Serum tryptase and SCORMA (SCORing MAstocytosis) Index as disease severity parameters in childhood and adult cutaneous mastocytosis.

BACKGROUND: Skin lesions are the predominant clinical feature of the commonest form of mastocytosis. Mastocytosis is classified according to World Health Organization criteria. Determination of the levels of mast-cell mediators or their metabolites reflects the mast-cell burden. The extent of cutaneous mastocytosis can be assessed clinically using a scoring system (SCORing MAstocytosis; SCORMA Index) that we have developed. OBJECTIVE: Serum tryptase levels were compared with the SCORMA Index in a large group of paediatric and adult patients to investigate whether there was any correlation between the two. METHODS: The SCORMA Index in 64 patients (31 children and 33 adults) was compared with serum tryptase levels. The results of the first visit at which SCORMA and tryptase were evaluated were analysed. RESULTS: There was a positive correlation between the SCORMA Index and serum tryptase levels, indicating the value of the SCORMA Index in the assessment of mastocytosis with skin involvement. CONCLUSION: The results of this study showed that the SCORMA Index is a useful tool for evaluating the severity of cutaneous mastocytosis. The correlation between the SCORMA Index and serum tryptase levels underlines the benefit of the SCORMA Index as a clinical tool. Repeated SCORMA Index measurements can provide a rapid impression of changes in the clinical state of mastocytosis. This is particularly relevant in children, because taking blood samples from this group is much more difficult. The well-established methods for evaluation of disease severity may be expanded by the rapid SCORMA Index method.

Mast cell distribution in normal adult skin.

AIMS: To investigate mast cell distribution in normal adult skin to provide a reference range for comparison with mastocytosis. METHODS: Mast cells (MCs) were counted in uninvolved skin adjacent to basal cell carcinomas and other dermatological disorders in adults. RESULTS: There was an uneven distribution of MCs in different body sites using the anti-tryptase monoclonal antibody technique. Numbers of MCs on the trunk, upper arm, and upper leg were similar, but were significantly different from those found on the lower leg and forearm. Two distinct groups were formed--proximal and distal. There were 77.0 MCs/mm2 at proximal body sites and 108.2 MCs/mm2 at distal sites. Adjusted for the adjacent diagnosis and age, this difference was consistent. The numbers of MCs in uninvolved skin adjacent to basal cell carcinomas and other dermatological disorders were not different from those in the control group. Differences in the numbers of MCs between the distal and the proximal body sites must be considered when MCs are counted for a reliable diagnosis of mastocytosis. A pilot study in patients with mastocytosis underlined the variation in the numbers of MCs in mastocytosis and normal skin, but showed a considerable overlap. The observed numbers of MCs in adults cannot be extrapolated to children. CONCLUSIONS: MC numbers varied significantly between proximal and distal body sites and these differences must be considered when MCs are counted for a reliable diagnosis of mastocytosis. There was a considerable overlap between the numbers of MCs in mastocytosis and normal skin.

Characterization of the mononuclear infiltrate in basal cell carcinoma: a predominantly T cell-mediated immune response with minor participation of Leu-7+ (natural killer) cells and Leu-14+ (B) cells.

We investigated the peritumoral inflammatory infiltrate in 22 basal cell carcinoma (BCC) from 18 patients using a series of monoclonal antibodies. In all the 22 BCC the infiltrate consisted mainly of T cells (55 +/- 15%) and only in three cases an invasion of the tumor nests by these cells was observed. The T helper (TH) subset predominated over the T suppressor/cytotoxic (TS/C) subset (TH/TS/C ratio of 1.9 +/- 0.8). In 8 of 22 BCC mild infiltrate was observed with 48 +/- 13% T cells and a TH/TS/C ratio of 1.5 +/- 0.6. In 14 of 22 BCC moderate to heavy infiltrate with 59 +/- 15% T cells and a TH/TS/C ratio of 2.0 +/- 1.0 was observed. There was a significant difference in the percentage of T cells in BCC with moderate to heavy infiltrate and that in BCC with mild infiltration. The mean percentage of HLA-DR+ cells was 54 +/- 11%; Langerhans cells (LC) 4 +/- 5%; and Leu-M5+ (monocytes and macrophages) 16 +/- 11%. Less than 2% Leu-14+(B) cells were seen in the infiltrate. The mean percentage of Leu-7+ (natural killer) cells was 4 +/- 4%, and only 1 of 22 BCC Leu-7+ cells invaded tumor nests, contacting with tumor cells. From these results we concluded that T cells play a major role in the defence against BCC proliferation. The main role of Langerhans cells and Leu-M5+ cells may be that of antigen presentation. B cells and NK cells probably play a minor role in the local defence against BCC proliferation.

Optimizing the solid-phase immunofiltration assay. A rapid alternative to immunoassays.

The technical variables of the solid-phase immunofiltration assay (SPIA) for the detection of antibodies bound to antigens on a solid-phase filter have been investigated. The binding to solid-phase filters of 125I-labelled axial filament proteins derived from Treponema phagedenis and the optimal conditions for blocking non-specific protein binding were analysed. Axial filament was applied to nitrocellulose, Hybond Nylon and Zeta Probe. After extensive rinsing, the highest amount (68%) of axial filament was observed bound to Zeta Probe. However, blocking non-specific protein binding by pre-wetting the filter with rinsing buffer containing 0.5% Tween 20, prevented the binding of protein to the filter only when nitrocellulose was used as solid phase. Tween 20 (0.5%) in the rinsing and incubation solutions was found to be necessary for the reduction of non-specific binding of contaminants in turbid sera. However, the use of such solutions resulted in a substantial leakage of antigen (47%) during rinsing procedures. Binding of antigen-specific antibody was analysed using 125I-labelled protein A. The maximal possible binding of the antibody occurred within 5 min when the antibody solution was filtered. For optimal binding of 125I-labelled protein A an incubation time of 1 h was needed. It is suggested that solid-phase immunofiltration may provide a rapid alternative for radioimmunoassays or enzyme immunoassays for the detection of specific antibodies.

The major histocompatibility complex of rhesus monkeys. VIII. Isolation and partial characterization of SD antigens.

The structure and chemical nature of the serologically defined (SD) antigens coded by the major histocompatibility complex (RhLA) of rhesus monkeys was studied. The use of specific anti-SD sera allowed the selective isolation of the corresponding antigens from crude antigen preparations. These were obtained by detergent solubilization after incorporation of 3H, 35S, and 14C amino acids in lymphocytes or mitogen-stimulated lymphoblasts. The results indicate that the SD antigens are of proteinaceous nature and are composed of two polypeptide chains with molecular weights of 44,000 and 12,000, the latter being beta 2-microglobulin. No differences in molecular size and subunit composition were detected between antigens of both segregant series. The results are discussed in relation to similar data available for the analogous human and murine histocompatibility antigens.

Prolongation of heterotropic heart allograft survival in rats by use of antigen-antibody complexes.

Treatment of BN recipients with soluble WAG/Rij histocompatibility antigens (HCA) did not produce prolongation of (WAG/Rij X BN) F1 heart allografts but resulted in specific sensitization of the recipients in most cases. Three different anti-WAG/Rij sera (ADS) were tested, either alone or complexed in equivalence to 0.5 mg of HCA. Depending on the serum used, ADS alone produced only a moderate prolongation of heart allograft survival or had no effect at all. Antigen-antibody complexes given i.v. on days 0, 2, 4, 6, and 8 induced a prolonged or indefinite cardiac graft survival (greater than 200 days), as well as accelerated graft rejection, depending on the source of the ADS used in immune complex formation. One month after heart transplantation, BN recipients, treated with antigen-antibody complexes, demonstrated normal graft-versus-host reactivity of peripheral blood lymphocytes, but rejected donor-type skin grafts in a slightly delayed fashion.

Expression of E-cadherin, alpha- & beta-catenin, and CD44V6 and the subcellular localization of E-cadherin and CD44V6 in normal epidermis and basal cell carcinoma.

Basal cell carcinoma (BCC) of the skin is a locally invasive, rarely metastasizing epithelial tumor. In the current study, the expression of E-cadherin, alpha- and beta-catenin and CD44V6 in normal epidermis and on BCC cells were investigated. A significantly reduced expression of alpha-catenin and CD44V6 and a slightly reduced expression of E-cadherin on BCC cells were observed compared with the overlying epidermis. Immunoelectron microscopy was used to investigate whether the decreased expression of E-cadherin and CD44V6 was due to either an absence or downregulation of specific membrane structures or due to an overall downregulation of these adhesion molecules in all membrane structures in BCC. E-cadherin and CD44V6 were expressed in adherens junctions, desmosomes, and complex interdigitating membrane structures both in normal epidermis and in BCC. A quantitative analysis showed that only a percentage of desmosomes was stained. In addition, the effect of pro-inflammatory cytokines, such as interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha), was investigated in biopsy specimens of normal skin and BCC, using a biopsy culture system and immunohistochemistry. The expression of E-cadherin and CD44V6 was not significantly decreased after culturing BCC or normal skin biopsy specimens for 48 hours with or without recombinant human (rHu)IFN-gamma or rHuTNF-alpha. It may be concluded that the decreased expression of both E-cadherin and CD44V6, observed in light microscopy, was not attributable to the absence of specific specialized structures in BCC and most likely also not caused by downregulation by local cytokines, but rather by generic downregulation of both of these adhesion molecules during malignant transformation.

Leukocyte adherence inhibition in patients with nonmalignant disorders of the colon and colorectal cancer.

The tube leukocyte adherence inhibition (LAI) technique was used to investigate the antitumor immunity in two groups of patients generally considered to be at "high risk" of developing colorectal cancer. The first group comprised 21 patients with colorectal polyps and the second 12 patients with various forms of colitis. Also 29 patients with histologically confirmed colorectal cancer were tested. The tube LAI assay was performed using peripheral blood leukocytes from individual patients and crude extracts of colorectal and breast cancers. Positive LAI reactions were observed in 18 out of 29 (62%) patients with colorectal cancer, in 1 out of 21 (5%) patients with colorectal polyps and in 1 out of 12 (8%) patients with colitis. The results indicate that in confirmed cases of malignancies, sensitization to colon tumor-associated antigens could be detected in the tube LAI test, whereas, premalignant sensitization to these antigens in "high risk" groups of patients could not be demonstrated.

Expression of cytokeratin 8 and low molecular weight cytokeratins in human basal cell carcinoma.

The expression of low molecular weight cytokeratins (Cks) 7,8,18, 18 and high molecular weight cytokeratin 10 in 23 basal cell carcinomas (BCCs) was investigated using a panel of 14 different commercially available monoclonal antibodies (MoAbs) with specific anti-cytokeratin activity. Four of these MoAbs were directed against Ck 8. The results showed that Ck 8 was detected in all 23 BCCs using MoAb 4.1.18. Two of the MoAbs showed inconsistent staining for Ck 8 and one of them did not show any staining at all. Cytokeratins 7 and 19 were detected incosistently. Cytokeratins 18 and 10 were not detected in any of the 23 BCCs that were examined. The incosistent observations on the expression of Ck 8 in BCCs in this study could have been due to different epitopes of the different cytokeratins that were detected by the different MoAbs. The results of this study lead to recommendation that whenever possible a panel of different MoAbs directed against the same Ck(s) should be used in order to minimize the risk of obtaining incorrect experimental results.

Absence of cytokeratin 8 and inconsistent expression of cytokeratins 7 and 19 in human basal cell carcinoma.

The expression of the low molecular weight cytokeratins (K) 7, 8, 18, 19 and the high molecular weight cytokeratin 10 in 21 basal cell carcinoma (BCC) was studied using seven different monoclonal antibodies (MoAbs) with specific anti-cytokeratin activity. MoAbs RCK 105 (anti-K7), RPN 1164 (anti-low molecular weight cytokeratins of basic group), Ks 19.1 (anti-K19) and Cam 5.2 (anti-K8, K18, K19) reacted positively but inconsistently in the BCC that were examined. MoAbs 1166 (anti-K8) and RGE53 (anti-K18) did not react at all. MoAb RKSE 60 (anti-K10) did not react with the tumor cells. From the results of this study, it can be concluded that cytokeratins 7 and 19 are expressed in BCC (43% and 71%, respectively), whereas cytokeratin 8 is not expressed.

Expression of cytokeratin 8 in basal cell carcinoma: a comparative immunohistochemical and immunoelectron microscopy study.

The comparative study reported here was undertaken in order to resolve the discrepancies in the detection of cytokeratin (Ck) 8 reported in previous studies. The expression of Ck 8 was compared in 6 basal cell carcinomas (BCCs) using immunohistochemical and immunoelectron microscopic techniques and a panel of 4 different commercially available monoclonal antibodies (MoAbs). The results of this comparative study demonstrated not only that the consistent expression of Ck 8 using one of the MoAbs in immunohistochemistry was confirmed by immunoelectron microscopy, but that the inconsistent expression of Ck 8 observed using two other MoAbs was also confirmed. One of the MoAbs did not show any staining at all. The inability of this MoAb to detect the expression of Ck 8 using either of the techniques also indicated that this MoAb may be directed against an epitope of Ck8 that is not detectable in BCC in situ.

Immunoelectron microscopic studies on cytokeratins in human basal cell carcinoma.

Ultrastructural investigations into the location and the expression of the cytokeratins 7, 8, 10 and 19 were undertaken on ultrathin cryosections of 8 basal cell carcinomas (BCC) using a high resolution immunogold labeling method and five different monoclonal antibodies against specific cytokeratins. The results showed that cytokeratin 10 was expressed only in the differentiated keratinocytes. In contrast to the previously reported biochemical and immunohistochemical studies, in this study cytokeratin 19 was expressed not only in the tumor cells of BCC but also in the normal epidermal keratinocytes. The expression of cytokeratin 7 in BCC could not be confirmed but the lack of expression of cytokeratin 8 was confirmed, excluding its potential role as a specific histopathological marker for BCC.

Adhesion molecules and IL-1 costimulate T lymphocytes in the autologous MECLR in psoriasis.

Membrane molecules such as CD36 (OKM5), intercellular adhesion molecule-1 (ICAM-1, CD54), gamma interferon-induced protein 10 (gamma-IP10) and IL-1 are induced and/or upregulated in psoriatic epidermis. These molecules have important accessory, trafficking or signalling functions in the immune system and also play a role in the pathophysiology of psoriasis. The relevance of adhesion molecules, CD36 and epidermal IL-1 in psoriasis was studied in vitro in the autologous mixed epidermal cell - T lymphocyte reaction (MECLR). Their level of expression was quantitated in epidermal cell suspensions (ECS) from patients with psoriasis and their function was assessed by blocking with specific mAbs and antisera or by depleting CD36+ cells from the ECS prior to the MECLR. ECS from psoriatic lesions contained increased numbers of CD36+ (23 +/- 12%), ICAM-1(+) (31 +/- 14%) and IL-1(+) (57 +/- 21%) cells. The autologous MECLR was inhibited in samples from all patients by mAb to CD2 (LFA-2), CD11a (LFA-1alpha), CD18 (LFA-1beta), ICAM-1, CD58 (LFA-3) and an antiserum to IL-1beta. Thus, adhesion molecules facilitate inflammation in psoriasis not only via adhesion and recruitment of T lymphocyte in psoriatic lesions, but also via activation of T cells. Furthermore CD36 molecules on psoriatic epidermal cells do not costimulate autologous T lymphocytes in psoriasis. The observed costimulatory function of IL-1beta in the MECLR emphasizes its relevance in psoriasis.

Changes in the pressure and the dynamic stiffness index of medical elastic compression stockings after having been worn for eight hours: a pilot study.

OBJECTIVES: There is no data available at present on the changes in the exerted pressure together with the dynamic stiffness index (DSI) of medical elastic compression stockings (MECS). The objective of this pilot study was to measure the pressure and calculate the DSI of 12 different brands of MECS before and after having been worn for eight hours. METHODS: In all, 12 different commercially available brands of MECS that were divided into two categories (class I round-knitted and class II flat-knitted MECS) were tested. The pressure was measured, and the DSI of the MECS was first calculated at the B1 level before wearing in the morning and again eight hours after they had been worn. All laboratory measurements were performed using a newly developed dynamic leg-segment model. RESULTS: The pressure at the B1 level dropped significantly in all 12 brands of MECS after having been worn for eight hours, whereas the DSI remained unchanged. CONCLUSION: The DSI of MECS reflects an important and particularly consistent therapeutic effect. As the pressure drops during the day, the pressure amplitude or pulsations remain the same. The pressure drop may be due to fatigue of the elastic material. The DSI would therefore form a valuable indicator for prescribing the most effective MECS for the patient.

Variation in the dynamic stiffness index of different types of medical elastic compression stockings.

OBJECTIVES: To calculate the dynamic stiffness index (DSI) of 18 different brands of medical elastic compression stockings (MECS). METHODS: In all, 18 different brands of MECS that were divided into five categories (class II round-knitted, class II flat-knitted, class III round-knitted, class III flat-knitted and class IV flat-knitted MECS) were tested. The static pressure and dynamic pressure pulsations at the B1 level were measured with a newly developed dynamic pressure-determining device. The DSI was calculated. RESULTS: The DSI of all 18 brands of MECS showed higher values compared with the static stiffness. A wide range of dynamic stiffness indices was observed not only between all brands of MECS, but also within the five categories. CONCLUSIONS: The DSI of MECS is independent of the compression class and the type of knit. The variation in the DSIs between MECS is not because of any measurement error and would indicate that different therapeutic effectiveness may be expected within one compression class. Therefore, a refinement in the current classification system for MECS with other characteristics such as the DSI is warranted.