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.     

Effects of a novel Nodal-targeting monoclonal antibody in melanoma

Nodal is highly expressed in various human malignancies, thus supporting the rationale for exploring Nodal as a therapeutic target. Here, we describe the effects of a novel monoclonal antibody (mAb), 3D1, raised against human Nodal. In vitro treatment of C8161 human melanoma cells with 3D1 mAb shows reductions in anchorage-independent growth and vasculogenic network formation. 3D1 treated cells also show decreases of Nodal and downstream signaling molecules, P-Smad2 and P-ERK and of P-H3 and CyclinB1, with an increase in p27. Similar effects were previously reported in human breast cancer cells where Nodal expression was generally down-regulated; following 3D1 mAb treatment, both Nodal and P-H3 levels are reduced. Noteworthy is the reduced growth of human melanoma xenografts in Nude mice treated with 3D1 mAb, where immunostaining of representative tumor sections show diminished P-Smad2 expression. Similar effects both in vitro and in vivo were observed in 3D1 treated A375SM melanoma cells harboring the active BRAF(V600E) mutation compared to treatments with IgG control or a BRAF inhibitor, dabrafenib. Finally, we describe a 3D1-based ELISA for the detection of Nodal in serum samples from cancer patients. These data suggest the potential of 3D1 mAb for selecting and targeting Nodal expressing cancers.     

Increased HLA-G Expression in Tissue-Infiltrating Cells in Inflammatory Bowel Diseases

Background

Since HLA-G is an immune checkpoint molecule and since Crohn’s disease (CD) and ulcerative colitis (UC) exhibit deregulated immune-mediated mechanisms, we aimed to evaluate intestinal HLA-G expression and soluble HLA-G (sHLA-G) levels in CD/UC patients stratified according to the CD phenotype/localization and UC extension.

Methods

HLA-G tissue expression was assessed by immunohistochemistry in biopsies collected from 151 patients (90 CD, 61 UC) and in surgical resection specimens (28 CD, 12 UC). Surgical material from 24 healthy controls was also assessed. Plasma sHLA-G levels (97 CD, 81 UC, and 120 controls) were evaluated using ELISA.

Results

HLA-G expression was similarly observed in the intestinal epithelial cells of control and CD/UC specimens. However, in biopsies, the plasma cells/lymphocytes infiltrating the lamina propria in CD/UC presented (1) increased HLA-G expression compared to controls (P?<?0.0001), (2) greater cell staining in UC cells than in CD cells irrespective of disease extent (P?=?0.0011), and (3) an increased number of infiltrating cells in the inflammatory CD phenotype compared to that in the stenosing and fistulizing phenotypes (P?=?0.0407). In surgical specimens, CD/UC patients exhibited higher infiltrating cell HLA-G expression in lesion areas than in margins. sHLA-G levels were higher in UC/CD patients (P?<?0.0001) than in controls, but no difference was observed between diseases.

Conclusions

Increased infiltrating cell HLA-G expression associated with increased sHLA-G levels in CD/UC patients may reflect ongoing host strategies to suppress chronic inflammation.

     

Blood dendritic cells in patients with acute lymphoblastic leukaemia

Myeloid and plasmacytoid dendritic cells (MDCs, PDCs) play a key role in the initiation of immune responses. In this study, we show a severe reduction of MDCs and PDCs in patients with B lineage acute lymphoblastic leukaemia (B-ALL; P = 0.01 vs. controls). DCs from patients with T lineage ALL (T-ALL) were quantitatively and functionally comparable to healthy donors, as demonstrated by secretion of interleukin (IL)-12p70 and interferon-alpha. In vitro, the circulating CD34(+) fraction of B-ALL cases did not generate either CD1a(+) MDCs or PDCs, suggesting that DC development is probably affected in B-ALL, but not in T-ALL.     

Local adjunct effect of antimicrobial photodynamic therapy for the treatment of chronic periodontitis in type 2 diabetics: split-mouth double-blind randomized controlled clinical trial

Diabetes has become a global epidemic. Its complications can have a significant impact on quality of life, longevity, and public health costs. The presence of diabetes might impair the prognosis of periodontal treatments due to its negative influence on wound healing. Antimicrobial photodynamic therapy (aPDT) is a local approach that can promote bacterial decontamination in periodontal pockets. The aim of this study was to investigate the local effect of adjunct aPDT to ultrasonic periodontal debridement (UPD) and compare it to UD only for the treatment of chronic periodontitis in type 2 diabetic patients. Twenty type 2 diabetic patients with moderate to severe generalized chronic periodontitis were selected. Two periodontal pockets with probing depth (PD) and clinical attachment level (CAL) ≥5 mm received UPD only (UPD group) or UPD plus adjunct aPDT (UPD?+?aPDT group). Periodontal clinical measures were collected and compared at baseline and 30, 90, and 180 days. After 180 days of follow-up, there were statistically significant reductions in PD from 5.75?±?0.91 to 3.47?±?0.97 mm in the UPD group and from 6.15?±?1.27 to 3.71?±?1.63 mm in the UPD?+?aPDT group. However, intergroup analysis did not reveal statistically significant differences in any of the evaluated clinical parameters (p?>?0.05). The adjunct application of aPDT to UPD did not present additional benefits for the treatment of chronic periodontitis in type 2 diabetic patients. The ClinicalTrials.gov identifier of the present study is NCT02627534.     

Impaired expression of perforin and granulysin in CD8+ T cells at the site of infection in human chronic pulmonary tuberculosis

Protective immunity in tuberculosis is dependent on the coordinated release of cytolytic effector molecules from effector T cells and the subsequent granule-associated killing of infected target cells. In this study, we investigated the expression of cytolytic (perforin and granzyme A) and antimicrobial (granulysin) molecules at the single-cell level in cryopreserved lung tissue from patients with chronic, progressive tuberculosis disease. Quantification of protein-expressing cells was performed by in situ imaging, while mRNA levels in the infected tissue were analyzed by real-time PCR. Persistent inflammation, including excessive expression of inducible nitric oxide synthase in CD68+ macrophages and significant infiltration of CD3+, CD8+ and CD4+ T cells, was evident in tuberculosis lesions in all patients. However, despite the accumulation of CD3+ T cells, perforin- and granulysin-expressing CD3+ T cells were detected at two- to threefold-lower ratios in the tuberculosis lesions than in distal lung parenchyma and uninfected control lungs, respectively. This was evident at both the protein and mRNA levels. Moreover, perforin- and granulysin-expressing CD8+ T cells were scarce in individual granulomas within the tuberculosis lesions. In contrast, significant up-regulation of granzyme A-expressing CD3+ T cells was evident in the lesions from all patients. Confocal microscopy revealed coexpression of perforin and granulysin, primarily in CD8+ T cells; however, this expression was lower in the tuberculosis lesions. These findings suggest that symptomatic, chronic tuberculosis disease is associated with insufficient up-regulation of perforin and granulysin coexpression in CD8+ T cells at the local site of infection.     

Analysis of differentiation of stem/progenitor cells in tissue culture of neocortical primordium of mouse brain

Differentiation of neural stem/progenitor cells from neocortical primordium of the brain from 14-day mouse embryos was studied by immunohistochemical methods during their culturing. Non-differentiated cells expressing nestin and vimentin persisted in freely floating neurospheres throughout the experiment. Glioblasts, neuroblasts, and differentiated neurons were found in neurospheres cultured in differentiating medium. However, neurons disappeared with increasing the number of passages, the formation of neuroblasts was terminated, and only astrocytes and nestin-positive cells were seen in the culture. It was found that cells of mouse embryonic neocortex lose the capacity for spontaneous multipotent differentiation during culturing. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 4, pp. 189–195, October, 2007     

HbA1c screening in the community: Lessons for safety and quality management of a point of care programme

Aim

To describe quality management processes and appropriate interpretation with respect to HbA_1c point-of-care (POC) testing in a national diabetes and cardiovascular risk screening programme.

Treatment of type I gastric neuroendocrine tumors with somatostatin analogs

Background and Aim: There are limited data on response and long‐term follow‐up of octreotide therapy in type‐I gastric neuroendocrine tumors. The objective of the present study was to assess the response of type‐I gastric neuroendocrine tumors to octreotide‐long acting, repeatable (LAR) therapy and evaluate long‐term follow up of such patients after therapy. Methods: Three patients with documented type‐I gastric neuroendocrine tumors from a tertiary gastroenterology centre were studied. Octreotide‐LAR therapy 20 mg intramuscularly every 28 days was administered for one year. Serum gastrin and chromogranin levels, gastroscopies and biopsies from tumor nodules at 6 months and one year on therapy and every 6 months after completion of drug therapy were taken. Follow‐up after completion of therapy extended for 3 years in two and 2.5 years in one patient. Results: During octreotide therapy there was normalization of serum gastrin levels and serum chromogranin levels. Tumors in all three patients had regressed at 6 months of treatment. Following cessation of therapy, there was progressive rise of serum gastrin to pre‐treatment levels. Serum chromogranin levels remained within normal limits. Gastroscopic and histologic examination of gastric biopsies did not reveal recurrence of tumors in any patients. All patients tolerated therapy well and became asymptomatic soon after drug therapy. Conclusions: Octreotide‐LAR therapy causes regression of type‐I gastric neuroendocrine tumors. After completion of drug therapy there was no recurrence of tumors even with continued hypergastrinemia. Octreotide therapy should be considered as one of the treatment options in such patients.     

Biliary disease after liver transplantation: the experience of the King Faisal Specialist Hospital and Research Center, Riyadh

BACKGROUND AND AIM: The biliary tract has been referred to as the "Achilles heel" of liver transplantation. The aim of this study was to document the frequency, clinical presentation and management of biliary complications after liver transplantation in the King Faisal Specialist Hospital and Research Center (KFSH&RC), Riyadh, Saudi Arabia. METHODS: The liver transplant clinic at KFSH&RC has registered and followed 220 patients (150 male and 70 female patients; age 40.6 +/- 18.6 years; pediatric 33, adult 187) during the period from 1987 to June 2003. A total of 235 transplants were carried out on these patients. Cadaveric liver transplants had been carried out on 202 patients, non-heart beating liver transplant in three patients, live donor liver transplants in 11 and split transplant in four. Biliary reconstruction was duct-to-duct anastomosis in 147 patients and Roux-en-Y in 73. Biliary complications were suspected on clinical and biochemical parameters and confirmed using imaging techniques. RESULTS: Forty patients (18.2%) developed 53 biliary complications. These included bile leak in 16, strictures in 25, calculi in eight, and sphincter of Oddi dysfunction and possible recurrence of primary sclerosing cholangitis in the donor duct in two patients each. Bile leaks were observed in the early postoperative period (median period 30 days, range 1-150 days, 95% confidence interval [CI] 8-51). Leakage occurred at the anastomotic site in 13 patients. Patients presented with bilious drainage (n = 6), abdominal pain at T-tube removal (n = 3), fever (n = 2), sepsis (n = 1), dyspnea (n = 1) and abnormal liver tests (n = 3). Eleven patients had intra-abdominal bilious collections. Two patients were treated conservatively, eight patients had ultrasound-guided aspiration of biloma, five had biliary stenting at endoscopic retrograde cholangiopancreatography and two patients needed surgery. There were four deaths, two of which were related to bile leak, one patient was left with permanent external biliary drainage and four patients had biliary strictures in the follow-up period. Biliary strictures occurred at a median period of 360 days (range 4-2900 days; 95% CI 50-670) after the transplant. Hepatic artery thrombosis caused biliary strictures in three, while 21 strictures were localized to the anastomotic site. Biliary strictures presented with elevated liver tests in five patients, progressive cholestasis in five, cholangitis (with septicemia in five) in 11, abdominal pain in two and acute pancreatitis in three patients. Repeat sessions of endoscopic or percutaneous dilatation and stenting (mean sessions 4.4/patient, range 3-7) were attempted in 20 patients to relieve strictures, with success in only nine patients. Seven patients had surgery. Four patients with biliary strictures died. Biliary calculi developed late in the follow-up period and had the appearance of biliary casts in five and sludge in three patients. Eleven (27.5%) patients with biliary disease died compared with 35 (19.4%) patients without biliary disease. CONCLUSIONS: Biliary complications occurred in 18.2% of patients after liver transplantation and included biliary leak and biliary strictures with or without calculi. Management involved a combination of endoscopic, radiologic and operative procedures. Biliary complications caused considerable morbidity and mortality in liver transplant patients.