To Örebro University

Purpose: The knowledge of somatic mutations that arise in penile cancer is limited. We examined the dysregulation of components in the phosphatidylinositol 3-kinase and Ras pathways.

Materials and Methods: Using single stranded conformational analysis and direct sequencing we performed mutational analysis of the PIK3CA, PTEN, HRAS, KRAS, NRAS and BRAF genes in 28 penile tumors.

Results: We identified somatic missense mutations in 11 of the 28 penile cancer samples (39%). In the PIK3CA gene 8 mutations (29%) were identified that were E542K or E545K. In the HRAS gene a G12S and a Q61L mutation were found (7%). The KRAS gene contained 1 mutation (3%), that is a G12S change. PIK3CA mutations were found in all grades and stages, whereas HRAS and KRAS mutations were found in larger and more advanced tumors. The mutations were mutually exclusive, suggesting that dysregulation of either pathway is sufficient for the development and progression of penile carcinoma.

Conclusions: The high frequency of mutations in the PIK3CA, HRAS and KRAS genes leads us to believe that dysregulation of the phosphatidylinositol 3-kinase or Ras pathway is significant for the development and progression of penile carcinoma.

The NLRP3 inflammasome is an intracellular multi-protein complex that triggers caspase-1 mediated maturation of interleukin-1β (IL-1β); one of the most potent mediators of inflammation and a major cytokine produced during severe infections, like sepsis. However, the excessive cytokine levels seem to stage for tissue injury and organ failure, and high levels of IL-1β correlates with severity and mortality of sepsis. Instead, recent data suggest caspase-1 to function as a guardian against severe infections. CARD8 has been implied to regulate the synthesis of IL-1β via interaction to caspase-1. In recent years, polymorphism of CARD8 (C10X) per se or in combination with NLRP3 (Q705K) has been implicated with increased risk of inflammation. The aim was to investigate the correlation of these polymorphisms with severe blood stream infection. Human DNA was extracted from blood culture bottles that were found to be positive for microbial growth (i.e. patients with bacteraemia). Polymorphisms Q705K in the NLRP3 gene and C10X in the CARD8 gene were genotyped using TaqMan genotyping assay. The results were compared to healthy controls and to samples from patients with negative cultures. The polymorphism C10X was significantly over-represented among patients with bacteraemia as compared to healthy controls, whereas patients with negative blood culture were not associated with a higher prevalence. No association was observed with polymorphism Q705K of NLRP3 in either group of patients. Patients carrying polymorphism C10X in the CARD8 gene are at increased risk of developing bacteraemia and severe inflammation.

The aim of the present study was to extend the creatinine-based Lund-Malmö GFR equation for use with rescaled cystatin C (r-LMRCys) and validate it against measured GFR (mGFR) in the EKFC cystatin C cohort of children (n = 2,293) and adults (n = 7,727). Rescaling was obtained by dividing each biomarker by a Q-value, representing the population-specific median biomarker level among healthy individuals. Validation included median bias/precision/accuracy (percent estimates within ±30% of mGFR, P30). Performance was compared with the EKFC-equation (EKFCCys), the CAPA cystatin C equation, the corresponding equations based on rescaled creatinine (r-LMRCr and EKFCCr) and the arithmetic mean of r-LMRCr and CAPA (r-LMRCr+CAPA), r-LMRCr and r-LMRCys (r-LMRMean), and EKFCCr and EKFCCys (EKFCMean). The overall P30 of r-LMRCys in adults was 86.2% (95% CI 85.4%-86.9%), which was 6.6 percentage points (pp; 95% CI 5.8-7.4 pp) higher than for CAPA and similar to r-LMRCr (P30 87.4%, 95% CI 86.6%-88.1%). r-LMRCys and EKFCCys exhibited similar performance both overall and across subgroups of age, sex, GFR and BMI and in children. All three arithmetic mean equations had similar P30-accuracy and generally performed better than the corresponding single-marker equations. Our results show that the Lund-Malmö GFR equation can be adapted for use with rescaled cystatin C with performance that is similar to the best-performing equations based on rescaled creatinine. The generality of the applied biomarker rescaling principle implies that the future demand for population- and biomarker-specific GFR estimating equations can be expected to decrease substantially.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950 Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each lab using a different lipidomics workflow. A total of 1527 unique lipids were measured across all laboratories, and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and inter-laboratory quality control and method validation. These analyses were performed using non-standardized laboratory-independent workflows. The consensus locations were also compared to a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Introduction: Reduced monocyte human leukocyte antigen (mHLA)-DR surface expression in the late phase of sepsis is postulated as a general biomarker of sepsis-induced immunosuppression and an independent predictor of nosocomial infections. However, traditional monitoring of mHLA-DR by flow cytometry has disadvantages due to specific laboratory requirements. An mRNA-based HLA-DR monitoring by polymerase chain reaction (PCR) would improve the clinical usage and facilitate conduction of large multicenter studies. In this study, we evaluated an mRNA-based HLA-DR monitoring by quantitative real-time PCR (qRT-PCR) as an alternative method to traditional flow cytometry.

Methods: Fifty-nine patients with sepsis and blood culture growing pathogenic bacteria were studied. Blood samples were collected at day 1 or 2 after admission, for measurement of mHLA-DR by flow cytometry and mRNA expression of HLA-DRA and class II transactivator (CIITA) by qRT-PCR. Blood samples from blood donors were used as controls (n = 30).

Results: A significant reduced expression of mHLA-DR, HLA-DRA, and CIITA was seen in septic patients compared with controls. HLA-DRA mRNA level in whole blood was highly correlated with surface expression of mHLA-DR.

Conclusions: Patients with sepsis display a diminished expression of HLA-DR at the monocyte surface as well as in the gene expression at the mRNA level. The mRNA expression level of HLA-DRA monitored by qRT-PCR correlates highly with surface expression of HLA-DR and appears to be a possible future biomarker for evaluation of immunosuppression in sepsis.

Eosinophils have been linked to functional dyspepsia; however, less is known about their role in irritable bowel syndrome (IBS). This study tested the hypothesis of alterations in levels of fecal eosinophil-derived neurotoxin (F-EDN) and eosinophil density and degranulation within the colonic mucosa of IBS patients compared with healthy controls (HC). Colonic biopsies were collected from 37 IBS patients and 20 HC and analyzed for eosinophil numbers and local degranulation of eosinophil cationic protein (ECP) by histologic procedures. Fecal samples were collected for F-EDN and microbiota analysis. Differentiated 15HL-60 cells were used in vitro to investigate the direct effect of live bacteria on eosinophil activation measured by a colorimetric assay with o-phenylenediamine (OPD) substrate. We observed a higher number of eosinophils and increased extracellular ECP in the mucosa of IBS patients compared with HC. Moreover, F-EDN levels in IBS samples were elevated compared with HC and positively correlated to extracellular ECP. Metagenomic analysis showed significant correlations between bacterial composition and eosinophil measurements in both HC and IBS patients. In vitro experiments revealed an increased degranulation of 15HL-60 after stimulation with Salmonella typhimurium, Salmonella enterica, and Yersinia enterocolitica. To conclude, we could demonstrate alterations related to eosinophils in IBS, and, for the first time, a positive correlation between F-EDN levels and degranulated eosinophils in the colonic mucosa of IBS patients. Together our results suggest that eosinophils play a role in the pathophysiology of IBS and the mechanisms might be linked to an altered microbiota.

Objectives: Endometrial homeostasis, indicated as the balance between apoptosis and proliferation, was studied with regard to endometrial safety and bleeding disturbances.

Materials and Methods: The quantitatively sufficient endometrial biopsies of 92 postmenopausal women enrolled in the study were investigated. The participants were divided into two groups, each receiving a continuous combined HRT regimen with either conjugated estrogen (CE) 0.625 mg + 5 mg medroxyprogesterone acetate (MPA) (=CE/MPA) or 17-beta-estradiol (E2) 2 mg + 1 mg norethisterone acetate (NETA) (=E2/NETA). These were evaluated according to apoptotic index (Ai) and proliferation marker Ki-67 index. Estrogen receptor alpha (ER) and progesterone receptor (PR) expression were also monitored, as well as endometrial thickness. Quantitative in situ techniques were used.

Results: Ai and Ki-67 index were unchanged in epithelial glands of endometrium from baseline to second biopsy obtained after 1 year of combined continuous HRT. In stromal tissue, Ki-67 index was increased, while Ai was on the same level. PR expression in both epithelium and stroma was unchanged. Endometrial thickness was unaffected during therapy, and the histopathological evaluation showed no development of hyperplasia or carcinoma.

Conclusions: The unaffected homeostasis in endometrial epithelium contributes to endometrial safety and is in accordance with the histopathological findings of no hyperplasia. The homeostasis of stroma was transformed to be more proliferative. Increased stromal proliferation may be of importance for stromal support of the veins and for decreasing breakthrough bleeding during HRT. The increased stromal proliferation, as well as the decreased ER expression both in epithelium and stroma, could be an effect of progesterone.

Interleukins 6 (IL-6) and 12 (IL-12), and the chemoattractant chemokine RANTES were studied in ethmoidal mucosa, using reverse transcriptase polymerase chain reaction. The 49 patients had chronic sinusitis or nasal/paranasal polyposis, and some also allergy. To the best of our knowledge, this is the first study that demonstrates RANTES and IL-12 on mRNA level in human sinonasal mucosa in situ. mRNA for IL-6, IL-12 and RANTES were detected in 2, 8 and 6 patients with chronic sinusitis, respectively, and in mucosa from patients with polyposis a positive expression was observed in 4, 14 and 10 cases. There were no statistically significant differences. Analysing the entire group of 49 patients, disregarding type of mucosal disease, the number of patients with positive RANTES was significantly higher than that for IL-6. Similarly, IL-12 positivity was more frequently expressed than IL-6. mRNA for IL-6 was expressed in only 2 of the allergic patients. The cytokine production studied thus seems to be unrelated to the clinically defined entities. There is thus a local production in human diseased sinonasal mucosa of RANTES, as well as of IL-6 and IL-12. The local production of RANTES is an important prerequisite for recruitment and migration of inflammatory cells into the tissue. IL-12 is a co-stimulator of antigen-specific responses of established T helper 1 (Th1) clones, and regulates the responsiveness of the clones to a number of T cell growth factors. The study supports a shift towards Th1 cells in these disease entities.

We investigated sub-populations of B-lymphocytes in nasal mucosa and peripheral blood of 17 patients with seasonal allergic rhinitis (birch pollen) and 10 controls. The study included provocation with allergen during the non-pollen season, during which no participant used medication. Samples were also taken during the pollen season. Subsets of B-cells as expressed by different CD antigens were investigated by immunohistochemistry on frozen sections and by flow cytometry of peripheral blood. Nasal CD23+ B-cells decreased in allergic patients during provocation, indicating that mature virgin CD23+ B-cells switch into a memory B-cell phenotype with loss of CD23 expression. This indicates differentiation towards cells that can represent a local source for IgE synthesis. No decrease was observed during the pollen season when the patients used medication. Serum IgE was significantly higher in allergic patients on all occasions. The observed up-regulation of CD40 expression on peripheral blood B-cells in allergic patients during the pollen season clearly indicate B-cell activation. Furthermore, a relative increase of CD19+ B-cells was observed in peripheral blood during provocation. Upregulation (by IL-4) of CD40 on B-cells which then may be stimulated by gp39 (CD40 ligand) can constitute an early and important event in the IgE-mediated allergic reaction.

Inadequate and delayed antibiotic treatment of extended spectrum beta-lactamase (ESBL)-producing isolates have been associated with increased mortality of affected patients. The purpose of this study was to compare the host response of human renal epithelial cells and polymorphonuclear leucocyte (PMN) cells when infected by ESBL-producing uropathogenic Escherichia coli (UPEC) isolates in the presence or absence of ineffective antibiotics.

The renal epithelial cell line A498 and PMN cells were stimulated with ESBL-producing UPEC isolates in the presence or absence of three different antibiotics (trimetoprim, ceftibuten and ciprofloxacin). Host cell responses were evaluated as release of cytokines (IL-6, IL-8), reactive oxygen species (ROS), ATP and endotoxins. Bacterial morphology and PMN phagocytosis were evaluated by microscopy.

In the presence of ceftibuten, 2 out of 3 examined ESBL-isolates changed their morphology into a filamentous form. The presence of ceftibuten enhanced IL-6, IL-8 and ROS-production from host cells, but only from cells stimulated by the filamentous isolates. The bacterial supernatant and not the filamentous bacteria per se was responsible for the increased release of IL-6, IL-8 and ROS. Increased endotoxin and ATP levels were found in the bacterial supernatants from filamentous isolates. Apyrase decreased IL-6 secretion from A498 cells and polymyxin B abolished the increased ROS-production from PMN cells. PMN were able to inhibit the bacterial growth of some ESBL-isolates in the presence of ceftibuten.

In conclusion, antibiotic-induced filamentation of ESBL-producing UPEC isolates and the associated release of ATP and endotoxins can alter the host cell response in the urinary tract.

Free milk-type oligosaccharides are produced during pregnancy and lactation and may have an impact on several cells in the immune system. Our aim was to investigate if patients with isolated hyperprolactinaemia, not related to pregnancy, also have increased synthesis and urinary excretion of milk-type oligosaccharides and to compare the excretion pattern with that found during pregnancy. Urine samples were collected as morning sample from 18 patients with hyperprolactinaemia, 13 healthy controls with normal prolactin levels and four pregnant women. After purification, lactose and free oligosaccharides were analysed and quantified by high-performance anion-exchange chromatography with pulsed amperometric detection. The identity of peaks was confirmed by exoglycosidase treatment and comparison with oligosaccharide standards. Prolactin was measured in serum collected between 09 and 11 a.m. by a standardized immunochemical method. Patients with hyperprolactinaemia had higher urinary excretion of lactose than normoprolactinemic controls and urinary lactose correlated positively to prolactin levels (r = 0.51, p < 0.05). Increased levels of the fucosylated oligosaccharides 2-fucosyl lactose and lacto-di-fucotetraose were found in urine from three and two patients, respectively. The acidic oligosaccharide 3-sialyl lactose was found in high amount in urine from two patients with prolactin of >10,000 mU/l. However, pregnant women in their third trimester had the highest concentration of all these oligosaccharides and excretion increased during pregnancy. This study is first to show that both lactose and certain fucosylated and sialylated milk-type oligosaccharides are increased in some patients with hyperprolactinaemia. It remains to elucidate the functional importance of these findings.

Mitral annulus motion (MAM) has recently been introduced as an index of left ventricular function. Previous studies have shown a good agreement between MAM (mm) x 5 and ejection fraction in middle-aged and elderly patients. These studies only included patients with sinus rhythm, while patients with atrial fibrillation were excluded. In the present study, MAM was reduced in patients with atrial fibrillation while ejection fraction (EF) did not differ from age-matched control patients with sinus rhythm. The 'conversion factor' (EF/MAM) was 7.2 in the group with atrial fibrillation and 5. 1 in controls with sinus rhythm. This difference must be taken into account when MAM is used to estimate left ventricular function in patients with atrial fibrillation. Patients with atrial fibrillation had lower stroke volume and higher heart rate than patients with sinus rhythm. A decreased systolic long-axis shortening was found (P<0.005) compared to patients with sinus rhythm, but no difference in short-axis diameter shortening.

Alpha-naphthyl acetate esterase (ANAE) and CD14 expression, used for determination of monocytic cells, were compared and related to prognosis in 65 AML patients. Bone marrow aspiration material from AML patients has been used for the cytochemistry as well as flow cytometry. All non-erythroid cells have been included in the evaluation in both methods. 17/65 cases showed at least 15% difference between the proportion CD14 and ANAE positive cells. Cases with 20% or more CD14 positivity had poorer prognosis. For FAB classes M0-M3, presence of 10% or more CD14 was negative for overall survival (P = 0.01). ANAE did not show significant prognostic influence.

The survival and outcome rates of 284 patients who underwent surgical treatment for non-small cell lung cancer were assessed retrospectively. Resectability rate was 94.1%, hospital mortality 3.9% (n = 11) and the mortality rates in patients who underwent pneumonectomy or lobectomy were 8.9% and 0.6%, respectively. The overall 5-year survival was 43.6%. Female gender, earlier stages of disease and a complete resection were strongly predictive for a long-term survival. Women in stage IA disease had a 5-year survival rate of 92.7%. The 5-year survival rate for patients in stages IIIA and N2 disease who underwent a complete resection was 21.9%, and 9% for those who did not undergo a complete resection. It is concluded that the best surgical results were observed in women who were operated on at an early stage of disease. A complete resection also contributed to a better outcome, even for patients in stage IIIA and N2 disease.

The present study was designed to analyze the relationship of human papillomavirus (HPV)-DNA, microvessel density, and their impact on clinical outcome in early cervical carcinoma. HPV-DNA was evaluated in 171 cases of cervical carcinoma treated from 1965 to 1990. In 110 cases, the analyses could be performed. A polymerase chain reaction technique was used on paraffin-embedded specimens obtained before the start of therapy. HPV-DNA of any type was detected in 78% (86/110) of all evaluable tumors. HPV16 was the predominant type and was detected in 56% (62/110), HPV18 in 8% (9/110), and HPV35 in 21% (23/110). Patients with tumors containing HPV16 or HPV18 were significantly (P = 0.011) younger than patients with tumors not containing either of these two subtypes. Vascular space invasion and lymph node metastases were observed more frequently in tumors expressing HPV16 and HPV18 (P = 0.002, P = 0.047) than in tumors negative for these HPV strains. Tumors containing HPV16 and HPV18 were significantly (P = 0.012) larger and more frequently (P = 0.005) associated with higher FIGO stages. The cancer-specific survival rate was lower for patients with HPV16- and HPV18-positive tumors, but the difference was not statistically significant. The microvessel density was a non-significant prognostic factor. The overall 5-year survival rate of the complete series was 91%. It was concluded that HPV-DNA was a prognostic factor in early-stage cervical cancer and was associated with the age of the patient, vascular space invasion, lymph node metastases, tumor size, and FIGO stage.

The purpose of the present study was to analyze the relation between the expression of p53, bcl-2, p21WAF1, MIB-1, HER-2/neu, DNA ploidy and HPV16 or 18 infections with clinical parameters. HPV-DNA was evaluated in 171 early cervical carcinomas treated from 1965 to 1990 and detected by PCR (polymerase chain reaction) on paraffin specimens obtained before therapy was started. HPV-DNA of any type was detected in 78% (86/110) of all tumors, HPV16 was the predominant type and was seen in 56% (62/110), HPV18 in 8% (9/110) and HPV35 in 21% (23/110). Patients with HPV16 or 18 were significantly (P=0.011) younger than patients with tumors not containing these two HPV subtypes. Lymph node metastases were seen more frequently (P=0.047) in tumors expressing HPV16 or 18. Tumor size was associated with the HPV-type. The frequency of DNA aneuploidy was lower in high-risk HPV tumors than in tumors with other HPV subtypes (P=0.014). MIB-1 expression was highly significantly (P=0.00007) associated with presence of HPV16 or 18. The cancer-specific survival rate was lower for patients with HPV16 and 18 positive tumors, but the difference was not statistically significant. The overall 5-year survival rate of the complete series was 91%. In conclusion, the HPV DNA subtype was a prognostic factor in early stage cervical cancer and it was associated with age, positive lymph nodes, tumor size, DNA ploidy and the proliferation marker MIB-1.

This study evaluated the prognostic importance of a new grading system focusing on the invasive tumor front, DNA profile, and the proliferation marker MIB-1. A complete geographic series of 172 women treated with radical hysterectomy (Wertheim-Meigs) for FIGO stage I-II cervical carcinomas was the target population. The analyses were performed on 141 (82%) squamous cell carcinomas of the complete series. During the period of observation (mean 222 months), 17 recurrences (12.1%) were encountered. Prognostic factors for disease-free survival were lymph node status (P < 0.000001), radical surgical margins (P = 0.00004), and tumor size (P = 0.002). The complete score of the invasive front grading system (IFG), and the individual scores of two variables-pattern of invasion and host response-were all significantly (P = 0.002, P = 0.007, P = 0.0001) associated with pelvic lymph node metastases. Host response was the single most important factor in the IFG system, and it was superior to the complete score in predicting lymph node metastases. The total IFG score was also a significant (P = 0.003) prognostic factor for disease-free survival. DNA ploidy, S-phase fraction, and MIB-1 expression were nonsignificant factors in predicting pelvic lymph node metastases and disease-free survival of the patient. The IFG in the original or modified versions could predict low- and high-risk groups of tumors and therefore be of value in treatment planning for these patients.

The purpose of this study was to investigate the prognostic importance of clinical and histopathologic factors, including malignancy grading systems (MGS), partial index (PI), invasive front grading (IFG), and microvessel density. A complete geographic series of 172 early stage (FIGO I-II) cervical carcinomas treated by Wertheim-Meigs surgery during the period 1965-1990 was studied. The patients were followed up for at least 10 years. Significant prognostic factors for disease-free survival were lymph node status (P < 0.0000001), radical surgical margins (P = 0.00003), and tumor size (P = 0.008). In a multivariate Cox analysis it was shown that lymph node status was the single most important prognostic factor with regard to disease-free survival. The total MGS and the PI scores were highly significantly (P = 0.0001) associated with pelvic lymph node metastases and disease-free survival rate in squamous cell carcinomas. The MGS and the PI systems were superior to the IFG system in predicting lymph node metastases. The total IFG score was also a statistically highly significant (P = 0.003) prognostic factor with regard to disease-free survival in both univariate and multivariate analyses. Microvessel density was a nonsignificant prognostic factor. There was a highly significant (P = 0.002) association between vascular space invasion of tumor cells and the presence of lymph node metastases. In conclusion, histopathologic malignancy grading systems provide valuable prognostic information in patients with early stage squamous cell carcinomas of the uterine cervix.

The objective of this study was to assess the value of p53, bcl-2, and p21(WAF1/CIP1) immunoreactivity as predictors of pelvic lymph node metastases (LNM), recurrences, and death due to the disease in early stage (FIGO I-II) cervical carcinomas. FIGO stage, type of histopathology, and tumor grade were also evaluated in this series of patients treated by radical hysterectomy (Wertheim-Meigs) between 1965 and 1990. A total of 172 patients were included. A tumor was regarded as positive when more than 30% of the neoplastic cells exhibited immunoreactivity. Positive immunostaining was found in 8.9% for p53, in 43.5% for bcl-2, and in 25.0% for p21(WAF1/CIP1). None of them was able to predict LNM or clinical outcome. Presence of LNM, tumor recurrence, and death from disease were significantly associated with the FIGO stage (P = 0.014, P = 0.009, and P = 0.001, respectively). The 5-year cancer-specific survival rate was 91.6% and the overall survival rate was 90.5%. It was concluded that immunohistochemically detected p53, bcl-2, and p21(WAF1/CIP1) appeared to be of no predictive value with regard to LNM, tumor recurrences, or long-term survival in early cervical carcinomas.

A complete series of 40 cervical carcinomas with pelvic lymph node metastases were analysed immunohistochemically for prognostic markers. The aims of this study were to examine whether the detection of MIB-1, p53, bcl-2, and WAF-1 could be used as a prognostic marker for tumor recurrence and survival rate. During the period of observation (mean 222, range 72-360 months) 22 (55%) recurrences were encountered and 20 patients died of the disease. There were 35 squamous cell carcinomas (87.5%), 2 adenosquamous carcinomas (5.0%), and 3 pure adenocarcinomas (7.5%). One tumor (2.5%) was well differentiated, 12 tumors (30%) were moderately differentiated, and 27 tumors (67.5%) were poorly differentiated. The primary tumor grade (P=0.037) and radicality of the surgical margins (P=0.021) were significant prognostic factors with regard to tumor recurrence. The site and number of lymph nodes with metastases had no prognostic value. P53, bcl-2, and WAF-1 were not predictive factors for recurrences or the cancer-specific survival rate. The concordant expression of WAF-1 in the primary tumor and in lymph node metastases was lower than for p53 and bcl-2. The proliferative activity (MIB-1) seemed to be lower in tumor cells metastasized to the pelvic lymph nodes than in cells of the primary tumor. Expression of MIB-1 in lymph nodes was predictive of disease-free survival in both univariate and multivariate proportional hazard Cox analyses.

Background: A girl suffering from a rare syndrome of unknown aetiology, termed hypercalprotectinaemia, was evaluated for tissue zinc status, because calprotectin is a protein which chelates Zn at multiple binding-sites, which might have affected the distribution of Zn in her body.

Methods: Measurement of serum, urine, hair and nail zinc (Zn) concentration, complemented with measurement of total Zn in ultrafiltrates of plasma.

Results: Her serum Zn concentration was 105-133 mu mol/L. Zn levels in her hair (102 mu g/g), nail (90 mu g/g) and urine (3-12 mu mol/L; 20-80 mu g/dL) were all at the lower end of the reference intervals described in the sparse literature. Zn concentrations in ultrafiltrates of plasma were below the detection limit (<100 nmol/L). Thus, the elevated serum Zn did not translate into a similarly increased level of Zn in any of the tissues tested, nor in free Zn concentrations. Instead it appeared to be a result of Zn being chelated to binder proteins, most probably calprotectin.

Conclusion: Her grossly elevated serum calprotectin concentration is probably able to raise circulating total Zn concentrations without raising ionized concentrations, but this Zn remains confined to the circulating blood as well as to excreted body fluids, particularly faeces.

This paper reports the presence of memory T cells in the nasal mucosa of allergic patients. The demonstration of CD4+/CD29+ (CD4+/CD45RO+) T lymphocytes, which are capable of interleukin-4 production, can indicate a complementary cell-mediated regulatory mechanism for mast cell proliferation and IgE synthesis in human nasal allergy. No substantial IgE production can be obtained in the absence of IL-4. Therefore, the existence of IL-4 producing cells on site in the nasal mucosa of allergic subjects probably implies a complementary interaction between cytokines and different immunocompetent nasal cells in the regulation of B cells and IgE synthesis.

Certain cytokines are involved in the generation of natural killer (NK) cells and participate in the regulation of the proto-oncogene bcl-2. We aimed to study the mRNA expression of interleukin (IL)-2, IL-4 and IL-5, the composition of the tumour infiltrating lymphocytes (TIL), and the expression of bcl-2 in 14 benign and malignant human parotid tumours. T IL were predominantly composed of T lymphocytes and NK cells. We found evidence for the homing of T cells, and for generation of NK cells in the vicinity of the tumours. mRNA for IL-2 and IL-12, were identified but IL-4 mRNA was not found. The cytokine profiles and the composition of TIL of the two tumour categories were indistinguishable, suggesting that these host-response variables do not explain the differences in biological behaviour of these particular tumours. The results support a shift towards Th 1 (T helper 1) cells and interferon-gamma production, and that IL-12 also in vivo may play an important role in the regulatory interaction between innate resistance and adaptive immunity in tumour diseases. Most infiltrating lymphocytes showed strong expression of bcl-2; an interesting observation with regard to lymphocytic apoptosis in neoplastic diseases. The immunoreactivity for the bcl-2 protein varied considerably between and within tumours, and almost all benign tumours showed strong bcl-2 positively whereas several of the malignant tumours showed weak or absent staining. The variable expression of bcl-2 protein suggests a different susceptibility of tumour cells to apoptosis. The results also indicate that bcl-2 cannot pla a major role as protective agent in the specific apoptotic pathway induced by NK cells.

Six patients with grass-pollen allergy were provoked with water-soluble grass pollen until a pronounced allergic reaction occurred. This was performed outside the grass-pollen season, and the allergen was administered on the edge of the inferior turbinate. Biopsies were taken both before provocation and during the reaction, 15-30 minutes after provocation. The nasal population of immunohistochemically positive cells for HLA-DR, CD1, interleukin-2-receptor, IgE, CD4 and CD8 were studied. There was a marked increase of IL2-R-positive cells (activated T lymphocytes) in the nasal mucosa after provocation, whilst the other cell populations approximately remained unchanged (apart from a certain increase of IgE). The increase of activated T lymphocytes may imply that certain subsets of T cells play a role in the allergic response, and that the role of helper T cells very likely is much more complex than the regulation of mast cells and eosinophils. The concomitant presence of Langerhans' cells (CD1-positive) and activated T lymphocytes may indicate a possible association on site between an antigen-presenting cell and both effector as well as memory cells in allergic reactions.

The clinicopathological and immunocytochemical features of nine cases of salivary duct carcinoma are described. This relatively rare tumour, which only recently has been widely recognized as a separate entity, is highly malignant and caused the death in eight of the patients. The tumour cells are arranged in cribriform and solid growth patterns, where the solid tumour nests frequently have comedo necrosis, and a fibrous, often sclerotic, stroma is present. The infiltrating desmoplasmic component and the diffuse invasive growth into adjacent adipose parotid tissue have similarities to ductal breast carcinoma. Immunocytochemical investigation of salivary duct carcinoma showed constant overexpression of c-erbB-2 as detected by membrane accentuation, and high proliferative activity as detected by nuclear positivity for MIB 1 (Ki-67). Changes in the expression of p53 and retinoblastoma gene product do not constitute a constant event in salivary duct carcinoma. A few of the tumours showed scattered cells with distinct nuclear positivity for both progesterone and oestrogen receptors. We emphasize that this highly malignant salivary gland tumour has a characteristic morphology, may not be as rare as previously considered, and that prompt and aggressive therapy is needed.

Culture is commonly regarded as the gold standard for diagnosis of Neisseria gonorrhoeae. However, nucleic acid amplification tests (NAATs) have rapidly replaced culture for diagnostics in many settings. The aim of the present study was to investigate the appropriate time for test-of-cure (TOC) when NAATs are used for diagnosis of gonorrhoea. In total, 30 patients (28 men and 2 women) provided urethral, cervical, rectal or pharyngeal specimens for TOC. All included patients, except one who did not return for second TOC before day 19, tested negative within 2 weeks after treatment with cefixime 400 mg x 1. Antimicrobial susceptibility testing showed that 68% of the culture-positive strains were resistant to ciprofloxacin. Thus, the recommended empirical treatment with ciprofloxacin in Norway should be changed immediately. TOC can be performed 2 weeks after treatment when NAATs are used for diagnosis of gonorrhoea.

Aims

Interaction between blood and bio-surfaces is important in many medical fields. With the aim of studying blood-mediated reactions to cellular transplants, we developed a whole-blood model for incubation of small volumes for up to 48 h.

Methods

Heparinized polyvinyl chloride tubing was cut in suitable lengths and sealed to create small bags. Multiple bags, with fresh venous blood, were incubated attached to a rotating wheel at 37°C. Physiological variables in blood were monitored: glucose, blood gases, mono- and divalent cations and chloride ions, osmolality, coagulation (platelet consumption, thrombin-antithrombin complexes (TAT)), and complement activation (C3a and SC5b-9), haemolysis, and leukocyte viability.

Results

Basic glucose consumption was high. Glucose depletion resulted in successive elevation of extracellular potassium, while sodium and calcium ions decreased due to inhibition of energy-requiring ion pumps. Addition of glucose improved ion balance but led to metabolic acidosis. To maintain a balanced physiological environment beyond 6 h, glucose and sodium hydrogen carbonate were added regularly based on analyses of glucose, pH, ions, and osmotic pressure. With these additives haemolysis was prevented for up to 72 h and leukocyte viability better preserved. Despite using non-heparinized blood, coagulation and complement activation were lower during long-term incubations compared with addition of thromboplastin and collagen.

Conclusion

A novel whole-blood model for studies of blood-mediated responses to a cellular transplant is presented allowing extended observations for up to 48 h and highlights the importance of stringent evaluations and adjustment of physiological conditions.

The association between known prognostic variables and altered immunostaining for the nuclear proteins retinoblastoma (Rb) and p53 was studied in a homogeneous series of locally advanced bladder cancer. The predictive value of this immunostaining for the local response to intended radical radiotherapy was investigated. Among 262 patients treated with intended radical radiotherapy between 1967 and 1986, a total of 154 patients were evaluable with respect to local response to treatment. The paraffin-embedded specimen from the tumour prior to irradiation was immunostained with the monoclonal antibodies PMG3-245 for Rb and 1801 for p53 nuclear proteins after heating in a microwave oven for 40 min at 650 W. An altered expression of Rb and p53 was observed in 18 and 42% of the tumours, respectively. p53 overexpression was associated with higher tumour grade. However, the results of the p53 and Rb immunostaining procedures had no predictive value for tumor response to radiation treatment, local control or cancer-specific mortality.

Purpose: We elucidate the association between altered immunostaining for retinoblastoma gene protein (pRb) and p53 nuclear proteins, and cancer specific death in patients treated with cystectomy for locally advanced bladder cancer.

Materials and Methods: The hospital records of 173 patients treated with cystectomy for advanced urothelial bladder cancer between 1967 and 1992 were retrospectively reviewed. Representative biopsies obtained before treatment were sectioned and stained using the standard immunohistochemical technique with antibody DO-7 (p53) and antibody PMG3-245 (pRb). A tumor was considered to have an altered p53 expression if 20% or more of tumor cells exhibited nuclear staining. Similarly, if no tumor cell had nuclear immunostaining the tumor was considered to have an altered pRb expression.

Results: An altered expression was observed for p53 in 98 tumors (57%) and for pRb in 60 (35%). In a proportional hazards analysis no association was found between an altered expression of pRb or p53 and cancer specific death. This finding was also true in another analysis when the results of immunostaining for pRb and p53 were combined.

Conclusions: An altered expression for pRb and/or p53 was not correlated to cancer specific death. Thus, these parameters could not be used as predictors of treatment outcome after cystectomy for locally advanced bladder cancer.

Background: The aim of this study was to elucidate the associations among immunostaining for p53, p21, and mdm2; their respective expression within each tumor; and the value of these variables for predicting treatment outcome after cystectomy for patients with locally advanced bladder carcinoma.

Methods: The hospital records from all 173 patients treated with cystectomy for locally advanced urothelial bladder carcinoma between 1967 and 1992 were retrospectively reviewed. Three consecutive sections from biopsies taken before any treatment were stained using the standard immunohistochemical technique for p53, p21, and mdm2, respectively. The cutoff limit was 20% or more for positive p53 expression and 10% or more for positive p21 and mdm2 expression.

Results: Positive immunostaining was observed for p53 in 98 tumors (57%), for p21 in 89 tumors (51%), and for mdm2 in only 16 tumors (9%). The only association found between immunostaining for the three antibodies was that most mdm2-positive tumors had positive p21 expression. Tumor mapping of regional immunostaining showed no association between immunostaining for p53 and p21. In a proportional hazards analysis, no association was found between the results of immunostaining for the three antibodies and treatment outcome.

Conclusions: Positive or negative expression of p53, p21, or mdm2, or combinations of these, was not associated with cancer specific mortality after cystectomy for bladder carcinoma. There was no association between immunostaining for p21 and p53, whereas positive immunostaining for mdm2 was observed in a minority of the tumors. These results indicate that, in addition to p21, p53, and mdm2, there are other oncoproteins and tumor suppressor proteins along the p53 pathway that are involved in tumor development and progression.

Compared to the 1990s when HbA1c was established as a marker for glycemic control, the accuracy of the HbA1c assays has greatly improved and HbA1c is currently used also for the diagnosis of type 2 diabetes. For most patients, the agreement is excellent between glycemic status and HbA1c results achieved by various methods. However, for patients with increased erythrocyte turnover, HbA1c does not reflect the glycemic status. For patients with rare haemoglobin variants the HbA1c value might be falsely decreased or increased, depending on which HbA1c assay has been used. Recently, falsely increased HbA1c results due to aspirin interference in an ion-exchange method were reported. When misleading results are suspected comparison of results from different HbA1c methods using different analytical principles or continuous glucose monitoring might be useful. HbA1c is likely to remain the most important marker for glycemic control, but complementary tests have to be established.

Immunosuppressive (IS) medication is needed to avoid graft rejection in porcine transplantation models. An ideal IS therapy should have no side-effects, but increased susceptibility to infections, disturbed intestinal microflora and toxic effects on organs and tissues are commonly reported. The aim of the present study was to design an IS protocol with tacrolimus and mycophenolic acid to be used for maintenance therapy in the post-transplant period. An eligible whole blood trough value for tacrolimus was 5-15 mu g/L. Conventional specific pathogen-free pigs were fitted with an indwelling catheter under general anaesthesia, and after the acclimatization period three groups were formed: group A (n = 4) received 0.15 mg/kg body weight (BW) twice daily tacrolimus and 500 mg twice daily mycophenolic acid; group B (n = 4) received 0.3 mg/kg BW twice daily tacrolimus and 500 mg twice daily mycophenolic acid; group C (n = 2) did not receive any medication. Daily clinical examinations and analyses of blood concentrations of tacrolimus and glucose were performed. Total and differential white blood cell counts, enzyme activities, bilirubin and electrolyte concentrations were measured every fourth day. At the end of the experiment, the pigs were killed with an overdose of pentobarbital intravenously and a necropsy was performed immediately. All animals seemed to tolerate the IS treatment well. No alterations in their clinical state of health were observed throughout the study and daily weight gain was similar for the three groups. The necropsy did not reveal any pathological findings related to medication. The study showed that 0.25 mg/kg BW twice daily tacrolimus and 500 mg twice daily mycophenolic acid would be an appropriate maintenance dosage for conventional pigs.

Diagnostic testing for Clostridium difficile infection (CDI) has, in recent years, seen the introduction of rapid dual-EIA (enzyme immunoassay) tests combining species-specific glutamate dehydrogenase (GDH) with toxin A/B. In a prospective study, we compared the C. DIFF Quik Chek Complete test to a combination of selective culture (SC) and loop-mediated isothermal amplification (LAMP) of the toxin A gene. Of 419 specimens, 68 were positive in SC including 62 positive in LAMP (14.7%). The combined EIA yielded 82 GDH positives of which 47 were confirmed toxin A/B positive (11%) corresponding to a sensitivity and specificity of 94% for GDH EIA compared to SC and for toxin A/B EIA a sensitivity of 71% and a specificity of 99% compared to LAMP. Twenty different PCR ribotypes were evenly distributed except for UK 081 where only 25% were toxin A/B positive compared to LAMP. We propose a primary use of a combined GDH toxin A/B EIA permitting a sensitive 1-h result of 379 of 419 (90%, all negatives plus GDH and toxin EIA positives) referred specimens. The remaining 10% being GDH positive should be tested for toxin A/B gene on the same day and positive results left to a final decision by the physician.

A methodological approach by computerized image analysis to quantify immunostained objects in histological sections is described. We have investigated antibodies against CD4, CD8, CD20, CD23 and CD25 in frozen sections of human nasal mucosa; however, the methodology of standardization is of general validity. The study was designed particularly to investigate the following points: 1) light intensity, 2) the grey level for counter staining intensity, 3) the grey level threshold value for positive objects, 4) the minimal acceptable size of a positive object, 5) the influence of the brightness of the light on both the number and the area of objects. Furthermore, random sampling and determination of 6) the area per section, and 7) the number of histological sections to be measured per biopsy. Finally, a study of reproducibility of immunostaining intensity was performed. The influence of the different parameters mentioned above was studied and the values (eg. threshold value) for our particular setting of microscope, image analysis equipment, computer software etc, were defined. The method was then tested for intra- and interindividual variation which was found to be less than 5%. Correlation analysis of the reproducibility gave coefficients of correlation of 0.99, both concerning number of immunopositive objects and immunopositive area. We emphasize the importance of a highly standardized methodology if the numeric data obtained from computer assisted image analysis are to be more accurate than semiquantitative assessments by experienced observers. With a thorough standardization as described in this method it is possible to obtain numeric values, and data with low deviations, which are two obvious and important advantages.

By means of immunocytochemistry we have investigated subsets of T lymphocytes in frozen sections of nasal mucosa from patients with seasonal allergic rhinitis and healthy control persons. All participants were subjected to time-course provocation during the non-pollen season, and samples were taken during provocation as well as during the natural pollen season. Computerized image analysis was applied for evaluation of the immunostained lymphocytes. CD45RO+ memory T cells outnumbered the remaining leukocyte populations in the mucosa of both allergic patients and controls on all occasions. During the repeat provocation there was no difference in numerical values, with respect to any of the five leukocyte subpopulations studied (CD4, CD8, CD25, CD45RA and CD45RO), between patients and controls. However, during continuous exposure in the pollen season a significant increase in CD4+ cells was observed in allergic patients compared to before provocation (p < 0.05). No changes were observed with respect to CD8+ and CD25+ cells. Similarly, an increase in CD45RO+ memory was found in allergic patients during the pollen season compared to the non-pollen season (p < 0.02). This latter finding was, however, only evident in the patients who did not use nasal corticosteroids. Hence the present investigation has demonstrated an allergen-induced increase in CD4+ and CD45RO+ memory T cells in the mucosa of allergic patients during the pollen season. These events may constitute a cellular basis for local continuous production of certain cytokines, particularly interleukin-4, which is essential for IgE synthesis.

In nasal biopsies from 17 adult patients with seasonal allergic rhinitis and from 10 healthy controls, cytokines were analyzed by reverse-transcriptase polymerase chain reaction (RT-PCR). The time-course study during winter included repeated local allergen provocation with subsequent nasal biopsies as well as biopsies taken during pollen season. The RT-PCR for CD44 yielded positive bands in 65 of 71 cases, in which cases mRNA for interleukins 2, 4, and 5 (IL-2, IL-4, and IL-5) were thus investigated by means of seminested PCR. IL-4 mRNA was found almost exclusively in the allergic patients. During provocation a significant increase in IL-4 was noticed compared with controls (p = 0.043). Equally, during the natural pollen season, IL-4 mRNA expression was significantly higher in patients not using nasal corticosteroids compared with those who did (p = 0.011). No differences in IL-2 or IL-5 were observed between the groups. These findings also indicate, together with earlier observations of T-cell activation, a phenotype switch toward T-helper 2 (Th2) cells, and the accumulation (homing) of these T cells in the nasal mucosa, that T cells constitute the main source for IL-4 in the nasal mucosa. Therefore, allergic patients have an increased synthesis of IL-4 when provoked with the allergen, and during natural pollen season this synthesis can be downregulated by corticosteroids. Furthermore, this study exemplifies the versatility of molecular biology in surgical pathology and that even low-copy-number cytokine mRNA can be examined in routinely snap-frozen surgical specimens.

An association has previously been reported between exposure to medical diagnostic ionizing radiation and papillary thyroid cancer in women. To further evaluate potential mechanisms in carcinogenesis, the expression of p53, c-erbB-2, as well as Ki-67, estrogen and progesterone receptors were analyzed by immunohistochemistry in 19 women exposed to X-rays and for comparison in nine women without such reported exposure. They all had papillary thyroid cancer. No difference was found between these groups. The results of this study showed that p53, c-erbB-2, Ki-67, estrogen and progesterone receptors are not involved in papillary thyroid cancer associated with exposure to medical diagnostic ionizing radiation.

During the allergic reaction mucosal T cells are activated and a local increase in numbers occurs. In peripheral blood, a concomitant T cell activation and switch towards memory phenotype appears. E-selectin, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 were studied in nasal mucosal biopsies taken during a time-course provocation study, including patients with seasonal allergic rhinitis and healthy controls. Allergic patients were also studied during the natural pollen season with particular attention to the influence of local corticosteroid treatment. Before provocation allergic patients and controls did not differ concerning the expression of endothelial adhesion molecules. However, the epithelial ICAM-1 expression was increased among allergics (P < 0.05). Repetitive allergen provocation induces an increased endothelial expression of VCAM-1 in allergic patients (P < 0.01). Similarly, VCAM-1 expression was increased during the natural pollen season (P < 0.05). Interestingly, the increased VCAM-1 expression was inhibited by the use of local corticosteroids. The present data demonstrate a putative integrin-VCAM-1 mechanism for selective homing of T memory cells to the allergic nasal mucosa and new in vivo effects of local corticosteroid treatment are demonstrated.

The peripheral blood of 17 patients with seasonal allergic rhinitis (birch pollen) and of 10 healthy subjects were analysed before and during a provocation study in the non-pollen season as well as during the pollen season. Analytical flow cytometry comprised a panel of monoclonal antibodies investigating helper/suppressor T cells, activated T cells, and naive/memory T helper cells. Allergic patients showed no increase in the amount of T lymphocytes but an increased proportion of CD4+ helper T cells early during pronounced exposure (provocation) but not during natural antigen exposure (pollen season). Allergic patients showed a significant increase in activated T cells during the non-pollen season compared with healthy subjects, and furthermore, the nasal allergen provocation induced an additional increase in activated T cells among allergics. The T cell activation mainly affected helper T cells (85%) rather than suppressor T cells. Furthermore, allergic patients showed a significant increase in naive T helper cells during the pollen season. The presence of a double-positive subpopulation indicates an activated T helper subpopulation that switches its phenotype from naive (CD45RA) to memory (CD29). The results indicate at least two important differences between patients with allergic rhinitis and healthy controls. In allergic patients T helper cells become activated upon allergen exposure, and circulate in the blood and switch their phenotype. These T cells have a potential homing tendency to the nasal mucosa. These two events do not occur in non-allergic individuals and may thus constitute new insights into the basic mechanisms of allergic rhinitis.

Introduction: Prosthetic joint infections (PJIs) caused by Propionibacterium acnes account for a larger proportion of the total number of PJIs than previously assumed and thus knowledge of the antimicrobial susceptibility patterns of P. acnes is of great value in everyday clinical practice.

Materials and methods: Using Etest, the present study investigated the susceptibility of 55 clinical isolates of P. acnes, obtained from orthopaedic implant-associated infections of the knee joint (n = 5), hip joint (n = 17), and shoulder joint (n = 33), to eight antimicrobial agents: benzylpenicillin, clindamycin, metronidazole, fusidic acid, doxycycline, moxifloxacin, linezolid and rifampicin. Synergy testing was also conducted, in which rifampicin was combined with each of the remaining seven antibiotics.

Results: All isolates (n = 55) were susceptible to most of the antibiotics tested, with the exception of 100% resistance to metronidazole, five (9.1%) isolates displaying decreased susceptibility to clindamycin, and one (1.8%) to moxifloxacin. None of the antimicrobial agents investigated were synergistic with each other when combined and nine isolates were antagonistic for various antimicrobial combinations. The majority of the antimicrobial combinations had an indifferent effect on the isolates of P. acnes. However, the combination of rifampicin and benzylpenicillin showed an additive effect on nearly half of the isolates.

Conclusion: Almost all P. acnes, isolated from orthopaedic implant-associated infections, predominantly PJIs, were susceptible to the antibiotics tested, with the exception of complete resistance to metronidazole. Synergy test could not demonstrate any synergistic effect but additive effects were found when combining various antibiotics. Antagonistic effects were rare.

BACKGROUND: In human blood, there are several molecular variants of prolactin with different biological effects. There is a need for new methods to detect and quantify these variants in order to fully understand the pathophysiological role of prolactin.

METHODS: A method based on ultrafiltration was optimized, validated and compared to PEG precipitation. Serum samples from 84 patients were analyzed before and after pre treatment on two immunoassays, Elecsys (Roche) and Access (Beckman). Protein G precipitation was used to confirm presence of macroprolactin.

RESULTS: The recovery of prolactin after ultrafiltration was lower than after PEG precipitation. A limit of 40% recovery after PEG precipitation corresponded to 27% recovery after ultrafiltration. Using these limits there were total agreement regarding detection of macroprolactin (r(s)=0.96). In contrast, recovery of prolactin in samples without macroprolactin showed a considerable disagreement between ultrafiltration and PEG precipitation (r(s)=0.48). Within-run CV was 4% for the ultrafiltration method. The correlation coefficient (r) between the immunoassays was 0.96 after ultrafiltration.

CONCLUSIONS: Ultrafiltration can be used to compare different prolactin immunoassays and to detect macroprolactin in assays with interference from PEG. For samples without macroprolactin ultrafiltration may give additional information reflecting individual variations of other molecular variants of prolactin.

Introduction: The aim of the study was to investigate whether biobanked liquid-based cytology (LBC) vaginal samples could be reanalyzed for the biomarkers HPV DNA and mRNA without loss of sensitivity.

Methods: One hundred LBC samples with ASCUS or CIN1 were tested for HPV DNA and mRNA before and after biobanking. DNA analysis targeted the viral genes E6 and E7, 12 high-risk and 2 low-risk HPV types together with the human control gene HBB, using real-time PCR. The Aptima HPV assay was used for mRNA analysis of 14 high-risk HPV types.

Results: With Aptima there was 84% agreement between results before and after biobanking. The sensitivity and specificity were 0.79 (95% CI, 0.68-0.88) and 0.94 (95% CI, 0.80-0.99), respectively. With the DNA-based method, the agreement between results was 87%, the sensitivity 0.85 (95% CI, 0.75-0.92) and the specificity 0.95 (95% CI, 0.77-1.00). Both methods presented a significant difference between positive results before and after biobanking; McNemar test: p = 0.004, p = 0.003, Cohen's kappa: 0.67 (95% CI, 0.53-0.81), 0.68 (95% CI, 0.52-0.84). Cycle threshold values for the DNA method were higher for all genotypes after biobanking, except for HPV-59. Some loss of sensitivity was seen after biobanking but the concordance between HPV detection before and after biobanking was good for both evaluated methods.

Conclusions: Biobanking of LBC vaginal samples offers a good platform for HPV testing and could be extended to further molecular analyses. However, in order to ensure a valid test result a larger portion needs to be analyzed from the biobanked sample.

Objective: To study the expression of thymidine phosphorylase (TP), thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) mRNA in breast cancer and its correlation with prognosis.

Methods: Expression levels of TP, TS and DPD mRNA in 86 micro-selected breast cancer tissues and 9 normal breast tissues were detected by real-time quantitative PCR.

Results: The median expression levels of TP, TS and DPD mRNA in tumor tissue and in normal tissues were 16.54, 0.38, 2.47 and 11.75, 0.25, 8.33, respectively, there were no significant differences (P >0.05). The expression levels of TP, TS and DPD mRNA showed no association with tumor size, lymph node metastasis, pathological grade and clinical stage, except that of DPD showed a negative association with patients' ages. There was no significant difference in disease-free survival or overall survival between the patients with high and low TP or DPD mRNA levels. Disease-free survival tends to be better in the patients with low TS mRNA level than those with high TS mRNA, but the difference was not significant (P=0.069), while the overall survival showed a statistically difference (59.00 month and 70.30 month) (P=0.0496).

Conclusion: The expression level of TS mRNA may serve as a prognostic marker for breast cancer patients.

Human papillomavirus (HPV) testing and genotyping of FFPE tissue samples is important in epidemiological investigations. Here, we compare four different HPV genotyping methods for use in FFPE clinical samples. Comparative testing was performed on 99 samples with a clinical suspicion of HPV. Specimens were analyzed with Anyplex II HPV28 detecting 28 genotypes using real-time PCR and melting curve analysis, CLART HPV2 detecting 35 genotypes using PCR and microarray detection, and MGP5+/6+ consensus primer system together with pyrosequencing. Results were compared to a real-time PCR reference protocol detecting 14 genotypes. In total, 68% of the samples were positive for an HPV genotype using the reference protocol and MGP5+/6+ primer system. Anyplex II HPV28 analysis and CLART HPV2 had 82% and 72% positive samples, respectively. All four methods showed good agreement when comparing the 14 genotypes included in the reference protocol. When evaluating all genotypes, the Anyplex II HPV28 assay and the CLART assay changed the status of the sample (individually or together) from negative with respect to the reference protocol to positive for either a Group 1 (n = 4) or Group 2 (n = 6) genotype. We conclude from this study that for an extended genotyping approach with a high sensitivity for FFPE specimens, both the Anyplex II HPV28 and CLART HPV2 assays are suitable alternatives despite minor intra-assay differences.

Panton-Valentine leukocidin (PVL) is a virulence factor of Staphylococcus aureus, which is associated with skin and soft-tissue infections and necrotizing pneumonia. To develop a rapid phenotypic assay, recombinant PVL F component was used to generate monoclonal antibodies by phage display. These antibodies were spotted on protein microarrays and screened using different lukF-PV preparations and detection antibodies. This led to the identification of the optimal antibody combination that was then used to establish a lateral flow assay. This test was used to detect PVL in S. aureus cultures. The detection limit of the assay with purified native and recombinant antigens was determined to be around 1 ng/ml. Overnight cultures from various solid and liquid media proved suitable for PVL detection. Six hundred strains and clinical isolates from patients from America, Europe, Australia, Africa, and the Middle East were tested. Isolates were genotyped in parallel by DNA microarray hybridization for confirmation of PVL status and assignment to clonal complexes. The sensitivity, specificity, and positive and negative predictive values of the assay in this trial were 99.7, 98.3, 98.4, and 99.7%, respectively. A total of 302 clinical isolates and reference strains were PVL positive and were assigned to 21 different clonal complexes. In summary, the lateral flow test allows rapid and economical detection of PVL in a routine bacteriology laboratory. As the test utilizes cultures from standard media and does not require sophisticated equipment, it can be easily integrated into a laboratory's workflow and might contribute to timely therapy of PVL-associated infections.