Mast cells are upregulated in hidradenitis suppurativa tissue, associated with epithelialized tunnels and normalized by spleen tyrosine kinase antagonism.

Mast cells have traditionally been associated with allergic inflammatory responses; however, they play important roles in cutaneous innate immunity and wound healing. The Hidradenitis Suppurativa tissue transcriptome is associated with alterations in innate immunity and wound healing-associated pathways; however, the role of mast cells in the disease is unexplored. We demonstrate that mast cell-associated gene expression (using whole tissue RNAseq) is upregulated, and in-silico cellular deconvolution identifies activated mast cells upregulated and resting mast cells downregulated in lesional tissue. Tryptase/Chymase positive mast cells (identified using IHC) localize adjacent to epithelialized tunnels, fibrotic regions of the dermis and at perivascular sites associated with Neutrophil Extracellular Trap formation and TNF-alpha production. Treatment with Spleen Tyrosine Kinase antagonist (Fostamatinib) reduces the expression of mast cell-associated gene transcripts, associated biochemical pathways and the number of tryptase/chymase positive mast cells in lesional hidradenitis suppurativa tissue. This data indicates that although mast cells are not the most abundant cell type in Hidradenitis Suppurativa tissue, the dysregulation of mast cells is paralleled with B cell/plasma cell inflammation, inflammatory epithelialized tunnels and epithelial budding. This provides an explanation as to the mixed inflammatory activation signature seen in HS, the correlation with dysregulated wound healing and potential pathways involved in the development of epithelialized tunnels.

Exercise training-induced modification of the gut microbiota persists after microbiota colonization and attenuates the response to chemically-induced colitis in gnotobiotic mice.

Exercise reduces the risk of inflammatory disease by modulating a variety of tissue and cell types, including those within the gastrointestinal tract. Recent data indicates that exercise can also alter the gut microbiota, but little is known as to whether these changes affect host function. Here, we use a germ-free (GF) animal model to test whether exercise-induced modifications in the gut microbiota can directly affect host responses to microbiota colonization and chemically-induced colitis. Donor mice (n = 19) received access to a running wheel (n = 10) or remained without access (n = 9) for a period of six weeks. After euthanasia, cecal contents were pooled by activity treatment and transplanted into two separate cohorts of GF mice. Two experiments were then conducted. First, mice were euthanized five weeks after the microbiota transplant and tissues were collected for analysis. A second cohort of GF mice were colonized by donor microbiotas for four weeks before dextran-sodium-sulfate was administered to induce acute colitis, after which mice were euthanized for tissue analysis. We observed that microbial transplants from donor (exercised or control) mice led to differences in microbiota ß-diversity, metabolite profiles, colon inflammation, and body mass in recipient mice five weeks after colonization. We also demonstrate that colonization of mice with a gut microbiota from exercise-trained mice led to an attenuated response to chemical colitis, evidenced by reduced colon shortening, attenuated mucus depletion and augmented expression of cytokines involved in tissue regeneration. Exercise-induced modifications in the gut microbiota can mediate host-microbial interactions with potentially beneficial outcomes for the host.

The phototoxicity of vemurafenib: An investigation of clinical monochromator phototesting and in vitro phototoxicity testing.

BACKGROUND: Vemurafenib is a targeted therapy approved for the treatment of patients with metastatic melanoma harbouring the BRAF V600E mutation. Photosensitivity has been reported in over 50% of patients and has been demonstrated to involve at least the broadband UVA spectrum in most patients. Erythrocyte protoporphyrin levels have also been reported as elevated in some patients. OBJECTIVES: We report the results of monochromator phototesting in one patient recorded before and while taking vemurafenib. Analysis of porphyrin levels was also conducted. RESULTS: After one month of vemurafenib therapy the patient demonstrated markedly increased light sensitivity in the UVA spectrum between 335 ± 27 nm, 365 ± 27 nm and 400 ± 27 nm. However responses in the UVB (305 ± 5 nm) and blue light (430 ± 27 nm) regions were normal. There was no abnormal immediate erythemal response. Pre-vemurafenib baseline phototesting was normal, as was repeat testing two months later when the patient was taking high doses of systemic steroid. No abnormal porphyrins were detected and the antinuclear antibody test was normal. In parallel studies, HaCaT keratinocytes incubated with vemurafenib were killed by UVA but not by visible (blue) light and did not show evidence of detectable intracellular porphyrin in the presence of the drug. CONCLUSION: These data confirm vemurafenib induced UVA photosensitivity with a probable phototoxic mechanism not mediated via enhanced porphyrin.

Porphyrin profile in four human cell lines after supplementation with 5-aminolaevulinic acid and its methyl ester.

Multiple factors can affect the synthesis of the prodrugs aminolaevulinic acid and its methyl ester to protoporphyrin. These may ultimately influence the efficacy of ALA-induced porphyrin as a photosensitiser for photodynamic therapy or fluorescence diagnosis. This study demonstrates the variation in total amount of porphyrin produced and cellular porphyrins synthesised in four different human cell lines after supplementation with these prodrugs. A non-invasive optical biopsy system was able to detect spectral changes associated with the more carboxylated porphyrins accumulating in oesophageal (OE19) and bladder (HT1197) carcinoma cells, and to a lesser extent neuroblastoma (SH-SY5Y) cells after a 24h incubation with the prodrugs. If the porphyrin profile changes during disease progression, or between normal and cancerous cells clinically, then the demonstrated non-invasive spectral analysis may be exploitable in distinguishing between normal, dysplastic and tumour tissue. Finally, the OE19 cell line was insensitive to photo-inactivation under the experimental conditions used, despite accumulating more porphyrin than the other cells lines.

Plasma ropivacaine concentrations during bilateral transversus abdominis plane infusions.

BACKGROUND: The transversus abdominis plane (TAP) block is a regional anaesthetic technique that blocks abdominal wall somatic afferent nerves. We conducted a prospective observational study to evaluate the venous plasma concentrations of ropivacaine during a continuous TAP infusion. METHODS: Twenty patients who were planned to undergo intra-abdominal cavity surgery requiring a mid-line laparotomy incision were enrolled. Patients were excluded if they had a history of chronic pain, opioid tolerance, renal or hepatic impairment, or contraindication to study medications. Subjects received a standardized general anaesthetic, and at the completion of surgery, ultrasound-guided subcostal or posterior TAP blocks and catheters. A TAP infusion of 2 mg ml(-1) ropivacaine was administered for 72 h after operation. Data collection during the 72 h included morphine requirements, pain scores, and plasma ropivacaine levels. RESULTS: TAP blocks and catheters were successfully inserted in all recruited subjects. The fourth subject experienced neurological symptoms attributed to local anaesthetic toxicity, but did not have high plasma ropivacaine concentrations. However, the protocol was amended for the subsequent 16 subjects, to a weight-based dosing regimen. The range of total plasma ropivacaine concentrations was 0.98-3.41 mg litre(-1) for posterior infusions and 0.96-3.48 mg litre(-1) for subcostal infusions. Four subjects had total ropivacaine levels >3.4 mg litre(-1). The range of unbound plasma ropivacaine concentrations was 0.022-0.135 mg litre(-1) for posterior infusions and 0.031-0.120 mg litre(-1) for subcostal infusions. CONCLUSION: Given the potential for high plasma concentrations from a bilateral TAP infusion technique, attention should be paid to individualized dosing strategies.

Effect of sex on wasted left ventricular effort following maximal exercise.

Wasted left ventricular effort (∆Ew) refers to work required of the left ventricle to eject blood that does not result in increased stroke volume and is related to left ventricular hypertrophy. Literature shows that men and women have differing ventricular and vascular responses to and following exercise. Our purpose was to determine how ∆Ew changes post-exercise in men and women and examine potential mechanisms. We hypothesized a reduction in ∆Ew that would be greater in men and that central pulse wave velocity and wave intensity (WIA) would be related to ∆Ew. Blood pressures, central pulse wave velocity (cPWV), and WIA were obtained at rest, 15 and 30 min after maximal exercise. Both sexes reduced ∆Ew post-maximal exercise (p>0.05 for interaction), but women had higher ∆Ew at each time point (p<0.05). The first peak of WIA increased 15 min post-exercise only in women (p<0.05). cPWV was attenuated (p<0.05) in women at 15 min and men at 30 min (p<0.05) post-exercise with a significant time by sex interaction (p<0.05). WIA (1st peak) was correlated (p<0.05) to ∆Ew in both sexes before and 15 min post-exercise, but cPWV was only associated with ∆Ew in men at 30 min post-exercise. We conclude that both sexes decrease ∆Ew after maximal exercise, but vascular and ventricular changes associated with the attenuation of ∆Ew are not uniform between sexes.

Effect of resistance training on biomarkers of vascular function and oxidative stress in young African-American and Caucasian men.

African Americans (AA) have an earlier onset of hypertension and a different vascular profile than their Caucasian (Cau) peers. Research suggests that biological mediators of vascular inflammation are different among these groups in hypertensive populations. Resistance training (RT) is an important exercise modality that improves the vascular profile of young AA men. We examined the role of RT on biomarkers of vascular function and oxidative stress in body mass index-matched AA and Cau men. RT for 6 weeks elicited significant changes in circulating matrix metalloprotease-9 (MMP-9) and 8-Isoprostane (8-IsoP) in young AA men (n=14, AA; n=18, Cau; 18-35 years). MMP-9 was lower and decreased in AA (pre: P=0.02; post: P<0.001) and a time × group interaction for MMP-9 (F(1, 30)=4.81; P=0.036) and 8-IsoP (F(1, 24)=7.09; P=0.014) was detected. 8-IsoP decreased in AA (P=0.026) but did not change in Cau (P=0.309). Notably, the increase in strength (1-repetition maximum (1-RM)) was correlated with the decrease in MMP-9 (r=-0.398; P=0.022). Furthermore, these adaptations were independent of any improvement in cardiorespiratory fitness. We demonstrate that RT effectively reduces matrix remodeling proteins and oxidative stress in young AA men. Increasing strength may be beneficial for improving vascular health and offsetting novel cardiovascular risk factors of hypertension in young AA men.

Ultraviolet A1 phototherapy: a British Photodermatology Group workshop report.

Whole-body ultraviolet (UV)A1 (340-400 nm) phototherapy was first introduced 30 years ago, but is currently available in the UK in only three dermatology departments. A workshop to discuss UVA1 was held by the British Photodermatology Group in May 2009, the aim of which was to provide an overview of UVA1 phototherapy and its role in practice, and to identify areas in which further studies are required. The conclusions were that UVA1 phototherapy is an effective treatment in several inflammatory skin diseases, including localized scleroderma and atopic eczema (AE); however, deficiencies and limitations exist in the published evidence base. For most diseases, such as AE, other treatments also exist, which are generally more effective than UVA1. However, for some diseases, particularly morphoea, the evidence of efficacy is stronger for UVA1 than for other treatments. Acute adverse effects of UVA1 are minimal. The risk of long-term adverse effects, particularly skin cancer, is unknown. Medium to high doses of UVA1 are needed for efficacy in most situations, but the equipment to deliver such doses is large, expensive and difficult to install. UVA1 is currently underprovided, and the recommendation of the workshop is that more tertiary centres should have access to UVA1 phototherapy in the UK.

Cyclobutane pyrimidine dimers are photosensitised by carprofen plus UVA in human HaCaT cells.

Every year in the UK about 75,000 cases of non-melanoma skin cancer (NMSC) are registered, and about 9500 people are diagnosed with cutaneous melanoma (CM). The main risk factor for these cancers is exposure to sunlight. The effects of light on skin are wavelength dependent, with wavelengths in the UVB waveband (280-315 nm) being the most carcinogenic. UVB is directly absorbed by DNA, producing dimeric pyrimidine photoproducts including cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimodone photoproducts (6-4PP). However UVA (315-400 nm) can also produce CPD, induce skin tumours in mice, and has been shown to be mutagenic in cell culture. Although the precise role of UVA in human skin cancer remains to be elucidated, it comprises the major portion of solar UV radiation, transmits through window glass and can be delivered in high doses from tanning lamps. Non-steroidal anti-inflammatory drugs (NSAIDs), in particular the 2-aryl propionic acid derivatives, are a well-documented group of photosensitising chemicals producing clinical phototoxic and photoallergic reactions. We have used carprofen, a model compound from this group to see if it could amplify the effects of UVA and contribute to the formation of CPD by UVA. Preliminary work has shown that carprofen combined with low doses of UVA (lambda(max): 365 nm; 5 J/cm(2)) can produce both strand breaks (SB) and CPD in human skin or blood cells. CPD were detected indirectly by both an immunofluorescence method and as T4 endonuclease V sensitive sites in the comet assay. These findings show that compounds other than fluoroquinolones and psoralen derivatives may contribute to CPD formation in skin cells in combination with UVA.

Reduction in trunk fat predicts cardiovascular exercise training-related reductions in C-reactive protein.

C-reactive protein (CRP) is an independent risk factor for cardiovascular disease. We sought to determine (1) if 10 months of cardiovascular exercise training (Cardio) reduces CRP in a group of older adults, (2) if such a reduction is related to improvements in trunk fat, fitness, and/or psychosocial variables, and (3) if the effect of Cardio on CRP differs between men and women. Community-dwelling residents (n=127; 60-83 yrs) were randomized to a Flex group (n=61) where they participated in 2-75 min supervised sessions per wk during which they performed non-cardiovascular flexibility and balance exercises or a Cardio group (n=66) where they participated in three supervised sessions per wk during which they performed cardiovascular exercises for approximately 45-60 min at 60-70% maximal oxygen uptake. The main outcome measures were serum CRP, cardiovascular fitness, total and central adiposity, and self-reported psychosocial function. Cardio experienced a reduction in CRP (-0.5mg/L), as well as improvements in fitness (+7%) and total (-1.5%) and central (i.e., trunk) (-2.5%) adiposity. These relationships were not modified by sex. Regression analyses indicated that only the reduction in trunk fat was significantly related to the reduction in CRP. Ten months of cardiovascular exercise training reduced CRP in previously sedentary older adults and this effect was partially mediated by a reduction in trunk fat.

Cardiovascular exercise intervention improves the primary antibody response to keyhole limpet hemocyanin (KLH) in previously sedentary older adults.

Based upon a prior cross-sectional study, we hypothesized that an aerobic exercise intervention in sedentary older adults would improve a primary T cell-dependent immune response. Participants were a subset of older subjects from a large, ongoing exercise intervention study who were randomly assigned to either an aerobic exercise (Cardio, n=30, 68.9+0.8 years) or flexibility/balance (Flex, n=20, 69.9+1.2 years) intervention. The intervention consisted of either three aerobic sessions for 30-60 min at 55-70% VO(2 max) or two 60 min flexibility/balance sessions weekly for 10 months. Eight months into the intervention, samples were collected before intramuscular administration of KLH (125 microg), followed by sampling at 2, 3, and 6 weeks post-KLH. Serum anti-KLH IgM, IgG1, and IgG2 was measured by ELISA. Physiological and psychosocial measures were also assessed pre- and post-intervention. While there was no difference in the anti-KLH IgG2 response between groups, Cardio displayed significantly (p<0.05) higher anti-KLH IgG1 (at weeks 2, 3, and 6 post) and IgM responses when compared to Flex. Despite cardiovascular intervention-induced improvement in physical fitness (approximately 11% vs. 1% change in VO(2 peak) in Cardio vs. Flex, respectively), we found no relationship between improved fitness and enhanced anti-KLH antibody responses. Optimism, perceived stress, and affect were all associated with enhanced immune response. We have shown for the first time that cardiovascular training in previously sedentary elderly results in significantly higher primary IgG1 and IgM antibody responses, while having no effect on IgG2 production.

Can St John's wort (hypericin) ingestion enhance the erythemal response during high-dose ultraviolet A1 therapy?

BACKGROUND: St John's wort (SJW) is widely used as a treatment for depression. A phototoxic reaction, due to its content of hypericin, can occur in animals and in cell culture, and has been reported in humans. Hypericin displays absorption within the ultraviolet (UV) A1 spectrum and there may therefore be a potential for phototoxicity if taken during high-dose UVA1 therapy. OBJECTIVES: To assess the phototoxicity risk of SJW ingestion. METHODS: Eleven adult volunteers of skin types I and II were exposed to a geometric dose series of UVA1 irradiation from a high-output source (Dermalight Ultra 1; Dr Hönle, Martinsreid, Germany; irradiance 70-77 mW cm(-2)) on the photoprotected lower back skin at eight 1.5-cm(2) test areas. Irradiation was carried out at baseline and after 10 days of SJW extract 1020 mg (equivalent to 3000 microg of hypericin) daily. Four, 8, 24 and 48 h after each exposure, the minimal erythema dose (MED) and the presence or absence of pigmentation were recorded visually and erythema was assessed objectively with an erythema meter. RESULTS: The median MED and D(0.025), an objective measure of MED, were lower at all time-points after SJW ingestion. The visual erythemal peak (lowest median MED), which was seen at 8 h postirradiation, was lower after SJW (median 14 J cm(-2), range 10-56) than at baseline (median 20 J cm(-2), range 14-56) (P = 0.047). Similarly, the median D(0.025) at 8 h postirradiation was lower after SJW (median 22.0 J cm(-2), range 15.2-53.9) than at baseline (median 33.7 J cm(-2), range 22.9-136.0) (P = 0.014). The MED and D(0.025) were also significantly different at the 48-h and 4-h time-points, respectively. Significance was not reached at the 24-h time-point. Median intensity of postirradiation erythema increased at all time-points after ingestion of SJW. Despite these differences, the maximum slope of the dose-response curve was not increased after SJW ingestion. CONCLUSIONS: These data suggest that SJW extract has the potential to lower the erythemal threshold to UVA1 irradiation in a significant proportion of individuals and highlight the importance of ascertaining a full drug history, including herbal remedies, before initiating UVA1 phototherapy.

Photogenotoxicity of hypericin in HaCaT keratinocytes: implications for St. John's Wort supplements and high dose UVA-1 therapy.

Extract of St. John's Wort (Hypericum perforatum) is commonly used as natural remedy for treatment of mild to moderate depression. However, it contains a powerful photoactive component, hypericin, which can cause a severe photodermatitis when eaten by grazing animals (hypericism). In humans, there is evidence that supplementation with St. John's Wort can reduce the minimal erythemal dose (MED) in patients undergoing high dose UVA-1 phototherapy. This is a recent development in phototherapy where the most erythemogenic parts of the UVA spectrum are filtered out, allowing delivery of higher doses of the longer wavelengths of UVA. Although current published evidence suggests that the plasma levels of hypericin are unlikely to cause clinical phototoxicity, it has been established that photoactive compounds can cause DNA damage at sub-toxic and sub-erythemal doses, the effects of which might not be apparent for many years after the event. The present study used HaCaT keratinocytes to investigate the photoclastogenic ability of hypericin on irradiation with UVA. The results show that although the combination of hypericin and UVA light increased the genotoxic burden, when all factors are taken into account, the risk of significant photogenotoxic damage incurred by the combination of Hypericum extracts and UVA phototherapy may be low in the majority of individuals.

Selected hormonal and immunological responses to strenuous live-fire firefighting drills.

This study investigated the effects of strenuous live-fire firefighting drills and a 90 min recovery period on selected hormonal, immunological and psychological variables. Apparently healthy, male, professional firefighters (n = 11) performed three trials of standardized firefighting tasks in a live-fire training structure. There was significant leukocytosis immediately post firefighting activity that persisted following recovery, although there was a variable response among the leukocyte subsets. Most notable was the decrease in number and percentage of lymphocytes following 90 min of recovery. Plasma levels of ACTH and cortisol were significantly elevated post firefighting activity and cortisol remained elevated following 90 min of recovery. Elevated cortisol immediately following activity was related to reduced feelings of energy. These data demonstrate the magnitude of the physiological and psychological disruption following strenuous firefighting activity and suggest that immune function may be altered following such activity. This is a finding that may have practical consequences for this group of first responders.

Can a positive photopatch test be elicited by subclinical irritancy or allergy plus suberythemal UV exposure?

Photopatch test (PhPT) interpretation is difficult and clinical relevance is not always apparent. A positive PhPT may reflect photocontact allergy or phototoxicity. We hypothesized that it may also reflect the additive or synergistic effects of a suberythemal reaction to a contact irritant [e.g. sodium lauryl sulfate (SLS)] or allergen (e.g. nickel) and suberythemal UV exposure. 10 nickel allergic volunteers had duplicate SLS and nickel series applied on either side of the back for 24 h and 48 h, respectively. After removal, one side was irradiated with 5 J/cm(2) UVA or the dose below the minimal erythema dose for solar-simulated radiation (SSR). The minimal irritancy dose (MID) for SLS and the minimal allergenic dose (MAD) for nickel were determined visually and objectively by erythema meter. While photoaugmentation of subclinical contact allergy or irritancy occurred in some subjects, photosuppression occurred in roughly an equal number. UVA changed the nickel MAD at 48 h in 2 of 5 volunteers but not the SLS MID. SSR changed the nickel MAD in 4 of 5 and the SLS MID in 3 of 5. 2 subjects (none after UVA) showed erythema only in the irradiated set of patches, which could have been interpreted as a positive PhPT. We have demonstrated photoaugmentation and photosuppression of contact allergy and irritancy, which could result in false-positive or false-negative interpretation of PhPTs.

Macrophage hypo-responsiveness to interferon-gamma in aged mice is associated with impaired signaling through Jak-STAT.

Since macrophages (Mphis) are a first line of defense against pathogens, and are involved in both innate and adaptive immunity, understanding the impact of aging on Mphi function is important. In the past studies, we and others have shown that aging decreases Mphi responsiveness to classical activating signals (e.g. IFN-gamma and lipopolysaccharide, LPS). In this study, we examined the impact of aging on Mphi signaling through the IFN-gamma receptor pathway. Mphis from male Balb/c mice aged 2 (young) and 18-24 (old) months were purified and then stimulated with IFN-gamma. Western blotting revealed a significant reduction ( approximately 50%) in IFN-gamma-stimulated tyrosine phosphorylation of signal transducer and activator of transcription-1 (STAT-1) alpha and beta in Mphis from aged, when compared with young mice. This reduction in phospho-STAT-1 was associated with a significant constitutive reduction ( approximately 80%) in total STAT-1alpha protein and a complete inhibition of STAT-1 gene expression in response to IFN-gamma in old compared to young mice. These data may, in part, explain why classical Mphi responses like reactive nitrogen and oxygen species generation, tumor killing and microbicidal activity are lower in Mphis from aged subjects. We conclude that peritoneal Mphis from aged mice have an intrinsic defect in Jak-STAT signaling which prevents them from fully responding to IFN-gamma.

The effect of photofrin on DNA strand breaks and base oxidation in HaCaT keratinocytes: a comet assay study.

Photodynamic therapy (PDT) kills cells via the production of singlet oxygen and other reactive oxygen species. PDT causes chromosomal damage and mutation to cultured cells. However, DNA damage does not contribute to the phototoxic effect. To study the effect of Photofrin-PDT-induced DNA damage, we used the comet assay in combination with endonuclease III and formamidopyrimidine DNA glycosylase and a human keratinocyte cell line to investigate photogenotoxicity and its prevention by tocopherol (TOC). This study shows that PDT induced DNA damage in HaCaT cells at doses allowing cells to survive 7 days after irradiation. alpha-TOC did not prevent the acute cell lysis caused by Photofrin-PDT but did prevent Photofrin-PDT-induced DNA damage. However, the concentration of TOC that conferred protection (100 microM) was higher than is detected in human serum. Base oxidation was also measured using the comet assay. Although TOC could prevent frank DNA strand breaks caused by PDT, it was unable to decrease the level of base oxidation as revealed by enzyme-sensitive sites. It is suggested that the potential genotoxic risk from laser-PDT could be low, and that topical micro-TOC at a high concentration may be useful in preventing some types of DNA damage without preventing acute photolysis after Photofrin-PDT.

Photodynamic therapy in dermatology: Dundee clinical and research experience.

Topical photodynamic therapy (PDT) is increasingly accepted and used as a highly effective treatment for superficial non-melanoma skin cancer and dysplasia. We describe the developments in topical PDT for the treatment of skin diseases in our own PDT Centre in Dundee, both clinically and from a research base. Improvements in PDT could be achieved by optimisation of photosensitiser and light delivery, and these goals underpin the aims of our centre. We hope to facilitate the dissemination of use of PDT in dermatology throughout Scotland and outline some of the progress in these areas.

Exercise training increases the näive to memory T cell ratio in old mice.

Aging is associated with changes in T cells including involution of the thymus gland and an imbalance in the proportion of näive (CD44lo) and memory (CD44hi) T cells in the periphery. Reversal of these changes may improve immunity in the aged. We sought to determine whether 4 months of moderately intense treadmill running (EXC; 5 days/week, 45 min/day, 13-22 m/min) in 2 month (Y) and 18 month (O) old male Balb/c mice would alter T lymphocyte profiles in the thymus and spleen when compared to sedentary controls (CON). Splenocytes and thymocytes were harvested 24-48 h after the last exercise session and analyzed using immunofluorescence and flow cytometry. While there were significant age-related changes (lower cell number, altered subsets) in the thymuses of O when compared to Y mice, exercise training failed to affect any of these measures in mice of either age. Aged mice exhibited a significantly (p < .05) higher percentage of splenic memory cells and a lower percentage of näive cells in both the CD4 and CD8 T cell subsets. Interestingly, exercise training significantly (p < .05) increased the percentage of näive and decreased the percentage of memory cells in both the CD4+ (69.6+/-1.7% näive and 30.4+/-1.7% memory for OCON vs. 75.0+/-1.5% näive and 25.0+/-1.5% memory in OEXC) and CD8+ (60.0+/-2.6% näive and 40.0+/-2.6% memory in OCON vs. 76.7+/-2.7% näive and 23.3+/-2.7% memory in OEXC) T cells subsets in O, but not Y, mice. This effect was due to a decrease in the absolute number of memory cells and not an increase in the absolute number of näive cells. We conclude that 4 months of EXC has little restorative effect on the thymus in aged mice, but can restore the percentages of näive and memory cells in the spleen towards that of young mice, perhaps due to removal of memory cells.

Toxicity of cholesterol oxidation products to Caco-2 and HepG2 cells: modulatory effects of alpha- and gamma-tocopherol.

Cholesterol can be oxidized to form a variety of cholesterol oxidation products also known as oxysterols. The aims of the present study were to compare the cytotoxic effects of four oxysterols, namely 25-hydroxycholesterol (25-OHC), 7beta-hydroxycholesterol (7beta-OHC), cholesterol-5beta,6beta-epoxide (beta-epox) and cholesterol-5alpha,6alpha-epoxide (alpha-epox), in two human cell culture models. Further, the ability of 10 and 100 micro m alpha- and gamma-tocopherol (alpha-TOC and gamma-TOC, respectively) to protect against oxysterol-induced cytotoxicity was also assessed. Human colonic adenocarcinoma Caco-2 and human hepatoma HepG2 cells were supplemented with increasing concentrations of 25-OHC, 7beta-OHC, beta-epox and alpha-epox (0-25 micro g ml(-1)) for 24, 48 or 96 h. Following 24-h and 48-h exposure, test media were replaced with normal growth media and the cells were maintained for 72 and 48 h, respectively. The 96-h exposure represented a constant challenge to the cells. Cytotoxicity was assessed using the neutral red uptake assay. The concentration of compound that inhibited cell viability by 50% (ic(50) value) was calculated. All four oxysterols investigated induced the greatest cytotoxic effects following 96 h of exposure. 25-Hydroxycholesterol exhibited the greatest cytotoxicity in both cell lines. Both beta-epox and alpha-epox were more toxic to HepG2 cells than to Caco-2 cells after the 48-h exposure. Pretreatment of cells with either alpha- or gamma-TOC did not protect against oxysterol-induced cytotoxicity. The caco-2 cells treated with the high concentration (100 micro m) of gamma-TOC were found to be more susceptible to oxysterol-induced toxicity under the conditions employed in this study.