Hypertrophic scars and keloids: Overview of the evidence and practical guide for differentiating between these abnormal scars.

Although hypertrophic scars and keloids both generate excessive scar tissue, keloids are characterized by their extensive growth beyond the borders of the original wound, which is not observed in hypertrophic scars. Whether or not hypertrophic scars and keloids are two sides of the same coin or in fact distinct entities remains a topic of much debate. However, proper comparison between the two ideally occurs within the same study, but this is the exception rather than the rule. For this reason, the goal of this review was to summarize and evaluate all publications in which both hypertrophic scars and keloids were studied and compared to one another within the same study. The presence of horizontal growth is the mainstay of the keloid diagnosis and remains the strongest argument in support of keloids and hypertrophic scars being distinct entities, and the histopathological distinction is less straightforward. Keloidal collagen remains the strongest keloid parameter, but dermal nodules and α-SMA immunoreactivity are not limited to hypertrophic scars alone. Ultimately, the current hypertrophic scars-keloid differences are mostly quantitative in nature rather than qualitative, and many similar abnormalities exist in both lesions. Nonetheless, the presence of similarities does not equate the absence of fundamental differences, some of which may not yet have been uncovered given how much we still have to learn about the processes involved in normal wound healing. It therefore seems pertinent to continue treating hypertrophic scars and keloids as separate entities, until such a time as new findings more decisively convinces us otherwise.

Correction to: Autologous tumor cell vaccination combined with systemic CpG-B and IFN-α promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial.

In the original publication of the article the following abstract and keywords were inadvertently omitted.

Autologous tumor cell vaccination combined with systemic CpG-B and IFN-α promotes immune activation and induces clinical responses in patients with metastatic renal cell carcinoma: a phase II trial.

Background: In this study the toxicity and efficacy of an irradiated autologous tumor cell vaccine (ATV) co-injected with a class-B CpG oligodeoxynucleotide (CpG-B) and GM-CSF, followed by systemic CpG-B and IFN-α administration, were examined in patients with metastatic renal cell carcinoma (mRCC). Methods: A single-arm Phase II trial was conducted, in which patients with mRCC were intradermally injected with a minimum of three whole-cell vaccines containing 0.7­1.3 × 107 irradiated autologous tumor cells (ATC), admixed with 1 mg CpG-B and 100 µg GM-CSF, followed by bi-weekly s.c. injections with 8 mg CpG-B and s.c. injections with 6 MU IFN-α three times per week. Results: Fifteen patients were treated according to the protocol. Treatment was well tolerated. Objective clinical responses occurred in three patients, including one long-term complete response. Disease stabilization occurred in another three patients. Positive delayed type hypersensitivity (DTH) responses to ATC were absent before treatment but present in 13 out of 15 patients during treatment. Immune monitoring revealed activation of plasmacytoid dendritic cells, non-classical monocytes and up-regulation of both PD-1 and CTLA4 on effector T cells upon treatment. Moreover, a pre-existing ex vivo IFN-γ response to ATC was associated with clinical response. Conclusions: ATV combined with systemic CpG-B and IFN-α is tolerable, safe, immunogenic and able to elicit anti-tumor responses in patients with mRCC. Immune activation and treatment-induced up-regulation of PD-1 and CTLA4 on circulating T cells further suggest an added benefit of combining this approach with immune checkpoint blockade [added]

Proteasome inhibition and mechanism of resistance to a synthetic, library-based hexapeptide.

Background The hexapeptide 4A6 (Ac-Thr(tBu)-His(Bzl)-Thr(Bzl)-Nle-Glu(OtBu)-Gly-Bza) was isolated from a peptide library constructed to identify peptide-based transport inhibitors of multidrug resistance (MDR) efflux pumps including P-glycoprotein and Multidrug Resistance-associated Protein 1. 4A6 proved to be a substrate but not an inhibitor of these MDR efflux transporters. In fact, 4A6 and related peptides displayed potent cytotoxic activity via an unknown mechanism. Objective To decipher the mode of cytotoxic activity of 4A6. Methods Screening of 4A6 activity was performed against the NCI60 panel of cancer cell lines. Possible interactions of 4A6 with the 26S proteasome were assessed via proteasome activity and affinity labeling, and cell growth inhibition studies with leukemic cells resistant to the proteasome inhibitor bortezomib (BTZ). Results The NCI60 panel COMPARE analysis revealed that 4A6 had an activity profile overlapping with BTZ. Consistently, 4A6 proved to be a selective and reversible inhibitor of ß5 subunit (PSMB5)-associated chymotrypsin-like activity of the 26S proteasome. This conclusion is supported by several lines of evidence: (i) inhibition of chymotrypsin-like proteasome activity by 4A6 and related peptides correlated with their cell growth inhibition potencies; (ii) 4A6 reversibly inhibited functional ß5 active site labeling with the affinity probe BodipyFL-Ahx3L3VS; and (iii) human myeloid THP1 cells with acquired BTZ resistance due to mutated PSMB5 were highly (up to 287-fold) cross-resistant to 4A6 and its related peptides. Conclusion 4A6 is a novel specific inhibitor of the ß5 subunit-associated chymotrypsin-like proteasome activity. Further exploration of 4A6 as a lead compound for development as a novel proteasome-targeted drug is warranted.

Immunostimulatory capacity of dental casting alloys on endotoxin responsiveness.

STATEMENT OF PROBLEM: Oral metal exposure has been associated with systemic and local adverse reactions, probably due to elemental release from the alloys. Although supraphysiological concentrations of salts from dentally applied metals can activate innate cells through TLR4 (Ni, Co, Pd) and TLR3 (Au), whether direct exposure to solid alloys can also trigger innate immune reactivity is still unknown. PURPOSE: The purpose of this in vitro study was to determine whether dental cast alloy specimens can activate innate cells and influence their responsiveness to bacterial endotoxin. MATERIAL AND METHODS: Human monocyte-derived dendritic cells (MoDC) and THP-1 cells were cultured on top of different alloy specimens (Ni-Cr, Co-Cr, Pd-Cu, Pd-Ag, Ti-6Al-4V, amalgam, gold, and stainless steel) or in alloy-exposed culture medium with or without endotoxin (lipopolysaccharide [LPS]; Escherichia coli 055:B5). Interleukin-8 (IL-8) production was used as the parameter for innate stimulation and evaluated by enzyme-linked immunosorbent assay after 24 hours of culture. The statistical significance of the effects of various casting alloys on the secretion of IL-8 was analyzed by using the nonparametric Wilcoxon rank sum test (α=.05). RESULTS: Dental cast alloys induced IL-8 production in MoDC and THP-1 cells, with Au and Pd-Cu providing the strongest stimulation. The alloy-exposed culture media tested contained sufficient stimulatory metal ions to induce detectable IL-8 production in THP-1 cells, except for the Ni-Cr and stainless steel exposed media. Au and Pd-Cu alloys were also most effective in potentiating LPS responsiveness as measured by IL-8 production. CONCLUSIONS: Using an in vitro culture system to expose MoDC and THP-1 cells to different alloy specimens this study showed that contact with the solid alloys, in particular when they contain Pd or Au, can trigger innate immune responses and augment responsiveness to bacterial endotoxin.

Local delivery of CpG-B and GM-CSF induces concerted activation of effector and regulatory T cells in the human melanoma sentinel lymph node.

Impaired immune effector functions in the melanoma sentinel lymph node (SLN) may allow for early metastatic events. In an effort to determine the optimal way to strengthen immune defenses, 28 clinical stage I-II melanoma patients were randomized in a 3-arm Phase II study to receive, prior to excision and sampling of the SLN, i.d. injections of saline or low-dose CpG-B (CpG), alone or combined with GM-CSF (GM), around the melanoma excision site. We previously described the combined administration of these DC-targeting agents to result in activation and recruitment of potentially cross-presenting BDCA3(+) DCs to the SLN. In this report we describe the effects on effector and regulatory T and NK cell subsets. Local low-dose CpG administration resulted in lower CD4/CD8 ratios, Th1 skewing, increased frequencies of melanoma-specific CD8(+) T cells and possible recruitment of effector NK cells, irrespective of GM co-administration. These immune-potentiating effects were counterbalanced by increased IL-10 production by T cells and significantly higher levels of FoxP3 and CTLA4 in regulatory T cells (Tregs) with correspondingly higher suppressive activity in the SLN. Notably, CpG ± GM-administered patients showed significantly lower numbers of SLN metastases (saline: 4/9, CpG + GM: 1/9, CpG: 0/10, p = 0.04). These findings indicate that i.d. delivery of low-dose CpG ± GM potentially arms the SLN of early-stage melanoma patients against metastatic spread, but that antitumor efficacy may be further boosted by counteracting the collateral activation of Tregs.

Palladium-based dental alloys are associated with oral disease and palladium-induced immune responses.

BACKGROUND: Palladium (Pd) and gold (Au) based dental alloys have been associated with oral disease. OBJECTIVES: This study was designed to explore possible associations between the presence of Au-based and Pd-based dental alloys, and oral lesions, systemic complaints, and specific in vivo and in vitro immune responses. METHODS: The investigated population consisted of three groups: 26 non-metal-allergic volunteers, 25 metal-allergic patients, and 20 oral disease patients. Medical histories were taken, oral examinations were carried out, and compositions of all dental alloys were determined. Then, Au and Pd patch tests and in vitro assays were performed, revealing cytokine production by peripheral blood mononuclear cells [T helper (Th)1, interferon-γ; Th2, interleukin (IL)-5 and IL-13] and lymphocyte proliferation (LTT-MELISA(®) ). RESULTS: Non-plaque-related gingivitis was associated with the presence of Pd-based dental alloys, and Pd-positive patch tests and in vitro assays. Collectively, participants with Pd-based dental alloys showed increased Pd patch test reactivity (p < 0.05) and lymphoproliferation (p < 0.05). In contrast, oral lichenoid lesions were associated with Au-based alloys (p < 0.05), but this was not reflected by Au-specific immunoreactivity. CONCLUSIONS: Oral lesions and Pd-induced immune responses are associated with the presence of dental alloys. However, most oral disease patients did not show positive patch test results or in vitro signs of specific immunoreactivity, suggesting local toxic reactions or the involvement of innate immune responses.

T cell profiling reveals high CD4+CTLA-4 + T cell frequency as dominant predictor for survival after prostate GVAX/ipilimumab treatment.

Immune checkpoint blockade enhances antitumor responses, but can also lead to severe immune-related adverse events (IRAE). To avoid unnecessary exposure to these potentially hazardous agents, it is important to identify biomarkers that correlate with clinical activity and can be used to select patients that will benefit from immune checkpoint blockade. To understand the consequences of CTLA-4 blockade and identify biomarkers for clinical efficacy and/or survival, an exploratory T cell monitoring study was performed in a phase I/II dose escalation/expansion trial (n = 28) of combined Prostate GVAX/ipilimumab immunotherapy. Phenotypic T cell monitoring in peripheral blood before and after Prostate GVAX/ipilimumab treatment revealed striking differences between patients who benefited from therapy and patients that did not. Treatment-induced rises in absolute lymphocyte counts, CD4(+) T cell differentiation, and CD4(+) and CD8(+) T cell activation were all associated with clinical benefit. Moreover, significantly prolonged overall survival (OS) was observed for patients with high pre-treatment frequencies of CD4(+)CTLA-4(+), CD4(+)PD-1(+), or differentiated (i.e., non-naive) CD8(+) T cells or low pre-treatment frequencies of differentiated CD4(+) or regulatory T cells. Unsupervised clustering of these immune biomarkers revealed cancer-related expression of CTLA-4(+) in CD4(+) T cells to be a dominant predictor for survival after Prostate GVAX/ipilimumab therapy and to thus provide a putative and much-needed biomarker for patient selection prior to therapeutic CTLA4 blockade.

IL-21 promotes the expansion of CD27+ CD28+ tumor infiltrating lymphocytes with high cytotoxic potential and low collateral expansion of regulatory T cells.

BACKGROUND: Adoptive cell transfer of tumor infiltrating lymphocytes has shown clinical efficacy in the treatment of melanoma and is now also being explored in other tumor types. Generation of sufficient numbers of effector T cells requires extensive ex vivo expansion, often at the cost of T cell differentiation and potency. For the past 20 years, IL-2 has been the key cytokine applied in the expansion of TIL for ACT. However, the use of IL-2 has also led to collateral expansion of regulatory T cells (Tregs) and progressive T cell differentiation, factors known to limit in vivo persistence and activity of transferred TIL. The use of alternative T cell growth factors is therefore warranted. Here, we have compared the effects of IL-2, -15 and -21 cytokines on the expansion and activation of TIL from single-cell suspensions of non-small cell lung cancer, ovarian cancer and melanoma. METHODS: We applied the K562-based artificial APC (aAPC) platform for the direct and rapid expansion of tumor infiltrating lymphocytes isolated from primary cancer specimens. These aAPC were engineered to express the Fc-γ receptor CD32 (for anti-CD3 antibody binding), the co-stimulatory molecule 4-1BBL, and to secrete either IL-2, IL-15 or IL-21 cytokine. RESULTS: Although IL-2 aAPC induced the greatest overall TIL expansion, IL-21 aAPC induced superior expansion of CD8+ T cells with a CD27+ CD28+ "young" phenotype and superior functional cytotoxic effector characteristics, without collateral expansion of Tregs. CONCLUSION: Our data rationalize the clinical application of IL-21-secreting aAPC as a standardized cell-based platform in the expansion of "young" effector TIL for ACT.

Characterization of four conventional dendritic cell subsets in human skin-draining lymph nodes in relation to T-cell activation.

To increase (tumor) vaccine efficacy, there is an urgent need for phenotypic and functional characterization of human dendritic cell (DC) subsets residing in lymphoid tissues. In this study we identified and functionally tested 4 human conventional DC (cDC) subsets within skin-draining sentinel lymph nodes (SLNs) from early-stage melanoma patients. These SLNs were all tumor negative and were removed on average 44 days after excision of the primary melanoma. As such, they were considered representative of steady-state conditions. On comparison with skin-migrated cDC, 2 CD1a(+) subsets were identified as most likely skin-derived CD11c(int) Langerhans cells (LC) with intracellular langerin and E-cadherin expression or as CD11c(hi) dermal DCs with variable expression of langerin. Two other CD1a(-) LN-residing cDC subsets were characterized as CD14(-)BDCA3(hi)CD103(-) and CD14(+)BDCA3(lo)CD103(+), respectively. Whereas the CD1a(+) skin-derived subsets displayed greater levels of phenotypic maturation, they were associated with lower levels of inflammatory cytokine release and were inferior in terms of allogeneic T-cell priming and IFNγ induction. Thus, despite their higher maturation state, skin-derived cDCs (and LCs in particular) proved inferior T-cell activators compared with the CD1a(-) cDC subsets residing in melanoma-draining LNs. These observations should be considered in the design of DC-targeting immunotherapies.

Autologous skin substitute for hard-to-heal ulcers: retrospective analysis on safety, applicability, and efficacy in an outpatient and hospitalized setting.

Chronic ulcers ((arterio)venous, decubitus, or postoperative) have no tendency to heal within a period of at least 3 months despite optimal therapy according to internationally accepted guidelines. This retrospective study evaluates the safety and efficacy of an autologous, dermal-epidermal skin substitute (SS) for treating ulcers of various origins. Ulcers were treated within 7 Dutch centers over 5 years. Sixty-six ulcers (size: 0.75-150 cm²; duration: 0.25-32 years) with a follow-up time of 24 weeks after a single-skin substitute application were assessed. Wound-bed preparation consisted of vacuum-assisted-closure-therapy (5 days, hospitalized) or application of acellular dermis (5-7 days, outpatient). Time to heal, adverse events, and recurrence 1 year after complete healing were recorded. Complete ulcer healing occurred in 36 of 66 ulcers (55%) at 24 weeks. At that time point, a further 29% of ulcers showed decrease in ulcer size between 50 and 99%. No difference was observed between the hospitalized vs. outpatient treatment with complete healing. There were 32 of 36 healed ulcers that were available for follow-up 1 year after complete closure, of which 27 (84%) were still closed. Only two minor/moderate possibly related adverse events were recorded. This retrospective analysis shows that SS provides a safe and successful treatment for particularly chronic ulcers of various origins.

Transition metal sensing by Toll-like receptor-4: next to nickel, cobalt and palladium are potent human dendritic cell stimulators.

BACKGROUND: Nickel was recently identified as a potent activator of dendritic cells through ligating with human Toll-like receptor (TLR)-4. OBJECTIVES: Here, we studied an extended panel of transition metals neighbouring nickel in the periodic table of elements, for their capacity to activate human monocyte-derived dendritic cells (MoDCs). METHODS: The panel included chromium, cobalt, and palladium, all of which are known to be frequent clinical sensitizers. MoDC activation was monitored by assessment of release of the pro-inflammatory mediator interleukin (IL)-8, a major downstream result of TLR ligation. Results The data obtained in the present study show that cobalt and palladium also have potent MoDC-activating capacities, whereas copper and zinc, but not iron and chromium, have low but distinct MoDC-activating potential. Involvement of endotoxin contamination in MoDC activation was excluded by Limulus assays and consistent stimulation in the presence of polymyxin B. The critical role of TLR4 in nickel-induced, cobalt-induced and palladium-induced activation was confirmed by essentially similar stimulatory patterns obtained in an HEK293 TLR4/MD2 transfectant cell line. CONCLUSIONS: Given the adjuvant role of costimulatory danger signals, the development of contact allergies to the stimulatory metals may be facilitated by signals from direct TLR4 ligation, whereas other metal sensitizers, such as chromium, may rather depend on microbial or tissue-derived cofactors to induce clinical sensitization.

Combined immunotherapy with granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells and ipilimumab in patients with metastatic castration-resistant prostate cancer: a phase 1 dose-escalation trial.

BACKGROUND: The granulocyte-macrophage colony-stimulating factor-transduced allogeneic prostate cancer cells vaccine (GVAX) has antitumour activity against prostate cancer; preclinical studies have shown potent synergy when combined with ipilimumab, an antibody that blocks cytotoxic T-lymphocyte antigen 4. We aimed to assess the safety of combined treatment with GVAX and ipilimumab in patients with metastatic castration-resistant prostate cancer (mCRPC). METHODS: We did an open-labelled, single-centre, dose-escalation study of ipilimumab concurrent with a fixed dose of GVAX, with a subsequent expansion phase, both at the VU University Medical Centre (Amsterdam, Netherlands). Eligible patients had documented mCRPC and had not been previously treated with chemotherapy. All patients received a 5×10(8) cell priming dose of GVAX intradermally on day 1 with subsequent intradermal injections of 3×10(8) cells every 2 weeks for 24 weeks. The vaccinations were combined with intravenous ipilimumab every 4 weeks. We enrolled patients in cohorts of three; each cohort received an escalating dose of ipilimumab at 0·3, 1·0, 3·0, or 5·0 mg/kg. Our primary endpoint was safety. This study is registered with ClinicalTrials.gov, number NCT01510288. FINDINGS: We enrolled 12 patients into our dose-escalation cohort. We did not record any severe immune-related adverse events at the first two dose levels. At the 3·0 mg/kg dose level, one patient had grade 2 and two patients grade 3 hypophysitis; at the 5·0 mg/kg dose level, two patients had grade 3 hypophysitis and one patient developed grade 4 sarcoid alveolitis (a dose-limiting toxic effect). Due to observed clinical activity and toxic events, we decided to expand the 3·0 mg/kg dose level, rather than enrol a further three patients at the 5·0 mg/kg level. 16 patients were enrolled in the expansion cohort, two of whom developed grade 2 hypophysitis, three colitis (one grade 1 and two grade 2), and one grade 3 hepatitis--all immune-related adverse events. The most common adverse events noted in all 28 patients were injection-site reactions (grade 1-2 events seen in all patients), fatigue (grade 1-2 in 20 patients, grade 3 in two), and pyrexia (grade 1-2 in 15 patients, grade 3 in one). 50% or greater declines in prostate-specific antigen from baseline was recorded in seven patients (25%); all had received 3·0 mg/kg or 5·0 mg/kg ipilimumab. INTERPRETATION: GVAX combined with 3·0 mg/kg ipilimumab is tolerable and safe for patients with mCRPC. Further research on the combined treatment of patients with mCRPC with vaccination and ipilimumab is warranted. FUNDING: Cell Genesys Inc, Prostate Cancer Foundation, Dutch Cancer Society (KWF-VU 2006-3697), and Foundation Stichting VUmc Cancer Center Amsterdam.

Activated iNKT cells promote Vγ9Vδ2-T cell anti-tumor effector functions through the production of TNF-α.

Vγ9Vδ2-T cells constitute a proinflammatory lymphocyte subpopulation with established antitumor activity. Phosphoantigens activate Vγ9Vδ2-T cells in vivo and in vitro. We studied whether the antitumor activity of Vγ9Vδ2-T cells can be potentiated by invariant NKT cells (iNKT), an important immunoregulatory T cell subset. When activated by the glycolipid α-galactosylceramide (α-GalCer), iNKT produce large amounts of cytokines involved in antitumor immune responses. Monocyte-derived dendritic cells were loaded with both phosphoantigens (using aminobisphosphonates) and α-GalCer during maturation and subsequently co-cultured with Vγ9Vδ2-T and iNKT cells. Aminobisphosphonates dose-dependently enhanced Vγ9Vδ2-T cell activation, and this was potentiated by α-GalCer-induced iNKT co-activation. iNKT co-activation also enhanced the IFN-γ production and cytolytic potential of Vγ9Vδ2-T cells against tumor cells. Using transwell experiments and neutralizing antibodies cross-talk between iNKT and Vγ9Vδ2-T cells was found to be mediated by TNF-α. Our data provide a rationale for combining both activating ligands to improve Vγ9Vδ2-T cell based approaches in cancer-immunotherapy.

CD40-targeted adenoviral cancer vaccines: the long and winding road to the clinic.

The ability of dendritic cells (DCs) to orchestrate innate and adaptive immune responses has been exploited to develop potent anti-cancer immunotherapies. Recent clinical trials exploring the efficacy of ex vivo modified autologous DC-based vaccines have reported some promising results. However, in vitro generation of autologous DCs for clinical administration, their loading with tumor associated antigens (TAAs) and their activation, is laborious and expensive, and, as a result of inter-individual variability in the personalized vaccines, remains poorly standardized. An attractive alternative approach is to load resident DCs in vivo by targeted delivery of TAAs, using viral vectors and activating them simultaneously. To this end, we have constructed genetically-modified adenoviral (Ad) vectors and bispecific adaptor molecules to retarget Ad vectors encoding TAAs to the CD40 receptor on DCs. Pre-clinical human and murine studies conducted so far have clearly demonstrated the suitability of a 'two-component' (i.e. Ad and adaptor molecule) configuration for targeted modification of DCs in vivo for cancer immunotherapy. This review summarizes recent progress in the development of CD40-targeted Ad-based cancer vaccines and highlights pre-clinical issues in the clinical translation of this approach.

Exposure of CD34+ precursors to cytostatic anthraquinone-derivatives induces rapid dendritic cell differentiation: implications for cancer immunotherapy.

Appropriate activation of dendritic cells (DC) is essential for successful active vaccination and induction of cell-mediated immunity. The scarcity of precursor cells, as well as long culture methods, have hampered wide-scale application of DC vaccines derived from CD34(+) precursors, despite their suggested superior efficacy over the more commonly applied monocyte-derived DC (MoDC). Here, employing the CD34(+)/CD14(+) AML-derived human DC progenitor cell line MUTZ3, we show that cytostatic anthraquinone-derivatives (i.e., the anthracenedione mitoxantrone and the related anthracyclin doxorubicin) induce rapid differentiation of CD34(+) DC precursors into functional antigen-presenting cells (APC) in a three-day protocol. The drugs were found to act specifically on CD34(+), and not on CD14(+) DC precursors. Importantly, these observations were confirmed for primary CD34(+) and CD14(+) DC precursors from peripheral blood. Mitoxantrone-generated DC were fully differentiated within three days and after an additional 24 h of maturation, were as capable as standard 9-day differentiated and matured DC to migrate toward the lymph node-homing chemokines CCL19 and CCL21, to induce primary allogeneic T cell proliferation, and to prime functional MART1-specific CD8(+) T lymphocytes. Our finding that anthraquinone-derivatives like mitoxantrone support rapid high-efficiency differentiation of DC precursors may have consequences for in vitro production of DC vaccines as well as for novel immunochemotherapy strategies.

Preferential Langerhans cell differentiation from CD34(+) precursors upon introduction of ABCG2 (BCRP).

Epidermal Langerhans cells (LC) and dermal interstitial dendritic cells (IDC) were found to express the ATP-binding cassette (ABC) transporter breast cancer resistance protein (BCRP; ABCG2). Also, low BCRP expression was present on CD34(+) blood DC precursors and expression was increased upon their differentiation to LC. The CD34(+) acute myeloid leukemia-derived DC cell line MUTZ3 can be cultured into LC or IDC, depending on the cytokine cocktail used. Introduction of functional BCRP in MUTZ3 progenitor cells through retroviral transduction resulted in the emergence of typical LC-characteristics in IDC cultures; the majority of cells remained negative for the IDC-specific C-type lectin DC-SIGN, but rather displayed enhanced expression of the LC-specific C-type lectin Langerin and characteristic high expression levels of CD1a. BCRP-induced skewing toward LC-like differentiation coincided with early RelB expression in 'IDC', derived from MUTZ3-BCRP, and depended on endogenous transforming growth factor beta (TGF-ß) production. Intriguingly, cellular BCRP localization differed between skin LC and IDC, and a more cytoplasmic BCRP localization, as observed in primary skin LC, seemed to relate to LC-like differentiation in IDC cultures upon BCRP introduction in MUTZ3 progenitors. Together these data support a role for BCRP in preferential LC differentiation from CD34(+) myeloid DC progenitors.

Inactivating PSMB5 mutations and P-glycoprotein (multidrug resistance-associated protein/ATP-binding cassette B1) mediate resistance to proteasome inhibitors: ex vivo efficacy of (immuno)proteasome inhibitors in mononuclear blood cells from patients with rheumatoid arthritis.

Bortezomib (BTZ), a registered proteasome inhibitor (PI) for multiple myeloma, has also been proposed as a potential antirheumatic agent. Its reported side effects, however, make it unappealing for long-term administration, and resistance may also develop. To overcome this, second-generation PIs became available. Here, we investigated whether a novel class of peptide epoxyketone-based PIs, including carfilzomib, N-((S)-3-methoxy-1-(((S)-3-methoxy-1-(((S)-1-((R)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)amino)-1-oxopropan-2-yl)amino)-1-oxopropan-2-yl)-2-methylthiazole-5-carboxamide (ONX0912), and (S)-3-(4-methoxyphenyl)-N-((S)-1-((S)-2-methyloxiran-2-yl)-1-oxo-3-phenylpropan-2-yl)-2-((S)-2-(2-morpholinoacetamido)propanamido)propanamide (ONX0914), might escape two established BTZ-resistance mechanisms: 1) mutations in the proteasome ß5 subunit (PSMB5) targeted by these PIs, and 2) drug efflux mediated by ATP-binding cassette transporters. THP1 myeloid sublines with acquired resistance to BTZ (54- to 235-fold) caused by mutations in the PSMB5 gene displayed marked cross-resistance but less pronounced cross-resistance to carfilzomib (9- to 32-fold), ONX0912 (39- to 62-fold), and ONX0914 (27- to 97-fold). As for ATP-binding cassette transporter-mediated efflux, lymphoid CEM/VLB cells with P-glycoprotein (Pgp)/multidrug resistance 1 overexpression exhibited substantial resistance to carfilzomib (114-fold), ONX0912 (23-fold), and ONX0914 (162-fold), whereas less resistance to BTZ (4.5-fold) was observed. Consistently, ß5 subunit-associated chymotrypsin-like proteasome activity was significantly less inhibited in these CEM/VLB cells. Ex vivo analysis of peripheral blood mononuclear cells from therapy-naive patients with rheumatoid arthritis revealed that, although basal Pgp levels were low, P-glycoprotein expression compromised the inhibitory effect of carfilzomib and ONX0914. However, the use of P121 (reversin 121), a Pgp transport inhibitor, restored parental cell inhibitory levels in both CEM/VLB cells and peripheral blood mononuclear cells. These results indicate that the pharmacologic activity of these PIs may be hindered by drug resistance mechanisms involving PSMB5 mutations and PI extrusion via Pgp.

The ABC of dendritic cell development and function.

ATP-binding cassette (ABC) transporters are known for their involvement in clinical multidrug resistance (MDR) and their physiological defensive functions in barrier organs. More recently, attention has been focused on their possible involvement in the regulation of immune responses following the identification of their substrates as known immunomodulating agents (e.g. prostaglandins, leukotrienes and cyclic nucleotides) and their functional expression in various immune effector cells, most notably in dendritic cells (DCs). This review addresses the possible roles of ABC transporters in DC development and function, as well as the putative immunostimulatory potential of their cytostatic substrates and how this knowledge might benefit DC-based chemo-immunotherapies.

Simple wound exudate collection method identifies bioactive cytokines and chemokines in (arterio) venous ulcers.

A major challenge for clinicians treating (arterio) venous leg ulcers is to decide between standard therapy and advanced interventions. Here, we developed a simple method to collect human material representative of the ulcer wound bed, which can be used to identify biomarkers for prognostic test development. Superficial surgical debridement was performed using a small vidal curette during the weekly visit to the outpatient clinic. Moist, easily removable debridement material essentially blood free (including necrotic and nonviable slough) was collected from the surface of the ulcer. The amount ranged from 5.5 mg to 78 mg material per ulcer. Seventeen cytokines, chemokines, and growth factors were extracted and analyzed by enzyme-linked immunosorbent assay (concentration range: 0.0005-78 ng/mg total protein). Notably, CXCL8 was by far the most abundant protein present. Inflammatory mediators were more abundant than anti-inflammatory mediators (e.g., interleukin (IL)-10 and transforming growth factor-ß1). Bioactivity assays showed chronic wound extracts to be capable of stimulating fibroblast migration in a chemokine-dependent manner and also capable of stimulating healthy cells within skin substitutes to secrete wound healing mediators (CCL2, CXCL1, CXCL8, IL-6) in an IL-1α dependent manner. Collection of debridement tissue enables investigation of the ulcer environment in an easy noninvasive manner that may be suitable for prognostic test development.