Innovative restriction site created PCR-RFLP for detection of benzimidazole resistance in Teladorsagia circumcincta

Benzimidazole compounds, especially albendazole, are the most commonly used anthelmintics for deworming of small ruminants in Iran. It is believed that the therapeutic effects of the benzimidazoles (BZs) come through their binding capacity to the β-tubulin isotype 1. Substitution of phenylalanine to tyrosine at position 200 of this polypeptide confers resistance to BZs. Several investigators developed different biological- and molecular-based techniques to demonstrate the occurrence of resistance in helminthes against BZs. To address the determination of resistance at position 200 of β-tubulin isotype 1, we developed an innovative restriction site created polymerase chain reaction-restriction fragment length polymorphism, in which nucleotide A at the position of 637 upstream flanked by the first two coding sequences of the phenylalanine (TT) triplet was substituted through the nucleotide G. The introduced modification in forward primer (UTvet MF-primer) leads to the creation of restriction site (AACGTT) for PSP1. Therefore, in the case of normal allele only, PSP1 can cut the corresponding PCR product. In the first step, the genomic DNA was isolated from each single Teladorsagia circumcincta collected either from the abomasa of untreated (n = 35) or of 5 mg/kg BW 2.5% albendazole suspension-treated (n = 40) sheep. It was amplified with the primer pair, creating PCR product of 403 bp in length. In the second step, the PCR product was extracted from agarose gel and amplified with the modified forward primer (UTvet MF-primer) and the same reverse primer as in step 1, creating a PCR product of 222 bp. The PCR product was then cut with PSP1 to obtain in the case of normal allele two DNA products (183 and 39 bp). Eight of the 35 worms collected from the untreated sheep were BZ^SS homozygotes, and the rest (27) were BZ^RS heterozygotes. In our preliminary experiment, we could not find a BZ^RR homozygote form within the examined samples. Five out of 40 worms collected from the albendazole-treated sheep were BZ^RR homozygotes, whereas the rest (35) were BZ^RS heterozygotes. No BZ^SS homozygote form was detected within this group.     

Characterization and investigation of single nucleotide polymorphisms and a novel TLR2 mutation in the human TLR2 gene

In the innate immune system, TLR2 plays a central role for the response to a wide variety of microbial and endogenous danger signals. A considerable number of genetic polymorphisms within the human TLR2 gene have been reported in non-coding and coding sequences. Except for the Arg753Gln variant, however, their clinical relevance is unclear and the assessment of the effects of amino acid substitutions on receptor function is lacking. In the present study, we have characterized all known single nucleotide polymorphisms (SNPs) of TLR2 for their functional relevance in transiently transfected HEK293 cells subsequently exposed to a specific stimulus. Among the known non-synonymous SNPs in the TLR2 coding sequence, four SNPs (Thr411Ile, Tyr715stop, Tyr715Lys and Arg753Gln) were found to be functionally relevant in our experimental setting. In addition, we identified a new mutation Arg447stop leading to a premature stop codon in the extracellular portion of the receptor. TLR2-specific stimulation of whole blood from two heterozygote donors of this mutation resulted in a reduced secretion of pro-inflammatory cytokines. Finally, we tested the prevalence of these functional genetic variants in 169 healthy individuals of Caucasian origin for the mutations in the extracellular domain and 106 individuals for the mutations in the intracellular domain of the receptor. Except for 10 heterozygote donors of the Arg753Gln variant determined to be prevalent in 9.4% of the tested individuals, none of the other SNPs was found in this population.     

Increase in Fibrinogen and Fibrin-Related Antigen in Human Serum Due to In Vitro Lysis of Fibrin by Thrombin

In vitro lysis of fibrin, as indicated by increased fibrinogen-fibrin-related antigen (FR-antigen) in serum is usually seen when whole blood, or plasma, or highly purified fibrinogen prepared by several different procedures is clotted and kept at temperatures above 0 degrees C. This increase is both time and temperature dependent, occurs despite the addition of various plasmin and cathepsin inhibitors, and is probably caused by thrombin evolved during clotting and/or added in vitro. In these experiments, the FR-antigen was measured by a sensitive, reproducible hemagglutination inhibition immunoassay adapted to the AutoAnalyzer. Serum from whole blood contained more than serum from plasma, and fibrin rather than fibrinogen proved to be essential for the in vitro lysis. The phenomenon was also caused by Arvin or Reptilase, suggesting that splitting of one or more arginine or lysine bonds in fibrin may be at least partially responsible. To obtain minimal levels of FR-antigen (< 0.5 mug/ml), plasma is clotted for 4 hr at 0 degrees C with 1.0-5.0 U/ml thrombin, CaCl(2) (0.0125 mole/liter), and epsilon aminocaproic acid (0.05 mole/liter). Slightly higher levels, probably adequate for clinical diagnosis, are obtained by 10-30 min clotting at room temperature. Since endogenous and/or exogenous thrombin is essential for the collection of serum FR-antigen, all the FR-antigen found in normal serum probably results from an irreducible amount of in vitro lysis rather than from continuous intravascular clotting and fibrinolysis.     

Inherited disorders of gamma‐aminobutyric acid metabolism and advances in ALDH5A1 mutation identification

Inherited disorders of gamma‐aminobutyric acid (GABA) metabolism include succinic semialdehyde dehydrogenase (SSADH) and gamma‐aminobutyric acid transaminase (GABA‐T) deficiencies. The clinical features, pathophysiology, diagnosis, and management of both, and an updated list of mutations in the ALDH5A1 gene, which cause SSADH deficiency, are discussed. A database of 112 individuals (71 children and adolescents, and 41 adults) indicates that developmental delay and hypotonia are the most common symptoms arising from SSADH deficiency. Furthermore, epilepsy is present in two‐thirds of SSADH‐deficient individuals by adulthood. Research with murine genetic models and human participants, using [¹¹C] flumazenil positron emission tomography (FMZ‐PET) and transcranial magnetic stimulation, have led to therapeutic trials, and the identification of additional disruptions to GABA metabolism. Suggestions for new therapies have arisen from findings of GABAergic effects on autophagy, with enhanced activation of the mammalian target of rapamycin (mTOR) pathway. Details of known pathogenic mutations in the ALDH5A1 gene, three of which have not previously been reported, are summarized here. Investigations into disorders of GABA metabolism provide fundamental insights into the mechanisms underlying epilepsy, and support the importance of developing biomarkers and clinical trials. Comprehensive definition of phenotypes arising as a result of deficiencies in both SSADH and GABA‐T may increase our understanding of the neurophysiological consequences of a hyper‐GABAergic state.     

Action of topical thyroid hormone analogue, triiodothyroacetic acid in reversing glucocorticoid-induced skin atrophy in humans.

The present study concerns the effect of topical treatment with a cream formulation of triiodothyroacetic acid (TRIAC) in comparison with a placebo preparation in producing a reversal of skin atrophy induced by long-term employment of topical glucocorticoid therapy in humans. A total of 39 patients with clinically verified skin atrophy due to long-term use of topical potent glucocorticoids were randomized. The changes in skin thickness, elastic fibers, and hyaluronic acid were evaluated by means of sonography and histology. After 8 weeks' treatment, the skin thickness measured by sonography increased by 16% in the epidermis, 8% in the dermis, and epidermis + dermis in the placebo group. In the TRIAC 0.1% group, the corresponding values were 24% ( p=0.063) in the epidermis, 28% ( p=0.042) in the dermis, and 25% ( p=0.039) in the epidermis + dermis. After 8 weeks, in the placebo group, the skin thickness measured by biopsy increased by 5% in the epidermis, epidermis + dermis, and 6% in the dermis. In the TRIAC 0.1% group, the corresponding values were 31% ( p=0.041) in the epidermis, 46% ( p=0.041) in the dermis and 44% ( p=0.043) in the epidermis + dermis. After 8 weeks, the elastic fibers of moderately irregular and thickened fibers increased by 56% in the placebo group and 100% ( p=0.043) in the TRIAC 0.1 group. This study indicates that topical treatment with TRIAC appears to reverse glucocorticoid-induced skin atrophy under the narrow conditions tested.     

The evaluation of hepatitis C virus core antigen in immunized BALB/c mice

Background

Hepatitis infection represents one of the important causes of morbidity and mortality in developing countries, however there is not any effective vaccine against hepatitis C which is one of the significant problems in vaccine project.

Objectives

The aim of the present study is to evaluate the role of HCV core protein in inducing IFN-Gamma secretion and TCL activities as a vaccine in Balb/C mice.

Material and Methods

Our previous cloned plasmid (HCV Core gene into pETDuet-1) applied for protein expression in bacteria. The expressed and purified recombinant protein together with Freund’s adjuvant was injected to 15 Balb/c mice. The total IgG and IgG2a of immunized mice sera were evaluated after a week. Two weeks after booster injection, we studied the proliferation and IFNγ secretion of spleens, inguinal and popliteal lymph nodes lymphocytes by ELISA and ELISPOT.

Results

The FSFC (Frequency of spot forming cells) of secreting cells of immunized mice with HCV/Core protein and sera IgG2a were considerably higher than the control groups.

Conclusions

The core protein together with proper adjuvant can be a candidate vaccine against of HCV infection.     

The challenge of developing a new predictive formula to estimate energy requirements in ventilated critically ill children

Background: Traditionally, energy requirements have been calculated using predictive equations. These methods have failed to calculate energy expenditure accurately. Routine indirect calorimetry has been suggested, but this method is technically demanding and costly. This study aimed to develop a new predictive equation to estimate energy requirements for critically ill children. Methods: This prospective, observational study on ventilated children included patients with an endotracheal tube leak of <10% and fractional inspired oxygen of <60%. An indirect calorimetry energy expenditure measurement was performed and polynomial regression analysis was used to develop new predictive equations. The new formulas were then compared with existing prediction equations. Results: Data from 369 measurements were included in the formula design. Only weight and diagnosis influenced energy expenditure significantly. Three formulas (A, B, C) with an R (2) > 0.8 were developed. When we compared the new formulas with commonly used equations (Schofield, Food and Agriculture Organization/World Health Organization/United Nations University, and White equation), all formulas performed very similar, but the Schofield equation seemed to have the lowest SD. Conclusions: All 3 new pediatric intensive care unit equations have R (2) values of >0.8; however, the Schofield equation still performed better than other predictive methods in predicting energy expenditure in these patients. Still, none of the predictive equations, including the new equations, predicted energy expenditure within a clinically accepted range, and further research is required, particularly for patients outside the technical scope of indirect calorimetry.     

ErbB2 growth factor receptor, a marker for neuroendocrine cells?

BACKGROUND/AIMS: The overexpression of ErbB2 in pancreatic cancer has been reported with a varying incidence ranging between 1 and 80%. Our routine examination, however, revealed a consistently strong immunoreactivity of three anti-ErbB2 growth factor receptor antibodies in pancreatic islets and intrapancreatic ganglia. To validate our findings and to understand the reasons for the reported differences in the frequency of ErbB2 overexpression in pancreatic cancer, the following studies were performed. MATERIALS AND METHODS: Tissue samples from 12 normal pancreata, 7 surgical chronic pancreatitis cases, 21 primary pancreatic adenocarcinomas, 9 metastatic pancreatic adenocarcinomas, and 4 islet cell tumors were subjected to immunohistochemical examination using antibodies from three manufacturers. Cultured human islet cells and pancreatic cancer cell lines, as well as samples from the gastrointestinal tract, the CNS, and the adrenal gland were included in the study. For comparison, mammary cancer tissue and mammary cancer cells, as well as selected tissues from Syrian golden hamsters, were used. To verify the results, Western blot and Northern slot-blot analyses were performed. RESULTS: Pancreatic cancer cells, in vitro and in vivo, showed a remarkable heterogeneity in the immunostaining of ErbB2, ranging from very faintly to strongly stained. On the other hand, in both humans and hamsters, a consistently strong immunostaining was found in the Langerhans' islets, in the ganglia of intrapancreatic and extrapancreatic nerves, as well as in the CNS, spinal cord and adrenal gland. CONCLUSIONS: ErbB2 appears to play an important role in neuroendocrine tissues and is probably involved in the development and functional regulation of these cells. The concomitant expression of these factors and islet cell hormones very likely results in the activation of multiple growth-promoting pathways in pancreatic cancer and its aggressive behavior.