Nuclear imaging to support anti-inflammatory drug discovery and development.

Nuclear medicine contributes important tools to support anti-inflammatory drug discovery and development. The support provided is manifold: new molecular entities (NME, either small molecules or biologics) labeled with radioisotopes can be applied in animal models and humans to measure biodistribution, target engagement, and pharmacokinetics. In addition, nuclear imaging techniques can be used to select or enrich the patient populations in clinical trials, to assess disease activity, target status and distribution and to quantify response to therapeutic interventions. In the first part of this review we will outline how nuclear imaging techniques can be applied to support informed decision making in drug development. In the second part, we will briefly high-light the use of nuclear imaging of inflammation in drug development in selected diseases, specifically rheumatoid arthritis (RA), inflammatory bowel diseases (IBD), atherosclerosis and - as an emerging topic - cancer.

Nuclear imaging to support anti-inflammatory drug discovery and development.

Nuclear medicine contributes important tools to support antiinflammatory drug discovery and development in many ways. The support provided is manifold: new molecular entities (NME, either small molecules or biologics) labeled with radioisotopes can be applied in animal models and humans to measure biodistribution, target engagement, and pharmacokinetics. In addition, nuclear imaging techniques can be used to select or enrich the patient populations in clinical trials, to assess disease activity, target status and distribution and to quantify response to therapeutic interventions. In the first part of this review we will outline how nuclear imaging techniques can be applied to support informed decision making in drug development. In the second part, we will briefly highlight the use of nuclear imaging of inflammation in drug development in selected diseases, specifically rheumatoid arthritis (RA), inflammatory bowel diseases (IBD), atherosclerosis (ATS) and as an emerging topic cancer.

A role for T-bet-mediated tumour immune surveillance in anti-IL-17A treatment of lung cancer.

Lung cancer is the leading cause of cancer deaths worldwide. The cytokine interleukin-17A supports tumour vascularization and growth, however, its role in lung cancer is unknown. Here we show, in the lungs of patients with lung adenocarcinoma, an increase in interleukin-17A that is inversely correlated with the expression of T-bet and correlated with the T regulatory cell transcription factor Foxp3. Local targeting of interleukin-17A in experimental lung adenocarcinoma results in a reduction in tumour load, local expansion of interferon-γ-producing CD4(+) T cells and a reduction in lung CD4(+)CD25(+)Foxp3(+) regulatory T cells. T-bet((-/-)) mice have a significantly higher tumour load compared with wild-type mice. This is associated with the local upregulation of interleukin-23 and induction of interleukin-17A/interleukin-17R-expressing T cells infiltrating the tumour. Local anti-interleukin-17A antibody treatment partially improves the survival of T-bet((-/-)) mice. These results suggest that local anti-interleukin-17A antibody therapy could be considered for the treatment of lung tumours.

Old proteins - new locations: myoglobin, haemoglobin, neuroglobin and cytoglobin in solid tumours and cancer cells.

AIM: The unexpected identification of myoglobin (MB) in breast cancer prompted us to evaluate the clinico-pathological value of MB, haemoglobin (HB) and cytoglobin (CYGB) in human breast carcinoma cases. We further screened for the presence of neuroglobin (NGB) and CYGB in tumours of diverse origin, and assessed the O(2) -response of HB, MB and CYGB mRNAs in cancer cell lines, to better elicit the links between this ectopic globin expression and tumour hypoxia. METHODS: Breast tumours were analysed by immunohistochemistry for HB, MB and CYGB and correlated with clinico-pathological parameters. Screening for CYGB and NGB mRNA expression in tumour entities was performed by hybridization, quantitative PCR (qPCR) and bioinformatics. Hypoxic or anoxic responses of HB, MB and CYGB mRNAs was analysed by qPCR in human Hep3B, MCF7, HeLa and RCC4 cancer cell lines. RESULTS: 78.8% of breast cancer cases were positive for MB, 77.9% were positive for HB and 55.4% expressed CYGB. The closest correlation with markers of hypoxia was observed for CYGB. Compared to the weakly positive status of MB in healthy breast tissues, invasive tumours either lost or up-regulated MB. Breast carcinomas showed the tendency to silence CYGB. HB was not seen in normal tissues and up-regulated in tumours. Beyond breast malignancies, expression levels of NGB and CYGB mRNAs were extremely low in brain tumours (glioblastoma, astrocytoma). NGB was not observed in non-brain tumours. CYGB mRNA, readily detectable in breast cancer and other tumours, is down-regulated in lung adenocarcinomas. Alpha1 globin (α1 globin) and Mb were co-expressed in MCF7 and HeLa cells; CYGB transcription was anoxia-inducible in Hep3B and RCC4 cells. CONCLUSIONS: This is the first time that HB and CYGB are reported in breast cancer. Neither NGB nor CYGB are systematically up-regulated in tumours. The down-regulated CYGB expression in breast and lung tumours is in line with a tumour-suppressor role. Each of the screened cancer cells expresses at least one globin (i.e. main globin species: CYGB in Hep3B; α1 globin + MB in MCF7 and HeLa). Thus, globins exist in a wide variety of solid tumours. However, the generally weak expression of the endogenous proteins in the cancer argues against a significant contribution to tumour oxygenation. Future studies should consider that cancer-expressed globins might function in ways not directly linked to the binding and transport of oxygen.

Tumor-associated CpG demethylation augments hypoxia-induced effects by positive autoregulation of HIF-1α.

Hypoxia-inducible factor 1α (HIF-1α) is frequently overexpressed in human cancers and controls the expression of several genes that have been implicated in tumor growth and progression. Activity of HIF-1α in cancer cells is regulated at the transcriptional, translational and posttranslational level by multiple inter- and coacting molecular pathways. In this report, we reveal for the first time that tumor-associated CpG demethylation facilitates positive autoregulation of HIF-1α, resulting in amplification of hypoxia-induced transactivation of HIF-1α target genes. The HIF-1α promoter harbors a hypoxia response element that is normally repressed by methylation of a CpG dinucleotide located in the core element. In colon cancer cell lines and in primary colon cancer specimens, however, we found frequent aberrant demethylation of this element, enabling binding of HIF-1α to its own promoter resulting in autotransactivation of HIF-1α expression. Our results provide novel and highly unexpected insights into the complexity of HIF-1α regulation in cancer cells and implicate that tumor-associated CpG demethylation augments HIF-1α-mediated effects on malignant cell growth.

A novel role for constitutively expressed epithelial-derived chemokines as antibacterial peptides in the intestinal mucosa.

Intestinal-derived chemokines have a central role in orchestrating immune cell influx into the normal and inflamed intestine. Here, we identify the chemokine CCL6 as one of the most abundant chemokines constitutively expressed by both murine small intestinal and colonic epithelial cells. CCL6 protein localized to crypt epithelial cells, was detected in the gut lumen and reached high concentrations at the mucosal surface. Its expression was further enhanced in the small intestine following in vivo administration of LPS or after stimulation of the small intestinal epithelial cell line, mIC(c12), with IFNgamma, IL-4 or TNFalpha. Recombinant- and intestinal-derived CCL6 bound to a subset of the intestinal microflora and displayed antibacterial activity. Finally, the human homologs to CCL6, CCL14 and CCL15 were also constitutively expressed at high levels in human intestinal epithelium, were further enhanced in inflammatory bowel disease and displayed similar antibacterial activity. These findings identify a novel role for constitutively expressed, epithelial-derived chemokines as antimicrobial peptides in the intestinal mucosa.

Influence of standard treatment on ileal and colonic antimicrobial defensin expression in active Crohn's disease.

BACKGROUND: Crohn's Disease (CD), a chronic intestinal inflammation, is currently treated primarily by therapeutics which are directed against inflammatory responses. Recent findings though suggest a central role of the innate immune barrier in the pathophysiology. Important factors providing this barrier are antimicrobial peptides like the alpha- and beta-defensins. Little is known about in vivo effects of common drugs on their expression. AIM: To analyse the influence of corticosteroids, azathioprine and aminosalicylate treatment on ileal and colonic antimicrobial peptides in active CD and also assess the role of inflammation. METHODS: We measured the expression of antimicrobial peptides and pro-inflammatory cytokines in 75 patients with active CD. RESULTS: Ileal and colonic alpha- and beta-defensins as well as LL37 remained unaffected by corticosteroids, azathioprine or aminosalicylate treatment. Additionally, we did not observe a negative coherency between Paneth cell alpha-defensins and any measured cytokines. HBD2 and LL37 unlike HBD1 levels were linked to inflammatory cytokines and increased in highly inflamed samples. CONCLUSIONS: Current oral drug treatment seems to have no major effect on the expression of antimicrobial peptides. In contrast to HBD2 and LL37, ileal levels of HD5 and HD6 and colonic HBD1 level are independent of current inflammation. Innovative drugs should aim to strengthen protective innate immunity.

Barrier dysfunction due to distinct defensin deficiencies in small intestinal and colonic Crohn's disease.

Defensins are endogenous antibiotics with broad microbicidal activity. A disturbed antimicrobial defense, as provided by Paneth and other epithelial defensins, seems to be a critical factor in the pathogenesis of inflammatory bowel diseases. Conspicuously, there is a relative lack of Paneth-cell alpha-defensins in ileal Crohn's disease (CD), both in the absence of a pattern recognition receptor nucleotide-binding oligomerization domain 2 (NOD2) frameshift mutation and, even more pronounced, in its presence. This deficit is independent of concurrent active inflammation and cannot be seen in active small intestinal ulcerative colitis (UC; pouchitis) as well as NOD2 wild-type graft vs. host ileitis. After intestinal transplantation, in case of NOD2 mutation, defensins are decreased before the onset of inflammation. In the majority of patients, the Paneth-cell deficiency is mediated by Wnt-TCF4, which suggests a disturbed Paneth-cell differentiation. In contrast, colonic CD is characterized by an impaired induction of mucosal beta-defensins, partly because of a low copy number of the beta-defensin gene cluster. In both ileal and colonic CD, the lack in defensins results in a broadly diminished antibacterial killing by the mucosa, which can also be found independent of inflammation. In summary, the main disease locations can be linked to distinct mechanisms of epithelial barrier dysfunction.

Is levonorgestrel intrauterine system effective for treatment of early endometrial cancer? Report of four cases and review of the literature.

BACKGROUND: Intrauterine progesterone therapy potentially provides a simple alternative treatment for women with Stage I Grade I endometrial cancers who are at high risk for surgery. The case histories of four women with early endometrial cancer primarily treated with levonorgestrel intrauterine system (Mirena) are reported and the literature reviewed. CASES: Four women had Stage I grade 1 endometrial adenocarcinoma with positive progesterone receptor. All were assessed to be in American Society of anaesthesiologists risk class IV. After insertion of mirena intrauterine system, one woman (25%) had complete histological regression of disease within 6 months. One of three women who did not respond to treatment subsequently had a vaginal hysterectomy, which showed endometrial cancer with superficial myometrial invasion. CONCLUSION: This report raises doubts about the effectiveness of intrauterine progesterone therapy as a definitive alternative for the treatment of early endometrial cancer.

Expression of cancer testis genes in human brain tumors.

Cancer-testis (CT) genes are expressed in a variety of human cancers but not in normal tissues, except for testis tissue, and represent promising targets for immunotherapeutic and gene therapeutic approaches. Because little is known about their composite expression in human brain tumors, we investigated the expression of seven CT genes (MAGE-3, NY-ESO-1, HOM-MEL-40/SSX-2, SSX-1, SSX-4,HOM-TES-14/SCP-1, and HOM-TES-85) in 88 human brain tumor specimens. Meningiomas expressed only HOM-TES-14/SCP-1 (18% of meningiomas were HOM-TES-14/SCP-1 positive) and did not express any other CT genes. One ependymoma was negative for all CT genes tested. SSX-4 was the only CT gene expressed in oligodendrogliomas (2 of 5 cases), and it was also expressed in oligoastrocytomas (3 of 4 cases) and astrocytomas (10 of 37 cases). Astrocytomas were most frequently positive for HOM-TES-14/SCP-1 (40%) and SSX-4 (27%), followed by HOM-TES-85 (13%), SSX-2 (11%), and MAGE-3 (7%). Whereas MAGE-3 was detected only in grade IV astrocytomas, the expression of the other CT genes showed no clear correlation with histological grade. Of 39 astrocytomas, 60% expressed at least one CT gene, 21% expressed two CT genes, and 8% coexpressed three CT genes of the seven CT genes investigated. We conclude that a majority of oligoastrocytomas and astrocytomas might be amenable to specific immunotherapeutic interventions. However, the identification of additional tu-mor-specific antigens with a frequent expression in gliomas is warranted to allow for the development of widely applicable polyvalent glioma vaccines.

Identification of a meiosis-specific protein as a member of the class of cancer/testis antigens.

Little is known about the function of human cancer/testis antigens (CTAs), such as MAGE, BAGE, GAGE, HOM-MEL-40, and NY-ESO-1, the expression of which is restricted to human malignancies and testis. When screening a cDNA expression library enriched for testis-specific representative long transcripts for reactivity with high-titered IgG antibodies from the serum of a patient with renal cell carcinoma, one repeatedly detected antigen, designated HOM-TES-14, turned out to be encoded by the synaptonemal complex protein 1 (SCP-1) gene. SCP-1 is known to be selectively expressed during the meiotic prophase of spermatocytes and is involved in the pairing of homologous chromosomes, an essential step for the generation of haploid cells in meiosis I. Investigation of a broad spectrum of normal and malignant tissues revealed expression of SCP-1 transcripts and antigen selectively in a variety of neoplastic tissues and tumor cell lines. Immunofluorescence microscopy analysis with specific antiserum showed a cell cycle phase-independent nuclear expression of SCP-1 protein in cancer cells. SCP-1 differs from other members of the class of CTA by its localization on chromosome 1 and its frequent expression in malignant gliomas, breast, renal cell, and ovarian cancer. The aberrant expression of SCP-1 in tumors might contribute to their genomic instability and suggests that the functional role of other CTA might also relate to meiosis.

Alpha-smooth muscle actin distribution in the pulmonary vasculature comparing hypoplastic and normal fetal lungs.

We investigated the intra-acinar pulmonary vascular muscularization in the developing human fetal lung between the 17th and 24th gestational weeks, that is, during the canalicular phase of lung development. Fifteen hypoplastic and 25 normal developed lungs were included in this study using monoclonal alpha-smooth muscle (sm) actin antibodies for smooth muscle detection. Computer-aided image analysis was performed for morphometrical measurements and statistical evaluation. Alpha-sm-actin-immunoreactive intra-acinar vessels down to a luminal diameter of less than 10 microns were detected in hypoplastic as well as in normally developed lungs. Crucial differences presented as follows: significantly higher density of intra-acinar vessels, especially due to alpha-sm-actin-negative vessels less than 30 microns in luminal diameter, in the control group; significantly higher alpha-sm-actin immunore-activity per section unit as well as per vessel in the hypoplastic lung group. As suggested by others, alpha-sm-actin-positive cells of the intra-acinar vessel wall in the developing human lung were demonstrated to be smooth muscle cells, their immediate precursors, and pericytes. We conclude that the increased alpha-sm-actin immunoreactivity represents muscularization of the vessel wall in functional terms and may be regarded as one structural cause among others for the establishment of persistent fetal circulation in hypoplastic lungs.

The SSX-2 gene, which is involved in the t(X;18) translocation of synovial sarcomas, codes for the human tumor antigen HOM-MEL-40.

Using autologous serum for the serological analysis of recombinantly expressed clones (SEREX) from a cDNA derived from a human melanoma, several new melanoma antigens were identified that are immunogenic in the autologous host. Sequence analysis revealed that one of these antigens, HOM-MEL-40, was coded for by the SSX2 gene, which has recently been described to be involved in the t(X;18) translocation of human synovial sarcomas. Expression analysis performed by Northern blot and RT-PCR demonstrated the presence of HOM-MEL-40 transcripts in a significant proportion of human melanomas (50%), colon cancers (25 %), hepatocarcinomas (30%), and breast carcinoma (20%) but not in normal tissues except for testis. Sequence comparison with transcripts cloned from testis ruled out mutations in the melanoma-derived HOM-MEL-40. Antibodies against HOM-MEL-40 were found in 10 of 89 patients with melanoma, including 3 of 8 patients with HOM-MEL-40-positive tumors, but not in 41 apparently healthy controls. In view of the specific expression pattern and immunogenicity in cancer patients, HOM-MEL-40 holds promise as a target for immune interventions in a considerable population of patients with HOM-MEL-40-positive tumors.

[Psychiatric morbidity following psychic trauma of combat origin].

Psychiatric morbidity following trauma of combat origin was studied in 60 Israeli veterans who applied for treatment 4-6 years after the war in Lebanon. Diagnostic tools were structured interviews for psychiatric disorders. In all subjects multiple psychiatric disorders had been diagnosed, most of which developed following the traumatic experience. Post-traumatic stress disorder (PTSD) was the most prevalent condition (100%-ever; 87%-present). Comorbidity included major depression with a very high prevalence (95%-ever; 50%-present) and anxiety disorders: phobic disorder, obsessive-compulsive disorder, panic attacks and generalized anxiety (prevalence range 12-25%-ever and 8-18%-present). The prevalence of minor affective disorders (specifically alcoholism or drug abuse) was considerably less. These findings suggest a possible link between PTSD and depressive and anxiety disorders, beyond a mere sharing of common symptoms. They also support the diagnostic validity of PTSD as an independent nosologic category. In addition, the lack of specific pharmacotherapy for PTSD on the one hand and the high prevalence of depressive and anxiety comorbidity on the other, support a special focus on antidepressive and anxiolytic medication for PTSD.