Post-synthetic benzylation of the mRNA 5' cap via enzymatic cascade reactions.

mRNAs are emerging modalities for vaccination and protein replacement therapy. Increasing the amount of protein produced by stabilizing the transcript or enhancing translation without eliciting a strong immune response are major steps towards overcoming the present limitations and improving their therapeutic potential. The 5' cap is a hallmark of mRNAs and non-natural modifications can alter the properties of the entire transcript selectively. Here, we developed a versatile enzymatic cascade for regioselective benzylation of various biomolecules and applied it for post-synthetic modification of mRNA at the 5' cap to demonstrate its potential. Starting from six synthetic methionine analogues bearing (hetero-)benzyl groups, S-adenosyl-l-methionine analogues are formed and utilized for N7G-cap modification of mRNAs. This post-synthetic enzymatic modification exclusively modifies mRNAs at the terminal N7G, producing mRNAs with functional 5' caps. It avoids the wrong orientation of the 5' cap-a problem in common co-transcriptional capping. In the case of the 4-chlorobenzyl group, protein production was increased to 139% during in vitro translation and to 128-150% in four different cell lines. This 5' cap modification did not activate cytosolic pathogen recognition receptors TLR3, TLR7 or TLR8 significantly more than control mRNAs, underlining its potential to contribute to the development of future mRNA therapeutics.

Evaluation of the Ocular Surface, Conjunctival Impression Cytology and Meibomian Gland Dropout in Patients with Familial Mediterranean Fever.

PURPOSE: Familial Mediterranean fever (FMF) is an idiopathic disease with chronic inflammation. We aimed to determine the changes caused by the chronic inflammatory nature of FMF on the ocular surface, meibomian glands (MG), and conjunctiva via conjunctival impression cytology (CIC). MATERIAL-METHOD: Forty-two FMF patients with a mean age of 11.93±3.92 years and 36 control patients with a mean age of 11.83±3.38 years were included in the study. Ocular surface anomalies of the patients were evaluated using Schirmer II, TBUT and OSDI. MG function (meibum quality), morphology (meiboography), and CIC were evaluated. RESULTS: Although there was a significant difference between the groups in terms of Schirmer II and TBUT, OSDI scores did not significantly differ (P=0.022, 0.010, and 0.099 respectively), and no significant dry eye sign was observed in either group. There was significant difference between the groups in terms of the percent area of MG dropout, MG density, meiboscore (P=0.020, 0.023, and 0.031 respectively), but no significant difference was observed in relation to MG quality (P=0.650). Although conjunctival impression cytology was of a higher grade in the patients with FMF according to Nelson's classification, no significant difference was observed between the groups (P=0.109). CONCLUSION: Although there was a decrease in the number of MGs in FMF patients, no significant deterioration was observed in conjunctival cytology. In these patients, tear film stability may deteriorate in particular. Clinicians should be aware of the possibility of ocular surface disease secondary to MG dropout in patients with FMF.

The impact of pretreatment with simvastatin on kidney tissue of rats with acute sepsis.

It has been reported that changes in cytokine levels affect mitochondrial functions, levels of hypoxia-inducible factor α (HIF-1α), and tissue damage during sepsis. We aimed to investigate the effects of simvastatin pretreatment on mitochondrial enzyme activities, and on levels of ghrelin, HIF-1α, and thiobarbituric acid reactive substances (TBARS) in kidney tissue during sepsis. Rats were separated into four groups, namely, control, lipopolysaccharides (LPS) (20 mg/kg), simvastatin (20 mg/kg), and simvastatin + LPS. We measured the levels of mitochondrial enzyme activities and TBARS in the kidney using spectrophotometry. The histological structure of the kidney sections was examined after staining with hematoxylin and eosin. Tumor necrosis factor α (TNF-α), IL-10, HIF-1α, and ghrelin immunoreactivity were examined using proper antibodies. In tissue, TNF-α (p < 0.01) and HIF-1α (p < 0.05) levels were increased in the simvastatin + LPS and LPS groups. TBARS levels were higher in the LPS group than in the other groups (p < 0.01), but they were similar in the simvastatin + LPS and control groups (p > 0.05). Ghrelin immunoreactivity was lower in the LPS group (p < 0.05) and higher in the simvastatin + LPS group than in the LPS group (p < 0.01). We observed tubular damage in the sections of the LPS group. There were no differences in mitochondrial enzyme activities between the groups (p > 0.05). We observed that pretreatment of simvastatin caused favorable changes on ghrelin and TBARS levels in rats with sepsis.

Bone mineralization defects after treatment of acute lymphoblastic leukemia in children.

AIM: Aim of the study was to assess bone metabolism disturbances in children with acute lymphoblastic leukemia following cessation of chemotherapy. For this purpose we measured bone mineral density (BMD) and evaluated bone metabolism markers. METHODS: Seventy-five patients (37 female, 38 males, mean age 10.77±3.80 years) were included. Lumbar spine BMD was measured by dual energy X-ray absorptiometry and serum calcium, phosphorus, magnesium, alkaline phosphatase, parathyroid hormone and 25OH vitamin D levels were analyzed. For characteristics of all patients at diagnosis data were retreived from hospital records and analyzed retrospectively. RESULTS: A total of 18.66% (14 patients) of patients were osteoporotic (z score <-2 SD), 22.67% (17 patients) were osteopenic (z-score between -2 and -1 SD) and 58.67% (44 patients) presented normal z-scores (>-1 SD). There were no statisticaly significant differences between normal, osteopenic and osteoporotic groups for mean serum vitamine D (P=0.677), calcium (P=0.280), phosphorus (P=0.179), magnesium (P=0.675), ALP (P=0.092) and serum PTH (P=0.915) levels. According to ages (P=0.745) and gender (P=0.810) there were no significant differences in BMD. There were no significant differences between normal, osteopenic and osteoporotic patients for the total dose of prednisolone (P=0.334), dexamethasone, (P=0.734), methotrexate (P=0.911), granulocyte colony-stimulating factor (P=0.173) and cranial irradiation (P=0.912) they have received during chemotherapy. Bone fracture and aseptic necrosis rates were 12%, 8%, respectively. CONCLUSION: Osteoporosis and osteopenia are still observed in high rates after chemotherapy. We must be aware of this morbidity and must screen the patients for decreased BMD during the long duration of leukemia treatment. Supportive treatments should be evaluated to minimize these serious complications.

Saccharomyces boulardii CNCM I-745 reduces the duration of diarrhoea, length of emergency care and hospital stay in children with acute diarrhoea.

Evidence from the literature has shown that Saccharomyces boulardii provides a clinically significant benefit in the treatment of acute infectious diarrhoea in children. In this multicentre, randomised, prospective, controlled, single blind clinical trial performed in children with acute watery diarrhoea, we aimed to evaluate the impact of S. boulardii CNCM I-745 in hospitalised children, in children requiring emergency care unit (ECU) stay and in outpatient settings. The primary endpoint was the duration of diarrhoea (in hours). Secondary outcome measures were duration of hospitalisation and diarrhoea at the 3(rd) day of intervention. In the whole study group (363 children), the duration of diarrhoea was approximately 24 h shorter in the S. boulardii group (75.4±33.1 vs 99.8±32.5 h, P<0.001). The effect of S. boulardii (diarrhoea-free children) was observed starting at 48 h. After 72 h, only 27.3% of the children receiving probiotic still had watery diarrhoea, in contrast to 48.5% in the control group (P<0.001). The duration of diarrhoea was significantly reduced in the probiotic group in hospital, ECU and outpatient settings (P<0.001, P<0.01 and P<0.001, respectively). The percentage of diarrhoea-free children was significantly larger after 48 and 72 h in all settings. The mean length of hospital stay was shorter with more than 36 h difference in the S. boulardii group (4.60±1.72 vs 6.12±1.71 days, P<0.001). The mean length of ECU stay was shorter with more than 19 h difference in the probiotic group (1.20±0.4 vs 2.0±0.3 days, P<0.001). No adverse effects related to the probiotic were noted. Because treatment can shorten the duration of diarrhoea and reduce the length of ECU and hospital stay, there is likely a social and economic benefit of S. boulardii CNCM I-745 in adjunction to oral rehydration solution in acute infectious gastroenteritis in children.

Evaluation of Health-related Quality of Life in Paediatric Patients with Acute Lymphoblastic Leukaemia.

OBJECTIVE: Health-related quality of life (HRQOL) measurement is used for assessing the impact of diseases and medical treatments on physical, psychological and social aspects of an individual's health and life. The Paediatric Quality of Life Inventory™ (PedsQL™) is a widely used instrument to measure paediatric HRQOL in children. The aim of this study is to investigate the HRQOL in paediatric patients with acute lymphoblastic leukaemia and determine the precautions for improving the quality of their life. METHODS: Paediatric Quality of Life Inventory™ 4.0 was administered to 75 paediatric patients with acute lymphoblastic leukaemia, and 50 healthy age- and gender-matched children. RESULTS: Health-related quality of life scores were significantly lower in patients with acute lymphoblastic leukaemia than in healthy controls in this study. CONCLUSIONS: It is thought that determination of the psychosocial, as well as the physical impacts of the disease on the child, will positively influence the treatment given by improving the quality of life of both the child and the family.

MEFV mutations in systemic onset juvenile idiopathic arthritis.

OBJECTIVES: Autoinflammatory diseases constitute a large spectrum of monogenic diseases like FMF or cryopyrin-associated periodic syndromes (CAPS) and complex genetic trait diseases such as systemic onset juvenile idiopathic arthritis (SoJIA). An increased rate of MEFV mutations has been shown among patients with PAN and HSP, in populations where FMF is frequent. The aim of the study is to search for MEFV mutations in our patients with SoJIA and see whether these mutations had an effect on disease course or complications. METHODS: Thirty-five children with the diagnosis of SoJIA were screened for 12 MEFV mutations. The control data were obtained from a previous study of our centre determining the carrier frequency in Turkish population. RESULTS: Two patients were homozygous and three patients were heterozygous for the M694V mutation. One patient was a compound heterozygote for the M680I/V726A mutations. Heterozygous V726A mutation was found in one patient. The overall mutation frequency of patients was 14.28%. This figure had been compared with the previously published rate of disease-causing mutations in this country, which is 5%. Disease-causing mutations were found to be significantly more frequent in the SoJIA patients than the population (P < 0.01). Among these, M694V was the leading mutation with a frequency of 10% in SoJIA. Six patients carrying MEFV mutations were among the most resistant cases requiring biological therapy. CONCLUSION: SoJIA patients had a significantly higher frequency of MEFV mutations but clinical studies with large number of patients are needed to confirm the association of MEFV mutations with SoJIA and its course.

Spontaneous remission of acute lymphoblastic leukemia with mediastinal mass.

Spontaneous remission/regression of cancer is defined as partial or complete disappearance of malignant disease temporarily or permanently in the absence of medical treatment. This event is named as spontaneous regression for solid tumors and spontaneous remission for leukemia. The authors report the case of a girl aged 4 years and 3 months, who presented with mediastinal mass and leukemic findings in the bone marrow both of which reappeared after spontaneous regression and remission, respectively.

Sublingual immunotherapy to house dust mite in pediatric patients with allergic rhinitis and asthma: a retrospective analysis of clinical course over a 3-year follow-up period.

BACKGROUND AND OBJECTIVE: Specific allergen immunotherapy is believed to be the only treatment able to change the natural history of allergic airway diseases. Sublingual immunotherapy (SLIT) is especially preferred because of its easy application and safety. The aim of this study was to describe the effect of SLIT in pediatric patients who have allergic airway disease. METHODS: Children with asthma and rhinitis who were allergic to house dust mite were evaluated. The effect on clinical course of 3 years of SLIT with 50% Dermatophagoides pteronyssinus and 50% Dermatophagoides farinae in a standardized extract was assessed retrospectively. RESULTS: The records of 39 patients (23 boys, 16 girls) were studied. The mean (+/- SD) age for starting SLIT was 8.8 +/- 2.3 years. The mean number of acute asthma attacks at the onset of the disease was 8.18 +/- 3.05. The mean number of attacks after 3 years of SLIT was 0.44 +/- 0.79. There was a statistically significant difference in the number of acute asthma attacks before and after therapy (P < .001). Complete clinical remission of asthma was recorded in 37 (95%) patients. Similarly, complete clinical remission of allergic rhinitis was recorded in 32 (82%) patients. CONCLUSION: This retrospective study shows that SLIT is effective in children who have allergic airway disease which cannot be controlled effectively with allergen avoidance measures only.

Mushroom poisoning.

OBJECTIVE: We aimed to review characteristics of mushrooms and mushroom poisoning and compare clinical picture, laboratory data, treatment modalities and prognostic factors in children with amanita intoxication and non-amanita mushroom poisoning. METHODS: We analyzed 39 pediatric patients through 1994-2004, retrospectively from the patient files and evaluated the patients in two groups as patients with amanita intoxication and patients with non-amanita mushroom poisoning. All of the cases were admitted to the hospital in autumn. Twenty three (59%) of the patients were female and 16 (41%) were male. Mean age of the patients was 8.05 +/- 2.10 years. RESULTS: Amanita phalloides toxin was detected in the serum in 8 patients. Eleven (28%) of the cases were strongly suggestive of amanita poisoning but alpha amanitin level could not be studied. The average time of appearance of symptoms after mushroom consumption, duration of symptoms, hospital stay, serum AST, ALT, PT and creatinine levels were significantly higher in patients with amanita poisoning (p<0.01). Conventional therapy, antidote therapy together with hemoperfusion were carried out in 16 (41%) of the patients. Four of the patients in whose blood amatoxin was detected (50%) and 3 of the patients highly suggestive of amanita poisoning (30%), totally 7 patients died of hepatic coma. The average time of admission to hospital, mean AST, ALT, creatinine and PT values at 3rd day were significantly higher in patients who died of hepatic coma. Prognosis was better in case of early admittance to hospital in patients with amanita poisoning. CONCLUSION: Early diagnosis and treatment in mushroom poisoning can be life saving. Public awareness is very important in prevention of intoxication as well as encouraging early admission to hospitals.