Development and validation of a gene expression test to identify hard-to-heal chronic venous leg ulcers.

BACKGROUND: Chronic venous leg ulcers pose a significant burden to healthcare systems, and predicting wound healing is challenging. The aim of this study was to develop a genetic test to evaluate the propensity of a chronic ulcer to heal. METHODS: Sequential refinement and testing of a gene expression signature was conducted using three distinct cohorts of human wound tissue. The expression of candidate genes was screened using a cohort of acute and chronic wound tissue and normal skin with quantitative transcript analysis. Genes showing significant expression differences were combined and examined, using receiver operating characteristic (ROC) curve analysis, in a controlled prospective study of patients with venous leg ulcers. A refined gene signature was evaluated using a prospective, blinded study of consecutive patients with venous ulcers. RESULTS: The initial gene signature, comprising 25 genes, could identify the outcome (healing versus non-healing) of chronic venous leg ulcers (area under the curve (AUC) 0·84, 95 per cent c.i. 0·73 to 0·94). Subsequent refinement resulted in a final 14-gene signature (WD14), which performed equally well (AUC 0·88, 0·80 to 0·97). When examined in a prospective blinded study, the WD14 signature could also identify wounds likely to demonstrate signs of healing (AUC 0·73, 0·62 to 0·84). CONCLUSION: A gene signature can identify people with chronic venous leg ulcers that are unlikely to heal.

The clinical significance and impact of interleukin 15 on keratinocyte cell growth and migration.

Chronic wounds represent a significant burden to health services and are associated with patient morbidity. Novel methods to diagnose and/or treat problematic wounds are needed. Interleukin (IL)-15 is a cytokine involved in a number of biological processes and disease states such as inflammation, healing and cancer progression. The current study explores the expression profile of IL-15 and IL-15 receptor α (IL-15Rα) in chronic wounds and its impact on keratinocytes. IL-15 and IL-15Rα expression were examined in healing and non-healing chronic wounds using qPCR and immunohistochemical analysis. The impact of recombinant IL-15 (rhIL-15) on human adult low calcium temperature (HaCaT) keratinocyte growth and migratory potential was further examined. IL-15 transcript expression was slightly, though non-significantly elevated in healing chronic wounds compared with non-healing chronic wounds. IL-15 protein staining was minimal in both subtypes of chronic wounds. By contrast, IL-15Rα transcript and protein expression were both observed to be enhanced in non-healing chronic wounds compared with healing chronic wounds. The treatment of HaCaT cells with rhIL-15 generally enhanced cell growth and promoted migration. Analysis with small molecule inhibitors suggested that the pro-migratory effect of rhIL-15 may be associated with ERK, AKT, PLCγ and FAK signalling. IL-15 may promote healing traits in keratinocytes and the differential expression of IL-15Rα is observed in chronic wounds. Together, this may imply a complex role for this interleukin in wound healing.

Reversal of Pathological Features of Graves' Orbitopathy by Activation of Forkhead Transcription Factors, FOXOs.

CONTEXT: Graves' orbitopathy (GO) is a disfiguring/distressing, inflammatory autoimmune condition. This intractable problem is caused by expansion of the orbital contents around the eye by excessive fat generation (adipogenesis) and overproduction of extracellular matrix components, especially hyaluronan (HA) from preadipocytes/fibroblasts (PFs). Current immunosuppressive/antiinflammatory treatments are largely ineffective and have unpleasant side effects, and a better therapeutic strategy through understanding GO-associated pathological features is needed. OBJECTIVE: Previously we identified depot-specific HA synthase 2 regulation (HAS2; major source of HA), which facilitates orbit-specific HA accumulation during adipogenesis, and targeting phosphatidylinositol-3-kinase/mechanistic target of rapamycin-complex-1 pathways blocked both pathological features. The current study revealed low expression levels of Forkhead box O (FOXOs; critical downstream effectors of phosphatidylinositol-3-kinase) in orbital PFs through adipogenesis compared with sc levels. We aimed to dissect the role of FOXOs in GO pathogenesis to identify nonimmunosuppressive targets for GO treatment. DESIGN/SETTING/PARTICIPANTS: Human orbital and sc primary PFs were treated with small interfering RNA/chemical inhibitor (AS1842856) of FOXOs or FOXO enhancer trifluoperazine hydrochloride (TFP; Food and Drug Administration approved drug), in serum-free medium for 24 hours, or TFP treatment in adipogenic medium for 15 days. MAIN OUTCOME MEASURES: Quantitative PCR was used to measure HAS2 transcripts and the terminal adipogenesis differentiation marker lipoprotein lipase. HA accumulation in the medium was measured by an ELISA. RESULTS: Substantially increased or decreased HAS2/HA production was observed by inhibiting (small interfering RNA or chemical inhibitor) or enhancing (TFP) FOXO expression, respectively. TFP treatment is also sufficient to counteract thyrotropin receptor-activated HAS2/HA production and block adipogenesis in orbital PFs. CONCLUSIONS: FOXOs play a crucial repressor role in the regulation of HAS2/HA production and adipogenesis in orbital PFs. Our data reveal for the first time that resetting GO-associated pathological features through drug-targeted activation of FOXOs could provide a feasible nonimmunosuppressive therapeutic strategy for GO.

Inflammatory-cell subpopulations in keloid scars.

The aim of this study was to investigate the contribution of lymphocytes and macrophages to keloid scarring by morphologically characterising inflammatory cell subpopulations in keloid scars in comparison with normal skin. We took 3mm punch biopsies from the anterior forearms of eight normal healthy volunteers. Eight keloid scars were excised using an intralesional technique. All tissue was snap frozen in liquid nitrogen and serial sections were stained with a panel of anti-inflammatory cell monoclonal antibodies. The numbers of macrophages and lymphocytes and the proportions of the subpopulations were compared. Higher numbers of both macrophages and lymphocytes were found in keloid dermis (P=0.01 and P=0.02, respectively (Mann-Whitney U -test)). There was no significant increase in the expression of the lymphocyte-activation markers, CD25 and CD27. However, there was a significantly higher CD4(+):CD8(+)(Th:Ts) ratio (P= 0.046) in keloid tissue. This suggests that an imbalance in these inflammatory cell subpopulations may contribute to keloid scarring in man.

The role of lymphocytes in human dermal wound healing.

BACKGROUND: Animal experimental data indicate a requirement for functionally active T lymphocytes to allow optimal healing of dermal wounds. Little evidence exists to confirm that this is the case in humans. Lymphocyte involvement in regulation of healing is probably mediated by release of secreted cytokines/growth factors, and we hypothesize that the cytokine profile requirement will be modulated as healing progresses. OBJECTIVES: As this is likely to be reflected in lymphocyte subset changes over the course of normal healing, we investigated the immunophenotype of lymphocyte subpopulations during wound healing. METHODS: Sequential biopsies were taken over 42 days from the margin of 12 wounds healing by secondary intention after pilonidal sinus excision. Serial biopsy sections were analysed by immunohistochemistry using lymphocyte-specific monoclonal antibodies, and lymphocytes were counted microscopically. RESULTS: Within 42 days, the mean decrease in wound volume was 87.5%. This was accompanied by significant changes in the wound margin lymphocyte population. Total numbers (mean +/- SEM) of T lymphocytes decreased from 36.8 +/- 9.8 cells per field at inclusion in the study to 25.9 +/- 3.0 immediately prior to wound closure, with a concomitant increase in B lymphocytes from 1 +/- 0.4 to 9.5 +/- 3.6 cells per field. The CD4/CD8 T-lymphocyte ratio fell from an initial level of 3.6 +/- 0.3 to 2.1 +/- 0.3 (mean +/- SEM) prior to closure. CONCLUSIONS: These data indicate that human wound-associated lymphocyte populations are modulated during healing; the increase in numbers of CD8+ T-suppressor lymphocytes is in accordance with previous animal data, indicating a role for these cells in downregulating healing as the wound closes. This study also documents an associated increase in B lymphocytes and healing of human wounds, with an as yet undefined role.

T lymphocytes and the lack of activated macrophages in wound margin biopsies from chronic leg ulcers.

The objective of this study was to characterize the leucocyte infiltrate which accumulates at the margin of chronic wounds. These leucocytes are a rich source of cytokines and growth factors, and an inappropriate function of these cells may contribute to the maintenance of wound chronicity. The leucocyte populations were stained immunohistochemically with monoclonal antibodies specific for surface receptors which give an indication of cellular function. Wound margin biopsies taken from chronic leg ulcers exhibited a localized infiltrate of CD45+ leucocytes associated with vascularized tissue in the dermis adjacent to the wound margin. Lymphocytes were identified in highest numbers in this area and CD45RO+ T lymphocytes predominated over B lymphocytes, which were either absent or present in very low numbers. In the majority of chronic wounds examined, CD4+ T lymphocytes were present in greater numbers than CD8+ T lymphocytes with a mean (+/-SD) ratio of CD4+:CD8+ of 1.5 +/- 0.6. CD68+ macrophages were identified in all layers of the dermis at the chronic wound margin. In 60% of wounds examined, macrophages were negative for the activation associated markers CD16 (Fc gamma III receptor) and CD35 (C3b receptor). In those biopsies where CD16 and CD35 positive macrophages were observed these were preferentially located in the perivascular regions. These data indicate that as monocytes extravasate into chronic wound tissue they may be subjected to microenvironmental influences which either suppress or do not induce macrophage activation. Suppression of macrophage activation may lead to an inappropriate cytokine/growth factor secretion and contribute to the maintenance of wound chronicity.

Axon-glial relationships in the anterior medullary velum of the adult rat.

The anterior medullary velum is a thin sheet of CNS tissue which roofs the rostral part of the IVth ventricle and contains fascicles of myelinated fibres which, in part, arise from the nucleus of the IVth cranial nerve. This study used histochemical, immunohistochemical, and intracellular dye-injection techniques to describe cellular interrelationships in the velum in whole-mounts and in sections. Rip antibody-stained whole mounts provided a unique description of both oligodendrocyte units (defined as an oligodendrocyte and the complement of myelinated internodal segments it forms), and consecutive myelin sheaths along the same axon. A broad range of unit morphologies was categorised into four arbitrary groups, according to classical criteria, which comprised small cells supporting the short, thin myelin sheaths of 15-30 small diameter axons (Type I), through intermediate types (II & III), to the largest cells forming the long, thick myelin sheaths of 1-3 large diameter axons. Rip antibody and ferric ion-ferrocyanide staining, together with intracellular dye injection, revealed oligodendrocyte process branching patterns and their mode of engagement of myelin sheaths, nodes of Ranvier, and the spatial disposition of the outer cytoplasmic rims of myelin sheaths. The latter formed a conspicuous spiral ridge on the exterior surface of myelin sheaths which connected with the paranodal loops at each heminode. Large bundles of axons decussated through the velum, the bulk of which were IVth nerve fibres which constituted the IVth nerve rootlet. The PNS/CNS transitional zone of the IVth nerve was located 0.25-0.50 mm along the root, where astrocytic end-feet defined an abrupt margin, convex towards the periphery, where the heminodes of central and peripheral myelin were apposed, and where the basal lamina tubes of the Schwann cell units were discontinued. The basal processes of ependymal cells lining the ventricular wall of the velum, passed between axon bundles before abutting on the basal lamina of the pia. Many of these processes branched and ran along the axonal bundles. A monolayer of microglia occupied a subependymal stratum in which the non-overlapping dendritic territories of each cell formed a regular mosaic throughout the velum without any obvious interaction with either axons or other glial cells. Astrocytes were also uniformly distributed; their fine processes made up a dense lattice amongst axons, often running parallel and within the fibre bundles; stouter ones had terminal end-feet which undercoated the basal lamina of both the glia limitans externa and the blood vessels in the velum.

Biochemical subtypes of oligodendrocyte in the anterior medullary velum of the rat as revealed by the monoclonal antibody Rip.

Oligodendrocytes were studied in the anterior medullary velum (AMV) of the rat using the monoclonal antibody Rip, an oligodendrocyte marker of unknown function. Confocal microscopic imaging of double immunofluorescent labelling with antibodies to Rip and carbonic anhydrase II (CAII) revealed two biochemically and morphologically distinct populations of oligodendrocyte which were either Rip+CAII+ or Rip+CAII-. Double immunofluorescent labelling with Rip and myelin basic protein (MBP) or glial fibrillary acidic protein (GFAP) provided direct evidence that Rip-labelled cells were phenotypically oligodendrocytes and confirmed that Rip did not recognise astrocytes. Oligodendrocytes which were Rip+CAII+ supported numerous myelin sheaths for small diameter axons, whilst Rip+CAII- oligodendrocytes supported fewer myelin sheaths for large diameter axons. Morphologically, Rip+CAII+ oligodendrocytes corresponded to types I or II of classical nomenclature, whilst Rip+CAII- oligodendrocytes corresponded to types III and IV. The results demonstrated a biochemical difference between oligodendrocytes which myelinated small and large diameter fibres.

In vitro allergenicity of cows' milk substitutes.

There are numerous alternatives to cows' milk formula for allergic children. We have investigated the allergenicity of several of these using RAST and RAST inhibition on serum from 16 patients with a known history of cows' milk protein intolerance (CMPI) and 16 atopic controls. A RAST grade of > or = 3 for cows' milk was present in all those with CMPI, whilst all the controls gave RAST of < or = 1. Modified cows' milk formula, goats' infant formula, sheep and goats' milk produced similar results to cows' milk. Only two patients had RAST > or = 3 for soya milk and the soy/beef hydrolysate gave positive results in only three patients. One had positive RAST to Nutramigen and two to Pregestimil. Of the whey hydrolysates investigated, Pepti-junior gave seven positive RASTs whilst we were unable to bind Alfare to the sepharose in sufficient quantities to interpret the results which were negative in all cases. RAST inhibition data on pooled sera from the same patients agreed with the RAST results. The inhibition curves showed high inhibition with goats', sheep, modified cows' milk formula and the casein formula, AL110 (50%). Soy and soy/beef hydrolysate showed a much lower inhibition pattern. Casein hydrolysates showed low inhibition while the whey hydrolysate produced higher inhibition. We have shown that despite claims of low allergenicity, some of these alternative formulae are antigenically recognized in vitro by some cows' milk intolerant patients.