Anders Bjartell

Expression of the neutrophil-activating CXC chemokine ENA-78/CXCL5 by human eosinophils.

Background Eosinophils are seen at sites of inflammation in diseases such as helminthic infestation, asthma, ulcerative colitis and some neoplastic diseases. They are also associated with connective tissue remodelling, for example in longstanding asthma. In the present study, we investigated whether eosinophils express the CXC chemokine epithelial cell‐derived neutrophil activating peptide (ENA‐78/CXCL5), a chemokine that can activate neutrophils and in addition possesses angiogenic properties.

Expression and characterization of trypsinogen produced in the human male genital tract.

Trypsinogen is a serine proteinase produced mainly by the pancreas, but it has recently been found to be expressed also in several cancers such as ovarian and colon cancer and in vascular endothelial cells. In this study, we found that trypsinogen-1 and -2 are present at high concentrations (median levels, 0.4 and 0.5 mg/L, respectively) in human seminal fluid and purified them to homogeneity by immunoaffinity and anion exchange chromatography. Purified trypsinogen isoenzymes displayed a M(r) of 25 to 28 kd in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Most of the trypsinogen-1 purified from seminal fluid was enzymatically active whereas trypsinogen-2 occurred as the proform, which could be activated by enteropeptidase in vitro. Immunohistochemically, trypsinogen protein was detected in the human prostate, urethra, utriculus, ejaculatory duct, seminal vesicles, deferent duct, epididymal glands, and testis. Expression of trypsinogen mRNA in the same organs was demonstrated by in situ hybridization. Trypsinogen mRNA was also detected in the prostate and seminal vesicles by reverse transcriptase-polymerase chain reaction and Northern blotting. Isolated trypsin was shown to activate the proenzyme form of prostate-specific antigen. These results suggest that trypsinogen isoenzymes found in seminal fluid are produced locally in the male genital tract and that they may play a physiological role in the semen.

Localization and mRNA expression of somatostatin receptor subtypes in human prostatic tissue and prostate cancer cell lines.

Somatostatin (SST) plays an important regulatory role in the physiological control of various organs including the prostate. Somatostatin receptors (SSTRs) and SST analogs are potential targets for prostate cancer treatment, especially since it has been shown that SST analogues are clinically effective in the treatment of advanced prostate cancer. The presence of SST containing neuroendocrine (NE) cells in the epithelium of the human prostate and their suggested role in the paracrine regulation of this gland prompted us to study the potential expression of somatostatin receptors (SSTRs) in human prostatic tissue and prostate cancer cell lines. Using the reverse transcriptase polymerase chain reaction (RT-PCR), we found the SSTR subtypes 1-3 in stromal cells and in prostate cancer cell lines LNCaP, PC-3 and DU 145. Immunohistochemical analysis of 27 radical prostatectomy specimens demonstrated the presence of hSSTR1 in a subpopulation of cancerous and NE cells, whereas hSSTR2 was found in the stroma, peritumoral blood vessels and tumor cells. Receptor subtype 3 was demonstrated to be present on the cell membrane of BPH and malignant areas. A strong immunoreaction (IR) of hSSTR4 was found in tumor cells, as compared with a less intense IR in adjacent BPH areas. Somatostatin receptor subtype 5 was not detectable. Western blot analysis revealed immunoreactive bands of molecular weight between 44-60 kDa. In summary, the present study clearly demonstrates the presence of hSSTR1-3 in tumoral and nontumoral epithelial cells as well as in the stromal compartment, whereas hSSTR4 was found to be confined to epithelial cells, and SSTR5 was not detectable.

Human antimicrobial peptide LL-37 is present in atherosclerotic plaques and induces death of vascular smooth muscle cells: a laboratory study

BackgroundDeath of smooth muscle cells in the atherosclerotic plaques makes the plaques more prone to rupture, which can initiate an acute ischemic event. The development of atherosclerosis includes the migration of immune cells e.g. monocytes/macrophages and T lymphocytes into the lesions. Immune cells can release antimicrobial peptides. One of these, human cathelicidin antimicrobial peptide hCAP-18, is cleaved by proteinase 3 generating a 4.5 kDa C-terminal fragment named LL-37, which has been shown to be cytotoxic. The aim of the study was to explore a potential role of LL-37 in the pathophysiology of atherosclerosis.MethodsWe investigated the presence of LL-37 in human atherosclerotic lesions obtained at autopsy using immunohistochemistry. The direct effects of LL-37 on cultured vascular smooth muscle cells and isolated neutrophil granulocytes were investigated with morphological, biochemical and flow cytometry analysis.ResultsThe neointima of atherosclerotic plaques was found to contain LL-37-like immunoreactivity, mainly in macrophages. In cultured smooth muscle cells, LL-37 at 30 μg/ml caused cell shrinkage, membrane blebbing, nuclear condensation, DNA fragmentation and an increase in caspase-3 activity as studied by microscopy, ELISA and enzyme activity assay, respectively. Flow cytometry demonstrated that LL-37 in a subset of the cells caused a small but rapidly developing increase in membrane permeability to propidium iodide, followed by a gradual development of FITC-annexin V binding. Another cell population stained heavily with both propidium iodide and FITC-annexin V. Neutrophil granulocytes were resistant to these effects of LL-37.ConclusionThis study shows that LL-37 is present in atherosclerotic lesions and that it induces death of vascular smooth muscle cells. In a subset of cells, the changes indicate the development of apoptosis triggered by an initial mild perturbation of plasma membrane integrity. The findings suggest a role for LL-37 as a mediator of immune cell-induced death of vascular smooth muscle cells in atherosclerosis.

Time-resolved fluorescence imaging for quantitative histochemistry using lanthanide chelates in nanoparticles and conjugated to monoclonal antibodies

Tissue and cell examinations have a potential to produce extremely valuable information about antigen quantities in samples. Using currently available methods, a truly quantitative analysis is nearly impossible. We have previously shown that immunohistochemical (IHC) detection of prostate-specific antigen and human glandular kallikrein from prostatic tissue, together with time-resolved fluorescence imaging (TRFI), is a suitable method for obtaining quantitative data from biological samples and that the signal response is linear. In this paper we show that Eu-chelate containing particles in the nanometer range are suitable labels for quantitative IHC. Even single nanoparticle molecules can be detected by TRFI and the signals measured can be readily quantitated. The signal intensity correlates very well with the amount of bound label, and the use of nanoparticles could markedly improve the sensitivity of quantitative IHC methods. TRFI provides a powerful tool for providing quantitative data about antigens or transcripts in tissue sections or cultured cells. It is also of major importance in standardization and optimization of protocols for fixation and tissue preparation, including antigen retrieval methods.

Human eosinophils produce the T cell-attracting chemokines MIG and IP-10 upon stimulation with IFN-{gamma}

Eosinophils participate in allergic inflammation, where expression of T helper cell type 2 (Th2) cytokines such as interleukin (IL)‐4 and IL‐5 are seen. However, eosinophils sometimes accumulate during disease with expression of Th1 cytokines [i.e., interferon‐γ (IFN‐γ), tumor necrosis factor α (TNF‐α), and IL‐1β]. In this study, we investigated whether eosinophils can respond with expression of the IFN‐inducible C–X–C chemokines monokine induced by IFN‐γ [MIG; CXC chemokine ligand 9 (CXCL9)], IFN‐γ‐inducible protein (IP‐10/CXCL10), and IFN‐inducible T cell α chemoattractant (I‐TAC/CXCL11). These chemokines share the ability to recruit and activate T cells and natural killer cells to sites of inflammation. We found that IFN‐γ induced rapid and sustained gene expression of MIG, IP‐10, and I‐TAC in eosinophils, as detected by quantitative reverse transcriptase‐polymerase chain reaction. During incubation, IFN‐γ‐stimulated eosinophils released MIG and IP‐10, as detected by enzyme‐linked immunosorbent assay, while I‐TAC could not be detected in the medium. TNF‐α but not IL‐1β enhanced the IFN‐γ‐induced production of MIG and IP‐10. Conversely, addition of the Th2 cytokine IL‐4 down‐regulated IFN‐γ‐induced synthesis of MIG and IP–10 in eosinophils. Crohns disease is characterized by a Th1‐polarized inflammation and presence of eosinophils. In lesions from this disease, MIG was detected in eosinophils by immunohistochemistry. Taken together, the results point to immunoregulatory roles for eosinophils during some diseases with Th1‐polarized inflammation.

Expression and production of the CXC chemokine growth-related oncogene-alpha by human eosinophils.

Eosinophils are seen together with neutrophils at sites of inflammation. However, their roles are not clear. In addition, eosinophils infiltrate tumor tissue in some neoplastic diseases. In this study, we show that large amounts of the neutrophil-activating CXC chemokine growth-related oncogene (GRO)-α can be produced by human eosinophils. Eosinophils showed presence of preformed GRO-α in the crystalloid-containing specific granules (190 pg/2 × 106 cells). During incubation, a strong increase in GRO-α gene expression was seen. At a low cell density, addition of TNF-α or IL-1β increased the production of GRO-α in eosinophils, which was not the case at a higher cell density. Eosinophils can produce TNF-α themselves, and neutralizing Abs against TNF-α significantly inhibited GRO-α production. This suggests that autocrine and paracrine effects from TNF-α can be important when up-regulating GRO-α gene expression. In contrast, IFN-γ, a prototypic Th1-cytokine, down-regulated expression of GRO-α. This may be important during resolution of inflammation but also suggests different roles for eosinophils depending on the inflammatory context. Tumor-infiltrating eosinophils in Hodgkin’s disease of the nodular sclerosing type are associated with a poor prognosis. Eosinophils from such tumor tissue showed an abundant expression of GRO-α. The GRO-α receptor CXCR2 was also detected in tumor tissue, proposing interactions between eosinophils and the tumor. Our findings suggest that eosinophils can promote inflammation through recruitment of CXCR2-bearing cells. In addition, this feature of the eosinophils indicates a role for these cells in the biology of certain tumors.

Male infertility and prostate cancer risk: a nested case-control study.

The pathogenesis of prostate cancer is unclear, although experimental evidence implicates androgens as playing an important role. Infertile men frequently suffer from some degree of hypogonadism and may hence be hypothesized to be at lower risk of developing prostate cancer than fertile men. To test this hypothesis, we conducted a case–control study nested within “the Malmö Diet and Cancer Study” cohort in Sweden, inviting 661 prostate cancer cases and 661 age-matched controls to participate. Of the 975 (74%) respondents, we excluded 84 childless men with unknown fertility status. Thus, 891 men were included, providing 445 prostate cancer cases and 446 controls. Of these, 841 (94%) men were biological fathers and 50 (6%) men were infertile. Logistic regression showed that the infertile men were at significantly lower risk of being diagnosed with prostate cancer than the fertile men (odds ratio, 0.45; 95% confidence interval, 0.25–0.83). Conditional and unconditional multivariate models, adjusting for socioeconomic, anthropometric, and health-status-related factors, provided similar estimates. We conclude that enduring male infertility is associated with a reduced prostate cancer risk, thus corroborating the theory that normal testicular function, and hence most probably sufficient steroidogenesis, is an important contributing factor to the later development of this malignancy.

Serotonin activates MAP kinase and PI3K/Akt signaling pathways in prostate cancer cell lines

PURPOSEnThis study was conducted to examine the effects of 5-HT on extracellular signal-regulated kinase 1/2 (Erk1/2) and Akt pathways in prostate cancer (PC) cells.nnnMETHODSnPC cell lines PC-3, Du145, and LNCaP stimulated with 5-HT in the presence of MEK or PI3K inhibitors and 5-HT receptor subtype 1A antagonist were analyzed by Western blotting and immunofluorescence. The proliferation assay BrdU and Boyden chamber were used to determine proliferation and migration, respectively.nnnRESULTSn5-HT dose-dependently induced rapid activation of Erk1/2 in PC-3 and Du145 cells, whereas in LNCaP cells, Erk1/2 phosphorylation was slow and sustained for up to 18 h. Similarly, 5-HT induced phosphorylation of Akt within 1 hour of stimulation, however, Akt phosphorylation was more pronounced in Du145 cells compared with PC-3 or LNCaP cells. The action of 5-HT was inhibited to varying degrees by inhibitors of MAPK and PI3K as well as by a 5-HT receptor subtype 1A antagonist. In addition to proliferation, 5-HT induced migration of PC-3 and Du145 cells, which were alleviated by the aforementioned inhibitors. The effects of 5-HT on LNCaP cells appeared to be related to neuroendocrine-phenotype acquisition and chromogranin A and neuron specific enolase expression.nnnCONCLUSIONSnThis study addresses the role of 5-HT in Erk1/2 and Akt activation in PC cells. The data presented here identify 5-HT receptors as a novel target in castration-resistant PC. Furthermore, our observations are in line with previous studies, which point towards neuroendocrine factors facilitating progression and migration of prostatic cancer cells in an androgen-deficient environment. Nonetheless, additional studies are warranted to corroborate the role of 5-HTR antagonists as a potential target for anticancer therapy.

Trypsin-2 degrades human type II collagen and is expressed and activated in mesenchymally transformed rheumatoid arthritis synovitis tissue

It has traditionally been believed that only the human collagenases (matrix metalloproteinase-1, -8, and -13) are capable of initiating the degradation of collagens. Here, we show that human trypsin-2 is also capable of cleaving the triple helix of human cartilage collagen type II. We purified human trypsin-2 and tumor-associated trypsin inhibitor by affinity chromatography whereas collagen type II was purified from cartilage extracts using pepsin digestion and salt precipitation. Degradation of type II collagen and gelatin by trypsin-2 was demonstrated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, and mass spectrometry, and tumor-associated trypsin inhibitor specifically inhibited this degradation. Although human trypsin-2 efficiently digested type II collagen, bovine trypsin did not. Furthermore, immunohistochemical staining detected trypsin-2 in the fibroblast-like synovial lining and in stromal cells of human rheumatoid arthritis synovial membrane. These findings were confirmed by reverse transcriptase-polymerase chain reaction and nucleotide sequencing. Trypsin-2 alone and complexed with alpha(1)-proteinase inhibitor were also detected in the synovial fluid of affected joints by time-resolved immunofluorometric assay, suggesting that trypsin-2 is activated locally. These results are the first to assess the ability of human trypsin to cleave human type II collagen. Thus, trypsin-2 and its regulators should be further studied for use as markers of prognosis and disease activity in rheumatoid arthritis.