Differences in the abilities of human tau isoforms to promote microtubule assembly.

Three isoforms of human tau protein were compared for their abilities to induce microtubule assembly. The three isoforms, tau 3 (tau containing three microtubule-binding domains), tau 4 (tau containing four microtubule-binding domains) and tau 4L (tau containing four microtubule binding domains plus a 58-amino-acid insert near the N-terminus) were expressed in E. coli and purified using ammonium sulfate precipitation, ion exchange, and size exclusion chromatography. All three isoforms induced microtubule assembly at micromolar concentrations and showed similar critical concentrations for assembly of 0.4-0.45 microM. However, tau 4 induced microtubule formation at a rate five- to tenfold faster than either tau 3 or tau 4L. The rate of microtubule elongation seen with tau 4 was twofold greater than with tau 3 or tau 4L, suggesting that the faster rate of microtubule assembly seen with tau 4 was due, at least in part, to faster elongation. Tau 4 induced a greater number of microtubules to form at steady state than did tau 3 or tau 4L. The microtubules generated with each tau isoform had similar steady-state length distributions and were equally susceptible to cold-induced disassembly. These results indicate that the additional microtubule-binding domain in tau 4 enhances microtubule assembly, while the 58-amino-acid insert negates the stimulatory effect of the fourth microtubule-binding domain.     

Calcium mobilization and entry by thapsigargin in neuronal tumor cell line, SK-N-SH.

Using fura-2 loaded neural tumour cells, SK-N-SH, we demonstrate that receptor-mediated activation of phosphoinositide hydrolysis not only causes the release of Ca2+ from intracellular stores but also causes a concomitant influx of extracellular Ca2+. Thapsigargin (TG), a sesquiterpene lactone, causes a sustained elevation of intracellular Ca2+ and depletion of the inositol 1, 4, 5-trisphosphate-sensitive intracellular Ca2+ stores. In the absence of extracellular Ca2+, the increase in intracellular Ca2+ concentration ([Ca2+]i) was transient, suggesting that thapsigargin activates both intracellular mobilization and the influx of Ca2+ from extracellular space. These results are consistent with the proposal that the depletion of the inositol 1, 4, 5-trisphosphate-sensitive intracellular Ca2+ pool serves as a signal for Ca2+ influx.     

Application of intermaxillary fixation screws in maxillofacial trauma.

PURPOSE: The use of intermaxillary fixation (IMF) in the treatment of maxillofacial trauma represents the cornerstone of fracture reduction and immobilization. Many modalities of IMF have been described; recently IMF screws have been introduced into clinical practice, however, hardware failure can occur. We performed a retrospective study evaluating hardware-associated complications for self-drilling/tapping IMF screws. MATERIALS AND METHODS: A retrospective study on 49 patients requiring IMF was performed. The diagnosis, duration of IMF, screw site, use of elastic or wire fixation, and associated complications were recorded. IMF screws were used to adjunct open reduction techniques, for definitive closed reduction, or fracture prevention following dentoalveolar surgery. Follow-up examinations were performed until fracture healing was complete (6 to 8 weeks). RESULTS: A single adverse event occurred in 19 patients (39%) while 4 patients (8%) had more than 1 complication. The most common event was screw loosening; 29% of patients had at least 1 screw dislodged in the treatment period. Of the total number of screws placed (229), 15 (6.5%) became loose, and were equally distributed among the mandible and maxilla. The remaining complications noted were root fracture, 4% (2 of 49); loosened wires, 6% (3 of 49); screw shear, 2% (1 of 49); malocclusion, 2% (1 of 49); and ingested hardware, 2% (1 of 49). CONCLUSIONS: Overall the IMF self-drilling/tapping screws have been shown to be a useful modality to establish maxillomandibular fixation. It is a safe, and time-sparing technique; however, it is not without limitations or potential consequences which the surgeon must be aware of in order to provide safe and effective treatment.     

Autoantibodies and drug-or metabolite-dependent antibodies in patients with diclofenac-induced immune haemolysis

Two patients with acute immune haemolytic anaemia caused by diclofenac are described. Both patients had developed IgG drug-independent autoantibodies and drug-dependent antibodies. The drug-dependent antibodies in one patient reacted with red blood cells (RBC) only in the presence of urine from patients receiving diclofenac (ex vivo antigen) but not in the presence of the drug itself or its known metabolites. This antibody appeared to recognize a trace metabolite which has not yet been identified. The drug-dependent antibodies in the second patient reacted with RBC in the presence of urine as ex vivo antigen as well as in the presence of the drug itself and its main metabolites. Incorrect diagnosis of such cases often is due to the occurrence of autoantibodies and/or unusual drug metabolism and excretion.     

Long‐standing ulcerative colitis complicated by right subclavian artery occlusion and upper limbs arteriolitis with digital ischemia

A 48‐year‐old male patient with long‐standing ulcerative colitis since February 2001 which was diagnosed by endoscopy, developed acute digital ischemia affecting both hands with fixed colour changes in the left index finger which was followed shortly by digital ulceration. Magnetic resonance angiography (MRA) of both upper limbs showed evidence of vasculitis affecting digital arterioles on both sided and right subclavian occlusion. The patient received pulse methylprednisolone followed by cyclophosphamide pulse therapy, the latter continuing on a monthly basis for 6 months with appreciable improvement and remission of the vasculitic process; follow‐up MRA showed reperfusion of the previously occluded subcalvian artery. To the authors’ knowledge vasculitis complicating the course of ulcerative colitis is a rare association and is only sporadically reported in the literature. This rare entity should be diagnosed early and aggressively treated; MRA is a very promising diagnostic tool that is suitable for both diagnosis and follow‐up of patients with this rare entity.     

Mechanisms of Tolerance Induced by Donor-Specific Transfusion and ICOS-B7h Blockade in a Model of CD4+ T-Cell-Mediated Allograft Rejection

The inducible co-stimulatory molecule (ICOS) has been shown to play a critical role in T-cell activation and differentiation, and the regulation of alloimmune responses in vivo. Using an MHC class II mismatched model of CD4(+) T-cell-mediated rejection, we found that treatment of mice with DST and ICOS-B7h blockade induced long-term skin allograft survival and donor-specific transplantation tolerance. ICOS blockade, either during antigen priming or during the effector phase, previously shown to alter the outcome of the immune response, had a similar effect on graft survival. DST and anti-B7h mAb reduced the frequency of IFN-gamma-producing allospecific cells but did not produce deviation to a T(H)2 phenotype. In an adoptive transfer model using ABM TCR transgenic mice directly reactive to I-A(bm12), DST and anti-B7h mAb reduced the number of allospecific CD4(+) T cells and increased CD4(+) T-cell apoptosis. These data demonstrate that DST and anti-B7h mAb induces transplantation tolerance to MHC class II mismatched skin grafts by a reduction of the alloreactive clone size that is, at least in part, dependent on apoptosis of host alloantigen-specific CD4(+) T cells.     

Telepathology for routine light microscopic and frozen section diagnosis

Telepathology (TP) uses telecommunication linkages to electronically capture, store, retrieve, and transmit images to distant sites. We assessed the feasibility of a dynamic real-time TP system for light microscopic (LM) diagnosis of anatomic pathology specimens, including frozen sections. Six pathologists, in 2 separate periods, read a set of 160 retrospectively retrieved slides (80 of which were frozen sections) by TP and LM. Reading times were recorded. Diagnoses were compared with the reference diagnosis (established by a group of 5 independent pathologists) and graded on a scale of 0 to 2 (2, correct; 1, incorrect but no clinical impact; 0, incorrect with clinical impact). Overall, LM was more accurate than TP compared with the reference diagnosis (score, 1.68 vs 1.54). There was no difference in accuracy between frozen section and paraffin-embedded tissue. Intraobserver agreement ranged from 82.5% to 88.2%. The average reading time was 6.0 minutes for TP and 1.4 minutes for LM. During the study, reading time decreased for TP but not for LM. These results show that despite marginally lower accuracy and longer reading times, TP isfeasible for routine light microscopic diagnosis, including frozen sections.     

Value of whole-cell antigen extracts for serologic detection of Helicobacter pylori.

Whole-cell protein extracts of Helicobacter pylori strains were evaluated by enzyme-linked immunosorbent assay to detect immunoglobulin G antibody against H. pylori in 113 patients with upper gastrointestinal complaints. These antigen preparations were of value for detecting infection by H. pylori in patients with high antibody titers (> or = 12,800), whereas for patients with lower titers, the results were inconclusive.     

Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis

Abstract: A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.