Protease‐activated receptor 2 suppresses lymphangiogenesis and subsequent lymph node metastasis in a murine pancreatic cancer model

Protease‐activated receptor‐2 (PAR‐2) is a G protein‐coupled receptor that functions as a cell‐surface sensor for coagulation factors and other proteases associated with the tumour microenvironment. Pancreatic cancer cells express high levels of PAR‐2 and activation of PAR‐2 may induce their proliferation and migration. Interestingly, however, PAR‐2 expression is increased in stroma‐rich pancreatic cancer regions, suggesting a potential role of PAR‐2 in the tumour microenvironment. Here, we assessed the importance of PAR‐2 in the stromal compartment by utilizing an orthotopic pancreatic cancer model, in which tumour cells are PAR‐2‐positive, whereas stromal cells are PAR‐2‐negative. We assessed tumour weight and volume and analysed proliferation and (lymph)angiogenesis both in vivo and in vitro. We show that genetic ablation of PAR‐2 from the stromal compartment inhibits primary tumour growth, which is accompanied by reduced vascularization in primary tumours and reduced in tube formation of vascular endothelial cells in vitro. In contrast to smaller primary tumours, the number of lymph node metastases was increased in PAR‐2‐deficient animals, which was accompanied by an increased number of lymphatic vessels. In vitro tube‐formation assays show that PAR‐2 does not inhibit the intrinsic tube‐forming capacity of lymphatic endothelial cells, but that PAR‐2 actually inhibits cancer cell‐induced tube formation. Overall, stromal PAR‐2 thus plays a dual role in pancreatic cancer development by potentiating primary tumour growth but limiting lymphangiogenesis and subsequent lymph node metastasis. Our data identify a novel role of PAR‐2 in the tumour microenvironment and pinpoint PAR‐2 as a negative regulator of lymphangiogenesis. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Efficacy and safety of rifampin containing regimen for staphylococcal prosthetic joint infections treated with debridement and retention

The aim of this study was to evaluate the efficacy and safety of rifampin for Staphylococcus aureus (SA) or coagulase negative staphylococci (CNS) prosthetic joint infection (PJI) treated with debridement and retention (D/R). We calculated the treatment failure cumulative incidence (TF) of a cohort of 101 patients with SA or CNS PJI treated with D/R and antimicrobial therapy. The effect of the use of a rifampin-based regimen was evaluated. Cox proportional hazards regression evaluated the association between treatment and time-to-TF controlling for the propensity to treat with rifampin and temporal confounders. Seven percent (1/14) of the prospective rifampin-treated patients, 32% (10/31) of the historical rifampin-treated patients and 38% (21/56) of the historical non-rifampin treated patients developed TF. After controlling for the propensity to treat with rifampin and American Society of Anesthesia scores, patients in the prospective cohort had a lower risk of TF compared to patients in the historical cohort not treated with rifampin (HR 0.11; 95%CI 0.01–0.84). None (0/14) of the patients in the prospective study developed hepatotoxicity. The outcome of staphylococcal PJI treated with D/R and rifampin-based regimens was better when compared with a historical cohort treated without rifampin.     

Immunology of gene therapy with adenoviral vectors in mouse skeletal muscle

Skeletal muscle is an attractive target for somatic gene transfer of both acquired and inherited disorders. Direct injection of adenoviral vectors in the skeletal muscle leads to recombinant gene expression in a large number of muscle fibers. Transgene expression has been transient in most organs and associated with substantial inflammation when experiments are performed in adult immune competent mice. In this report, we utilize a variety of in vivo and in vitro models of T and B cell function to characterize the nature of the immune response to adenoviral vectors injected into murine skeletal muscle. Cellular immunity dependent on CD4+ and CD8+ T cells contributes to the loss of recombinant gene expression and the development of localized inflammation. Antigen specific activation of T cells occurs to both viral proteins and the reporter gene beta-galactosidase. Systemic levels of neutralizing antibody to the capsid proteins of the vector are also generated. Destructive immune responses responsible for loss of transgene expression are largely directed against beta-galactosidase in that transgene expression was stable when beta-galactosidase was eliminated as a neoantigen in mice transgenic for lacZ. A strategy to prevent the cellular and humoral immunity to this therapy was developed based on transiently ablating CD4+ T cell activation at the time of vector delivery. Encouraging results were obtained when vector was administered with one of several immune modulating agents including cyclophosphamide, mAb to CD4+ cells, and mAb to CD40 ligand. These studies indicate that cellular and humoral immune responses are elicited in the context of gene therapy directed to skeletal muscle with adenoviral vectors. Transient ablation of CD4+ T cell activation prevents the effects responses of the CD8+ T and B cells.      

Microsatellite instability and mutation analysis of hMSH2 and hMLH1 in patients with sporadic, familial and hereditary colorectal cancer

To date, at least four genes involved in DNA mismatch repair, hMSH2, hMLH1, hPMS1 and hPMS2, have been demonstrated to be altered in the germline of patients with hereditary nonpolyposis colorectal cancer (HNPCC). Additionally, defective mismatch repair is thought to account for the observation of microsatellite instability (MIN) in tumors from these patients. The genetic defect responsible for the MIN+ phenotype in sporadic colorectal cancer, however, has yet to be clearly delineated. In order to better understand the role of somatic and germline alterations within hMSH2 and hMLH1 in the process of colorectal tumorigenesis, we examined the entire coding regions of both of these genes in seven patients with MIN+ sporadic colorectal cancer, 19 patients with familial colorectal cancer, and 20 patients meeting the strict Amsterdam criteria for HNPCC. Thirteen germline, two somatic, and four neutral alterations were identified. The two somatic mutations occurred in patients having familial cancer, while the germline mutations were distributed among one sporadic (14%), three familial (16%), and nine HNPCC (45%) cases. All patients with identified mutations in the mismatch repair genes, whose tumors were available for analysis, demonstrated MIN. On the other hand, we could not identify mutations in the subset of clinically defined HNPCC patients with MIN negative tumors nor in the majority (6/7) of MIN+ sporadic tumors.      

Phenotypic and genotypic mupirocin resistance among Staphylococci causing prosthetic joint infection

Mupirocin MICs and mupA presence were determined in 108 staphylococci causing prosthetic joint infection. Zero of 35 isolates (0%) of methicillin-susceptible Staphylococcus aureus, 4/15 (27%) methicillin-resistant S. aureus isolates, 3/16 (19%) methicillin-susceptible coagulase-negative staphylococci, and 11/42 (26%) methicillin-resistant coagulase-negative staphylococci were mupirocin resistant. mupA was detected in all five high-level mupirocin-resistant staphylococci and one mupirocin-susceptible staphylococcus.     

Reevaluation of Streptococcus bovis endocarditis cases from 1975 to 1985 by 16S ribosomal DNA sequence analysis

Studies that detected an association between Streptococcus bovis endocarditis and colon carcinoma have not taken into account the recently identified genetic diversity among organisms historically classified as S. bovis. With near full-length 16S ribosomal DNA sequence analysis, organisms cultured from the blood of endocarditis patients at the Mayo Clinic from 1975 to 1985 and previously identified as S. bovis or streptococcus group D nonenterococci were shown to represent S. bovis biotypes I (11 isolates) and II/2 (1 isolate), S. salivarius (1 isolate), and S. macedonicus (1 isolate). Two of the S. bovis biotype I cases were associated with colon cancer. Whether S. bovis biotype II or other organisms closely related to and historically identified as S. bovis (e.g., S. macedonicus) are associated with malignant (or premalignant) colon lesions in humans remains to be definitively determined.     

The molecular basis of medium-chain acyl-CoA dehydrogenase (MCAD) deficiency in compound heterozygous patients: is there correlation between genotype and phenotype?

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most commonly recognized defect of mitochondrial beta-oxidation. It is potentially fatal, but shows a wide clinical spectrum. The aim of the present study was to investigate whether any correlation exists between MCAD genotype and disease phenotype. We determined the prevalence of the 14 known and seven previously unknown non-G985 mutations in 52 families with MCAD deficiency not caused by homozygosity for the prevalent G985 mutation. This showed that none of the non-G985 mutations are prevalent, and led to the identification of both disease- causing mutations in 14 families in whom both mutations had not previously been reported. We then evaluated the severity of the mutations identified in these 14 families. Using expression of mutant MCAD in Escherichia coli with or without co-overexpression of the molecular chaperonins GroESL we showed that five of the missense mutations affect the folding and/or stability of the protein, and that the residual enzyme activity of some of them could be modulated to a different extent depending on the amounts of available chaperonins. Thus, some of the missense mutations may result in relatively high levels of residual enzyme activity, whereas the mutations leading to premature stop codons will result in no residual enzyme activity. By correlating the observed types of mutations identified to the clinical/biochemical data in the 14 patients in whom we identified both disease-causing mutations, we show that a genotype/phenotype correlation in MCAD deficiency is not straightforward. Different mutations may contribute with different susceptibilities for disease precipitation, when the patient is subjected to metabolic stress, but other genetic and environmental factors may play an equally important role.      

Cardiac characterization of 16 patients with large NF1 gene deletions

The aim of this study was to characterize cardiac features of patients with neurofibromatosis 1 (NF1) and large deletions of the NF1 gene region. The study participants were 16 patients with large NF1 deletions and 16 age‐ and sex‐matched NF1 patients without such deletions. All the patients were comprehensively characterized clinically and by echocardiography. Six of 16 NF1 deletion patients but none of 16 non‐deletion NF1 patients have major cardiac abnormalities (p = 0.041). Congenital heart defects (CHDs) include mitral insufficiency in two patients and ventricular septal defect, aortic stenosis, and aortic insufficiency in one patient each. Three deletion patients have hypertrophic cardiomyopathy. Two patients have intracardiac tumors. NF1 patients without large deletions have increased left ventricular (LV) diastolic posterior wall thickness (p < 0.001) and increased intraventricular diastolic septal thickness (p = 0.001) compared with a healthy reference population without NF1, suggestive of eccentric LV hypertrophy. CHDs and other cardiovascular anomalies are more frequent among patients with large NF1 deletion and may cause serious clinical complications. Eccentric LV hypertrophy may occur in NF1 patients without whole gene deletions, but the clinical significance of this finding is uncertain. All patients with clinical suspicion for NF1 should be referred to a cardiologist for evaluation and surveillance.     

Rapid Molecular Microbiologic Diagnosis of Prosthetic Joint Infection

We previously showed that culture of samples obtained by prosthesis vortexing and sonication was more sensitive than tissue culture for prosthetic joint infection (PJI) diagnosis. Despite improved sensitivity, culture-negative cases remained; furthermore, culture has a long turnaround time. We designed a genus-/group-specific rapid PCR assay panel targeting PJI bacteria and applied it to samples obtained by vortexing and sonicating explanted hip and knee prostheses, and we compared the results to those with sonicate fluid and periprosthetic tissue culture obtained at revision or resection arthroplasty. We studied 434 subjects with knee (n = 272) or hip (n = 162) prostheses; using a standardized definition, 144 had PJI. Sensitivities of tissue culture, of sonicate fluid culture, and of PCR were 70.1, 72.9, and 77.1%, respectively. Specificities were 97.9, 98.3, and 97.9%, respectively. Sonicate fluid PCR was more sensitive than tissue culture (P = 0.04). PCR of prosthesis sonication samples is more sensitive than tissue culture for the microbiologic diagnosis of prosthetic hip and knee infection and provides same-day PJI diagnosis with definition of microbiology. The high assay specificity suggests that typical PJI bacteria may not cause aseptic implant failure.     

Illness behaviour and antibiotic prescription in patients with respiratory tract symptoms.

BACKGROUND: Although the vast majority of respiratory tract symptoms are self-limiting, many patients visit their GP for these symptoms and antibiotics are over-prescribed. AIM: To explore determinants of patients visiting GPs for recent cough, sore throat, or earache; for being prescribed antibiotics; and for patients' satisfaction with visiting the GP. Design of the study: Second Dutch National Survey of General Practice (DNSGP-2) with a health interview and an additional questionnaire. SETTING: A total of 7057 adult patients of 163 GPs in the Netherlands. METHOD: Characteristics of patients and GPs as well as morbidity data were derived from the DNSGP-2 and a health interview. Characteristics of the symptoms, GPs' management and patients' satisfaction were measured by an additional written questionnaire. Data were analysed by means of multivariate logistic regression. RESULTS: About 40% of the responders (n = 1083) reported cough, sore throat, or earache in the 2 weeks preceding the interview and, of them, 250 visited their GP. Of this latter group, 97 patients were prescribed antibiotics. Apart from non-medical reasons, relevant medical factors played an important role in deciding to visit the GP. Smokers and patients with cardiac disease or diabetes mellitus were not especially inclined to see their GP. Smoking behaviour, fever, and views on respiratory tract symptoms and antibiotics of patients and GPs were associated with being prescribed antibiotics. Patients' perception of having been carefully examined was associated with their satisfaction, while receiving antibiotics was not. CONCLUSION: GPs should inform patients with clear elevated risk when to visit their GP in cases of cough, sore throat, or earache. There is still a need for GPs and patients to be better informed about the limited significance of single inflammation signs (for example, fever and green phlegm) as an indication for antibiotics. Careful examination of the patient contributes to patient satisfaction.