IL-7 overexpression in transgenic mouse keratinocytes causes a lymphoproliferative skin disease dominated by intermediate TCR cells: evidence for a hierarchy in IL-7 responsiveness among cutaneous T cells

IL-7 is a keratinocyte-derived lymphocyte growth factor critical for the development of gammadelta T cells including murine dendritic epidermal T cells (DETC). We derived transgenic mice that overexpress IL-7 in basal keratinocytes under the control of the human K14 promoter. These K14/IL-7 mice develop dermal and epidermal T cell infiltrates associated with alopecia. This lymphoproliferative skin disease is substantially more severe in mice homozygous for the K14/IL-7 transgene. Conventional DETC expressing a Vgamma5 Vdelta1 TCR are rare or absent among the cutaneous T cells in these mice. The T cells in the skin infiltrates of young K14/IL-7 mice are predominantly gammadelta T cells that express intermediate levels of TCR, are negative for E-cadherin, often lack expression of CD2, and include cells that coexpress NK1.1. T cells expressing intermediate levels of a TCR-alphabeta are also present in transgenic skin, and progressively increase in number as the mice age. Phenotypically similar intermediate gammadelta and alphabeta T cell subsets also constitute the major lymphocyte populations recovered from organ culture of normal mouse skin in the presence of IL-7, suggesting that the T cells that accumulate in the epidermis of K14/IL-7 mice are derived from precursors normally resident in skin. We conclude that intermediate TCR cells, some of which coexpress NK1.1, can be selectively expanded in skin under the influence of IL-7 produced locally. Our results also suggest that features of the epidermal microenvironment besides keratinocyte-derived IL-7 account for the normal predominance of Vgamma5 Vdelta1 DETC in mouse epidermis.     

Misinterpretation of body sensations in panic disorder

Cognitive accounts of panic predict that panic disorder patients will be particularly prone to misinterpret autonomic sensations. Several studies have produced results consistent with this prediction, but each is open to alternative interpretation. To clarify matters, 2 studies administered the Body Sensations Interpretation Questionnaire (BSIQ) to panic patients and controls. Panic patients were more likely to interpret ambiguous autonomic sensations as signs of immediately impending physical or mental disaster and were more likely than other anxiety disorder patients and nonpatients to believe these interpretations. In a 3rd study, a brief version of the BSIQ was shown to have satisfactory test-retest reliability, to change with treatment, and to discriminate treatments that varied in their effects on panic.     

Effects of molecular crowding on the interaction between DNA and the Escherichia coli regulatory protein TyrR

Fluorescence quenching has been used to measure quantitatively the effects of sucrose and triethylene glycol on the interaction between the Escherichia coli regulatory protein TyrR and a 30-basepair oligonucleotide containing the strong TyrR box of the TyrR operon. It was observed that the apparent binding constant increased in the presence of co-solutes, the dependence of the logarithm of the apparent binding constant on molar concentration being indistinguishable and essentially linear for both co-solutes. This activation of the TyrR-oligonucleotide interaction is attributed to thermodynamic nonideality arising from molecular crowding, an interpretation which is supported by the reasonable agreement observed between the experimental extent of reaction enhancement and that predicted on the statistical-mechanical basis of excluded volume.     

A novel application of the infrared coagulator in hair transplantation. Significant reduction of width of the donor site wound

BACKGROUND: The infrared coagulator, a by-product of laser technology, has been used in dermatology in a variety of settings. During hair transplantation sessions, we observed a significant reduction of the donor ellipse width while performing hemostasis with the infrared coagulator. OBJECTIVE: Quantitative assessment of the donor wound width after infrared coagulator use, and correlation to the number of previous transplant sessions and patients' age. METHODS: Twenty-four patients (22 men, two women) underwent hair transplantation. The infrared coagulator was utilized for hemostasis with a pulse duration of 2.5 seconds. RESULTS: The infrared coagulator produced an average donor area decrease of 42%, while achieving rapid hemostasis. No correlation was demonstrated to number of previous transplant sessions or patients' age. CONCLUSIONS: The infrared coagulator significantly decreases the donor wound width while providing hemostasis. Advantages include the potential of larger donor strip harvest, minimal tissue manipulation, and less traumatic closure.     

Characterisation of erythrocyte shapes and sizes by NMR diffusion-diffraction of water: correlations with electron micrographs

Irradiation of the mammalian foetus produces a broad spectrum of congenital abnormalities, growth retardations, developmental delays, and functional deficits, depending upon the dose and the specific gestational phase of irradiation. The developing brain is particularly susceptible to production of deleterious effects, with decreased brain size, behavioural alterations, and mental retardation having been documented. Supplementing the limited human data, rodent models have been extensively used to investigate the specific processes by which relatively low doses, with correspondingly minor cellular damage to the developing neocortex, can produce dramatic postnatal consequences in brain structure and function. The effects of a variety of physical (dose, linear energy transfer, dose rate, fractionation) and biological (species, strain, gestational age, time course post-irradiation) parameters have been examined in an attempt to provide much needed information on such critical aspects as dose response, threshold doses for effect, and extrapolation to human risk estimates. Various acute cellular responses (e.g. appearance of pyknotic cells and macrophages) observed in the developing neocortex 0-24 h after in utero irradiation can be associated with postnatal effects. Moreover, it is possible to correlate thinning of specific layers of the cerebral cortex with specific behavioural aberrations, allowing prediction of brain structural changes from functional alterations, and vice versa. Thus, it is possible to speculate as to the mechanisms and targets for extremely sensitive, radiation-induced cellular damage in the developing foetal brain, that will interfere with the orderly and precisely programmed development of the mammalian brain, leading finally to postnatal expression as delays in growth and development, perturbations in behaviour, and alterations in brain structure.     

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.     

Investigation of the N-substituent conformation governing potency and mu receptor subtype-selectivity in (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine opioid antagonists

A study of the binding site requirements associated with the N-substituent of (+)-(3R,4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) derivatives was undertaken using a set of rigid vs flexible N-substituents. The study showed that compounds 7-9 bearing the trans-cinnamyl N-substituent most closely reproduced the potency at the opioid receptor of the flexible N-propylphenyl or N-propylcyclohexyl analogues previously reported. Neither the N-substituted cis-cinnamyl nor the cis-phenylcyclopropylmethyl compounds 10 and 11, respectively, showed high affinity for the opioid receptor. However, the N-trans-phenylcyclopropylmethyl compound 12 closely approximated the affinity of compounds 7-9. Additionally, we found that free rotation of the phenyl ring is necessary for high affinity binding and mu receptor subtype selectivity as the planar N-substituted thianaphthylmethyl and benzofuranylmethyl compounds 13 and 14 had significantly lower binding affinities. Altogether, these findings suggest that the high binding affinity, selectivity, and antagonist potency of N-propylphenyl or N-propylcyclohexyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) are achieved via a conformation wherein the connecting chain of the N-substituents is extended away from piperidine nitrogen with the appended ring system rotated out-of-plane relative to the connecting chain atoms. This conformation is quite similar to that observed in the solid state for 5, as determined by single crystal X-ray analysis. Additionally, it was found that, unlike naltrexone, N-substituents bearing secondary carbons attached directly to the piperidine nitrogen of 4 suffer dramatic losses of potency vs analogues not substituted in this manner. Using a functional assay which measured stimulation or inhibition of [35S]GTP-gamma-S binding, we show that the trans-cinnamyl analogues of (+)-(3R, 4R)-dimethyl-4-(3-hydroxyphenyl)piperidine (4) retain opioid pure antagonist activity and possess picomolar antagonist potency at the mu receptor.     

Immunohistochemical video-microdensitometry of myocardial collagen type I and type III

Collagen is an essential part of the cardiac interstitium. Collagen subtypes, their location, total amount and the architecture of the fibrillar network are of functional importance. Architecture in terms of density of the fibrillar network is assumed to be reflected by the intensity of immunohistochemical staining of collagen. The aim of this study was to evaluate a video-based microdensitometric method for quantifying density expressed as absorbance of collagen subtypes I and III stained with an indirect immunoperoxidase method in myectomy specimens of patients with hypertrophic obstructive cardiomyopathy. Various factors influencing the immunohistochemical staining product and the technical properties of the image analysis system were investigated. Linearity between collagen concentration and the absorbance of the immunohistochemical staining product was demonstrated for collagen I using a dot-blot technique. Immunohistochemical collagen staining and density measurement were easily reproducible. The cardiac disability of the patients was assessed according to the New York Heart Association (NYHA) criteria. There was a significant increase in collagen type I density with higher NYHA class, whereas no significant association was found for total collagen area fraction. Thus, video-based microdensitometry gives further insight into the structural remodelling of myocardial collagens and reveals their significance in the process of heart failure in hypertrophic cardiomyopathy.     

Induction of plasminogen activator inhibitor type 1 and type 1 collagen expression in rat cardiac microvascular endothelial cells by interleukin-1 and its dependence on oxygen-centered free radicals

BACKGROUND: Ischemia with or without reperfusion induces the release of diverse products from monocytes, including cytokines such as interleukin-1 (IL-1). To determine whether these phenomena modulate fibrinolysis and potentially exacerbate impairment of the macrocirculation, microcirculation, or both, we characterized the effects of IL-1 on the expression of fibrinolytic system and matrix proteins in rat cardiac microvascular endothelial cells (CMECs). METHODS AND RESULTS: Confluent CMECs were exposed to IL-1 in serum-free medium for 24 hours, and cell-conditioned medium was assayed for plasminogen activator inhibitor type 1 (PAI-1), the primary physiological inhibitor of plasminogen activators, and for type 1 collagen with Western blotting. IL-1 (2 ng/mL) specifically increased the accumulation of PAI-1 (4.4 +/- 0.6-fold; mean +/- SD; n = 9) without affecting tissue plasminogen activator (t-PA) or urokinase plasminogen activator (u-PA) levels, which remained unchanged. IL-1 increased the accumulation of collagen in conditioned media by 3.5 +/- 0.7-fold (n = 6). Conversely, the accumulation of both PAI-1 and collagen induced by IL-1 was inhibited with an IL-1 receptor antagonist (200 ng/mL; n = 6) and with cycloheximide (10 micrograms/mL; n = 6), implying that protein synthesis was a requirement for the effect. To determine whether the IL-1 effect was mediated by induction of oxygen-centered free radical production, known to be induced by IL-1, we exposed the cells to the hydroxyl radical scavenger tetramethylthiourea (10 mmol/L) and observed abolition of the IL-1-induced increase in the expression of PAI-1 and collagen (n = 6). Conversely, superoxides (generated with 10 mU/mL xanthine oxidase plus 0.6 mmol/L hypoxanthine, and 100 mumol/L hydrogen peroxide) induced the accumulation of PAI-1 and collagen (n = 6). IL-1 (1 microgram/kg body wt) and lipopolysaccharide (50 micrograms/kg body wt) administered in vivo increased PAI-1 protein in rat hearts as detected with Western blotting and PAI-1 immunostaining of rat heart microvessels, indicating the effects delineated in vitro were paralleled by effects in vivo. CONCLUSIONS: These results indicate that IL-1-induced oxygen-centered free radicals stimulate elaboration of PAI-1 and collagen by CMECs. Accordingly, microvascularly mediated inhibition of fibrinolysis may predispose to the persistence of microvascular thrombi, thereby contributing to impaired microcirculatory function, the no-reflow phenomenon, and cardiac dysfunction after ischemia and reperfusion.