Infiltration of NK and plasma cells is associated with a distinct immune subset in non-small cell lung cancer.

Immune cells of the tumor microenvironment are central but erratic targets for immunotherapy. The aim of this study was to characterize novel patterns of immune cell infiltration in non-small cell lung cancer (NSCLC) in relation to its molecular and clinicopathologic characteristics. Lymphocytes (CD3+, CD4+, CD8+, CD20+, FOXP3+, CD45RO+), macrophages (CD163+), plasma cells (CD138+), NK cells (NKp46+), PD1+, and PD-L1+ were annotated on a tissue microarray including 357 NSCLC cases. Somatic mutations were analyzed by targeted sequencing for 82 genes and a tumor mutational load score was estimated. Transcriptomic immune patterns were established in 197 patients based on RNA sequencing data. The immune cell infiltration was variable and showed only poor association with specific mutations. The previously defined immune phenotypic patterns, desert, inflamed, and immune excluded, comprised 30, 13, and 57% of cases, respectively. Notably, mRNA immune activation and high estimated tumor mutational load were unique only for the inflamed pattern. However, in the unsupervised cluster analysis, including all immune cell markers, these conceptual patterns were only weakly reproduced. Instead, four immune classes were identified: (1) high immune cell infiltration, (2) high immune cell infiltration with abundance of CD20+ B cells, (3) low immune cell infiltration, and (4) a phenotype with an imprint of plasma cells and NK cells. This latter class was linked to better survival despite exhibiting low expression of immune response-related genes (e.g. CXCL9, GZMB, INFG, CTLA4). This compartment-specific immune cell analysis in the context of the molecular and clinical background of NSCLC reveals two previously unrecognized immune classes. A refined immune classification, including traits of the humoral and innate immune response, is important to define the immunogenic potency of NSCLC in the era of immunotherapy. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

COX-2 expression and effects of celecoxib in addition to standard chemotherapy in advanced non-small cell lung cancer.

AIM: Inhibition of cyclooxygenase-2 (COX-2) is proposed as a treatment option in several cancer types. However, in non-small cell lung cancer (NSCLC), phase III trials have failed to demonstrate a benefit of adding COX-2 inhibitors to standard chemotherapy. The aim of this study was to analyze COX-2 expression in tumor and stromal cells as predictive biomarker for COX-2 inhibition. METHODS: In a multicenter phase III trial, 316 patients with advanced NSCLC were randomized to receive celecoxib (400 mg b.i.d.) or placebo up to one year in addition to a two-drug platinum-based chemotherapy combination. In a subset of 122 patients, archived tumor tissue was available for immunohistochemical analysis of COX-2 expression in tumor and stromal cells. For each compartment, COX-2 expression was graded as high or low, based on a product score of extension and intensity of positively stained cells. RESULTS: An updated analysis of all 316 patients included in the original trial, and of the 122 patients with available tumor tissue, showed no survival differences between the celecoxib and placebo arms (HR 1.01; 95% CI 0.81-1.27 and HR 1.12; 95% CI 0.78-1.61, respectively). High COX-2 scores in tumor (n = 71) or stromal cells (n = 55) was not associated with a superior survival outcome with celecoxib vs. placebo (HR =0.96, 95% CI 0.60-1.54; and HR =1.51; 95% CI 0.86-2.66), and no significant interaction effect between COX-2 score in tumor or stromal cells and celecoxib effect on survival was detected (p = .48 and .25, respectively). CONCLUSIONS: In this subgroup analysis of patients with advanced NSCLC treated within the context of a randomized trial, we could not detect any interaction effect of COX-2 expression in tumor or stromal cells and the outcome of celecoxib treatment in addition to standard chemotherapy.

PD-L1 immunohistochemistry in clinical diagnostics of lung cancer: inter-pathologist variability is higher than assay variability.

Assessment of programmed cell death ligand 1 (PD-L1) immunohistochemical staining is used for decision on treatment with programmed cell death 1 and PD-L1 checkpoint inhibitors in lung adenocarcinomas and squamous cell carcinomas. This study aimed to compare the staining properties of tumor cells between the antibody clones 28-8, 22C3, SP142, and SP263 and investigate interrater variation between pathologists to see if these stainings can be safely evaluated in the clinical setting. Using consecutive sections from a tissue microarray with tumor tissue from 55 resected lung cancer cases, staining with five PD-L1 assays (28-8 from two different vendors, 22C3, SP142, and SP263) was performed. Seven pathologists individually evaluated the percentage of positive tumor cells, scoring each sample applying cutoff levels used in clinical studies: <1% positive tumor cells (score 0), 1-4% (score 1), 5-9% (score 2), 10-24% (score 3), 25-49% (score 4), and >50% positive tumor cells (score 5). Pairwise analysis of antibody clones showed weighted kappa values in the range of 0.45-0.91 with the highest values for comparisons with 22C3 and 28-8 and the lowest involving SP142. Excluding SP142 resulted in kappa 0.75-0.91. Weighted kappa for interobserver variation between pathologists was 0.71-0.96. Up to 20% of the cases were differently classified as positive or negative by any pathologist compared with consensus score using ≥1% positive tumor cells as cutoff. A significantly better agreement between pathologists was seen using ≥50% as cutoff (0-5% of cases). In conclusion, the concordance between the PD-L1 antibodies 22C3, 28-8 and SP263 is relatively good when evaluating lung cancers and suggests that any one of these assays may be sufficient as basis for decision on treatment with nivolumab, pembrolizumab, and durvalumab. The scoring of the pathologist presents an intrinsic source of error that should be considered especially at low PD-L1 scores.

An Overview of Multimodal Neuroimaging Using Nanoprobes.

Nanomaterials have gained tremendous significance as contrast agents for both anatomical and functional preclinical bio-imaging. Contrary to conventional medical practices, molecular imaging plays an important role in exploring the affected cells, thus providing precision medical solutions. It has been observed that incorporating nanoprobes improves the overall efficacy of the diagnosis and treatment processes. These nano-agents and tracers are therefore often incorporated into preclinical therapeutic and diagnostic applications. Multimodal imaging approaches are well equipped with nanoprobes to explore neurological disorders, as they can display more than one type of characteristic in molecular imaging. Multimodal imaging systems are explored by researchers as they can provide both anatomical and functional details of tumors and affected tissues. In this review, we present the state-of-the-art research concerning multimodal imaging systems and nanoprobes for neuroimaging applications.

Reduction of nitrous oxide emission by using stearic acid combined zinc coated urea in silty clay and sandy loam soils under bare and planted conditions.

Overuse of chemical fertilizers in agroecosystems leads to the increased economic burden, low crop production in terms of input and environmental pollution. Due to its improved nutrient management and degrading properties, synthetic slow release fertilizers have become a significant advancement in the fertilizer sector. In this study we evaluated the effect of slow release urea on nitrous oxide (N2O) emission, crop growth and crop nutrient contents. Measurements were carried out in two different texture soils (sandy loam and silty clay) under two different conditions (bare soil and planted). The N2O emission was measured for 15 days from bare soils and 48 days from planted soil. Plant fresh weight, dry weight, chlorophyll contents, N and Zn were measured in the end of the experiment. The results showed that N2O emission was reduced 33-39 % from coated urea as compared to conventional urea in bare soil. In planted soil, the coated urea reduced the N2O emission 29-33 %. The deep placement of urea in silty clay soil reduced the N2O emission up to 22.8 % as compared to surface placement. Plant fresh matter, dry matter, N and Zn contents were significantly (p ≤ 0.05) higher with coated urea as compared to conventional urea. It is concluded that the coating of urea with hydrophobic materials like stearic acid, along with Zn sources i.e. Zn fortified nano-bentonite or the ZnO nanoparticles (NPs) presents opportunities to overcome the environmental pollution and increasing the crop production and quality.

High iNOS and IL-1ß immunoreactivity are features of colitis-associated colorectal cancer tumors, but fail to predict 5-year survival.

Background: Inflammatory bowel disease (IBD; mainly ulcerative colitis and Crohn's disease) is associated with the development of colorectal cancer (CRC) referred to as colitis-associated colorectal cancer (CAC). In inflammatory flares of IBD, the production of luminal nitric oxide (NO) increases due to the increased inducible nitric oxide synthase (iNOS) activity in inflamed tissue. It is believed that iNOS parallels pro-inflammatory interleukin-1ß (IL-1ß). How these biomarkers relate to CAC pathogenesis or survival is unknown. Aim: The primary aim of this study was to investigate iNOS and IL-1ß immunoreactivity in CAC tumors in comparison with CRC and normal colonic mucosa, and the secondary aim was to determine if immunoreactivity correlates with 5-year survival of CAC. Methods: Immunohistochemistry was performed on tissue sections as follows: CAC (n = 59); sporadic CRC (sCRC) (n = 12); colonic mucosa >2 cm outside sCRC margin (normal mucosa) (n = 22); paracancerous IBD (pIBD) (n = 12). The expression of iNOS and IL-1ß was quantified separately for epithelium and stroma. Data were evaluated using the Mann-Whitney U-test and the log-rank test for 5-year Kaplan-Meier survival curves. Results were compared with online mRNA databases. Results: Immunoreactivity occurred predominantly in epithelial cells and to lesser extent in stroma. Compared with normal mucosa, immunoreactivity for iNOS (P < 0.01) and IL-1ß (P < 0.005) was higher in CAC epithelium. In CAC stroma, iNOS immunoreactivity was lower than normal mucosa (P < 0.001), whereas IL-1ß was higher (P < 0.05). Immunoreactivity differences of iNOS or IL-1ß among CAC patients failed to correlate with 5-year survival. These findings were supported by online mRNA databases. Conclusion: Consistent with high NO production in IBD, there is more iNOS in CAC epithelium, albeit not in stroma. This immunoreactivity difference exists for IL-1ß in both epithelium and stroma. The intervention of arginine or iNOS activity for CAC chemotherapy is not straightforward.

Spatial immunophenotyping of the tumour microenvironment in non-small cell lung cancer.

INTRODUCTION: Immune cells in the tumour microenvironment are associated with prognosis and response to therapy. We aimed to comprehensively characterise the spatial immune phenotypes in the mutational and clinicopathological background of non-small cell lung cancer (NSCLC). METHODS: We established a multiplexed fluorescence imaging pipeline to spatially quantify 13 immune cell subsets in 359 NSCLC cases: CD4 effector cells (CD4-Eff), CD4 regulatory cells (CD4-Treg), CD8 effector cells (CD8-Eff), CD8 regulatory cells (CD8-Treg), B-cells, natural killer cells, natural killer T-cells, M1 macrophages (M1), CD163+ myeloid cells (CD163), M2 macrophages (M2), immature dendritic cells (iDCs), mature dendritic cells (mDCs) and plasmacytoid dendritic cells (pDCs). RESULTS: CD4-Eff cells, CD8-Eff cells and M1 macrophages were the most abundant immune cells invading the tumour cell compartment and indicated a patient group with a favourable prognosis in the cluster analysis. Likewise, single densities of lymphocytic subsets (CD4-Eff, CD4-Treg, CD8-Treg, B-cells and pDCs) were independently associated with longer survival. However, when these immune cells were located close to CD8-Treg cells, the favourable impact was attenuated. In the multivariable Cox regression model, including cell densities and distances, the densities of M1 and CD163 cells and distances between cells (CD8-Treg-B-cells, CD8-Eff-cancer cells and B-cells-CD4-Treg) demonstrated positive prognostic impact, whereas short M2-M1 distances were prognostically unfavourable. CONCLUSION: We present a unique spatial profile of the in situ immune cell landscape in NSCLC as a publicly available data set. Cell densities and cell distances contribute independently to prognostic information on clinical outcomes, suggesting that spatial information is crucial for diagnostic use.

Comparison of ROS1-rearrangement detection methods in a cohort of surgically resected non-small cell lung carcinomas.

Background: Patients with non-small cell lung cancer (NSCLC) harboring a ROS proto-oncogene 1 (ROS1)-rearrangement respond to treatment with ROS1 inhibitors. To distinguish these rare cases, screening with immunohistochemistry (IHC) for ROS1 protein expression has been suggested. However, the reliability of such an assay and the comparability of the antibody clones has been debated. Therefore we evaluated the diagnostic performance of current detection strategies for ROS1-rearrangement in two NSCLC-patient cohorts. Methods: Resected tissue samples, retrospectively collected from consecutive NSCLC-patients surgically treated at Uppsala University Hospital were incorporated into tissue microarrays [all n=676, adenocarcinomas (AC) n=401, squamous cell carcinomas (SCC) n=213, other NSCLC n=62]. ROS1-rearrangements were detected using fluorescence in situ hybridization (FISH) (Abbott Molecular; ZytoVision). In parallel, ROS1 protein expression was detected using IHC with three antibody clones (D4D6, SP384, EPMGHR2) and accuracy, sensitivity, and specificity were determined. Gene expression microarray data (Affymetrix) and RNA-sequencing data were available for a subset of patients. NanoString analyses were performed for samples with positive or ambiguous results (n=21). Results: Using FISH, 2/630 (0.3% all NSCLC; 0.5% non-squamous NSCLC) cases were positive for ROS1 fusion. Additionally, nine cases demonstrated ambiguous FISH results. Using IHC, ROS1 protein expression was detected in 24/665 (3.6% all NSCLC; 5.1% non-squamous NSCLC) cases with clone D4D6, in 18/639 (2.8% all NSCLC; 3.9% non-squamous NSCLC) cases with clone SP384, and in 1/593 (0.2% all NSCLC; 0.3% non-squamous NSCLC) case with clone EPMGHR2. Elevated RNA-levels were seen in 19/369 (5.1%) cases (Affymetrix and RNA-sequencing combined). The overlap of positive results between the assays was poor. Only one of the FISH-positive cases was positive with all antibodies and demonstrated high RNA-expression. This rearrangement was confirmed in the NanoString-assay and also in the RNA-sequencing data. Other cases with high protein/RNA-expression or ambiguous FISH were negative in the NanoString-assay. Conclusions: The occurrence of ROS1 fusions is low in our cohorts. The IHC assays detected the fusions, but the accuracy varied depending on the clone. The presumably false-positive and uncertain FISH results questions this method for detection of ROS1-rearrangements. Thus, when IHC is used for screening, transcript-based assays are preferable for validation in clinical diagnostics.

Difficulties in diagnostics of lung tumours in biopsies: an interpathologist concordance study evaluating the international diagnostic guidelines.

AIMS: Accurate and reliable diagnosis is essential for lung cancer treatment. The study aim was to investigate interpathologist diagnostic concordance for pulmonary tumours according to WHO diagnostic criteria. METHODS: Fifty-two unselected lung and bronchial biopsies were diagnosed by a thoracic pathologist based on a broad spectrum of immunohistochemical (IHC) stainings, molecular data and clinical/radiological information. Slides stained with H&E, thyroid transcription factor-1 (TTF-1) clone SPT24 and p40 were scanned and provided digitally to 20 pathologists unaware of reference diagnoses. The pathologists independently diagnosed the cases and stated if further diagnostic markers were deemed necessary. RESULTS: In 31 (60%) of the cases, ≥80% of the pathologists agreed with each other and with the reference diagnosis. Lower agreement was seen in non-small cell neuroendocrine tumours and in squamous cell carcinoma with diffuse TTF-1 positivity. Agreement with the reference diagnosis ranged from 26 to 45 (50%-87%) for the individual pathologists. The pathologists requested additional IHC staining in 15-44 (29%-85%) of the 52 cases. In nearly half (17 of 36) of the malignant cases, one or more pathologist advocated for a different final diagnosis than the reference without need of additional IHC markers, potentially leading to different clinical treatment. CONCLUSIONS: Interpathologist diagnostic agreement is moderate for small unselected bronchial and lung biopsies based on a minimal panel of markers. Neuroendocrine morphology is sometimes missed and TTF-1 clone SPT24 should be interpreted with caution. Our results suggest an intensified education need for thoracic pathologists and a more generous use of diagnostic IHC markers.

Localized IgG4-related disease manifested on the tongue: a case report.

Immunoglobulin G4-related disease (IgG4-RD) is an immune-mediated fibroinflammatory condition that can affect multiple organs. IgG4-RD may show a variety of initial symptoms. In the oral mucosa, lesions present as inflammatory fibrosis with a large number of IgG4-positive plasma cells. Evaluating treatment is a well-known problem in IgG4-RD due to the absence of an established assessment system. There are difficulties in defining the severity of the disease, which is why treatment is primarily based on its clinical manifestations. We present a case report of localized IgG4-RD with ulcerative and proliferative manifestations on the tongue, which clinically mimicked oral squamous cell carcinoma. A tumor-like lesion on the tongue can indicate something else other than the malignant or reactive changes commonly found in the oral mucosa. Multiple differential diagnoses of these atypical oral lesions, including localized IgG4-RD, should be considered.

High Expression of PPM1D Induces Tumors Phenotypically Similar to TP53 Loss-of-Function Mutations in Mice.

PPM1D is a negative regulator of p53 and genomic aberrations resulting in increased activity of PPM1D have been observed in cancers of different origins, indicating that PPM1D has oncogenic properties. We established a transgenic mouse model overexpressing PPM1D and showed that these mice developed a wide variety of cancers. PPM1D-expressing mice developed tumors phenotypically and genetically similar to tumors in mice with dysfunctional p53. T-cell lymphoblastic lymphoma was the most frequent cancer observed in these mice (55%) followed by adenocarcinomas (24%), leukemia (12%) and other solid tumors including neuroblastoma. Characterization of T-cell lymphomas in mice overexpressing PPM1D demonstrates Pten-deletion and p53-accumulation similar to mice with p53 loss-of-function. Also, Notch1 mutations which are recurrently observed in T-cell acute lymphoblastic lymphoma (T-ALL) were frequently detected in PPM1D-transgenic mice. Hence, PPM1D acts as an oncogenic driver in connection with cellular stress, suggesting that the PPM1D gene status and expression levels should be investigated in TP53 wild-type tumors.

Blood brain barrier: A tissue engineered microfluidic chip.

With the increasing concern of neurological diseases, the improvised therapy for neurodegenerative disorders such as Alzheimer's disease is crucial. Yet, the efficacious delivery of drug across blood-brain barrier (BBB) remains a formidable challenge. BBB acts as a gate keeper to prevent the ingress of harmful foreign agents into the brain. It has built a great interest in designing BBB models to boost the field of neurotherapeutics. Recently, microfluidic systems are gaining ground in cell culture and bio-system analysis. It creates a new era of micro engineered laboratory onto a chip by combining the benefits of both in vitro and in vivo models. The high-fidelity microfluidic BBB-on-a-Chip possess the engineered physiological microenvironment for real time monitoring of barrier properties with human derived stem cells. These emerging models have intrinsic merits of regulating micro-scale fluid delivery and versatile fabrication. Moreover, the progress of 3D printing technology and versatility of stem cells assist in fabricating these robust and reproducible models. This review revolves around the various approaches of modelling microfluidic BBBs and emphasises on the limitations of existing models and technology. It contributes to the interdisciplinary engineering aspects of BBB research and its magnificent impact on drug development.

Targeted pancreatic beta cell imaging for early diagnosis.

Pancreatic beta cells are important in blood glucose level regulation. As type 1 and 2 diabetes are getting prevalent worldwide, we need to explore new methods for early detection of beta cell-related afflictions. Using bioimaging techniques to measure beta cell mass is crucial because a decrease in beta cell density is seen in diseases such as diabetes and thus can be a new way of diagnosis for such diseases. We also need to appraise beta cell purity in transplanted islets for type 1 diabetes patients. Sufficient amount of functional beta cells must also be determined before being transplanted to the patients. In this review, indirect imaging of beta cells will be discussed. This includes membrane protein on pancreatic beta cells whereby specific probes are designed for different imaging modalities mainly magnetic resonance imaging, positron emission tomography and fluorescence imaging. Direct imaging of insulin is also explored though probes synthesized for such function are relatively fewer. The path for successful pancreatic beta cell imaging is fraught with challenges like non-specific binding, lack of beta cell-restricted targets, the requirement of probes to cross multiple lipid layers to bind to intracellular insulin. Hence, there is an urgent need to develop new imaging techniques and innovative probing constructs in the entire imaging chain of bioengineering to provide early detection of beta cell-related pathology.

Use of cholesterol and soluble tumour markers CEA and syndecan-2 in pleural effusions in cases of inconclusive cytology.

AIMS: In order to improve diagnostics in pleural effusions, additional value of effusion cholesterol, carcinoembryonic antigen (CEA) and syndecan-2 assays to cytology was studied. METHODS: Biomarkers were measured in effusion supernatants from 247 patients, of whom 126 had malignant pleural involvement, and their additional diagnostic efficacy to cytology was assessed. RESULTS: Syndecan-2 measurement, although gave detectable concentrations in all effusions with highest median value in mesotheliomas, was non-discriminative between different pathological conditions. CEA concentrations exceeding 5 ng/mL cut-off point indicated carcinomas, regardless of pleural involvement, which gave a sensitivity of 62% and specificity of 100% for carcinoma. Cholesterol concentration over 1.21 mmol/L cut-off value indicated neoplastic pleural involvement with 99% sensitivity and 'merely' 69% specificity, the latter mainly due to raised levels being associated also with benign inflammatory effusions. Combined CEA and cholesterol determinations increased the sensitivity for diagnosing carcinomatosis from 70% with cytology alone to 84% and established the correct diagnosis in 16 of 31 carcinomatosis cases with inconclusive cytology. Cholesterol measurement alone, with elevated level, in combination with absence of substantial number of inflammatory cells in effusion sediment proved to be a magnificent marker for neoplastic pleural involvement with 99% efficacy, and recognised all 36 such cases with inconclusive cytology. CONCLUSIONS: Simultaneous measurement of CEA and cholesterol concentrations in effusion, or at least cholesterol alone, in combination with non-inflammatory fluid cytology, provides additional specific information about neoplastic pleural involvement, and can therefore be used as an adjunct to cytology, above all, in inconclusive cases.

Proteasome inhibitor PSI induces apoptosis in human mesothelioma cells.

Malignant mesothelioma is an increasingly common tumor with an almost 100% mortality rate. It is refractory to conventional treatment. We have previously shown with SSH and microarray that the mRNA expression level of proteasome is higher in epithelioid mesothelioma cell lines than in sarcomatoid ones. This study evaluates the differential apoptotic effect of proteasome inhibitors on both of these mesothelioma sub-lines. Proteasome inhibitors show substantial anti-tumor activity in some tumor cells in vitro and in vivo, but the effects on mesothelioma cells has not been studied. The viability of mesothelioma cells was reduced in a dose- and time-dependent manner by the proteasome inhibitors tested; PSI was effective with a low dose, but higher concentrations were needed for calpain inhibitor I. The epithelioid mesothelioma cells are more sensitive to the inhibitors than the sarcomatoid ones, their IC50 after 24 h of treatment with PSI being 4 and 16 microm, respectively. Other mesothelioma cell lines show similar sensitivity. PSI seemed to decrease mesothelioma viability by inducing apoptosis, as verified by cell morphology, Western blotting analysis of caspase 3 cleavage, and flow-cytometric analysis. In conclusion, PSI, a representative agent that reduces viability and induces apoptosis of mesothelioma cells, might be useful in the treatment of patients with mesothelioma, especially of epithelioid phenotype.

Prognostic impact of COX-2 in non-small cell lung cancer: a comprehensive compartment-specific evaluation of tumor and stromal cell expression.

Cyclooxygenase-2 (COX-2) is an enzyme that has been extensively investigated as a prognostic marker in cancer. In non-small cell lung cancer (NSCLC) previous results regarding the prognostic impact of COX-2 expression are inconsistent. Therefore we evaluated the association between transcript levels and overall survival in nine publicly available gene expression data sets (total n = 1337) and determined in situ compartment-specific tumor and stromal cell protein expression in two independent cohorts (n = 616). Gene expression did not show any correlation with clinical parameters or with overall survival. Protein expression in tumor and stromal cells did not correlate with any clinical parameter or with overall survival in one of the analyzed cohorts, while a significant association of high stromal expression with longer survival was observed in both univariate and multivariate analysis in the other cohort. Stromal expression of COX-2 has not been separately evaluated in NSCLC previously and may be a subject of further investigation, whereas the presented findings from this comprehensive compartment specific evaluation clearly reject the hypothesis of COX-2 tumor cell expression having a prognostic value in NSCLC.

A sandwich ELISA for the estimation of human syndecan-2 and syndecan-4 in biological samples.

Syndecans are integral membrane proteoglycans containing both chondroitin and heparan sulphate side chains. Syndecans are shed from the cell surface, and may be detected in cellular supernatant or body fluids. In this study, we wished to design ELISA methods for syndecan-2 and syndecan-4, with the future aim of analyzing syndecans in large number of patient materials. Briefly, sandwich ELISAs for human syndecan-2 and syndecan-4 were designed employing monoclonal mouse antibodies as capture and commercially available polyclonal rabbit anti-syndecan as secondary antibodies. Although no quantified standard was available, relative estimation of syndecan-2 and syndecan-4 levels was possible in a small pilot material. The paper presents the ELISA method for these proteoglycans. We suggest that this may be a useful tool in the analysis of patient materials and in the detection of syndecans during proteoglycan purification.

Early events in xenograft development from the human embryonic stem cell line HS181--resemblance with an initial multiple epiblast formation.

Xenografting is widely used for assessing in vivo pluripotency of human stem cell populations. Here, we report on early to late events in the development of mature experimental teratoma from a well-characterized human embryonic stem cell (HESC) line, HS181. The results show an embryonic process, increasingly chaotic. Active proliferation of the stem cell derived cellular progeny was detected already at day 5, and characterized by the appearance of multiple sites of engraftment, with structures of single or pseudostratified columnar epithelium surrounding small cavities. The striking histological resemblance to developing embryonic ectoderm, and the formation of epiblast-like structures was supported by the expression of the markers OCT4, NANOG, SSEA-4 and KLF4, but a lack of REX1. The early neural marker NESTIN was uniformly expressed, while markers linked to gastrulation, such as BMP-4, NODAL or BRACHYURY were not detected. Thus, observations on day 5 indicated differentiation comparable to the most early transient cell populations in human post implantation development. Confirming and expanding on previous findings from HS181 xenografts, these early events were followed by an increasingly chaotic development, incorporated in the formation of a benign teratoma with complex embryonic components. In the mature HS181 teratomas not all types of organs/tissues were detected, indicating a restricted differentiation, and a lack of adequate spatial developmental cues during the further teratoma formation. Uniquely, a kinetic alignment of rare complex structures was made to human embryos at diagnosed gestation stages, showing minor kinetic deviations between HS181 teratoma and the human counterpart.

A recruitment breath manoeuvre directly after endotracheal suction improves lung function: an experimental study in pigs.

BACKGROUND: Atelectasis occurs after a well performed endotracheal suction. Clinical studies have shown that recruitment manoeuvres added after endotracheal suction during mechanical ventilation restore lung function. Repetitive lung over-distension is, however, harmful for the lung, and the effects of adding a larger breath, recruitment breath, directly after repeated endotracheal suction were therefore investigated. METHODS: Twelve healthy anaesthetized pigs were randomized into two groups: one without and one with a recruitment breath manoeuvre (RBM), i.e. a breath 15 cmH(2)O above inspiratory pressure for 10 s during pressure-controlled ventilation. The pigs were suctioned every hour for 4 hours with an open suction system. RESULTS: At the end of the study there was a statistically significant difference between the group given RBM and that without with respect to PaCO(2), tidal volume (V(T)), and compliance (Crs). Without RBM, the PaCO(2) increased from 4.6+/-0.4 to 6.1+/-1.5 kPa, V(T) decreased from 345+/-39 to 247+/-71 mL, and Crs decreased from 28+/-6 to 18+/-5 mL/cmH(2)O. There was no change in PaCO(2) or Crs when a RBM was given. Morphological analysis revealed no differences in aeration of apical and central lung parenchyma. In the basal lung parenchyma there were, however, greater areas with normal lung parenchyma and less atelectasis after RBM. CONCLUSIONS: Atelectasis created by endotracheal suction can be opened by inflating the lung for a short duration with low pressure, without over-distension, immediately after suction.

Species-specific in vivo engraftment of the human BL melanoma cell line results in an invasive dedifferentiated phenotype not present in xenografts.

For clinically relevant studies on melanoma progression and invasiveness, in vivo experimental systems with a human cellular microenvironment would be advantageous. We have compared tumor formation from a human cutaneous malignant melanoma cell line (BL), after injection as conventional xenografts in the mouse, or when injected into a predominantly species-specific environment of human embryonic stem cell-derived teratoma induced in the mouse (the hEST model). The resulting melanoma histology was generally analogous, both systems showing delimited densely packed areas with pleomorphic cells of malignant appearance. A specificity of the integration process into the human embryonic teratoma tissues was indicated by the melanoma exclusively being found in areas compatible with condensed mesenchyme, similar to neural crest development. Here, also enhanced neovascularization was seen within the human mesenchymal tissues facing the BL melanoma growth. Furthermore, in the hEST model an additional melanoma cell phenotype occurred, located at the border of, or infiltrating into, the surrounding human loose mesenchymal fibrous stroma. This BL population had a desmoplastic spindle-like appearance, with markers indicative of dedifferentiation and migration. The appearance of this apparently more aggressive phenotype, as well as the induction of human angiogenesis, shows specific interactions with the human embryonic microenvironment in the hEST model. In conclusion, these data provide exciting options for using the hEST model in molecular in vivo studies on differentiation, invasiveness, and malignancy of human melanoma, while analyzing species-specific reactions in vivo.