Transgenic mice containing a human heavy chain immunoglobulin gene fragment cloned in a yeast artificial chromosome.

We have developed a method for the introduction of yeast artificial chromosomes (YACs) into transgenic mice. An 85 kilobase (kb) fragment of the human heavy chain immunoglobulin gene was cloned as a YAC, and embryonic stem cell lines carrying intact, integrated YACs were derived by co-lipofection of the YAC with an unlinked selectable marker. Chimaeric founder animals were produced by blastocyst injection, and offspring transgenic for the YAC were obtained. Analysis of serum from these offspring for human heavy chain antibody subunits demonstrated expression of the YAC-borne immunoglobulin gene fragment. Co-lipofection may prove to be a highly-successful means of producing transgenic mice containing large gene fragments in YACs.

Human GM-CSF: molecular cloning of the complementary DNA and purification of the natural and recombinant proteins.

Clones of complementary DNA encoding the human lymphokine known as granulocyte-macrophage colony-stimulating factor (GM-CSF) were isolated by means of a mammalian cell (monkey COS cell) expression screening system. One of these clones was used to produce recombinant GM-CSF in mammalian cells. The recombinant hematopoietin was similar to the natural product that was purified to apparent homogeneity from medium conditioned by a human T-cell line. The human T-cell GM-CSF was found to be 60 percent homologous with the GM-CSF recently cloned from murine lung messenger RNA.

Impact of tranexamic acid use on blood loss and transfusion rates following femoral varus derotational osteotomy in children with cerebral palsy.

PURPOSE: Previous studies have established the safety and efficacy of tranexamic acid (TXA) in reducing blood loss after total joint arthroplasty and spinal fusion surgery; however, literature regarding the effectiveness of intraoperative TXA in children with cerebral palsy (CP) is limited. The aim of this study was to investigate the safety and efficacy of intraoperative TXA in reducing blood loss and transfusion requirements for children with CP undergoing a proximal femoral varus derotational osteotomy (VDRO). METHODS: This is a retrospective review of 258 children with CP who underwent VDRO performed at the author's institution between 2004 and 2017. In all, 36 subjects underwent VDRO surgery with administration of intravenous TXA and 222 subjects underwent VDRO without administration of TXA. Outcome measures including blood loss, transfusion requirements and venous thromboembolic events were compared between groups using t-tests and chi-squared tests. RESULTS: No significant differences were seen in the rates of transfusion between groups for the entire hospitalization (TXA group: 11.1% versus No TXA group: 19.8%), intraoperatively (TXA: 2.8% versus No TXA: 9.0%) or postoperatively (TXA: 8.3% versus No TXA: 14.4%). Intraoperative estimated blood loss (TXA: 144.4 mL versus No TXA: 159.0 mL) and percentage blood loss (TXA: 8.9% versus No TXA: 9.2%) were similar between groups. No major thromboembolic complications events occurred in either group. CONCLUSION: The use of TXA was not associated with thromboembolic complications in this series of children with CP undergoing VDRO surgery. Though there was a trend toward lower rates of intraoperative and postoperative blood transfusion with TXA use in these patients, the differences were not significant, possibly due to low estimated blood loss in both groups and sample size. LEVEL OF EVIDENCE: III- retrospective comparative study.

Synovial fluid cell counts and its role in the diagnosis of paediatric septic arthritis.

PURPOSE: Clinical presentation of paediatric septic arthritis (SA) can be similar to other joint pathologies. Despite potential for infection in all major joints, most diagnostic criteria are based on values from the hip. This study identifies the best joint aspirate values in diagnosing SA in all joints. METHODS: In all, 166 patients who underwent 172 joint aspirations at the authors' institution between 01 September 2004 and 01 September 2014 were retrospectively identified. Recorded measures included age, sex, duration of symptoms, fever history, weight-bearing status, aspiration results, serum results and antibiotic administration. Patients were placed in the following four categories: 'culture confirmed SA' (C-SA), 'suspected SA' (S-SA), 'Other' and 'Other-rheumatologic' (Other-R), a subcategory of 'Other'. RESULTS: Most common sites of aspiration were the knee (55%) and hip (29%). Diagnostic grouping was as follows: C-SA = 44, S-SA = 45, Other = 83 (Other-R = 21). Fever and non-weight-bearing prior to admission were useful predictors of SA, though in C-SA patients, 21% did not have a fever and 23% could weight bear at the time of admission. Aspirate white blood cell (WBC) count was significantly greater in both C-SA (92 000 cells/hpf) and S-SA (54 000) than in Other (10 000) and Other-R (18 000) patients. The percentage of polymorphonuclear (%PMN) was also significantly greater in C-SA (81.1%) and S-SA (80.9%) than in Other (57.9%) and Other-R (63.3%). CONCLUSION: Joint aspirate values, especially %PMN, are valuable in diagnosing SA. Additionally, antibiotics pre-aspiration did not affect %PMN, facilitating subsequent diagnosis of infection. Lastly, while aspirate WBC count was a valuable indicator of SA, this finding is not as definitive as previous research suggests. LEVEL OF EVIDENCE: IV Case Series.

Coamplification and coexpression of human tissue-type plasminogen activator and murine dihydrofolate reductase sequences in Chinese hamster ovary cells.

Expression of human tissue-type plasminogen activator (t-PA) at high levels has been achieved in Chinese hamster ovary (CHO) cells by cotransfection and subsequent coamplification of the transfected sequences. Expression vectors containing the t-PA cDNA gene and dihydrofolate reductase (DHFR) cDNA gene were cotransfected into CHO DHFR-deficient cells. Transformants expressing DHFR were selected by growth in media lacking nucleosides and contained low numbers of t-PA genes and DHFR genes. Stepwise selection of the DHFR+ transformants in increasing concentrations of methotrexate generated cells which had amplified both DHFR genes and t-PA genes over 100-fold. These cell lines expressed elevated levels of enzymatically active t-PA. To optimize both t-PA sequence amplification and t-PA expression, various modifications of the original procedure were used. These included alterations to the DHFR expression vector, optimization of the molar ratio of t-PA to DHFR sequences in the cotransfection, and modification of the methotrexate resistance selection procedure. The structure of the amplified DNA, its chromosomal location, and its stability during growth in the absence of methotrexate are reported.

High-avidity human IgG kappa monoclonal antibodies from a novel strain of minilocus transgenic mice.

Human immunoglobulin transgenic mice provide a method of obtaining human monoclonal antibodies (Mabs) using conventional hybridoma technology. We describe a novel strain of human immunoglobulin transgenic mice and the use of this strain to generate multiple high-avidity human sequence IgG kappa Mabs directed against a human antigen. The light chain transgene is derived in part from a yeast artificial chromosome clone that includes nearly half of the germline human V kappa region. In addition, the heavy-chain transgene encodes both human mu and human gamma 1 constant regions, the latter of which is expressed via intratransgene class switching. We have used these animals to isolate human IgG kappa Mabs that are specific for the human T-cell marker CD4, have high binding avidities, and are immunosuppressive in vitro. The human Mab-secreting hybridomas display properties similar to those of wild-type mice including stability, growth, and secretion levels. Mabs with four distinct specificities were derived from a single transgenic mouse, consistent with an extensive diversity in the primary repertoire encoded by the transgenes.

Complete division of the radial nerve associated with a closed fracture of the humeral shaft in a child.

The management of closed fractures of the humerus with an associated nerve palsy remains controversial. With very little written about this injury in children, we present the case of a three-year-old child with a closed humeral shaft fracture in whom surgical exploration and reconstruction of the radial nerve with a sural nerve graft was performed three months after injury. The child regained full function. To the best of our knowledge, this is the first such case to be reported in the English literature.

Reliability of patellar height indices in children with cerebral palsy and spina bifida.

BACKGROUND: The Koshino (KI) and Caton-Deschamps (CDI) indices are used to measure patellar height in children, with the CDI showing excellent reliability in typically developing (TD) children. Reliability of such measures in children with cerebral palsy (CP) and spina bifida (SB) is unknown. METHODS: Lateral knee radiographs were reviewed retrospectively for children with TD (n = 49), CP (n = 48) and SB (n = 42). Five raters took measurements from radiographs twice, at least two weeks apart. Measurements included the CDI, Insall-Salvati Index (ISI) and KI. Systematic variability (bias) and random variability were examined using repeated measures ANOVA, 95% limits of agreement (LOA) and coefficients of variation (CV). RESULTS: Mean values of all three indices differed among raters (p < 0.0001). A significant difference was seen between the first and second measurements for CDI and KI indicating a learning effect. LOA ranges were large for the CDI (intra-rater: 0.37-0.95, inter-rater: 0.60-1.04) and ISI (intra-rater: 0.25-0.49, inter-rater: 0.51-0.57) for all patient groups. The KI showed a clinically acceptable range for TD participants (intra-rater: 0.14-0.16, inter-rater: 0.11-0.14) with larger ranges for CP (intra-rater: 0.26-0.33, inter-rater 0.0.2-0.35) and SB patients (intra-rater: 0.23-0.27, inter-rater: 0.19-0.25). CVs were lowest (best) for KI (3.8% to 7.4%) and highest (worst) for CDI (14.7% to 23.1%) for all three groups. Results were similar for patients with both open and closed physes. CONCLUSIONS: The KI is the most reliable patellar height measure for paediatric patients with TD, CP and SB, with either open or closed physes. The KI is more complex and experience may be important for valid, reliable measurement.

Treatment of multidirectionally unstable supracondylar humeral fractures in children. A modified Gartland type-IV fracture.

BACKGROUND: There is an uncommon subset of supracondylar humeral fractures in children that are so unstable they can displace into both flexion and extension. The purposes of this study were to describe this subset of supracondylar fractures and to report a new technique of closed reduction and percutaneous pinning for their treatment. METHODS: In a retrospective review of 297 consecutive displaced supracondylar humeral fractures in children treated operatively at our institution, we identified nine that were completely unstable with documented displacement into both flexion and extension as seen on fluoroscopic examination with the patient under anesthesia. We used a new technique for closed reduction and fixation of these fractures, and then we assessed fracture-healing and complications from the injury and treatment. RESULTS: All nine fractures were treated satisfactorily with closed reduction and percutaneous pinning. The complication rate associated with these unstable fractures was no higher than that associated with the 288 more stable fractures. Seven of the nine fractures were stabilized with lateral entry pin placement, and two fractures were stabilized with crossed medial and lateral pins. None of the patients had a nonunion, cubitus varus, malunion, additional surgery, or loss of motion. CONCLUSIONS: In rare supracondylar fractures in children, multidirectional instability results in displacement into flexion and/or extension. This fracture can be classified as type IV according to the Gartland system, as it is less stable than a Gartland type-III extension supracondylar fracture. These fractures can be treated successfully with a new technique of closed reduction and percutaneous pinning, thus avoiding open reduction.

Effects of diet on the fecal excretion and bacterial modification of acidic and neutral steroids, and implications for colon carcinogenesis.

Diet-induced changes in the excretion and fecal concentration of bile acids have been associated with enhancement or reduction of chemically initiated colon carcinogenesis in experimental animals. Dietary lipid increases the excretion and fecal concentration of bile acids. Certain types of dietary fiber such as pectin and lignin also increase fecal sterol output. However, the predominant effect of food-derived fiber (e.g., wheat brain) is to increase stool bulk and, hence, reduce the concentration of fecal sterols. Although it has been suggested that the tumor-promoting activity of bile acids is enhanced following bacterial dehydroxylation, dietary factors appear to have limited effect on bacterial flora or acidic sterol degradation in the colon.

An hypothesis for the action of dietary fiber along the gastrointestinal tract.

Dietary fiber consists of a sponge matrix with specific physicochemical properties. These properties are dependent on the structure and composition of the fiber components and determine the physiological effects of different food fibers in the upper and lower gastrointestinal tract.

Effect of citrus pectin on blood lipids and fecal steroid excretion in man.

Citrus pectin (15 g/day) was added for 3 weeks to metabolically controlled diets in nine subjects. Pectin was consumed with fruit and sugar as a gel in divided doses with meals. Plasma cholesterol concentrations were reduced by a mean of 13% (P less than 0.001). Fecal fat excretion increased by 44% (P less than 0.001); neutral steroids by 17% (P less than 0.001) and fecal bile acids by 33% (P less than 0.02). Plasma triglyceride levels did not change.

Multivariate analysis of diet and serum lipids in normal men.

The relationships among intake of dietary fiber, fat, and serum lipids were examined in a population of 200 normal men. Univariate analyses indicated that men with lower serum cholesterol and triglyceride concentrations were consuming significantly more dietary fiber and proportionately fewer fat calories. However, higher fiber consumption was also associated with differences in relative body weight, fat intake, and cigarette smoking; the relationship of dietary fiber intake to serum lipids was largely mediated by those coexisting differences in other environmental variables. In contrast, the percentage of total calories consumed as fat was independently positively related to both serum cholesterol and triglyceride levels.

Oat-bran intake selectively lowers serum low-density lipoprotein cholesterol concentrations of hypercholesterolemic men.

To evaluate selected metabolic effects of plant fibers, we fed control and oat-bran diets in an alternating sequence to eight men with previously documented hypercholesterolemia. The two solid diets differed only in the inclusion of 100 g of oat bran in the test diet. We randomized diet sequences and the measured intakes of carbohydrate, protein, fat, and cholesterol were virtually identical on the two diets. Serum total cholesterol concentrations were stable on control diets whereas a progressive reduction was observed in seven men on oat-bran diets. On oat-bran diets, average reductions in serum total cholesterol concentrations were 13% (p less than 0.01, N = 8); plasma low-density lipoprotein cholesterol concentrations were 14% lower (p less than 0.05) while high-density lipoprotein cholesterol concentrations were not changed. Fasting and postprandial serum glucose, insulin, and triglyceride concentrations were similar on the two diets. Fecal excretion of total bile acids was 54% higher (p less than 0.001) on oat-bran diets than on control diets but neutral steroid excretion was slightly lower while on oat bran. Palatable and inexpensive high-fiber foods such as oat bran may have a role in the treatment of certain patients with hypercholesterolemia.

Sustained alterations in lipoprotein cholesterol concentrations dependent on the daily distribution of lipid intake.

Since the fat content of a single meal influences chylomicron size and hence intestinal apoprotein synthesis, we determined the chronic effects of the daily distribution of fat intake on plasma concentrations of total cholesterol (TC), high density lipoprotein cholesterol (HDL-C) and low density lipoprotein cholesterol (LDL-C). Eight normal male subjects ingested 100 g of fat (a) as a bolus at the evening meal (SL) or (b) equally distributed over 4 meals (q4h) (DL). Each diet was consumed for 7 days; studies were performed 14 days apart using a crossover design and paired comparisons. Nutrient intake and body weight were held constant. At the end of the DL dietary regimen, fasting plasma concentrations of TC, LDL-C and HDL-C were significantly increased as compared to the SL phase of study (TC: 174 +/- 2.9 (mean +/- SEM) vs 161 +/- 2.7; LDL-C: 108 +/- 3.2 vs 98 +/- 3.3 and HDL-C: 53 +/- 1.1 vs 48 +/- 0.8) (P less than 0.05). The consumption of 100 g/day of fat in several small meals results in a sustained increase in LDL-C and HDL-C. This may be due to increased synthesis of lipoprotein components (e.g. apoprotein A-I) or to altered metabolism of intestinal and hepatic TG-rich lipoproteins dependent on size, number and apoprotein composition.

Acute effects of the pattern of fat ingestion on plasma high density lipoprotein components in man.

Since Apoprotein A-I (apo A-I) is partially synthesized in the intestine and is secreted into plasma via chylomicrons, we have tested the effects of the daily distribution of fat intake on the concentration and composition of plasma high density lipoprotein (HDL). Ten normal subjects ingested 136 g fat either as a single load (SL) at 0 h or in 6 divided doses from 0 to 10 h (DL). Serial blood samples were obtained over a 24-h period. Studies were performed 7 days apart using a double crossover design and paired comparisons. HDL apo A-I increased during DL (+11% at 9 h, P < 0.01) but was not significantly altered after SL. The HDL Apo A-II concentration did not change. HDL cholesterol decreased significantly (-4 to -7%) during postprandial lipaemia in both phases of the study. Apo A-I and A-II were detected in lipoproteins of d < 1.006 during alimentary lipaemia but not in fasting plasma. These results indicate that HDL apo A-I increases when fat intake is distributed throughout the day (DL), perhaps due to intestinal production of particles with a higher protein/lipid ratio during DL than after a large bolus of oral lipid (SL). Alimentary lipaemia is associated with acute alterations in HDL components including a transient reduction in HDL cholesterol and an increase in the apo A-I/A-II ratio during DL.

Relationship between changes in plasma lipoprotein concentrations and fecal steroid excretion in man during consumption of four experimental diets.

Limited information is available on the mechanism by which changes in nutrient intake influence plasma lipids. We compared the effects on plasma lipoprotein levels of 3 dietary modifications involving changes in total fat intake (27-40% of calories), cholesterol intake (100-250 mg/1000 kcal), the dietary polyunsaturated to saturated fatty acid ratio (0.3-1.0) and intake of vegetable-derived fiber and protein. On these 3 diets, plasma low density lipoprotein was reduced by 26-34%. Fecal bile acid excretion was similar on all diets (363-379 mg/day). There was no alteration in fecal bile acid output associated with an increase in polyunsaturated or total fat intake. Sterol balance became significantly more negative during consumption of only 1 of the 3 cholesterol-lowering diets. The observed reduction in plasma cholesterol levels was not associated with an increase in fecal bile acid output suggesting that diet-induced changes in circulating cholesterol are not maintained by an increase in sterol turnover but may reflect alterations in hepatic cholesterol and lipoprotein synthesis.

Operative treatment of supracondylar fractures of the humerus in children. The consequences of pin placement.

BACKGROUND: The commonly accepted treatment of displaced supracondylar fractures of the humerus in children is fracture reduction and percutaneous pin fixation; however, there is controversy about the optimal placement of the pins. A crossed-pin configuration is believed to be mechanically more stable than lateral pins alone; however, the ulnar nerve can be injured with the use of a medial pin. It has not been proved that the added stability of a medial pin is clinically necessary since, in young children, pin fixation is always augmented with immobilization in a splint or cast. METHODS: We retrospectively reviewed the results of reduction and Kirschner wire fixation of 345 extension-type supracondylar fractures in children. Maintenance of fracture reduction and evidence of ulnar nerve injury were evaluated in relation to pin configuration and fracture pattern. Of 141 children who had a Gartland type-2 fracture (a partially intact posterior cortex), seventy-four were treated with lateral pins only and sixty-seven were treated with crossed pins. Of 204 children who had a Gartland type-3 (unstable) fracture, fifty-one were treated with lateral pins only and 153 were treated with crossed pins. RESULTS: There was no difference with regard to maintenance of fracture reduction, as seen on anteroposterior and lateral radiographs, between the crossed pins and the lateral pins. The configuration of the pins did not affect the maintenance of reduction of either the Gartland type-2 fractures or the Gartland type-3 fractures. Ulnar nerve injury was not seen in the 125 patients in whom only lateral pins were used. The use of a medial pin was associated with ulnar nerve injury in 4% (six) of 149 patients in whom the pin was applied without hyperflexion of the elbow and in 15% (eleven) of seventy-one in whom the medial pin was applied with the elbow hyperflexed. Two years after the pinning, one of the seventeen children with ulnar nerve injury had persistent motor weakness and a sensory deficit. CONCLUSIONS: Fixation with only lateral pins is safe and effective for both Gartland type-2 and Gartland type-3 (unstable) supracondylar fractures of the humerus in children. The use of only lateral pins prevents iatrogenic injury to the ulnar nerve. On the basis of our findings, we do not recommend the routine use of crossed pins in the treatment of supracondylar fractures of the humerus in children. If a medial pin is used, the elbow should not be hyperflexed during its insertion.

Complications of posterior iliac crest bone grafting in spine surgery in children.

STUDY DESIGN: The perioperative and postoperative complications associated with harvesting posterior iliac crest bone graft in children were reviewed. A retrospective study was performed and a questionnaire interview conducted. OBJECTIVES: To determine the morbidity associated with posterior iliac crest bone graft in children. SUMMARY OF BACKGROUND DATA: Iliac crest bone is commonly used as a source of bone graft in spine surgery. Although there are multiple reports of complications in adults, there are no reports in children. METHODS: A retrospective chart review was performed of 214 consecutive children who underwent spinal fusion with posterior iliac crest bone graft from 1990 through 1996. An interview was conducted of 87 patients with normal mental status, predominantly those with idiopathic scoliosis with a minimum of 2 years' follow-up (mean, 55 months). RESULTS: The review showed one (0.5%) instance of arterial injury in the sciatic notch. Two (1%) patients had infections, both of which resolved with a single irrigation and débridement. There was one documented instance of sacroiliac penetration that did not cause clinical problems. The chart review showed three (1.4%) instances of continued pain and one (0.5%) of numbness. By contrast to the few reports of pain in the chart review, responses to an interview of 87 patients showed 21 (24%) children reporting pain at the iliac crest site, with 13 (15%) reporting problems with daily activities. The self-reported pain, on a scale of 1 to 10, ranged from 1 to 10 with a mean of 4. Nonsteroidal anti-inflammatory drugs (NSAIDS) were taken by eight (9%) children for pain at the bone graft site. Five (6%) reported skin irritation, and 18 (20%) mentioned numbness surrounding the scar. CONCLUSION: The perioperative rate of complications in iliac crest bone grafting in children is low (2%). The complication of pain (24%) and pain that is severe enough to interfere with daily activity (15%) is significant at a mean follow-up of more than 4 years. The true extent of pain and numbness after posterior iliac crest bone grafting in children was severely underreported in the medical records and may be underrecognized.