False positive rate of thoracic outlet syndrome diagnostic maneuvers.

OBJECTIVE: In this study, we aim to determine the false-positive rate & specificity in normal subjects and carpal tunnel syndrome (CTS) patients of five provocative maneuvers used to diagnose thoracic outlet syndrome (TOS). DESIGN/METHODS: We prospectively evaluated subjects with clinical and electrophysiological evidence of CTS as well as normal subjects. All subjects underwent provocative testing by a blinded physician, which included the Adson A & B tests, Costoclavicular maneuver (CCM), Elevated arm stress test (Roos), and Supraclavicular pressure (SCP). RESULTS: In the CTS group, false positive tests were observed in 42% in the Adson A test, 45% in the Adson B test, 48% in the CCM, 77% in the Roos test, and 61% in the SCP 94% of the CTS patients had at least 1 positive TOS diagnostic maneuver. In the normal group, false positive tests were observed in 9% in the Adson A test, 20% in the Adson B test, 16% in the CCM, 47% in the Roos test, and 30% in the SCP 56% of the normal patients had at least 1 positive TOS diagnostic maneuver. CONCLUSIONS: We conclude that current provocative maneuvers used to diagnose TOS result in a high false-positive rate in normal subjects and an even higher false-positive rate in CTS patients.

Binding proteins selected from combinatorial libraries of an alpha-helical bacterial receptor domain.

Small protein domains, capable of specific binding to different target proteins have been selected using combinatorial approaches. These binding proteins, called affibodies, were designed by randomization of 13 solvent-accessible surface residues of a stable alpha-helical bacterial receptor domain Z, derived from staphylococcal protein A. Repertoires of mutant Z domain genes were assembled and inserted into a phagemid vector adapted for monovalent phage display. Two libraries, each comprising approximately 4 x 10(7) transformants, were constructed using either an NN(G/T) or an alternative (C/A/G)NN degeneracy. Biopanning against the target proteins Taq DNA polymerase, human insulin, and a human apolipoprotein A-1 variant, showed that in all cases significant enrichments were obtained by the selection procedures. Selected clones were subsequently expressed in Escherichia coli and analyzed by SDS-PAGE, circular dichroism spectroscopy, and binding studies to their respective targets by biospecific interaction analysis. The affibodies have a secondary structure similar to the native Z domain and have micromolar dissociation constants (KD) for their respective targets.

Recurrent abdominal pain and lactose absorption in children.

The association of lactase deficiency with recurrent abdominal pain was investigated. One hundred three white children between the ages of 6 to 14 years with recurrent abdominal pain were evaluated. Sixty-nine underwent lactose tolerance tests and 26 had intestinal biopsies with lactase determinations; 21 of 69 (30.4%) had abnormal lactose tolerance tests and eight of 26 (31%) were lactase deficient. However, 16 of 61 (26.4%) control subjects matched for age and ethnic background exhibited lactase deficiency. Thus, a similar prevalence of lactase deficiency was found in the control and the recurrent abdominal pain groups. Thirty-eight patients with recurrent abdominal pain completed three successive six-week diet trials conducted in a double-blind fashion. An increase above base line value in pain frequency was seen in ten of 21 (48%) lactose malabsorbers and four of 17 (24%) lactose absorbers. After a 12-month milk elimination diet, six of 15 (40%) malabsorbers and five of 13 (38%) absorbers had elimination of their pain. This result compared with improvement occurring in five of 12 (42%) absorbers with recurrent abdominal pain who received a regular diet for one year and suggests that the elimination of lactose will not affect the overall frequency of improvement in recurrent abdominal pain. In addition, the recovery rate from recurrent abdominal pain is similar in both lactose absorbers and nonabsorbers independent of dietary restrictions.

Extent and duration of small intestinal mucosal injury in intractable diarrhea of infancy.

Thirty infants with intractable diarrhea of infancy (IDI) underwent small bowel biopsies in order to determine the extent and duration of small intestinal mucosal injury. The onset of the persistent diarrhea occurred prior to 3 months of age and continued for an average of 48 days prior to investigation. In 18 cases, no associated entities were found. Mucosal injury was invariably found in all 30 infants: grade IV injury in 11, grade III in eight, grade II in nine, and grade I atrophy in one. Disaccharidase activities were diminished and corresponded to the degree of atrophy. Lactase activity was diminished to a greater extent than sucrase and maltase. Significant, persistent mucosal injury existed for an average of six months in 16 of the 23 (70%) repeat biopsies. All infants were given an elemental diet (ED). Twelve of the 30 infants required parenteral nutrition (PN). These infants were gradually advanced to an oral elemental diet and maintained on this diet until histologic findings and disaccharidase levels were normal. Eighteen infants were fed and maintained on an elemental diet by mouth from time of admission until normal histologic findings and disaccharidases were found. No mortality occurred during management and follow-up. Twenty-two of the 28 infants in whom follow-up growth data were available excelled in weight and height velocity. The data suggest that prolonged injury to the small intestinal mucosa is a common finding in many cases of intractable diarrhea of infancy. Elemental diets should be started early in the course of protracted diarrhea in young infants, and may need to be continued for several months since histologic and enzymatic changes of the small intestine may persist for extended periods.

Ligands selected from combinatorial libraries of protein A for use in affinity capture of apolipoprotein A-1M and taq DNA polymerase.

Here we show that robust and small protein ligands can be used for affinity capture of recombinant proteins from crude cell lysates. Two ligands selectively binding to bacterial Taq DNA polymerase and human apolipoprotein A-1(M), respectively, were used in the study. The ligands were selected from libraries of a randomized alpha-helical bacterial receptor domain derived from staphylococcal protein A and have dissociation constants in the micromolar range, which is typical after primary selection from these libraries consisting of approximately 40 million different members each. Using these ligands in affinity chromatography, both target proteins were efficiently recovered from crude cell lysates with high selectivities. No loss of column capacity or selectivity was observed for repeated cycles of sample loading, washing and low pH elution. Interestingly, column sanitation could be performed using 0. 5 M sodium hydroxide without significant loss of ligand performance. The results suggest that combinatorial approaches using robust protein domains as scaffolds can be a general tool in the process of designing purification strategies for biomolecules.

Evaluation of treatment modalities for septic arthritis with histological grading and analysis of levels of uronic acid, neutral protease, and interleukin-1.

We compared the effectiveness of antibiotics alone and in combination with arthroscopy, arthroscopy with debridement, arthrotomy, or needle aspiration for the treatment of septic arthritis. Each modality has its proponents, but, to our knowledge, no comparative studies have been conducted in animals. We used biochemical and histological analysis to compare these methods of treatment in an experimental model. The right hind knee of thirty goats was injected with 1 x 10(5) Staphylococcus aureus bacilli. The left hind knee was not inoculated and served as the normal control. Seventy-two hours after inoculation, a two-week course of treatment with intramuscular administration of cefuroxime sodium, either alone or in combination with another mode of treatment, was initiated in each of five groups. The cartilage was evaluated histologically with biochemical, enzymatic, and interleukin-1 analyses. Despite the early therapeutic intervention, on the average, there was a 25 per cent loss of uronic acid (t test, p < 0.001) and a 43 per cent increase in neutral protease activity (signed-rank test, p = 0.003) in the treatment groups. There were no significant intergroup differences with regard to the histochemical-histological rating or the levels of uronic acid, neutral protease, or interleukin-1.

Peptic ulcer disease in the pediatric population.

Peptic ulcer disease occurs in infants, children, and adolescents. Primary and stress ulcers pose a challenge to the pediatrician, who needs to arrange for appropriate diagnostic and therapeutic services. Highlights of our understanding of the pathophysiology, genetics, natural history, diagnosis, and therapy of peptic ulcer disease are presented.

Faecal microflora and urease activity during the first six months of infancy.

Gastrointestinal degradation of urea might, according to a new hypothesis, have consequences for the regulation of acid-base balance as well as control of breathing during infancy. Thirteen infants were investigated from their first few days of life to the age of 6 months by collecting faecal samples at the age of 3 days, 2, 3, and 6 months, respectively. The faecal microflora was determined after aerobic and anaerobic cultivation and the faecal urease activity was assessed after 36 h aerobic and anaerobic preincubation. The infants were mostly breast fed and had a faecal microflora containing anaerobic bacteria such as Bifidobacteria, Bacterioides and Lactobacilli but also aerobics such as Escherichia coli, Enterococci and sometimes Klebsiella. The faecal pH increased from approximately 5.30 to 5.90, the pH after anaerobic preincubation being on an average 0.2 pH units lower than after aerobic preincubation. Simultaneously the nitric oxide production of the faecal specimens increased approximately 10-fold and the urease activity decreased by a factor of 3 to 5. We also found an inhibitory action of nitrate, nitrite (in mumolar concentration) and nitric oxide (in parts per million concentration) on the faecal urease activity. Hence, the present results warrant further research in order to determine more precisely the action of different concentrations of various nitrous oxides on individual bacterial species, and furthermore, to assay the faecal urease activity in victims of sudden infant death syndrome as well as in infants dead due to other causes.

Photoreactivity of biologically active compounds, XIV: influence of oxygen on light induced reactions of primaquine.

The influence of molecular oxygen and oxygen radicals on the photoreactivity of the antimalarial drug primaquine (PQ) has been investigated. Oxygen is directly involved in photodecomposition of the drug. Flushing with helium gas prior to and during irradiation to suppress the oxygen level of the medium, retards the degradation rate of PQ (followed by HPLC) and leads to the formation of only two degradation products (identified by MS) compared to eight main- and several minor products under normal atmospheric conditions. Flushing with oxygen gas prior to and during irradiation to increase the oxygen content of the medium accelerates the degradation rate of PQ. PQ produces oxygen radicals (hydroxyl and superoxide) during photolysis, while the photoproducts of PQ seem likely to induce singlet oxygen formation (detected by addition of radical scavengers). Sensitization reactions involving singlet oxygen lead to decomposition of PQ (followed by HPLC). On the basis of our results, photochemical reaction mechanisms of PQ are postulated and discussed. At physiological conditions (aqueous, neutral pH, oxygen rich) PQ has a large potential to decompose after light absorption. The photoreaction seems to be initiated at the quinoline nitrogen. The ability to form an intramolecular hydrogen bond seems to be essential for the luminescence properties of the drug. Phosphorescence lifetime of PQ is about 5 microseconds. Fast chemical reactions may occur from the short-lived triplet state of the drug, but the excited compound can diffuse only a limited distance prior to deexcitation. This can be important concerning light-induced adverse effects which may appear after medication with PQ.

Control of caprine arthritis-encephalitis virus and Corynebacterium pseudotuberculosis infection in a Norwegian goat herd.

A control programme for caprine arthritis-encephalitis virus (CAEV) and Corynebacterium pseudotuberculosis (C. pseudotuberculosis) infection was established in a Norwegian goat herd comprising approximately 100 milking goats. The herd seroprevalences of antibodies against CAEV and C. pseudotuberculosis were 97% and 94%, respectively. Kids were removed from the infected flock at birth, avoiding any contact between dam and kid. The kids were kept completely segregated from the seropositive flock and fed cow's colostrum and milk. A seronegative flock was established, based on the removed kids and their offspring. Goasts belonging to the seronegative flock were allowed to kid naturally and to mother their kids. The seropositive flock was slaughtered during the second year of the control programme. After washing and disinfection, housing systems and nearby outdoor premises were left empty for 3 months. Of 230 goats examined for antibodies against CAEV with ELISA regularly during 3 years of the control program, altogether 6 were found to be seropositive, while for 10 the result was indeterminate. All 16 animals were immediately culled. During the third year of the control programme, all goats were examined and proved negative for antibodies against C. pseudotuberculosis by a haemolysis inhibition test. Clinical examination revealed no signs of CAE or caseous lymphadenitis.

Effects of infection by caprine arthritis-encephalitis virus on milk production of goats.

The effects of caprine arthritis-encephalitis virus on lactational performance of goats were examined. The results of an ELISA for antibodies against caprine arthritis-encephalitis virus were compared with milk production records. Mean production of milk, protein, fat, and lactose and somatic cell counts were compared for seropositive and seronegative goats of similar ages. The results from 1799 lactating goats from 66 herds suggested that milk production was similar for 1-yr-old goats that tested seropositive and those that tested seronegative. For 900 of those goats for which data permitted comparison, milk fat and protein were also similar. A comparison of 331 goats showed that lactose contents did not differ between 1- and 2-yr-old goats, but somatic cell counts were higher in 2-yr-old seropositive goats.

Early infant feeding and micro-ecology of the gut.

Newborn infants are rapidly colonized by both aerobic and and anaerobic bacteria, initially with about 50% of each type. Several factors related both to the infant and its environment influence the composition of the intestinal microflora quantitatively as well as qualitatively. Major ecological disturbances are observed in newborn infants treated with antimicrobial agents. One way of minimizing the ecological disturbances, which may be seen in infants treated in neonatal intensive care units, is to provide them with fresh breast milk from their mothers and to use antimicrobial therapy only under strict clinical indications.

Peptic ulcer in children: the predominance of gastric ulcers.

Thirty-two children with ulcer disease were seen over a four-year period. Twenty-seven children had a primary ulcer and five had an ulcer associated with an acute or chronic illness (secondary ulcer). Antral ulcer was diagnosed most commonly, followed by duodenal ulcer and gastric body ulcer. The ratio of gastric ulcer to duodenal ulcer was 17:11. Diagnosis of ulcer was accomplished by endoscopy in 97% of the patients and by radiography in 70% of those studied. Radiologic accuracy was obtained in 89% with duodenal ulcer but in only 50% of those with gastric ulcer. Children with primary gastric ulcer presented with no evidence of chronicity and 12% had persistence or recurrence of ulcer during follow-up. Eighty-two percent of the children with primary duodenal ulcer presented with chronic symptoms consisting of abdominal pain, nausea, vomiting or recurrent bleeding and 45% had persistence or recurrence of ulcer during follow-up. Children with secondary ulcer all presented with acute symptoms and none had persistence or recurrence. Twenty children were treated prospectively with cimetidine and 11 were treated with antacids. Repeat endoscopy was employed in 16 as a measure of healing. All children with isolated antral ulcer did well clinically, regardless of mode of therapy and of those studied by re-endoscopy all showed complete or substantial healing at six to eight weeks. Treatment of a small group of children with primary duodenal ulcer using cimetidine was initially efficacious, although recurrence of ulcer was noted after cessation of treatment in four of six children given cimetidine. In addition, cimetidine appears to offer no advantage compared to antacids in the treatment of uncomplicated antral ulcer in children.

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling.

The possibility of increasing the affinity of a Taq DNA polymerase specific binding protein (affibody) was investigated by an alpha-helix shuffling strategy. The primary affibody was from a naive combinatorial library of the three-helix bundle Z domain derived from staphylococcal protein A. A hierarchical library was constructed through selective re-randomization of six amino acid positions in one of the two alpha-helices of the domain, making up the Taq DNA polymerase binding surface. After selections using monovalent phage display technology, second generation variants were identified having affinities (K(D)) for Taq DNA polymerase in the range of 30-50 nM as determined by biosensor technology. Analysis of binding data indicated that the increases in affinity were predominantly due to decreased dissociation rate kinetics. Interestingly, the affinities observed for the second generation Taq DNA polymerase specific affibodies are of similar strength as the affinity between the original protein A domain and the Fc domain of human immunoglobulin G. Further, the possibilities of increasing the apparent affinity through multimerization of affibodies was demonstrated for a dimeric version of one of the second generation affibodies, constructed by head-to-tail gene fusion. As compared with its monomeric counterpart, the binding to sensor chip immobilized Taq DNA polymerase was characterized by a threefold higher apparent affinity, due to slower off-rate kinetics. The results show that the binding specificity of the protein A domain can be re-directed to an entirely different target, without loss of binding strength.

Recombinant human factor VIII-specific affinity ligands selected from phage-displayed combinatorial libraries of protein A.

Factor VIII-specific affibodies were selected from phage displayed libraries constructed by combinatorial mutagenesis of an alpha helical bacterial receptor domain derived from staphylococcal protein A. Bead-immobilized recombinant human factor VIII (rVIII) (80 and 90 kDa chains) protein was used during competitive biopannings in the presence of free 80-kDa chain protein, resulting in the selection of several binders that showed dissociation constants (Kd) in the range 100-200 nM as determined by biosensor analyses. One variant (Z[rVIII:3], 90-kDa chain specific) was further characterized in small-scale affinity chromatography experiments, and showed efficient and selective recovery of biologically active rVIII from Chinese hamster ovary cell supernatant-derived feed stocks. The purity of the enriched rVIII was comparable with rVIII material purified by immunoaffinity chromatography using a 90-kDa chain-specific monoclonal antibody. Interestingly, epitope mapping showed that the monoclonal antibody and the affibody ligand competed for the same or at least overlapping epitopes on rVIII. In addition, the Z[rVIII:3] variant was produced by peptide synthesis with a C-terminal cysteine to enable directed coupling to solid supports. This 59-residue protein was analyzed by circular dichroism and showed a secondary structure content similar to that of the parental Z domain used as scaffold. In biosensor studies, the synthetic affibody was immobilized recruiting the C-terminal cysteine residue, and demonstrated to bind both recombinantly produced and plasma-derived factor VIII. From a secondary library, constructed by re-randomization of relevant positions identified after alignment of the first-generation variants, a panel of affinity-improved second-generation affibodies were selected of which one clone showed a dissociation constant (Kd) for rVIII of 5 nM. Several of these variants also showed higher apparent binding efficiencies towards rVIII when analyzed as immobilized ligands in biosensor experiments. Taken together, the results suggest that affibody ligands produced by bacterial or synthetic routes could be of interest as an alternative to monoclonal antibodies in purification processes or as diagnostic or monitoring tools.