Monitoring of serologic immune parameters in inflammatory skin diseases

This paper deals with the correlation of clinical scoring and serologic markers of inflammation in atopic dermatitis and psoriasis. Serum eosinophil cationic protein (ECP), soluble interleukin-2 receptor (sIL-2R), total serum IgE, IgG and IgM anti-IgE antibodies, and IgE immune complexes were evaluated in monitoring inflammatory skin diseases such as atopic dermatitis and psoriasis. Well-established clinical activity scores were used as standards in recording skin improvement under treatment in a clinical setting. Serum ECP was found to be increased in both atopic dermatitis and psoriasis patients compared to normal controls; sIL-2R and IgE immune complexes were increased only in atopics with increased serum IgE. Anti-IgE antibodies did not show any deviation in both groups of patients. There was a significant elevation of sIL-2R and IgE immune complexes and a nonsignificant elevation of ECP in high-IgE atopics in comparison to those with normal serum IgE. In both groups of patients, there was a significant reduction of ECP and sIL-2R accompanying the improving skin condition. Serum IgE and the other immune parameters failed to respond. In contrast to other studies, serum ECP failed to correspond significantly with disease activity in our study. Our results showed measurable changes of ECP and sIL-2R for atopic dermatitis and/or psoriasis under treatment, but comparison to clinical scores remains difficult due to the different basis of the two systems. The only significant correlation was established for relative changes in sIL-2R and psoriasis area and intensity (PASI), a correlation which might be a useful approach in psoriasis.     

In vitro culture of human peritoneal fluid cells

The author has studied the behaviour of cells from human ascitic fluid in long-term culture (5-6 months). Three cellular types are described with different morphological features, namely the cellular shape, the fashion in which the cell spreads on the glass, the nucleocytoplasmic ratio, the chromatin appearance and the abundance of mitochondria. The three cellular types can phagocytose, but each one in a different way. The first one phagocytoses exclusively erythrocytes 'by contact' without emission of pseudopods; the second one phagocytoses degenerating nucleated cells in the same way as the first; the third type phagocytoses degenerating nucleated cells by emission of long pseudopods. The origin of these three cellular types is discussed; it is felt that they are transformed mesothelial cells. According to this study, it cannot be excluded, especially for the second and the third type, that they are histiocytes coming from serous membranes. The life in vitro of the three cellular types is depending upon the composition of nourishing medium. Cells can divide by mitosis only during the first 10 - 15 days of culture (mitotic index 0.1-3.0(0/00). Nuclear amitosis, nucleolus expulsion into cytoplasm and cytoplasmatic DNA synthesis can be observed in healthy cells.     

An in vitro tissue culture bilayer model to examine early events in Mycobacterium tuberculosis infection

A tissue culture bilayer system that mimics some aspects of early alveolar infection by Mycobacterium tuberculosis was developed. This model incorporates human lung epithelial type II pneumocyte (A549) (upper chamber) and endothelial cell (lower chamber) layers separated by a microporous membrane. This construction makes it possible to observe and quantify the passage of bacteria through the two layers, to observe the interaction of the bacteria with the various cell types, and to examine the basic mechanisms of immune cell recruitment to the site of infection. After 10(7) organisms were added to the upper chamber we microscopically observed large numbers of bacteria attached to and within the pneumocytes and we determined by viable-cell counting that a small percentage of the inoculum (0.02 to 0.43%) passed through the bilayer into the lower chamber. When peripheral blood mononuclear cells were added to the lower chamber, microscopic examination indicated a migration of the mononuclear cells through the bilayer to the apical surface, where they were seen associated with the mycobacteria on the pneumocytes. The added complexity of the bilayer system offers an opportunity to define more precisely the roles of the various lung cell types in the pathogenesis of early tuberculosis.     

In vitro activity of levofloxacin against a selected group of anaerobic bacteria isolated from skin and soft tissue infections

The in vitro activity of levofloxacin was compared to the activities of ofloxacin, ciprofloxacin, ampicillin-sulbactam (2:1), cefoxitin, and metronidazole for a selected group of anaerobes (n = 175) isolated from skin and soft tissue infections by using the National Committee for Clinical Laboratory Standards-approved Wadsworth method. Ampicillin-sulbactam and cefoxitin inhibited 99% of the strains of this select group, levofloxacin and ofloxacin inhibited 73 and 50%, respectively, at 2 microg/ml, and ciprofloxacin inhibited 51% at 1 microg/ml. The geometric mean MIC of levofloxacin was lower than those of ofloxacin and ciprofloxacin for every group except Veillonella.     

Immunity at the surface: homeostatic mechanisms of the skin immune system

The coordinated function of multiple epidermal and dermal cell populations allows the skin immune system to respond rapidly and effectively to a wide variety of insults occurring at the interface of the organism and its environment. Keratinocytes are the first line of defense in the skin immune system, and keratinocyte-derived cytokines are pivotal in mobilizing leukocytes from blood and signaling other cutaneous cells. Cytokine-mediated cellular communication also enables dermal fibroblasts and endothelial cells lining the cutaneous vasculature to participate in immune and inflammatory responses. Skin is an important site for antigen presentation, and both epidermal Langerhans cells and dermal dendritic cells play pivotal roles in T cell-mediated immune responses to antigens encountered in skin. Proinflammatory signaling pathways are necessarily balanced by a variety of regulatory pathways that help maintain the homeostatic functioning of the skin immune system.     

In vitro fertilization, culture, and transfer of rabbit ova

Ovulated rabbit oocytes were fertilized in vitro in chemically defined media supplemented with bovine serum albumin and either cultured up to the expanding blastocyst stage or transferred to recipients after varying periods of culture. Embryos transferred after up to 72 hours of in vitro culture were born as viable young. Oocytes from young virgin does were superior to oocytes from nonvirgin does for the purpose of in vitro fertilization (54% versus 26% fertilized, P less than 0.01). Capacitated sperm from artificially inseminated capacitators resulted in fertilization rates slightly lower than those from naturally mated does (46% versus 57% fertilized, P less than 0.025). Removal of cumulus and corona cells from oocytes with hyaluronidase and repeated aspiration through a fine pipette resulted in lowered fertilization rates (51% versus 73%, P less than 0.025). Linbro Disposo Tray wells were as good as glass tissue-culture dishes for the in vitro mixing of gametes and were more convenient to use. Modified Ham's F10 medium was used to culture the in vitro-fertilized embryos. However, when a modified Brackett's medium was used instead of modified Ham's F10 for the initial 4-hour period after mixing gametes, more oocytes were fertilized (52% versus 28%, P less than 0.01).     

Human keratinocytes express cellular prion-related protein in vitro and during inflammatory skin diseases

OBJECTIVE: Our purpose was to study the neonatal effects of red blood cell alloimmunization in a rabbit model. STUDY DESIGN: Eighteen does were alloimmunized to incompatible red blood cells. Does were bred twice, once with a homozygous buck of incompatible blood type and once with a homozygous buck of compatible blood type. Fetal blood sampling was undertaken on day 27 of gestation (term 28 to 31 days). Does were delivered on day 30 and the neonatal pups were anesthetized. Direct cardiac samplings were performed for hemoglobin, reticulocyte count, and direct Coombs' test. Hepatic, splenic, and renal wet weights were measured. RESULTS: Twenty-two pregnancies (12 compatible and 10 incompatible) were studied. Neonatal hemoglobin was higher in the compatible litters (11.1 gm/dL [7.7 to 12.6 gm/dL] vs 4.9 gm/dL [2.1 to 9.1 gm/dL], P <.001), whereas no difference could be detected between the respective reticulocyte counts (34.0/100 red blood cells [27.3 to 36.1/100 red blood cells] vs 32.6/100 red blood cells [26.8 to 43.5/100 red blood cells], P =.55). The direct Coombs' assay was negative in 23 pups from 8 compatible litters and false positive (weakly positive result) in 2 pups of a ninth compatible litter. The Coombs' assay was positive in all 22 incompatible pups tested. Hepatosplenomegaly was noted in affected pups but not in controls. CONCLUSIONS: A disease analogous to human hemolytic disease of the newborn can be induced in the rabbit neonate.     

Eosinophil-enriched inflammatory response to schistosomula in the skin of mice immune to Schistosoma mansoni

Exposure of the mouse skin to Schistosoma mansoni cercariae gives rise to acute, exudative inflammation in both normal and immune mice, but the immune response is anamnestically accelerated and is oesinophil-enriched, thereby enhancing opportunities for tegumental contact of schistosomula with host leukocytes, particularly with eosinophils. Many of the inflammatory changes occurring within the first 48 hours after exposure are due to cercarial products, e.g., "penetration tracts," but some remain demonstrable when schistosomula metamorphosed in vitro are injected intradermally and are therefore directed against the schistosomula themselves, such as the leukocyte "streaming patterns" seen in their pathways. In contrast to earlier observations in primates, cellular responses to schistosomula in the mouse lung 4 days after penetration are minimal in either normal or immune mice. Thus, immune cellular responses to schistosomula in mice are limited to an early time period after cercarial penetration and are morphologically suggestive of an antibody-mediated response rather than of delayed hypersensitivity. Our observations complement earlier evidence suggesting that antibody-mediated host leukocyte contact with schistosomula initiates the killing of challenge parasites in immune mice, with the eosinophil probably playing a crucial role.     

Mechanisms of immune cell-mediated tissue injury in inflammatory bowel disease (Review)

This review discusses the mechanisms and pathways of immune cell-mediated intestinal inflammation and tissue injury in inflammatory bowel disease (IBD). Our lack of understanding of how the mucosal immune system normally functions to maintain the balance between tolerance and immunity to innumerable dietary and bacterial constituents of the gut is perhaps the biggest obstacle to understanding the cause(s) of IBD, and to developing more effective treatments for these debilitating disorders. Evidence that abnormalities or disruptions in the interaction of immune cells and gut bacteria can trigger or contribute to changes in the composition, regulation and activity of the mucosal immune system that result in inflammatory immune responses and tissue injury are discussed. Based upon these studies, we propose a model to explain how a breakdown in regulation and failure to resolve immune responses in the gut mucosa results in persistent activation of T lymphocytes and other immune cells and the uncontrolled production of soluble inflammatory mediators that directly or indirectly produce the pathophysiological changes and tissue injury characteristic of IBD.     

In vitro production of parathyroid hormone-related protein by cholesteatoma and normal skin

Severe inflammation and infection of the middle ear occasionally results in clinical evidence of bone resorption but with the addition of the cholesteatoma epithelium it becomes inevitable. This study examined production by the cholesteatoma epithelium of a bone resorbing factor, namely parathyroid hormone-related protein (PTH-rP), which would not be expected in inflammatory states alone. PTH-rP was detected in the conditioned medium of primary and secondary cell cultures derived from 12 cholesteatoma biopsies. The levels of PTH-rP were significantly greater than in control cultures of cells derived from normal scalp tissue. Production by the cholesteatoma epithelium may explain the increased incidence of bone resorption in this disease, particularly in cases where inflammation is minimal.     

The effect of immune and inflammatory mediators on growth of Lucilia cuprina larvae in vitro

Immune and inflammatory responses occurring during dermal infestation by larvae of Lucilia cuprina can retard larval growth and development. This study examined the effect of 4 classes of humoral inflammatory mediators on larval growth in an in vitro assay. Mediators of plasma leakage (histamine, bradykinin, platelet-activating factor and serotonin), leucocyte chemotactic agonists (activated complement, leukotriene B4 and interleukin-8), effector molecules of immune responses (interleukin-1 beta, tumour necrosis factor-alpha and interferon-gamma) and endotoxin all failed to inhibit larval growth. In contrast, immunoglobulins isolated from immune serum caused marked retardation of larval growth. The results suggest that humoral mediators of inflammatory and immune responses do not play a role in immune defence against Lucilia cuprina.     

In vitro isolation and cultivation of Cowdria ruminantium under serum-free culture conditions

Imagined halitosis is poorly documented in the psychiatric literature. It is perhaps best thought of as a symptom rather than as a specific syndrome (a collection of symptoms that co-vary together). Many of the cases with imagined halitosis described in the literature resemble the psychiatric syndrome of social phobia.     

Comparison of an in vitro skin model to normal human skin for dermatological research

EpiDerm, an in vitro human skin equivalent (HSE), was compared to normal human breast skin (NHS) to morphologically and biochemically assess its feasibility for dermatological research. Intralot and interlot variability was studied in day 0, 1, 2, and 3 in vitro cultures and in day 0, 3, 5, and 7 NHS. For NHS, light microscopy (LM) at day 0 showed stratified epidermis which exhibited an increase in vacuoles and dark basal cells as storage increased to 3, 5, and 7 days. Transmission electron microscopy (TEM) revealed typical organelles in the epidermis and a convoluted basement membrane at day 0. With increased storage, vacuoles and paranuclear clefts became numerous, necrosis increased, tonofilaments became less organized, and overall cellular integrity decreased. Biochemical data showed consistent MTT and glucose utilization (GU) through day 5, while lactate production decreased to 75% by day 3. By LM, day 0 HSE consisted of a thick, compact, stratum corneum that sent projections between the stratum granulosum cells. By TEM, the configuration organization, differentiation, distribution, and frequency of the organelles differed slightly from NHS. In addition, the basement membrane of the HSE was not completely differentiated, and the dermis was thin and acellular. Although day 1 and 2 cultures showed little change, day 3 exhibited an overall degeneration. Biochemical analysis showed GU and the lactate production decreased through day 3. In conclusion, the EpiDerm HSE, although exhibiting slight differences, was morphologically and biochemically similar to normal human epidermis and may be a valuable model in assessing the toxicology, metabolism, or pharmacology of nonvesicating compounds.     

In vitro screening of biochemical activity of folic acid antagonists in skin

Dihydrofolate reductase (DHFR) inhibitors, which differ from the classical folate antagonists in physicochemical and pharmacologic parameters such as lipid solubility and mechanisms of cellular transport, were screened for DHFR inhibitory activity and biologic activity in newborn rat skin. The most effective drugs from this screen were tested for their effects on de novo DNA synthesis in psoriatic epidermis in vitro. Of the 24 compounds studied, methotrexate (MTX) was the most potent inhibitor of rat skin DHFR (I50=8.6 X 10(-9) M). Methotrexate-dimethylester, methasquin-diethylester, DDEP (2,4-diamino-5-(3',4'-dichlorophenyl)-6-ethylprimidine), and Baker's triazine antifolate (NSC 139105), while less effective than MTX as DHFR inhibitors, were more effective than MTX as inhibitors of de novo DNA synthesis in rat skin in vitro. Baker's antifolate was the only compound tested which was considerably more effective than MTX as an inhibitor of de novo DNA synthesis in psoriatic epidermis in vitro.     

Haemophilus ducreyi infection causes basal keratinocyte cytotoxicity and elicits a unique cytokine induction pattern in an In vitro human skin model

Haemophilus ducreyi is the etiologic agent of the sexually transmitted genital ulcer disease chancroid. Predominantly a cutaneous pathogen, H. ducreyi is present in chancroid ulcers that are characterized by extensive neutrophil accumulation in intraepidermal lesions accompanied by a mononuclear infiltrate in the dermis. We used an in vitro human skin model composed of foreskin fibroblasts and keratinocytes to examine host skin cell interactions with H. ducreyi 35000. Bacteria replicated and persisted in artificial skin for at least 14 days. We observed H. ducreyi inside suprabasal keratinocytes using transmission electron microscopy. Although no bacteria were seen in the basal keratinocyte region, these cells were disrupted in infected cocultures. H. ducreyi infection stimulated increased secretion of interleukin-6 (IL-6) and IL-8 by skin cells. Conversely, tumor necrosis factor alpha and IL-1alpha levels were not elevated. IL-8 produced in response to H. ducreyi infection may be involved in recruiting polymorphonuclear leukocytes and other inflammatory cells, thereby contributing to the tissue necrosis and ulcer formation characteristic of chancroid.     

In vitro training model for endovascular embolization of cerebral aneurysms

We present a new in vitro model for the training of endovascular embolizations of cerebral aneurysms. The technique and laboratory set-up described allow the embolization of thin-walled, transparent silicone aneurysm models under "semi-physiologic" conditions, i.e., in a closed circulation with pulsatile flow and pressure, simulating the hemodynamic conditions encountered in biologic systems.     

Enhanced growth of murine melanoma in ultraviolet-irradiated skin is associated with local inhibition of immune effector mechanisms

We have developed a model for studying the role of local immunologic mechanisms in tumor development, in which injection of K1735 melanoma cells into the UV-irradiated ears of C3H mice results in a significantly higher incidence of tumors than injection into unirradiated ears. This effect of UV irradiation is immunologically mediated. We hypothesized that UV blocks the efferent arm of the immune response, thereby facilitating tumor development within the irradiated site. We demonstrate that elicitation of a delayed type hypersensitivity response to alloantigen is diminished in UV-irradiated ears. in addition, tumor rejection is impaired in melanoma-immune mice challenged in UV-irradiated ears, even though such mice exhibit systemic immunity when challenged in a nonirradiated site. The ability of immune lymphoid cells to inhibit melanoma growth when mixed with tumor cells and injected into the ears was inhibited by prior UV irradiation of the ears, indicating that the activity of immune effector cells is abrogated in the UV-irradiated microenvironment. Analysis of lymphoid cells in growing tumors indicated that the number of CD8+ T lymphocytes was reduced in the UV-irradiated site. We conclude that efferent immune responses are impaired in UV-irradiated tissue and suggest that the impairment may involve reductions in both the number and the activity of immune effector cells. These studies illustrate that conditions in the local microenvironment during the early stages of tumor growth may profoundly influence the outcome of the host-tumor interaction.     

Relationship between the skin permeation movement of propranolol and skin inflammatory reactions

We studied inflammatory reactions induced by dermal application of the beta-blocker propranolol (PRL) in ethanol to guinea pigs in order to elucidate the relation of the reactions with the cumulative PRL permeating amount through the stratum corneum or the PRL content in the stripped skin, and to investigate the chemical mediators responsible for the reactions. The cumulative PRL permeating amount through the stratum corneum increased rapidly up to 2 h after dermal application, then increased linearly with time up to 24 h after application. Visual observation revealed formation of erythema and edema at the applied site of PRL, and histopathological examination revealed infiltration of pseudoeosinophiles of dermis and epidermis and degeneration/necrosis of epidermis. In general, it was considered that the duration and the extent of these reactions were dependent on the PRL dosage and application time. It was expected that the cumulative PRL permeating amount through the stratum corneum could be used to predict possible inflammatory reactions during development of transdermal drug delivery systems. On the other hand, contact of PRL with guinea pig skin tissues released histamine, and intradermal injection of PRL caused an increase of capillary permeability at the site of application. Also, the inhibitory effects of anti-inflammatory agents (diphenhydramine, dexamethasone, indomethacin, cyproheptadine hydrochloride, CV3988 and AA-861) to PRL-induced erythema formation demonstrated that histamine and prostaglandins were responsible for the inflammatory reactions induced by PRL.