Daniël Broekaert

Identification of the Y985 and Y1077 motifs as SOCS3 recruitment sites in the murine leptin receptor

The leptin system provides a link between adipose mass and the central nervous system. The appetite suppressing effects of leptin are impaired in most obese patients and some mutant mice strains. Herein we describe how suppressor of cytokine signalling 3 (SOCS3), a potential mediator of this leptin resistance is recruited into the activated murine leptin receptor complex. Using a functional assay based on inhibition of leptin mediated reporter induction, and using phosphopeptide affinity chromatography we show binding of SOCS3 to the highly conserved phosphorylated Tyr‐985 and Tyr‐1077 motifs within the mouse leptin receptor.

A Role for Leptin in the Systemic Inflammatory Response Syndrome (SIRS) and in Immune Response, an Update

Leptin was originally identified as an adipocyte-derived cytokine with a key role in the regulation of the energy balance. Subsequent research revealed that leptins biological action is not restricted to its effects on appetite and food intake, but instead has a much more pleiotropic character. There is now ample evidence that leptin has important functions in reproduction, hematopoiesis, HPA-axis endocrinology and angiogenesis. In this review we have focused on the effects of leptin in the antigen-specific immunity and in the inflammatory effector system.

Neutralizing monoclonal antibodies can potentiate IL-5 signaling.

IL‐5 is a major determinant in the survival, differentiation and effector‐functions of eosinophils. It mediates its effect upon binding and activation of a membrane bound receptor (R), composed of a ligand‐specific α‐chain and a β‐chain, shared with the receptors for IL‐3 and granulocyte‐macrophage colony‐stimulating factor. We have generated and mapped the epitopes of three monoclonal antibodies (mAb) directed against this cytokine: the strong neutralizing mAb 5A5 and 1E1, and the very weak neutralizing mAb H30. We found that H30 as well as 5A5 can increase proliferation above the level induced by human (h)IL‐5 alone, in a JAK‐2‐dependent manner, and at every sub‐optimal hIL‐5 concentration analyzed. This effect is dependent on mAb‐mediated cross‐linking of IL‐5R complexes, and is only observed on cell lines expressing a hybrid human/mouse IL‐5Rα‐chain. We discuss these findings in view of the stoichiometric and topological requirements for an activated IL‐5R. Since humanized anti‐IL‐5 mAb are currently in clinical testing, our findings imply that such mAb should be carefully evaluated for their potentiating effects.

Cytokeratin expression patterns in the human tympanic membrane and external ear canal.

SummaryImmunohistochemical investigations were carried out to further reveal the pattern of cytokeratin (CK) expression in middle ear cholesteatoma. Using chain-specific monoclonal antibodies and the indirect immunoperoxidase technique, 10 out of 19 CK polypeptides were screened in cryoslices of fresh postmortem eardrums and external ear canal specimens. Our data, combined with those published before, indicate an intimate relationship between middle ear cholesteatoma lesions and epidermal tissues in the immediate vicinity. Our CK data do not favor the metaplastic origin of cholesteatoma, because the CK complement of cholesteatoma lesions does not include major and typical CK constituents of the middle ear mucosa.

THE PROLIFERATIVE CAPACITY OF THE KERATINIZING ANNULAR EPITHELIUM.

Using monoclonal antibodies specific for CK chains and the indirect immunoperoxidase technique, the expression of 10 CK polypeptides was investigated. The external stratified squamous epithelium of the tympanic membrane generally expressed CKs 5, 10 and 14. In addition, basal keratinocytes in the annular region, both tympanic and deep meatal, expressed CK19 (a simple epithelium marker). Suprabasally the hyperproliferative marker CK16 (known to have a limited distribution in healthy epidermis) was expressed in the same area. These data reflect the unusually proliferative nature of this transitional area.

A role for leptin in the systemic inflammatory response syndrome (SIRS) and in immune response.

Leptin was originally identified as an adipocyte-derived cytokine with a key role in the regulation of the energy balance. Subsequent research has, however, revealed that leptins biological action is not restricted to its effects on appetite and food intake, but rather has a much more pleiotropic character. Evidence is now accumulating that it has important functions in reproduction, hematopoiesis, HPA-axis endocrinology and angiogenesis. In this review, we have focused on the effects of leptin in the immune system, which can be found in both the antigen-specific immunity and in the inflammatory effector system.

Differentiation of Nuclei during Keratinization in Middle Ear Cholesteatoma: DNA Cytophotometry Completed by Computerized Image Analysis

Quantitative DNA cytophotometric techniques were applied to judge the alteration (differentiation) and ultimate fate of nuclei during keratinization in human middle ear cholesteatoma. Compared with a healthy epidermis, a tendency towards postponed nuclear degradation was noticed. Two patterns governing the loss of DNA are recognized. In one group, the mean nuclear DNA content declines continuously, starting in the nearest suprabasal layers and continuing throughout the prickle and granular cell stages, where the ultimate degeneration of nuclei takes place. This pathway corresponds to that observed in epidermis, but evolves more slowly. In another group of samples, the onset of the DNA decline is delayed to the upper prickle cells, exceptionally to more terminal stages of keratinization. During matrix keratinization, a profound nuclear remodelling takes place, similar to that in epidermal tissues, as far as eu- and heterchromatin DNA and area data are concerned. However, euchromatinization of nuclei in matrix prickle cells is more pronounced than in epidermal tissues. The topography of residual heterochromatic clumps does not reflect a persistent margination as in epidermal nuclei, but is the result of more individualized rearrangements. The changes in karyotype are less elaborate when the complete decline of the nuclear DNA content only occurs during terminal keratinization.

Keratin pattern in hyperkeratotic and ulcerated gastric Pars oesophagea in pigs.

Ulceration of the gastric pars oesophagea is a serious problem in the pig industry, and in spite of numerous studies the underlying mechanisms of the development of such ulcers remains largely unknown. The present study was designed first to test the hypothesis that the epithelium of the pars oesophagea of affected pigs would be more susceptible to the irritating action of acidic gastric content owing to a change in the pattern of expression of keratin, and second to look for a member of the keratin family that could be a suitable indicator of early lesions. Samples were collected from the gastric pars oesophagea of slaughter pigs with and without grossly visible mucosal changes, and the keratin patterns of normal and hyperkeratotic and ulcerated epithelium were compared immunohistochemically. The keratin pairs K 4/K 13, and K 5/K 14 were present in both normal and affected epithelia, and had a similar pattern of expression in both conditions. K 4 and K 13 were expressed in all the suprabasal layers, and K 5 and K 14 were expressed only in the basal and epibasal cells. Immunological reactivity with the monoclonal antibodies LL020 and LHK6-markers for hyperproliferative conditions-was present in the suprabasal layers of the epithelium of the hyperkeratotic and the ulcerated pars oesophagea but not in the normal epithelium. These results indicate that K 6 is expressed in association with the mucosal changes. The pattern of the intermediate filaments of keratin suggests that in basic to gastric ulcers in pigs there is an epithelial proliferation leading to visible hyperkeratosis.

Keratinization of middle ear cholesteatomas

SummaryA histochemical study was performed to clarify further the role played by epidermal transglutaminase (ETgase) in the keratinization of aural cholesteatoma. Weakly and strongly keratinized epidermal tissues and healthy middle ear mucosa were included as references. A first assay revealed the distribution of non-specified acyl donor substrates. In a second assay, the topography of involucrin was assessed immunohistochemically. In both epidermal and cholesteatoma matrix tissues, the presence of acyl donors was not restricted to the sites of (E)Tgase activity, but was almost uniformly extended throughout living layers. In reference tissues, residual acyl donors were poorly detected in horny layers, while they were more abundant in the stratum corneum of the cholesteatomas studied. The presence of involucrin along the cell membrane was observed at varying distances throughout the spinous and granular layers, depending upon the epidermal and matrix configurations. In thick epithelia, involucrin rapidly became concentrated at the cell periphery (in spinous kerationcytes), while in thin epithelia it was usually associated with cell flattening. This latter staining profile was observed more frequently in cholesteatomatous tissues. In addition, we regularly noticed an immediately suprabasal accumulation of involucrin, suggesting a locally hyperproliferative state of the matrix. An insufficient availability of acyl donors, especially involucrin, could not be used to explain the defective ETgase-mediated cross-linking of cholesteatoma cell membranes during terminal stages of differentiation. The present investigation may be the first to demonstrate the presence of involucrin in middle ear mucosa.

Further research on solubilization of cholesteatoma keratins.

In the search for a better protein extraction medium for soft keratins and cholesteatomas, we found that the best results were obtained with N-(2 mercapto-propionyD-glycine, Thiola. Thiola also seems to be a better local disruptive agent of keratin deposits such as cholesteatomas in middle ears and operative cavities.