An in vivo model of postinflammatory hyperpigmentation and erythema: clinical, colorimetric and molecular characteristics.

BACKGROUND: Postinflammatory hyperpigmentation (PIH) is a common, acquired pigmentary disorder of the skin associated with significant quality-of-life impairment, especially in individuals with skin of colour. Current treatment for PIH is limited, largely due to a poor understanding of disease pathogenesis and the lack of a representative disease model. OBJECTIVES: This study is intended to further develop, update and validate our previously designed in vivo model of acne-induced PIH/postinflammatory erythema (PIE) using different concentrations of trichloroacetic acid (TCA), a medium-depth chemical peel. METHODS: Twenty-nine patients with skin types II-VI and clinician-confirmed presence of two or more truncal acne pustules and PIH/PIE were included. On the basis of Investigator's Global Assessment (IGA), clinical polarized photography (CPP), colorimetry and Skindex, we experimentally determined an optimum TCA concentration and assessed our model's ability to exhibit a dose-response relationship between degree of inciting insult and severity of resulting pigmentation. We also performed differential microRNA profiling and pathway analysis to explore the potential of microRNAs as molecular adjuncts to our model. RESULTS: Application of TCA 30% produced lesions indistinguishable from acne-induced PIH and PIE lesions on the basis of colorimetry data without causing epidermal necrosis. Application of progressively increasing TCA doses from 20% to 30% resulted in concentration-dependent increases in CPP, IGA and colorimetry scores at all timepoints during the study. miRNA-31 and miRNA-23b may play a role in PIH pathogenesis, although further validation is required. CONCLUSIONS: Our TCA-based in vivo model, using TCA concentrations between 20% and 30% with an optimum of 30%, enables the quantitative assessment of the pigmentary response to varying degrees of cutaneous inflammation in a fashion that mirrors natural acne-induced PIH and PIE.

Assessment of inter-rater reliability of clinical hidradenitis suppurativa outcome measures using ultrasonography.

BACKGROUND: Hidradenitis suppurativa (HS) staging and severity is typically based upon physical examination findings, which can result in misclassification of severity based on subclinical disease activity and significant variation between healthcare providers. Ultrasonography (US) is an objective tool to help evaluate subclinical disease and to more accurately classify disease severity. AIM: To evaluate inter-rater reliability in HS disease severity assessment using clinical and US techniques. METHODS: In total, 20 subjects underwent clinical evaluation of HS, independently by two physicians, using clinical outcome measures, including Hurley, Sartorius, HS Physician Global Assessment (HS-PGA) and Hidradenitis Suppurativa Clinical Response (HiSCR). US was subsequently performed, and clinical assessments were repeated. Intraclass correlation coefficients (ICC) were obtained to evaluate inter-rater agreement of each outcome measure before and after US. RESULTS: Pre-US to post-US improvement in ICC was seen with the Sartorius, HiSCR nodule and abscess count, and the HiSCR draining fistula count. The scores went from having 'good' rater agreement for Sartorius and HiSCR nodule and abscess count, to 'poor' rater agreement for HiSCR draining fistula count, to 'excellent' rater agreement among these scores. CONCLUSION: US improved inter-rater agreement and should be used in conjunction with physical examination findings to evaluate disease severity to ensure uniform staging of HS.

B cell differentiation and isotype switching is related to division cycle number.

The mature, resting immunoglobulin (Ig) M, IgD+ B lymphocyte can be induced by T cells to proliferate, switch isotype, and differentiate into Ig-secreting or memory cells. Furthermore, B cell activation results in the de novo expression or loss of a number of cell surface molecules that function in cell recirculation or further interaction with T cells. Here, a novel fluorescent technique reveals that T-dependent B cell activation induces cell surface changes that correlate with division cycle number. Furthermore, striking stepwise changes are often centered on a single round of cell division. Particularly marked was the consistent increase in IgG1+ B cells after the second division cycle, from an initial level of < 3% IgG1+ to a plateau of approximately 40% after six cell divisions. The relationship between the percentage of IgG1+ B cells and division number was independent of time after stimulation, indicating a requirement for cell division in isotype switching. IgD expression became negative after four divisions, and a number of changes centered on the sixth division, including the loss of IgM, CD23, and B220. The techniques used here should prove useful for tracking other differentiation pathways and for future analysis of the molecular events associated with stepwise differentiation at the single cell level.

The fate of self-reactive B cells depends primarily on the degree of antigen receptor engagement and availability of T cell help.

Self-reactive B cells from tolerant double-transgenic (Dbl-Tg) mice coexpressing hen egg lysozyme (HEL) and rearranged anti-HEL immunoglobulin genes have a relatively short life span when compared to normal B cells, irrespective of whether they are exposed to antigen in multivalent membrane-bound form (mHEL-Dbl-Tg mice) or soluble form (sHEL-Dbl-Tg mice). The factors responsible for determining the fate of these B cells after encounter with self-antigen were investigated using a cell-tracking technique in which anti-HEL Ig-Tg spleen cells were labeled with the intracellular dye 5-carboxyfluorescein diacetate-succinimidyl ester (CFSE) and injected either into non-Tg recipients or a variety of HEL-Tg hosts. In non-Tg recipients, HEL-binding B cells persisted in the circulation and could be detected in the follicles of the spleen for at least 5 d. On transfer into either mHEL-Tg or sHEL-Tg hosts, they underwent activation and then rapidly disappeared from the blood and spleen over the next 3 d, consistent with the short life span reported previously. Immunohistology of spleens from sHEL-Tg recipients indicated that the transferred B cells had migrated to the outer margins of the periarteriolar lymphoid sheath (PALS), where they were detectable for 24 h before being lost. The positioning of B cells in the outer PALS depended on a critical threshold of Ig receptor binding corresponding to a serum HEL concentration between 0.5 and 15 ng/ml, but was not restricted to endogenously expressed HEL in that the same migratory pattern was observed after transfer into non-Tg recipients given exogenous (foreign) HEL. Moreover, bone marrow-derived immature Ig-Tg B cells homed to the outer PALS of sHEL-Tg mice and then disappeared at the same rate as mature B cells, indicating that the stage of maturation did not influence the fate of self-reactive B cells in a tolerant environment. On the other hand, HEL-binding B cells transferred into sHEL-Dbl-Tg recipients persisted over the 3-d period of study, apparently due to insufficient availability of antigen, as indicated by the fact that the degree of Ig receptor downregulation on the transferred B cells was much less than in sHEL-Tg recipients. If T cell help was provided to Ig-Tg B cells at the time of transfer into sHEL-Tg recipients in the form of preactivated CD4+ T cells specific for major histocompatibility complex-peptide complexes on the B cell surface, HEL-binding B cells migrated through the outer PALS of the spleen to the follicle, where they formed germinal centers, or to adjacent red pulp, where they formed proliferative foci and secreted significant amounts of anti-HEL antibody. Taken together, these results indicated that the outcome of the interaction between self-antigen and B cells is largely determined by a combination of the degree of receptor engagement and availability of T cell help.

Surgical correction of the medial rotation contracture in obstetric brachial plexus palsy.

The medial rotation contracture caused by weak external rotation secondary to obstetric brachial plexus injury leads to deformation of the bones of the shoulder. Scapular hypoplasia, elevation and rotation deformity are accompanied by progressive dislocation of the humeral head. Between February and August 2005, 44 children underwent a new surgical procedure called the 'triangle tilt' operation to correct this bony shoulder deformity. Surgical levelling of the distal acromioclavicular triangle combined with tightening of the posterior glenohumeral capsule (capsulorrhaphy) improved shoulder function and corrected the glenohumeral axis in these patients. The posture of the arm at rest was improved and active external rotation increased by a mean of 53 degrees (0 degrees to 115 degrees ) in the 40 children who were followed up for more than one year. There was a mean improvement of 4.9 points (1.7 to 8.3) of the Mallet shoulder function score after surgical correction of the bony deformity.

Imatinib inhibits the in vitro development of the monocyte/macrophage lineage from normal human bone marrow progenitors.

The antileukaemic tyrosine kinase inhibitor, imatinib, has been reported to inhibit specifically the growth of bcr-abl expressing CML progenitors at levels of 0.1-5.0 microM, by blocking the ATP-binding site of the kinase domain of bcr-abl. Inhibition of the c-abl, platelet-derived growth factor receptor and stem cell factor receptor (c-kit) tyrosine kinases by imatinib has also been reported. Here, we demonstrate that imatinib significantly inhibits in vitro monocyte/macrophage development from normal bone marrow progenitors, while neutrophil and eosinophil development was less affected. Monocyte/macrophage inhibition was observed in semisolid agar and liquid cultures at concentrations of imatinib as low as 0.3 microM. The maturation of monocytes into macrophages was also found to be impaired following treatment of cultures with 1.0 microM imatinib. Imatinib blocked monocyte/macrophage development in cultures stimulated with and without M-CSF, suggesting that inhibition of the M-CSF receptor, c-fms, by imatinib was unlikely to be responsible. Imatinib may therefore have an inhibitory activity for other kinase(s) that play a role in monocyte/macrophage differentiation. This inhibition of normal monocyte/macrophage development was observed at concentrations of imatinib achievable pharmacologically, suggesting that imatinib or closely related derivatives may have potential for the treatment of diseases where monocytes/macrophages contribute to pathogenesis.

Evidence for induction of humoral and cytotoxic immune responses against devil facial tumor disease cells in Tasmanian devils (Sarcophilus harrisii) immunized with killed cell preparations.

Tasmanian devils (Sarcophilus harrisii) risk extinction from a contagious cancer, devil facial tumour disease (DFTD) in which the infectious agent is the tumor cell itself. Because devils are unable to produce an immune response against the tumor cells no devil has survived 'infection'. To promote an immune response we immunized healthy devils with killed DFTD tumor cells in the presence of adjuvants. Immune responses, including cytotoxicity and antibody production, were detected in five of the six devils. The incorporation of adjuvants that act via toll like receptors may provide additional signals to break 'immunological ignorance'. One of these devils was protected against a challenge with viable DFTD cells. This was a short-term protection as re-challenge one year later resulted in tumor growth. These results suggest that Tasmanian devils can generate immune responses against DFTD cells. With further optimization of immune stimulation it should be possible to protect Tasmanian devils against DFTD with an injectable vaccine.

Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution.

Since its introduction in 1994 (J. Immunol. Methods 171 (1994) 131), the flow cytometric analysis of lymphocyte proliferation by serial halving of the fluorescence intensity of the vital dye CFSE (carboxyfluorescein diacetate, succinimidyl ester or CFDA-SE) has become widely used in immunological laboratories around the world. This technique allows the visualisation of eight to 10 discrete cycles of cell division by flow cytometry, both in vitro and in vivo. Appropriately conjugated antibodies can be used to probe surface marker changes as cells divide, or changes in expression of internal molecules such as cytokines when appropriate fixation and permeabilisation protocols are used. An added advantage of the technique is the ability to recover viable cells which have undergone defined numbers of cell divisions by flow cytometric sorting, allowing functional studies to be performed. Other commonly used assays of cell proliferation give only limited information, as they usually measure division at a population level. The CFSE technique can be used to determine kinetics of immune responses, track proliferation in minor subsets of cells and follow the acquisition of differentiation markers or internal proteins linked to cell division.

Determination of lymphocyte division by flow cytometry.

Techniques currently available for determining cell division are able to show one or, at best, a limited number of cell divisions. Other methods exist which can quantify overall division, but tell nothing about the division history of individual cells. Here we present a new technique in which an intracellular fluorescent label is divided equally between daughter cells upon cell division. The technique is applicable to in vitro cell division, as well as in vivo division of adoptively transferred cells, and can resolve multiple successive generations using flow cytometry. The label is fluorescein derived, allowing monoclonal antibodies conjugated to phycoerythrin or other compatible fluorochromes to be used to immunophenotype the dividing cells.

The importance of efficacy and partial agonism in evaluating models of B lymphocyte activation.

Immunologists have developed a range of in vitro techniques for probing the receptor mediated response of cells comprising the immune system. An important and ubiquitous method is the use of antibodies in either soluble or aggregated form to engage cell surface receptors and transmit a signal. Models of cell and molecular interactions, derived from the use of these antibodies, form the basis of our efforts to understand and explain the corresponding in vivo systems. However, interpreting in vitro experiments and distinguishing between alternative models is difficult. This complexity is illustrated here using B cell stimulation by surface immunoglobulin and CD40. The fluorescent cell labelling dye carboxyfluorescein, diacetate, succinimidyl ester (CFSE) is used to show that many anti-Ig and CD40 stimulatory agents, used to assess the role of B cells and lymphokines, are partial agonists. By modelling each step in B cell signalling, activation and division it is possible to show that small changes in signal contributed by a second receptor can generate numerous distinct dose response curves that are highly dependent on the "efficacy" of signal transmission by the primary ligand and the number of cell divisions taken in culture. Differences in dose response curves become particularly striking if the primary activating stimulus is a partial agonist. Although exemplified here with B cell stimulation the conclusions are applicable to other in vitro activation systems and suggest ways to improve both the design and interpretation of in vitro experiments.

The effect of recombinant cytokines on the proliferative potential and phenotype of cells of the human myelomonocytic leukaemia line, RC-2A.

Cells of the human myelomonocytic line RC-2A can be induced to differentiate towards mature monocytes by culture in the presence of phytohaemagglutinin-treated lymphocyte conditioned medium (Lyons and Ashman, Leukemia Res. 11, 797, 1987). We have now examined the effect on RC-2A cells of some (recombinant) cytokines which might be present in conditioned medium. Gamma interferon most closely mimicked the effect of conditioned medium in inducing clonogenic suppression and the induction of monocytic maturation over 7 days of culture. Granulocyte colony stimulating factor induced enhancement of proliferation followed by clonogenic suppression, while granulocyte-macrophage colony stimulating factor had a purely stimulatory effect on proliferation over a 7-day period. Tumour necrosis factor alpha failed to affect cell proliferation or to induce characteristic monocytic differentiation, but did increase the expression of C3bi receptors. We conclude that RC-2A cells have receptors for all four cytokines studied, and that gamma interferon is a major differentiation-inducing stimulus for these cells.

Studies on the differentiation of the human myelomonocytic cell line RC-2A in response to lymphocyte-derived factors.

Cells of the human myelomonocytic line RC-2A were induced to differentiate toward macrophages by culturing for up to 12 days in the presence of supernatant from phytohaemagglutinin stimulated human peripheral blood mononuclear cells (PHA-LCM). The process of differentiation was monitored by changes in expression of two-macrophage related enzymes (alpha-naphthol butyrate esterase and acid phosphatase), the changes in expression of the monocyte-macrophage cell surface markers detected by the monoclonal antibodies anti-Mo1 and anti-Mo2, HLA class 2 antigen detected by FMC-14, and alteration in cell morphology. Maturation induced by PHA-LCM was accompanied by a marked decrease in the proliferative potential of the cell population, and a reduced ability to form colonies in semi-solid medium. Induced RC-2A cells were able to stimulate in one-way mixed leukocyte culture more effectively than control cells.

Discrete subpopulations, defined by CD45 isoforms, coexist within the leukaemic cells of B-chronic lymphocytic leukaemia patients.

The expression of CD45 isoforms by B-CLL leukaemic lymphocytes has been analysed by 2 colour flow cytometry and vectorial iodination. The cytometry has demonstrated the presence of cells with different CD45 phenotypes which vary in the relative expression of the CD45RA and CD45RO determinants. Discrete populations have been detected which can coexist within an individual patient. One of these populations which is CD45RO-negative consists of cells expressing only the 230 kD isoform, in the others the smaller isoforms are expressed, the appearance of the CD45RO determinant of 180 kD being accompanied by the appearance of the 190 kD isoform. The relative proportion of cell populations is stable within patients but can be altered by phorbol ester which enhances CD45RO expression and diminishes CD45RA expression. The populations may be partially resolved by the density gradient fractionation.

A monoclonal antibody to a human mast cell/myeloid leukaemia-specific antigen binds to normal haemopoietic progenitor cells and inhibits colony formation in vitro.

An antigen identified by murine monoclonal antibody YB5.B8 has previously been detected only on acute non-lymphoblastic leukaemia (ANLL) cells and tissue mast cells. We now report that the YB5.B8 antigen is present on a minor population (up to 3%) of normal bone marrow mononuclear cells which overlaps the set of progenitor cells capable of forming haemopoietic colonies in vitro. The results indicate that the antigen is a normal haemopoietic progenitor cell marker which is selectively retained on mast cells during maturation, and that leukaemias which express the antigen are not necessarily committed to the mast cell lineage. Furthermore, the antibody was capable of partially inhibiting the formation of haemopoietic colonies in vitro, indicating an important functional role for the antigen. This is consistent with the observation, reported in the accompanying paper, that expression of the YB5.B8 antigen is strongly correlated with poor response to therapy in patients with ANLL.

Human myeloid differentiation antigens identified by monoclonal antibodies to the myelomonocytic leukemia cell line RC-2A.

Murine monoclonal antibodies to human myeloid cell surface differentiation antigens were prepared using the myelomonocytic leukemia cell line RC-2A as immunogen. Using a highly sensitive colorimetric assay, antibodies were selected as myeloid-associated based on their binding to RC-2A cells, but not to cells of the autologous EBV-transformed* B cell line Cess-B. Antibodies to five distinct cell surface antigens were extensively characterized for their binding to normal and leukemic hemopoietic cells, and to tissue sections. Three antibodies may identify antigens previously described in the International Leucocyte Typing Workshops (CD14, CD11b and CD31). The other two antigens appear to be expressed at low levels on the surface of RC-2A cells, and do not correspond to existing CD groups. One of these is also present on monocytes and neutrophils. Both were present on myeloid progenitor cells, as judged by depletion experiments with antibody and complement, although neither bound appreciably to myeloid leukemic cells as judged by indirect immunofluorescence. The other three antibodies bound preferentially to leukemic specimens displaying monocytic differentiation. Four of the antibodies could be demonstrated to bind to cells in frozen sections of tonsil and small intestine and all gave distinct patterns of reactivity. In particular, these antibodies differed markedly in their binding to endothelium, follicular dendritic cells and various types of tissue macrophages. These antibodies may be useful in the study of the differentiation of myeloid cells and in studies of immunologically mediated disease such as allograft rejection.

Pertussis toxin pretreatment alters the in vivo cell division behaviour and survival of B lymphocytes after intravenous transfer.

Pertussis toxin (PT), produced by the causative agent of whooping cough, Bordetella pertussis, contributes to the immune dysfunction seen in infected patients. Treatment of laboratory animals with purified toxin reproduces many of the biological effects exhibited in the disease state, which include lymphocytosis, adjuvant effects for IgE secretion and delayed-type hypersensitivity reactions. In previous studies, we have demonstrated that PT pretreatment of intravenously transferred lymphocytes not only results in them being held up in the blood, but also causes a profound alteration in their positioning within the spleen. Pertussis toxin pretreated lymphocytes fail to traverse the layer of marginal zone macrophages encircling the white pulp, resulting in their exclusion from the lymphoid area of the spleen. Using a novel flow cytometric assay of cell division, the studies presented here show that a significant proportion of B, but not T, lymphocytes underwent proliferation after intravenous transfer of donor splenic lymphocytes to syngeneic recipients. This proliferation was markedly reduced by PT pretreatment of lymphocytes before transfer. In contrast, the in vitro proliferative responses of B lymphocytes to anti-IgM, LPS and antibody engagement of CD40 were unimpaired by exposure to the same levels of PT. Furthermore, the rate of in vivo decay of transferred B cells was accelerated by pretreatment with PT. Together, these data suggest PT impairs the receipt of signals which promote survival and proliferation of B cells, due to altered recirculation and positioning of lymphocytes.

Divided we stand: tracking cell proliferation with carboxyfluorescein diacetate succinimidyl ester.

Most techniques for assessing cell division can either detect limited numbers of cell divisions (bromodeoxyuridine incorporation) or only quantify overall proliferation (tritiated thymidine incorporation). In the majority of cases, viable cells of known division history cannot subsequently be obtained for functional studies. The cells of the immune system undergo marked proliferation and differentiation during the course of an immune response. The relative lack of an organized structure of the lymphohaemopoietic system, in contrast with other organ systems, makes lineage interrelationships difficult to study. Coupled with the remarkable degree of mobility engendered by recirculation, the differentiation occurring along with cell division in the immune system has not been readily accessible for investigation. The present article reviews the development of a cell division analysis procedure based on the quantitative serial halving of the membrane permeant, stably incorporating fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE or CFDA, SE). The technique can be used both in vitro and in vivo, allowing eight to 10 successive divisions to be resolved by flow cytometry. Furthermore, viable cells from defined generation numbers can be sorted by flow cytometry for functional analysis.

The Rose Bengal assay for monoclonal antibodies to cell surface antigens: comparisons with common hybridoma screening methods.

An automated colorimetric procedure for detection of antibodies specific for cell surface antigens (1) has been compared for specificity and sensitivity to other methods of hybridoma supernatant screening. The Rose Bengal colorimetric assay (RBA) compared favourably in these respects with whole cell radioimmunoassay and indirect immunofluorescence with manual or flow cytometric analysis (FACS). A major advantage of the method is that it allows a large number of samples to be screened in a comparatively short time. Unlike other semi-automated colorimetric assays, such as ELISA, the procedure does not require cell fixation, which can destroy some antigenic determinants. The original assay of O'Neill and Parish (1) has been modified to give increased sensitivity and also to enable the detection of erythrocyte specific antibodies by elimination of the dye staining step and direct measurement of haemoglobin by spectrophotometry. The RBA allows detection of monoclonal antibodies (MoAb's) binding to only a proportion of the cells in a sample, which is an important feature when hybridoma supernatants are screened for reactivity to a minor cell population, for example against leukaemic cell samples with low percentages of blast cells.