IL6/JAK1/STAT3 Signaling Blockade in Endometrial Cancer Affects the ALDHhi/CD126+ Stem-like Component and Reduces Tumor Burden.

Cancer stem-like cells (CSC) may be critical to maintain the malignant behavior of solid and hematopoietic cancers. Recently, patients with endometrial cancer whose tumors expressed high levels of aldehyde dehydrogenase (ALDH), a detoxifying enzyme characteristic of many progenitor and stem cells, exhibited a relative reduction in survival compared with patients with low levels of ALDH. Given evidence of its role as a CSC marker, we hypothesized that high level of ALDH activity (ALDH(hi)) in a tumor might positively correlate with the presence of stem- and progenitor-like tumor cells in this disease setting. In support of this hypothesis, ALDH could be used to enrich for CSC in endometrial cancer cell lines and primary tumors, as illustrated by the increased tumor-initiating capacity of ALDH(hi) cells in immunodeficient mice. ALDH(hi) cells also exhibited greater clonogenic and organoid-forming capacity compared with ALDH(lo) cells. Notably, the number of ALDH(hi) cells in tumor cell lines and primary tumors inversely correlated with differentiation grade. Expression analysis revealed upregulation of IL6 receptor subunits and signal transducers CD126 and GP130 in ALDH(hi) endometrial cancer cells. Accordingly, targeted inhibition of the IL6 receptor and its downstream effectors JAK1 and STAT3 dramatically reduced tumor cell growth. Overall, our results provide a preclinical rationale to target IL6 or its effector functions as a novel therapeutic option in endometrial cancer.

A mouse model for endometrioid ovarian cancer arising from the distal oviduct.

Ovarian cancer is the deadliest gynecological malignancy in Western countries. Early detection, however, is hampered by the fact that the origin of ovarian cancer remains unclear. Knowing that in a high percentage of endometrioid ovarian cancers Wnt/ß-catenin signaling is activated, and in view of the hypothesis that ovarian cancer may originate from the distal oviduct, we studied mice in which Wnt/ß-catenin signaling was activated in Müllerian duct-derived tissues. Conditional adenomatous polyposis coli (Apc) knockout mice were used to study the activation of Wnt/ß-catenin signaling in Müllerian duct-derived organs. These Pgr(Cre/+);Apc(ex15lox/lox) mice (n = 44) were sacrificed at 10, 20, 40 and 80 weeks and uterus, oviduct, ovaries and surrounding fat tissues were assessed using immunohistochemistry. Using nuclear ß-catenin staining, Wnt/ß-catenin signaling activation was confirmed in the entire epithelium of the adult Müllerian duct (fimbriae, oviduct and endometrium), but was absent in ovarian surface epithelium cells (OSEs). Besides endometrial hyperplasia, in 87.2% of mice intraepithelial lesions of the distal oviduct were found, whereas OSEs remained unaffected. In addition, 62.5% of mice developed tumors in the distal and fimbrial part of the oviduct. In the ovaries, mainly at young age, in 16.3% of mice, simple epithelial cysts were noted, which developed further into endometrioid ovarian tumors, resembling human endometrioid ovarian cancer (27.9% of mice). Next to this, locoregional growth in the utero-ovarian ligament was also shown. Here, for the first time, mutations (activation of Wnt/ß-catenin) in the distal oviduct result in precursor lesions that develop into ovarian tumors, resembling human endometrioid ovarian cancer.

Alterations in Wnt-ß-catenin and Pten signalling play distinct roles in endometrial cancer initiation and progression.

Endometrioid endometrial cancer arises through a gradual series of histological changes, each accompanied by specific alterations in gene expression and activity. Activation of the Wnt-ß-catenin pathway and loss of PTEN activity are frequently observed in endometrial cancers. However, the specific roles played by alterations in these pathways in the initiation and progression of endometrial cancer are currently unclear. Here, we investigated the effects of loss of Pten and Apc gene function in the mouse endometrium by employing tissue-specific and inducible mutant alleles, followed by immunohistochemical (IHC) and loss of heterozygosity (LOH) analysis of their corresponding cancerous lesions. Loss of the Apc function in the endometrium leads to cytoplasmic and nuclear ß-catenin accumulation in association with uterine hyperplasia and squamous cell metaplasia, but without malignant transformation. Loss of Pten function also resulted in squamous metaplasia but, in contrast to loss of Apc function, it initiates endometrial cancer. On the other hand, loss of Apc function in the endometrium accelerates Pten-driven endometrial tumourigenesis. Analysis of compound heterozygous mice confirmed that somatic loss of the wild-type Pten allele represents the rate-limiting initiation step in endometrial cancer. Simultaneous loss of Pten and Apc resulted in endometrial cancer characterized by earlier onset and a more aggressive malignant behaviour. These observations are indicative of the synergistic action between the Wnt-ß-catenin and Pten signalling pathways in endometrial cancer onset and progression.

An autoimmune phenotype in vulvar lichen sclerosus and lichen planus: a Th1 response and high levels of microRNA-155.

Vulvar lichen sclerosus and lichen planus are T-cell-mediated chronic skin disorders. Although autoimmunity has been suggested, the exact pathogenesis of these disorders is still unknown. Therefore, the aim of the current study was to investigate the molecular and immunological mechanisms critical to the pathogenesis of vulvar lichen sclerosus and lichen planus. By using gene expression profiling and real-time RT-PCR experiments, we demonstrated a significantly increased expression of the pro-inflammatory cytokines (IFNγ, CXCR3, CXCL9, CXCL10, CXCL11, CCR5, CCL4, and CCL5) specific for a Th1 IFNγ-induced immune response. In addition, BIC/microRNA-155 (miR-155)--a microRNA involved in regulation of the immune response--was significantly upregulated in lichen sclerosus and lichen planus (9.5- and 17.7-fold change, respectively). Immunohistochemistry showed a significant T-cell response, with pronounced dermal infiltrates of CD4(+), CD8(+), and FOXP3(+) cells. In conclusion, these data demonstrate an autoimmune phenotype in vulvar lichen sclerosus and lichen planus, characterized by increased levels of Th1-specific cytokines, a dense T-cell infiltrate, and enhanced BIC/miR-155 expression.

Different DNA damage and cell cycle checkpoint control in low- and high-risk human papillomavirus infections of the vulva.

Human papillomavirus (HPV) infections may result in benign hyperplasia, caused by low-risk HPV types, or (pre)malignant lesions caused by high-risk HPV types. The molecular basis of this difference in malignant potential is not completely understood. Here, we performed gene profiling of different HPV infected vulvar tissues (condylomata acuminata (n = 5), usual type vulvar intraepithelial neoplasia (uVIN) (n = 9)) and control samples (n = 14) using Affymetrix Human U133A plus 2 GeneChips. Data were analyzed using OmniViz®, Partek® and Ingenuity® Software. Results were validated by real-time RT-PCR and immunostaining. Although similarities were observed between gene expression profiles of low- and high-risk HPV infected tissues (e.g., absence of estrogen receptor in condylomata and uVIN), high-risk HPV infected tissues showed more proliferation and displayed more DNA damage than tissues infected with low-risk HPV. These observations were confirmed by differential regulation of cell cycle checkpoints and by increased expression of DNA damage-biomarkers p53 and γH2AX. Furthermore, FANCA, FANCD2, BRCA1 and RAD51, key players in the DNA damage response, were significantly upregulated (p < 0.05). In addition, we compared our results with publicly available gene expression profiles of various other HPV-induced cancers (vulva, cervix and head-and-neck). This showed p16(INK4a) was the most significant marker to detect a high-risk HPV infection, but no other markers could be found. In conclusion, this study provides insight into the molecular basis of low- and high-risk HPV infections and indicates two main pathways (cell cycle and DNA damage response) that are much stronger affected by high-risk HPV as compared to low-risk HPV.

Treatment of vulvar intraepithelial neoplasia with topical imiquimod: seven years median follow-up of a randomized clinical trial.

OBJECTIVE: Recently we reported on the efficacy of imiquimod for treating vulvar intraepithelial neoplasia (VIN) in a placebo-controlled, double-blinded randomized clinical trial (RCT). Four weeks after treatment, a complete response was observed in 35% of patients and a partial response in 46%. All complete responders remained disease-free at 12 months follow-up. In the current investigations, we assessed long-term follow-up at least 5 years after the initial RCT. METHODS: Twenty-four of 26 imiquimod-treated patients who had participated in the initial RCT were seen for follow-up. Primary endpoint was durability of clinical response to imiquimod assessed by naked eye vulvar examination and histology. Long-term clinical response was correlated to lesion size before start of the initial RCT. Secondary endpoints were mental health, global quality of life, body image and sexual function in relation with long-term clinical response. RESULTS: Median follow-up period was 7.2 years (range 5.6-8.3 years). VIN recurred in one of nine complete responders. Of the initial partial responders, two became disease-free after additional imiquimod treatment. In the other partial responders, VIN recurred at least once after the initial RCT. In long-term complete responders, lesion size at study entry was smaller and these patients had a significantly better global quality of life at follow-up than patients with residual disease and/or recurrence after imiquimod treatment. CONCLUSIONS: In case of a complete response, imiquimod is effective in the long-term. Furthermore, patients with a long-term complete response had a significantly better global quality of life than patients who recurred after imiquimod treatment.

Nonsteroidal anti-inflammatory drugs do not interfere with imiquimod treatment for usual type vulvar intraepithelial neoplasia.

Imiquimod has been shown to be an effective treatment for usual type vulvar intraepithelial neoplasia (uVIN). Since local inflammation and burning are common side effects, patients often use nonsteroidal anti-inflammatory drugs (NSAIDs). Our study investigated whether NSAID-use, which has been documented to inhibit the cell-mediated immune response, interferes with the outcome of imiquimod treatment. Monocyte-derived dendritic cells (moDCs) and Langerhans cells (moLCs) were cultured in the presence of NSAIDs. The expression of relevant surface markers (CD80, CD86, CD40, HLA-DR, CCR6 and CCR7), stimulatory function, and cytokine production were evaluated. Furthermore, we analyzed in uVIN patients whether frequent NSAID-use had an effect on the clinical response and on immunocompetent cell counts before and after imiquimod treatment. Although an effect was observed on the expression of moDC and moLC maturation markers, NSAIDs did not affect the ability of moDCs and moLCs to stimulate allogeneic T-cell proliferation, or the production of cytokines in an allogeneic T-cell stimulation assay. In agreement with this, in uVIN patients treated with imiquimod, no interference of frequent NSAID-use with clinical outcome was observed. However, we did notice that high CD1a(+) and CD207(+) cell counts in frequent NSAID-users before treatment seemed to predict a favourable response to imiquimod treatment. Our data indicate that NSAID-use does not seem to interfere with moDC and moLC function and does not interfere with immunomodulatory properties of imiquimod in uVIN patients. Therefore, NSAIDs can safely be used to reduce imiquimod side effects in uVIN patients during treatment.

Imiquimod-induced clearance of HPV is associated with normalization of immune cell counts in usual type vulvar intraepithelial neoplasia.

Recently, we reported on the efficacy of imiquimod for treatment of usual type vulvar intraepithelial neoplasia (uVIN). A histologic regression of uVIN to normal tissue was observed in 58% of patients. As success of treatment is related to clearance of high-risk human papilloma virus (HPV), the aim of our study was to assess differences in immune cell counts and in the expression of p16(INK4a) in VIN tissue before and after imiquimod treatment, in relation to HPV clearance and clinical response. Vulvar tissue samples taken prior to imiquimod treatment and 4 weeks after treatment were tested for the presence of HPV. Previously determined immune cell counts (CD1a, CD207, CD208, CD123/CD11c, CD94, CD4, CD8 and CD25/HLA-DR) in epidermis and dermis of 25 VIN patients and 19 healthy controls were completed with the counts for CD14 and CD68. The expression of p16(INK4a) was investigated by immunohistochemistry in 15 patients. Before imiquimod treatment, both HPV cleared and HPV noncleared patients showed mainly in the dermis significantly upregulated immune cell counts compared to healthy controls. However, in patients that cleared HPV and showed histologic regression already 4 weeks after imiquimod treatment, immune cell counts and p16(INK4a) expression were normalized. In conclusion, our data indicate that imiquimod-induced clearance of HPV results in normalization of counts for certain immune cells and is strongly correlated with histologic regression of the disease.

Inflammatory profile of awake function-controlled craniotomy and craniotomy under general anesthesia.

BACKGROUND: Surgical stress triggers an inflammatory response and releases mediators into human plasma such as interleukins (ILs). Awake craniotomy and craniotomy performed under general anesthesia may be associated with different levels of stress. Our aim was to investigate whether those procedures cause different inflammatory responses. METHODS: Twenty patients undergoing craniotomy under general anesthesia and 20 patients undergoing awake function-controlled craniotomy were included in this prospective, observational, two-armed study. Circulating levels of IL-6, IL-8, and IL-10 were determined pre-, peri-, and postoperatively in both patient groups. VAS scores for pain, anxiety, and stress were taken at four moments pre- and postoperatively to evaluate physical pain and mental duress. RESULTS: Plasma IL-6 level significantly increased with time similarly in both groups. No significant plasma IL-8 and IL-10 change was observed in both experimental groups. The VAS pain score was significantly lower in the awake group compared to the anesthesia group at 12 hours postoperative. Postoperative anxiety and stress declined similarly in both groups. CONCLUSION: This study suggests that awake function-controlled craniotomy does not cause a significantly different inflammatory response than craniotomy performed under general anesthesia. It is also likely that function-controlled craniotomy does not cause a greater emotional challenge than tumor resection under general anesthesia.

Disturbed patterns of immunocompetent cells in usual-type vulvar intraepithelial neoplasia.

Genital infection with human papillomavirus (HPV) is usually transient, as the immune system is capable of eliminating the virus. When immunity "fails" and the infection persists, vulvar intraepithelial neoplasia (VIN) may develop. In this study, we examined the distribution of inflammatory cells in 51 patients with HPV-associated usual-type VIN and in 19 healthy controls. Frozen vulvar tissue samples were tested for the presence of HPV-DNA, and immunohistochemical staining for the markers CD1a, CD207, CD208, CD123/CD11c, CD94, CD4, CD8, and CD25/HLA-DR was performed. Cells were counted in both the epidermis and dermis over at least 2 mm of basal membrane length. In the epidermis of VIN patients, CD1a(+) and CD207(+) (Langerin) dendritic cells (DC) and CD8(+) T cells were significantly lower than in controls, whereas the number of CD123(+)/CD11c(-) plasmacytoid DCs (pDC) was significantly increased. No significant changes were observed for CD208(+) DCs, CD94(+) natural killer (NK) cells, CD4(+) T cells, and CD25(+)/HLA-DR(+) regulatory T cells. In the dermis of VIN patients, elevated numbers of CD208(+), CD123(+)/CD11c(-), CD94(+), CD4(+), CD8(+), and CD25(+)/HLA-DR(+) cells were observed when compared with healthy controls. The numbers of CD1a(+) and CD207(+) DCs were not different between groups. In summary, high-risk HPV-related usual-type VIN lesions are characterized by an immunosuppressive state in the epidermis, showing a reduction of immature myeloid DCs (mDC) and CD8(+) T cells. In the dermis, inflammatory activation is reflected by the influx of mature mDCs and pDCs, NK cells, and T cells, suggesting that the cellular immune response on viral HPV infection occurs in the dermis of VIN patients.

Expression of endothelial nitric oxide synthase and endothelin-1 in skin tissue from amputated limbs of patients with complex regional pain syndrome.

BACKGROUND AND OBJECTIVES: Impaired microcirculation during the chronic stage of complex regional pain syndrome (CRPS) is related to increased vasoconstriction, tissue hypoxia, and metabolic tissue acidosis in the affected limb. Endothelial dysfunction is suggested to be the main cause of diminished blood flow. The aim of this study was to examine the distribution of endothelial nitric oxide synthase (eNOS) and endothelin-1(ET-1) relative to vascular density represented by the endothelial marker CD31-immunoreactivity in the skin tissue of patients with chronic CRPS. METHODS: We performed immunohistochemical staining on sections of skin specimens obtained from the amputated limbs (one arm and one leg) of two patients with CRPS. RESULTS: In comparison to proximal specimens we found an increased number of migrated endothelial cells as well as an increase of eNOS activity in distal dermis specimens. CONCLUSIONS: We found indications that endothelial dysfunction plays a role in chronic CRPS.

Six years follow-up of the levels of TNF-alpha and IL-6 in patients with complex regional pain syndrome type 1.

In an earlier study, levels of the proinflammatory cytokines TNF-alpha and IL-6 are higher in blisters fluid from the complex regional pain syndrome type 1 (CRPS1) side obtained at 6 and 30 months (median) after the initial event. The aim of this follow-up study is to determine the involvement of these cytokines in long lasting CRPS1. Twelve CRPS1 patients, with median disease duration of 72 months, participated. The levels of TNF-alpha and IL-6 were measured in blister fluid; disease activity was reevaluated by measuring pain and differences in temperature, volume, and mobility between both extremities. Differences in levels of IL-6 and TNF-alpha and mobility between both sides were significantly decreased. Pain and differences in temperature and volume were not significantly altered. No correlation was found between the cytokines and the disease characteristics. These results indicate that IL-6 and TNF-alpha are only partially responsible for the signs and symptoms of CRPS1.

Reduced local immunity in HPV-related VIN: expression of chemokines and involvement of immunocompetent cells.

Usual type VIN is a premalignant disorder caused by persistent HPV infection. High prevalence of VIN in immuno-suppressed women suggests that a good innate and adaptive immune response is important for defense against HPV. Here, we explored expression levels of chemokines and related these to the presence or absence of immuno-competent cells (dendritic and T-cells) in affected (HPV-positive VIN) and non-affected (HPV-negative) vulvar tissues from the same patients. Combining microarray data with quantitative real-time RT-PCR, it was observed that several important chemokines were differentially expressed between VIN and control samples (up-regulation of IL8, CXCL10, CCL20 and CCL22 and down-regulation of CXCL12, CCL21 and CCL14). Furthermore, an increased number of mature dendritic cells (CD208+) seemed to be bottled up in the dermis, and although a T-cell response (increased CD4+ and CD8+ cells) was observed in VIN, a much larger response is required to clear the infection. In summary, it seems that most mature dendritic cells do not receive the proper chemokine signal for migration and will stay in the dermis, not able to present viral antigen to naive T-cells in the lymph node. Consequently the adaptive immune response diminishes, resulting in a persistent HPV infection with increased risk for neoplasia.

Treatment of vulvar intraepithelial neoplasia with topical imiquimod.

BACKGROUND: Alternatives to surgery are needed for the treatment of vulvar intraepithelial neoplasia. We investigated the effectiveness of imiquimod 5% cream, a topical immune-response modulator, for the treatment of this condition. METHODS: Fifty-two patients with grade 2 or 3 vulvar intraepithelial neoplasia were randomly assigned to receive either imiquimod or placebo, applied twice weekly for 16 weeks. The primary outcome was a reduction of more than 25% in lesion size at 20 weeks. Secondary outcomes were histologic regression, clearance of human papillomavirus (HPV) from the lesion, changes in immune cells in the epidermis and dermis of the vulva, relief of symptoms, improvement of quality of life, and durability of response. Reduction in lesion size was classified as complete response (elimination), strong partial response (76 to 99% reduction), weak partial response (26 to 75% reduction), or no response (< or =25% reduction). The follow-up period was 12 months. RESULTS: Lesion size was reduced by more than 25% at 20 weeks in 21 of the 26 patients (81%) treated with imiquimod and in none of those treated with placebo (P<0.001). Histologic regression was significantly greater in the imiquimod group than in the placebo group (P<0.001). At baseline, 50 patients (96%) tested positive for HPV DNA. HPV cleared from the lesion in 15 patients in the imiquimod group (58%), as compared with 2 in the placebo group (8%) (P<0.001). The number of immune epidermal cells increased significantly and the number of immune dermal cells decreased significantly with imiquimod as compared with placebo. Imiquimod reduced pruritus and pain at 20 weeks (P=0.008 and P=0.004, respectively) and at 12 months (P=0.04 and P=0.02, respectively). The lesion progressed to invasion (to a depth of <1 mm) in 3 of 49 patients (6%) followed for 12 months (2 in the placebo group and 1 in the imiquimod group). Nine patients, all treated with imiquimod, had a complete response at 20 weeks and remained free from disease at 12 months. CONCLUSIONS: Imiquimod is effective in the treatment of vulvar intraepithelial neoplasia. (Current Controlled Trials number, ISRCTN11290871 [controlled-trials.com].).

Tumor necrosis factor-alpha and interleukin-6 are not correlated with the characteristics of Complex Regional Pain Syndrome type 1 in 66 patients.

It was previously shown in a group of 9 patients with complex regional pain syndrome type 1 (CRPS1) that levels of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) are higher in blister fluid from the involved side. We hypothesize that local inflammation is responsible for the characteristics of CRPS1. The aim of this study was to confirm the previous observation in a large group of CRPS1 patients, repeating the measurement of TNF-alpha and IL-6 in blister fluid. Furthermore, we sought to determine whether these cytokines are responsible for the characteristics of CRPS1 and characterize the relationship between cytokine levels and duration of the disease. Sixty-six patients with CRPS1 participated. Skin blisters were artificially induced for measurement of cytokines in both extremities. The following disease characteristics were assessed: pain and differences in temperature, volume, and mobility between the extremities. TNF-alpha and IL-6 levels were significantly higher in blister fluid from the involved side. However, cytokine levels did not correlate with the characteristics or duration of the disease. Our findings confirm the presence of local inflammation in a population of 66 patients in the first 2 years of CRPS1. Proinflammatory cytokines seem to be only partly involved in the pathophysiology of CRPS1, as indicated by the lack of coherence between TNF-alpha and IL-6 levels and the signs and symptoms of inflammation and disease duration. Other inflammatory mediators and mechanisms, such as central sensitization, are probably involved in the early stages of CRPS1.

HPV related VIN: highly proliferative and diminished responsiveness to extracellular signals.

UNLABELLED: Vulvar intraepithelial neoplasia (VIN) is a premalignant disorder caused by human papillomaviruses. Basic knowledge about the molecular pathogenesis of VIN is sparse. Therefore, we have analyzed the gene expression profile of 9 VIN samples in comparison to 10 control samples by using genome wide Affymetrix Human U133A plus2 GeneChips. Results were validated by quantitative real-time RT-PCR analysis and immunostaining of a few representative genes (TACSTD1, CCNE2, AR and ESR1). Significance analysis of microarrays (SAM) showed that 1,497 genes were differentially expressed in VIN compared to controls. By analyzing the biological processes affected by the observed differences, we found that VIN appears to be a highly proliferative disease; many cyclins (CCNA, CCNB and CCNE) and almost all prereplication complex proteins are upregulated. Thereby, VIN does not seem to depend for its proliferation on paracrine or endocrine signals. Many receptors (for example ESR1 and AR) and ligands are downregulated. Furthermore, although VIN is not an invasive disease, the inhibition of expression of a marked number of cell-cell adhesion molecules seems to indicate development towards invasion. Upon reviewing apoptosis and angiogenesis, it was observed that these processes have not become significantly disregulated in VIN. IN CONCLUSION: although VIN is still a premalignant disease, it already displays several hallmarks of cancer.

Increased endothelin-1 and diminished nitric oxide levels in blister fluids of patients with intermediate cold type complex regional pain syndrome type 1.

BACKGROUND: In complex regional pain syndrome type 1 (CRPS1) pro-inflammatory mediators and vascular changes play an important role in the sustained development and outcome of the disease. The aim of this study was to determine the involvement of vasoactive substances endothelin-1 (ET-1) and nitric oxide (NO) during early chronic CRPS1. METHODS: Included were 29 patients with CRPS 1 who were diagnosed during the acute stage of their disease and observed during follow-up visits. Disease activity and impairment were determined and artificial suction blisters were made on the CRPS1 and the contralateral extremities for measurements of IL-6, TNF-alpha, ET-1 and nitrate/nitrite (NOx). RESULTS: The levels of IL-6, TNF-alpha and ET-1 in blister fluid in the CRPS1 extremity versus the contralateral extremity were significantly increased and correlated with each other, whereas NOx levels were decreased. CONCLUSION: The NOx/ET-1 ratio appears to be disturbed in the intermediate stage of CRPS, resulting in vasoconstriction and consequently in a diminished tissue blood distribution.

Multiplex bead array assay for detection of 25 soluble cytokines in blister fluid of patients with complex regional pain syndrome type 1.

Inflammatory processes are known to be involved at least in the early phase of complex regional pain syndrome type 1 (CRPS1). Blister fluid obtained from the involved extremities displayed increased amounts of proinflammatory cytokines IL-6 and TNFalpha compared with the noninvolved extremities. The aim of this paper is to investigate the involvement of mediators by measurement of several other cytokines using new detection techniques that enable multiple cytokine measurement in small samples. The use of a multiplex-25 bead array cytokine assay and Luminex technology enabled simultaneous measurement of representative (1) proinflammatory cytokines such as GM-CSF, IL-1beta, IL-1RA, IL-6, IL-8, and TNF-alpha; (2) Th1/Th2 distinguishing cytokines IFN-gamma, IL-2, IL-2R, IL-4, IL-5, and IL-10; (3) nonspecific acting cytokines IFN-alpha, IL-7, IL-12p40/p70, IL-13, IL-15, and IL-17; and (4) chemokines eotaxin, IP-10, MCP-1, MIP-1alpha, MIP-1beta, MIG, and RANTES. Although minimal detection levels are significantly higher in the bead array system than those in common ELISA assays, in blister fluid, IL-1RA, IL-6, IL-8, TNF-alpha, IL-12p40/p70, MCP-1, and MIP-1beta were detectable and increased in CRPS1 affected extremities. Levels of IL-6 and TNF-alpha simultaneously measured by ELISA (Sanquin Compact kit) and by multiplex-25 bead array assay (Biosource) were highly correlated (r = 0.85, P < .001 for IL-6 and r = 0.88, P < .001 for TNF-alpha). Furthermore, IP-10 and eotaxin were detectable but diminished in CRPS1, whereas detectable amounts of IL-10 were similar in involved and noninvolved extremities. Multiplex bead array assays are useful systems to establish the involvement of cytokines in inflammatory processes by measurements in blister fluids of CRPS1. Ten representative cytokines were detectable. However, detection levels and amounts measured are at least 3 times higher in the multiplex-25 array assay than in the ELISA assays used simultaneously for the measurement of cytokines.

Detection of human papillomavirus (HPV) 16-specific CD4+ T-cell immunity in patients with persistent HPV16-induced vulvar intraepithelial neoplasia in relation to clinical impact of imiquimod treatment.

PURPOSE: Topical application of the immune response modifier imiquimod is an alternative approach for the treatment of human papillomavirus (HPV)-positive vulvar intraepithelial neoplasia (VIN) and aims at the immunologic eradication of HPV-infected cells. We have charted HPV16-specific immunity in 29 patients with high-grade VIN and examined its role in the clinical effect of imiquimod treatment. EXPERIMENTAL DESIGN: The magnitude and cytokine polarization of the HPV16 E2-, E6-, and E7-specific CD4+ T-cell response was charted in 20 of 29 patients by proliferation and cytokine bead array. The relation between HPV16-specific type 1 T-cell immunity and imiquimod treatment was examined in a group of 17 of 29 patients. RESULTS: HPV16-specific proliferative responses were found in 11 of the 20 patients. In eight of these patients, T-cell reactivity was associated with IFNgamma production. Fifteen of the women treated with imiquimod were HPV16+, of whom eight displayed HPV16 E2- and E6-specific T-cell immunity before treatment. Imiquimod neither enhanced nor induced such immunity in any of the subjects. Objective clinical responses (complete remission or >75% regression) were observed in 11 of the 15 patients. Of these 11 responders, eight patients displayed HPV16-specific type 1 CD4+ T-cell immunity, whereas three lacked reactivity. Notably, the four patients without an objective clinical response also lacked HPV16-specific type 1 T-cell immunity. CONCLUSIONS: HPV16-specific IFNgamma-associated CD4+ T-cell immunity, although not essential for imiquimod-induced regression of VIN lesions, may increase the likelihood of a strong clinical response (P = 0.03).

Reduced hematopoietic reserves in DNA interstrand crosslink repair-deficient Ercc1-/- mice.

The ERCC1-XPF heterodimer is a structure-specific endonuclease involved in both nucleotide excision repair and interstrand crosslink repair. Mice carrying a genetic defect in Ercc1 display symptoms suggestive of a progressive, segmental progeria, indicating that disruption of one or both of these DNA damage repair pathways accelerates aging. In the hematopoietic system, there are defined age-associated changes for which the cause is unknown. To determine if DNA repair is critical to prolonged hematopoietic function, hematopoiesis in Ercc1-/- mice was compared to that in young and old wild-type mice. Ercc1-/- mice (3-week-old) exhibited multilineage cytopenia and fatty replacement of bone marrow, similar to old wild-type mice. In addition, the proliferative reserves of hematopoietic progenitors and stress erythropoiesis were significantly reduced in Ercc1-/- mice compared to age-matched controls. These features were not seen in nucleotide excision repair-deficient Xpa-/- mice, but are characteristic of Fanconi anemia, a human cancer syndrome caused by defects in interstrand crosslink repair. These data support the hypothesis that spontaneous interstrand crosslink damage contributes to the functional decline of the hematopoietic system associated with aging.