A two-component model of hair cortisol concentration in fibromyalgia: Independent effects of pain chronicity and severity.

BACKGROUND: Fibromyalgia (FM) is a chronic pain disorder of unknown aetiopathogenesis, in which the role of activity of the hypothalamic-pituitary-adrenal (HPA) axis is not clearly established. METHODS: This study analysed the modulatory effects of disease chronicity and severity on cortisol levels. Hair cortisol concentrations (HCC) and clinical evaluation data (pain severity, impact of FM on daily activities, depression, anxiety, fatigue and insomnia) were collected from 47 female patients with FM and 36 healthy women (HW). RESULTS: The results showed that disease chronicity, with a negative effect, and symptom severity, with a positive effect, were independent predictors of HCC. Patients with a shorter disease duration had higher HCC than patients with a longer disease duration and healthy participants. Furthermore, patients with greater symptom severity had higher HCC than those patients with lower clinical severity and healthy participants. While disease chronicity in FM was associated with a decrease in HCC, clinical severity increased HCC. CONCLUSIONS: These results support the existence of a dysfunction in the regulation of the HPA axis in FM and its possible contribution to chronic pain development. SIGNIFICANCE: This is the first study to assess hair cortisol concentrations in a specific sample of patients with fibromyalgia (FM). This method is especially useful for the assessment of long-term regular cortisol excretion. Results showed a two-component model for explaining cortisol levels: disease chronicity, with a negative effect, and symptom severity, with a positive effect. This suggests that severe pain/stress evokes higher cortisol levels at earlier stages of FM, while in the longer term a decrease in cortisol levels was observed.

Variation in the Presence of Anti-Batrachochytrium dendrobatidis Bacteria of Amphibians Across Life Stages and Elevations in Ecuador.

Amphibian populations are decreasing worldwide due to a variety of factors. In South America, the chytrid fungus Batrachochytrium dendrobatidis (Bd) is linked to many population declines. The pathogenic effect of Bd on amphibians can be inhibited by specific bacteria present on host skin. This symbiotic association allows some amphibians to resist the development of the disease chytridiomycosis. Here, we aimed (1) to determine for the first time if specific anti-Bd bacteria are present on amphibians in the Andes of Ecuador, (2) to monitor anti-Bd bacteria across developmental stages in a focal amphibian, the Andean marsupial tree frog, Gastrotheca riobambae, that deposits larvae in aquatic habitats, and (3) to compare the Bd presence associated with host assemblages including 10 species at sites ranging in biogeography from Amazonian rainforest (450 masl) to Andes montane rainforest (3200 masl). We sampled and identified skin-associated bacteria of frogs in the field using swabs and a novel methodology of aerobic counting plates, and a combination of morphological, biochemical, and molecular identification techniques. The following anti-Bd bacteria were identified and found to be shared among several hosts at high-elevation sites where Bd was present at a prevalence of 32.5%: Janthinobacterium lividum, Pseudomonas fluorescens, and Serratia sp. Bd were detected in Gastrotheca spp. and not detected in the lowlands (sites below 1000 masl). In G. riobambae, recognized Bd-resistant bacteria start to be present at the metamorphic stage. Overall bacterial abundance was significantly higher post-metamorphosis and on species sampled at lower elevations. Further metagenomic studies are needed to evaluate the roles of host identity, life-history stage, and biogeography of the microbiota and their function in disease resistance.

Dimorphic HLA-B signal peptides differentially influence HLA-E- and natural killer cell-mediated cytolysis of HIV-1-infected target cells.

As a mechanism of self-protection, signal peptides cleaved from human leukocyte antigen (HLA) class I products bind to HLA-E before the complex interacts with the natural killer (NK) cell receptor CD94/NKG2A to inhibit NK-mediated cell lysis. Two types of the signal peptides differ in their position 2 (P2) anchor residue, with P2-methionine (P2-M) having higher HLA-E binding affinity than P2-threonine (P2-T). All HLA-A and HLA-C molecules carry P2-M, whereas HLA-B products have either P2-M or P2-T. Epidemiological evidence suggests that P2-M is unfavourable in the context of HIV-1 infection, being associated with accelerated acquisition of HIV-1 infection in two African cohorts. To begin elucidating the functional mechanism, we studied NK-mediated killing of CD4(+) T cells and monocyte-derived macrophages infected with two laboratory-adapted HIV-1 strains and two transmitted/founder (T/F) viruses. In the presence of target cells derived from individuals with the three HLA-B P2 genotypes (M/M, M/T and T/T), NK-mediated cytolysis was elevated consistently for P2-T in a dose-dependent manner for all cell and virus combinations tested (P = 0·008-0·03). Treatment of target cells with an anti-HLA-E monoclonal antibody restored NK-mediated cytolysis of cells expressing P2-M. Observations on cell lysis were also substantiated by measurements of HIV-1 p24 antigen in the culture supernatants. Overall, our experiments indicate that the anti-HIV-1 function mediated by NK cells is compromised by P2-M, corroborating the association of HLA-B genotype encoding P2-M with accelerated HIV-1 acquisition.

Overexpression of matrix metalloproteinases and their inhibitors in mononuclear inflammatory cells in breast cancer correlates with metastasis-relapse.

An immunohistochemical study was performed using tissue microarrays and specific antibodies against matrix metalloproteinase (MMP)-1, -2, -7, -9, -11, -13 and -14, tissular inhibitors of metalloproteinase (TIMP)-1, -2 and -3. More than 2600 determinations on cancer specimens from 131 patients with primary ductal invasive tumours of the breast were performed. To identify specific groups of tumours with distinct expression profiles the data were analysed by unsupervised hierarchical cluster analysis by each cellular type. We did not find well-defined cluster of cases for tumour cells or fibroblastic cells. However, for mononuclear inflammatory cells the dendogram shows a first-order division of the tumours into two distinct MMP/TIMP molecular profiles, designated group 1 (n=89) and group 2 (n=42). Matrix metalloproteinase-7, -9, -11, -13 and -14, and TIMP-1 and -2, were identified as showing significant high expression in group 2 compared with group 1. Multivariate analysis demonstrated that clustering for mononuclear inflammatory cells was the most potent independent factor associated with distant relapse-free survival (group 2: 5.6 (3.5-9.6), P<0.001). We identify a phenotype of mononuclear inflammatory cells infiltrating tumours, which is associated with the development of distant metastasis. Therefore, this finding suggests that these host inflammatory cells could be a possible target for inhibition of metastasis.

Study of matrix metalloproteinases and their inhibitors in breast cancer.

An immunohistochemical study was performed using tissue microarrays and specific antibodies against matrix metalloproteinases (MMPs) 1, 2, 7, 9, 11, 13, 14, and their tisullar inhibitors (TIMPs) 1, 2, and 3. More than 2600 determinations on cancer specimens from 131 patients with primary ductal invasive tumours of the breast (65 with and 66 without distant metastasis) and controls were performed. Staining results were categorised using a score based on the intensity of the staining and a specific software program calculated the percentage of immunostained cells automatically. We observed a broad variation of the total immunostaining scores and the cell type expressing each protein. There were multiple and significant associations between the expression of the different MMPs and TIMPs evaluated and some parameters indicative of tumour aggressiveness, such as large tumour size, advanced tumour grade, high Nottinham prognostic index, negative oestrogen receptor status, peritumoural inflammation, desmoplastic reaction, and infiltrating tumoural edge. Likewise, the detection of elevated immunohistochemical scores for MMP-9, 11, TIMP-1, and TIMP-2, was significantly associated with a higher rate of distant metastases. The expression of MMP-9 or TIMP-2 by tumour cells, MMP-1, 7, 9, 11, 13, or TIMP-3 by fibroblastic cells, and MMP-7, 9, 11, 13, 14, TIMP-1, or TIMP-2 by mononuclear inflammatory cells, was also significantly associated with a higher rate of distant metastases.

Clinical significance of pepsinogen C tumor expression in patients with stage D2 prostate carcinoma.

Pepsinogen C is an aspartyl protease mainly involved in the digestion of proteins in the stomach, and an androgen-inducible protein in breast cancer cells. The aims of this study were to evaluate the expression and clinical significance of this enzyme in the primary tumors of prostate cancer patients with bone metastasis who were scheduled to receive antiandrogenic therapy. This study was prospectively performed in 28 stage D2 prostate cancer patients who, after diagnosis, received maximum androgen blockade. Pepsinogen C tumor expression was analyzed in samples (24 from needle biopsy cylinders and four from transurethral resection specimens) from primary tumors using an immunohistochemical assay. Twelve prostate carcinomas (42.8%) were positive for pepsinogen C. Pepsinogen C was a significant prognostic factor to predict a longer overall survival in the patients of our study (p<0.01). Pepsinogen C can be a new prognostic factor and a useful biological marker of androgen dependency in prostate cancer.

Lysozyme expression by breast carcinomas, correlation with clinicopathologic parameters, and prognostic significance.

BACKGROUND: Here we evaluate the expression and prognostic value of lysozyme, a milk protein that is also synthesized by a significant percentage of breast carcinomas, in women with breast cancer. METHODS: Lysozyme expression was examined by immunohistochemical methods in a series of 177 breast cancer tissue sections. Staining was quantified by using the HSCORE system, which considers both the intensity and the percentage of cells staining at each intensity. The prognostic value of lysozyme was retrospectively evaluated by multivariate analysis that took into account conventional prognostic factors. RESULTS: A total of 126 of 177 carcinomas (69.4%) stained positive for this protein, but there were clear differences among them with regard to the intensity and percentage of stained cells. Lysozyme values were higher in well-differentiated and moderately differentiated tumors than in poorly differentiated tumors (P < .05). Similarly, lysozyme levels were higher in small and node-negative tumors than in large and node-positive tumors (P < .05). Moreover, results indicated that low lysozyme content predicted shorter relapse-free survival and overall survival (P < .005). Separate Cox multivariate analysis in subgroups of patients as defined by node status showed that lysozyme expression was an independent prognostic factor able to predict both relapse-free survival and overall survival in node-negative patients (P < .05). CONCLUSIONS: Tumoral expression of lysozyme is associated with lesions of favorable evolution in breast cancer. This milk protein may be a new prognostic factor in patients with breast cancer.

Immunohistochemical study of pepsinogen C expression in cutaneous malignant melanoma: association with clinicopathological parameters.

BACKGROUND: The aim of this study was to evaluate the pepsinogen C expression in malignant cutaneous melanomas and analyze its possible relationship to clinical and pathological parameters. Pepsinogen C is an aspartyl proteinase primarily involved in the digestion of proteins in the stomach and represents one of the main androgen-inducible proteins in breast cancer cells. METHOD: Tumoral pepsinogen C expression was retrospectively analyzed in 35 paraffin-embedded tissues from patients with primary malignant cutaneous melanoma and in 10 samples from 10 benign lesions (4 dermal melanocytic nevi, 4 compound melanocytic nevi and 2 dysplastic melanocytic nevi), using immunohistochemical methods. RESULTS: The benign lesions were consistently negative for pepsinogen C, whereas 20 of the 35 malignant melanomas (57%) showed positive immunostaining for pepsinogen C. The percentage of pepsinogen C-positive tumors was significantly higher in men than in women (p=0.01) and in epithelioid melanomas than in fusocellular or mixed type melanomas (p=0.003). In addition, the percentage of pepsinogen-C positive tumors was positively and significantly correlated with lesion thickness (p=0.003), Clark's level of invasion (p=0.028) and tumor stage (p<0.001). CONCLUSION: Pepsinogen C could be a new prognosticator of unfavorable outcome in cutaneous malignant melanoma.

Expression and prognostic significance of pepsinogen C in gastric carcinoma.

BACKGROUND: In this study we evaluated the expression and clinical significance of pepsinogen C, an aspartic proteinase involved in the digestion of proteins in the stomach, in patients with gastric cancer. METHODS: Pepsinogen C expression was examined by immunohistochemical methods in a series of 95 gastric carcinomas. The prognostic value of pepsinogen C was retrospectively evaluated by multivariate analysis taking into account conventional prognostic parameters. Follow-up period of patients was 21.4 months. RESULTS: A total of 25 (26.3%) gastric carcinomas stained positively for pepsinogen C. The percentage of pepsinogen C-positive tumors was higher in well-differentiated (50%) than in moderately differentiated (19.5%) and poorly differentiated (21.9%) tumors (P < .05). Similarly, significant differences in pepsinogen C immunostaining were found between node-negative and node-positive tumors (47.1% vs. 14.7%; P < .001). In addition, statistical analysis revealed that pepsinogen C expression was associated with clinical outcome in gastric cancer patients. Low pepsinogen C levels predicted short overall survival periods in the overall group of patients with gastric cancer (P < .001), and in 71 patients with resectable carcinomas (P < .005). Multivariate analysis according to Cox's model indicated that pepsinogen C immunostaining was an independent predictor of outcome for both overall and resectable gastric cancer patients (P < .05, for both). CONCLUSIONS: The expression of pepsinogen C in gastric cancer may represent a useful biological marker able to identify subgroups of patients with different clinical outcomes.

Pepsinogen C expression in tumors of extragastric origin.

We have examined by immunohistochemistry the ability of human carcinomas of various origin to produce pepsinogen C, an aspartyl proteinase mainly involved in the digestion of proteins in the stomach and recently found to be associated with breast carcinomas. Of the 268 tumors analyzed 80 (29.8%) showed positive staining for pepsinogen C. These positive tumors included 12 gastric (38.7% of the 31 examined cases), nine pancreatic (42.8%), two renal (20%), 12 prostatic (40%), three bladder (27.3%), 14 endometrial (29.7%) and 18 ovarian (40%) carcinomas. We also detected 10 melanomas (50%) that were positive for pepsinogen C. By contrast, immunohistochemical staining for the proteinase was not detected in colorectal, cervical, lung and basal cell skin carcinomas. These results demonstrate that pepsinogen C, a proteolytic enzyme of highly restricted expression in human tissues, can also be expressed by a wide variety of human carcinomas. In addition, and similar to pepsinogen C expression in breast carcinomas, the production of this enzyme by different human tumors might be related to putative hormonal alterations associated with the development and progression of these tumors.

Comparative study of two androgen-induced markers (apolipoprotein D and pepsinogen C) in female and male breast carcinoma.

BACKGROUND: Male breast cancer (MBC) is a rare tumor in comparison to the same disease in the female population (FBC). Classical prognostic factors (tumor size, node status, estrogen receptor positivity, histological grade) have a similar prognostic value in both tumors, but MBC seems to have a worse outcome. Several markers under estrogen control have been shown with a similar incidence in breast tumors of both sexes, while androgen-induced markers have been detected in a higher percentage of breast tumors in males. AIMS AND METHODS: Our purpose was to compare in 68 MBC and in 68 FBC the expression of Pepsinogen C (Pep C) and Apolipoprotein D (Apo D), two proteins under androgen control, by immunohistochemical methods. RESULTS: Pep C was expressed by 52 of 68 (76.4%) MBC patients, whereas 34 of 68 (50%) FBC showed positive staining. Apo D was expressed by 57 of 68 (83.8%) MBC patients, while 40 of 68 (41.2%) FBC stained positively. Differences between percentages of positive expression were significant (p<0.005 for Pep C; p<0.0001 for Apo D). Moreover, differences between expression levels of Pep C and Apo D in both populations of patients were significant. The mean value of Pep C was significantly higher in male breast tumors (HSCORE = 141.3) than in the females (HSCORE = 80.3) (p<0.0001). Similarly, Apo D mean value was significantly higher in MBC (HSCORE = 161.5) than in FBC (HSCORE = 102.3) (p=0.006). CONCLUSION: These differences can open the field of a more selective hormonal therapy that should not be based on estrogen receptor status only, but also on androgen receptor status.

Apolipoprotein D expression in metastasic lymph nodes of breast cancer.

BACKGROUND: Apolipoprotein D is a glycoprotein of the human plasma whose functional role remains unclear. On the other hand, this protein is also produced by breast carcinomas and is positively associated with a favorable outcome of patients. However, none study has focused on metastasic lesions. AIM: To analyze apolipoprotein D expression in breast cancer patients and their synchronous metastasic axillary lymph nodes. METHODS: We analyzed by immunohistochemical assay both, the tumoral expression of apolipoprotein D in primary tumors and in their synchronous metastasic axillary lymph nodes of 30 node-positive breast cancer patients. RESULTS: Of the primary tumors, 28 (93.3%) showed a positive immunostaining for apolipoprotein D, although there was wide variability immunostaining values. On the other hand, 16 (53.3%) patients showed a positive immunostaining for the protein in their tumoral lymph nodes. In addition, there was a significant positive relationship between the tumoral expression of apolipoprotein D in primary tumors and metastasic lymph nodes (P < 0.05). However, only immunostaining values of the protein in primary tumors achieve statistical signification (P < 0.05) as prognostic factor of favorable evolution to predict overall survival from patients. CONCLUSIONS: Apolipoprotein D is also expressed in metastasic lymph nodes of breast carcinomas, but with a different pattern of immunostaining and less clinical significance than in primary tumors.

Apolipoprotein D expression in benign and malignant prostate tissues.

BACKGROUND: Apolipoprotein D (ApoD), a protein component of the human plasma lipid transport system, is an androgen regulated protein in both breast and prostate cancer cell lines. METHODS: Here we examined, by immunohistochemical assay, the ApoD expression in normal, benign and malignant prostate tissues. RESULTS: ApoD was detected in the two analyzed normal prostate tissues, in 16 (94.1%) of 17 benign prostatic hyperplasias, and in 36 (63.1%) of 57 prostate carcinomas. In prostate carcinomas, ApoD immunostaining values did not show significant relationship neither with age of the patients, stage and histologic grade of the tumors, nor with preoperative serum levels of prostatic specific antigen (PSA). However, ApoD-negative immunostaining predicted shorter survival duration in the subgroup of 46 patients with nonresectable prostate cancer (p = 0.02). CONCLUSIONS: Our results led us to consider that Apo D expression by prostate carcinomas migth be of clinical usefulness for identificating lesions with different evolution.

Expression of pepsinogen C in gynecomastias and male breast carcinomas.

Pepsinogen C is a proteolytic enzyme involved in the digestion of proteins in the stomach; it is also synthesized by a significant percentage of female breast carcinomas. In addition, it has been demonstrated that pepsinogen C is one of the few proteins induced by androgens in breast carcinoma cells. Here we evaluate the expression of pepsinogen C by immunoperoxidase staining in normal breast tissue from 3 male patients, 15 gynecomastia tissues, 2 male in situ breast carcinomas, and 68 male invasive breast carcinomas. Pepsinogen C immunostaining values were quantified in male breast tumors using the HSCORE system, which considers both the intensity and the percentage of cells staining at each intensity. The results indicated positive immunohistochemical staining for pepsinogen C in all gynecomastia tissues, the two in situ ductal carcinomas, and 52 of 68 invasive breast carcinomas (76.4%). The three normal breast tissues analyzed showed negative staining for pepsinogen C, whereas invasive tumors showed clear differences among them with regard to the intensity and percentage of staining cells. In addition, pepsinogen C scores were significantly higher in well-differentiated (grade I, 188.7) and moderately differentiated (grade II, 145.8) tumors than in poorly differentiated (grade III, 98.5) tumors (p = 0. 032). Similarly, significant differences in pepsinogen C content were found between estrogen receptor (ER)-positive tumors and ER-negative tumors (158.5 vs. 44.3, respectively; p = 0.009). Patients with pepsinogen C-positive tumors reached longer relapse-free and overall survival periods than did those with tumors with negative staining, but no statistical differences were observed between survival curves calculated for these two groups of patients. This results demonstrate expression of pepsinogen C by gynecomastias and by a high percentage of male breast carcinomas and may indicate an important role of pepsinogen C in the pathophysiology of male breast diseases.

Pepsinogen C is a new prognostic marker in primary breast cancer.

PURPOSE: Here we evaluate in breast cancer patients the prognostic value of pepsinogen C, a proteolytic enzyme involved in the digestion of proteins in the stomach that is also synthesized by a significant percentage of breast carcinomas. PATIENTS AND METHODS: Pepsinogen C expression was examined by immunoperoxidase staining in a series of 243 breast cancer tissue sections, and results obtained were quantified using the HSCORE system, which considers both the intensity and the percentage of cells staining at each intensity. Evaluation of the prognostic value of pepsinogen C was performed retrospectively in corresponding patients by multivariate analysis that took into account conventional prognostic factors. The mean follow-up period was 48.5 months. RESULTS: A total of 113 carcinomas (46.5%) stained positively for this proteinase, but there were clear differences among them with regard to the intensity and percentage of stained cells. Pepsinogen C values were significantly higher in well differentiated (grade I, 89.1) and moderately differentiated (grade II, 88.5) tumors than in poorly differentiated (grade III, 27.7) tumors (P < .001). Similarly, significant differences in pepsinogen C content were found between estrogen receptor (ER)-positive tumors and ER-negative tumors (85.9 v 41.2, respectively; P < .05). Moreover, results indicated that low pepsinogen C content predicted shorter relapse-free survival duration and overall survival duration (P < .0001). Separate Cox multivariate analysis for relapse-free survival and overall survival in subgroups of patients as defined by node status showed that pepsinogen C expression was the strongest factor to predict both relapse-free survival and overall survival in node-positive patients (P < .0001 for both) and node-negative patients (P < .005 and P < .01, respectively). CONCLUSION: Pepsinogen C is a new prognostic factor for early recurrence and death in both node-positive and node-negative breast cancer. In addition, and in contrast to most studies that concern the prognostic significance of proteolytic enzymes in cancer, pepsinogen C production by breast cancer cells is associated with lesions of favorable evolution.

Expression and prognostic significance of apolipoprotein D in breast cancer.

Apolipoprotein D (apo D) is a glycoprotein involved in the human plasma lipid transport system and present at large amounts in cyst fluid from women with gross cystic disease of the breast. Apo D expression in breast carcinomas was examined by immunoperoxidase staining of a series of 163 tumors. A total of 60 (36.8%) tumors were negative for apo D immunostaining, 28 (17.2%) carcinomas were weakly positive, 33 (20.2%) were moderately stained, whereas the remaining 42 (25.8%) tumors were strongly stained with the specific antibodies. No significant correlation was found between apo D content and tumor size, lymph node involvement, or biochemical parameters such as estrogen receptors, cathepsin D, or pS2 protein. However, the finding of a significant association between apo D and menopausal status of patients or differentiation grade of tumors, with apo D values being lower in tumors from premenopausal women or in poorly differentiated carcinomas, suggested a potential value of this glycoprotein as a prognostic factor in breast cancer. Preliminary analysis of relapse-free survival and overall survival in a subgroup of 152 women with a mean follow-up of 42 months confirmed that low apo D values were significantly associated to a shorter relapse-free survival and poorer survival. According to these data, we propose that apo D in combination with other well-established prognostic factors may contribute to more accurately identify subgroups of breast cancer patients with low or high risk for relapse and death.

Expression of pepsinogen C in human breast tumours and correlation with clinicopathologic parameters.

We have examined by immunohistochemistry the ability of breast carcinomas to produce pepsinogen C, an aspartyl proteinase usually involved in the digestion of proteins in the stomach. A total of 113 out of 245 breast tumours (46%) were positive for pepsinogen C immunostaining. There was a significant association between pepsinogen C and oestrogen receptors with proteinase levels higher (HSCORE) in oestrogen receptor positive tumours than in oestrogen receptor negative. There was also a significant association between pepsinogen C and histological grade, pepsinogen C levels being higher in well and moderately differentiated breast carcinomas than in poorly differentiated tumours. On the basis of these results, we suggest that pepsinogen C may be useful as a marker of good prognosis in breast cancer.

Isolation and characterization of a pepsin C zymogen produced by human breast tissues.

An aspartic proteinase present in cyst fluid from women with gross cystic breast disease was purified by a procedure involving affinity chromatography on pepstatin-agarose and size-exclusion high performance liquid chromatography. The amino-terminal sequence of the purified breast proteinase was identical to that corresponding to gastric pepsinogen C. Additional data on cleavage specificity, pH optimum, and immunological properties supported the close relationship between both molecules. Northern blot analysis and polymerase chain reaction amplification studies performed on RNAs obtained from normal and pathological breast tissues demonstrated that the protein is produced by mammary carcinomas and cysts, but not by the normal resting mammary gland. Immunohistochemical staining of paraffin-embedded tissue sections confirmed the existence of a subset of tumors that have the ability to synthesize and secrete this pepsin zymogen. On the basis of these results, we suggest that pepsinogen C expression by human mammary epithelium may be involved in the development of breast diseases, being also of potential interest as a biochemical marker of the hormonal imbalance underlying these pathologies.