The effects of oxidized low density lipoproteins on inducible mouse macrophage gene expression are gene and stimulus dependent.

Oxidized LDL has been previously reported to suppress the expression of genes induced in mononuclear phagocytes by inflammatory stimuli. In this study we extend these findings to demonstrate that the suppressive effects of oxidized LDL vary depending upon the gene being monitored and the stimulus being used to induce or enhance its expression. The expression of a selection of LPS-inducible genes exhibited differential sensitivity to pretreatment with oxidized LDL. Furthermore, the ability of oxidized LDL to suppress gene expression varied markedly with the inducing stimulus used. TNF alpha and IP-10 mRNA expression induced by IFN gamma and IL-2 was markedly more sensitive to suppression by oxidized LDL than that induced by LPS. The cooperative effects of IFN gamma and LPS on the expression of the inducible nitric oxide synthase gene were suppressed by oxidized LDL while the antagonistic effect of IFN gamma on LPS-induced expression of the TNF receptor type II mRNA was not altered. The suppressive activity of LDL was acquired only after extensive oxidation and was localized in the extractable lipid component. These results suggest a potent and direct connection between the oxidative modification of LDL and the chronic inflammation seen in atherogenic lesions. Furthermore, the appreciable selectivity of oxidized LDL in mediating secondary control of cytokine gene expression demonstrates that the active material(s) is targeted to disrupt specific intracellular signaling pathways.

A chemokine-to-cytokine-to-chemokine cascade critical in antiviral defense.

Macrophage inflammatory protein 1alpha (MIP-1alpha) promotes natural killer (NK) cell inflammation in livers during murine cytomegalovirus (MCMV) infections, and NK cell-produced interferon gamma (IFN-gamma) contributes to defense against MCMV infections. A specific role for local NK cell IFN-gamma production, however, has not been established. The importance of MIP-1alpha and NK cell-produced IFN-gamma in shaping endogenous immune responses and defense in different compartments was examined. MIP-1alpha deficiency profoundly decreased resistance to MCMV and was associated with dramatically reduced NK cell accumulation and IFN-gamma production in liver. MIP-1alpha-independent IFN-gamma responses were observed in serum and spleen, and infection-induced elevations in blood NK cell populations occurred in absence of the factor, but peak liver expression of another chemokine, the monokine induced by IFN-gamma (Mig), depended upon presence of MIP-1alpha, NK cells, and IFN-gamma. The Mig response was also important for viral resistance. Thus, serum cytokine responses are insufficient; MIP-1alpha is critical for NK cell migration and IFN-gamma delivery to mediate protection; and Mig induction in tissues is a downstream protective response resulting from the process. These results define a critical chemokine-to-cytokine-to-chemokine cascade required for defense during a viral infection establishing itself in tissues.

Combination therapy with adriamycin and interleukin 2 augments immunity against murine renal cell carcinoma.

The mechanism(s) through which combination treatment with Adriamycin and recombinant interleukin 2 (rIL2) affects murine renal cell carcinoma was investigated. A single dose of Adriamycin (ADM) administered 1 day after implantation of tumor cells s.c. delayed the formation of tumor and significantly extended the median survival time. About 40% of the treated mice remained tumor free for more than 6 months. Treating mice with a similar dose of ADM 12 to 16 days after implantation of tumor caused complete regression of established tumors within 10 to 12 days in more than 90% of the mice. However, after a transient tumor free period of 15 to 20 days recurrence of tumor was observed in all treated mice. In contrast, a regimen that included treating tumor bearing mice with a single dose of ADM followed by a daily i.p. injection of rIL2 5 x 10(4) units for 10 days delayed the recurrence of tumors and significantly prolonged the survival time compared to the median survival time of mice treated with ADM alone. About 20 to 30% tumor bearing mice remained tumor free for 4 months following treatment with ADM and rIL2. Treatment with rIL2 alone produced no antitumor response. In addition, rIL2 itself was not inhibitory for tumor cell growth nor did it modulate the cytotoxic response of renal cell carcinoma cells to ADM in vitro. Mice that were cured following treatment with ADM and rIL2 were resistant to a rechallenge with viable tumor cells, and cured mice also expressed a tumor specific T-cell mediated delayed hypersensitivity reaction. The immune cells that mediate tumor rejection were identified as Thy-1.2+ T-cells. Taken together, these results indicate that antitumor activity of combined Adriamycin/rIL2 treatment is at least partly attributable to the production of tumor specific immunity.

T-cells infiltrating renal cell carcinoma display a poor proliferative response even though they can produce interleukin 2 and express interleukin 2 receptors.

The fact that progressing tumors contain a significant infiltrate of T-cells brings into question the competency of the infiltrating T-lymphocytes (T-TIL). We have examined the role of the T-cell receptor/CD3 complex and/or the interleukin 2 receptor (IL2R) in responsiveness of T-cells that infiltrate human renal cell carcinoma. T-TIL display a poor proliferative response to interleukin 2 (IL2) alone, IL2 in combination with antibody to CD3, or mitogen stimulation. The proliferative unresponsiveness was not related to low expression of CD3 or IL2R beta as the percentage of T-cells expressing CD3 and IL2R beta were comparable in both T-TIL and peripheral blood T-cells obtained from the same patient. In contrast to the lack of proliferative activity, stimulation of T-TIL or peripheral blood lymphocytes with phytohemagglutinin or anti-CD3 resulted in comparable levels of both IL2 and gamma-interferon mRNA and protein expression. While levels of IL2R alpha were low in unstimulated T-TIL and peripheral blood lymphocytes, anti-CD3 antibody or IL2 were capable of inducing surface expression of this protein in both cell populations. IL2R alpha mRNA levels were comparable in T-cells from the tumor and peripheral blood although in some experiments both the percentage of IL2R alpha-positive cells and the density of surface expression per cell were reduced in T-TIL. This reduced IL2R alpha expression on T-TIL was not responsible for the proliferative unresponsiveness since T-TIL that expressed both IL2R alpha and/or IL2R beta still failed to respond to high doses of IL2. Thus T-TIL display a selective loss of response to at least two well defined extracellular stimuli. While T-TIL exhibit a poor proliferative response regardless of the form of stimulation these cells remain sensitive to both anti-CD3 and IL2 in terms of IL2 and gamma-interferon or IL2R alpha expression, respectively. The fact that proliferative unresponsiveness exists even though T-TIL can produce IL2 and express IL2R alpha/beta suggests that T-TIL have a selective loss of a common intracellular signaling pathway which is requisite to proliferation but not other aspects of response to antigenic stimulation.

T cells from renal cell carcinoma patients exhibit an abnormal pattern of kappa B-specific DNA-binding activity: a preliminary report.

Recent data suggest that the poor induction of a T-cell response to human renal cell carcinoma (RCC) may be related to alterations in signal transduction pathways. We report that T cells from RCC patients have two alterations in kappa B motif-specific DNA-binding activity. The first alteration involves the constitutive expression of substantial kappa B-binding activity in nuclear extracts, which was observed in the electrophoretic mobility shift assay. The magnitude of kappa B activity in unstimulated patient T cells was similar to that observed in T cells from normal individuals that had been activated in vitro. On the basis of Western blotting experiments using antibodies to kappa B/Rel family proteins, the kappa B-binding activity constitutively expressed in T cells from RCC patients is composed mostly of the NF-kappa B1 (p50) subunit. The second abnormality in kappa B-binding activity in T cells from these patients is that RelA, a member of the Rel homology family which is part of the normal NF-kappa B complex, was not induced in the nucleus following activation. Western blotting analysis did not detect any RelA in nuclear extracts either before or after stimulation of T cells. The altered kappa B-binding activity in T cells from RCC patients may impair their capacity to respond normally to various stimuli.

Biosynthesis of mammalian transfer RNA. Evidence for regulation by deacylated transfer RNA.

The rate of tRNA synthesis in cultured Friend leukemia cells has been examined as a function of the variation in polyribosome structure produced by treatment with a variety of inhibitors of protein synthesis. The results indicate, in contrast to the conclusions of Bölcsföldi (Bölcsföldi, G. (1974) Exp. Cell Res., 88, 231--240), that no necessary relationship exists between the ribosome distribution and the rate of tRNA synthesis. Alternatively, it is observed that inhibitors of tRNA aminoacylation cause, in all cases, a decrease in the rate of tRNA synthesis whereas drugs which may stimulate the aminoacylation of tRNA cause, in all cases, an elevation of the rate of tRNA synthesis. It is concluded that tRNA synthesis in mammalian cells may be regulated by the relative levels of acylated and deacylated tRNA.

Thrombin-induced expression of the KC gene in cultured aortic endothelial cells. Involvement of proteolytic activity and protein kinase C.

The KC gene, first identified in platelet-derived growth factor-stimulated BALB/c 3T3 cells, shares structural similarities with a new family of genes that code for secreted cytokines which appear to be involved in wound healing and inflammation. Thrombin is a coagulation system proteinase likely to be present in vivo at sites of tissue injury. This enzyme is known to stimulate multiple responses in cultured endothelial cells (EC), including the production of eicosanoids, the expression of growth factor genes and the adhesion of leukocytes. The present experiments were designed to examine the effect of thrombin on KC mRNA expression in EC and to explore the molecular mechanisms involved. Thrombin caused a marked concentration-dependent increase in the steady state level of KC mRNA in confluent porcine aortic EC. The level of KC mRNA reached a peak 2 h after thrombin treatment and returned to near control levels by 8 h. Thrombin that was pretreated with phenylmethylsulfonyl fluoride (PMSF) to block proteolytic activity did not stimulate KC gene expression. Trypsin (2 micrograms/ml) but not PSMF-trypsin also caused a substantial increase in the level of KC mRNA. We postulated a role for protein kinase C in thrombin-induced KC gene expression since previous work had demonstrated a similar EC response to phorbol esters. This hypothesis was further supported by the finding that thrombin-induced KC expression was suppressed by the C kinase inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, but not by its structural analogue. The results of the present study demonstrate that thrombin augments KC mRNA expression by vascular EC in a process that requires intact proteinase activity. The activation of protein kinase C may be a necessary component of the intracellular signalling pathway involved in this response.

Regulation of macrophage gene expression by T-cell-derived lymphokines.

Cytokines secreted from antigen-specific T lymphocytes provide important positive and negative control of inflammation through their effects on non-antigen-specific inflammatory leukocytes. These effects often involve modulation of gene expression. Lymphokine-inducible macrophage gene expression is largely controlled at the level of transcription. Multiple cis-acting sequence motifs cooperate with one another to produce patterns of expression that are relatively unique to individual genes. Members of trans-acting transcription factor families, which recognize related regulatory sequence elements, participate frequently in complex protein-protein interactions that generate remarkable complexity in terms of the number of potential combinations and the consequential functional differences exhibited by each combination. Thus, the remarkable plasticity of immune-mediated inflammation derives from combinations of finite numbers of options at several points in the cellular and molecular sequence.

Interferon-gamma and CXC chemokine induction by interleukin 12 in renal cell carcinoma.

Interleukin 12 (IL-12) is known to play an important role in the development of an antitumor response. Its activity has been shown to be dependent upon the intermediate production of IFN-gamma and the influx into the tumor of CD8 lymphocytes. In a murine model, tumor regression induced by IL-12 treatment correlated with IFN-gamma, IP-10, and Mig expression in the tumor bed and was abrogated by antibodies to both chemokines. Here we examined the effects of rHuIL-12 on IFN-gamma and CXC chemokine gene expression in patients with renal cell carcinoma (RCC) in an attempt to determine whether a similar series of molecular events leading to IL-12-mediated tumor regression in mice is also detectable in humans. As in the murine RENCA model, cultured RCC cells themselves could be induced by IFN-gamma to synthesize IP-10 and Mig mRNA. Explanted RCC produced IFN-gamma and IP-10 mRNA in response to IL-12 treatment, which was consistent with the finding that biopsied RCC tumors from IL-12-treated patients also variably expressed augmented levels of those molecules after therapy. Although Mig mRNA was present in the majority of biopsied tumors prior to treatment, both the Mig and IP-10 chemokines as well as IFN-gamma were induced in the peripheral blood mononuclear cells of IL-12-treated patients. Skin biopsies of IL-12-treated patients also all synthesized IP-10 mRNA. This study demonstrates that recombinant human IL-12 therapy of patients with RCC has the potential to induce the expression of gene products within the tumor bed that may contribute to the development of a successful antitumor response.

Oxidized LDL potentiates LPS-induced transcription of the chemokine KC gene.

Oxidized low-density lipoprotein (LDL) has been shown to modulate the expression of multiple gene products associated with inflammation in several different cell types including mononuclear phagocytes. The reported effects vary dramatically, however, depending upon cell type, stimulus, and degree of LDL oxidation. In the present report, oxidized LDL has been found to markedly potentiate expression of the KC chemokine gene in lipopolysaccharide (LPS)-stimulated macrophages. Pretreatment of elicited mouse peritoneal macrophages with oxidized LDL but not native LDL produced a significant enhancement of LPS-induced KC mRNA expression, whereas levels of IP-10 mRNA, another CXC chemokine, were altered in opposite fashion. The alteration in KC mRNA expression was dependent upon the dose, exposure time, and extent of LDL oxidation. Oxidized LDL selectively prolonged the expression of KC mRNA. Surprisingly this was not a consequence of altered mRNA stability, but rather of prolonged transcription. These effects on KC gene transcription were in marked contrast to previous reports demonstrating inhibitory effects of oxidized LDL on LPS-induced macrophage chemokine expression. Thus extensively oxidized LDL acts on the transcriptional control process in macrophages in both positive and negative fashion on separate members of the same gene family.

Requirement for STAT1 in LPS-induced gene expression in macrophages.

This study examines the role of the signal transducer and activator of transcription 1 (STAT1) in induction of lipopolysaccharide (LPS)-stimulated gene expression both in vitro and in vivo. LPS-induced expression of an interferon (IFN)-inducible 10-kDa protein (IP-10), IFN regulatory factor-1 (IRF-1), and inducible nitric oxide synthase (iNOS) mRNAs was severely impaired in macrophages prepared from Stat1-/- mice, whereas levels of tumor necrosis factor alpha and KC (a C-X-C chemokine) mRNA in LPS-treated cell cultures were unaffected. A similar deficiency in LPS-induced gene expression was observed in livers and spleens from Stat1-/- mice. The reduced LPS-stimulated gene expression seen in Stat1-/- macrophages was not the result of reduced activation of nuclear factor kappaB. LPS stimulated the delayed activation of both IFN-stimulated response element and IFN-gamma-activated sequence binding activity in macrophages from wild-type mice. Activation of these STAT1-containing transcription factors was mediated by the intermediate induction of type I IFNs, since the LPS-induced IP-10, IRF-1, and iNOS mRNA expression was markedly reduced in macrophages from IFN-alpha/betaR-/- mice and blocked by cotreatment with antibodies against type I IFN. These results indicate that indirect activation of STAT1 by LPS-induced type I IFN participates in promoting optimal expression of LPS-inducible genes, and they suggest that STAT1 may play a critical role in innate immunity against gram-negative bacterial infection.

A lipopolysaccharide-inducible macrophage gene (D3) is a new member of an interferon-inducible gene cluster and is selectively expressed in mononuclear phagocytes.

We previously reported the isolation and characterization of cDNA clones encoding novel lipopolysaccharide (LPS)-inducible mRNAs from murine peritoneal macrophages. We now present the complete coding sequence of a cDNA previously termed D3. Analysis of multiple clones from a murine macrophage cDNA library provided a complete cDNA sequence of approximately 1.6 kb. The corresponding RNA contains a single open reading frame encoding a hydrophilic protein composed of 425 amino acids and is characterized by a region including three perfect and two imperfect repeats of a seven-amino-acid sequence. Based on nucleotide and deduced amino acid sequence, this mRNA is a new member of a previously described multigene cluster of interferon-inducible genes termed the Mouse 200 series genes. This new sequence most closely resembles gene 204 because both D3 and 204 genes have segments containing the seven-amino-acid repeat sequence. The Mouse 202 and 204 genes, however, have an approximately 200-amino-acid carboxyl-terminal domain that is absent in the LPS-inducible macrophage-derived cDNA. In addition, D3, 202, and 204 can all be distinguished from one another by virtue of unique 3' noncoding regions 200-300 base pairs in length. The D3 unique sequence is largely restricted to the smallest of the three size classes of this gene family expressed in macrophages and is not detected in interferon- or platelet-derived growth factor-stimulated fibroblasts. Overall, three separate mRNAs have now been described, each of which has three or more of a possible seven nucleotide sequence domains. Although the function(s) of the members of this gene family remains unknown, the multiple forms inducible by diverse stimuli and their restricted cell type expression suggest diverse and important physiologic roles for their products in inflammation.

Oxidized LDL modulates activation of NFkappaB in mononuclear phagocytes by altering the degradation if IkappaBs.

Oxidized low density lipoprotein (oxLDL) is known to alter the expression of inflammatory gene products in mononuclear phagocytes. The mechanisms involved in this effect were studied by examining the activation of nuclear factor kappaB (NFkappaB), a transcription factor known to be important in controlling the expression of such genes. Pretreatment of peritoneal macrophages with oxLDL modulated the activation of NFkappaB in response to either lipopolysaccharide (LPS) or the combination of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). In macrophages pretreated with oxLDL the nuclear translocation of Rel family members (RelA and c-Rel) is delayed (LPS) or markedly diminished (IFN-gamma/IL-2) and results in delayed or reduced appearance of kappaB binding activity within the nucleus. These changes in NFkappaB activation result from alterations in the stimulus-dependent degradation of IkappaB alpha and IkappaB beta. The effects of oxLDL on NFkappaB activation depend both on the degree of LDL oxidation (most potent with extensive oxidation) and on the time of exposure of the cells to the lipoprotein preparation (a minimal exposure of 6 h is required before inhibitory effects are observed). The modulation of IkappaB/NFkappaB function in cells exposed to oxLDL appears to be responsible for previously reported effects of oxLDL on chemoattractant cytokine gene expression where both inhibition and delay of such stimulus-dependent events has been observed.

IL-10 suppresses LPS-induced KC mRNA expression via a translation-dependent decrease in mRNA stability.

This study examines the mechanism of interleukin-10 (IL-10)-mediated suppression of KC chemokine gene expression in mouse macrophages. Suppression of KC mRNA levels by IL-10 occurred late in the time course of response to lipopolysaccharide (LPS). Equivalent IL-10-mediated suppression was observed when the agent was added 1 h before, simultaneous with, or 1 h after LPS. IL-10 did not inhibit KC gene transcription but rather produced a decrease in the stability of KC mRNA. The suppressive action of IL-10 was prevented in macrophages that were also treated with inhibitors of protein synthesis even when added 2 h after LPS and IL-10. These results suggest that IL-10 acts to destabilize LPS-induced KC mRNA through a process that depends on coincident KC mRNA translation.

Chemokine expression in trinitrochlorobenzene-mediated contact hypersensitivity.

The expression of the murine IP-10 and MCP-1 genes has been examined in the skin of mice during contact hypersensitivity reactions to the hapten trinitrochlorobenezene (TNCB). In both naive and passively sensitized animals, challenge with TNCB resulted in elevated expression of both genes as early as 4 h as detected by Northern hybridization analysis. Twenty-four hours after challenge, expression was markedly reduced in naive animals but remained elevated in sensitized animals. This prolonged expression of chemokine gene products correlates with the tissue swelling response generally used as a measure of delayed-type hypersensitivity (DTH) in this model and suggests that the continued expression of these genes results from the stimulation of hapten-specific T helper cells. Examination of cell type expression patterns by in situ hybridization using 3H-radiolabeled riboprobes confirmed the results of Northern hybridization experiments. Both genes were expressed predominantly in cells exhibiting the morphology of connective tissue fibroblasts, although the distribution of cells positive for IP-10 mRNA expression differed from that of cells expressing MCP-1 mRNA. IP-10 expression was localized almost exclusively to a population of connective tissue cells surrounding the fur follicle. MCP-1 expression was rarely found associated with fur follicles but instead was distributed throughout the dermis in cells embedded in the collagenous extracellular matrix. Surprisingly, neither endothelial cells lining the small vessels located deep within the dermis nor epidermal keratinocytes were positive under any of the conditions utilized in the present study. Expression of both IP-10 and MCP-1 has been previously reported in a variety of distinct cell types in vitro. The present results indicate that only a subset of the cell types with such potential are stimulated to express these chemokine genes in vivo during hapten-mediated DTH responses, implying the presence of subtle cell type- and tissue-specific control mechanisms.

Tissue-specific expression of murine IP-10 mRNA following systemic treatment with interferon gamma.

We have examined the tissue distribution of 10-kd inflammatory protein (IP-10) mRNA expression in C57Bl/6 mice injected intravenously (i.v.) with various inflammatory stimuli. IP-10 mRNA was strongly induced by interferon-gamma (IFN-gamma) in liver and kidney but only poorly in skin, heart, and lung. IFN-gamma had nearly equivalent access to these tissues as indicated by the distribution of radiolabeled recombinant IFN-gamma 1 h after injection. The time course of IP-10 mRNA appearance was rapid and transient in both liver and kidney; maximal expression in the liver (2 h) preceded that in the kidney (3 h) and declined rapidly thereafter in both tissues. Expression of IP-10 mRNA in the liver and kidney was highly sensitive to IFN-gamma treatment; nearly maximal stimulation occurred with injection of 500 U of IFN-gamma per mouse. Comparable stimulation of IP-10 mRNA expression in splenic macrophages required 10,000 U of IFN-gamma administered i.v., indicating that liver and kidney responses are 10- to 20-fold more sensitive. IP-10 mRNA expression in both tissues was not restricted to stimulation by IFN-gamma but was also seen with injection of lipopolysaccharide (LPS) (25 micrograms/mouse) or IFN-beta (100,000 U/mouse). Two other members of the IP-10 gene family, KC (gro) and JE (MCP-1), were expressed at lower levels under similar treatment conditions. Analysis of IP-10 mRNA distribution in the liver and kidney by in situ hybridization indicated that expression in both tissues was most prominent in the reticuloendothelial cell system, particularly in the endothelial lining of the microvascular circulation. Although the function of the IP-10 gene product has not been defined, these results suggest that it may play an important role in the response of both the liver and kidney to systemic inflammation.

Comparison of minimal phototoxic dose and skin type for determining initial UVA dose in oral liquid methoxsalen photochemotherapy for the treatment of psoriasis.

Twenty-five patients with extensive psoriasis were randomly assigned into one of three groups, each receiving 0.5 mg/kg of oral liquid methoxsalen photochemotherapy followed 1 h later by exposure to long-wave ultraviolet light (UVA). The sole difference between the three groups was the method used to determine the initial UVA dose, which was either based on skin type, 25% of the minimal phototoxic dose (MPD), or 50% of the MPD. All patients were treated in the Phototherapy Unit at the Massachusetts General Hospital. Data were obtained until reaching the endpoint of clearance. At clearance, the results of the number of treatments required to clear, final UVA dose, cumulative UVA dose, and side effects were tabulated, compared, and analyzed for each of the three groups. The 25% and 50% MPD groups required a mean of 15.0 +/- 1.7 and 13.4 +/- 1.9 treatments to clear, respectively, as compared to the skin type group, which needed an average of 17.6 +/- 2.5 treatments. The mean final UVA dose was 7.4 +/- 0.9 J/cm2 and 8.4 +/- 1.4 J/cm2 for the 25% and 50% MPD groups, respectively, in contrast to 11.6 +/- 1.4 J/cm2 for the skin type group. The mean cumulative UVA dose at clearance for the 25% and 50% MPD groups was 79 +/- 16 J/cm2 and 87 +/- 27 J/cm2, respectively, versus 136 +/- 30 J/cm2 for the skin type group. The comparisons between the individual MPD groups and the skin type group did not achieve statistical significance with the exception of a marginally significant difference in final dose between the skin type group and the 25% MPD group (p = 0.06). However, the results of the two MPD groups were then pooled and the mean final (7.9 +/- 0.8 J/cm2) and cumulative (83 +/- 15 J/cm2) UVA doses were significantly (p = 0.04) and marginally significantly (p = 0.07) lower than the respective means of the skin type group. The number of treatments to clear, although lower in the pooled MPD groups (14.2 +/- 1.3) than in the skin type group, did not attain statistical significance (p = 0.19). Our data suggest that the MPD measurement may be superior to the skin-typing system when calculating the initial UVA dose in oral liquid methoxsalen photochemotherapy for the treatment of psoriasis.

Reduced type I and type III procollagens in photodamaged adult human skin.

We have quantitatively assessed the relation between type I and type III procollagen precursor levels and the severity of clinical photodamage in human skin. Levels of procollagen, pN collagen (collagen without the carbroxypropeptide), and/or pC collagen (collagen without the aminopropeptide) were determined by radioimmunoassay, Western blot, and immunohistology in punch biopsy specimens from mildly and severely photodamaged forearm skin and from sunprotected underarm and buttock skin of the same subjects. Collagen precursor levels in forearm and underarm skin were expressed relative to buttock levels for comparison. In the mildly photodamaged group, collagen precursors in the forearm did not differ from those in the underarm by any measurement, except for type I collagen precursors measured by radioimmunoassay, which were reduced 16%. In severely photodamaged forearm skin, both type I and type III collagen precursor levels, measured by radioimmunoassay, were significantly reduced (approximately 40%). Western analysis revealed similar significant reductions in type I and type III collagen precursor levels in severely photodamaged forearm skin compared with the sun-protected underarm. Immunohistology localized both type I and III pN collagens predominantly to the extracellular papillary dermis. Relative staining intensities of type I and type III pN collagen were also significantly reduced in severely photodamaged forearm skin. Multiple linear regression modeling of all data demonstrated that reductions in collagen precursor levels were significantly correlated (p < 0.03) with the severity of photodamage, but not with chronologic age. These data demonstrate, by three independent methods, coordinate reductions of both type I and type III collagen precursors in photodamaged human skin, and the degree of reduction correlated with the degree of photodamage. It is likely that such changes in collagen precursors lead to reduced levels and/or altered organization of fibrillar collagen, and thus may contribute to the wrinkled appearance of photodamaged human skin.

The erythromycin breath test as a predictor of cyclosporine blood levels.

The daily dose of cyclosporine required to attain a desired blood level can vary greatly among patients. Because elimination of cyclosporine depends on its metabolism in the liver by an enzyme (cytochrome P-450IIIA) that also demethylates erythromycin, we reasoned that the ability of patients to demethylate a test dose of erythromycin might be useful in estimating their appropriate daily doses of cyclosporine. Accordingly, the [14C-N-methyl] erythromycin breath test was administered to 32 patients before they received 3.0, 5.0, or 7.5 mg/kg/day cyclosporine to treat psoriasis. We found that a simple mathematical equation incorporating just the 14CO2 production, the age of the patient, and the daily dose of cyclosporine accounted for almost 80% (R2 = 0.78) of the interpatient variability in cyclosporine blood levels we observed. Our data indicate that P-450IIIA activity largely accounts for the relationship between dose of cyclosporine and blood levels for an individual patient. We conclude that the erythromycin breath test may be a convenient guide for cyclosporine dosing.

Immunohistochemical studies of basement membrane proteins and proliferation and apoptosis markers in sulfur mustard induced cutaneous lesions in weanling pigs.

Sulfur mustard (2,2-dichlorodiethyl sulfide, HD) is a chemical warfare agent that is a threat to both troops and civilians. The focus of HD research has been on intracellular adduct formation leading to apoptosis and/or necrosis in cutaneous lesions. However, there is work which suggests that HD may have a more direct effect on the basement membrane zone. Immunohistochemical staining to desmosomal proteins, cellular fibronectin, laminin 1, laminin 5, collagen IV, collagen VII, p53, Bcl-2, and PCNA was performed on weanling pig skin exposed to vesicating doses of HD, GB3, an antibody to laminin 5, showed a progressive decrease with loss of expression during the time period of clinical vesiculation. The other basement membrane proteins showed no change or inconsistent changes. PCNA, and p53 staining increased in the overlying epidermis in areas of vesiculation without significant necrosis. Bcl-2 positive cells were decreased or absent after exposure. This study implicates laminin 5 as the main basement membrane protein affected acutely by HD exposure. The patterns of staining of PCNA, Bcl-2, and p53 within the epidermis suggest that apoptosis and cellular necrosis both may play a role in cell death secondary to HD.