Evidence for effect of l-serine, a novel therapy for GRIN2B-related neurodevelopmental disorder.

RATIONALE: To date, causal therapy is potentially available for GRIN2B-related neurodevelopmental disorder (NDD) due to loss-of-function (LoF) variants in GRIN2B, resulting in dysfunction of the GluN2B subunit-containing N-methyl-d-aspartate receptor (NMDAR). Recently, in vitro experiments showed that high doses of NMDAR co-agonist d-serine has the potential to boost the activity in GluN2B LoF variant-containing NMDARs. Initial reports of GRIN2B-NDD patients LoF variants, treated with l-serine using different regimens, showed varying effects on motor and cognitive performance, communication, behavior and EEG. Here, this novel treatment using a standardized protocol with an innovative developmental outcome measure is explored further in an open-label observational GRIN2B-NDD study. METHODS: Initially, in vitro studies were conducted in order to functionally stratify two de novo GRIN2B variants present in two female patients (18 months and 4 years old). Functional studies showed that both variants are LoF, and thus the patients were treated experimentally according to an approved protocol with oral l-serine (500 mg/kg/day in 4 doses) for a period of 12 months. Both patients showed a heterogeneous clinical phenotype, however overlapping symptoms were present: intellectual developmental disability (IDD), behavioral abnormalities and hypotonia. Outcome measures included laboratory tests, quality of life, sleep, irritability, stool, and performance skills, measured by, among others, the Perceive-Recall-Plan-Perform System of Task Analysis (PRPP-Assessment). RESULTS: Both patients tolerated l-serine without adverse effects. In one patient, improvement in psychomotor development and cognitive functioning was observed after 12 months (PRPP mastery score 10% at baseline, 78% at twelve months). In the most severe clinically affected patient no significant objective improvement in validated outcomes was observed. Caregivers of both patients reported subjective increase of alertness and improved communication skills. CONCLUSION: Our observational study confirms that l-serine supplementation is safe in patients with GRIN2B-NDD associated with LoF variants, and may accelerate psychomotor development and ameliorate cognitive performance in some but not all patients. The PRPP-Assessment, a promising instrument to evaluate everyday activities and enhance personalized and value-based care, was not performed in the severely affected patient, meaning that possible positive results may have been missed. To generate stronger evidence for effect of l-serine in GRIN2B-NDD, we will perform placebo-controlled n-of-1 trials.

A self-centring osteotomy of the tibial tubercle for patellar maltracking or instability: results with ten-years' follow-up.

An increased tibial tubercle-trochlear groove (TT-TG) distance is related to patellar maltracking and instability. Tibial tubercle transfer is a common treatment option for these patients with good short-term results, although the results can deteriorate over time owing to the progression of osteoarthritis. We present a ten-year follow-up study of a self-centring tibial tubercle osteotomy in 60 knees, 30 with maltracking and 30 with patellar instability. Inclusion criteria were a TT-TG ≥ 15 mm and symptoms for > one year. One patient (one knee) was lost to follow-up and one required total knee arthroplasty because of progressive osteoarthritis. Further patellar dislocations occurred in three knees, all in the instability group, one of which required further surgery. The mean visual analogue scores for pain, and Lysholm and Kujala scores improved significantly and were maintained at the final follow-up (repeated measures, p = 0.000, intergroup differences p = 0.449). Signs of maltracking were found in only a minority of patients, with no difference between groups (p > 0.05). An increase in patellofemoral osteoarthritis was seen in 16 knees (31%) with a maximum of grade 2 on the Kellgren-Lawrence scale. The mean increase in grades was 0.31 (0 to 2) and 0.41 (0 to 2) in the maltracking and instability groups respectively (p = 0.2285) This self-centring tibial tubercle osteotomy provides good results at ten years' follow-up without inducing progressive osteoarthritis.

Total parenteral nutrition after surgery rapidly increases serum leptin levels.

OBJECTIVE: In humans, leptin is regulated by long-term changes in energy intake. However, short-term regulation of serum leptin by nutrients has been difficult to show. The aim of this study was to investigate whether short periods of fasting and stress sensitise the leptin response to nutrients. SUBJECTS AND EXPERIMENTAL PROTOCOL: Fourteen patients of normal weight undergoing elective open cholecystectomy were randomised into two groups. One group received saline infusion during surgery and for 24 h postoperatively. The other group also received saline during the surgical procedure, but total parenteral nutrition (TPN) was started immediately after surgery. Blood samples were drawn before as well as 2, 4, 8, 16, and 24 h after the start of surgery to determine the serum levels of leptin and other hormones. RESULTS: Postoperative TPN induced a significant rise in serum leptin within 6 h, reaching a more than fourfold increase within 14 h (P<0.001). Serum glucose and insulin levels increased within 2 h. Growth hormone and IGF-1 serum levels also increased significantly in the group receiving TPN. Serum cortisol levels increased postoperatively in both groups, which may explain why no significant reduction in serum leptin was observed in the group receiving saline. Free tri-iodothyronine (T3) decreased in both groups, while catecholamines were similar in the groups. CONCLUSION: During fasting and surgical stress, nutrients rapidly increased the serum leptin levels in humans in a manner similar to that previously reported in rodents. This may be mediated by increases in serum glucose, insulin and cortisol.

Growth hormone together with glutamine-containing total parenteral nutrition maintains muscle glutamine levels and results in a less negative nitrogen balance after surgical trauma.

BACKGROUND: Muscle protein catabolism, reflected by a decrease in glutamine (GLN), a decrease in muscle protein synthesis, and a negative nitrogen balance can be reduced by either administration of GLN or growth hormone (GH). In this study, the effects of a combination of GH and GLH were studied. METHODS: Patients (n = 16) undergoing abdominal operation were given total parenteral nutrition (TPN) containing either GLN alone or GLN together with GH (GH/GLN) during 3 postoperative days. The amino acid concentration and protein synthesis in muscle tissue and the nitrogen balance were measured. RESULTS: GH/GLN reduced nitrogen losses compared with GLN alone (-5.8 +/- 1.4 g nitrogen versus -10.6 +/- 1.1 g nitrogen, P <.05). GH/GLN maintained muscle GLN at preoperative levels compared with a 47.5% +/- 6.3% decline in the GLN group. A similar decrease was seen in the fractional synthesis rate of muscle protein postoperatively in both groups. CONCLUSIONS: GH has an additive effect given together with GLN on muscle amino acid metabolism, preventing the decrease in the GLN concentration in skeletal muscle and diminishing the loss of whole body nitrogen. However, the improvements in muscle amino acid concentrations and nitrogen loss were not associated with differences between the groups in muscle protein synthesis postoperatively.

Growth hormone does not affect albumin synthesis in the critically ill.

OBJECTIVE: To study the effect of growth hormone (GH) on albumin synthesis in critically ill patients. DESIGN: Prospective randomized controlled study. SETTING: Two intensive care units, university hospital and county hospital, respectively. PATIENTS: Twenty-two critically ill patients in the intensive care unit. INTERVENTIONS: Albumin synthesis was measured twice in each patient, with a 5-day interval. The patients in the control group (n = 11) received standard intensive care unit (ICU) treatment between measurements, whereas those in the GH group (n = 11) also received 0.3 U/kg daily of human recombinant GH. MEASUREMENTS AND RESULTS: Albumin synthesis was measured by labeling with L-[2H5]phenylalanine. In the control group, the fractional synthesis rate (FSR) of albumin was 16.3+/-4.1%/day (mean and SD) in the first measurement and 15.7+/-4.2%/day 5 days later (NS), whereas in the GH group the corresponding values were 17.0+/-4.7%/day and 16.7+/-5.5%/day (NS). The calculated absolute synthesis rates of albumin, based on FSR and intravascular albumin mass, also showed no effect of GH. CONCLUSION: Albumin synthesis rates were consistently higher in the two groups of critically ill patients than previously reported values in healthy subjects. However, GH treatment for 5 days neither stimulated nor inhibited albumin synthesis rates in these critically ill patients.

Longitudinal changes of biochemical parameters in muscle during critical illness.

The study was undertaken to characterize the time course of biochemical parameters in skeletal muscle during critical illness to gain information for the design of a suitable protocol for interventional studies using metabolic or nutritional manipulation. Critically ill patients in our intensive care unit ([ICU] N = 9) were investigated on two separate sampling occasions with percutaneous muscle biopsies for determination of protein, nucleic acids, free amino acids, energy-rich phosphates, fat, water, and electrolytes. The first biopsy specimen was taken 3 to 11 days after admission and the second biopsy specimen 3 to 7 days later. Protein concentration, expressed as alkali-soluble protein (ASP)/DNA, decreased by 12% (P < .02) between the two biopsies. The total free amino acid content was only 50% of normal, but remained unaltered over time. In particular, the concentration of glutamine remained low, approximately 25% of normal. In contrast, branched-chain amino acid (BCAA) increased by 25% (P < .05) and phenylalanine by 55% (P < .05) between biopsies. The fat content related to fat-free solid (FFS) increased by 130% (P < .001) between the two biopsies. Muscle water did not change during the study period. The extracellular portion was double the normal value when related to FFS. Intracellular water, on the other hand, was outside the 95% confidence interval for normal values in the second biopsy. The concentrations of adenosine triphosphate (ATP), creatine, phosphocreatine, and the phosphorylated fraction of total creatine remained at the same level between the two biopsies. We conclude that in critically ill patients, there is a decrease in protein content over time and increases in BCAA, phenylalanine, and fat content, while the low glutamine level and high extracellular water content remain unaltered. The temporal alterations were well characterized after a 5-day study period.

A combined stress hormone infusion decreases in vivo protein synthesis in human T lymphocytes in healthy volunteers.

In vivo protein synthesis decreases in mononuclear cells following a combined stress hormone infusion given to healthy volunteers as a human trauma model. Here, the purpose was to further investigate this finding and to measure in vivo protein synthesis in isolated T lymphocytes. Furthermore, the effects of stress hormones on the lymphocyte subpopulations and mononuclear cells, characterized by flow cytometry and phytohemagglutinin (PHA)-induced and unstimulated proliferative responses in vitro, were elucidated. Healthy volunteers (n = 16) were randomized into 2 groups to receive either a stress hormone or a saline infusion for 6 hours. In vivo protein synthesis was studied before and after the treatment by measuring the incorporation of stable isotopically-labeled phenylalanine into lymphocyte and mononuclear cell proteins. Protein synthesis decreased after stress hormone infusion in both cell populations: in T lymphocytes from 13.0% +/- 0.7%/d (mean +/- SD) to 8.6% +/- 2.1%/d (P <.01) and in mononuclear cells from 13.3% +/- 1.2%/d to 6.3 +/- 2.0%/d (P <.001). No change in proliferative responsiveness in vitro was observed. The stress hormone infusion produced a decrease in the percentage of T helper CD3/CD4 from 41% to 18% (P <.001), T cytotoxic CD3/CD8 from 27% to 15% (P <.001), as well as total T CD3 cells from 69% to 35% (P <.001). There was an increase in the percentage of natural killer (NK) cells CD16/CD56 from 17% to 55% (P <.001). Determination of phenotypes expressed on activated T lymphocytes showed that CD3/HLA-DR was unchanged and CD3/CD25 decreased from 14% to 7% (P <.01) in the stress hormone group. The study showed that the decrease of in vivo protein synthesis was 34% in T lymphocytes as compared with 53% in mononuclear cells, when determined immediately after a 6-hour stress hormone infusion. This change was associated with a pronounced decrease in all lymphocyte subpopulations, except for the NK cells, which increased substantially.

Protein synthesis rates of skeletal muscle, lymphocytes, and albumin with stress hormone infusion in healthy man.

The rate of protein synthesis was assessed in muscle, lymphocytes, and albumin in healthy volunteers administered an infusion of 6.0 micrograms cortisol +3.0 ng glucagon +0.5 nmol epinephrine min-1.kg-1. Protein synthesis in muscle tissue was not sensitive to the immediate effects of hormone infusion, but decreased significantly by 18 hours after the infusion had ceased (1.77% +/- 0.12% per day v 1.29% +/- 0.10%, P < .05). The rate of protein synthesis in lymphocytes was acutely sensitive to the effect of the hormone infusion, decreasing from 7.15% +/- 1.02% per day to 2.47% +/- 0.5% (P < .05). However, measurements made 18 hours after the end of the hormone infusion indicated that lymphocyte protein synthesis returned to the preinfusion rates. The rate of albumin synthesis was unaltered during infusion of the stress hormones, but was significantly increased when measured 18 hours after ending the hormone infusion (6.84% +/- 0.43% per day v 7.99% +/- 0.45%, P < .05). Thus, tissues respond differently to stress hormone infusion, demonstrating the importance of studying multiple organ systems when assessing the regulation of protein metabolism.

Stimulation of human albumin synthesis and gene expression by growth hormone treatment.

BACKGROUND AND AIMS: In this study the effects of acute (5 h) and short-term (5 days) GH treatment on albumin synthesis rates in man were investigated and related to changes in the availability of hepatic albumin mRNA. METHODS: 30 patients undergoing elective laparoscopic cholecystectomy were randomized into controls (n=10) or GH-treatment (12 U/dose) for 5 h or 5 days (n=10 in each group). Albumin mRNA levels (in liver biopsy specimens) were measured employing a quantitative polymerase chain reaction assay developed specifically for this purpose, whereas albumin synthesis was measured using [(2)H(5)]phenylalanine. RESULTS: The fractional synthesis rate of albumin was 6.0+/-0.9 %/day in the control group and 8.0+/-1.8 %/day and 8.3+/-1.7 %/day in the GH-treated groups, respectively (P<0.05 vs controls in both cases). The corresponding values for the concentration of albumin mRNA were 2.6+/-1.1 ng/microg total RNA, 2.9+/-0.8 ng/microg total RNA (NS) and 4.7+/-1.8 ng/microg total RNA in the "GH 5" group (P<0.01 vs controls). The changes in albumin synthesis were only partly explained by the differences in hepatic albumin mRNA levels (r=0.5, P<0.01). CONCLUSION: These results suggest that GH may induce a quick, gene expression-independent increase in albumin synthesis, which is sustained by a later-occurring increase in albumin gene expression.

Effect of a short-term infusion of glutamine on muscle protein metabolism postoperatively.

The acute effect of a short-term postoperative infusion of glucose supplemented with glutamine (0.285 g/kg body weight), on muscle protein metabolism, was studied by analyses of free amino acid concentrations and determinations of protein synthesis. A glutamine-glucose infusion was given for 5.5 h to 6 patients 2-3 days after elective surgery for colon cancer. The free glutamine concentration was 5.72 +/- 0.96 mmol/kg wet weight (ww) before and 6.14 +/- 1.10 mmol/kg ww 4 h after the glutamine infusion. The rate of protein synthesis was 1.26 +/- 0.15%/24 h before the infusion and 1.12 +/- 0.16%/24 h during its latter part. The percentage of polyribosomes was 42.2 +/- 3.4% before and 40.9 +/- 1.3% after the infusion. The results showed no difference in these biochemical parameters, indicating that a short-term infusion of glutamine given postoperatively is insufficient to affect protein metabolism in human skeletal muscle.

Response of biting midges (Diptera: Ceratopogonidae) to carbon dioxide, octenol, and light in southeastern Queensland, Australia.

The effect of 1-octen-3-ol (octenol) on catches of estuarine biting midges in encephalitis vector surveillance (EVS) traps was examined in southeastern Queensland. Octenol by itself was not attractive but appeared to act as a synergist with CO2 to increase catches of most species. For four of six species tested, a medium (about 6 mg/h) release rate of octenol captured the most individuals. Addition of light also increased the catch size of most species. Octenol in combination with CO2 could be used to enhance biting midge catch size, improving the sensitivity of surveillance for midges and the pathogens they vector.

The effects of short-term parenteral nutrition on human liver protein and amino acid metabolism during laparoscopic surgery.

BACKGROUND: This study was undertaken to elucidate the specific effects of short-term artificial nutrition on human liver protein metabolism. METHODS: Thirty patients undergoing elective laparoscopic cholecystectomy were studied: a control group (n = 16) and a group that received total parenteral nutrition (TPN; n = 14). The nutrition consisted of a balanced i.v. solution of nutrients (17.5 nonprotein kcal/kg body wt, 50% fat, 50% carbohydrates, and 0.1 gN/kg) that was discontinued when the investigation was finished, after a total infusion time of 8.6 +/- 1.0 hours. A liver biopsy specimen was taken as soon as possible after surgery was started, for the determination of the free hepatic amino acid concentrations. In 16 of the patients, L[2H5]phenylalanine was given by i.v. to determine the fractional synthesis rate of total liver protein in a second liver biopsy specimen taken approximately 30 minutes later. RESULTS: The fractional synthesis rate of total liver protein was 15.2% +/- 4.7%/d in the TPN group (n = 7), which was not different from that of the control group (17.7% +/- 3.8%/d, n = 9). However, the free hepatic concentrations of alanine (p < .05) and the essential amino acids increased (p < .001) in the TPN group, whereas the total hepatic amino acid concentrations were comparable between the groups. CONCLUSION: Thus short-term TPN induced specific changes of the free hepatic amino acid concentrations, whereas total liver protein synthesis remained unaffected by the nutrition.

Muscle protein synthesis rate decreases 24 hours after abdominal surgery irrespective of total parenteral nutrition.

BACKGROUND: Muscle protein synthesis rate is known to decrease postoperatively as a part of the catabolic response to trauma. Conventional total parenteral nutrition (TPN) in the postoperative period does not seem to counteract the decrease in protein synthesis. However, it is still unclear if ongoing TPN given continuously after surgery would inhibit this fall in muscle protein synthesis. METHODS: The rate of protein synthesis in skeletal muscle was determined before and 24 hours after open cholecystectomy, used as a standardized human model of trauma. Patients (n = 14) were randomized to receive either TPN continuously throughout the postoperative period or saline as postoperative fluid therapy. The protein synthesis rate was calculated from the increase in enrichment of labeled phenylalanine in protein after an IV flooding dose of [2H5] phenylalanine, 45 mg/kg body weight. RESULTS: The fractional synthesis rate decreased by 31% from 1.74 +/- 0.13% to 1.15 +/- 0.10% per 24 hours in the saline group (p < .02) and by 23% from 1.59 +/- 0.10% to 1.22 +/- 0.07% per 24 hours in the group receiving TPN (p < .01), showing no significant difference between the two groups. CONCLUSION: A continuous and ongoing infusion of conventional TPN started immediately after surgery did not counteract the obligatory decline of muscle protein synthesis, observed 24 hours postoperatively.

Effect of hemodialysis on protein synthesis.

BACKGROUND: Earlier studies have shown that hemodialysis (HD) treatment stimulates net protein catabolism. Several factors associated with HD affect protein catabolism, such as an inflammatory effect due to blood-membrane contact and loss of amino acids and glucose into the dialysate. SUBJECTS, MATERIAL AND METHODS: We have studied protein synthesis in skeletal muscle of healthy volunteers (n = 9) before and after a single heparin-free HD. Protein synthesis (PS) was studied, using 2 independent techniques: the incorporation of labeled 2H5-phenylalanine into muscle protein, which gives a quantitative measure of the fractional synthesis rate of muscle proteins, and the concentration and size distribution of ribosomes, which gives a qualitative estimate of protein synthesis. Furthermore, free amino acid concentrations were determined in muscle and plasma. RESULTS: The rate of PS, expressed as the fractional synthesis rate, decreased by 13% during HD (p < 0.02). The capacity for PS, as reflected by the total concentration of ribosomes, was reduced by 22% (p < 0.02) and the activity of PS, expressed as the relative proportion of polyribosomes, decreased from 48.4 +/- 0.9% to 44.8 +/- 0.8% after dialysis (p < 0.01). There was a total loss of 5.8 +/- 0.3 g amino acid to the dialysate. Plasma and muscle free amino acid concentrations were determined at four time points; before and after the phenylalanine incorporation period, before dialysis and before and after the second incorporation period after dialysis. Immediately after dialysis, there was a decrease in plasma asparagine, histidine, alanine, taurine, valine and tryptophane. In muscle, no changes occurred except for a slight increase in leucine after dialysis. In blood, the glucose concentration decreased and the total amount of glucose lost to the dialysate was 21 +/- 3.0 g. In summary, one single hemodialysis treatment decreases fractional protein synthesis rate in skeletal muscle. CONCLUSION: The results demonstrate substantial losses of amino acids and glucose to the dialysate and decreased amino acid concentrations in plasma, but only minimal changes in the intracellular amino acid concentrations in muscle, suggesting that the decreased PS is caused not by lack of amino acid precursors at the site of the synthesis activity, but by other mechanisms.

Response of mosquitoes to carbon dioxide and 1-octen-3-ol in southeast Queensland, Australia.

Encephalitis vector surveillance (EVS) traps were used to study the attractant effect of CO2 and 1-octen-3-ol (octenol) on mosquitoes at 2 different locations in southeast Queensland. Octenol alone was only slightly attractive for Aedes vigilax. There was a significant increase in the numbers of Ae. vigilax and Aedes funereus caught when octenol was added to CO2, although catches of Culex annulirostris and Culex sitiens did not change significantly. The size and age compositions of Ae. vigilax attracted by CO2 and by octenol were comparable. These data suggest that octenol should be considered as a supplement to CO2-baited EVS traps for mosquito-based arbovirus surveillance in southeast Queensland.

Autophagy activity is up-regulated in adipose tissue of obese individuals and modulates proinflammatory cytokine expression.

Autophagy, an evolutionary conserved process aimed at recycling damaged organelles and protein aggregates in the cell, also modulates proinflammatory cytokine production in peripheral blood mononuclear cells. Because adipose tissue inflammation accompanied by elevated levels of proinflammatory cytokines is characteristic for the development of obesity, we hypothesized that modulation of autophagy alters adipose tissue inflammatory gene expression and secretion. We tested our hypothesis using ex vivo and in vivo studies of human and mouse adipose tissue. Levels of the autophagy marker LC3 were elevated in sc adipose tissue of obese vs. lean human subjects and positively correlated to both systemic insulin resistance and morphological characteristics of adipose tissue inflammation. Similarly, autophagic activity levels were increased in adipose tissue of obese and insulin resistant animals as compared with lean mice. Inhibition of autophagy by 3-methylalanine in human and mouse adipose tissue explants led to a significant increase in IL-1ß, IL-6, and IL-8 mRNA expression and protein secretion. Noticeably, the enhancement in IL-1ß, IL-6, and keratinocyte-derived chemoattractant (KC) by inhibition of autophagy was more robust in the presence of obesity. Similar results were obtained by blocking autophagy using small interfering RNA targeted to ATG7 in human Simpson-Golabi-Behmel syndrome adipocytes. Our results demonstrate that autophagy activity is up-regulated in the adipose tissue of obese individuals and inhibition of autophagy enhances proinflammatory gene expression both in adipocytes and adipose tissue explants. Autophagy may function to dampen inflammatory gene expression and thereby limit excessive inflammation in adipose tissue during obesity.