Disulfide bond mapping and structural characterization of spruce budworm antifreeze protein

We have previously reported that bryostation 1 (Bryo 1) induces differentiation of chronic lymphocytic leukemia (CLL) in vitro to a hairy cell (HC) stage. This study tests the hypothesis that Bryo 1-differentiated CLL cells are more susceptible to 2-chlorodeoxyadenosine (2-CdA) than parent CLL cells. A recently established EBV-negative CLL line (WSU-CLL) from a patient resistant to chemotherapy including fludarabine was used to test this hypothesis. Both Bryo 1 (10-1000 nM) and 2-CdA (5.6-22.4 microM) exhibited a dose-dependent growth inhibitory effect on the WSU-CLL cell line. In vitro, the sequential exposure to Bryo 1 (100 nM for 72 h) followed by 2-CdA (11.2 microM) resulted in significantly higher rates of growth inhibition than either agent alone. Changes in immunophenotype, enzymes, lipids, proteins, and the DNA of WSU-CLL cells were studied before and after Bryo 1 treatment. Bryo 1 induced a positive tartrate-resistant acid phosphatase reaction and two important markers, CD11c and CD25, after 72 h of culture, confirming the differentiation of CLL to HC. The Fourier transformation infrared spectroscopic analysis showed that the amount of membrane lipids significantly increased in Bryo 1-treated cells compared to controls after 24 h, whereas the protein content, as well as the DNA content, decreased. This finding supports the change of CLL to HC. To evaluate the in vivo efficacy of Bryo 1 and 2-CdA, we used a xenograft model of CLL in WSU-CLL-bearing mice with severe combined immune deficiency. s.c. tumors were developed by injection of 10(7) WSU-CLL cells, and fragments were then transplanted into a new batch of severe combined immunodeficient mice. Bryo 1 and 2-CdA at the maximum tolerated doses (75 micrograms/kg i.p. and 30 mg/kg s.c., respectively) were administered to the mice at different combinations and schedules. The survival in days, the tumor growth inhibition ratio, the tumor growth delay, and the log10 kill of the mice treated with Bryo 1 followed by 2-CdA were significantly better than the control and other groups. We conclude that the sequential treatment with Bryo 1 followed by 2-CdA resulted in higher antitumor activity and improved animal survival.     

Predictive value of monitoring parameters in fetal surgery

BACKGROUND/PURPOSE: The choice of monitoring parameters in fetal surgery has thus far been based on feasibility rather than on predictability. Ideally, monitoring should be noninvasive, have a rapid response time and high sensitivity, and be applicable to open and endoscopic techniques. Herein, the authors studied the response of several parameters to standardized episodes of fetal ischemia and stress. METHODS: Eight time-dated fetal lambs (110 days, term, 145 days) were used. Under general anesthesia, a balloon occluder was placed around the umbilical cord. Pulse oximetry (POx + heart rate, HR), electrocardiography (ECG), direct oximetry (DOx), and blood pressure (BP) were recorded continuously. After stabilization, the umbilical cord was completely occluded for 5 seconds, then released. False-negative recordings were defined as failure of a parameter to respond to umbilical occlusion; false-positive episodes were defined as 10% change in value over < or = 10 seconds during stabilization (baseline) period. RESULTS: The fetuses were monitored for an aggregate of 358 minutes. Baseline DOx was 64%+/-5%, POx, 66%+/-16%; HR, 141+/-18 beats per minute (bpm); systolic BP (SBP), 51+/-3 torr; and diastolic BP (DBP), 38+/-2 torr. During umbilical occlusion (n=15), SBP increased to 56+/-3 torr and DBP to 43+/-2 torr at 0.5 seconds, then returned to baseline at 8.0 seconds. A decrease was seen in DOx (start at 3.5s, maximum delta 9.9+/-1.5% at 10.5 seconds) and POx (start at 4.2 seconds, maximum delta 7.3+/-2.4% at 20.5 seconds). Heart rate showed <10% decrease (start at 8.5 seconds, nadir 131+/-14 bpm at 19.5 seconds). No ECG changes were noted. Sensitivity was 100% for DOx, POx, and BP, but only 14% for HR; specificity was 97% for DOx and 88% for POx; positive predictive value was 58% for DOx and 37% for POx; negative predictive value was 100% for DOx and POx. CONCLUSIONS: Direct intravascular oximetry and blood pressure provide a prompt and reliable response to acute fetal stress, but are too invasive for routine use. Bradycardia is an insensitive and late sign of fetal distress. Pulse oximetry has a rapid response time (<5 seconds), high sensitivity, and negative predictive value. In addition, its application is noninvasive and has proven to be feasible in open and endoscopic fetal surgical procedures. It therefore appears to be the monitoring parameter of choice for fetal surgery.     

Management and after-care of extensor tendon injuries of the hand

The investigation and management of extensor tendons play a minor role compared to those of flexor tendons. The finger extension does not seem to be as important as the flexion. But the practical value of the hand is determined by both. Different managements are used depending on the level of extensor tendon injury. More distal located injuries require a longer restraint than those, which are located more proximal. On the one hand this depends on the blood supply of the extensor tendon, which is by far better in the lower arm and dorsal hand than peripheral. On the other hand the reason is the different amplitude of gliding of extensor tendons, which decreases from proximal to distal to lower than 1 mm. Therefore the tension on extensor tendon sutures increases from proximal to distal. The varies techniques of extensor tendon reconstruction will be described.     

Effects of hypesthesia on oral behaviors of the orthognathic surgery patient

PURPOSE: The purpose of this study was to compare orthognathic surgery patients with and without significant hypesthesia with respect to perceived problems with specific oral behaviors. PATIENTS AND METHODS: Data from 116 patients 6 months after bilateral sagittal split osteotomy (BSSO) and mandibular advancement were analyzed. Tactile sensation in the right and left mental nerve areas was determined using monofilaments and brush strokes (von Frey hairs). The right infraorbital region was used as a control. A difference of 450 mg of force between the control and test sites was considered significant hypesthesia. Patients rated their level of subjective problems with swallowing liquids or solids, smiling, spitting, kissing, speaking, eating, and drooling on a scale from 1 (none to mild) to 7 (extreme). A value of 5 or greater was considered significant impairment. RESULTS: Hypesthesia was shown in 23 patients (19.8%) with the monofilaments and in 29 patients (25.0%) using brush stroke direction. In each of these two groups, a significant correlation was observed between hypesthesia and difficulty in chewing and kissing. No correlation was observed between any of the remaining seven oral behaviors and hypesthesia. CONCLUSION: These findings suggest that only certain oral behaviors are affected by hypesthesia of the mental nerve.     

Energy substrates and amino acids provided during in vitro maturation of bovine oocytes alter acquisition of developmental competence

Clinicians commonly include an assessment of leg length inequality (LLI) as a component of a musculoskeletal examination. Little research is available, however, documenting reliability and validity of clinical methods for assessing LLI. The purpose of this study was to determine the reliability and validity of assessing functional LLI using a pelvic leveling device. Subjects were 19 women and 13 men between the ages of 18 and 55 who reported having a diagnosed or suspected LLI. Clinical determination of LLI was made by placing rigid lifts under the suspected shorter lower extremity until the leveling device indicated that the iliac crests were level. This measurement was made twice by one investigator and once by a second investigator. Standing radiographic measurements of LLI using rigid lifts were used to establish validity of the clinical method. Intraclass correlation coefficients [ICC(2,1)] and absolute difference values were computed to assess reliability and validity. The mean absolute difference between the two clinical measurements of LLI by the same investigator was 0.29 cm (+/- 0.52), with an ICC = 0.84. The mean absolute difference between clinical measurements of LLI by the two investigators was 0.49 cm (+/- 0.46), with an ICC = 0.77. The ICC and mean absolute difference reflecting agreement between radiographic measurements and clinical measurements of LLI was 0.64 and 0.58 cm (+/- 0.58), respectively, for one investigator and 0.76 and 0.55 cm (+/- 0.37), respectively, for the second investigator. The intratester reliability, intertester reliability, and validity assessments included instances in which paired observations disagreed regarding which lower extremity was the shorter lower extremity. Factors that may be associated with the unacceptable reliability and validity of the clinical assessment method include asymmetric positioning of the ilia, body composition of the patient, and design of the clinical instrument. The authors discuss clinical implications related to assessment of LLI.     

Enterogastric bile reflux during technetium-99m-sestamibi cardiac imaging

Twenty-six patients with squamous cell cancer of the cervix were treated with i.v. paclitaxel, 250 mg/m2 over 3 h every 21 days. They received steroid, H1 and H2 blocker premedications, and granulocyte-colony-stimulating factor (G-CSF) support (5 microgram/kg/day). No prior chemotherapy, except as a radiation sensitizer, was allowed. The median age was 50 (range, 36-81) years, and performance status Zubrod was 1 (range, 0-2). Eight (33%) patients had prior surgery, and 22 (92%) had prior radiation therapy. Twenty-four patients were evaluable for response; 2 were later found to be ineligible. Five patients had partial responses (21%; 95% confidence interval, 6-40%), and 14 (58%; 95% confidence interval, 35-78%) had stable disease. The median duration of response was 10 (range, 3-27+) weeks. The responses were within the radiation port (four responses) and outside of it (one response). The median interval from the start of irradiation to the start of paclitaxel in responding patients was 94 weeks, whereas in patients with stable disease it was 68 weeks, and in patients whose disease progressed it was 46 weeks. Eighty-eight percent of the 105 cycles of paclitaxel were administered at a dose of 250 mg/m2 or higher. Granulocytopenia was brief and noncumulative, with grades 3 and 4 experienced by 5 and 3 patients, respectively. G-CSF was used for a median of 7 (range, 2-14) days/cycle. Anemia was mild, with G3 noted in 3 patients, and thrombocytopenia was not significant. Infections and musculoskeletal pain were mild and infrequent. Sensory (14 patients G1 or G2 and 2 patients G3) and motor (4 patients G1 or G2 and 1 patient G3) neurotoxicity was noted. There was no significant cardiovascular toxicity. Paclitaxel is active in patients with squamous cell cancer of the cervix and is well tolerated at this dose schedule with G-CSF support.     

Effect of tissue-plasminogen activator on leukocyte-endothelial interactions at the microcirculatory level

In free tissue transfer and replantation surgery, there is a debate over whether any pharmacologic agents should be used to improve vessel patency and tissue survival. Because tissue-plasminogen activator (t-PA) is a highly effective and safe fibrinolytic, it may be useful in obtaining and maintaining vessel patency. The direct effects of t-PA on skeletal muscle hemodynamics and leukocyte activation at the microcirculatory level were investigated. Male Sprague-Dawley rats (n = 20) were divided into three experimental groups: control (n = 8), vehicle (n = 6), and t-PA (n = 6). Using the cremaster muscle flap model and intravital microscopy, red blood cell velocity, vessel diameter, capillary perfusion, endothelial edema index, and leukocyte-endothelial interactions (rolling, adhering, and transmigrating leukocytes) in postcapillary venules were measured. In the vehicle and t-PA groups, vehicle or t-PA was infused by means of a catheter inserted into the lower abdominal aorta for local infusion. Except for a significant reduction in the diameter of the first order arterioles from 117 microm to 82 microm (medians; p = 0.026), t-PA did not significantly affect red blood cell velocity, vessel diameter, or capillary perfusion compared with vehicle. However, leukocyte-endothelial interactions did differ significantly in postcapillary venules. Adhering leukocytes counted per visual field decreased from 4.67 in the vehicle group and 3.50 in the control group to 1.67 in the t-PA group (medians; p = 0.015 and p = 0.005, respectively); transmigrating leukocytes in the t-PA group decreased from 4.75 in the vehicle group and 3.50 in the control group to 1.67 in the t-PA group (medians; p = 0.002 and p = 0.043, respectively). t-PA treatment significantly decreased the number of both adhering and transmigrating leukocytes. These novel findings on leukocyte-endothelial interactions suggest that t-PA has anti-inflammatory effect.     

Immunological and molecular characterization of three variant subtype P1.14 strains of Neisseria meningitidis

Epidemic outbreaks of group B meningococcal disease exhibit a clonal nature consisting of a common serotype-subtype. Subtype-specific monoclonal antibodies (MAbs) directed toward two variable regions (VR1 and VR2) of the class 1 protein of Neisseria meningitidis are used in this classification scheme. A new MAb was developed to classify a nonsubtypeable (NST) strain of N. meningitidis, 7967. This MAb bound to both the NST strain and the prototype subtype P1. 14 strain, S3446, by dot blot analysis. However, a MAb produced to the prototype P1.14 strain did not bind to strain 7967. Sixteen additional strains were further identified as P1.14 with the prototype MAb; of these, 15 strains bound both MAbs. Differences in the characteristics of binding of both antibodies to the three apparently diverse P1.14 strains were studied further by using outer membrane complex proteins, immobilized peptides, and soluble peptides. Deduced amino acid analysis suggested that both MAbs bind to VR2 and that single amino acid changes within VR2 (KM, NM, or KK) might explain the differences in binding characteristics. These results demonstrated that minor variations which exist within subtype variable regions may be clearly identified only by a combination of molecular and immunologic testing. The impact of subtype variation will become more evident as subtype-specific vaccines are developed and tested for efficacy.     

Combined oral positive inotropic and beta-blocker therapy for treatment of refractory class IV heart failure

OBJECTIVES: We sought to assess the effects of combined oral positive inotropic and beta-blocker therapy in patients with severe heart failure. BACKGROUND: Patients with severe, class IV heart failure who receive standard medical therapy exhibit a 1-year mortality rate >50%. Moreover, such patients generally do not tolerate beta-blockade, a promising new therapy for chronic heart failure. Positive inotropes, including phosphodiesterase inhibitors, are associated with increased mortality when administered over the long term in these patients. The addition of a beta-blocker to positive inotropic therapy might attenuate this adverse effect, although long-term oral inotropic therapy might serve as a bridge to beta-blockade. METHODS: Thirty patients with severe heart failure (left ventricular ejection fraction [LVEF] 17.2+/-1.2%, cardiac index 1.6+/-0.1 liter/min per m2) were treated with the combination of oral enoximone (a phosphodiesterase inhibitor) and oral metoprolol at two institutions. Enoximone was given at a dose of < or = 1 mg/kg body weight three times a day. After clinical stabilization, metoprolol was initiated at 6.25 mg twice a day and slowly titrated up to a target dose of 100 to 200 mg/day. RESULTS: Ninety-six percent of the patients tolerated enoximone, whereas 80% tolerated the addition of metoprolol. The mean duration of combination therapy was 9.4+/-1.8 months. The mean length of follow-up was 20.9+/-3.9 months. Of the 23 patients receiving the combination therapy, 48% were weaned off enoximone over the long term. The LVEF increased significantly, from 17.7+/-1.6% to 27.6+/-3.4% (p=0.01), whereas the New York Heart Association functional class improved from 4+/-0 to 2.8+/-0.1 (p=0.0001). The number of hospital admissions tended to decrease during therapy (p=0.06). The estimated probability of survival at 1 year was 81+/-9%. Heart transplantation was performed successfully in nine patients (30%). CONCLUSIONS: Combination therapy with a positive inotrope and a beta-blocker appears to be useful in the treatment of severe, class IV heart failure. It may be used as a palliative measure when transplantation is not an option or as a bridge to heart transplantation. Further study of this form of combined therapy is warranted.     

Magnetic resonance imaging of the pelvic floor in the postpartum patient

Magnetic resonance imaging (MRI) was used to assess anatomical changes in the pelvic floor after childbirth. Six women underwent serial MRI examination within 30 hours and at 1 week, 2 weeks, 6 weeks and 6 months after delivery; 8 additional women were studied only within 30 hours of delivery. T-1 and T-2-weighted images of the pelvis in the transverse and sagittal planes with a 1.5-T MR imager were obtained. In the sagittal section we assessed the urethrovesical angle, urethral length, distance from the symphysis to the proximal and distal vagina, vaginal length, width and length of the sphincters, and the presence of sphincter defects. Axial sections were assessed for sphincter defects for the distance between the symphysis and midurethra, vagina and rectum. Only one parameter (distance between symphysis and distal vagina) changed significantly over time, without a clear trend in direction. Interobserver variation was reasonable (<15%) except for anal canal length, urethral length and distance between symphysis and anus. There were no significant correlations between birthweight and MRI parameters. There was a non-significant association (P = 0.09) between the sole combined sphincter defect and rectal injury, but not with episiotomy or parity. We concluded that it is feasible to determine multiple measurements on MR images to evaluate structures of the pelvic floor.