A comparative analysis of oncofetal fibronectin and tenascin-C incorporation in tumour vessels using human recombinant SIP format antibodies

Tumour angioneogenesis is associated with the reexpression of oncofetal fibronectin (oncFn) and tenascin-C (oncTn-C) splice variants, which may serve as targets for antibody-based pharmacodelivery. Knowledge of the vascular distribution and organization in different tumours is of importance for the understanding of tumour vessel formation and might be crucial for therapy. Therefore, human SIP format antibodies against Fn ED-A, Fn ED-B and Tn-C A and C splice domains were used for immunofluorescence labelling in renal, lung, oral, colon, breast and urinary bladder carcinoma specimens and in a renal carcinoma xenograft. The spatial relation to stroma, vessels and vascular basement membrane (vBM) was analysed including CD31 and laminin α4 chain antibodies. Renal cell carcinomas and atypical carcinoid of the lung revealed vessel-restricted oncFn and/or oncTn-C depositions; all other entities showed a variable stroma positivity including vessels. The individual pattern of oncFn/oncTn-C incorporation in the vBM depended on tumour type, vessel size and intratumoural heterogeneity. There was a stratification of the vessel wall showing luminal oncFn and extraluminal oncTn-C depositions. As shown in the xenograft, perivascular oncTn-C is provided by carcinoma cells. In conclusion, tumours differ in the pattern of Fn or Tn-C isoform positivity in the vessel wall, potentially representing a tumour type specific endothelial cell–tumour cell–stromal cell interaction. Carcinoma cells themselves are involved in vascular Tn-C matrix organization. Up to antigen distribution, Fn and Tn-C domain antibodies may serve as vehicles for antiangiogenetic and antifibrotic agents; oncFn/oncTn-C based targeting should be adapted individually.     

Effect of Genetic Variation in the Human Fatty Acid Synthase Gene (FASN) on Obesity and Fat Depot‐Specific mRNA Expression

Inhibition of fatty acid synthase (FASN) induces a rapid decline in fat stores in mice, suggesting a role for this enzyme in energy homeostasis. To investigate the potential role of FASN in the pathophysiology of human obesity, the FASN gene was sequenced in 48 German whites. Thirty‐five single‐nucleotide polymorphisms (SNPs) were identified. Eight SNPs representative for their linkage disequilibrium groups and the Val1483Ile (rs2228305) substitution were genotyped for subsequent association analyses in 1,311 adults from Germany. Further, the tagging SNPs were genotyped also in German childhood cohorts (738 schoolchildren, 205 obese children). Effects of genetic variation on FASN mRNA expression in visceral and subcutaneous adipose tissue from a subgroup of 172 subjects were analyzed. Several polymorphisms in the FASN (rs62078748, rs2229422, rs2229425, and rs17848939) were nominally associated with obesity in case–control studies including 446 obese subjects (BMI ≥30 kg/m²) and 389 lean controls (BMI ≤25 kg/m²) (adjusted P < 0.05). The strongest significant effect was found for rs2229422 (P = 1.3 × 10^?5 adjusted for age, sex, type 2 diabetes status), which was supported by associations with BMI, waist‐to‐hip ratio (WHR), fasting plasma insulin and glucose infusion rate (adjusted P < 0.05). Subjects with the Val1483Ile substitution appeared to be protected against obesity. In addition, rs17848939 was nominally significantly associated with the ratio of visceral/subcutaneous FASN mRNA expression (adjusted P = 0.04). No effect of genetic variation in FASN on obesity was found in children. In conclusion, our data indicate a role of FASN genetic variation in susceptibility to obesity in adults.     

Depletion of the high-abundance plasma proteins

Summary. Body fluids, like plasma and urine, are comparatively easy to obtain and are useful for the detection of novel diagnostic markers by applying new technologies, like proteomics. However, in plasma, several high-abundance proteins are dominant and repress the signals of the lower-abundance proteins, which then become undetectable either by two-dimensional gels or chromatography. Therefore, depletion of the abundant proteins is a prerequisite for the detection of the low-abundance components. We applied affinity chromatography on blue matrix and Protein G and removed the most abundant human plasma proteins, albumin and the immunoglobulin chains. The plasma proteins, prior to albumin and immunoglobulin depletion, as well the eluates from the two chromatography steps were analyzed by two-dimensional electrophoresis and the proteins were identified by MALDI-TOF-MS. The analysis resulted in the identification of 83 different gene products in the untreated plasma. Removal of the high-abundance proteins resulted in the visualization of new protein signals. In the eluate of the two affinity steps, mostly albumin and immunoglobulin spots were detected but also spots representing several other abundant plasma proteins. The methodology is easy to perform and is useful as a first step in the detection of diagnostic markers in body fluids by applying proteomics technologies.Current address: Foundation for Biomedical Research of the Academy of Athens, Greece     

Chlamydiae in oviducts and uteri of repeat breeder pigs

Chlamydial infections of the genital organs cause reproductive failure in female pigs, and the uterus is recognized a target tissue for an infection. In contrast, information on the effect of chlamydiae on the porcine oviduct is patchily and inconclusive, although the bacteria are known to cause severe tubal defects in humans and laboratory animals. The aim of this study was to examine the segments ampulla (A), isthmus (I) and utero-tubal junction of the left (n=20) or both (n=22) oviducts, and uteri (U) from 42 culled repeat breeder pigs for chlamydiae using ompA-PCR, partial ompA gene sequencing, immunohistochemistry (IHC) and microscopy of tissue specimens for histopathology. As revealed by PCR, among a total of 26 chlamydia-positive females, 19 were tested positive in one or more segments of one or both oviducts, 14 were found positive in the uterus, and concomitant infections of both organs were observed in 7 of them. Sequencing of 33 PCR products revealed the following chlamydial species: Chlamydophila (Cp.) psittaci (n=18), Cp. abortus (n=2), Chlamydia (C.) suis (n=10), and C. trachomatis (n=3). Immunopositive staining was observed within the surface epithelium (in A, I, U), stromal tissue (in I, U) and muscular layer (in A, I, U). A total of 24 females had inflamed oviductal segments (in A and/or I) and 36 inflamed uteri. However, there was no relationship between histopathology and results of PCR or IHC. In conclusion, chlamydiae were found to infect oviducts and uteri of pigs. Further studies are required to clarify whether chlamydial infection causes specific histopathology and alters tubal function.     

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