Quantitative correlative proton and electron microprobe analysis of biological specimens

To investigate the possibility of quantitative correlative proton microprobe (PMP) and electron microprobe (EMP) analysis of biological soft tissue, model specimens were analyzed by both techniques. The specimens consisted of freeze-dried sections of gelatin containing known concentrations of nickel chloride. Both for PMP and for EMP, the signal was expressed as the ratio of the characteristic intensity and the continuum intensity in a peak-free region of the spectrum. With both techniques, calibration curves (signal versus known concentration) obtained, showed a deviation from linearity at high nickel concentrations. However, a linear relation (correlation coefficient 0.996) was obtained in a plot of EMP signal versus PMP signal. This indicates that quantitative correlative PMP and EMP analysis can be carried out by using the same standard for both analytical techniques.     

Quantitative correlative proton and electron microprobe analysis of biological specimens

Summary To investigate the possibility of quantitative correlative proton microprobe (PMP) and electron microprobe (EMP) analysis of biological soft tissue, model specimens were analyzed by both techniques. The specimens consisted of freeze-dried sections of gelatin containing known concentrations of nickel chloride. Both for PMP and for EMP, the signal was expressed as the ratio of the characteristic intensity and the continuum intensity in a peak-free region of the spectrum. With both techniques, calibration curves (signal versus known concentration) obtained, showed a deviation from linearity at high nickel concentrations. However, a linear relation (correlation coefficient 0.996) was obtained in a plot of EMP signal versus PMP signal. This indicates that quantitative correlative PMP and EMP analysis can be carried out by using the same standard for both analytical techniques.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

Dolichos biflorus agglutinin (DBA) reveals a similar basal cell differentiation in normal and psoriatic epidermis

Summary Binding of N-acetyl galactosamine (GalNAc)-specific Dolichos biflorus agglutinin (DBA) conjugates to frozen sections of normal epidermis and of psoriatic uninvolved and lesional skin was studied in fluorescence microscopy. The DBA conjugates bound only to single basal cell layer in normal and uninvolved psoriatic epidermis from patients with different blood group status. In the lesional area of psoriatic skin a similar reaction with a single basal cell layer was revealed. Other lectin-conjugates applied, presenting also GalNAc specificity, reacted with most cell layers of normal and both uninvolved and lesional psoriatic epidermis and gave an attenuated reaction with the middle epidermal layers. The results show that the basal cell characteristics are confined only to the cells along the basal membrane also in psoriatic epidermis, although cells in three lowest layers may be able to proliferate.     

Micro-chemical imaging of cesium distribution in Arabidopsis thaliana plant and its interaction with potassium and essential trace elements

Cesium as an alkali element exhibits a chemical reactivity similar to that of potassium, an essential element for plants. It has been suggested that Cs phytotoxicity might be due either to its competition with potassium to enter the plant, resulting in K starvation, or to its intracellular competition with K binding sites in cells. Such elemental interactions can be evidenced by chemical imaging, which determines the elemental distributions. In this study, the model plant Arabidopsis thaliana was exposed to 1 mM cesium in the presence (20 mM) or not of potassium. The quantitative imaging of Cs and endogenous elements (P, S, Cl, K, Ca, Mn, Fe, and Zn) was carried out using ion beam micro-chemical imaging with 5 microm spatial resolution. Chemical imaging was also evidenced by microfocused synchrotron-based X-ray fluorescence (microXRF) which presents a better lateral resolution (<1 microm) but is not quantitative. Cesium distribution was similar to potassium which suggests that Cs can compete with K binding sites in cells. Cesium and potassium were mainly concentrated in the vascular system of stems and leaves. Cs was also found in lower concentration in leaves mesophyll/epidermis. This late representing the larger proportion in mass, mesophyll/epidermis can be considered as the major storage site for cesium in A. thaliana. Trichomes were not found to accumulate cesium. Interestingly, increased Mn, Fe, and Zn concentrations were observed in leaves at high chlorosis. Mn and Fe increased more in the mesophyll than in veins, whereas zinc increased more in veins than in the mesophyll suggesting a tissue specific interaction of Cs with these trace elements homeostasis. This study illustrates the sensitivity of ion beam microprobe and microfocused synchrotron-based X-ray fluorescence to investigate concentrations and distributions of major and trace elements in plants. It also shows the suitability of these analytical imaging techniques to complement biochemical investigations of metallic stress in plants.     

Transforming growth factor-beta 1 localization in normal and psoriatic epidermal keratinocytes in situ.

Transforming growth factor-beta 1 (TGF beta 1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGF beta 1-specific polyclonal antibodies (anti-LC and anti-CC) to determine the presence of TGF beta 1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti-TGF beta 1 staining was observed primarily in the epidermis. Anti-LC TGF beta 1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti-CC TGF beta 1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti-LC and anti-CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGF beta 1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGF beta 1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGF beta 1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGF beta 1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.     

A mathematical model of paracrine regulation of the proliferative activity of epidermis with the participation of T lymphocytes

A mathematical model of paracrine regulation of the proliferative activity of epidermis in the foci of skin lesions in psoriatic patients is presented. The model simulates the participation of skin-infiltrating activated T lymphocytes as cellular regulators of apoptosis of proliferating keratinocytes. It describes all the main phases of the development of a psoriatic lesion: the onset of formation, a long stable or periodic manifestation, resolution of the lesion and its involution to clinically uninvolved and normal skin. The model shows also the possibility of jumpwise (trigger) transition of a pathological focus from remission to a new exacerbation and vice versa, which is a characteristic feature of the disease.     

Biological applications of X-ray fluorescence microscopy: exploring the subcellular topography and speciation of transition metals

Synchrotron X-ray fluorescence microscopy (SXRF) is a microanalytical technique for the quantitative mapping of elemental distributions. Among currently available imaging modalities, SXRF is the only technique that is compatible with fully hydrated biological samples such as whole cells or tissue sections, while simultaneously offering trace element sensitivity and submicron spatial resolution. Combined with the ability to provide information regarding the oxidation state and coordination environment of metal cations, SXRF is ideally suited to study the intracellular distribution and speciation of trace elements, toxic heavy metals and therapeutic or diagnostic metal complexes.     

The tetraspan protein epithelial membrane protein-2 interacts with beta1 integrins and regulates adhesion

The growth arrest-specific-3 (GAS3)/PMP22 proteins are members of the four-transmembrane (tetraspan) superfamily. Although the function of these proteins is poorly understood, GAS3/PMP22 proteins have been implicated in the control of growth and progression of certain cancers. Epithelial membrane protein-2 (EMP2), a GAS3/PMP22 family member, was recently identified as a putative tumor suppressor gene. Here, we addressed the normal function of EMP2 by testing the prediction that it influences integrin-related cell functions. We observed that EMP2 associates with the beta(1) integrin subunit. Co-immunoprecipitation and immunodepletion experiments indicated that approximately 60% of beta(1) integrins and EMP2 can be isolated in common protein complexes. Whereas this association between EMP2 and beta(1) integrin may be direct or indirect, it has features of integrin heterodimer selectivity. Thus, by laser confocal microscopy, EMP2 colocalized with alpha(6)beta(1) but not alpha(5)beta(1) integrin. Increased expression of EMP2 also influenced the integrin heterodimer repertoire present on the plasma membrane. EMP2 specifically increased the surface expression of the alpha(6)beta(1) integrin while decreasing that of the alpha(5)beta(1) protein. Reciprocally, reduction in EMP2 expression using a specific ribozyme decreased surface expression of alpha(6)beta(1) integrin. Accordingly, these EMP2-mediated changes resulted in a dramatic alteration in cellular adhesion to extracellular matrix proteins. This study demonstrates for the first time the interaction of a GAS3/PMP22 family member with an integrin protein and suggests that such interactions and their functional consequences are a physiologic role of GAS3/PMP22 proteins.     

Simulating psoriasis by altering transit amplifying cells

Computational models of tissue homeostasis will facilitate a deeper understanding of many diseases. They link molecular networks, cellular differentiation and the spatial and temporal organization of tissues. Here we show an approach which is able to computationally turn a healthy in silico epidermis into one with four central properties of psoriatic epidermis. We achieve this by altering a single simulation parameter in the cellular differentiation program of the simulated epidermal keratinocytes: the fractional time period during which transit amplifying cells proliferate (tau). Prolonging tau results in the four main pathological characteristics of psoriatic skin: (1) an absolute increase of the germinative compartment, (2) an absolute increase of the differentiated compartment, (3) a higher proportion of germinative cells and (4) a marked reduction in turnover time. The prolongation of tau is able to increase the proliferation capacity of the epidermal tissue without altering the cell cycle frequency.     

Plasminogen activator inhibitor-1 fused with erythropoietin (EPO) mimetic peptide (EMP) enhances the EPO activity of EMP.

Erythropoietin (EPO) mimetic peptide (EMP) encoding sequence was inserted into the gene of plasminogen activator inhibitor-1 (PAI-1) between Ala348 and Pro349 (P2'-P3'), generating a novel gene, PAI-1/EMP (PMP). This was cloned into pET32a expression vector, fused with TrxA peptide in the vector, and a 63-kDa protein was expressed in inclusion bodies with an expression level >50%. The TrxA/PMP protein was purified by Ni-NTA-agarose metal-ligand affinity chromatography to a purity >90%, showing a single, silver-stained band on SDS-PAGE. Using a reticulocyte counting assay, the EPO activity of PMP was determined to be 5,000 IU/mg, 2,500-fold that of EMP.     

Micron-scale assessment of molecular lipid organization in human stratum corneum using microprobe X-ray diffraction

Lipid and protein components of the stratum corneum (SC) are organized in complex supramolecular arrangements. Exploring spatial relations between various possible substructures is important for understanding the barrier function of this uppermost layer of epidermis. Here, we report the first study where micro-focus X-ray scattering was used for assessing fine structural variations of the human skin barrier with micrometer resolution. We found that the scattering profiles were unchanged when scanning in the direction parallel to the SC surface. Furthermore, small-angle scattering profiles did not change as a function of depth in the SC, confirming that the lipid lamellar spacings remained the same throughout the SC. However, the wide-angle scattering data showed that the orthorhombic phase was more abundant in the middle layers of the SC, whereas the hexagonal phase dominated in the surface layers both at the external and the lowest part of the SC; i.e., the lipids were most tightly packed in the middle region of the SC. Taken together, our results demonstrate that microprobe X-ray diffraction provides abundant information about spatial variations of the SC lipid structure and thus may be a promising tool for assessing the effects of topical formulations on the barrier function of skin.     

Use of laser microprobe mass analysis (LAMMA) for localizing multiple elements in soft and hard tissues

The potential of laser microprobe mass analysis (LAMMA) as a sensitive microanalytical technique was explored in applications relevant to nephrology. Aluminum and associated elements, such as iron, were localized in fresh tissue biopsies obtained from uremic patients treatment by chronic hemodialysis. The LAMMA was applied to serum, liver, bone, and parathyroid glands of such patients. In addition, we used LAMMA to evaluate the specificity and sensitivity of routine histochemistry, in particular on human bone sections stained by the aluminon method. The high, multielemental sensitivity and molecular microprobe potential of LAMMA established important advantages over other microchemical methods forin situ analysis at the micron level in histological sections.     

Histochemical analysis of macrophage migration inhibitory factor in psoriasis vulgaris

Psoriasis is a persistent cutaneous disease characterized by skin inflammation and infiltration of immunocytes such as lymphocytes and monocytes/macrophages, concomitant with abnormal epidermal hyperproliferation. We previously showed that the serum level of macrophage migration inhibitory factor (MIF) and its production by peripheral blood mononuclear cells of patients with psoriasis were closely correlated with the severity of clinical symptoms; however, the precise role of MIF in psoriatic epidermis remains to be clarified. The current study was carried out to elucidate the possible involvement of MIF in psoriasis, using immunohistochemistry and in situ hybridization. In contrast to elevated serum MIF in psoriasis, MIF-positive staining in the lesional psoriatic epidermis was significantly decreased, as demonstrated by immunohistochemical analysis using an anti-MIF antibody. Consistent with this finding, we found, by in situ hybridization, that MIF mRNA concomitantly decreased in the psoriatic lesions. Although the reason for the different MIF levels in the psoriatic epidermis and in the circulation remains unknown, it is hypothesized that MIF, a potential growth factor, might be decreased in psoriatic lesions to counterregulate the abnormal epidermal proliferation caused by dysregulation of cytokines and growth factors.     

Cellular retinoic acid-binding protein type 2 mRNA is overexpressed in human psoriatic skin as shown by in situ hybridization.

In situ hybridization with full length mouse cellular retinoic acid-binding protein type 1 and cellular retinoic acid-binding protein type 2 cDNA derived RNA probes showed overexpression of cellular retinoic acid-binding protein type 2 mRNA in lesional hyperplastic psoriatic skin whereas cellular retinoic acid-binding protein type 1 mRNA was undetectable. This suggests that the previously reported increase of cellular retinoic acid-binding protein in psoriatic epidermis corresponds to increased translation of cellular retinoic acid-binding protein type 2 gene. Cellular retinoic acid-binding protein types 1 and 2 mRNAs were not detectable in normal epidermis; however, type 2 message was detected in non-hyperplastic, non-lesional skin of psoriatic patients thus before altered epidermal differentiation and hyperplasia are morphologically detectable.     

Elevated interleukin-18 protein expression in early active and progressive plaque-type psoriatic lesions

Psoriasis is a T cell-mediated inflammatory skin disease characterized by an elevated IFN-gamma and IL-12p70 expression in skin lesions. Interleukin-18 (IL-18) synergizes with IL-12 to induce IFN-gamma production and a strong T-helper-1-mediated immune response, or to induce Th2 polarization depending on the immunological context. We have previously shown that keratinocytes in normal skin produce and store large amounts of pro-IL-18. In this study, we hypothesized that the expression of IL-18 in psoriatic lesional skin might be altered compared to normal skin. Therefore, IL-18 expression was assessed in psoriatic, stable, plaque-type lesions and early active and progressive lesions. IL-18 mRNA and protein concentrations were constitutively high, and did not differ between normal and stable, plaque-type epidermis. In active and progressive lesions an elevated expression of total IL-18 protein relative to normal and stable, plaque-type epidermis was detected using ELISA, while on Western blot, the differences in pro- or mature IL-18 were less clear. Our results indicate that the role of IL-18 in the pathogenesis of early phases of psoriasis may be more prominent than in established psoriatic lesions.     

On the existence of non-cycling germinative cells in human epidermis in vivo and cell cycle aspects of psoriasis

Abstract. This report deals with the controversies of whether all germinative epidermal cells in human epidermis are in the cycling state and whether stimulated hyperproliferation of psoriatic epidermis is due to a shortening of the cell cycle time or to a recruitment of non-cycling germinative epidermal cells. Experiments were performed on human subjects in vivo . Continuous infusion of [³H]thymidine for 8½ days indicated that 40% of germinative epidermal cells reside in the non-cycling state. Proliferative stimulation by tape stripping indicated recruitment of non-cycling (G₀) germinative epidermal cells in both normal and psoriatic skin, and a prolongation (rather than a shortening) of cell cycle traverse in activated psoriatic epidermal cells.     

Stereospecificity of the products of the fatty acid oxygenases derived from psoriatic scales

The principal in vivo oxygenase products of arachidonic acid and linoleic acid in psoriatic skin scales are 12-hydroxyeicosatetraenoic acid (R/S ratio = 5.7), 13-hydroxyoctadecadienoic acid (S/R = 1.9), and 9-hydroxyoctadecadienoic acid (R/S = 2.4). Definition of the enzymatic origin of these fatty acid derivatives is an important step in assessing their possible role in the pathogenesis of psoriasis. Psoriatic skin scales were incubated with radiolabeled arachidonic acid and linoleic acid and the monohydroxylated derivatives produced in vitro were characterized. The products of incubation with [3H]arachidonic acid were an enantiopure 15(S)-[3H]hydroxyeicosatetraenoic acid and a nonracemic mixture of the 12-[3H]hydroxyeicosatetraenoic acid steroisomers (R/S ratio = 4.5). An enantiopure 13(S)-[14C]hydroxyoctadecadienoic acid was produced from [14C]linoleic acid. No radiolabeled products were derived from incubations with heat-denatured scales. These results provide evidence for two distinct oxygenase activities that are preserved in psoriatic skin scales. One is that of an omega-6 oxygenase with strict (S) stereospecificity, consistent with the activity of a lipoxygenase. This enzyme activity appears to be similar to that of the 15-lipoxygenase which has been described in cultured human keratinocytes. The second activity is that of an arachidonic acid 12(R)-oxygenase that has not been observed in normal human epidermis but which appears to be expressed in psoriatic epidermis.