The human OX40/gp34 system directly mediates adhesion of activated T cells to vascular endothelial cells.

Fresh leukemic cells from patients with adult T cell leukemia (ATL) and some ATL-derived T cell lines show adhesion to human umbilical vein endothelial cells (HUVECs) mainly through E-selectin, but a proportion of this binding remains unaffected by the addition of combinations of antibodies against known adhesion molecules. By immunizing mice with one of such cell lines, we established monoclonal antibodies (mAbs), termed 131 and 315, that recognize a single cell surface antigen (Ag) and inhibit the remaining pathway of the adhesion. These mAbs did not react with normal resting peripheral blood mononuclear cells (PBMC) or most of the cell lines tested except for two other human T cell leukemia virus type I (HTLV-I)-infected T cell lines. After stimulation with phytohemagglutinin (PHA), PBMC expressed Ag 131/315 transiently, indicating that these mAbs define a T cell activation Ag. Western blotting and immunoprecipitation revealed that Ag 131/315 has an apparent molecular mass of 50 kD. Expression cloning was done by transient expression in COS-7 cells and immunological selection to isolate a cDNA clone encoding Ag 131/315. Sequence analysis of the cDNA indicated that it is identical to human OX40, a member of the tumor necrosis factor/nerve growth factor receptor family. We then found that gp34, the ligand of OX40, was expressed on HUVECs and other types of vascular endothelial cells. Furthermore, it was shown that the adhesion of CD4+ cells of PHA-stimulated PBMC to unstimulated HUVECs was considerably inhibited by either 131 or 315. Finally, OX40 transfectants of Kit 225, a human interleukin 2-dependent T cell line, were bound specifically to gp34 transfectants of MMCE, a mouse epithelial cell line, and this binding was blocked by either 315 or 5A8, an anti-gp34 mAb. These results indicate that the OX40/gp34 system directly mediates adhesion of activated T cells or OX40+-transformed T cells to vascular endothelial cells.

CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNF alpha.

Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34+ hematopoietic progenitors in presence of GM-CSF+TNF alpha for 12 d. The present study demonstrates that cord blood CD34+ HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CD1a and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CD80, CD83, CD86, CD58, high HLA class II). CD1a+ precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14+ progenitors mature into CD1a+ DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIIIa described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA+ naive T cells. Interestingly, the CD14+ precursors, but not the CD1a+ precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T cells. Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14(+)-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.

Identification and characterization of antimicrobial peptide, defensin, in the taiga tick, Ixodes persulcatus.

Ixodes persulcatus is the primary vector for human tick-borne diseases in Japan. A cDNA library was constructed from whole body homogenates of fed nymphs of I. persulcatus. From this library, one cDNA encoding defensin-like antimicrobial peptide was identified. The amino-acid sequence showed high similarity to those of the defensins of other ticks and arthropods. I. persulcatus defensin mRNA transcripts were detected at all life cycle stages of fed ticks and found to be predominantly expressed in the midguts of adult female ticks, but not in the salivary glands, a finding corroborated by Western blotting analysis. To investigate the function of I. persulcatus defensin, we examined its antibacterial activity by evaluation of growth of several bacterial strains in the presence of the synthetic peptide. The defensin from I. persulcatus markedly inhibited the growth of Gram-positive bacteria including Staphylococcus aureus, Bacillus subtilis and Corynebacterium renale, but not Gram-negative bacteria except Escherichia coli O157. In conclusion, these results suggest that I. persulcatus defensin may be playing a significant role in the defence against microbes from bloodmeals.

Analysis of N-methyl-N-nitrosourea-induced mutations in a shuttle vector plasmid propagated in mouse O6-methylguanine-DNA methyltransferase-deficient cells in comparison with proficient cells.

A shuttle vector plasmid, pYZ289, was constructed from the pZ189 plasmid and polyoma virus DNA. The plasmid contains a supF gene as a marker of mutation and can replicate in both Escherichia coli and mouse cells. The pYZ289 plasmids treated with N-methyl-N-nitrosourea were passed through mouse cells originating from skin tumors, which are either proficient (HL18) or deficient (HL8) in O6-methylguanine-DNA methyltransferase activity, and mutations in the supF gene were analyzed. In the repair-deficient HL8 cells, N-methyl-N-nitrosourea-treated pYZ289 showed lower plasmid survival and higher mutation frequency than in the repair-proficient HL18 cells. DNA sequence analysis in the mutated supF gene revealed that most mutations occurred in G:C base pairs (86% for HL8, 76% for HL18), and the frequency of G:C----A:T transition was higher in HL8 cells (69%) than in HL18 cells (31%). G:C----T:A transversions occurred more frequently in HL18 cells (31%) than in HL8 cells (12%). Mutations occurred frequently at the base pair positions of 123 and 159 of supF gene in HL18 cells and at 169 in HL8 cells. Analysis of the bases neighboring the mutations appeared to be related to the mutability of the base pairs with the sequence of 5'-purine-G-G-3' being the most frequently mutated. These results show that the new pYZ289 plasmid is useful for the analysis of mutations and that a deficiency in O6-methylguanine-DNA methyltransferase enhances the N-methyl-N-nitrosourea-induced mutation with significant specificity.

Establishment and characterization of a tumorigenic murine vascular endothelial cell line (F-2).

A new murine cell line, designated F-2, was established from an ultraviolet light-induced tumor which developed on the back skin of a BALB/c x C57BL/6 F1-nu/nu nude mouse. More than 1 x 10(5) F-2 cells injected into nude mouse skin produced rapidly developing hemangiomatous lesions. F-2 had a 14-h doubling time under 10% fetal calf serum-containing Dulbecco's modified Eagle's medium culture conditions without any cell growth factor supplementation, and it showed "cobblestone" appearance at confluency. F-2 was able to rapidly differentiate on Matrigel with resultant fine network structure and tubule formation. Ultrastructural observations of the tubule demonstrated that the F-2 cells were connected to each other by intermediate junctions and arranged to form spaces, but that no Weibel-Palade bodies were found in the cytoplasm. F-2 also showed the active uptake of fluorescent-labeled acetylated low-density lipoprotein at 37 degrees C but not at 4 degrees C, and it showed prominent binding to the lectin, Griffonia simplicifolia I agglutinin. The addition of fibroblast growth factor did not facilitate the growth of F-2 cells. These findings strongly suggest that F-2 is a transformed cell line with tumorigenicity and vascular endothelial cell properties, and it may be useful in the study of vascular tumor biology.

ADP-ribosylation of the rhoA gene product by botulinum C3 exoenzyme causes Swiss 3T3 cells to accumulate in the G1 phase of the cell cycle.

Using botulinum C3 exoenzyme, which specifically ADP-ribosylates the rho gene products (rho proteins), we examined the role of these proteins in cell cycle progression in Swiss 3T3 cells. Incubation of cell lysates with C3 exoenzyme revealed a single [32P]ADP-ribosylated protein with an M(r) of 23K. This protein was identified as rhoA protein by isoelectric focusing and peptide mapping. When C3 exoenzyme was added to the culture, it ADP-ribosylated the substrate protein in the cells and reduced their growth rate and saturation density. The reduction was dependent on the amount of C3 exoenzyme and on the extent of ADP-ribosylation of the rho protein in the cells. Flow cytometric analysis of logarithmically growing cells showed that the enzyme treatment concentration-dependently accumulated the cells in the G1 phase of the cell cycle. When G1-enriched cells were treated with C3 exoenzyme and cell cycle progression initiated by the addition of serum was monitored, inhibition of G1-S transition was clearly observed. These results suggest that the rhoA gene product plays a critical role in G1-S progression in cultured Swiss 3T3 cells and that the ADP-ribosylation abolishes this activity and causes the cells to accumulate in G1 phase.

Novel mechanism associated with an inherited cardiac arrhythmia: defective protein trafficking by the mutant HERG (G601S) potassium channel.

BACKGROUND: The congenital long-QT syndrome (LQTS) is an inherited disorder characterized by a prolonged cardiac action potential and a QT interval that leads to arrhythmia. Mutations in the human ether-a-go-go-related gene (HERG), which encodes the rapidly activating component of the delayed rectifier current (IKr), cause chromosome 7-linked LQTS (LQT2). Studies of mutant HERG channels in heterologous systems indicate that the mechanisms mediating LQT2 are varied and include mutant subunits that form channels with altered kinetic properties or nonfunctional mutant subunits. We recently reported a novel missense mutation of HERG (G601S) in an LQTS family that we have characterized in the present work. METHODS AND RESULTS: To elucidate the electrophysiological properties of the G601S mutant channels, we expressed these channels in mammalian cells and Xenopus oocytes. The G601S mutant produced less current than wild-type channels but exhibited no change in kinetic properties or dominant-negative suppression when coexpressed with wild-type subunits. To examine the cellular trafficking of mutant HERG channel subunits, enhanced green fluorescent protein tagging and Western blot analyses were performed. These showed deficient protein trafficking of the G601S mutant to the plasma membrane. CONCLUSIONS: Our results from both the Xenopus oocyte and HEK293 cell expression systems and green fluorescent protein tagging and Western blot analyses support the conclusion that the G601S mutant is a hypomorphic mutation, resulting in a reduced current amplitude. Thus, it represents a novel mechanism underlying LQT2.

Conditional expression of anti-apoptotic protein p35 by Cre-mediated DNA recombination in cardiomyocytes from loxP-p35-transgenic mice.

p35, a viral inhibitor of caspase, prevents cell death induced by various stimuli. We established an experimental system to study the involvement of caspases in cell death, using primary cultured cells from p35 transgenic mice in which the p35 open reading frame (ORF) had been disrupted by the insertion of a DNA segment flanked by loxP sites, the Cre recognition sites. In this system, p35 expression can be initiated by Cre recombinase. Cardiomyocytes, which are highly sensitive to hypoxic stress, were infected with an adenovirus carrying the cre gene (AxCANCre). Expression of p35 by infection with AxCANCre resulted in inhibition of caspase-3 activation and resistance to hypoxia-induced cell death. Hypoxia-induced cytochrome c release was also attenuated in p35-expressing cardiomyocytes. Our transgenic mice can be used as an experimental model for studying the involvement of caspases in various degenerative diseases as well as programmed cell death both in vitro and in vivo.

Selective gentamicin uptake by cytochemical subpopulations of guinea-pig geniculate ganglion cells.

Cytochemical subpopulations of geniculate ganglion (GG) cells were identified in guinea-pigs using immunohistochemistry and selective gentamicin accumulation. Two subpopulations of GG cells were evident based upon their location and immunoreactivity for peptide 19 (PEP 19), for plasma membrane Ca2+-ATPase (PMCA-ATPase), and for neurofilament proteins. Cells within the posterior part of GG were positive for PEP 19 and PMCA-ATPase, but not for 68 kD or 160 kD neurofilament proteins. Cells within the anterior part showed complementary staining properties. Cells within these populations showed differences in accumulation of gentamicin, depending upon the administration route. Cells within the posterior part showed avid accumulation of gentamicin when animals received the drug systemically. When the drug was administered directly into the middle ear, cells within the anterior part showed avid gentamicin accumulation. Immunostaining for gentamicin in both cell populations was much more extreme and remained so for longer post-administration times when compared with spiral ganglion and vestibular ganglion cells. The results suggest that cells in the anterior part of GG have little exposure to gentamicin in the serum and that perhaps they innervate the middle ear mucosa or they absorb the drug through their axons within the middle ear. In contrast, cells in the posterior part of GG have greater access to systemically administered gentamicin either directly or via their axon terminals.

Monocyte-derived dendritic cells have a phenotype comparable to that of dermal dendritic cells and display ultrastructural granules distinct from Birbeck granules.

Most monocyte-derived dendritic cells (DC) display CD1a, like Langerhans cells (LC) and some dermal DC, but their relationship with these skin DC remains unclear. To address this issue, we studied the expression of different antigens characteristic of skin DC and of monocyte/macrophages in CD1a+ and CD1a- monocyte-derived DC. Their phenotype indicated that they may be related to dermal DC rather than to LC, i.e., they were all CD11b-positive, and 72% were Factor XIIIa-positive, but they did not express E-cadherin nor VLA-6. It is interesting that CD1a+ and CD1a-cells showed intracytoplasmic granules that were different from LC Birbeck granules. These phenotypical and ultrastructural features are comparable to those of CD14-derived DC obtained from cord blood precursors [C. Caux et al. J. Exp. Med. 184, 695-706]. These results show a close relationship between these two in vitro models, which are both related to dermal DC.

Stress-induced ceramide generation and apoptosis via the phosphorylation and activation of nSMase1 by JNK signaling.

Neutral sphingomyelinase (nSMase) activation in response to environmental stress or inflammatory cytokine stimuli generates the second messenger ceramide, which mediates the stress-induced apoptosis. However, the signaling pathways and activation mechanism underlying this process have yet to be elucidated. Here we show that the phosphorylation of nSMase1 (sphingomyelin phosphodiesterase 2, SMPD2) by c-Jun N-terminal kinase (JNK) signaling stimulates ceramide generation and apoptosis and provide evidence for a signaling mechanism that integrates stress- and cytokine-activated apoptosis in vertebrate cells. An nSMase1 was identified as a JNK substrate, and the phosphorylation site responsible for its effects on stress and cytokine induction was Ser-270. In zebrafish cells, the substitution of Ser-270 for alanine blocked the phosphorylation and activation of nSMase1, whereas the substitution of Ser-270 for negatively charged glutamic acid mimicked the effect of phosphorylation. The JNK inhibitor SP600125 blocked the phosphorylation and activation of nSMase1, which in turn blocked ceramide signaling and apoptosis. A variety of stress conditions, including heat shock, UV exposure, hydrogen peroxide treatment, and anti-Fas antibody stimulation, led to the phosphorylation of nSMase1, activated nSMase1, and induced ceramide generation and apoptosis in zebrafish embryonic ZE and human Jurkat T cells. In addition, the depletion of MAPK8/9 or SMPD2 by RNAi knockdown decreased ceramide generation and stress- and cytokine-induced apoptosis in Jurkat cells. Therefore the phosphorylation of nSMase1 is a pivotal step in JNK signaling, which leads to ceramide generation and apoptosis under stress conditions and in response to cytokine stimulation. nSMase1 has a common central role in ceramide signaling during the stress and cytokine responses and apoptosis.

Expression of four myosin heavy chain genes in developing blood vessels and other smooth muscle organs in rabbits.

At least two smooth muscle myosin heavy chain (MHC) isoforms (SM1, SM2) and two non-muscle MHC isoforms (NMA, NMB) have been detected in smooth muscles. We used the S-1 nuclease mapping procedure to study the expression of these four types of MHC mRNAs in various rabbit blood vessels, such as the aorta (Ao), pulmonary artery (PA), inferior vena cava (IVC) and ductus arteriosus (DA), and in various rabbit tissues and organs. The results demonstrated that in the blood vessels, the four types of MHC mRNA were expressed during all developmental stages in Ao and PA and that SM2 expression appeared to increase dramatically after birth. Compared to the other fetal vessels, the fetal DA contained considerably higher amounts of the four types of MHC mRNAs. SM2 was more prevalent than SM1 in the esophagus, stomach, small intestine and urinary bladder, which are often stretched and show vigorous contractile activity. SM2 expression in a smooth muscle cell appears to correlate well with the contractile ability and/or activity of the cell. Our data show that among the four types of MHC mRNAs, the expression of SM2 MHC mRNA shows great variation among different organs, blood vessel types and stages of development of Ao and PA.

The localization and distribution of gram-positive cocci in normal skin and in lesions of acne vulgaris.

The localization of gram-positive cocci in the normal skin and in the lesions of acne vulgaris was investigated using fluorescein-labeled antiserum raised to gram-positive, coagulase-negative cocci. The cocci were found in 10 of 19 specimens from normal facial skin and in 3 of 11 specimens from the normal skin of the rest of the body. The bacteria were found mostly in the openings of follicles, but in 6 of 10 facial skin specimens, they were also present deeply in the lumina of the dilated sebaceous follicles near the sebaceous glands. Cocci were found in 5 of 6 noninflammatory acne comedones. In inflammatory acne they were demonstrated not only in the follicular canals but also sparsely in the infiltrate surrounding the follicles.

Discrepancy between the localization of in vivo bound immunoglobulins in the skin and in vitro binding sites of circulating anti-BMZ antibodies in bullous pemphigoid: immunoelectron microscopic studies.

The immunoelectron microscopic reaction products showing in vivo bound immunoglobulins or complement in the basement membrane zone (BMZ) of the lesional skin of 12 patients with bullous pemphigoid (BP) were homogeneously distributed in the lamina lucida in 3 cases, just beneath the basilar surface of the basal cells in 5 cases, and were associated with the basal lamina in 4 cases. No reaction products could be found beneath the melanocytes in any of the first 2 groups; however, positive reaction products were found on the basal lamina beneath some melanocytes, and were associated with the detached basal lamina in the dermis in the latter 4 cases. In vitro binding sites of circulating anti-BMZ antibodies in the sera from 7 patients with BP indicated that the antibodies reacted with the basilar surface of the hemidesmosomes, with a weak reaction on the basal lamina in 5 cases and with no reaction in 2 cases. No positive findings were obtained beneath the melanocytes or on the basal lamina under the melanocytes. The discrepancy between the antigenic sites and the localization of immune deposits in BP is assumed to be due to the renewal of the hemidesmosomes and constant clearance of immune deposits in vivo. The in vivo bound immunoglobulins and complement seen in the BP skin are the result of their accumulation in the lamina lucida or in association with the basal lamina.

Immunological studies on Pityrosporum genus and Malassezia furfur.

The antigenicity of Malassezia furfur from patients with tinea versicolor and 3 species of Pityrosporum was investigated using the antiserum against P. orbiculare. The Ouchterlony gel diffusion test revealed a considerable similarity between the antigenicities of P. orbiculare and P. ovale, and little similarity between P. orbiculare andP. canis. Similar results were obtained by immunofluorescence staining with the FITC-labeled P. orbiculare antiserum. Hyphae and round spores of M. furfur in the scales and biopsy specimens from the lesions in patients with tinea versicolor revealed specific fluorescence much the same as seen in cases of P. orbiculare or P. ovale. The FITC-labeled antiserum absorbed with P. orbiculare or P. ovale failed to give a positive reaction with hyphae and round spores of M. furfur. These findings suggest a similar antigenicity among P. orbiculare, P. ovale and M. furfur.

Experimental production of rabbit anti-guinea-pig epidermal cell sera. Comparison to pemphigus antibodies.

Anti-epidermal cell sera (AES) were obtained by immunizing rabbits with enzymatically dispersed, viable guinea-pig epidermal cells followed by absorption with guinea-pig red blood cells, spleen and thymus cells, and liver powders. Complement-mediated cytotoxicity and immunofluorescence demonstrated that AES were towards cell surface antigen specific for stratified squamous epithelia of guinea pigs, and cross reacted with the corresponding tissues of humans and monkeys. Immunofluorescence revealed that AES reacted with Hassall's corpuscles and surrounding epithelial cells of the guinea-pig thympus which seemed to share common antigens with the epidermis; AES gave no reaction with other organs. While antigens reactive with pemphigus antibodies (PA) were demonstrated by membrane immunofluorescence to be present on the epidermal cell surface, PA showed no cytotoxicity to guinea-pig and human epidermal cells. Re-treatment of isolated epidermal cells with trypsin showed that antigens ractive with PA were more susceptible to the enzyme than those reactive with AES. These findings suggest that the cell surface antigens binding AES are different from the antigens which bind PA.

Direct effects of cutaneous neuropeptides on adenylyl cyclase activity and proliferation in a keratinocyte cell line: stimulation of cyclic AMP formation by CGRP and VIP/PHM, and inhibition by NPY through G protein-coupled receptors.

Many neuropeptides are present in the peripheral nerves of human skin and are distributed from the intraepidermis to subcutaneous appendages, and those peptides are considered to be involved in the pathogenesis of various inflammatory dermatoses. In this investigation, we determined the effects of various neuropeptides on intracellular cyclic adenosine-5'-monophosphate (AMP) formation in cultured human keratinocytes. Among the many peptides tested, calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP), peptide histidine-methionine (PHM), and growth hormone releasing factor (GRF) stimulated a rapid and marked formation of intracellular cyclic AMP in keratinocytes in a dose-dependent manner. The direct association of the receptors for CGRP and VIP with adenylyl cyclase in keratinocytes was confirmed by the findings that CGRP and VIP stimulated the enzyme activity in membrane preparations derived from cultured keratinocytes in the presence of guanosine triphosphate (GTP). On the other hand, neuropeptide Y (NPY) showed an inhibitory effect on forskolin-induced cyclic AMP accumulation in keratinocytes. This inhibitory effect of NPY was completely eliminated by glucocorticoid pretreatment of cultured keratinocytes. Furthermore, the presence of peptides that substantially increase intracellular cyclic AMP accumulation also stimulated DNA synthesis and proliferation in a human keratinocyte cell line in a dose-dependent manner. These results suggest that neuropeptides work directly as biologic modulators of keratinocytes through the cyclic AMP cascade.

Decreased skin superoxide dismutase activity by a single exposure of ultraviolet radiation is reduced by liposomal superoxide dismutase pretreatment.

The effect of a single exposure to UV radiation on skin superoxide dismutase (SOD) activity was examined in mice. A significant decrease in SOD activity was observed 24 and 48 h after UV irradiation, returning to the normal level by 72 h after irradiation. Decreased SOD activity after UV exposure was reduced by pretreatment with liposomal SOD (L-SOD). This protective effect of L-SOD may have potential clinical application for photodermatologic reactions.