SPI-CI and SPI-6 cooperate in the protection from effector cell-mediated cytotoxicity

Tumors have several mechanisms to escape from the immune system. One of these involves expression of intracellular anticytotoxic proteins that modulate the execution of cell death. Previously, we have shown that the serine protease inhibitor (serpin) SPI-6, which inactivates the cytotoxic protease granzyme B (GrB), is capable of preventing cytotoxic T lymphocyte (CTL)-mediated apoptosis. Despite its potent antiapoptotic activity, SPI-6 does not prevent membranolysis induced by cytotoxic lymphocytes. We now provide evidence that several colon carcinoma cell lines do resist membranolysis and that this protection is dependent on SPI-6 but also requires expression of a closely related serpin called SPI-CI (serine protease inhibitor involved in cytotoxicity inhibition). Expression of SPI-CI is absent from normal colon but observed in placenta, testis, early during embryogenesis, and in cytotoxic lymphocytes. SPI-CI encodes a chymotrypsin-specific inhibitor and irreversibly interacts with purified granzyme M. Moreover, SPI-CI can protect cells from purified perforin/GrM-induced lysis. Our data therefore indicate that SPI-CI is a novel immune escape molecule that acts in concert with SPI-6 to prevent cytotoxic lymphocyte-mediated killing of tumor cells.     

Bleeding and bruising in Osteogenesis Imperfecta: International Society on Thrombosis and Haemostasis bleeding assessment tool and haemostasis laboratory assessment in 22 individuals

Osteogenesis imperfecta (OI) is characterized by susceptibility to bone fractures. Other symptoms, such as easy bruising and bleeding complications during surgery necessitating transfusions, have also been reported. The aim of the cross-sectional pilot study was to assess the bleeding and bruising tendency in OI patients and to screen for possible underlying haematological disorders. Bleeding tendency was investigated using the International Society on Thrombosis and Haemostasis bleeding assessment tool (ISTH-BAT) in 22 adult OI patients. Laboratory testing was performed to investigate for bleeding disorders or abnormal coagulation. Four patients [OI type 1(n = 3), OI type 4(n = 1)] had a bleeding score (BS) fitting with a bleeding tendency, but without test results pointing to a coagulopathy. Two patients [OI type 1(n = 1), OI type 3 (n = 1)] without a bleeding tendency according to the BS had increased fibrinolysis. This is the second largest study to date addressing bleeding tendency in OI and the first study to use ISTH-BAT and elaborate laboratory testing for coagulopathies. Four patients had an increased bleeding tendency. However, laboratory testing demonstrated no bleeding disorder or abnormal coagulation. Increased fibrinolysis was demonstrated in two patients without bleeding tendency on BS. Vascular fragility as a cause of bleeding tendency in OI has been suggested earlier. Further research on bleeding tendency in OI is important.     

Identification of citrullinated vimentin peptides as T cell epitopes in HLA–DR4–positive patients with rheumatoid arthritis

Objective

Antibodies directed against citrullinated proteins (ACPAs) are highly specific for rheumatoid arthritis (RA). The production of ACPAs is most likely dependent on the presence of T cells, since ACPAs undergo isotype switching and are associated with the shared epitope (SE)–containing HLA–DRB1 alleles. Vimentin is a likely candidate protein for T cell recognition, since >90% of patients positive for ACPAs that are reactive with (peptides derived from) citrullinated vimentin carry SE‐containing HLA–DRB1 alleles. The aim of this study was to identify citrullinated vimentin peptides that are presented to HLA–DRB1*0401–restricted T cells.

Methods

HLA–DR4–transgenic mice were immunized with all possible citrulline‐containing peptides derived from vimentin, and T cell reactivity was analyzed. Peptides recognized in a citrulline‐specific manner by T cells were selected and analyzed for their ability to be processed from the entire vimentin protein. A first inventory of the selected epitopes recognized by T cells was performed using peripheral blood mononuclear cells (PBMCs) from ACPA+, HLA–DR4+ patients with RA.

Results

A citrulline‐specific response was observed for 2 of the peptides analyzed in DR4‐transgenic mice. These peptides were found to be naturally processed from the vimentin protein, since citrullinated vimentin was recognized by peptide‐specific T cells. T cell reactivity against these peptides was also observed in cultures of PBMCs from RA patients.

Conclusion

This study identifies, for the first time, 2 naturally processed peptides from vimentin that are recognized by HLA–DRB1*0401–restricted T cells in a citrulline‐specific manner. These peptides can be recognized by T cells in ACPA+, HLA–DR4+ patients with RA, as shown in a first inventory.

     

Dose Dependency and Reversibility of Serotonin-Induced Valvular Heart Disease in Rats

Serotonergic drugs may lead to valvular heart disease in humans and more recently also in rats. Although clinical data suggest that dose dependency and reversibility after drug cessation might occur, proof of this is lacking. For that purpose, a total of 106 rats were prospectively enrolled: 22 control animals and 7 groups of 12 rats that received daily subcutaneous serotonin injections (5, 10, 20, 30, 40, 50 and 60 mg/kg respectively) for 12 weeks. At 12 weeks, half of the animals of each group were killed for histological analysis, whereas the remaining rats were further followed (without serotonin injections) for an additional 8 weeks. After 12 weeks of serotonin treatment, aortic and mitral regurgitation (AR, MR) were more frequently observed in the high dose groups (>30 mg/kg) compared to controls. Moreover, aortic and mitral valves were also thicker in the high dose groups compared to controls. After 8 weeks free of serotonin injections, AR and MR were no longer significantly higher than controls. Moreover, aortic and mitral valve thickness had normalized, returning to control levels. In conclusion, this study provides evidence for a dose-dependent valvular toxicity of serotonergic drugs, which appears to be reversible after drug withdrawal.     

Aortosternopexy for tracheomalacia following repair of esophageal atresia: evaluation by cine-CT and technical refinement

During the period of 1 year (1988 to 1989), five infants, aged 3 weeks to 10 months, presented with recurrent respiratory distress following repair of esophageal atresia with tracheoesphageal fistula (EA/TEF). These patients had associated congenital anomalies, including right aortic arch (1), biliary atresia (1), and a long gap that required esophageal elongation by spiral myotomy (1). The patients were evaluated for tracheomalacia using cinecomputed tomography (cine-CT; C-100 Scanner, Imatron, San Francisco, CA), which provides images of eight levels (8-mm interval) simultaneously with 0.7 second time intervals of cine-CT. Dynamic studies of the trachea by cine-CT showed tracheal collapse that was most significant during expiration in the segment immediately above and at the aortic arch. These patients underwent aortosternopexy. Via a right second intercostal approach, the ascending aorta and aortic arch were lifted anteriorly using two to four sutures of 5-0 Tevdek on pledgets placed between the tunica media and adventitia of the side walls of the aorta and the adjacent sternum. Respiratory distress was significantly improved postoperatively. Preliminary experience with these patients allows us to conclude that (1) cine-CT is a useful technique for diagnosing tracheomalacia; (2) it provides objective indication for its correction by aortosternopexy; and (3) the refined technique in placing sutures on the aorta may reduce the surgical risks of aortosternopexy.     

Automatic determination of left ventricular ejection fraction from gated blood-pool tomography.

The aim of this study was to develop and validate a new algorithm to automatically compute left ventricular ejection fraction (LVEF) from gated blood-pool tomography (GBPT). The results were compared with those of conventional planar radionuclide angiocardiography (PRNA). METHODS: Fifty-three consecutive patients received an injection of 740 MBq (99m)Tc-labeled human serum albumin. PRNA and GBPT were performed consecutively in a random sequence. PRNA served as the reference, and GBPT images were processed using a new edge detection algorithm. The algorithm is fast (<45 s), fully automatic, and works in three-dimensional space. The method includes identification of the valve plane and the septum. The left ventricular cavity at end-diastole is delineated by segmentation using an iterative threshold technique. An optimal threshold is reached when the corresponding isocontour best fits the first derivative of the end-diastolic count distribution in three dimensions. This optimal threshold is then applied to delineate the left ventricular cavity on the other time bins. The data are corrected for the partial-volume effect. Left ventricular volumes are determined using a geometry-based method and are used to calculate the ejection fraction. RESULTS: The success rate of the new algorithm was 94%. LVEFs calculated from GBPT agreed well with those calculated from PRNA (r = 0.78; GBPT = 0.94 PRNA + 6.33). The systematic error was 2.8%, and the random error was 8.8%. Excellent inter- and intraobserver reproducibility was found, with average differences of 1.1% +/- 4.6% and 1.1% +/- 5.0%, respectively, between the two measurements. CONCLUSION: This new algorithm provides a fast, automated, and objective method to calculate LVEF from GBPT.     

Contrast enema diagnosis of necrotizing enterocolitis

Contrast enema was performed in a select group of 126 neonates with ambiguous diagnosis of necrotizing enterolcolitis (NEC). Enema findings were compared with that on plain abdominal radiographs (AXR) and the clinical outcome. Thirty-one of the 34 patients with a discharge diagnosis of NEC were interpreted to have a positive contrast enema, and 91 of the 92 patients without a discharge diagnosis of NEC were interpreted to have a negative contrast enema. The AXR was interpreted as positive in 57 patients and as negative or indeterminate in 69. Not a single case of perforation was produced. Judicious use of contrast enema can improve specificity of diagnosis in cases with discordant clinical and plain radiographic findings.Presented at the Annual Meeting of The Society of Gastrointestinal Radiologists, Carlsbad, California, February 17–21, 1991     

Evaluation of a PACS workstation for assessment of body CT studies

Conventional hardcopy images from 266 body CT studies were compared with those provided at a commercially available picture archiving communication system (PACS) workstation. Unprocessed PACS images were larger but otherwise precisely duplicated hardcopy images. The PACS images were evaluated before and after application of various image processing/display features. Approximately three-quarters of the cases were depicted equally well with PACS and hardcopy, but in one quarter of the cases, diagnostic features were judged to be shown more clearly at the PACS workstation. When PACS images were viewed first, change in diagnosis after subsequent hardcopy inspection was infrequent (confidence change: 4%; different findings: 2%). Conversely, when hardcopy images were viewed first, change in diagnosis after subsequent PACS inspection was more frequent (confidence change: 19%; different findings: 8%). Specialized image manipulation available on PACS was critical for its performance. Review of cases with new findings discovered during the second inspection showed the majority of them to be clinically significant, true-positives discovered by PACS. We conclude that PACS is a useful modality for interpretation of body CT images.     

Sperm decondensation and semen parameters: utilization of a simple staining technique for the evaluation of human sperm decondensation

Summary. The ability of spermatozoa to fertilize an oocyte depends on a sequence of events ending ultimately in the decondensation of the sperm chromatin on penetration of the oocyte. Knowledge of what percentage of sperm decondenses is useful, especially in patients where other functional tests and sperm quality fail to explain the reported poor in vitro fertilization (IVF) rates. The objective of this study was (1) to compare sperm decondensation induced by either SDS/EDTA or heparin with semen parameters (volume, concentration, motility and morphology), and (2) to evaluate the use of a simplified staining technique (Diff Quik^R [DQ]) in comparison with the standard phase contrast method (Rose Bengal-[RB]). Randomly selected semen samples from 31 men attending an assisted reproductive programme were analysed for basic semen parameters and decondensation with SDS/EDTA and heparin. Two staining methods for the evaluation of decondensation were compared (phase contrast microscopy after Rose Bengal [RB] staining and light microscopy after Diff Quik^R (DQ) staining). Moderate and grossly swollen sperm heads were recorded. Semen samples included both fertile and unfertile semen parameters. Sperm decondensation results showed poor to moderate correlations with semen parameters. The SDS/EDTA (DQ) (moderate forms) showed a significant negative correlation (r = -0.46) with seminal volume and and a significant positive correlation (r = 0.41) with normal sperm morphology. The heparin (DQ) (moderate forms) decondensation showed a significant positive correlation with motility (r = 0.61) and sperm concentration (r = 0.43). The DQ method was preferred over the RB method due to its optical and storage advantage. Sperm decondensation by SDS/EDTA and heparin have limited use in the IVF laboratory as they correlate poorly with semen parameters. Future studies should investigate the use of an ooplasmic factor similar to nucleoplasmin in Xenopus laevis egg.