Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells

The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays. Three mAbs were generated and shown to react with unique determinants of PFN. All mAbs recognized intracellular PFN in human peripheral blood mononuclear cell (PBMC) as assessed by flow cytometry and immunohistochemistry. Functional PFN capture ELISA and ELISpot assays were developed utilizing two of the mAbs for capture and the third mAb for detection. When examining PFN release by the YT lymphoma cell line, the ELISpot displayed a greater detection sensitivity than the ELISA. Assessment of PFN release by a CTL clone using ELISpot gave results consistent with a parallel (51)Cr-release cytotoxicity assay. Moreover, PFN release by PBMC could be quantified by ELISpot and ELISA after ex vivo stimulation with defined CTL epitopes from common viruses. These novel immunoassays will be valuable for further investigations of the mechanisms underlying granule-mediated apoptosis. In addition, the capture immunoassays could provide tools for studying CTL responses in infectious and tumor diseases as well as for vaccine development.     

Malassezia furfur in a case of onychomycosis: colonizer or etiologic agent?

The etiologic role of Malassezia furfur in onychomycosis is a contentious diagnostic problem because its keratinolytic ability has never been verified. This case report describes the isolation of M. furfur from the infected nails of a child clinically diagnosed with onychomycosis, and discusses the role of this organism as an etiologic agent/colonizer. The patient presented with subungual hyperkeratosis and onycholysis without associated paronychia. Budding yeast cells compatible with M. furfur were repeatedly demonstrated in KOH wet mounts of damaged nails, histopathology of hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) stained sections showed penetration of fungal elements between deeper layers of keratin, and numerous colonies of M. furfur were isolated on three consecutive occasions from nail specimens collected from different areas of hand and toenail lesions. No evidence of nail invasion by dermatophytic or nondermatophytic filamentous fungi were found by direct microscopy or culture. Microscopy and culture were negative following 12 weeks of ketoconazole treatment, which resulted in growth of healthy nail plates with normal beds. We can infer from these observations that M.furfur was an etiologic agent rather than a colonizer in the patient's nails even though direct keratinolytic character of this fungus was not demonstrated.     

IL-4–BATF signaling directly modulates IL-9 producing mucosal mast cell (MMC9) function in experimental food allergy

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The Wnt pathway, epithelial-stromal interactions, and malignant progression in phyllodes tumours

In a previous study of phyllodes tumours, it has been shown that both the stroma and the epithelium can exhibit distinct molecular changes, suggesting that both are part of the neoplastic process. In view of this finding, it was decided to study stromal-epithelial interactions in these tumours by examining the Wnt-APC-beta-catenin pathway. Beta-catenin and cyclin D1 immunohistochemistry was performed on 119 phyllodes tumours. Eighty-six (72%) showed stromal nuclear beta-catenin localization and in 57% the staining was moderate or strong; however, of the eight malignant tumours in the series, seven showed absent or weak nuclear staining (p<0.025). In no tumour was nuclear beta-catenin staining seen in the epithelial component. Moderate or strong stromal cyclin D1 staining correlated with nuclear stromal beta-catenin staining (p<0.05). Forty-five of the tumours, including two malignant lesions, were screened for beta-catenin exon 3 mutations using SSCP and sequencing, but none was found. Loss of heterozygosity (LOH) of the marker D5S346 was used to infer APC mutation, but only one (benign) tumour showed LOH. Wnt2 and Wnt5a mRNA was localized by in situ hybridization in 13 cases (three malignant) chosen to reflect the different beta-catenin staining patterns. There was an association between strong nuclear beta-catenin staining of stromal cells and epithelial Wnt5a expression (p<0.0015). These data suggest that stromal proliferation in benign phyllodes tumours relies on abnormalities in the Wnt pathway which result not from mutation, but from Wnt5a expression in the epithelium. In the progression to malignancy, the stromal proliferation appears to become independent of the Wnt pathway and, presumably, of the epithelial component of these tumours.     

ω-123I-Hexadecanoic acid metabolic probe of cardiomypathy

The utility of -¹²³I-hexadecanoic acid myocardial scintigraphy as a metabolic probe of cardiomyopathies was investigated. Sixteen patients with a variety of cardiomyopathies and myopathies that involve cardiac muscle and ten volunteers were imaged in the postabsorptive state in a 40° LAO projection after a standard dose of -¹²³I-hexadecanoic acid. An elimination T_1/2 was calculated from the left ventricular myocardial time-activity curve. An uptake index, corrected for chest wall attenuation, was also computed in 7 of 10 volunteers and 8 of 16 patients.Of the 16 patients, only 2 had distinctly abnormal -¹²³I-hexadecanoic acid myocardial tracer kinetics. The first patient had a metabolic disorder of which cartine deficiency was one component. The second patient had endocardial fibroelastosis, a process which has been linked to disorders which deprive the myocardium of oxygen and energy. Therefore, the cardiomyopathy may have been caused by some abnormality of cardiac metabolism other than carnitine deficiency. Although of limited utility in the overall cardiomyopathic population, -¹²³I-hexadecanoic acid myocardial scintigraphy should be further investigated as a screening test for carnitine deficiency and related metabolic abnormalities in patients at risk.Supported by a Canadian Heart Foundation Research FellowshipSupported by an Australian Heart Foundation Travel Grant     

Selected conditions associated with an increased incidence of incisional hernia: A review of molecular biology

BackgroundIncisional hernias (IH) following a laparotomy, on average, occur in 10–20% of patients, however, little is known about its molecular basis. Thus, a better understanding of the molecular mechanisms could lead to the identification of key target(s) to intervene pre-and post-operatively.MethodsWe examined the current literature describing the molecular mechanisms of IH and overlap these factors with smoking, abdominal aortic aneurysm, obesity, diabetes mellitus, and diverticulitis.ResultsThe expression levels of collagen I and III, matrix metalloproteinases, and tissue inhibitors of metalloproteases are abnormal in the extracellular matrix (ECM) of IH patients and ECM disorganization has an overlap with these comorbid conditions.ConclusionUnderstanding the pathophysiology of IH development and associated risk factors will allow physicians to identify patients that may be at increased risk for IH and to possibly act preemptively to decrease the incidence of IH.     

Safety and Efficacy of a Steroid Avoidance Immunosuppression Regimen in Renal Transplant Patients With De Novo or Preformed Donor-Specific Antibodies: A Single-Center Study

Although interest in the role of donor-specific antibodies (DSAs) in kidney transplant rejection, graft survival, and histopathological outcomes is increasing, their impact on steroid avoidance or minimization in renal transplant populations is poorly understood. Primary outcomes of graft survival, rejection, and histopathological findings were assessed in 188 patients who received transplants between 2012 and 2015 at the Scripps Center for Organ Transplantation, which follows a steroid avoidance protocol. Analyses were performed using data from the United Network for Organ Sharing. Cohorts included kidney transplant recipients with de novo DSAs (dnDSAs; n = 27), preformed DSAs (pfDSAs; n = 15), and no DSAs (nDSAs; n = 146). Median time to dnDSA development (classes I and II) was shorter (102 days) than in previous studies. Rejection of any type was associated with DSAs to class I HLA (P < .05) and class II HLA (P < .01) but not with graft loss. Although mean fluorescence intensity (MFI) independently showed no association with rejection, an MFI >5000 showed a trend toward more antibody-mediated rejection (P < .06), though graft loss was not independently associated. Banff chronic allograft nephropathy scores and a modified chronic injury score were increased in the dnDSA cohort at 6 months, but not at 2 years (P < .001 and P < .08, respectively). Our data suggest that dnDSAs and pfDSAs impact short-term rejection rates but do not negatively impact graft survival or histopathological outcomes at 2 years. Periodic protocol post-transplant DSA monitoring may preemptively identify patients who develop dnDSAs who are at a higher risk for rejection.