation (peripheral myeloid cells (macrophages and neutrophils) and microglia) versus central myeloid TNF ablation (microglia) in a SCI contusion model. We show that TNF ablation in macrophages and neutrophils leads to reduced lesion volume and improved functional outcome after SCI. In contrast, TNF ablation in microglia only or TNF deficiency in all cells had no effect. TNF levels tended to be decreased 3 h post-SCI in mice with peripheral myeloid TNF ablation and was significantly decreased 3 days after SCI. Leukocyte and microglia populations and all other cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IFNγ) and chemokines (CCL2, CCL5, and CXCL1) investigated, in addition to TNFR1 and TNFR2, were comparable between genotypes. Analysis of post-SCI signaling cascades demonstrated that the MAPK kinase SAPK/JNK decreased and neuronal Bcl-XL levels increased post-SCI in mice with ablation of TNF in peripheral myeloid cells. These findings demonstrate that peripheral myeloid cell-derived TNF is pathogenic in SCI.

Immune therapy improves cancer outcomes, yet many patients do not respond. This pre-clinical study investigated whether vascular disrupting agents (VDAs) could convert an immune unresponsive tumor into a responder. CDF1 mice, with 200 mm 3 C3H mammary carcinomas in the right rear foot, were intraperitoneally injected with combretastatin A-4 phosphate (CA4P), its A-1 analogue OXi4503, and/or checkpoint inhibitors (anti-PD-1, PD-L1, or CTLA-4 antibodies), administered twice weekly for two weeks. Using the endpoint of tumor growth time (TGT5; time to reach five times the starting volume), we found that none of the checkpoint inhibitors (10 mg/kg) had any effect on TGT5 compared to untreated controls. However, CA4P (100 mg/kg) or OXi4503 (5-50 mg/kg) did significantly increase TGT5. This further significantly increased by combining the VDAs with checkpoint inhibitors, but was dependent on the VDA, drug dose, and inhibitor. For CA4P, a significant increase was found when CA4P (100 mg/kg) was combined with anti-PD-L1, but not with the other two checkpoint inhibitors. With OXi4503 (50 mg/kg), a significant enhancement occurred when combined with anti-PD-L1 or anti-CTLA-4, but not anti-PD-1. We observed no significant improvement with lower OXi4503 doses (5-25 mg/kg) and anti-CTLA-4, although 30% of tumors were controlled at the 25 mg/kg dose. Histological assessment of CD4/CD8 expression actually showed decreased levels up to 10 days after treatment with OXi4503 (50 mg/kg). Thus, the non-immunogenic C3H mammary carcinoma was unresponsive to checkpoint inhibitors, but became responsive in mice treated with VDAs, although the mechanism remains unclear., Competing Interests: All authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

This study evaluated whether the reduction of prefrontal cortex oxygenation (ScO 2 ) during maximal exercise depends on the hyperventilation-induced hypocapnic attenuation of middle cerebral artery blood velocity (MCA V mean ). Twelve endurance-trained males (age: 25 ± 3 years, height: 183 ± 8 cm, weight: 75 ± 9 kg; mean ± SD) performed in three separate laboratory visits, a maximal oxygen uptake (VO 2 max) test, an isocapnic (end-tidal CO 2 tension (PetCO 2 ) clamped at 40 ± 1 mmHg), and an ambient air controlled-pace constant load high-intensity ergometer cycling to exhaustion, while MCA V mean (transcranial Doppler ultrasound) and ScO 2 (near-infrared spectroscopy) were determined. Duration of exercise (12 min 25 s ± 1 min 18 s) was matched by performing the isocapnic trial first. Pulmonary VO 2 was 90 ± 6% versus 93 ± 5% of the maximal value (P = .012) and PetCO 2 40 ± 1 versus 34 ± 4 mmHg (P < .05) during the isocapnic and control trials, respectively. During the isocapnic trial MCA V mean increased by 16 ± 13% until clamping was applied and continued to increase (by 14 ± 28%; P = .017) until the end of exercise, while there was no significant change during the control trial (P = .071). In contrast, ScO 2 decreased similarly in both trials (-3.2 ± 5.1% and -4.1 ± 9.6%; P < .001, isocapnic and control, respectively) at exhaustion. The reduction in prefrontal cortex oxygenation during maximal exercise does not depend solely on lowered cerebral blood flow as indicated by middle cerebral blood velocity., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Telomerase is reactivated in most cancers and is possibly an early driver event in melanoma. Our aim was to test a novel in situ hybridization technique, RNAscope, for the detection of human telomerase reverse transcriptase (hTERT) mRNA in archival formalin-fixed, paraffin-embedded (FFPE) tissue and to compare the mRNA expression of melanomas and benign naevi. Furthermore, we wanted to see if hTERT mRNA could be a diagnostic or prognostic marker of melanoma. In situ hybridization for the detection of hTERT mRNA was performed on FFPE tissue of 17 melanomas and 13 benign naevi. We found a significant difference in the expression of hTERT mRNA between melanomas and benign naevi (P<0.001) and the expression of hTERT mRNA correlated with Breslow thickness (ρ=0.56, P=0.0205) and the Ki67 proliferation index (ρ=0.72, P=0.001). This study showed that RNAscope was a reliable in situ hybridization method for the detection of hTERT mRNA in FFPE tissue of melanomas and benign naevi. hTERT mRNA was more abundantly expressed in melanomas compared with benign naevi, but cannot be used solely as a diagnostic marker due to an overlap in expression. The hTERT mRNA expression in melanomas correlated with the prognostic markers Breslow thickness and the Ki67 index indicating a prognostic potential of hTERT mRNA.

The range of hosts exploited by a parasite is determined by several factors, including host availability, infectivity and exploitability. Each of these can be the target of natural selection on both host and parasite, which will determine the local outcome of interactions, and potentially lead to coevolution. However, geographical variation in host use and specificity has rarely been investigated. Maculinea (= Phengaris) butterflies are brood parasites of Myrmica ants that are patchily distributed across the Palæarctic and have been studied extensively in Europe. Here, we review the published records of ant host use by the European Maculinea species, as well as providing new host ant records for more than 100 sites across Europe. This comprehensive survey demonstrates that while all but one of the Myrmica species found on Maculinea sites have been recorded as hosts, the most common is often disproportionately highly exploited. Host sharing and host switching are both relatively common, but there is evidence of specialization at many sites, which varies among Maculinea species. We show that most Maculinea display the features expected for coevolution to occur in a geographic mosaic, which has probably allowed these rare butterflies to persist in Europe. This article is part of the theme issue 'The coevolutionary biology of brood parasitism: from mechanism to pattern'.

Putative cancer stem cell (CSC) markers have arisen from melanoma mouse and in vitro models, but their expression in paraffin embedded patient samples relative to clinical outcome remains largely unexplored. Rather than cells of the tumour bulk, conceivably, CSC drive tumour progression. Accordingly, complete eradication may prevent melanoma relapse. Because elevated tumour-cell proliferation is an established indicator of aggressive disease, this study aimed to investigate the correlation between melanoma recurrence and proliferation of putative CSC that express CD271, CD166, or CD20. Additionally, the expression of these markers was studied in naevi, melanomas, and their recurrence. In melanoma patients, 30 with relapse (cases) and 30 without (controls) were matched for tumour thickness, ulceration, Clark level, subtype, site, gender, and age. One paraffin-embedded section of the patients' primary melanoma (n = 60), relapse (n = 21), and naevus (n = 17) were immunohistochemically double-stained for Ki-67/MART1 and single-stained for CD271, CD166, and CD20. Their whole slide images were aligned as virtual quadruple stains. Image analysis established proliferation indices of each putative stem cell marker and the tumour bulk in addition to the markers' percentage level in tumour areas and the epidermis. In cases vs controls, median dermal proliferation indices (no./mm 2 ) were 211 vs 103 (p = 0.04) for CD271, 512 vs 227 (p = 0.3) for CD166, 184 vs 97 (p = 0.3) for CD20, and 95 vs 103 (p = 0.6) for the tumour bulk. Of additional interest, epidermal CD271 + keratinocytes totalled 8.8% in naevi and 0.98% in melanomas (p = 0.0007). Even though differences between naevi and melanomas also were observed for CD166 in both the epidermis (p = 0.002) and dermis (p = 0.006), they were visually less apparent. CD20 + MART1 + cells were absent in half of the melanomas, and all naevi and relapses. In conclusion, high levels of CD271 + Ki-67 + MART1 + cells were linked to melanoma relapse as opposed to common Ki-67 indices in this particular case-control study. With further investigation, such cells could be potential targets of therapy. Especially, loss of epidermal CD271 + keratinocytes seemed necessary for melanoma development; hence, identification may serve as a diagnostic tool with additional research., (Copyright © 2018 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.)

Interaction between tumour survival tactics and anti-tumour immune response is a major determinant for cancer growth. Regulatory T cells (T-regs) contribute to tumour immune escape, but their role in basal cell carcinoma (BCC) is not understood. The fraction of T-regs among T cells was analysed by immunohistochemistry followed by automated image analysis in facial BCC, peritumoural skin and normal, buttock skin. Quantitative real-time PCR (qRT-PCR) was performed for Foxp3 and cytokines involved in T-reg attraction and T-cell activation. T-regs comprised 45% of CD4-cells surrounding BCC. Foxp3 was highly expressed in BCC, but absent in buttock skin. Unexpectedly, expression of Foxp3 was increased in peritumoural skin, with the FoxP3/CD3 fractions exceeding those of BCC (p = 0.0065). Transforming growth factor (TGF)-β and T-reg chemokine expression was increased in BCC and peritumoural skin, but not in buttock skin, with expression levels correlating with Foxp3. T-regs are abundantly present both in BCC and in peritumoural skin, mediating an immunosuppressed microenvironment permissive for skin cancer

Background: Inability to distinguish melanomas from benign nevi is the most frequent reason for malpractice lawsuits in surgical pathology. Reliable diagnostic tools to support hematoxylin and eosin (H&E) stains and induce diagnostic vigilance are thus highly needed. Because high diagnostic performance recently was showed using automated image analysis, the immunohistochemical proliferation marker Ki67 seems a potential candidate. This study aimed to investigate if this previously presented automated algorithm could have prevented 10 false-negative melanoma diagnoses. In addition, diagnostic utility of another, but narrower, immunohistochemical proliferation marker, phosphohistone H3 (PHH3), was explored., Methods: A total of 10 formalin-fixed paraffin-embedded melanocytic tumors, initially classified as benign or dysplastic but revised as melanomas at metastatic debut, were dual-stained for Ki67/MART1 and PHH3/MART1. A Ki67 index was automatically calculated in epidermis, dermis, a combination of such, and a dermal hot spot. Dermal PHH3/MART1 scores were established semi-automatically., Results: The dermal Ki67 index identified all 10 melanomas, the hot-spot index 8 and the epidermal and combined indices only 2 and 5, respectively. Nine melanomas were PHH3 positive and scores correlated with Ki67., Conclusions: PHH3 added limited information, but supplemental automated Ki67 assessment could possibly have prevented the misdiagnosis of most melanomas had the algorithm been available at the time of diagnosis., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Staging of melanoma includes quantification of a proliferation index, i.e., presumed melanocytic mitoses of H&E stains are counted manually in hot spots. Yet, its reproducibility and prognostic impact increases by immunohistochemical dual staining for phosphohistone H3 (PHH3) and MART1, which also may enable fully automated quantification by image analysis. To ensure manageable workloads and repeatable measurements in modern pathology, the study aimed to present an automated quantification of proliferation with automated hot-spot selection in PHH3/MART1-stained melanomas., Methods: Formalin-fixed, paraffin-embedded tissue from 153 consecutive stage I/II melanoma patients was immunohistochemically dual-stained for PHH3 and MART1. Whole slide images were captured, and the number of PHH3/MART1-positive cells was manually and automatically counted in the global tumor area and in a manually and automatically selected hot spot, i.e., a fixed 1-mm(2) square. Bland-Altman plots and hypothesis tests compared manual and automated procedures, and the Cox proportional hazards model established their prognostic impact., Results: The mean difference between manual and automated global counts was 2.9 cells/mm(2) (P = 0.0071) and 0.23 cells per hot spot (P = 0.96) for automated counts in manually and automatically selected hot spots. In 77 % of cases, manual and automated hot spots overlapped. Fully manual hot-spot counts yielded the highest prognostic performance with an adjusted hazard ratio of 5.5 (95 % CI, 1.3-24, P = 0.024) as opposed to 1.3 (95 % CI, 0.61-2.9, P = 0.47) for automated counts with automated hot spots., Conclusions: The automated index and automated hot-spot selection were highly correlated to their manual counterpart, but altogether their prognostic impact was noticeably reduced. Because correct recognition of only one PHH3/MART1-positive cell seems important, extremely high sensitivity and specificity of the algorithm is required for prognostic purposes. Thus, automated analysis may still aid and improve the pathologists' detection of mitoses in melanoma and possibly other malignancies.

Background: Smoking affects the arterial wall and increases the risk of cardiovascular disease. It also affects the extracellular matrix in skin, causing impaired wound healing. However, little is known about putative molecular changes in the arterial wall. Our aim was to investigate the possible correlation between extracellular matrix content in arterial tissue and cigarette smoking., Methods: We studied the non-atherosclerotic arterial wall of the internal mammary artery from coronary artery by-pass surgery in 13 never-smokers and 11 active smokers. Using histomorphometric methods, the area fraction of collagen stainable material was determined. In addition, proteome analysis of matrix molecules and other proteins was performed., Results: The area fraction of collagen stainable material in smokers vs. never-smokers was 29.1% ± 3.8% vs. 43.3% ± 3.6% (mean ± SEM, p = 0.012) in tunica intima, 39.7% ± 5.5% vs. 56.8% ± 5.6% (mean ± SEM, p = 0.042) in tunica media, and 50.4% ± 3.9% vs. 61.0% ± 3.2% (mean ± SEM, p = 0.046) in tunica adventitia. We discovered significantly lower relative levels of collagen α1(I) (0.68 ± 0.048 vs. 1.02 ± 0.112, mean ± SEM, p = 0.013), collagen α2(I) (0.81 ± 0.046 vs. 1.14 ± 0.118, mean ± SEM, p = 0.038) and decorin (0.64 ± 0.04 vs. 0.98 ± 0.11, mean ± SEM, p = 0.009) in smokers., Conclusions: Arterial tissue from active smokers contains decreased amounts of collagen stainable material, as well as type 1 collagen and decorin. These findings may explain some effects of smoking on the development of cardiovascular disease including compromised remodelling and increased risk of aneurysms., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

The tumour microenvironment in classical Hodgkin's lymphoma (cHL) is characterised by a minor population of neoplastic Hodgkin and Reed-Sternberg cells within a heterogeneous background of non-neoplastic bystanders cells, including mast cells. The number of infiltrating mast cells in cHL has been reported to correlate with poor prognosis. We used immunohistochemistry to assess the degree of tumour-infiltrating mast cells in cHL tissue microarrays and correlated this with clinico-pathological features and prognosis in a cohort of homogeneously treated patients with Hodgkin's disease. A high degree of tumour mast cells was associated with nodular sclerosis (NS) subtype histology (P = 0.0002). Moreover, the number of mast cells was inversely correlated with the numbers of CD68+ and CD163+ macrophages (P = 0.0001 and P = 0.003, respectively) and with the number of granzyme+ cytotoxic cells (P = 0.004). The degree of mast cell infiltration was not a prognostic factor in cHL of nodular sclerosis subtype. In contrast, in mixed cellularity cHL a high number of intratumoral mast cells correlated with significantly poorer outcome both in terms of overall (P = 0.03) and event-free survival (P = 0.01). Further studies are warranted into the biological mechanisms underlying this adverse outcome and their possible therapeutic implications., (© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Cholangiocarcinoma (CCA) is a malignancy of bile duct with the difficulty in early diagnosis, poor prognosis and less alternation in therapy. S100P is a member of S100 family proteins and plays important roles in cancers. We investigated the S100P expression and its correlation with clinicopathology in 78 cases of opisthorchiasis-associated CCA, and the effects of S100P knockdown with shRNA interference on the proliferation, cell cycle, migration, apoptosis and sensitivity to anti-cancer drug. Extremely high expression of S100P mRNA was detected in the CCA tumor tissues. The increased S100P protein expression was immunohistochemically confirmed and localized in the CCA cytoplasm and/or nuclei as well as in the hyperneoplasia and dysplasia bile ducts, but not in normal bile ducts. The intensity of immunostaining was correlated with survival, tumor stage and metastasis, and the high expression could be an independent prognostic factor. High levels of S100P were detected in the serum and bile fluid of CCA patients. The shRNA-mediated knockdown of S100P expression inhibited the proliferation in vitro and in vivo, and migration of CCA cells, arrested cell cycle with the up-regulated expression of cell cycle arrest related factors, p21, p27, GADD45A, and 14-3-3 zeta. S100P knockdown also promoted CCA cell apoptosis by up-regulating expression of apoptosis related factors, DR5, TRADD, caspase 3 and BAX, and increased the sensitivity of CCA cells to the chemotherapeutic agents sunitinib and apigenin. Taken together, this study indicates that S100P might be a promising biomarker for the diagnosis, prognosis and therapy of CCA., (© 2015 UICC.)

Background: The increased risk of cardiovascular diseases in type 2 diabetes mellitus has been extensively documented, but the origins of the association remain largely unknown. We sought to determine changes in protein expressions in arterial tissue from patients with type 2 diabetes mellitus and moreover hypothesized that metformin intake influences the protein composition., Methods and Results: We analyzed nonatherosclerotic repair arteries gathered at coronary bypass operations from 30 patients with type 2 diabetes mellitus and from 30 age- and sex-matched nondiabetic individuals. Quantitative proteome analysis was performed by isobaric tag for relative and absolute quantitation-labeling and liquid chromatography-mass spectrometry, tandem mass spectrometry analysis on individual arterial samples. The amounts of the basement membrane components, α1-type IV collagen and α2-type IV collagen, γ1-laminin and β2-laminin, were significantly increased in patients with diabetes mellitus. Moreover, the expressions of basement membrane components and other vascular proteins were significantly lower among metformin users when compared with nonusers. Patients treated with or without metformin had similar levels of hemoglobin A1c, cholesterol, and blood pressure. In addition, quantitative histomorphometry showed increased area fractions of collagen-stainable material in tunica intima and media among patients with diabetes mellitus., Conclusions: The distinct accumulation of arterial basement membrane proteins in type 2 diabetes mellitus discloses a similarity between the diabetic macroangiopathy and microangiopathy and suggests a molecular explanation behind the alterations in vascular remodeling, biomechanical properties, and aneurysm formation described in diabetes mellitus. The lower amounts of basement membrane components in metformin-treated individuals are compatible with the hypothesis of direct beneficial drug effects on the matrix composition in the vasculature., (© 2015 American Heart Association, Inc.)

Post-transplant lymphoproliferative disorders (PTLDs) are potentially fatal, often Epstein-Barr virus (EBV)-driven neoplasias developing in immunocompromised hosts. Initial treatment usually consists of a reduction in immunosuppressive therapy and/or rituximab with or without chemotherapy. However, patients who relapse do poorly, and new treatment options are warranted. With the introduction of the immunoconjugate brentuximab vedotin, the CD30 antigen has become an effectively targetable molecule. Therefore, we investigated the frequency and level of CD30 expression in PTLDs. We identified 108 patients with PTLDs diagnosed during 1994-2011, of whom 62 had adequate paraffin-embedded tissue for tissue microarray construction. Immunohistochemical expression of CD30 was consistently detected in all types of PTLD (overall 85.25%), including the monomorphic subtypes, and was correlated with a more favorable outcome. For diffuse large B-cell lymphoma (DLBCL)-type PTLD this was regardless of EBV status, and remained significant in multivariate analysis. Cell-of-origin had no independent prognostic value in our series of DLBCL PTLD.

Background: Ki-67 immunohistochemical expression is a prognostic and predictive marker in many breast cancer studies. Instead of the conventional time-consuming score of Ki-67 single stains associated with low reproducibility, Ki-67/KL1 double stains may facilitate fast, repeatable quantification by digital image analysis. This study aims to detect the difference in accuracy and precision between manual indices of single and double stains, to develop an automated quantification of double stains, and to explore the relation between automated indices and tumor characteristics when quantified in different regions: hot spots, global tumor areas, and invasive fronts., Materials and Methods: Paraffin-embedded, formalin-fixed tissue from 100 consecutive patients with invasive breast cancer was immunohistochemically stained for Ki-67 and Ki-67/KL1. Ki-67 was manually scored in different regions by 2 observers and automated image analysis., Results: Indices were predominantly higher for single stains than double stains (P≤0.002), yet the difference between observers was statistically significant (P<0.001) for both stains. The Pearson correlation coefficient for manual and automated indices ranged from 0.69 to 0.85 (P<0.001). Hot spots were slightly superior to other regions when correlating automated indices with tumor characteristics, for example, tumor size (P<0.001), grade (P=0.009), and estrogen receptor status (P=0.04)., Conclusions: Although precision was unsatisfactory for manual indices of both stains, Ki-67 should be quantified on double stains to reach a higher accuracy. Automated indices correlated well with manual estimates and tumor characteristics, and they are thus possibly valuable tools in future exploration of Ki-67 in breast cancer.

Systematic validation of construction and analysis parameters when using tissue microarray (TMA) in rare, morphologically heterogenous entities such as peripheral T-cell lymphoma (PTCL) is not reported. We describe a tissue-saving virtual TMA to predetermine the number of cores needed to represent whole tissue sections (WTS) from the same biopsies, using automated and traditional manual methods for the quantification of immunohistochemical stains. Whole paraffin hematoxylin and eosin- and immunohistochemical (CD2, CD30, and Ki-67)-stained sections from 30 PTCLs were digitalized. A virtual TMA with six 1-mm cores per slide was designed to compare agreements in the immunohistochemical scoring. Using digital image analysis and manual stereological counting, immunohistochemical positivity was quantified. Associations were analyzed using the Bland-Altman and correlation plots. In PTCL, we report that 4 cores are required to represent WTS results (ie, agreement within ±10%). High concordance was demonstrated between digital results obtained with WTS compared with 4-core virtual TMA (correlation coefficients: 0.89-0.98), and in the comparative evaluation of 4-core virtual TMA by digital image analysis versus manual stereology (correlation coefficients: 0.91 to 0.99). Virtual TMAs provide an efficient tool for optimizing and validating TMA construction parameters when planning a study. The method can be applied to the same tissues used in a subsequent formal study, without wasting scarce tissue resources. In PTCL, TMAs constructed with four 1-mm cores are representative of WTS. In parallel tests using TMAs and WTS from PTCLs, there is a high level of agreement comparing automated digital with manual stereological methods for the quantification of immunohistochemical biomarker staining.

The MART1-verified Ki-67 proliferation index is a valuable aid to distinguish melanomas from nevi. Because such indices are quantifiable by image analysis, they may provide a novel automated diagnostic aid. This study aimed to validate the diagnostic performance of automated dermal Ki-67 indices and to explore the diagnostic capability of epidermal Ki-67 in lesions both with and without a dermal component. In addition, we investigated the automated indices' ability to predict sentinel lymph node (SLN) status. Paraffin-embedded tissues from 84 primary cutaneous melanomas (35 with SLN biopsy), 22 melanoma in situ, and 270 nevi were included consecutively. Whole slide images were captured from Ki-67/MART1 double stains, and image analysis computed Ki-67 indices for epidermis and dermis. In lesions with a dermal component, the area under the receiver operating characteristic (ROC) curve was 0.79 (95% confidence interval [CI], 0.72-0.86) for dermal indices. By excluding lesions with few melanocytic cells, this area increased to 0.93 (95% CI, 0.88-0.98). A simultaneous analysis of epidermis and dermis yielded an ROC area of 0.94 (95% CI, 0.91-0.96) for lesions with a dermal component and 0.98 (95% CI, 0.97-1.0) for lesions with a considerable dermal component. For all lesions, the ROC area of the simultaneous analysis was 0.89 (95% CI, 0.85-0.92). SLN-positive patients generally had a higher index than SLN-negative patients (P ≤ .003). Conclusively, an automated diagnostic aid seems feasible in melanocytic pathology. The dermal Ki-67 index was inferior to a combined epidermal and dermal index in diagnosis but valuable for predicting the SLN status of our melanoma patients., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Milk fat globule epidermal growth factor 8 (MFG-E8) is a pleiotropic secreted glycoprotein to play roles in mediating immune tolerance and homeostasis maintenance and enhancing angiogenesis. To evaluate its value as a biomarker in opisthorchiasis-associated cholangiocarcinoma (CCA), the present study investigated MFG-E8 expression kinetics during the tumorigenesis in Opisthorchis viverrini infection-induced CCA, and demonstrated its expression in the tumor tissues of CCA patients and its serum level among them. During the tumorigenesis of CCA, MFG-E8 expression was increased in a time-dependent manner with the pathological processes. Absolutely higher expression of MFG-E8 messenger RNA was detected in the tumor tissues from CCA patients, compared with those in adjacent tissues. Immunobiochemical analysis showed that more than 90% CCA cases were positive and the positive reaction located in the membrane and cytoplasm of the tumor cells. Moreover, the average serum level in the CCA patients was significantly higher than that in healthy individuals and those with O. viverrini infection or other parasitosis. Correlation analysis of MFG-E8 expression with CCA clinicopathology revealed that a high expression of MFG-E8 protein was significantly bound with a poor differentiation, pathological advanced stage, and metastasis of CCA. The multivariation analysis indicated that MFG-E8 was an independent prognostic factor. In addition, short hairpin RNA-mediated MFG-E8 knockdown in CCA cell line obviously suppressed the cell proliferation. Our results strongly suggested that MFG-E8 is a promising biomarker for the diagnosis, prognosis, and therapy target of opisthorchiasis-associated CCA.

Introduction: A tumor-promoting impact of neutrophils and macrophages has been demonstrated in some cancers. However, the prognostic significance of innate immune cells in patients with non-small cell lung cancer (NSCLC) is unclear., Methods: A total of 335 consecutive patients resected for stage I-IIIA NSCLC were assessed for CD66b(+) neutrophils and CD163(+) macrophages in the tumor nests and adjacent stromal tissue by immunohistochemistry in whole tissue sections using stereology as well as automatic computerized quantification. Findings were correlated with clinical and histopathological parameters, baseline blood inflammatory markers (C-reactive protein (CRP) and white blood cell count (WBC)). Endpoints were recurrence-free survival (RFS) and overall survival (OS)., Results: Elevated CRP above median (101 nmol/l) and WBC above median (8.6 × 10(9)cells/l) were associated with poor RFS (p ≤ 0.002) and poor OS (p ≤ 0.01). Higher density of CD66b(+) in tumor nests and stroma was associated with elevated CRP and WBC, squamous cell histology, tumor size, and necrosis (p ≤ 0.01). Higher density of CD163(+) macrophages in tumor nests and stroma was associated with elevated CRP and lymph node metastases (p ≤ 0.049). The densities of tumor nest CD66b(+) neutrophils and CD163(+) macrophages were not significantly correlated with RFS or OS, irrespective of assessment method., Conclusions: The densities of tumor-associated CD66b(+) neutrophils and CD163(+) macrophages in NSCLC were correlated with adverse prognostic factors and systemic blood inflammation markers, but not directly correlated with RFS or OS. Further research of chronic inflammation in NSCLC is warranted., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Cellular proliferation is correlated with the progression of melanoma. Accordingly, the proliferation index of H&E-stained thin melanomas was recently included in the staging system of the American Joint Committee on Cancer. Yet, the immunohistochemical markers of proliferation phosphohistone H3 and Ki67 may improve such indices. To accurately quantify these markers, they should be combined with a melanocytic marker, for example, MART1 in an immunohistochemical double stain; also enabling automated quantification by image analysis. The aim of the study was to compare the prognostic impact of phosphohistone H3/MART1, Ki67/MART1, and H&E stains in primary cutaneous melanoma, and to determine the difference between indices established in hot spots and the global tumor areas. The study included 153 consecutive stage I/II melanoma-patients. The follow-up time was 8-14 years for event-free melanoma. Recurrent disease occurred in 43 patients; 37 died of melanoma. Both events occurred in only three thin melanomas. Their paraffin-embedded tissue was stained for phosphohistone H3/MART1, Ki67/MART1, and with H&E. And proliferation indices were established in 1-mm(2) hot spots and in the global tumor areas. In multivariate Cox analyses, only hot spot indices of phosphohistone H3/MART1 and Ki67/MART1 were independent prognostic markers. Phosphohistone H3/MART1 tended to be better than Ki67/MART1 with adjusted hazard ratios of 3.66 (95% CI, 1.40-9.55; P=0.008) for progression-free survival and 3.42 (95% CI, 1.29-9.04; P=0.013) for melanoma-specific death. In all stains, prognostic performance was substantially improved by using hot spots instead of the global tumor areas. In conclusion, phosphohistone H3/MART1 and Ki67/MART1 were superior to H&E stains, and hot spots superior to the global tumor areas. Given the potential for automated analysis, these double stains seem to be robust alternatives to conventional mitotic detection by H&E in stage I/II melanomas in general. This was particularly true for thick melanomas whereas no specific analyses for thin melanomas only could be performed.

Our previous report showed that platelet-derived growth factor (PDGF) and related genes were upregulated in a Syrian hamster model and could be detected in all human cholangiocarcinoma (CCA) tissues. We therefore hoped that PDGF could be used as a diagnostic and prognostic marker. We analyzed 78 samples of human CCA and adjacent tissues for PDGF and related gene expression, and localized PDGF protein expression. The mechanism of anti-cancer drugs on PDGF and related genes or proteins in CCA cell lines (OCA17, M156, and KKU100) was studied through MTT cell viability assay, quantitative real-time PCR, and immunoblotting. Mutagenesis of the PDGFRA coding region was analyzed. Moreover, the PDGFRA in sera of CCA patients and healthy controls was investigated. PDGFA was found to be upregulated in CCA tissue (84.6 %). Positive PDGFA immunohistochemical staining was significantly correlated with status (P = 0.000), stage of CCA (P = 0.013), metastasis (P = 0.017), and short survival rate (P = 0.005), and the multivariate analysis confirmed that PDGFA positive immunostaining had a higher likelihood of the risk of death (HR = 2.907, P = 0.016). For DNA point mutation of the PDGFRA sequence, silent mutations were found at tyrosine kinase 2 V824V (exon 18) and A603A (exon 13), and a missense mutation in S478P (exon 10); there was only a missense mutation in S478P (29 %) that has significant correlation with the histopathological grading (P = 0.037) and positive immunoreactive PDGFA (P = 0.021). In vitro cell line study by immunowestern blotting found that sunitinib malate had an inhibitory effect on the PDGFA pathway by decreasing p-PDGFRA, AKT, and p-AKT expression. The serum level of PDGFA in CCA patients was significantly higher than those of healthy control by 1.4-fold (P = 0.014). The present results suggest that PDGFA and PDGFRA may be used for CCA prognosis and/or as diagnostic candidate markers.

Context: The proliferation marker Ki67 is an important diagnostic and prognostic aid in surgical pathology. However, manual quantification in a counting frame to accurately establish the proliferation rate (Ki67 index) is cumbersome and time-consuming. Instead, digital image analysis of Ki67/MART1 double stains may provide fast and novel index computations for entire tumor sections., Objectives: To design and compare image analysis protocols that compute Ki67 indices of Ki67/MART1 double stains, to compare automated indices with previously published manual indices, and to compare the total number of proliferating cells (mimicking a Ki67 single stain) with the number of MART1-verified proliferating cells., Design: Whole slide images were captured from 48 melanomas and 77 nevi stained with an immunohistochemical cocktail against Ki67 and MART1. Ki67 indices were determined by digital image analysis and different equations based on number or area., Results: The differences between mean indices of melanomas and nevi were significant (P < .001) in all index computations. Number-based image analysis of lesions with more than 250 melanocytic cells misclassified 1 of 42 melanomas and 4 of 53 nevi, numbers comparable with manual counting. Automated indices were significantly higher than manual indices, as were indices of mimicked Ki67 single stains compared with MART1-verified Ki67 indices (P < .001)., Conclusions: Ki67 indices established by digital image analysis of Ki67/MART1 double stains demonstrated excellent abilities to discriminate melanomas from nevi with diagnostic performances equal to manually performed indices. Testing different definitions of the automated MART1-verified Ki67 index, no single definition stood out; thus, a variety of definitions may be used.

Background: Fragmentation of terrestrial ecosystems has had detrimental effects on metapopulations of habitat specialists. Maculinea butterflies have been particularly affected because of their specialized lifecycles, requiring both specific food-plants and host-ants. However, the interaction between dispersal, effective population size, and long-term genetic erosion of these endangered butterflies remains unknown. Using non-destructive sampling, we investigated the genetic diversity of the last extant population of M. arion in Denmark, which experienced critically low numbers in the 1980s., Results: Using nine microsatellite markers, we show that the population is genetically impoverished compared to nearby populations in Sweden, but less so than monitoring programs suggested. Ten additional short repeat microsatellites were used to reconstruct changes in genetic diversity and population structure over the last 77 years from museum specimens. We also tested amplification efficiency in such historical samples as a function of repeat length and sample age. Low population numbers in the 1980s did not affect genetic diversity, but considerable turnover of alleles has characterized this population throughout the time-span of our analysis., Conclusions: Our results suggest that M. arion is less sensitive to genetic erosion via population bottlenecks than previously thought, and that managing clusters of high quality habitat may be key for long-term conservation.

Distinction between benign and malignant melanocytic lesions may be difficult by today's methods, even for highly skilled dermatopathologists, emphasizing the need for improved diagnostic tools. We have studied the discriminative abilities of immunohistochemical (IHC) double stains using the IHC markers Ki67 combined with MART1, and HMB45 combined with MITF. Paraffin-embedded tissue sections from 50 melanomas and 78 benign nevi were stained using a simple simultaneous IHC double staining technique. Both simple semiquantitative estimates of the immunopositivity in the deepest third of the lesions and full-scale quantitative measurements of the Ki67 and HMB45 indices were performed, and scores for melanomas and nevi were compared. The differences between melanomas and nevi were significant (P < 0.0001) using either analysis or stain. The misclassification rates for melanomas and nevi were generally lower for Ki67/MART1 stains than for HMB45/MITF stains. In the simple semiquantitative Ki67/MART1 analysis, the misclassification rates were 6% (2%-17%) for melanomas and 12% (6%-21%) for nevi. In full-scale quantitative analysis the corresponding rates were 4% (1%-14%) and 8% (4%-16%), and by combining Ki67 and HMB45 indices, the misclassification rates were 0% (0%-7%) for melanomas and 13% (7%-22%) for nevi. We conclude that both semiscale and fullscale quantitative analyses of Ki67/MART1 stains are valuable diagnostic tools to distinguish melanomas and nevi with a large degree of certainty. The HMB45/MITF stains may serve as adjuncts to predict malignancy and the diagnostic potential of combining the HMB45 and Ki67 indices are promising. The IHC double stains may potentially reduce misinterpretations of melanomas in histopathology.

This chapter describes a method for the isolation of intact polyadenylated mRNA using LNA oligo(T) capture. The method enables efficient isolation of poly(A)(+) RNA directly from guanidinium thiocyanate (GuSCN)-containing cell or tissue extract by combining the design of biotinylated LNA oligo(T) capture probes with subsequent immobilization of the captured poly(A)(+) RNA onto streptavidin-coated magnetic particles. In contrast to DNA oligo-dT and polyT PNA based mRNA isolation techniques, the LNA oligo(T) capture method allows poly(A) selection in the presence of 4 M GuSCN cell lysis buffer, which is needed for efficient inactivation of endogenous RNases. In addition, LNA oligo(T) facilitates highly efficient poly(A)(+) isolation at elevated temperatures compared to standard oligo(dT) technology. The successful use of the LNA oligo(T) capture method in recovery of mRNA from human cells and the subsequent use of the mRNA in northern blotting analysis, RT-PCR and qRT-PCR are demonstrated.

Digitization of histologic slides is associated with many advantages, and its use in routine diagnosis holds great promise. Nevertheless, few articles evaluate virtual microscopy in routine settings. This study is an evaluation of the validity and diagnostic performance of virtual microscopy in routine histologic diagnosis of skin tumors. Our aim is to investigate whether conventional microscopy of skin tumors can be replaced by virtual microscopy. Ninety-six skin tumors and skin-tumor-like changes were consecutively gathered over a 1-week period. Specimens were routinely processed, and digital slides were captured on Mirax Scan (Carl Zeiss MicroImaging, Göttingen, Germany). Four pathologists evaluated the 96 virtual slides and the associated 96 conventional slides twice with intermediate time intervals of at least 3 weeks. Virtual slides that caused difficulties were reevaluated to identify possible reasons for this. The accuracy was 89.2% for virtual microscopy and 92.7% for conventional microscopy. All κ coefficients expressed very good intra- and interobserver agreement. The sensitivities were 85.7% (78.0%-91.0%) and 92.0% (85.5%-95.7%) for virtual and conventional microscopy, respectively. The difference between the sensitivities was 6.3% (0.8%-12.6%). The subsequent reevaluation showed that virtual slides were as useful as conventional slides when rendering a diagnosis. Differences seen are presumed to be due to the pathologists' lack of experience using the virtual microscope. We conclude that it is feasible to make histologic diagnosis on the skin tumor types represented in this study using virtual microscopy after pathologists have completed a period of training. Larger studies should be conducted to verify whether virtual microscopy can replace conventional microscopy in routine practice., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Objective: High risks for locomotor diseases have been reported for bus and truck drivers in general; however, little is known about the specific risks of long-haul truck drivers., Methods: Standardised hospitalisation ratios (SHR) for different locomotor diseases of bus drivers, long-haul truck drivers and other truck drivers were compared with each other and with the general Danish working population., Results: Intervertebral disc disorders were more common among professional drivers (SHR: 119, 95% CI: 114-125) and of similar magnitude for cervical and non-cervical disorders. Both long-haul truck drivers and bus drivers had higher SHR for intervertebral disc disorders (SHR: 133, 95% CI: 114-155/SHR: 141, 95% CI: 129-154, respectively) than other truck drivers (SHR: 109, 95% CI: 102-116). The SHR for carpal tunnel syndrome was high among long-haul drivers (SHR: 163, 95% CI: 101-249) and for other truck drivers (SHR: 130, 95% CI: 108-156) compared to bus drivers (SHR: 110, 95% CI: 79-149). All drivers had high SHR for lesions of the ulnar nerve (SHR: 159, 95% CI: 119-207), especially bus drivers (SHR: 197, 95% CI: 116-311). Long-haul truck drivers had high SHRs for synovitis and bursitis (SHR: 150, 95% CI: 105-207), in contrast to other truck drivers and bus drivers (SHR: 103, 95% CI: 89-120/SHR: 87, 95% CI: 66-112, respectively)., Conclusion: All groups of professional drivers experience high risk of various disorders of the locomotor system but type of vehicle and specific working conditions are associated with different health effects on the locomotor system.

Aims: Noise-induced hearing loss (NIHL) is a leading occupational disease and some seafarers and fishermen may be at high risk. We present here standardized hospital contact ratios (SHCRs) for hearing loss among Danish seafarers and fishermen., Materials and Methods: Cohorts of all Danish seafarers registered by the Danish Maritime Authority (DMA) and fishermen retrieved from a 1989-1998 pension registry were linked to the nationwide Occupational Hospitalisation Registry (OHR) with follow-up for NIHL from 1994 to 2003, using rates specific for age and calendar time for the entire Danish workforce as a reference., Results: We found high SHCRs for NIHL: 165 [95% confidence interval (CI) 131-206] among officers, 113 (79-157) for nonofficers and 119 (85-162) for fishermen. The increased SHCR for hearing impairment among seafarers was solely found in engine room personnel (SHCR = 222; 95% CI 178-277). Compared to other seafarers, the engine room personnel had a relative risk ratio of 2.39 (95% CI: 1.74-3.26). Short-term employment is common in many trades. No duration response pattern was observed which may suggest a secondary healthy worker effect., Conclusions: These findings indicate that hearing problems are frequent among men who work in the engine rooms on ships. Long-term cumulative effects of employment were not shown.

Objectives: Seafarers' and fishermen's working conditions may impact on their lifestyle and health. Standardized hospital contact ratios (SHCRs) were compared in two time periods and the relative risks of hospital contact as a function of employment time were estimated., Methods: Cohorts of all Danish seafarers (officers and non-officers) registered by the Danish Maritime Authority (DMA) 1989-98 and fishermen retrieved from a pension registry 1989-98 were linked to the nationwide Occupational Hospitalization Registry (OHR) and followed up for incident diseases in two five-year time periods, from 1 January 1994 and 1 January 1999, respectively, using rates specific for age and calendar time for the entire Danish workforce as a reference., Results: The SHCRs for lung and cardiovascular diseases were high for non-officers. Among male officers, the SHCR for diabetes was high in the 1999 cohort and the SHCR for chronic heart diseases was statistically significantly higher in the 1999 than in the 1994 cohort. For both time periods high SHCR values were found for bronchitis, emphysema, cancer of the lung, alcohol-related liver diseases, and diabetes among male non-officers, and lung cancer among male officers. Among female non-officers, a high SHCR for skin melanomas was seen. Among fishermen high SHCRs for bronchitis, emphysema, lung cancer, and Raynaud's syndrome were found in both cohorts. No duration-response pattern was observed in any of the analyses, which may reflect health- and lifestyle-related selection into the trades or a healthy worker effect., Conclusions: Danish seafarers, especially short-term employees, had an elevated risk of hospitalization for lifestyle-related diseases.

In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues. This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)(+) RNA accumulation within the nucleus/ nucleolus of wild-type cells. LNA-based probes should be readily applicable to a diverse array of cells and tissue samples.

Aims: Seafarers aboard oil and chemical tankers may be exposed to many chemicals, including substances like benzene that are known to be carcinogenic. Other seafarers are exposed to engine exhaust, different oil products, and chemicals used aboard and some years ago asbestos was also used extensively in ships. The aim of this study was to study cancer morbidity among Danish seafarers in relation to type of ship and job title., Methods: A cohort of all Danish seafarers during 1986-1999 (33,340 men; 11,291 women) registered by the Danish Maritime Authority with an employment history was linked with the nationwide Danish Cancer Registry and followed up for cancer until the end of 2002. The number of person years at risk was 517,518. Standardised incidence ratios (SIR) were estimated by use of the corresponding national rates., Results: The SIR of all cancers combined was higher than expected: 1.26 (95% CI 1.19 to 1.32) for men and 1.07 (95% CI 0.95 to 1.20) for women. This was mainly due to an excess of cancer of the larynx, lung, tongue, mouth, pharynx, oesophagus, pancreas, kidney, urinary bladder, colon, and bone as well as skin melanomas among men (the three latter borderline significantly increased), and an excess of cancer of the lung, rectum, and cervix uteri among women. The differences in risk pattern for lung cancer between the different job categories among men ranged in terms of SIR from 1.2 (95% CI 0.9 to 1.7) (engine officers) to 2.3 (1.6 to 3.3) (engine room crew), and 4.1 (2.1 to 7.4) among maintenance crew. Non-officers had a 1.5 times higher lung cancer risk than officers. No increased occurrence of all lymphatic and haematopoietic malignancies combined was found for employees on tankers, but the number of cases was limited to a total of 7., Conclusions: Danish seafarers, especially men, face an increased overall cancer risk, in particular a risk for lung cancer and other tobacco associated cancers.

We have developed a ready-to-spot polymer microarray slide, which is coated with a uniform layer of reactive electrophilic groups using anthraquinone-mediated photo-coupling chemistry. The slide coating reduces the hydrophobicity of the native polymer significantly, thereby enabling robust and efficient one-step coupling of spotted 5' amino-linked oligonucleotides onto the polymer slide. The utility of the coated polymer slide in gene expression profiling was assessed by fabrication of spotted oligonucleotide microarrays using a collection of 5' amino-linked 70-mer oligonucleotide probes representing 96 yeast genes from Operon. Two-colour hybridizations with labelled cDNA target pools derived from standard grown and heat-shocked wild type yeast cells could reproducibly measure heat shock induced expression of seven different heat shock protein (HSP) genes. Moreover, the observed fold changes were comparable to those reported previously using spotted cDNA arrays and high-density 25-mer oligonucleotide arrays from Affymetrix. The low hybridization signals obtained from the DeltaSSA4 mutant cDNA target, together with the high signal detected in two-colour hybridizations with heat-shocked wild type yeast relative to the DeltaSSA4 mutant strain implies that unspecific binding of cDNA target to the SSA4-specific 70-mer oligonucleotide probes is negligible. Combined, our results indicate that the coated polymer microarray slide represents a robust and cost-effective array platform for pre-spotted oligonucleotide arrays.

Identification of regulatory elements directing definite and specific spatiotemporal expression patterns is a prerequisite to the next generation of transgenic plants with commercial and ethical feasibility for producing plantibodies or other pharmaceutically important compounds. Here we describe the functional dissection of the barley nonspecific lipid transfer protein gene promoter, HvLTP2. The gene is specifically expressed in aleurone cells of cereals and used as an aleurone marker in maize and rice. The transcript is uniformly localised in the barley aleurone cells from around 10 DAP. Patchy expression in aleurone cells of transgenic rice has been reported and explained by silencing of transgenes. We have performed deletion analyses of the 801-bp HvLTP2 promoter to gain insight into the molecular basis of its regulation and the presence of putative regulatory elements. From the deletion studies, a 49-bp promoter region directing aleurone-specific expression was identified. Simultaneously, in vivo footprinting was carried out to identify promoter elements bound by putative regulatory proteins. Within the 49-bp fragment, the most promising candidate for a minimal cis-acting regulatory region directing aleurone specificity is the ds-sequence. Based on our results, we hypothesise that the ds-sequence directs aleurone specificity, possibly through a concerted action with elements directing general expression in the seed. Moreover, we present an overview of LTP2 elements putatively involved in directing seed, endosperm, and aleurone expression. Additionally, we report HvLTP2 expression in the embryo, not previously detected. The regulatory element(s) directing expression in embryo is located downstream of the 49-bp fragment directing aleurone specificity, thus demonstrating independent control of aleurone and embryo-localised expression. Finally, we discuss the existence of several endosperm-specific boxes and whether alternative promoter elements and combinations of them may direct aleurone expression, explaining why comparing different genes expressed in aleurone fail to identify only one required, common promoter element.