Mona Ståhle-Bäckdahl

The Human Cationic Antimicrobial Protein (hCAP-18) Is Expressed in the Epithelium of Human Epididymis, Is Present in Seminal Plasma at High Concentrations, and Is Attached to Spermatozoa

ABSTRACT Innate immunity is important for the integrity of the host against potentially invasive pathogenic microorganisms in the environment. Antibiotic peptides with broad antimicrobial activity are part of the innate immune system. We investigated the presence of the cathelicidin, human cationic antimicrobial protein (hCAP-18), in the male reproductive system. We found strong expression of the hCAP-18 gene by in situ hybridization and hCAP-18 protein, as detected by immunohistochemistry, in the epithelium of the epididymis, but not in the testis. The highest expression in the epididymis was in the caudal part. Western blotting showed a doublet band, the upper part corresponding to the size of hCAP-18 in plasma and neutrophils. Using a specific enzyme-linked immunosorbent assay (ELISA), levels of 86.5 ± 37.8 μg/ml (mean ± standard deviation; range, 41.8 to 142.8 μg/ml; n = 10) were detected in seminal plasma from healthy donors, which is 70-fold higher than the level in blood plasma. Flow cytometry and immunocytochemistry revealed the presence of hCAP-18 on spermatozoa. ELISA measurement showed levels of 196 ng/106 spermatozoa, corresponding to 6.6 × 106 molecules of hCAP-18 per spermatozoon. Our results suggest a key role for hCAP-18 in the antibacterial integrity of the male reproductive system. The attachment of hCAP-18 to spermatozoa may implicate a role for hCAP-18 in conception.

Neutrophil gelatinase-associated lipocalin is a marker for dysregulated keratinocyte differentiation in human skin

Abstract: Neutrophil gelatinase‐associated lipocalin (NGAL) is a 25‐kDa protein initially isolated from the specific granules of human neutrophils. It is a member of the highly heterogeneous lipocalin protein family, which shares a common tertiary structure. Its synthesis is induced in gastrointestinal epithelium in association with inflammation and malignancy. To gain insight into its potential role in other epithelia we have investigated the expression of NGAL in human skin embryonic development, in normal adult skin, and in skin associated with inflammation and neoplastic transformation. In the present study we report that the embryonic expression of NGAL appears to be regulated in a spatio‐temporal pattern. It was induced in the interfollicular epidermis at 20–24 weeks of gestational age but thereafter progressively receded towards the hair follicles. In normal adult skin, NGAL was detected solely in association with hair follicles. However, strong induction of NGAL in the epidermis was seen in a variety of skin disorders characterized by dysregulated epithelial differentiation such as psoriasis, pityriasis rubra and squamous cell carcinoma. In these tissues production of NGAL was confined to spatially distinct subpopulations of keratinocytes underlying areas of parakeratosis, whereas skin samples lacking parakeratotic epithelium such as lichen ruber planus, acute contact eczema and basal cell carcinoma were negative for NGAL. Consistent with being a marker for disturbed terminal differentiation, NGAL immunoreactivity showed an inverse pattern when compared with that of the differentiation marker filaggrin. The biologic functions of NGAL in epithelia are not fully known, although an immunomodulatory role in host defense has been proposed. In addition, the transient interfollicular NGAL expression during skin embryogenesis along with the induction of NGAL in adult parakeratotic epidermis suggests it play a role in epithelial differentiation pathways.

Polymorphisms in the SEEK1 and SPR1 genes on 6p21.3 associate with psoriasis in the Swedish population.

Abstract:  Psoriasis is a chronic skin disease that results in red and scaly lesions. Several psoriasis susceptibility loci have been identified across the genome, of which PSORS1 on 6p21.3 is predominant. There is an ongoing debate regarding whether the HLA‐C allele, Cw*0602, can be considered the major predisposing factor in this region. Investigation of other genes in the PSORS1 region with regard to psoriasis may provide alternate candidates to HLA‐C. We have characterized two overlapping genes, SEEK1 and SPR1. SEEK1 encodes two putative protein isoforms: the first being one of 152 amino acids from the full‐length splice‐isoform (exon 1–6), and the second being one of 100 amino acids from an alternate splice‐isoform (exon 1 and 6). SPR1 encodes a highly conserved protein of 134 amino acids, and in addition to characterization of human SPR1 we report the cloning of its orthologs in mouse and pig. Both SEEK1 and SPR1 are expressed in normal and psoriasis skin. In a case–control study, five of the nine single nucleotide polymorphisms (SNPs) found in SEEK1 were associated with psoriasis, while one of the four SNPs found in SPR1 showed association. Testing the Cw*0602 confounding status revealed that two of the SEEK1 SNPs showed Cw*0602‐independent association, while the SPR1 SNP showed Cw*0602‐dependent association. The second exon of SEEK1, containing the two Cw*0602‐independent SNPs, showed the highest concentration of the psoriasis‐associating SNPs, but did not appear to be translated.

Polyclonal antibodies to tropoelastin and the specific detection and measurement of tropoelastin in vitro

Because tropoelastin is difficult to purify, most antibodies to elastin are raised against the insoluble form of the molecule. While these antibodies cross-react with tropoelastin, antigenic differences between insoluble and soluble elastin suggest that antibodies raised directly against tropoelastin might provide a more sensitive and specific reagent for evaluating tropoelastin production in elastin-producing systems. Using an improved method for purifying tropoelastin from tissue culture explants, we describe the generation and characterization of an antibody to bovine tropoelastin. This antibody was used to develop a sensitive, direct-binding immunoassay capable of quantifying small levels of tropoelastin in conditioned medium from cultured cells. This assay takes advantage of the propensity of tropoelastin to adsorb to vinyl microtiter plates, even in the presence of serum proteins. This property, in combination with the increased sensitivity obtained using antibodies to tropoelastin, provides for a direct-binding immunoassay that detects nanogram quantities of tropoelastin directly in cell culture medium, avoiding sample preparation steps that result in extensive loss of tropoelastin. In addition, this direct-binding assay is ten- to 30-fold more sensitive than the existing competitive ELISA assays.