IMPORTANCE OF JOINT LINE RESTORATION IN TOTAL KNEE ARTHROPLASTY.

The restoration of the joint line is important for a good functional outcome after a Total Knee Arthroplasty(TKA). Knee joint biomechanics need to be restored as near normal as possible. Joint line elevation leads to anterior knee pain, decrease in range of motion, patella baja ,mid-flexion instability and impingement of patellar tendon. Joint line depression on the other hand leads to patella alta, risk of patellar subluxation and mid-flexion instability of the knee. Various studies have demonstrated various range of acceptable joint line variation but there is no clear acceptable range of joint line variation. More studies are required for establishing the acceptable range of joint line variation and standard practices should be established for arthroplasty surgeons for preventing variation of joint line.

Evaluation of transforming growth factor-beta1 gene expression in pterygium tissue of atopic patients.

BACKGROUND: The exact pathogenesis of pterygium is still not fully understood. Growth factors are considered to play an important role in the formation of pterygium. Transforming growth factor (TGF)-ß1 is considered to be one of the main mediators of fibroblast stimulation and tissue remodeling in allergic conditions. The objective of the present study was to investigate the association between TGF-ß1 gene expression and pterygium in atopic and nonatopic participants. METHODS: We used questionnaires to record demographic and clinical information from patients who underwent pterygium excision surgery. Skin prick examination was done to confirm or rule out atopy in 30 patients with atopy (Case Group) and 30 individuals without atopy (Control Group). Additionally, measurement of serum immunoglobulin E, cytokines, including interleukin-4 and interferon-γ, and peripheral blood eosinophil count was performed to confirm atopy in 30 consecutive patients (Case Group). A semiquantitative reverse transcription polymerase chain reaction was performed to determine TGF-ß1 gene expression in all individuals. RESULTS: TGF-ß1 mRNA gene expression was significantly higher (p = 0.0001) in atopic patients 2.50 ± 1.11 compared to nonatopic individuals 1.40 ± 0.46. Eosinophil count and serum immunoglobulin E were significantly higher (p = 0.031 and p = 0.001, respectively) in atopic patients compared to the Control Group. Serum interleukin-4 was also significantly higher (p = 0.01) in atopic patients compared with nonatopic individuals. CONCLUSION: Excess expression of TGF-ß1 gene in pterygium tissue of atopic individuals suggests that growth factors play a role in the pathogenesis of pterygium.

Cell-SELEX-based selection and characterization of a G-quadruplex DNA aptamer against mouse dendritic cells.

Targeting of dendritic cells (DCs) by aptamers increases antigen capture and presentation to the immune system. Our aim was to produce aptamers against DC molecules using the cell-SELEX procedure. For this purpose, 18 rounds of cell-SELEX were performed on mouse macrophage J774A.1 and CT26 as target and control cells, respectively. The selected aptamers were truncated and their binding to mouse macrophages, and immature and mature DCs analyzed. Two macrophage-specific aptamers, Seq6 and Seq7, were identified. A truncated form of Seq7, Seq7-4, 33 nucleotides in length and containing the G-quadruplex, bound macrophages and immature DCs with KD values in the nanomolar range. We anticipate that Seq7-4 has potential as a therapeutic tool in targeting of mouse macrophages and immature DCs to efficiently improve different immunotherapy approaches.

Molecular, biochemical, and structural analysis of a novel mutation in patients with methylmalonyl-CoA mutase deficiency.

BACKGROUND: Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting from genetic defects in methylmalonyl-CoA mutase (MCM). This enzyme is encoded by the MUT gene and is required for the degradation of odd-chain fatty acids, the amino acids valine, isoleucine, methionine, and threonine, and cholesterol. METHOD: Three unrelated affected patients with isolated MMA and their parents were studied. The MUT gene was analyzed by PCR and sequencing of its entire coding region and the highly conserved exon-intron splice junctions. The homology modeling of the novel mutation found in the MUT gene was performed using the online Swiss-Prot server for automated modeling and then analyzed with special bioinformatics software to better study the structural effects caused by the mutation. RESULT: We found one homozygous nucleotide change in intron 12 of the MUT gene (c.2125-3 C>G). The variant is located near the highly conserved acceptor splice site of intron 12. A region at the C-terminus of the protein from ASP709 to GLN748 has been deleted by the alteration of c.2125-3 C>G in intron 12 of the MUT gene. Further studies of the novel mutation in the MUT gene by means of homology modeling revealed abnormalities in the protein's structure, which causes the protein to act malfunctioning and also the mRNA expression analysis of MUT gene confirmed these results. CONCLUSION: We report this novel mutation, including its clinical and biochemical features and genetic defects, in the MUT gene of three patients affected with isolated MMA. Structural analyses of the mutated protein identified changes in the energy and stereochemical features of the protein that unfortunately altered the protein's functionalities. Therefore, we demonstrate that a novel splice site mutation in intron 12 of the MUT gene is a potential highly pathogenic allele via inhibition of alternative splicing.

Aptamers: new arrows to target dendritic cells.

Aptamers, as a novel class of molecular probes for diagnosis, imaging and targeting therapy, have attracted increasing attention in recent years. Aptamers are generated from libraries of single-stranded nucleic acids against different molecules via the "systematic evolution of ligands by exponential enrichment" (SELEX) method. SELEX is a repetitive process of a sequential selection procedure in which a DNA or RNA library pool is incubated separately with target and control molecules to select specific oligonucleotide aptamers with high affinities and specificities. Cell-SELEX is a modified version of the SELEX process in which whole living cells are used as targets for the aptamers. Dendritic cell (DC) targeting, as a new therapeutic approach, can improve the efficiency of immunotherapy in the treatment of allergies and cancers. DCs use various receptors to continuously induce adaptive immunity via capture and presentation of antigens to naïve T cells. DCs are considered as the best targets in modulating immune responses against cancer, autoimmunity, allergy and transplantation. Aptamers, as a new agent, can be applied in DC targeting. The purpose of this review is to present some general concepts of aptamer production and DC targeting by aptamer molecules.

The expression of vascular endothelial growth factor in pterygium tissue of atopic patients.

The exact pathogenesis of pterygium has not been completely elucidated. Growth factors have been considered to play a role in pterygium formation. Vascular endothelial growth factor (VEGF) is one of the principal mediators of angiogenesis, fibroblast stimulation and tissue remodeling in allergic conditions. The aim of this study was to compare the association between pterygium and VEGF gene expression between atopic and non-atopic individuals. At first visit, all patients with pterygium underwent blood tests, serum immunoglobulin E (IgE), serum cytokines including interleukin-4 (IL-4) and interferon-γ (IFN-γ) and peripheral blood eosinophil count. After obtaining informed consents, questionnaires were used to obtain demographic and clinical data from patients who underwent pterygium excision surgery. Skin prick test was performed to confirm or rule out atopy in 30 patients with (case group) and 30 patients without (control group) atopy. Pterygium tissues were then removed by surgery. A semi-quantitative reverse transcriptase polymerase chain reaction was performed to determine VEGF gene expression in all patients. Our results illustrated that VEGF mRNA expression in atopic patients was significantly higher than in the non-atopic group (P = 0.01). Eosinophil count, serum IgE and IL-4 were also significantly higher in atopic patients than in the non-atopic group (P = 0.03, 0.001 and 0.001, respectively). However, no significant difference was noted in serum IFN-γ between the two groups (P = 0.06). The excessive expression of VEGF gene in pterygium tissue of patients with atopy suggests that growth factors may play a role in the pathogenesis of pterygium or accelerate its formation.

Expression of grape class IV chitinase in Spodoptera frugiperda (Sf9) insect cells.

INTRODUCTION: Most of pathogenesis related (PR) proteins possess complicated structures; hence their active recombinant forms are usually produced in eukaryotic systems. In this study, we employed an insect cell line to express a recombinant form of a previously identified grape PR3 allergen categorised as class IV chitinase. METHODS: Grape chitinase cDNA was amplified by RT-PCR and inserted into pFastBacHTA using restriction enzymes. The recombinant pFastBacHTA was applied for the transformation of Escherichia coli DH10Bac cells. The purified recombinant bacmid was used for transfection of Sf9 cells. Finally, the IgE-immunoreactivity of purified recombinant protein was evaluated using grape allergic patient's sera. Moreover, polyclonal anti-6His-tag and monoclonal anti-chitinase antibodies were used for further assessment of recombinant protein. RESULTS: SDS-PAGE analysis of the transfected Sf9 cells showed expression of a monomeric 25kDa and a dimeric 50 kDa recombinant protein. Western blotting revealed considerable IgE reactivity of the recombinant protein with grape allergic patients' sera. Furthermore, confirmatory assays showed specific reactivity of the recombinant protein with anti-His tag and anti-chitinase antibodies. CONCLUSION: This study showed that, in contrast to E. coli, insect cells are suitable hosts for the production of a soluble and IgE-reactive recombinant form of grape class IV chitinase. This recombinant allergen could be used for component resolved diagnosis of grape allergy or other immunodiagnostic purposes.

Monarch-1 Activation in Murine Macrophage Cell Line (J774 A.1) Infected with Iranian Strain of Leishmania major.

BACKGROUND: Leishmania major is an intracellular parasite transmitted through the bite of the female phlebotomine sand flies. Leishmania major is able to escape the host immune defense and survive within macrophages. Modulation of the NF-κB (Nuclear Factor-Kappa B) activation and suppression of the pro-inflammatory cytokines by L. major are the main evasion mechanisms that remain to be explored. This study aims to examine the expression level of the Monarch-1 in L. major-infected macrophages, as a negative regulator of the NF-κB activation. METHODS: Murine macrophage cell line (J774 A.1) was infected by metacyclic form of Leishmania promastigotes at macrophage/parasite ratio of 1:10. After harvesting infected cells at different times, total RNA was extracted and converted to cDNA. Semi-quantitative RT-PCR was performed for Monarch-1 by specific primers. Hypoxanthine Phospho-Ribosyl Transferase (HPRT) was used as an internal control to adjust the amount of mRNA in each sample. RESULTS: Semiquantitive analysis of Monarch-1 mRNA expression level showed a significant expression increase within 6 to 30 hours after L. major infection of macrophages when compared to the control macrophages. CONCLUSION: Monarch-1 expression level reveals a significant increase in the early phase of macrophage infection with L. major, which in turn may suppress IL-12 production in Leishmania infected macrophages and deeply influence the relationship between host and parasite.

Effect of Ramadan fasting on glycemic control in patients with Type 2 diabetes.

BACKGROUND: Although Muslim patients with Type 2 diabetes may be exempt from fasting during Ramadan for medical reasons, a high proportion of them fast. AIM: To investigate the association between Ramadan fasting and glycemic control in patients with Type 2 diabetes. SUBJECTS AND METHODS: A prospective cohort clinical trial was designed. Eighty-eight patients with Type 2 diabetes (45 male, 43 female, age 51±10 yr) who opted to fast for at least 10 days during the month of Ramadan were recruited. Fasting blood samples were taken at the beginning and end of Ramadan, and 1 month after Ramadan, to assess fasting blood glucose (FBG), fasting insulin, full blood count, glycated hemoglobin (HbA(1c)) and fasting lipid profile. Insulin resistance was estimated using the homeostatic model assessment. Anthropometrics and blood pressure were also measured. RESULTS: There was a significant deterioration in FBG and HbA(1c) (p=0.002 and p≤0.001, respectively) and significant improvements in HDL and LDL cholesterol and body mass index after Ramadan (p<0.001). Interestingly, HbA(1c) showed a reduction 1 month after Ramadan (9.4±2% at the end of Ramadan vs 8.4±2.5% 1 month after Ramadan; p<0.001). CONCLUSION: Results from this study showed that fasting during Ramadan deteriorated the glycemic control in Type 2 diabetes patients. This was more evident in patients using oral hypoglycemic medication than diet- controlled patients. However, Ramadan fasting had small positive effects on lipid profile and body weight.

Is C-reactive protein helpful for early diagnosis of acute appendicitis?

PURPOSE: Appendectomy is one of the most common surgical procedures all over the world. Although various laboratory tests and imaging studies are available to improve the accuracy of diagnosis, the rate of negative appendectomy is still about 15-30%. This study was designed to assess the diagnostic value of quantitative C-reactive protein (CRP) in patients suspected to acute appendicitis. MATERIALS AND METHODS: In a prospective study, blood samples of 102 patients were collected before appendectomy. CRP was measured by immunoturbidimetry and the data were compared with the final histopathologic reports. Diagnostic accuracy of the CRP test was analyzed by ROC curve. RESULTS: In histopathology, 83 patients (81/4%) had acute appendicitis and 19 (18/6%) had normal appendices. Considering 14 mg/lit as the cut-off point, this test shows 59% (95% CI, 48-69%) sensitivity and 68% (95% CI, 47-88%) specificity. The positive and negative predictive values were 89% (95% CI, 80-97%) and 27% (95% CI, 14-39%), respectively. CONCLUSIONS: The measurement of CRP levels is not an ideal diagnostic tool for ruling out or determination of acute appendicitis.

Generation and characterization of anti-chitinase monoclonal antibodies.

Class IV chitinase, an allergenic protein of Vitis vinifera (grape), was purified by anion exchange chromatography and used for immunization of Balb/c mice. Monoclonal antibodies (MAbs) were raised by hybridoma technology using Sp2/0 myeloma cells. Finally after three limiting dilutions, six stable clones were generated. Antibody isotyping showed that IgG(2a), IgG(2b), and IgM were produced by one, two, and three of the clones, respectively. All of the MAbs had kappa light chain. The affinities were in the range of 3 × 10(8) to 1.2 × 10(9) M(-1). The MAbs were specific for grape chitinase as confirmed by Western blotting. In conclusion, we successfully produced several MAbs against grape class IV chitinase, which could be used for assessment of this allergen in different grape cultivars.

Apoptosis of human lymphocytes after exposure to hydatid fluid.

BACKGROUND: Modulation of the immune response is an important strategy by which establishment and growth of hydatid cyst in the internal organs of human is warranted. Induction of apoptosis in the lymphocytes might be a considerable component. This study was designed to evaluate apoptotic impact of hydatid fluid (HF) on human lymphocytes. METHODS: Human lymphocytes were treated with hydatid fluid. After 6 hours of exposure, caspase-3 activity, the central enzyme of apoptosis cascade, was measured by fluorometric assay in the HF-treated lymphocytes and control cells. In addition, the expression of Bax (a pro-apoptotic protein) and Bcl-2 (an anti-apoptotic protein) mRNA was assessed by RT-PCR after 12 hours of exposure. RESULTS: Both the ratio of Bax/Bcl-2 mRNA expression and Caspase-3 activity were higher in the HF-treated lymphocytes relative to the control group. CONCLUSION: Apoptosis could be as a possible mechanism by which Echinococcus granulosus overwhelms host defenses.

Prevalence of work-related respiratory symptoms in Iranian farmers.

BACKGROUND: Animal exposure may be an important trigger for work-related symptoms among farmers. OBJECTIVE: To estimate the prevalence of work-related respiratory symptoms (WRS) in sheep breeders and agricultural farmers and to determine work-related risk factors. METHODS: A family doctor used a questionnaire to interview a cohort of 173 farmers comprised of 127 sheep breeders and 46 agricultural farmers in the rural area of Rokh (northeast Iran). The questionnaire pertained to recurrent wheezing, cough, breathlessness or chronic phlegm while at work (these symptoms define WRS), flu-like illness and physician-diagnosed asthma. RESULTS: There were 71 subjects (41%) with WRS: 10 of 46 agricultural farmers (21.7%) and 61 of 127 sheep breeders (48.0%). The proportions of sheep breeders with wheezing (16.5%), asthma (14%), cough (29%), breathlessness (31.5%) and flu-like illness (38%) were higher than in agricultural farmers. A significant dose-response relationship among the daily hours worked with animals, the number of animals and the prevalence of symptoms was established for sheep farmers. Sheep shearing and the use of pesticide were associated with an increased risk of wheezing and phlegm. CONCLUSIONS: The results suggest that sheep farmers in general have higher rates of work-related symptoms than agricultural farmers. The severity of work-related symptoms will increase with an increase in frequency of animal contact; therefore, these results may underestimate the impact of this exposure.

Cooperative alpha-helix formation of beta-lactoglobulin induced by sodium n-alkyl sulfates.

It is generally assumed that folding intermediates contain partially formed native-like secondary structures. However, if we consider the fact that the conformational stability of the intermediate state is simpler than that of the native state, it would be expected that the secondary structures in a folding intermediate would not necessarily be similar to those of the native state. beta-Lactoglobulin is a predominantly beta-sheet protein, although it has a markedly high intrinsic preference for alpha-helical structure. The formation of non-native alpha-helical intermediate of beta-lactoglobulin was induced by n-alkyl sulfates including sodium octyl sulfate, SOS; sodium decyl sulfate, SDeS; sodium dodecyl sulfate, SDS; and sodium tetradecyl sulfate, STS at special condition. The effect of n-alkyl sulfates on the structure of native beta-lactoglobulin at pH 2 was utilized to investigate the contribution of hydrophobic interactions to the stability of non-native alpha-helical intermediate. The addition of various concentrations of n-alkyl sulfates to the native state of beta-lactoglobulin (pH 2) appears to support the stabilized form of non-native alpha-helical intermediate at pH 2. The m values of the intermediate state of beta-lactoglobulin by SOS, SDeS, SDS and STS showed substantial variation. The enhancement of m values as the stability criterion of non-native alpha-helical intermediate state corresponded with increasing chain length of the cited n-alkyl sulfates. The present results suggest that the folding reaction of beta-lactoglobulin follows a non-hierarchical mechanism and hydrophobic interactions play important roles in stabilizing the non-native alpha-helical intermediate state.

An avidin-biotin ELISA for the measurement of mitochondrial aspartate aminotransferase in human serum.

We describe a new double-antibody ELISA system using avidin-biotin amplication for the mass measurement of mitochondrial aspartate aminotransferase (m-AST) in human serum. The assay is very sensitive and as little as 0.5 ng/ml of m-AST may be detected. The method is linear up to 100 micrograms/l. The within-day and day-to-day coefficients of variation were found to be 8.9% and 11.5% respectively for a low m-AST concentration (2.7 micrograms/l), and 5.9% and 8.0% respectively for a high level of m-AST (30 micrograms/l). The assay requires 100 microliters of serum and can be completed within 5 h. The ELISA procedure and a classical immunoprecipitation technique measuring the catalytic activity of the isoenzyme were applied simultaneously to the sera of 189 subjects. The protein levels determined by ELISA correlated poorly (r = 0.66) with the catalytic activity of m-AST.

Serum mitochondrial aspartate aminotransferase activity: not useful as a marker of excessive alcohol consumption in an unselected population.

Using an immunochemical method, we measured the activity of the mitochondrial isoenzyme (mAST) of aspartate amino-transferase (EC 2.6.1.1, AST) in the serum of 687 subjects attending the Centre for Preventive Medicine for a health examination. The distributions of the activities were asymmetrical, with mean values of 1.8 U/L (SD 2.0) for men and 1.4 U/L (SD 1.6) for women. The average ratio of mitochondrial to total AST activity was 0.051 (range 0-0.42). In this unselected population we found no change in the mitochondrial activity or in the mitochondrial-to-total ratio attributable to alcohol consumption, even in subjects who consumed more than 88 g per day. Of 35 men with an alcohol consumption greater than 88 g/d, 19 had a serum gamma-glutamyltransferase activity of greater than or equal to 60 U/L, 17 had glutamate dehydrogenase values greater than or equal to 5 U/L, and only nine had an mAST activity greater than or equal to 3 U/L (values corresponding to the 80th percentiles of the total population). We conclude that the test is not particularly useful as a screening procedure in an unselected population under present-day conditions of measurement.