Intestinal mast cells mediate gut injury and systemic inflammation in a rat model of deep hypothermic circulatory arrest.

OBJECTIVE: Cardiac surgery, especially when employing cardiopulmonary bypass and deep hypothermic circulatory arrest, is associated with systemic inflammatory responses that significantly affect morbidity and mortality. Intestinal perfusion abnormalities have been implicated in such responses, but the mechanisms linking local injury and systemic inflammation remain unclear. Intestinal mast cells are specialized immune cells that secrete various preformed effectors in response to cellular stress. We hypothesized that mast cells are activated in a microenvironment shaped by intestinal ischemia/reperfusion, and investigated local and systemic consequences. DESIGN: Rat model of deep hypothermic circulatory arrest. SETTING: University research laboratory. SUBJECTS: Twelve- to 14-week-old male Sprague-Dawley rats. INTERVENTIONS: Rats were anesthetized and cooled to 16°C to 18°C on cardiopulmonary bypass before instituting deep hypothermic circulatory arrest for 45 minutes. Specimens were harvested following rewarming and 2 hours of recovery. MEASUREMENTS AND MAIN RESULTS: Significant intestinal barrier disruption was found, together with macro- and microscopic evidence of ischemia/reperfusion injury in ileum and colon, but not in the lungs or kidneys. Immunofluorescence and toluidine blue staining revealed increased numbers of mast cells and their activation in the gut. In animals pretreated with the mast cell stabilizer, cromolyn sodium, mast cell degranulation was blocked, and intestinal morphology and barrier function were preserved following deep hypothermic circulatory arrest. Furthermore, cromolyn sodium treatment was associated with reduced intestinal neutrophil influx and blunted systemic release of proinflammatory cytokines. CONCLUSION: Our data provide primary evidence that intestinal ischemia/reperfusion is a leading pathophysiologic process in a rat model of deep hypothermic circulatory arrest, and that intestinal injury, and local and systemic inflammatory responses are critically dependent on mast cell activation. This identifies intestinal mast cells as central players in deep hypothermic circulatory arrest-associated responses, and opens novel therapeutic possibilities for patients undergoing this procedure.

Predictive testing of early cervical pre-cancer by detecting human papillomavirus E6/E7 mRNA in cervical cytologies up to high-grade squamous intraepithelial lesions: diagnostic and prognostic implications.

The type-specific persistence of oncogenic human papillomavirus (HPV) is considered to be the true precursor of cervical cancer at which the transcription of the viral oncogenes E6 and E7 is necessary for the malignant transformation and maintenance of the neoplastic state. In the present pilot study, a cohort of 66 women was investigated from a routine office-based screening population who had an index cytological result from normal to high-grade squamous intraepithelial lesions and who were also HPV-DNA positive for at least one of the following high-risk HPV types: HPV 16, 18, 31, 33 and 45 detected by MY09/MY11 consensus and GP5+/6+ general primers, followed by sequencing. The expression of E6/E7 transcripts from the same HPV types was detected by the PreTect HPV-Proofer. Cervical status was checked 18 months after the mRNA test. The expression of E6/E7 mRNA was found in 58% of the cases showing a 97% concordance with the HPV-DNA types and a positive correlation with increasing cytological and histological grade. All HPV-mRNA positive cases were also positive for HPV DNA whereas 25 (38%) of the HPV-DNA positive cases did not express the respective mRNA. The diagnostic validity of the PreTect assay for detecting histologically-proven prevalent CIN3 lesions were: sensitivity 95%, specificity 55%, positive predictive value (PPV) 81% and negative predictive value (NPV) 86%. The prognostic power of the PreTect test for predicting cytological disease progression was as follows: 78% sensitivity, 60% specificity, 37% PPV and 90% NPV. In conclusion, our results showed that the detection of oncogenic HPV E6/E7 mRNA in cervical smears in a routine screening setting identifies prevalent CIN3 lesions with nearly 100% sensitivity and has a very high negative predictive value for disease progression during the natural course of HPV infection. Thus, testing for HPV oncogenic activity may be used as a clinically predictive marker to enhance the net effectiveness of screening and enable the prognostication of prevalent cervical lesions.

The spectrum of cervical diseases induced by low-risk and undefined-risk HPVS: implications for patient management.

BACKGROUND: The natural history and carcinogenicity of rare and novel HPV types is unclear. MATERIALS AND METHODS: From a total of 5,964 women tested for HPV by PCR and sequence analysis, Pap smears from 293 patients harbouring mono-infection with low-risk, undetermined-risk or novel HPV genotypes were investigated. RESULTS: Sixty-three percent of patients had ASC-US, 23% LSIL, 9% were negative and 5% had HSIL in cytology. Of 30 HPV types detected, 19 were of unknown risk (UR)-types including 3 novel genotypes. Four of the UR-HPVs (HPV 69, 30, 67 and 34) could be assigned as probable high-risk types and eight as low-risk types based on phylogenetical relationship. Morphology was not discriminative with regard to HPV type, but non-classical HPV-signs were generally present even in "normal" cytologies. CONCLUSION: HPV-typing is important for risk-adapted individual patient management. Women harbouring novel high-risk or probably high-risk HPVs require more intensive care than those bearing non high-risk infections.

Predicting treatment outcome in cervical diseases using liquid-based cytology, dynamic HPV genotyping and DNA cytometry.

BACKGROUND: In this study, our prospective experience with a multimodal follow-up protocol is summarized, with special emphasis on predicting the treatment outcome of cervical diseases. MATERIALS AND METHODS: Liquid-based cytology samples (ThinPrep) from 209 women exhibiting the whole spectrum of human papilloma virus (HPV)-related cervical diseases were investigated by cytology, PCR-based HPV genotyping and DNA cytometry pre-surgery. The first control cytology and type-specific HPV tests were performed at 3 months post-surgery. RESULTS: The success rate of surgery was 95% in eradicating high-grade cervical disease and 90% in eliminating the baseline HPV genotype. Treatment failure was significantly correlated with baseline cytology (p=0.011), resection margin status (p=0.016) and HPV positivity at 3 months post-surgery (p=0.04). Multivariate logistic regression analysis showed that type-specific persistent HPV infection (p=0.028), baseline cytology (p=0.039) and histology (p=0.065) were independent predictors of residual cervical neoplasias. CONCLUSION: Our results showed that our multimodal surveillance protocol may help to individually assess the anticipated clinical outcome of cervical diseases post-surgery.

Quantitative molecular urinary cytology by fluorescence in situ hybridization: a tool for tailoring surveillance of patients with superficial bladder cancer?

OBJECTIVE: To determine whether it is possible to stratify patients with superficial bladder cancer into low- and high-risk groups for tumour recurrence/progression based on the chromosomal pattern detected by fluorescence in situ hybridization (FISH) in one urine cytology specimen used for follow-up testing. PATIENTS AND METHODS: Voided urine samples from 47 consecutive patients with urinary tract neoplasms (13 with no history of urothelial malignancy and 34 under follow-up after complete transurethral resection of superficial urothelial carcinoma of the bladder) were evaluated by liquid-based cytology (ThinPrep(R), CYTYC Corp., Boxborough, MA, USA) and UroVysion FISH (Vysis-Abbott, Downers Grove, IL). RESULTS: Of the 34 patients under surveillance, the UroVysion test was negative in four, 17 had loss of 9p21 sequences either alone or combined with low-frequency trisomy/ies or tetrasomy/ies of chromosomes 3, 7 and 17 in single cells (low-risk FISH), and 13 also had complex aneusomies of the remaining chromosomes (high-risk FISH). One of the four FISH-negative neoplasms, four of the 17 low-risk FISH cases and five of the 11 informative high-risk FISH-positive patients developed recurrence. Progression occurred only in patients with high-risk FISH results, showing high-frequency complex chromosomal polysomies (four of 11). CONCLUSION: The results from this pilot study indicate that the UroVysion FISH test may help to individually assess the clinical behaviour of superficial bladder cancer, based on the chromosomal pattern of exfoliated tumour cells in follow-up urinary cytology. It might be of use to identify those patients likely to progress at earlier and curable stages of disease, and lengthen the surveillance period in those with persistent or recurrent low-risk disease.

Validity of combined cytology and human papillomavirus (HPV) genotyping with adjuvant DNA-cytometry in routine cervical screening: results from 31031 women from the Bonn-region in West Germany.

Our aim was to improve the accuracy of routine cervical screening by a risk-adapted multimodal protocol with special focus on possible reduction and prognostic assessment of false positive results. A cohort of 31031 women from the Bonn-region in West Germany, median age 36 years, were screened by cytology (conventional or liquid-based), followed by PCR-based HVP detection with genotyping and adjuvant DNA image cytometry, if indicated, in a sequential manner. The true prevalence of high-grade cervical intraepithelial neoplasia and carcinoma (>/=CIN2) was 0.32% in the population as projected from cervical biopsies of 123 women (0.4%), of whom 100 showed >/=CIN2. Sensitivity of the cytology screening program at PapIIID/HSIL threshold for detecting histologically confirmed >/=CIN2 cases was 81%, with specificity, positive predictive value (PPV) and negative predictive value (NPV) of 99, 20.9 and 99.9%, respectively. Of 38 women receiving the complete screening protocol, all the 31 >/=CIN2 cases were correctly detected by cytology alone, 30 by positive high-risk HPV genotype and 30 by aneuploid DNA profile. The combination of the three methods resulted in an up to 6.9% increase in PPV for >/=CIN2 at practically unchanged detection rate with the additional benefit of being able to predict the probable outcome of CIN1 lesions detected as false positives with any single test. Multimodal cervical screening might permit identification of those women with low-grade squamous intraepithelial lesions likely to progress at an earlier and curable stage of disease and lengthen the screening interval in those with transient minor lesions caused by productive HPV infection.