Prevalence of Molecular Alterations in a Swiss Cohort of 512 Colorectal Carcinoma Patients by Targeted Next-Generation Sequencing Analysis in Routine Diagnostics.

INTRODUCTION: Colorectal carcinoma (CRC) is among the most common carcinomas in women and men. In the advanced stage, patients are treated based on the RAS status. Recent studies indicate that in the future, in addition to KRAS and NRAS, alterations in other genes, such as PIK3CA or TP53, will be considered for therapy. Therefore, it is important to know the mutational landscape of routinely diagnosed CRC. METHOD: We report the molecular profile of 512 Swiss CRC patients analyzed by targeted next-generation sequencing as part of routine diagnostics at our institute. RESULTS: Pathogenic and likely pathogenic variants were found in 462 (90%) CRC patients. Variants were detected in TP53 (54.3%), KRAS (48.2%), PIK3CA (15.6%), BRAF (13.5%), SMAD4 (10.5%), FBXW7 (7.8%), NRAS (3.5%), PTEN (2.7%), ERBB2 (1.6%), AKT1 (1.5%), and CTNNB1 (0.9%). The remaining pathogenic alterations were found in the genes ATM(n= 1), MAP2K1(n= 1), and IDH2(n= 1). DISCUSSION/CONCLUSIONS: Our analysis revealed the prevalence of potential predictive markers in a large cohort of CRC patients obtained during routine diagnostic analysis. Furthermore, our study is the first of this size to uncover the molecular landscape of CRC in Switzerland.

Stem cell secretome attenuates acute rejection in rat lung allotransplant.

OBJECTIVES: Stem cells secrete significant amounts of bioactive factors in their secretome that can be immunosuppressive. We studied the effect of the secretome obtained from bone marrow-derived mesenchymal stem cells (BMSC-sec) in combination with cyclosporine A following acute rejection of lung allografts in the rat. METHODS: Lung allotransplants were performed from male Brown Norway donor rats to recipient male Fisher 344 rats. Rat BMSC-sec was introduced intratracheally in the recipient every day after the transplant until the day the animal was sacrificed. Group A (n = 5) received control medium and cyclosporine A (2.5 mg/kg body weight intraperitoneally) for 5 days post-transplant and group B (n = 5) received BMSC-sec and cyclosporine A. Blood gas analysis was performed to assess graft function at day 5 only from the graft, and the tissue was sampled for measurement of the wet/dry ratio and histological grading of rejection. RESULTS: All control animals (group A) showed severe signs of rejection. At day 5 grafts in group B showed improved gas exchange (i.e. mean PaO2 mmHg 237.9 ± 130 mmHg vs 24.9 ± 7.8 mmHg in group A). Histological examination according to the International Society of Heart and Lung Transplantation (ISHLT) revealed moderate to severe rejection in all animals in group A (III B) and a significant improvement in group B (I-IIA). The wet/dry ratio was also reduced in group B to 6.19 ± 0.6 compared to 9.36 ± 2 in group A. Furthermore, in vitro T-cell proliferation was reduced after treatment with BMSC-sec for CD 3 cells (69.55 ± 07 vs 73 ± 0.84), for CD 4 (24.95 ± 1.2 vs 27.75 ± 0.21) and for CD 8 cells (3.75 ± 0.2 vs 5.68 ± 0.02). CONCLUSIONS: The BMSC-sec is a promising novel cell-based therapeutic option for acute rejection in a rat lung allograft model.

Bone marrow stem cells modified with human interleukin 10 attenuate acute rejection in rat lung allotransplantation.

OBJECTIVES: The aim of this study was to investigate new therapeutic options to attenuate acute rejection in a rat lung allograft model. Cell-based gene therapies have recently been reported as a novel curative option in acute and chronic diseases for which conventional treatments are not available. We studied the effect of human interleukin 10 (hIL-10) on expressing bone marrow-derived mesenchymal stem cells (BMSCs) in combination with cyclosporine A (CsA) on acute rejection of lung allografts in the rat. METHODS: Lung allotransplantation was performed from male Brown Norway donor to male Fisher (F344) rats. Rat BMSCs were transfected with hIL-10 in vitro and introduced in the graft prior to implantation. Group A (n = 5) received CsA intraperitoneally (2.5 mg/kg body weight) for 5 days post-transplant; Group B (n = 5) received BMSC and CsA and Group C (n = 5) received hIL-10-BMSC before implantation and CsA. Graft function was assessed by blood gas levels only from the graft on day 5; tissue was sampled for histological grading of rejection and measurement of the wet-to-dry ratio. RESULTS: All Group A control animals showed severe signs of rejection. On Day 5, all grafts in Group C showed improved gas exchange (mean arterial partial pressure of oxygen 222.2 ± 40.38 mmHg vs 92.36 ± 20.92 mmHg in Group B and 42.72 ± 18.07 mmHg in Group A). Histological examination revealed moderate-to-severe rejection in all animals in Group A [International Society for Heart and Lung Transplantation Level III B (ISHLT)] in contrast to low-to-moderate rejection in Group B (II-IIIA) and much improved histological grade in Group C (I-IIA). Moreover, the wet-to-dry ratio was also reduced in Group C (4.8 ± 1.19 compared with 4.78 ± 0.62 in Group B and 9.36 ± 0.90 in Group A). CONCLUSIONS: The hIL-10 BMSC represent a promising novel method for localized cell-based gene therapy for acute rejection in a rat lung allograft model.

Gene Network Analysis of Interstitial Macrophages After Treatment with Induced Pluripotent Stem Cells Secretome (iPSC-cm) in the Bleomycin Injured Rat Lung.

Idiopathic pulmonary fibrosis (IPF) is a complex disease involving various cell types. Macrophages are essential in maintenance of physiological homeostasis, wound repair and fibrosis in the lung. Macrophages play a crucial role in repair and remodeling by altering their phenotype and secretory pattern in response to injury. The secretome of induced pluripotent stem cells (iPSC-cm) attenuates injury and fibrosis in bleomycin injured rat lungs. In the current study, we evaluate the effect of iPSC-cm on gene expression and phenotype of interstitial macrophage in bleomycin injured rat lungs in vivo. iPSC-cm was intratracheally instilled 7 days after bleomycin induced lung injury and assessed 7 days later and single cell isolation was performed. Macrophages were FACS sorted and microarray analysis was performed. We characterized changes in the rat lung interstitial macrophages using transcriptional profiling. iPSC-cm reduced the total collagen content of the lung and reduced different macrophage populations. Gene set enrichment analysis revealed involvement of three essential pathways (a) immune modulation, (b) branching morphogenesis and (c) canonical Wnt signaling. This study demonstrates that iPSC-cm reduces fibrosis in bleomycin injured rat lung by partially altering the macrophages and regulating their gene expression.

MET overexpression and gene amplification: prevalence, clinico-pathological characteristics and prognostic significance in a large cohort of patients with surgically resected NSCLC.

The prevalence of overexpression and amplification of the proto-oncogene mesenchymal epithelial transition (MET) in non-small cell lung cancer (NSCLC) varies greatly in the literature. Since MET is a potential treatment target, knowledge of its prevalence and prognostic importance is crucial. We investigated MET expression and gene status in 735 NSCLC cases using tissue microarrays. Prognostic significance as well as correlations with various clinico-pathological parameters were evaluated. The prevalence of MET overexpression was 17% and MET amplification was present in 2.4% of cases. MET overexpression was found more frequently in adenocarcinomas (and TTF1-positive tumors) and female patients and was also associated with expression of members of the epidermal growth factor receptor (EGFR) signaling cascade. MET amplified tumors tended to express MET more frequently and more intensively. MET expression or gene status did not prove to be relevant prognostic factors. MET may not be an unequivocal prognostic parameter; however, its expression is associated with certain clinico-pathological characteristics and with EGFR and downstream EGFR effectors. This could be an important point for future studies addressing targeted MET therapy and should be considered as a possible means of optimizing the benefit and minimizing undesirable effects.

Glycine decarboxylase and HIF-1α expression are negative prognostic factors in primary resected early-stage non-small cell lung cancer.

Glycine decarboxylase (GLDC) was recently described as a critical enzyme of tumor-initiating cells and, thus, a driver of tumorigenesis in lung non-small cell cancer (NSCC). It is important in metabolism under hypoxic conditions. Hypoxia-inducible factor 1-alpha (HIF-1α) is the unique subunit that determines HIF system activity, thereby regulating the adverse effects of hypoxia on cancer outcome. We examined the expression and prognostic significance of GLDC and HIF-1α in primary resected stage I/II NSCC. Immunohistochemistry for GLDC and HIF-1α was validated on two lung NSCC cell lines (A549, NCI-H460) and evaluated on a tissue microarray with 428 lung NSCC: 184 adenocarcinomas, 211 squamous cell carcinomas, and 33 large cell carcinomas (LCC). The results were correlated with clinico-pathological parameters. High levels of GLDC expression were detected in 33/428 cases (7.7%). HIF-1α was expressed in 71 (16.6%) cases and more frequently in squamous cell carcinoma (p < 0.001). Significantly longer survival was seen in younger patients (p = 0.007), patients with non-LCC histology (p = 0.006), lower primary tumor category (p = 0.002), and Union for International Cancer Control (UICC) stage (p = 0.001). Both GLDC and HIF-1α were significantly associated with worse tumor-related survival (p = 0.013, p = 0.021, respectively), although not independent from each other in multivariate models. The combination of low-GLDC/negative HIF-1α expression was significantly prognostic for longer survival (p = 0.002) and emerged as an independent prognostic factor in multivariate analysis (p = 0.007, HR 2.052), next to UICC stage and age. We show that the combination of GLDC and HIF-1α expression is an independent prognostic factor in early-stage NSCC. Our results will assist future development of therapeutic approaches targeting GLDC or exploiting tumor hypoxia.

MicroRNA-106a targets autophagy and enhances sensitivity of lung cancer cells to Src inhibitors.

OBJECTIVES: Src tyrosine kinase inhibitors (TKIs) significantly inhibit cell migration and invasion in lung cancer cell lines with minor cytotoxic effects. In clinical trials, however, they show modest activity in combination with chemotherapeutic agents. Possible resistance mechanisms include the induction of cytoprotective autophagy upon Src inhibition. Autophagy is a cellular recycling process that allows cell survival in response to a variety of stress stimuli including responses to various treatments. MATERIAL AND METHODS: We screened autophagic activity in A549, H460, and H1299 NSCLC cell lines treated with two different Src-TKIs (saracatinib, dasatinib) or shRNA targeting SRC. The autophagy response was determined by LC3B-I to -II conversion, increased ULK1 epxression and increased GFP-LC3B dot formation. Autophagy was inhibited by pharmacological (bafilomycin A, chloroquine) or genetic (ULK1 shRNA) means. Expression of miR-106a and miR-20b was analyzed by qPCR, and we used different lentivral vectors for ectopic expression of either miR-106a mimetics, anti-sense miR-106a or different miR-106a-363 cluster constructs. RESULTS: In the current study we found that Src-TKIs induce autophagy in lung adenocarcinoma cell lines and that a combination of autophagy and Src tyrosine kinase inhibition results in cell death. Moreover, Src-TKI induced autophagy depends on the induction of the key autophagy kinase ULK1. This ULK1 upregulation is caused by downregulation of the ULK1-targeting microRNA-106a. An inverse correlation of miR-106a and ULK1 expression was seen in lung adenocarcinoma. Accordingly, ectopic expression of miR-106a in combination with Src-TKI treatment resulted in significant cell death as compared to control transduced cells. CONCLUSIONS: Autophagy protects lung adenocarcinoma cells from Src-TKIs via a newly identified miR-106a-ULK1 signaling pathway. The combined inhibition of Src and ULK1/autophagy might represent a promising treatment option for future clinical trials. Lastly, our data might challenge the term "oncogenic" miR-106a as it can promote sensitivity to Src-TKIs thereby underlining the context-dependent function of miRNAs.

Prognostic value of the autophagy markers LC3 and p62/SQSTM1 in early-stage non-small cell lung cancer.

Autophagy is a cellular degrading process that promotes tumor cell survival or cell death in cancer, depending on the progress of oncogenesis. Protein light chain 3 (LC3) and p62/SQSTM1 (p62) are associated with autophagosomal membranes that engulf cytoplasmic content for subsequent degradation. We studied LC3 and p62 expression using immunohistochemistry in a large cohort of 466 stage I/II non-small cell lung cancer (NSCLC) using a tissue microarray. We evaluated dot-like cytoplasmic expression of LC3 and dot-like, cytoplasmic and nuclear staining for p62 in relation to clinico-pathological parameters.LC3 expression correlated with all p62 patterns, as those correlated among each other (p < 0.001 each). There was no correlation with stage, age or gender. A combination of high LC3/high p62 dot-like staining (suggesting impaired autophagy) showed a trend for better outcome (p = 0.11). Interestingly, a combined low cytoplasmic/low nuclear p62 expression regardless of dot-like staining was an independent prognostic factor for longer survival (p = 0.006; HR=1.96), in addition to tumor stage (p = 0.004; HR=1.4).The autophagy markers LC3 and p62 are differentially expressed in NSCLC, pointing towards a biologically significant role. High LC3 levels seem to be linked to lower tumor aggressiveness, while high general p62 expression was significantly associated with aggressive tumor behavior.

Molecular profiling of lung adenosquamous carcinoma: hybrid or genuine type?

Lung adenosquamous carcinoma is a particular subtype of non-small cell lung carcinoma that is defined by the coexistence of adenocarcinoma and squamous cell carcinoma components. The aim of this study was to assess the mutational profile in each component of 16 adenosquamous carcinoma samples from a Caucasian population by a combination of next generation sequencing using the cancer hotspot panel as well as the colon and lung cancer panel and FISH. Identified mutations were confirmed by Sanger sequencing of DNA from cancer cells of each component collected by Laser Capture microdissection. Mutations typical for adenocarcinoma as well as squamous cell carcinoma were identified. Driver mutations were predominantly in the trunk suggesting a monoclonal origin of adenosquamous carcinoma. Most remarkably, EGFR mutations and mutations in the PI3K signaling pathway, which accounted for 30% and 25% of tumors respectively, were more prevalent while KRAS mutations were less prevalent than expected for a Caucasian population. Surprisingly, expression of classifier miR-205 was intermediate between that of classical adenocarcinoma and squamous cell carcinoma suggesting that adenosquamous carcinoma is a transitional stage between these tumor types. The high prevalence of therapy-relevant targets opens new options of therapeutic intervention for adenosquamous carcinoma patients.

Crizotinib inhibits migration and expression of ID1 in MET-positive lung cancer cells: implications for MET targeting in oncology.

AIMS: ID1 is an important component of the MET-SRC signaling pathway, which is a regulator of cell migration and invasion. We hypothesized that the ALK/MET inhibitor crizotinib inhibits migration via MET-SRC-ID1, rather than ALK. MATERIALS & METHODS: We used ALK fusion-positive and -negative lung cancer cell lines; crizotinib, PHA-665752, and saracatinib, and stable transfection with shMET. We performed western blotting for p-ALK, ALK, p-MET, MET, p-SRC, SRC and ID1, and quantitative real-time PCR for ID1. RESULTS: Crizotinib decreased p-MET, p-SRC and ID1 levels in ALK- and or MET-positive cell lines and inhibited cell migration. Knockdown of MET was comparable with the effect of crizotinib. CONCLUSION: The effects of crizotinib on ID1 expression and cancer cell migration were associated with the presence of activated MET, rather than ALK fusion.

Pulmonary aspergilloma: A rare differential diagnosis to lung cancer after positive FDG PET scan.

Early diagnosis and treatment of lung cancer, one of the leading causes of cancer-related death, is important to improve morbidity and mortality. Therefore any suspect solitary pulmonary nodule should prompt the pursuit for a definitive histological diagnosis. We describe the case of a 55-years-old male ex-smoker, who was admitted to our hospital due to recurrent hemoptysis and dry cough. A CT scan showed an irregular nodule of increasing size (28 mm in diameter) in the left lower lobe (LLL). A whole body PET-CT scan (643 MBq F-18 FDG i.v.) was performed and confirmed an avid FDG uptake of the nodule in the LLL, highly suspicious of lung cancer, without any evidence of lymphogenic or hematogenic metastasis. Bronchoscopy was not diagnostic and due to severe adhesions after prior chest trauma and the central location of the nodule, a lobectomy of the LLL was performed. Surprisingly, histology showed a simple aspergilloma located in a circumscribed bronchiectasis with no evidence of malignancy. This is a report of an informative example of an aspergilloma, which presented with symptoms and radiological features of malignant lung cancer.

HGF Expressing Stem Cells in Usual Interstitial Pneumonia Originate from the Bone Marrow and Are Antifibrotic.

BACKGROUND: Pulmonary fibrosis may result from abnormal alveolar wound repair after injury. Hepatocyte growth factor (HGF) improves alveolar epithelial wound repair in the lung. Stem cells were shown to play a major role in lung injury, repair and fibrosis. We studied the presence, origin and antifibrotic properties of HGF-expressing stem cells in usual interstitial pneumonia. METHODS: Immunohistochemistry was performed in lung tissue sections and primary alveolar epithelial cells obtained from patients with usual interstitial pneumonia (UIP, n = 7). Bone marrow derived stromal cells (BMSC) from adult male rats were transfected with HGF, instilled intratracheally into bleomycin injured rat lungs and analyzed 7 and 14 days later. RESULTS: In UIP, HGF was expressed in specific cells mainly located in fibrotic areas close to the hyperplastic alveolar epithelium. HGF-positive cells showed strong co-staining for the mesenchymal stem cell markers CD44, CD29, CD105 and CD90, indicating stem cell origin. HGF-positive cells also co-stained for CXCR4 (HGF+/CXCR4+) indicating that they originate from the bone marrow. The stem cell characteristics were confirmed in HGF secreting cells isolated from UIP lung biopsies. In vivo experiments showed that HGF-expressing BMSC attenuated bleomycin induced pulmonary fibrosis in the rat, indicating a beneficial role of bone marrow derived, HGF secreting stem cells in lung fibrosis. CONCLUSIONS: HGF-positive stem cells are present in human fibrotic lung tissue (UIP) and originate from the bone marrow. Since HGF-transfected BMSC reduce bleomycin induced lung fibrosis in the bleomycin lung injury and fibrosis model, we assume that HGF-expressing, bone-marrow derived stem cells in UIP have antifibrotic properties.

Targeted gene transfer of hepatocyte growth factor to alveolar type II epithelial cells reduces lung fibrosis in rats.

Inefficient alveolar wound repair contributes to the development of pulmonary fibrosis. Hepatocyte growth factor (HGF) is a potent growth factor for alveolar type II epithelial cells (AECII) and may improve repair and reduce fibrosis. We studied whether targeted gene transfer of HGF specifically to AECII improves lung fibrosis in bleomycin-induced lung fibrosis. A plasmid encoding human HGF expressed from the human surfactant protein C promoter (pSpC-hHGF) was designed, and extracorporeal electroporation-mediated gene transfer of HGF specifically to AECII was performed 7 days after bleomycin-induced lung injury in the rat. Animals were killed 7 days after hHGF gene transfer. Electroporation-mediated HGF gene transfer resulted in HGF expression specifically in AECII at biologically relevant levels. HGF gene transfer reduced pulmonary fibrosis as assessed by histology, hydroxyproline determination, and design-based stereology compared with controls. Our results indicate that the antifibrotic effect of HGF is due in part to a reduction of transforming growth factor-ß(1), modulation of the epithelial-mesenchymal transition, and reduction of extravascular fibrin deposition. We conclude that targeted HGF gene transfer specifically to AECII decreases bleomycin-induced lung fibrosis and may therefore represent a novel cell-specific gene transfer technology to treat pulmonary fibrosis.

MicroRNA-381 represses ID1 and is deregulated in lung adenocarcinoma.

INTRODUCTION: MicroRNAs are small, noncoding RNAs that suppress gene expression by binding to the 3' untranslated region (UTR) and thereby repress translation or decrease messenger RNA stability. Inhibitor of differentiation 1 (ID1) is a putative stem-cell gene involved in invasion and angiogenesis. We previously showed that ID1 is regulated by Src kinases, overexpressed in human lung adenocarcinoma, and targeted by Src-dependent microRNAs. The current study focused on the association between miR-381 and ID1 in lung adenocarcinoma. METHODS: An ID1 3'UTR-luciferase reporter assay was used to determine whether miR-381 directly targets ID1. Human lung cancer cell lines were stably transduced with a precursor of miR-381 to evaluate its role on ID1 expression and to investigate changes in cell migration and invasion. The Src tyrosine kinase inhibitors saracatinib and dasatinib were used to repress ID1 expression. MiR-381 expression was measured in 18 human lung adenocarcinomas and corresponding normal lung tissue by quantitative reverse-transcription polymerase chain reaction. RESULTS: ID1 is a direct target of miR-381 as shown by 3'UTR luciferase reporter assays. MiR-381 expression was negatively correlated with ID1 expression in lung cancer cell lines. Ectopic expression of miR-381 reduced ID1 mRNA and protein levels, and significantly decreased cell migration and invasion. Furthermore, miR-381 was significantly downregulated in human lung adenocarcinomas, and low miR-381 expression levels correlated with poor prognosis. CONCLUSION: These results suggest that downregulation of miR-381 and thus induction of its target ID1 may contribute to the metastatic potential of lung adenocarcinomas. Further studies to explore potential therapeutic strategies, including Src inhibitors, are ongoing.

[Glomerulonephritis and vasculitis as causes of arterial hypertension]. / Glomerulonephritis und Vaskulitis als Ursacheneiner arteriellen Hypertonie.

The various types of glomerulonephritis, including many forms of vasculitis, are responsible for about 15% of cases of end-stage renal disease (ESRD). Arterial hypertension represents a frequent finding in patients suffering from glomerulonephritis or vasculitis and hypertension also serves as an indicator for these severe types of diseases. In addition, there are symptoms and signs like hematuria, proteinuria and renal failure. Especially, rapidly progressive glomerulonephritis (RPGN) constitutes a medical emergency and must not be missed by treating physicians. This disease can either occur limited to the kidneys or in the context of a systemic inflammatory disorder, like a vasculitis. If left untreated, RPGN can lead to a necrotizing destruction of glomeruli causing irreversible kidney damage within several months or even weeks. With respect to the immunologically caused vasculitis, there are - depending upon the severity and type of organ involved - many clinical warning signs to be recognized, such as arterial hypertension, hemoptysis, arthalgias, muscle pain, palpable purpura, hematuria, proteinuria and renal failure. In addition, constitutional signs, such as fever and loss of body weight may occur concurrently. Investigations of glomerulonephritis or vasculitis must contain a careful and complete examination of family history and medications used by the respective patient. Thereafter, a thorough clinical examination must follow, including skin, joints and measurement of arterial blood pressure. In addition, a spectrum of laboratory analyses is required in blood, such as full blood screen, erythrocyte sedimentation rate, CRP, creatinine, urea and glucose, and in urine, including urinalysis looking for hematuria, red cell casts and proteinuria. Importantly, proteinuria needs to be quantified by the utilization of a random urine sample. Proteinuria > 3g/d is diagnostic for a glomerular damage. These basic tests are usually followed by more specialized analyses, such as a screening for infections, including search for HIV, hepatitis B or C and various bacteria, and for systemic inflammatory diseases, including tests for antibodies, such as ANA, anti-dsDNA, ANCA, anti-GBM and anti-CCP. In cases of membranous nephropathy, antibodies against phospholipase-A2-receptor need to be looked for. Depending upon the given clinical circumstances and the type of disease, a reasonable tumor screening must be performed, especially in cases of membranous and minimal-change nephropathy. Finally, radiological examinations will complete the initial work-up. In most cases, at least an ultrasound of the kidney is mandatory. Thereafter, in most cases a renal biopsy is required to establish a firm diagnosis to define all treatment options and their chance of success. The elimination of a specific cause for a given glomerulonephritis or vasculitis, such as an infection, a malignancy or a drug-related side-effect, remains the key principle in the management of these diseases. ACE-inhibitors, angiotensin receptor-blockers, aldosteron antagonists and renin-inhibitors remain the mainstay in the therapy of arterial hypertension with proteinuria. Only in cases of persistently high proteinuria, ACE-inhibitors and angiotensin receptor blockers can be prescribed in combination. Certain types of glomerulonephritis and essentially all forms of vasculitis require some form of more specific anti-inflammatory therapy. Respective immunosuppressive drug regimens contain traditionally medications, such as glucocorticoids (e. g. prednisone), cyclosporine A, mycophenolate mofetil, cyclophosphamide, and azathioprine. With respect to more severe forms of glomerulonephritis and vasculitis, the antibody rituximab represents a new and less toxic alternative to cyclophosphamide. Finally, in certain special cases, like Goodpasture's syndrome or severe ANCA-positive vasculitis, a plasma exchange will be useful and even required.

1,25-Dihydroxycholecalciferol with low-calcium diet reduces acute rejection in rat lung allotransplantation.

OBJECTIVES: The effect of 1,25-dihydroxycholecalciferol (calcitriol, vitamin D3) with a low-calcium diet on the acute lung allograft rejection in a rat unilateral left lung transplantation model was evaluated. METHODS: Three transplantation groups were studied (n = 5, male Brown-Norway to Fischer F344, 235 ± 15 g body weight): calcitriol and low-calcium diet, low-calcium diet and normal diet. Calcitriol (4 µg/kg/day) was injected intraperitoneally for 5 days, starting from the day of transplantation. In addition, two non-transplantation groups were compared: (n = 3, Brown-Norway) to measure the level of cytokines, and Fischer F344 receiving calcitriol and a low-calcium diet to measure the serum calcium level. The recipients of transplantation were killed on Day 5 post-transplant. The contralateral right main bronchus and the pulmonary artery were occluded for 5 min and blood was drawn for the blood gas analysis, and the grafts were assessed for histology (International Society for Heart and Lung Transplantation 1996/rank scale). Lung levels of interleukin (IL)-2, IL-6, IL-12 and tumour necrosis factor-α (TNF-α) were assessed within the calcitriol and low-calcium diet, low-calcium diet and Brown-Norway groups. The serum calcium level was assessed in the Fischer F344 group. An analysis of variance with Tukey's post hoc test was used to compare the arterial blood oxygen pressure and the lung cytokine expression between groups. A non-parametric Kruskal-Wallis test followed by the Siegel and Castellan post hoc test was used to assess the differences between the groups according to the lung graft rejection grading. Student's paired t-test was used to compare the serum calcium level. RESULTS: The arterial PaO(2) was significantly higher in the calcitriol and the low-calcium diet groups when compared with low-calcium diet or normal diet groups (356 ± 72 mmHg; P < 0.05 vs other groups). The arterial and bronchial rejection observed in calcitriol and low-calcium diet group was significantly milder than in the low-calcium diet or normal diet groups (A1-2, B1-2; P < 0.05 vs other groups). IL-2 and IL-6 levels were significantly higher in low-calcium diet vs calcitriol and low-calcium diet and Brown-Norway groups. IL-12 and TNF-α did not differ among the groups. There was no significant difference in serum calcium level before and after the treatment in the Fischer F344 group. CONCLUSIONS: Calcitriol with a low-calcium diet treatment improves lung function, reduces lung allograft acute rejection, decreases IL-2 and IL-6 allograft expression and does not change the serum calcium level significantly.

Meningial perineurioma: a benign peripheral nerve sheath tumor in a previously unrecognized central nervous system location, mimicking meningioma.

Perineurioma is an uncommon, mostly benign, spindle-cell tumor of peripheral nerve sheath origin with a predilection for the soft tissues. Although increasing awareness points to the sites of involvement by perineurioma possibly being as ubiquitous as those frequented by schwannian tumors, only one intracerebral example has been described to date. We report on a surgically resected perineurioma of the falx cerebri in an 86-year-old woman. Preoperative imaging showed an enhancing extraaxial mass of 6 cm × 5.7 cm × 3.7 cm. Histologically, the tumor consisted of a proliferation of spindle cells interwoven by a lattice of basal lamina. Alongside a prevailing soft tissue perineurioma pattern, sclerosing and reticular areas were seen as well. Tumor cells coexpressed EMA and GLUT-1, and a minority immunoreacted for smooth muscle actin. Pericellular basal lamina was decorated with collagen type IV. No staining for S100 protein was detected. Mitotic activity was virtually absent, and the MIB1 labeling index averaged 2%. Ultrastructural examination revealed abundant pinocytotic vesicles within and conspicuous tight junctions between slender cytoplasmic processes which, in turn, were encased by discontinuous basal lamina. FISH analysis confirmed loss of at least part of one chromosome 22q. This observation calls attention to perineurioma as a novel item in the repertoire of low-grade meningial spindle cell neoplasms, in the differential diagnostic context of which it is apt to being misconstrued as either meningioma, solitary fibrous tumor, or neurofibroma. Confusion with the latter bears the risk of overgrading innocuous features of perineurioma as criteria for malignancy.

Eculizumab in atypical hemolytic uremic syndrome: long-term clinical course and histological findings.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with defective regulation of the alternative complement pathway. The prognosis for patients with aHUS is poor, and plasma exchange represents the first-line therapy. Eculizumab is a humanized monoclonal anti-C5 antibody that prevents the activation of the terminal complement pathway. Here, we report the case of a 9-year-old girl with frequent relapsing aHUS due to heterozygous factor H mutation who was initially treated with plasma exchange three times per week with 150% plasma exchange volume. This treatment frequently caused allergic reactions and school absences. Because any reduction in the frequency of plasma exchange immediately induced relapses of the aHUS, treatment with eculizumab, 600 mg every 2 weeks, was started and plasma exchange completely stopped. On this drug regimen the patient showed no evidence of disease activity during a period of more than 24 months. Renal function improved, proteinuria disappeared, the number of antihypertensive medications could be decreased, and the quality of life increased substantially. The inhibition of the terminal complement pathway by eculizumab was also confirmed by renal biopsy, which showed the absence of thrombotic microangiopathy 2 months after the initiation of eculizumab therapy. This case illustrates the long-term favorable outcome of aHUS with eculizumab treatment.

The stem cell gene "inhibitor of differentiation 1" (ID1) is frequently expressed in non-small cell lung cancer.

AIMS: Inhibitor of differentiation 1 (ID1) plays a role in cellular differentiation, proliferation, angiogenesis and tumor invasion. As shown recently, ID1 is positively regulated by the tyrosine kinase SRC in lung carcinoma cell lines and with that appears as a potential new therapeutic target in non-small cell carcinoma (NSCLC). To substantiate this hypothesis we examined ID1, SRC and matrix metalloproteinase-9 (MMP-9) immunohistochemically in human NSCLC specimens. METHODS: From 61 consecutive patient tissue samples of a tumor tissue bank a one core tissue microarray (TMA) was produced and whole slide tissue samples of preinvasive lesions used. The staining of commercial antibodies was assessed by the H-score. Statistical analyses based on Spearman's rank correlation coefficient. RESULTS: ID1 was expressed in the nucleus in 70% of squamous cell carcinomas and 50% of non-squamous cell carcinomas and in vascular endothelium of non-tumor tissue. Cytoplasmic staining was found in all samples for SRC and in 93% for MMP-9. ID1-positive tissue samples co-expressed SRC and MMP-9 in 94%. In non-squamous cell carcinomas, H-scores of ID1 and SRC correlated with each other (p=0.04). H-score of MMP-9 correlated with tumor grade (p=0.04). The carcinoma findings were reflected in preinvasive lesions. CONCLUSIONS: We describe for the first time the immunohistochemical expression of ID1 in the majority of NSCLC samples. The almost general co-expression of ID1, SRC and MMP-9 supports their cooperation in vivo and warrants further investigation of ID1 as a therapeutic target.