Comparative study between direct mild acid extraction and immunobead purification technique for isolation of HLA class I-associated peptides.

Identification of tumour-specific peptide(s) hidden within the groove of human leucocyte antigens is a crucial prerequisite for peptide vaccine therapy. Conventionally, the peptide(s) are isolated by mild acid extraction (MA) technique followed by sequential ultra-filtrations. Here we describe a new approach for peptide isolation using the immunobead purification (IB-P) technique in conjunction with reverse-phase high-performance liquid chromatography. The obtained data were validated by SDS-PAGE followed by the silver staining technique. The results can be summarised as follows: (1) Comparison of class I-associated peptides isolated from a bladder cell line before and after the correction of class I antigens by gene transfection followed by IB-P technique showed the presence of peptides only from the class I-corrected cells. The data were confirmed using the silver staining technique as a way of detecting individual peptide bands. (2) Whilst peptides could be isolated by both techniques, the MA method led to the isolation of peptides from both class I-negative and class I-positive Fen cell lysates. (3) The IB-P approach could be used for isolation of class I-associated peptides from a normal kidney tissue. The data showed the high efficiency of the IB-P approach for isolation of class I-associated peptides. Unlike the MA technique, where the presence of non- class I-associated peptides was a problem, the IB-P approach isolated only peptides associated with the class I antigens. In addition, the data showed the feasibility of extracting peptides from tissue fragments by the IB-P method. The approach presented here may assist the future development of peptide vaccine therapy in urological cancers.     

Cytokines and the chronic inflammation of rheumatic disease. I. The presence of interleukin-1 in synovial fluids

Synovial fluids from patients with rheumatoid arthritis (13 cases), ankylosing spondylitis (six), psoriatic arthritis (two), osteoarthritis (three) and rubella arthritis (four) contain interleukin-1 (Il-1) activity as measured in the CH3/He mouse thymocyte assay. That this activity is Il-1 was shown by: (i) the time course of thymocyte stimulation; (ii) failure of PHA blast cells to absorb out the activity; (iii) molecular weight of 15,000-20,000 daltons, and (iv) ability to stimulate prostaglandin E2 production from synovial cells. These results indicate that IL-1 is an important mediator in the inflammatory pathway of different types of joint disease.     

The Effect of Intravenous Magnesium Sulphate as an Adjuvant in the Treatment of Acute Exacerbations of COPD in the Emergency Department: A Double-Blind Randomized Clinical Trial

BackgroundAcute exacerbations of chronic obstructive pulmonary disease (AECOPD) are serious complications that often require immediate intervention in an emergency department (ED). The aim of this study was to investigate the effect of intravenous magnesium sulphate as an adjuvant in the treatment of AECOPD in the ED.MethodsIn a double-blind, randomized clinical trial, a total of 60 patients with AECOPD presenting to the ED of Imam Khomeini Hospital in Sari, Iran, were included. The study was conducted between September 2016 and February 2018. Eligible patients were randomly allocated into two groups of intervention and control. Patients in the intervention and control groups received intravenous infusion of magnesium sulfate (2 gr) or normal saline over 30 minutes, respectively. For all patients, Borgdyspnea score, forced expiratory volume in one second (FEV1) result and clinical variables of interest were evaluated before the beginning of the intervention, and also 45 minutes and 6 hours after the commencement of intervention.ResultsRegardless of time of evaluation, pulse rate (PR), respiratory rate (RR) and Borg score in intervention group was lower than control group. Also, FEV1 and SPO2 were greater in intervention group compared to control group. However, these differences were not statistically significant (between-subject differences or group effect) (p<0.001). The trends of FEV1, SPO2, PR, RR and Borg score were similar between two groups of study (no interaction effect; P>0.05).ConclusionAccording to the results of this study, it seems that using intravenous magnesium sulfate has no significant effect on SPO2, FEV1, RR, and PR of patients with AECOPD who presented to ED.     

A randomized,double-blind placebo-controlled add-on trial to assess the efficacy,safety, and anti-atherogenic effect of spirulina platensis in patients with inadequately controlled type 2 diabetes mellitus

The efficacy of spirulina platensis (S. platensis) as an add-on therapy to metformin and its effect on atherogenic keys in patients with uncontrolled Type 2 Diabetes Mellitus (T2DM) was evaluated. Sixty patients were randomly assigned to S. platensis (2 g/day) or placebo group for three months while continuing metformin as their usual treatment. The efficacy of S. platensis was determined using the pre- and post-intervention HbA1c levels (primary outcome) as well as tracking FBS and lipid profiles levels (TC, LDL-C, TG, and HDL-C) as secondary outcomes at the different treatment time points (0,30,60,90 days). During the three–month intervention period, supplementation with S. platensis resulted in a significant lowering of HbA1c (↓1.43, p < 0.001) and FBS (↓ 24.94 mg/dL, p < 001) levels. Mean TG in the intervention group was found to be significantly lower in the intervention group than in controls (p < 0.001). Total cholesterol (TC) and its fraction, LDL-C, exhibited a fall (↓41.36 mg/dL and ↓38.4 mg/dL, respectively; p < 0.001) coupled with a marginal increase in the level of HDL-C (↑3 mg/dL; p < 0.001). Add-on therapy with S. platensis was superior to metformin regarding long-term glucose regulation and controlling blood glucose levels of subjects with T2DM. Also, as a functional supplement, S. platensis has a beneficial effect on atherogenic keys (TG and HDL-C) with no adverse events.     

The possible relevance of the expression of MHC antigens and of EGF receptor in aggressive oral tumours

In this study the intensity of expression of major histocompatibility complex (MHC) class I and II antigens, adhesion molecule i.e. ICAM-1, epidermal growth factor receptor i.e. EGFr, T cell marker and cytokeratin were compared in oral squamous cell carcinoma (OSCC) and in the benign ameloblastoma of the jaws. The results showed that: a) There was strong expression of both monomorphic and of polymorphic class I MHC antigens (90% of cases) in both basal and suprabasal cells of controls from normal mucose. b) Whereas up to 4% of OSCCs and 27% of ameloblastomas showed complete loss of monomorphic class I antigens, the frequency of polymorphic class I abnormalities was even more marked in both tumour types. c) Strong expression of class II MHC antigens and of ECFr was observed in the basal cells of most normal controls. d) Both class II (50% of cases) and ICAM-1 (30% of cases) showed strong expression in OSCC but not in ameloblastoma. The statistical values between OSCC and normal basal cells for class II and ICAM-1 were not significant whilst the corresponding values for OSCC compared with ameloblastoma were p<0.001 and p<0.001. In the case of OSCC, there were a large number of infiltrating T cells expressing activation marker i.e. class II antigen. e) Strong expression of EGFr was seen in more than 90% of the OSCC cases compared with only 16% of ameloblastomas (0.01

0.001). Our working hypothesis to explain these abnormalities is that although both tumour types (more so in the case of ameloblastoma) have in place an escape mechanism from the immune system, the overexpression of EGFr in OSCC may in part be responsible for the more aggressive behaviour of the malignancy compared with the locally invasive but benign ameloblastoma.     

Local anergy rather than systemic anti-tumour immunity to explain tumour growth in an animal model of oral squamous cell carcinoma

A chemically induced syngeneic hamster tumour model of human oral squamous cell carcinoma (OSCC) was used to investigate the possible influence of locally transplanted growing rumours on the immune system of the recipient. Cell activation and cell cytotoxicity assays were performed in vitro using the colorimetric MTT assay to measure any possible changes. The fast growing nature of the tumour model if grafted locally as a fragment was confirmed but not if injected as a single cell suspension (SCS). Stimulation (Concanavalin A) of spleen cells from normal and from tumour bearing animals showed that there was a minor though statistically significant decrease in the mitogenic response of the latter. Thus, the respective stimulatory indices (SI) were 4.06+/-1.61 and 2.06+/-0.87 (0.02

0.01). No significant difference was observed when spleen cells were stimulated with interleukin-2 (IL-2), although there was a similar trend. Pre-immunisation of animals with irradiated autologous SCS three weeks prior to grafting, resulted in a significant decrease in the tumour growth rate of subsequently grafted tumour. Thus, the mean If: SD (weight of takes in mg) for the successful takes of untreated (n=10) and treated (n=9) groups were 52.0+/-52.2 and 25.7+/-19.4 (0.02

0.05) respectively. The number of cases with no tumour takes were 2 of 10 (20%) and 6 of 9 (66%) respectively. In a separate experiment groups of 5 animals were immunized with an increasing number of cells as irradiated SCS, the results of which demonstrated an inverse correlation between the rate of tumour growth and the number of injected tumour cells. The addition of irradiated SCS to IL-2 activated normal spleen cells (LAK cells) in vitro led to a dose-related decrease in the efficiency of cytotoxicity of latter when tested against an xenogeneic super-sensitive surrogate tumour target cell line (Fen cells). Thus, the percent killing by IL-2-activated normal spleen cells was 56.4%. The corresponding mean values for IL-2 activated normal spleen cells in the presence of tumour SCS at 25/1 and 50/1 ratios were 35.9% (p<0.05) and 11.9% (p<0.001) respectively. Ln an attempt to establish the presence of T suppressor cells, spleen cells from tumour bearing animals were injected concomitantly with SCS into 5 recipients. After four weeks no tumour growth had occurred. In conclusion we demonstrated that the presence of injected or grafted tumour had only a minor effect on systemic immune function but induced a strong local anergic effect. This local anergic effect was demonstrable as blocking of LAK activity and thus perhaps allowed suppression of the functional activities of incoming immunocompetent cells.     

Standardization and potential use of HPLC for detection of cellular placental alkaline phosphatase using established tumour cell lines and fresh tumour biopsies

Placement alkaline phosphatase (PLAP) is one of the cellular phosphatases (ALP) expressed in patients with testis cancers, particularly in seminomas. Using various techniques including Western blot and high-performance liquid chromatography (HPLC) systems and ATC2, a newly developed specific anti-PLAP monoclonal antibody (Mab), the presence of active form of PLAP in lysates prepared from testis tumour fragment and tumour cell lines, was studied. This was carried out following isolation of PLAP from biological samples using CNBr Sepharose-conjugated ATC2 beads. The results showed that: (1) The target for the newly developed Mab ATC2 was PLAP. (2) The ATC2-conjugated bead system was an efficient method for isolating pure PLAP. (3) Diethylamine (DEA), in contrast to urea and glycine, was the most efficient for separation of PLAP from ATC2-conjugated beads, as the isolated molecule did not lose any phosphatase activity and there was very little uncoupling of the ATC2 Mab from the beads. (4) ATC2-conjugated CNBr beads could pick up PLAP from a solution containing standard PLAP and lysates prepared from tumour cell lines or testis tissue fragments positive for the PLAP. (5) HPLC profile of testis tumour lines and testis tumours showed two distinct peaks with ALP activity, one at retention time 7-8 min (corresponding to 95 kDa molecule) and one at 12-13 min corresponding to 70 kDa molecule). These data demonstrated the potential use of various biochemical methods in combination with HPLC for isolation of the fully functional molecules with ALP activity from different samples including lysates prepared from patients with testis cancer. The nature of ALP activity at 95 kDa is being investigated as no such molecule has been reported previously. These techniques might have an important implication for an early detection of germ cell tumours, particularly in patients with equivocal ultrasound.