High resolution crystal structure of a human tumor necrosis factor-alpha mutant with low systemic toxicity

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.     

A composite silent corticotroph pituitary adenoma with interspersed adrenocortical cells: case report

Recently, perfusion imaging has been of increasing interest in MRI. We applied this method for semiquantitative evaluation of hepatic parenchymal portal blood flow in patients with diffuse liver damage. Twenty patients with diffuse hepatic damage were divided according to the Child's Classification and studied. Gadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) was administered into the superior mesenteric artery (SMA), and a dynamic series of T2*-weighted fast low angle shot (FLASH) images was obtained. We evaluated relative regional portal blood volume (rrPBV), mean transit time (MTT), and relative regional portal blood flow (rrPBF). The relationship between the rrPBV, rrPBF, and plasma indocyanine green retention rate test at 15 minutes (ICGR15 was also evaluated in 12 patients. Both rrPBF and rrPBV are significantly decreased in Child B & C patients compared with Child A patients. On the other hand, the MTT is significantly prolonged in Child B & C patients compared with Child A patients. Significant correlations were also noted between rrPBV and ICGR15 and between rrPBF and ICGR15. By means of selective catheterization into the SMA, we were able to estimate rrPBV, rrPBF, and MTT. This method may play a clinical role for assessment of regional portal perfusion in various diseases with diffuse liver damage.     

Structure of poly d(AI).poly d(CT) in two different packing arrangements

X-ray diffraction analysis of poly d(AI).poly d(CT) in oriented and polycrystalline fibers has revealed the DNA structure to be a 10-fold, right-handed, antiparallel, Watson-Crick base paired double helix in two distinct packing arrangements corresponding to one and two helices, respectively, in the unit cell. The helix pitch is 32.1 A and 32.4 A in the two cases, almost 1.5 A shorter than in classical B-DNA. The resulting B'-DNA geometry, described in terms of a tetranucleotide repeat which is conformationally similar to B-DNA, has its minor groove closely shut and major groove correspondingly widened, thus striking a sharp morphological contrast to B-DNA. According to difference electron density maps, a spine of hydration along the minor groove connects both strands and provides structural stability; ordered sodium ions and water molecules are actively involved in bridging the phosphate groups of neighboring helices. The crystallographic R-values for these two allomorphs are 0.26 and 0.20, respectively, for data up to 3.0 A resolution.     

The identification of CD4+ T cell epitopes with dedicated synthetic peptide libraries

For a large number of T cell-mediated immunopathologies, the disease-related antigens are not yet identified. Identification of T cell epitopes is of crucial importance for the development of immune-intervention strategies. We show that CD4+ T cell epitopes can be defined by using a new system for synthesis and screening of synthetic peptide libraries. These libraries are designed to bind to the HLA class II restriction molecule of the CD4+ T cell clone of interest. The screening is based on three selection rounds using partial release of 14-mer peptides from synthesis beads and subsequent sequencing of the remaining peptide attached to the bead. With this approach, two peptides were identified that stimulate the beta cell-reactive CD4+ T cell clone 1c10, which was isolated from a newly diagnosed insulin-dependent diabetes mellitus patient. After performing amino acid-substitution studies and protein database searches, a Haemophilus influenzae TonB-derived peptide was identified that stimulates clone 1c10. The relevance of this finding for the pathogenesis of insulin-dependent diabetes mellitus is currently under investigation. We conclude that this system is capable of determining epitopes for (autoreactive) CD4+ T cell clones with previously unknown peptide specificity. This offers the possibility to define (auto)antigens by searching protein databases and/or to induce tolerance by using the peptide sequences identified. In addition the peptides might be used as leads to develop T cell receptor antagonists or anergy-inducing compounds.     

The incidence of artery puncture with central venous cannulation using a modified technique for detection and prevention of arterial cannulation

BACKGROUND: Cannulation of the central circulation is essential for management of patients who require major surgery, and for patients who are critically ill. Arterial puncture is the most frequent complication associated with central venous cannulation, and is potentially fatal. Detection of arterial puncture can be problematic, especially in patients with cyanotic congenital heart disease. METHODS: One thousand eleven consecutive cardiothoracic and vascular surgical patients who required central venous cannulation were studied using a new technique for detection of arterial puncture and prevention of arterial cannulation. This technique involves continuous pressure transduction of the steel introducer needle. Central venous cannulation was attempted in all patients. The sites of attempted catheterizations, number of arterial punctures and cannulations, and the number of successful catheterizations were noted. All patients were treated in accordance with standard anesthetic and surgical techniques in the institution. RESULTS: One thousand one hundred seventy-two central venous catheters were placed. The overall success rate was 99.6%. The incidence of arterial puncture was 9.3% for central venous cannulation attempts of the internal jugular, subclavian, and femoral veins. No arterial cannulation occurred, and none of the patients had significant complications. Congenital heart disease patients had a higher incidence of arterial puncture (14.1%) and a lower rate (96.8%) of successful cannulation. CONCLUSION: Pressure transduction of the steel needle is a useful technique for detecting arterial puncture and preventing arterial cannulation during attempts to achieve central venous cannulation.     

Centralized follow-up of patients treated for head and neck cancer

In a prospective study, the value of the clinical follow-up after treatment for head and neck cancer has been assessed. A total of 407 visits in 377 patients were recorded during a three month period in 1993 at the two major radiotherapy departments in Denmark. The results showed that 61% of follow-up visits included one or more problems either related to treatment morbidity or tumour recurrence. About 50% of all visits included treatment related normal tissue problems, and 30% had problems that required intervention. Although the majority of problems occurred within a few years after treatment, 47% of patients at three to four years observation time still had one or more problems. A total of 34 new tumour recurrences were found in the period, and of these 11 (32%) were asymptomatic. It is concluded that head and neck cancer patients have both tumour and normal tissue problems several years after the end of treatment. Since effective salvage treatment improves local control significantly, early detection of possible recurrence is important. A follow-up period of four to five years is recommended-preferably by qualified experts in the management of both recurrent disease and treatment morbidity.     

Time course of changes in lactate and free fatty acids after experimental brain injury and relationship to morphologic damage

Regional levels of lactate and free fatty acids (FFA) were measured after lateral fluid percussion (FP) brain injury in rats. At 5 min after injury, tissue concentrations of lactate were elevated in the cortices and hippocampi of both ipsilateral and contralateral hemispheres. Whereas lactate levels had returned to normal by about 20 min after injury in the penumbra and contralateral cortices, their elevation persisted in the ipsilateral injured cortex and hippocampus for 24 h after injury. Increases in the levels of FFA (particularly stearic and arachidonic acids) were observed in the cortices and hippocampi of both ipsilateral and contralateral hemispheres at 5 min after injury; these levels returned to normal in only the penumbra and contralateral cortices by 20 min after injury. Increased amounts of palmitic and oleic acids were also found only in the injured left cortex and ipsilateral hippocampus at 20 min or later after injury. In general, these elevations persisted for as long as 6 to 24 h in the injured cortex and for 2.5 to 24 h after injury in the ipsilateral hippocampus. Histologic studies revealed a similar extent of damage in the cortex between 5 min and 24 h after injury, whereas damage in the CA3 region of the ipsilateral hippocampus increased during that period. These findings suggest a role for lactic acid and FFA, two secondary injury factors, in neuronal cell loss after brain injury.     

Pharmacokinetics of gadodiamide injection in patients with severe renal insufficiency and patients undergoing hemodialysis or continuous ambulatory peritoneal dialysis

RATIONALE AND OBJECTIVES: The authors performed this study to evaluate the pharmacokinetics, dialysability, and safety of gadodiamide injection in patients with severely reduced renal function not treated with renal replacement therapy and patients undergoing hemodialysis or continuous ambulatory peritoneal dialysis. MATERIALS AND METHODS: Twenty-seven patients--nine with severely reduced renal function (glomerular filtration rate, 2-10 mL/min), nine undergoing hemodialysis, and nine undergoing continuous ambulatory peritoneal dialysis--were followed up for 5, 8, and 22 days, respectively, after receiving gadodiamide injection (0.1 mmol per kilogram body weight). RESULTS: Gadodiamide injection caused no changes in renal function. In patients with severely reduced renal function, the elimination half-life of gadodiamide injection was prolonged (34.3 hours +/- 22.9) compared with data in healthy volunteers (1.3 hours +/- 0.25). An average of 65% of the gadodiamide injected was eliminated during a hemodialysis session. After 22 days of continuous ambulatory peritoneal dialysis, 69% of the total amount of gadodiamide was excreted; this reflects the low peritoneal clearance. In all patients, no metabolism or transmetallation of gadodiamide was found. There were no contrast material-related adverse events. CONCLUSION: Gadodiamide is dialysable and can safely be used in patients with severely impaired renal function or those undergoing hemodialysis or continuous ambulatory peritoneal dialysis. No precautions to increase the elimination are necessary.