Permeabilities of alkyl p-aminobenzoates through living skin equivalent and cadaver skin

The in vitro permeabilities of alkyl p-aminobenzoates through living skin equivalent (LSE) and cadaver skin were compared. Methyl, ethyl, and butyl p-aminobenzoates were used as model compounds. The permeabilities of these compounds through LSE and cadaver skin from an aqueous drug suspension were determined with a flow-through diffusion cell. The permeability coefficients of these esters in LSE were an order of magnitude higher than in cadaver skin. This was primarily because of low resistances offered by the outermost layer (i.e., stratum corneum) of LSE. In the case of cadaver skin, the permeability coefficient increased as the carbon chain length increased, whereas no appreciable change in the permeability coefficients of these esters in LSE was observed. These results clearly suggest that the LSE membrane offered very little resistance as opposed to cadaver skin. Therefore, the LSE membrane may not quantitatively represent a good human skin model for evaluating skin permeation of a drug from topical or transdermal formulations.     

Microprocessor based spatial TENS (transcutaneous electric nerve stimulator) designed with waveform optimality for clinical evaluation in a pain study

1. The use of organs from animal donors (xenotransplantation) is a potential solution to the chronic shortage of allogeneic organs and currently the pig is thought to be the most suitable donor for man. However, porcine organs are rejected rapidly by a vascular process called hyperacute rejection which has so far prevented clinical xenotransplantation. Although it is likely that this barrier will be overcome in the near future by the application of novel strategies, probably involving the use of organs from transgenic pigs, data from animal models indicate that multiple other immune mechanisms will contribute to the rejection of xenografts. 2. We have described two aspects of these immune mechanisms. First, the phenomenon of 'accommodation', whereby xenografts acquire in vivo resistance to vascular rejection, has been explored in an in vitro model utilizing immortalized porcine endothelial cells. The results indicate that human anti-pig antibodies induce a concentration-dependent and time-dependent change in porcine endothelial cells compatible with the development of accommodation. 3. Secondly, the in vitro human anti-porcine T-cell response has been documented in detail, with particular emphasis on quantitative and qualitative comparisons with the in vitro T-cell alloresponse. The results of this work, which indicate that the response to porcine xenografts is likely to be significantly stronger than that against allografts, have important implications for the level of conventional immunosuppression that may be necessary to prevent xenograft rejection, and provide an important basis for the development of strategies to promote xenograft-specific immunosuppression and tolerance.     

An experimental model for ovarian tumor invasion of cultured mesothelial cell monolayer

BACKGROUND: Peritoneal spread of tumor cells is one of the characteristic features of biologic behavior of ovarian cancers. To understand the mechanism by which human tumor cell invasion takes place, we have tried to establish an in vitro experimental model for ovarian tumor cell invasion of the mesothelial cell monolayer. EXPERIMENTAL DESIGN: Mesothelial cells were isolated from normal rat mesentery by trypsin digestion and the cells (1 x 10(5)/dish) were cultured in Eagle's minimum essential medium supplemented with 10% fetal calf serum. Cultured mesothelial cells (M cells) grew forming a pavement-like monolayer. When M cells grew to a confluent state, tumor cells (1 x 10(5)/dish) were seeded on M cell monolayers and cultured. Four tumor cell lines derived from human ovarian cancers were tested for their invasive behaviors. The penetration of M cell monolayers by the tumor cells was confirmed by a perpendicular section of the cell layers. The number of penetrated single tumor cells and colonies/cm2 was counted under a phase contrast microscope after the tumor cell seeding. RESULTS: Several hours after the tumor cell seeding, the cells adhered to M cell monolayers and started to penetrate by extending pseudopodia-like cytoplasmic processes through junctional margins of neighboring M cells, resulting in the formation of penetrated single tumor cells that then proliferated to form colonies under the monolayer. The number of penetrated single tumor cells and colonies/cm2 increased up to 24 hours after the tumor cell seeding, and thereafter stayed almost constant. The number increased with the number of tumor cells seeded, when counted at 48 hours, and therefore was taken to be the number of tumor cells invaded. The in vitro invasiveness of tumor cells varied with the tumor cell lines examined. CONCLUSIONS: Application of this system appears to provide rapid determinations of the invasive potential of ovarian tumor cells and to make it easy to screen substances that modify the invasion of mesothelial cells.     

Large surface of cultured human epithelium obtained on a dermal matrix based on live fibroblast-containing fibrin gels

The aim of this study was to develop a new keratinocyte culture system on a dermal equivalent suitable for skin wound closure. Our dermal matrix is based on a fibrin gel from plasma cryoprecipitate containing live human fibroblast (from human foreskin). Keratinocytes obtained from primary culture according to the Rheinwald and Green method, were seeded on the gel at different seeding ratios. In all cases, the keratinocytes plated on the dermal equivalent grew to confluence and stratified epithelium was obtained within 10-15 days in culture. Early expression of basal membrane proteins was detected by immunostaining with laminin and type IV collagen antibodies. Cell proliferation was detected both in the epidermal layer and in the fibroblast embedded in the gel as assessed by BrdU incorporation. Detachment of composite cultures from dishes or flasks is a simple and quick procedure without the need for dispase treatment. Grafting of composite cultures to nude mice gave rise to an orderly stratified, orthokeratinized epithelium resembling human epidermis. A number of advantages including a large expansion factor without the need of 3T3 feeder layer, the availability of fibrin/plasma cryoprecipitate from blood banks and the versatile manipulation of composite cultures suggest that this system could be suitable for the definitive coverage of severely burned patients.     

Rat renal allograft tolerance is associated with local TGF-beta and absence of IL-2r expression within chimeric immunocytic foci

Tolerance was induced in Lewis (LEW) rat renal allograft recipients of Brown Norway kidneys by multiple pretransplant donor-blood transfusions and prior limited cyclosporine A. Rat renal allograft tolerance was associated with the induction of systemic donor T cells (10%), an early phase of nonspecific suppressor-cell generation, followed by maturation of systemic antigen-specific suppressor cells, and renal cellular infiltrates that develop long-term in situ in the kidney graft model. It was hypothesized that these infiltrates represent chimeric immunocytic foci that are locally regulated via a TGF-beta-dependent mechanism. Both immunohistochemical staining and digital image analysis for cellular and extracellular TGF-beta, IL-2 receptor (CD25), and the BN Class I-MHC marker (OX-27) were performed. Control rejecting (REJ) kidneys did not demonstrate any differences with respect to levels of infiltrating immunocyte area vs long-term surviving (TOL) kidneys (3.9% vs 4.5%, P = .303). Immunostaining with the BN Class I MHC marker (OX-27) demonstrated high levels of chimerism within immunocyte foci of the tolerant grafts (OX-27 BN+immunocytes 49.0% +/- 5.1%). In situ cellular IL-2 receptor (CD25) expression was demonstrated in REJ kidney infiltrates but not within TOL immunocytic infiltrating foci, when measured as percent of total lymphocytes (REJ = 5.0% vs TOL = 0.4%, P = .031). Conversely, TGF-beta expression was significantly higher in immunocytes of TOL kidneys when measured as the number of DAB chromogen-staining pixels per total immunocyte area (TOL = .076 vs REJ = .047, P = .003). In conclusion, these results suggested that stable mixed immune chimerism (SMIC) plays an important role in DST-CyA-induced tolerance in situ. SMIC-induced tolerance may involve a local TGF-beta-dependent mechanism that is associated with in situ TGF-beta (+) and IL-2r (-) immunocytes.     

Immunolocalization of acidic fibroblast growth factor and receptors in the tubulointerstitial compartment of chronically rejected human renal allografts

Tubular damage and loss associated with interstitial inflammation and fibrosis may be the most important determinants in chronic renal allograft rejection. To elucidate potential pathophysiologic mechanisms associated with tubulointerstitial lesions, we examined the expression of a fibrogenic cytokine, acidic fibroblast growth factor (FGF-1) and its high-affinity receptors, in both relevant renal transplant controls (n=5) and tissue from patients (n=19) who underwent nephrectomy after graft loss, secondary to chronic rejection. In situ hybridization and immunohistochemical analyses demonstrated minimal expression of FGF-1 mRNA and protein in the tubulointerstitial compartment of the normal human kidney. In contrast, tubulointerstitial lesions in kidney allografts experiencing chronic rejection demonstrated the exaggerated appearance of both FGF-1 protein and mRNA in resident inflammatory and tubular epithelial cells. Patterns of staining were consistent throughout tubular compartments and did not appear to be localized to any particular region. The tubulointerstitium in kidneys with findings of chronic rejection also exhibited increased immunodetection of proliferating cell nuclear antigen in the tubular epithelium, inflammatory cell infiltrate, and neovascular structures. The enhanced appearance of FGF-1 and readily detectable fibroblast growth factor receptors suggests that this polypeptide mitogen may serve as an important mediator of growth and repair responses, associated with development of angiogenesis and tubulointerstitial lesions during chronic rejection of human renal allografts.     

The chondrogenic potential of human bone-marrow-derived mesenchymal progenitor cells

Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage.     

Engraftment of human kidney tissue in rat radiation chimera: I. A new model of human kidney allograft rejection

BACKGROUND: We have recently shown that lethally irradiated normal strains of mice and rats, reconstituted with bone marrow from severe combined immune deficiency (SCID) mice, can be engrafted with human peripheral blood mononuclear cells (PBMC). METHODS: The feasibility of transplanting human renal tissue under the kidney capsule of the SCID/Lewis and SCID/nude radiation chimera and the effects of intraperitoneal infusion of allogeneic human PBMC on the human renal implants were investigated by histology, electron microscopy, immunohistochemistry, and fluorescence-activated cell sorter analysis. RESULTS: Sequential evaluation of the human renal implants from 10 days to 2 months after transplantation showed that human parenchymal elements survive in the implants up to 2 months after transplantation. The overall architecture of the transplanted kidney tissue and the normal structure of individual cells in the glomeruli and tubuli were preserved. Infusion of allogeneic human PBMC after kidney implantation resulted in patchy cellular infiltrates, composed mainly of activated human T cells, and led to prompt rejection of the human renal tissue, whereas no signs of inflammation were observed in human renal implants of chimeric rats that did not receive human PBMC. Treatment with OKT3 antibody, anti-human CD25 antibody, or CTLA4Ig fusion protein in vivo ameliorated the rejection process. CONCLUSIONS: Human adult kidney fragments transplanted into SCID-like rats transiently retain competent parenchymal structures. When these grafts are combined with allogeneic human PBMC, acute cellular rejection develops. We suggest that this chimeric model might be useful for the investigation of the effects of experimental manipulation on the kinetics of the inflammatory response during human renal allograft rejection.     

Tuberculous meningitis and co-infection with HIV

Sulfur mustard (HD) is a chemical warfare blister agent. The biochemical basis of HD-induced vesication is unknown, and no antidote currently exists. Basal epidermal cells are a major site of HD toxicity in vivo, with inflammation and HD-increased proteolytic activity implicated as factors that contribute to HD pathology. Fc receptors (FcR) bind to the Fc region of antibody to mediate many effector and regulatory functions that can influence inflammatory responses. FcR are found on all types of immune cells and are also expressed on the surface of human keratinocytes. Assay by fluorescent antibodies demonstrated significantly enhanced CD32 (FcRII) and CD16 (FcRIII) on human epidermal keratinocyte (HEK) cell cultures at 8 to 24 h after exposure to HD (50, 100 and 200 micromol/L). The enhanced CD32 was time- and concentration-dependent and agreed well with the time course of increased proteolysis and cutaneous pathology observed during HD vesication. HD-increased FcR on the surface of HEK might be a mechanism of vesication.     

The role of mast cell-derived histamine in the closure of an in vitro wound

We have previously reported that mast cells (MC) stimulate 3T3 fibroblast migration and proliferation into an in vitro model of wound obtained by producing in a confluent 3T3 monolayer, a midline cut and by scraping the cells from half of the monolayer. The purpose of the present study was to determine the contribution of mast cell-derived histamine to this MC increasing effect. Histamine levels in supernatants of MC/ 3T3 cultures unactivated or activated with either compound 48/80 or anti-IgE antibodies (10 min) did not correlate to the degree of fibroblast migration and proliferation into the wound space (42 h). Various concentrations of histamine were added to 3T3 fibroblast monolayers in the absence of cocultured MC, and fibroblasts beyond the wound line were counted (42 h). Addition of 100 ng/ml histamine had the highest stimulating effect on fibroblast numbers. This effect was abrogated by the addition of cimetidine (an H-2 antagonist). Addition of cimetidine to unactivated MC/ 3T3 cultures did not affect the increasing activity of MC presence on the wounded monolayer, although it diminished the enhancing effect obtained after MC activation with compound 48/80. These results indicate that histamine is partially responsible for the mast cell enhancing effect on fibroblast migration and proliferation in an in vitro model of wound.     

Photodynamic biologic tissue glue to enhance corneal wound healing after radial keratotomy

BACKGROUND: In this study, we describe the development of a novel experimental system in which rejection of porcine skin grafts by human peripheral blood cells can be studied directly in vivo in immunodeficient mice. METHODS: To construct a small animal model of discordant xenograft rejection, recombinase-activating gene-deficient mice (R-) lacking both mature B and T cells were grafted with porcine skin grafts and administered, by adoptive cell transfer, human cells stimulated in vitro with irradiated porcine peripheral blood cells to create Hu-R- mice. RESULTS: R- mice accepted porcine skin grafts indefinitely without the need for immunosuppression. In contrast, Hu-R- mice were able to reject porcine skin grafts. Immunohistochemical analysis of rejecting skin grafts revealed the accumulation of human T cells around dermal porcine vessels and focally in the epidermis. Graft rejection was manifested by vascular endothelial cell proliferation, edema at the dermal-epidermal border, and perivascular hemorrhage. The tissue damage observed in the rejecting grafts was similar to that observed in delayed primate anti-porcine cell-mediated rejection of vascularized organ xenografts. CONCLUSIONS: The development and characterization of a small animal model, to study cellular immune responses of human cells to discordant xenografts in vivo, should provide a convenient means for asking mechanistic questions related to discordant xenotransplantation, and may also provide a practical system for testing new approaches designed to prevent xenograft rejection.     

A rapid 2D time-resolved variable-rate k-space sampling MR technique for passive catheter tracking during endovascular procedures

The protease-activated receptor-2 (PAR-2) is a seven transmembrane domain receptor related to the thrombin receptor, which is activated in vitro by cleavage by trypsin. Affinity-purified rabbit IgG raised against a peptide corresponding to the trypsin cleavage site of PAR-2 was used for an immunohistochemical study of skin. The expression of PAR-2 in epidermis was striking, with keratinocytes showing abundant intercellular and cytoplasmic staining. Basal cells showed the strongest staining intensity and the stratum corneum was negative. Staining with control IgG used at the same concentration was consistently negative. The functional expression of PAR-2 by the simian virus transformed human skin keratinocyte cell line SVK14 was demonstrated by Northern blot analysis, flow cytometric analysis and the measurement of intracellular calcium. Treatment of SVK14 with trypsin or a receptor agonist peptide (SLIGKV-NH2) caused a dose-dependent increase in the secretion of the chemokine interleukin-8 (IL-8) in vitro. The effect of the peptide was specific, since control acetylated peptide was without activity. We conclude that PAR-2 is highly expressed by epidermal keratinocytes and receptor activation in vitro leads to increased IL-8 secretion by keratinocytes. These data raise the possibility that PAR-2 may play a role in epidermal homeostasis and inflammatory conditions.     

Chronic in vitro shear stress stimulates endothelial cell retention on prosthetic vascular grafts and reduces subsequent in vivo neointimal thickness

OBJECTIVE: The absence of endothelial cells at the luminal surface of a prosthetic vascular graft potentiates thrombosis and neointimal hyperplasia, which are common causes of graft failure in humans. This study tested the hypothesis that pretreatment with chronic in vitro shear stress enhances subsequent endothelial cell retention on vascular grafts implanted in vivo. METHODS: Cultured endothelial cells derived from Fischer 344 rat aorta were seeded onto the luminal surface of 1.5-mm internal diameter polyurethane vascular grafts. The seeded grafts were treated for 3 days with 1 dyne/cm2 shear stress and then for an additional 3 days with 1 or 25 dyne/cm2 shear stress in vitro. The grafts then were implanted as aortic interposition grafts into syngeneic rats in vivo. Grafts that were similarly seeded with endothelial cells but not treated with shear stress and grafts that were not seeded with endothelial cells served as controls. The surgical hemostasis time was monitored. Endothelial cell identity, density, and graft patency rate were evaluated 24 hours after implantation. Endothelial cell identity in vivo was confirmed with cells transduced in vitro with beta-galactosidase complementary DNA in a replication-deficient adenoviral vector. Histologic, scanning electron microscopic, and immunohistochemical analyses were performed 1 week and 3 months after implantation to establish cell identity and to measure neointimal thickness. RESULTS: The pretreatment with 25 dyne/cm2 but not with 0 or 1 dyne/cm2 shear stress resulted in the retention of fully confluent endothelial cell monolayers on the grafts 24 hours after implantation in vivo. Retention of seeded endothelial cells was confirmed by the observation that beta-galactosidase transduced cells were retained as a monolayer 24 hours after implantation in vivo. In the grafts with adherent endothelial cells that were pretreated with shear stress, immediate graft thrombosis was inhibited and surgical hemostasis time was significantly prolonged. Confluent intimal endothelial cell monolayers also were present 1 week and 3 months after implantation. However, 1 week after implantation, macrophage infiltration was observed beneath the luminal cell monolayer. Three months after the implantation in vivo, subendothelial neointimal cells that contained alpha-smooth muscle actin were present. The thickness of this neointima averaged 41 +/- 12 micrometer and 60 +/- 23 micrometer in endothelial cell-seeded grafts that were pretreated with 25 dyne/cm2 shear stress and 1 dyne/cm2 shear stress, respectively, and 158 +/- 46 micrometer in grafts that were not seeded with endothelial cells. CONCLUSION: The effect of chronic shear stress on the enhancement of endothelial cell retention in vitro can be exploited to fully endothelialize synthetic vascular grafts, which reduces immediate in vivo graft thrombosis and subsequent neointimal thickness.     

Aortic peri-valvular abscess, mycotic aneurysm of Valsalva sinus, and fistula to the right ventricle in a context of acute infectious endocarditis

OBJECTIVE: Techniques for in vitro culturing and autotransplantation have been developed for a variety of human cells and are used today in several fields of medicine. In reconstructive surgery within the genitourinary tract, autologous urothelial cells cultured in vitro could be of considerable value but have not yet been used clinically. The aim of this study was to facilitate transplantation of cultured urothelium by establishing a reliable method for culturing urothel on an immunologically inert and biodegradable structure. METHODS: Normal human urothelial cells were cultured in vitro using a feeder-cell system. To achieve an optimal carrier structure, cells were removed enzymatically from a split thickness skin graft. Human urothelial cells were then seeded on the cell-free dermis and incubated in vitro. The seeded dermis samples were investigated histologically and with immunohistochemical methods at days 7, 14 and 21. RESULTS: The human urothelial cells incubated in vitro reached confluence after 7-10 days and the cells could be cultured through 9 passages with preserved proliferative potential. When the cells were seeded on a cell-free dermis they attached, formed colonies and became confluent and stratified up to three cell layers after 21 days of incubation. The urothelial origin of the cells was confirmed by immunohistochemical staining against cytokeratin. CONCLUSION: The advantages of culturing the urothelial cells on a cell-free dermis include a short time lag until grafts are available, probably facilitated transplantation procedure, transplantation of undifferentiated cells and the formation of a vascularised base under the new urothelium. The method described in this study may be of great value in providing autologous urothelium for reconstructive surgery in the genitourinary tract.     

Induction of biologically active IL-1 beta-converting enzyme and mature IL-1 beta in human keratinocytes by inflammatory and immunologic stimuli

IL-1beta, a major mediator of inflammatory and immunologic skin disease, undergoes post-translational site-specific cleavage by a novel cysteine protease termed IL-1beta-converting enzyme (ICE). Although in human skin keratinocytes produce significant amounts of the 31-kDa IL-1beta precursor protein, they fail under nonpathologic conditions to convert it to the 17.5-kDa bioactive form. In this study, we examined whether haptens and inflammatory agents might serve as stimuli for ICE activity in human keratinocytes, and, if so, whether ICE activity might precipitate enzymatic processing of IL-1beta to its 17.5-kDa form. Baseline levels of ICE mRNA were detected in keratinocyte cultures devoid of Langerhans cells and were up-regulated by nontoxic concentrations of the reactive hapten urushiol and by the irritant chemicals sodium lauryl sulfate and PMA. Although untreated keratinocytes expressed the 31-kDa form of the protein, 17.5-kDa IL-1beta was easily detected in keratinocytes and keratinocyte supernatants treated with either urushiol or the irritant chemicals. Enzymatic conversion from the 31-kDa to the 17.5-kDa form of IL-1beta was blocked by addition of a highly specific aldehyde inhibitor that contained a tetrapeptide recognition sequence specific for ICE, but not by an aldehyde inhibitor of a related ICE-like cysteine protease. Induction of IL-1beta-converting enzyme by immunologic and inflammatory stimuli may be one of the key regulatory elements in the pathogenesis of allergic and irritant contact hypersensitivity.     

An in vitro blood-brain barrier model: cocultures between endothelial cells and organotypic brain slice cultures

This communication describes a novel in vitro blood-brain barrier (BBB) model: organotypic slice cultures from the central nervous system were overlaid on endothelial cell monolayers grown on permeable membranes. Morphological, electrophysiological, and microdialysis approaches were carried out to characterize and validate this model. After 10 days in coculture, morphological studies reveal the presence of tight junctions. Electrophysiological recordings of neuronal activity performed on organotypic cultures with or without an endothelial cell monolayer show that amplitude of evoked responses were comparable, indicating good viability of cocultures after 2 weeks. Perfusion of known BBB permeable or nonpermeable molecules was used to test the coculture tightness in conjunction with electrophysiological or microdialysis approaches: application of glutamate (Glu), which doesn't easily cross the BBB, triggers off rhythmic activity only in control cultures, whereas epileptogenic activity was observed in both control cultures and cocultures during perfusions with picrotoxin, a molecule that can diffuse through the BBB. Finally, the microdialysis technique was used to determine the permeability of molecules coming from the perfusion chamber: L-dopa, dopamine, and Glu were employed to assess the selective permeability of the coculture model. Thus, these results indicate that the in vitro model described possesses characteristics similar to those of the BBB in situ and that cocultures of organotypic slices and endothelial cell monolayers have potential as a powerful tool for studying biochemical mechanisms regulating BBB function and drug delivery to the central nervous system.     

Human cytomegalovirus infection in a retinoblastoma cell line in vitro

BACKGROUND: The focus of these studies was to determine whether the Y79 human retinoblastoma cell line could function as a good in vitro model system for studying human cytomegalovirus (HCMV) infection. METHODS: Y79 cells were exposed to an HCMV mutant carrying a LacZ gene, and the resulting beta-galactosidase expression in infected cells was assessed by flow cytometry. The extent to which the three classes of viral gene products immediate early, early, and late proteins - were expressed was analyzed by immunohistochemical staining and Western blotting. Infected Y79 cells were also co-cultivated on human foreskin fibroblast (SF cell) cultures to recover virus. RESULTS: Infection of Y79 cells with the virus resulted in beta-galactosidase expression as detected by flow-cytometric analysis. Immunohistochemical staining revealed that a portion of Y79 cells expressed antigens reactive to monoclonal antibodies against immediate early, early, and late HCMV proteins. The 43-kDa early gene product was also detected by Western blotting. Infected Y79 cells co-cultivated on SF cell cultures yielded infectious foci, which turned blue following X-gal staining, demonstrating productive HCMV infection in the Y79 cells. CONCLUSION: These results demonstrate that while HCMV can productively infect Y79 cultures, it does so in a highly inefficient manner, leading these authors to conclude that this cell line does not provide a particularly good model system to study HCMV infection.     

Low-grade mucoepidermoid carcinoma on the vermilion border of the lip

BACKGROUND AND PURPOSE: CPT-11 (7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin) is anew semisynthesized derivative of camptothecin. SN-38 (7-ethyl-10-hydroxycamptothecin), a metabolite of CPT-11, plays a key role in the action of CPT-11. MATERIALS AND METHODS: To determine whether SN-38 potentiates the cytotoxic effect of radiation, we investigated the interaction of SN-38 and radiation in vitro in monolayer cultures and multicellular spheroids of HT-29 human colon adenocarcinoma cells. RESULTS: HT-29 spheroids were more resistant to both SN-38 and irradiation than monolayer cells. SN-38 at a concentration of 2.5 microg/ml, which by itself was not cytotoxic, greatly increased the lethal effects of radiation in spheroids, but not in monolayer cultures. Exposure to SN-38 following irradiation inhibited the potentially lethal damage repair (PLDR) in spheroids. It is suggested that the mechanism of the radiosensitization by SN-38 is due to the PLDR inhibition. CONCLUSIONS: These results indicate that CPT-11 may play a role as radiosensitizer and that a combination of CPT-11 and irradiation could prove to be a particularly effective strategy with which to treat human colon adenocarcinoma.     

Different HPV16 E6/E7 oncogene expression patterns in epithelia reconstructed from HPV16-immortalized human endocervical cells and genital keratinocytes

Human papillomavirus type 16 (HPV16) E6/E7 oncogenes immortalize two types of human genital epithelial cells in vitro, endocervical cells and ectocervical or foreskin keratinocytes. Epithelia reconstructed in in vivo nude mouse implants or in vitro organotypic raft cultures from immortalized endocervical cells form higher grade dysplasia than those from keratinocytes. Here, we compared viral E6/E7 mRNA expression in immortalized cell lines of the three cell types using implants, rafts and in situ hybridization assays. Endocervical cells expressed E6/E7 throughout their reconstructed epithelia. In contrast, oncogenes were limited to basal cells for keratinocyte lower grade dysplasias. To study the role of the HPV16 promoter/enhancer in this repression in the upper layers of keratinocyte epithelia, new cell lines were established by immortalization with E6/E7 controlled by the SV40 promoter. The oncogenes were shown to be controlled from the SV40 elements after immortalization. Nevertheless, E6/E7 in the two cell types had the same cell-specific expression pattern as that controlled from the homologous HPV16 promoter. In addition, naturally occurring premalignant lesions having integrated HPV16 DNA expressed E6/E7 extensively in the high-grade dysplastic region of undifferentiated metaplasia. On the other hand, oncogene expression was restricted to lower layers in the lower grade dysplastic region of more mature differentiation. Our data suggest that keratinocytes have an inherent HPV16 promoter-nonspecific mechanism of repression. Apparently this mechanism, which can be acquired during maturation, is initially nonfunctional in in vitro and in vivo epithelia derived from metaplastic endocervical cells.     

EGF-receptor tyrosine kinase inhibition induces keratinocyte growth arrest and terminal differentiation

Epidermal keratinocyte growth and differentiation are regulated by specific families of growth factors and receptors. Peptide growth factors of the epidermal growth factor family stimulate proliferation of clonal density human keratinocytes and suppress markers of terminal differentiation in confluent cultures of human keratinocytes. We present evidence that selected inhibitors of activation of the type I human epidermal growth factor receptor (EGFR or HER-1), namely, neutralizing monoclonal antibody to HER-1/EGFR and the specific tyrosine kinase inhibitor PD 153035, potently inhibit proliferation of human keratinocytes in autonomously replicating subconfluent cultures. Coupled to growth arrest is the suppression of HER-1 tyrosine autophosphorylation in inhibitor-treated human keratinocytes. Proliferation and tyrosine autophosphorylation are initially reversible following removal of the inhibitor and restimulation of cells with epidermal growth factor. Sustained inactivation of HER-1 in autonomously replicating cultures of human keratinocytes induces expression of keratin 1 and keratin 10 genes, early markers of terminal differentiation. Reversal of growth inhibition by epidermal growth factor suppresses keratin 1 and keratin 10 expression. These results demonstrate that human keratinocyte terminal differentiation as well as proliferation are mediated by HER-1. Co-expression of autocrine epidermal growth factor-related ligands as well as HER-1 by human keratinocyte may function as part of the signal transduction network in epidermis to regulate cell number, replication rate, and terminal differentiation.