Drug-induced suppression of phosphorylase kinase activity correlates with resolution of psoriasis as assessed by clinical, histological and immunohistochemical parameters.

BACKGROUND: Phosphorylase kinase (PhK), also known as adenosine triphosphate (ATP)-phosphorylase b phosphotransferase, integrates multiple calcium/calmodulin-dependent signalling pathways, including those involved in cell migration and cell proliferation, while coupling these pathways to glycogenolysis and ATP-dependent phosphorylation, thus ensuring continuing energy supply for these activities. OBJECTIVES: Our laboratory recently reported correlation of elevated PhK activity with psoriatic activity. This study further evaluates the significance of drug-induced suppression of PhK activity on psoriatic activity. PATIENTS AND METHODS: PhK activity was assayed in four groups, each with 10 patients: (i) active untreated psoriasis; (ii) resolving psoriasis treated by calcipotriol (Dovonex(R), Bristol Myers Squibb, Princeton, NJ, U.S.A. ), a vitamin D3 analogue and an indirect inhibitor of PhK; (iii) curcumin (diferuloylmethane), a selective PhK inhibitor; and (iv) 10 normal non-psoriatic subjects. RESULTS: PhK activity in units mg-1 protein was highest in active untreated psoriasis (1204 +/- 804.3; mean +/- SD), lower in the calcipotriol-treated group (550.7 +/- 192. 9), lower in curcumin-treated group (207.2 +/- 97.6), and lowest in normal skin (105.4 +/- 44.6). One-way analysis of variance performed on log-transformed PhK activity measure showed significant differences among the four groups, F3,36 = 48.79, P < 0.0001. Decreased PhK activity in curcumin-and calcipotriol-treated psoriasis was associated with corresponding decreases in keratinocyte transferrin receptor (TRR) expression, severity of parakeratosis and density of epidermal CD8+ T cells. CONCLUSIONS: Our results demonstrate that drug-induced suppression of PhK activity is associated with resolution of psoriatic activity as assessed by clinical, histological and immunohistochemical criteria, and support the hypothesis that effective antipsoriatic activity may be achieved through modulation of PhK activity.

Involvement of luminal bacteria, heat shock protein 60, macrophages and gammadelta T cells in dextran sulfate sodium-induced colitis in rats.

The in vivo immunological events in dextran sulfate sodium (DSS) -induced colitis were evaluated. Rats were fed water (control) or 5% DSS. Colonic sections were assessed by light microscopy, Gram stain, immunohistochemistry, and electron microscopy. A progressive decline in number and increase in fragmentation of bacteria in the colonic lumen was observed over time. Luminal bacteria were the first to show heat shock protein 60 (HSP60) staining (day 3). Macrophages in close proximity to these bacteria were next to show such staining (day 6), and finally the damaged epithelial cells when colitis became severe (day 15). Ultrastructural assessment showed cell-cell contact interactions between macrophages and dendritic gammadelta T cells. An increase in the number of gammadelta T cells and ED1-positive macrophages in the affected colonic tissue over time was documented. These results suggest colonic bacteria, host macrophages, and gammadelta T cells play specific roles in the immunological reactions in DSS-induced colitis, possibly via an HSP60-mediated mechanism.

Enhanced healing and cost-effectiveness of low-pressure oxygen therapy in healing necrotic wounds: a feasibility study of technology transfer.

Recent advances in topical hyperbaric oxygen technology identified the use of low-pressure topical hyperbaric oxygen therapy in enhancing wound healing. This study prospectively examined the feasibility of technology transfer from university to Health Maintenance Organization personnel, using topical hyperbaric oxygen therapy to heal necrotic wounds. Fifteen patients with 24 gangrenous and/or necrotic wounds that did not improve or worsened after at least 6 weeks of standard wound care were treated with topical hyperbaric oxygen therapy by trained HMO personnel. Four patients underwent digital amputation for osteomyelitis and/or gangrene followed by topical hyperbaric oxygen therapy. Assessment parameters included wound healing and cost of wound care before and after topical hyperbaric oxygen therapy. Six of the six Level 2 wounds healed within 2 to 4 weeks, nine of the ten Level 3 wounds healed within 4 to 10 weeks, and seven of the eight Level 4 wounds healed within 4 to 12 weeks. The ulcers improved by a mean of 0.829 cm2 per day. T test (SSPS 7.5) showed significant improvement per day after topical hyperbaric oxygen therapy, t = 5.217, df = 24, P < 0.0001 (95% CI = 1.13-0.49). Wound healing with topical hyperbaric oxygen therapy was associated with decreased costs. The results of this support the feasibility of transfer of new wound healing technology from research to HMO personnel.

Angiogenesis in necrotic ulcers treated with hyperbaric oxygen.

Necrotic/gangrenous wounds lack adequate blood supply and develop further vascular damage from either reperfusion injury or oxygen toxicity when exposed to oxygen at the wrong pressures. A prospective randomized study was performed to confirm the efficacy of topical hyperbaric oxygen at 1.004 to 1.013 atmospheres (THOT) in stimulating angiogenesis and healing of necrotic/gangrenous wounds. Participants included 40 inpatients (79 ulcers) recruited over 12 months who were assigned to treatment by either THOT or standard wound care (SWC). The results showed that 90% of the wounds healed in the THOT group compared to 22% in the SWC controls. Repeated measures ANOVA on log (ulcer size at 4 weeks) showed a significant group by time interaction, F(1,55) = 68.2, P < 0.0001. The size of ulcers (at 4 weeks) was significantly smaller with THOT, but larger with SWC. Capillary density/hpf (high power field) was significantly higher in THOT wounds than in SWC wounds (P < 0.001). It was concluded that THOT is effective in stimulating angiogenesis with enhanced healing of necrotic wounds.

Metabolic alterations of zinc and prostaglandins in both human and animal colonic tumor cells.

OBJECTIVE: To understand the relationship between zinc and prostaglandin (PG) metabolisms in inducing colon cancer incidence in human and animals. METHODS: Human colonic tumor and normal cells were obtained from Departments of Surgery and Pathology at the Kaiser Permanente Medical Center, Los Angeles, CA and US VA Medical Center, North Hills, CA. Rat colonic tumor and normal cells were isolated from the rats that received two injections of 50 mg/kg of Azoxymethan (AOM) in 2 weeks and then kept 30 weeks in the animal facility. Then, the effects of zinc on the PGE2 synthesis and PGE2 on zinc metabolism in tumor and normal cells were determined. RESULTS: PGE2 concentrations in both human and AOM-induced rat colonic tumor cells increased compared to those in adjacent normal colonocytes, whereas PGF2 alpha concentrations did not change. Gene expression of inducible form of prostaglandin synthase (PGS-2) is stimulated in rat colonocytes by epidermal growth factor and by tetradecanoyl 13-phorbol acetate (a tumor promoter and mitogen) only in the presence of zinc. PGE2 binding activity of rat enterocytes was maximum at 15 microM of zinc (normal plasma zinc concentration), but PGE2 synthesis activity increased for the first 15 minutes when extracellular zinc concentrations were either higher or lower than the normal extracellular zinc concentration. However, variations in extracellular zinc concentrations did not change the rate of PGF2 alpha synthesis in the normal rat enterocytes. PGE2 significantly increased zinc uptake rates of colonic tumor cells but PGF2 alpha showed only moderate effect. CONCLUSIONS: These results suggest that zinc is required for PGS-2 gene expression, that maintaining an optimal zinc nutriture is important for normal PG synthesis of intestinal cells, and that only PGE2 synthesis mechanisms rather than PGS-2 gene expression are altered in colonic tumor cells resulting in the abnormal zinc nutriture of these cells.

Heat-shock protein 65 and activated gamma/delta T cells in injured arteries.

Because immune mechanisms are implicated in atherogenesis, we investigated the T-lymphocyte subset and factors related to its activation after acute arterial ligation (22 ligated and 13 non-ligated specimens). Ligated arteries produced heat-shock protein 65 (hsp65) and were infiltrated with activated T cells (mostly dendritic, CD3+, CD4-, CD8-, and gamma/delta T-cell-receptor bearing). The protein was found with dendritic T cells, with immunogold-labelled hsp65 beside the dendritic processes. Thus, the immune reaction after acute arterial injury may be associated with binding and recognition of in-situ hsp65 by dendritic gamma/delta T-cells.

Elevated phosphorylase kinase activity in psoriatic epidermis: correlation with increased phosphorylation and psoriatic activity.

To determine whether abnormal activity of a calmodulin-containing enzyme which catalyses phosphorylation reactions may play a pathogenetic role in psoriasis, the presence and activity of phosphorylase kinase (PK) in human epidermis were determined in patients with untreated/active psoriasis (n = 10), treated/resolving psoriasis (n = 10), and non-psoriatic controls (n = 10). Biopsies were taken from involved and uninvolved skin for PK, organic phosphorus, and inorganic phosphate estimation, and light and electron microscopy. The enzyme was present in involved and uninvolved skin of every patient in the study. PK activity (units/mg protein) was significantly higher in active psoriasis than in resolving psoriasis and controls. PK activity correlated directly with organic phosphorus levels, and inversely with the extent of cellular glycogenolysis measured by the depletion of glycogen granules within the keratinocytes. The study demonstrates that PK is present in both psoriatic and normal epidermis, with significantly higher levels in active psoriasis. Furthermore, higher levels of PK activity, glycogenolysis and phosphorylation are associated with increased clinical psoriatic activity. We conclude that PK, a calmodulin-containing enzyme, is involved in regulating calcium-dependent phosphorylation events in human epidermis, and disturbance of its activity may play a key role in the clinical manifestations of psoriasis.

Haemorrhagic cellulitis: a syndrome associated with tumour necrosis factor-alpha.

A newly defined clinical syndrome, haemorrhagic cellulitis, is described in 12 patients. The syndrome consists of an acute onset of extremely painful erythema affecting dependent areas, followed by dermal haemorrhage and sloughing of the overlying epidermis, and requiring both antibiotics and systemic corticosteroids for complete resolution. The patients usually have demonstrable Gram-negative or Gram-positive infection, of non-cutaneous origin, and underlying systemic disease. Vacuolopathic necrosis of epidermal keratinocytes, and damaged vascular endothelium of the dermal blood vessels can be demonstrated by light and electron microscopy, as well as by lectin studies. Immunocytochemical studies reveal the presence of activated macrophages and T lymphocytes. We believe the syndrome is due to lipopolysaccharide-induced or bacterial mitogen-induced tumour necrosis factor-alpha (TNF-alpha), secreted by previously primed activated macrophages in a second-set response. TNF-alpha characteristically injures endothelial cells and epidermal keratinocytes. It is thought to induce its cytotoxic effects partly via neutrophil degranulation, and partly via DNAase activation, with resultant DNA fragmentation and cell lysis. Corticosteroids have been shown not only to inhibit TNF-alpha secretion by activated macrophages, but also to block its cytotoxicity, thus accounting for the extremely rapid clinical response to this drug in conjunction with adequate and appropriate antibiotic therapy.

Co-infection and synergy of human immunodeficiency virus-1 and herpes simplex virus-1.

The human immunodeficiency virus (HIV-1) uses the CD4 molecule, expressed by T helper cells and activated macrophages, as a receptor for entry into host cells. In tissues co-infected with herpes simplex type 1 (HSV-1), HIV-1 virions were observed to infect keratinocytes, which, because they lack the CD4 molecule, are normally incapable of being infected by HIV-1. Although a number of other viruses have been reported to enhance HIV-1 viral transcription in vitro, this is the first in-vivo report to our knowledge of reciprocal enhancement of viral replication associated with co-infection of keratinocytes and macrophages by HIV-1 and HSV-1 in patients with AIDS and non-genital herpes simplex lesions. The virions in the co-infected cells were larger, morphologically atypical, and appear to be hybrids; most contain the HIV-1 envelope necessary for infectivity. The increased viral load and the proximity of the virions to the cutaneous surface may lead to increased risk of transcutaneous transmission of both viruses. These findings point to the need for incorporation of suppressive treatment for herpes simplex in the treatment of AIDS.

Reciprocity between tissue calmodulin and cAMP levels: modulation by excess zinc.

Signal transduction of many intracellular events is initiated by a minute influx of calcium ions into the cells, resulting in the formation of calcium-calmodulin complex and cAMP. Because zinc appears to have an inhibitory effect on a number of tissue reactions, it is postulated that this occurs through modulation of intracellular calcium influx. To test the hypothesis that the inhibitory effects of zinc are mediated through the calcium-calmodulin-cAMP pathway, zinc was administered by various routes to five groups of nude mice (control, intragastric, intraperitoneal, intradermal and oral groups), and calmodulin and cAMP concentrations were measured in the cytosol of epidermal cells. Calmodulin levels decreased significantly in the groups given intraperitoneal zinc (P < 0.025) and intradermal zinc (P < 0.001) injections. Significant elevations of cAMP levels were noted with intradermal zinc (P < 0.025). Overall, the relationship between calmodulin and cAMP appeared to be inversely logarithmic, with the lowest calmodulin levels associated with the highest cAMP concentrations. In addition, there was a significant trend towards a smaller calmodulin/cAMP ratio in all zinc-treated groups, except the mice fed dietary zinc. These results appear to correlate with tissue zinc levels obtained with these various forms of zinc administration. Our results therefore indicate that there is a reciprocity between epidermal calmodulin and cAMP levels, which may be modulated by external factors such as zinc.

Topical hyperbaric therapy for problem skin wounds.

BACKGROUND: Hyperbaric oxygen remains the sole treatment capable of inducing growth of new blood vessels. However, systemic hyperbaric oxygen therapy risks central nervous system and pulmonary toxicity. OBJECTIVE: To describe topical hyperbaric oxygen therapy for the treatment of recalcitrant open wounds. METHODS: Topical and systemic hyperbaric oxygen treatments are described and contrasted from one another. Applications of topical hyperbaric oxygen therapy are described. CONCLUSION: Topical hyperbaric oxygen therapy is useful only for open wounds. The advantages of topical hyperbaric oxygen therapy include low cost, the lack of systemic oxygen toxicity, and effectiveness, allowing this treatment to be prescribed for many patients early in the course of their disease rather than as a last resort.

Brefeldin A inhibits phosphate transport in opossum kidney cells.

Brefeldin A (BFA) is a fungal metabolite that blocks the transport processes between the endoplasmic reticulum and the Golgi apparatus. In the present study, we have tested the effect of BFA on phosphate transport in a kidney epithelial cell line, opossum kidney (OK) cells. Electron microscopy showed that exposure of OK cells to BFA caused a rapid and reversible disorganization of Golgi apparatus. Addition of BFA also caused a time (2-8 h)- and dose (1-10 micrograms/ml)-dependent inhibition of Na(+)-dependent cell phosphate uptake. The inhibition of cell phosphate uptake by BFA was reversible and was associated with a decrease in the maximum velocity of phosphate transport. Both the inhibition and the stimulation of cell phosphate uptake by parathyroid hormone and insulin, respectively, were not affected by BFA. BFA at 1 microgram/ml concentration did not affect protein synthesis as determined by [3H]leucine incorporation but diminished the adaptive increase in cell phosphate uptake in response to 2 or 8 h of incubation in nominally phosphate-free medium. On the other hand, inhibition of protein synthesis by cycloheximide (5 microM) abolished the adaptive increase in cell phosphate uptake in response to 8 but not 2 h of incubation in nominally phosphate-free medium, indicating the existence of an early response to phosphate deprivation, which does not require new protein synthesis but is sensitive to the effect of BFA. In summary, results of these studies show that, in OK cells, BFA inhibits phosphate uptake and curtails the adaptive response to phosphate deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the L-fucose moiety on epidermal keratinocytes in psoriasis induced by the Koebner phenomenon: a sequential study.

The expression of Ulex europaeus agglutinin (UEA I) binding sites on cell-surface glycoproteins has been used as a marker for terminal differentiation. Increased number of UEA I binding sites of L-fucose specificity have been demonstrated in psoriatic epidermis. The results of lectin-binding studies in a series of biopsies taken sequentially (0 min, 5 min, 24 h, 7 days and 8 weeks) after tape-stripping of uninvolved skin in 12 psoriatic patients (three of whom were taking diltiazem, a calcium blocker at the time of the study) and six controls are presented. UEA I binding sites, which were expressed on the granular layer and upper layers of the stratum spinosum of pre-tape stripped uninvolved skin in psoriatic individuals, were progressively more numerous, with the expression of the L-fucose moiety on the lower stratum spinosum keratinocytes in the 7-day post-tape-stripping biopsies and 8-week biopsies, correlating with a moderate and marked increase in the proliferative index, respectively. In the Koebner-negative and non-psoriatic individuals who failed to develop psoriasis after tape-stripping, the UEA I binding sites were not expressed on keratinocytes of the lower stratum spinosum in any of the biopsies, although a mild increase in the proliferative index was noted in the 7-day biopsies. Our data suggest that the increased commitment of keratinocytes to terminally differentiate may be involved in the psoriatic process.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of Ulex europaeus agglutinin I lectin-binding sites in squamous cell carcinomas and their absence in basal cell carcinomas. Indicator of tumor type and differentiation.

Lectins bind tightly to carbohydrate moieties on cell surfaces. Alterations in lectin binding have been reported to accompany epidermal cell differentiation, marking alterations in membrane sugars during this process. The presence of UEA I (Ulex europaeus agglutinin I) L-fucose-specific lectin-binding sites has been used as a marker for terminally differentiated (committed) keratinocytes. In this article, we report the presence of UEA-I-binding sites on squamous keratinocytes of well-differentiated squamous cell carcinomas, with patchy loss of UEA I positivity on poorly differentiated cells of squamous cell carcinomas, suggesting a possible use for this technique in the rapid assessment of less differentiated areas within the squamous cell tumor. The absence of UEA-I-binding sites on basal cell carcinomas may be related to an inability of cells comprising this tumor to convert the L-D-pyranosyl moiety on basal cells to the L-fucose moiety, resulting in an inability of basal cell carcinoma cell to undergo terminal differentiation into a committed keratinocyte.

Prostaglandin interacts with steroid sex hormones in the regulation of intestinal zinc transport.

1. The effects of prostaglandins (PGs) E1 and E2, testosterone, 17 beta-estradiol and indomethacin on the intestinal zinc transport rates of male and female rats were compared. 2. PGE1 stimulated Jms (the zinc flux rate from mucosa-to-serosa) of jejunal segments from male rats but inhibited Jms of those from female rats in Ussing chamber experiments. 3. 17 beta-Estradiol inhibited Jms of jejunal segments from male rats, while testosterone stimulated it in those from female rats. However, testosterone inhibited Jms of segments isolated from male rats and 17 beta-estradiol that of those from female rats, while in segments from ovariectomized rats, both of these steroid hormones stimulated Jms. 4. When PGE2 was added to an indomethacin containing medium, Jms significantly increased whereas Jsm (the zinc flux rate from serosa-to-mucosa) decreased further.

Expression of the L-fucose moiety on infrainfundibular follicular keratinocytes of terminal follicles, its decreased expression on vellus and indeterminate follicles of androgenetic alopecia, and re-expression in drug-induced hair regrowth.

The distribution of various glycoprotein molecules on the surface of follicular keratinocytes was studied with a panel of lectins with specificity for various sugar moieties on biopsy specimens from both bald/balding scalp and normal occipital scalp, of 23 patients with androgenetic alopecia as well as on biopsies of normal forearm skin of four patients. The most significant differences between bald and normal scalp biopsy were noted with Ulex europaeus agglutinin I (UEA I). We noted an increased (91.8% +/- 3.1; mean +/- SE) expression of UEA I binding sites on the infra-infundibular follicular keratinocytes in anagen terminal scalp hairs, compared to 28.5% +/- 5.2 in the indeterminate (anagen) hairs of balding scalps, and 23.2% +/- 6.3 in the anagen follicles of vellus fore-arm hairs. By contrast, the telogen hairs demonstrated minimal UEA I staining: 4.0% +/- 0.8, mean +/- SE in telogen scalp hairs, 1.8% +/- 0.5 in telogen hairs of balding scalps (0% in completely bald scalps, in which all the hairs were in the telogen phase), and 1.9% +/- 0.2 in telogen forearm hairs. The percentage of UEA I staining correlated with the length of the infra-infundibular follicles in all cases studied. In three cases of hair regrowth after hair growth promotors, the UEA I staining increased to 80.6% +/- 6.1 in anagen hairs and correlated with increased length of infra-infundibular follicles. Our data indicate that there are 1) marked differences between anagen and telogen follicles in UEA I binding to infra-infundibular follicular keratinocytes; 2) the percentage of UEA I staining reflects the size (length) of the infra-infundibular hair follicle; and 3) the anagen follicles of balding scalps (indeterminate hairs) show UEA I staining resembling that exhibited by anagen follicles of vellus hairs.

Electron microscopic and immunocytochemical studies of the sequence of events in psoriatic plaque formation following tape-stripping.

Electron microscopic and immunocytochemical studies were performed on sequential biopsies following the tape-stripping of the uninvolved skin in 12 patients with psoriasis. In the biopsies taken after 5 min for up to 7 days during the pre-psoriatic phase, there were initial lymphocyte-Langerhans cell interactions as well as interactions between lymphocytes and keratinocytes. In biopsies taken after 6-8 weeks during the proliferative phase there were lymphocyte-macrophage interactions. In the 24-h and 7-day biopsies there were close contacts between epidermal lymphocytes and keratinocytes via microvilli, with blebbing of the keratinocyte plasma membranes and granular cytoplasmic changes around these microvilli. Few basal keratinocyte herniations were noted during this phase. The 6-8-week biopsies of Köbner-positive patients were characterized by a marked increase in lymphocyte-macrophage interactions via similar microvillous processes with associated electronlucent areas suggestive of cytotoxicity.

Efficacy of cyclophosphamide in toxic epidermal necrolysis. Clinical and pathophysiologic aspects.

In this article we describe the immunocytochemical and electron microscopic findings in five patients with toxic epidermal necrolysis. They indicate the occurrence of necrotic keratinocytes with nuclear disintegration associated with apposed dendritic cells with the nuclear chromatin configuration of T lymphocytes. These findings, including the presence of blebbing of the keratinocytes and membrane defects associated with cytoplasmic processes from these apposed lymphoid cells, fit known electron microscopic criteria that suggest the involvement of T lymphocyte-mediated cytolysis of drug-altered target keratinocytes in toxic epidermal necrolysis. The effector cell appears to be a dendritic subset, with the phenotypic characteristics (CD3+, CD4-, CD8+, CD2+, DR+) of a T cell subset. There is some evidence that tumor necrosis factor alpha, secreted by activated macrophages, may play a role in necrolysis of the epidermis. The dramatic response of our patients to cyclophosphamide, which is known to inhibit cell-mediated cytotoxicity by inhibiting both the recognition and lethal hit stages, together with the rapid regrowth of the epidermis within 4 days to a week in patients who received adequate dosage of the drug, supports the preceding concepts.

Focal endothelial cell degeneration and proliferative endarteritis in trauma-induced early lesions of necrobiosis lipoidica diabeticorum.

Microangiopathy is an essential component in diabetic vascular pathology. We report ultrastructural observations of ballooning degeneration involving isolated endothelial cells of cutaneous capillaries, while leaving adjacent endothelial cells relatively intact in six diabetic patients with early lesions of necrobiosis lipoidica induced by trauma. Focal proliferation of endothelial cells encroaching upon the vascular lumina (obliterative endarteritis) was also observed. Lectin studies on biopsy specimens of older lesions of necrobiosis lipoidica revealed paucity of dermal blood vessels. These observations enable us to gain further insight into the pathophysiological mechanisms that underlie diabetic microvascular disease.