Constitutive abnormal expression of RasGRP-1 isoforms and low expression of PARP-1 in patients with systemic lupus erythematosus.

OBJECTIVE: Defective expression of Ras guanil releasing protein-1 (RasGRP-1) and increased apoptosis have been reported in lymphocytes from SLE patients. Whether these aberrations are correlated and linked to disease activity has not been elucidated. METHODS: Expression of normal 90 kDa RasGRP-1, its most prevalent 86 kDa isoform and full PARP-1 116 kDa and its cleavage fragment 84 kDa were determined in whole protein lysates of peripheral blood mononuclear cells (PBMC) in correlation with mitogen activated protein kinase (MAPK) activity and SLE clinical status in a large group of SLE patients during 1 year follow-up. RESULTS: Expression of normal 90 kDa RasGRP-1 was comparable in patients and controls. However, SLE patients demonstrated a constitutively increased 86 kDa/90 kDA ratio (p < 0.01) and decreased full poly (ADP-ribose) polymerase protein-1 (PARP-1) expression (p < 0.002) compared with controls who were disease-independent. A remission in disease activity was associated with decreased RasGRP-1 expression. Expression of 84 kDa PARP-1 cleavage fragment was found in 15% of patients but in none of the controls. In addition, expression of PARP-1 correlated positively with normal 90 kDa RasGRP-1 expression and negatively with the RasGRP-1 86 kDa/90 kDA ratio. CONCLUSIONS: These data suggest that constitutive aberrant expression of PARP-1 and RasGRP-1 ratio may act in concert to impair survival of lymphocytes in SLE patients.

Increased ERK and JNK activities correlate with disease activity in patients with systemic lupus erythematosus.

BACKGROUND: Aberrant signalling along the p21ras/MAP kinase pathway has been demonstrated in systemic lupus erythematosus (SLE). OBJECTIVE: To determine whether expression and activity of the MAP kinases ERK and JNK reflect disease activity in patients with SLE. METHODS: Blood samples of 42 outpatients with SLE were prospectively collected during four consecutive visits. The control group included 20 healthy subjects. Disease activity was assessed using the SLE Disease Activity Index (SLEDAI). Expression of total ERK and JNK kinases and their active forms (pERK and pJNK) was determined in whole protein lysates of peripheral blood mononuclear cells. RESULTS: The mean levels of the active kinases pERK and pJNK were significantly increased in patients with active disease (SLEDAI 4-20) as compared with patients with inactive disease (SLEDAI 0-3), p = 0.04, as well as with healthy controls, p = 0.03 and p = 0.003 for pERK and pJNK, respectively. The percentage of activated forms of ERK and JNK of the total expression of these MAP kinases was also gradually increased, reaching 50% for pERK and >40% for pJNK in patients with SLE with moderate-to-severe disease (SLEDAI 7-20), p = 0.005, p = 0.005 and p = 0.02, p = 0.05 as compared with controls and inactive patients, respectively. A decrease of more than three SLEDAI points was associated with a significant reduction in the expression of both total and activated forms of ERK and JNK, p = 0.03, p = 0.01, respectively. CONCLUSIONS: The results show that ERK and JNK activity reflects disease activity in patients with SLE. These MAP kinases may serve as additional tools for the evaluation of disease activity and management of these patients.

Evidence for aberrant regulation of MAP kinase signal transduction pathway in peripheral blood mononuclear cells in patients with active celiac disease.

BACKGROUND: Aberrant signaling via the p21/mitogen-activated proteins (MAP) kinase pathway has been described in lymphocytes of patients with various autoimmune diseases. There is little published data about the intracellular mediators and signals that regulate expression and activity of transcription factors and their effect on celiac disease induction and progression. AIM: To investigate the possible involvement of MAP kinase pathway in peripheral blood mononuclear cells (PBMC) in celiac disease and its correlation with disease activity. METHODS: Expression of the total and activated forms of two MAP kinases [extracellular response kinase (ERK) and c-Jun amino terminal kinase (JNK)] were studied by Western blots in PBMC of 17 untreated and 19 treated celiac patients, and 17 controls. Seven of these untreated celiac patients were studies before and after 6 months of gluten-free diet. RESULTS: Phosphorylated ERK of active celiac disease patients was significantly lower compared with controls (P < 0.01) and was increased towards normal after 6 month of gluten-free diet (P < 0.01). Phosphorylated JNK was increased significantly in the untreated celiac group (P < 0.01) and normalized towards the control level after 6 months of gluten-free diet (P < 0.04). CONCLUSIONS: Aberrant MAP-kinase pathway activity is associated with active celiac disease (CD). Further studies should examine the potential role of this aberration in pathogenesis of CD.

Clinical characteristics and diabetes associated autoantibodies in patients with both type 1 diabetes mellitus and asthma.

OBJECTIVE: Type 1 diabetes mellitus (DM1) and asthma are mediated by opposite arms of the cellular immune system, namely T helper (Th)1 and Th2 CD4+ cells, respectively. It is not known whether their coexistence affects their clinical manifestations. METHODS: The number of asthma exacerbations, frequency of hypoglycemic events, HbA1c levels, diabetes associated autoantibody status and diabetes associated late complications were determined in three paired groups of patients (n = 11) matched by gender and age: DM1 and asthma, asthma only, and DM1 only. RESULTS: Patients with both diseases had a higher prevalence of hypoglycemic events per month compared to patients with DM1 only: 5.67 +/- 4.27 vs 1.45 +/- 2.06, respectively (p = 0.008). The co-existence of the two diseases did not modify the remaining clinical and laboratory parameters. CONCLUSION: Patients with both DM1 and asthma have similar clinical characteristics to patients with only one of these diseases apart from a higher rate of hypoglycemic events compared to patients with DM1 without asthma.

Universal calibration of surgical instruments for spinal stereotaxy.

PURPOSE: To describe new software applications and interchangeable instrumentation enabling the use of standard surgical instruments with image-guided systems for stereotactic spinal procedures. CONCEPT: The ability to adapt essentially any surgical instrument for stereotactic procedures will improve the safety and accuracy of image-guided spinal surgery. RATIONALE: Using universal dynamic registration hardware and software, standard surgical instruments are adapted for real-time image-guided surgery. The Radionics Optical Tracking System (Radionics, Inc., Burlington, MA) has custom software applications and universal hardware adaptation devices for spinal stereotaxy that allows the use of standard instruments for intraoperative guidance. An array of light-emitting diodes can be attached to essentially any rigid instrument with a definable tip and can then be calibrated to the system for intraoperative use. Stereotactic guidance of a drill, tap, and screwdriver may improve screw placement accuracy in spinal surgery because every step of the procedure can be monitored in real time. DISCUSSION: Most stereotactic systems have only a standard probe or limited instruments for localization, targeting, and tracking a procedure. The surgeon then resumes the operation using standard surgical instruments without the benefit of image guidance for the key steps of the procedure. Because each surgical step for screw placement in the spine has a potential for error, use of multiple instruments that can be interchanged for real-time image-guided spinal surgery may increase the accuracy and safety of spinal instrumentation procedures. These techniques can also be applied to intracranial image-guided surgery.

Frameless stereotaxy for anterior spinal procedures.

OBJECT: Intraoperative image guidance provides real-time three-dimensional visualization and has been successfully applied in many posterior spinal procedures. The feasibility of applying these techniques to anterior spinal surgery has not been studied systematically because the anterior spine, in contrast to the posterior spine, lacks distinct anatomical landmarks for registration. The authors sought to evaluate the practicality of performing stereotaxy in the anterior spine in a cadaveric model. METHODS: Unilateral C4-L4 pedicle screws were placed posteriorly in three cadaveric specimens to serve as unknown markers within each vertebral body. The specimens then underwent computerized tomography (CT) scanning, and the CT data were transferred to an optical tracking system. The anterior surface of the spine was registered for use with the stereotactic system by using a paired point-matching technique. Attached to a surgical drill, K-wires were placed under stereotactic guidance in a tip-to-tip orientation with the posterior pedicle screws. A second postoperative CT scan was obtained, and accuracy was determined by measuring the distance between the tips of the K-wire and pedicle screw. The K-wires were placed tip to tip with pedicle screw markers in 57 vertebral levels. The mean registration error was 1.47+/-0.04 mm, and when combined with the universal instrument registration error of 0.7 mm yielded an overall registration error of 2.17+/-0.04 mm. The mean tip-to-tip distance for all K-wires placed was 2.46+/-0.23 mm. The difference between the mean tip-to-tip distance and overall registration error was not statistically significant (p > 0.05), indicating that the K-wires were placed within the expected range of error. CONCLUSIONS: The results of this study confirmed the feasibility of performing anterior stereotactic procedures throughout the spine. The accuracy of the findings in this study indicates that anterior stereotaxy should be applicable in clinical practice.

Effect of frameless stereotaxy on the accuracy of C1-2 transarticular screw placement.

OBJECT: In recent studies some authors have indicated that 20% of patients have at least one ectatic vertebral artery (VA) that, based on previous criteria in which preoperative computerized tomography (CT) and standard intraoperative fluoroscopic techniques were used. may prevent the safe placement of C1-2 transarticular screws. The authors conducted this study to determine whether frameless stereotaxy would improve the accuracy of C1-2 transarticular screw placement in healthy patients, particularly those whom previous criteria would have excluded. METHODS: The authors assessed the accuracy of frameless stereotaxy for C1-2 transarticular screw placement in 17 cadaveric cervical spines. Preoperatively obtained CT scans of the C-2 vertebra were registered on a stereotactic workstation. The dimensions of the C-2 pars articularis were measured on the workstation, and a 3.5-mm screw was stereotactically placed if the height and width of the pars interarticularis was greater than 4 mm. The specimens were evaluated with postoperative CT scanning and visual inspection. Screw placement was considered acceptable if the screw was contained within the C-2 pars interarticularis, traversed the C 1-2 joint, and the screw tip was shown to be within the anterior cortex of the C-1 lateral mass. Transarticular screws were accurately placed in 16 cadaveric specimens, and only one specimen (5.9%) was excluded because of anomalous VA anatomy. In contrast, a total of four specimens (23.5%) showed significant narrowing of the C-2 pars interarticularis due to vascular anatomy that would have precluded atlantoaxial transarticular screw placement had previous nonimage-guided criteria been used. CONCLUSIONS: Frameless stereotaxy provides precise image guidance that improves the safety of C1-2 transarticular screw placement and potentially allows this procedure to be performed in patients previously excluded because of the inaccuracy of nonimage-guided techniques.

New software applications for interchangeable instrumentation in spinal stereotaxis.

Computer image-guided surgery has been widely accepted because it allows the surgeon to track an instrument through unvisualized critical structures of a patient in real-time, thus minimizing the risk of injury. Current spinal and cranial image-guided surgery is, however, limited by the lack of surgical instruments and software applications that would allow rapid interchange of useful instruments to perform the procedures. Most image-guided systems utilize a single standard probe or a few pre-defined instruments that are not necessarily useful for performing the actual surgical procedure. Present image-guided technology for screw placement in spinal surgery utilizes the standard probe only to confirm the entry point location and view the planned trajectory of the screw. The surgeon then resumes the procedure using standard surgical instruments to drill, tap and place screws without the benefit of image guidance. Our clinical laboratory experience with spinal image-guided surgery indicates that there is potential for error between each of these procedural steps of screw placement. Despite accurately locating an entry point, any deviation in the trajectory during drilling of a pilot hole, tapping or screw placement may result in significant errors in screw placement and potential neurovascular injury. We have developed custom software applications and universal hardware adaptation devices for spinal image-guided surgery that allow the use of standard instruments for intraoperative guidance. Utilizing universal dynamic registration hardware and software, standard surgical instruments are adapted for real-time image guided surgery. An array of light emitting diodes can be attached to essentially any rigid instrument with a definable tip and then calibrated to the system for intraoperative use. Laboratory tests using a cadaveric model indicate a difference in accuracy of less than 1.0 mm between the standard probe and a dynamically registered custom instrument and an absolute mean error of less than 2.0 mm for the image-guided system which is clinically insignificant in most cases. This technology is a significant step forward as it allows the surgeon to use a full array of instruments with image guidance and will ultimately make spinal and intracranial surgery safer and more accurate.

Reduced GLP-1R expression in gastric glands of patients with type 2 diabetes mellitus.

BACKGROUND: The incretin effect is reduced in type 2 diabetes mellitus (T2DM) patients. Whether the impaired function of the enteropancreatic axis in these patients is due to defective GLP-1 receptor (GLP-1R) expression in extrapancreatic target organs is not known. AIMS AND METHODS: To compare the GLP-1R expression and distribution in gastric mucosa biopsies of patients with (n =22) and without (n =22) T2DM referred for routine esophagogastroduodenoscopies. GLP-1R mRNA levels were estimated by real-time PCR. The intensity of GLP-1R immunostaining, frequency, and types of glandular cells bearing GLP-1R and their glandular distribution in different stomach mucosa regions were evaluated by immunohistochemical morphological semiquantitative and quantitative analysis. RESULTS: Mean mRNA GLP-1R levels were significantly reduced in patients with T2DM compared with nondiabetic patients (P < .02). Immunohistochemical analysis revealed that the reduced GLP-1R expression in T2DM patients was due to a decreased intensity of immunostaining (P < .01). The number of glandular GLP-1R-bearing cells in both body and antrum mucosa was decreased in T2DM patients. Most notably, the frequency of GLP-1R immunoreactive acid-secreting parietal cells was reduced in the neck area of the gastric principal glands of T2DM patients (P < .01). No correlation was found between the reduced GLP-1R expression and clinical parameters including body mass index, age, glycosylated hemoglobin, and disease duration. CONCLUSION: This is the first evidence of reduced GLP-1R expression in gastric glands of T2DM patients. These data demonstrate that the defective function of the incretin axis in T2DM may also result from decreased GLP-1R expression in its extrapancreatic target organs.

Improved activity of streptozotocin-selected insulinoma cells following microencapsulation and transplantation into diabetic mice.

We have recently shown that repeated streptozotocin (STZ) treatment induces the selection of insulinoma cells (RINmS) with both improved resistance to diabetogenic toxins and functional activity, compared to parental RINm cells. The aim of the present study was to estimate the potential of RINmS cells to maintain their engineered characteristics during in vivo hyperglycemic conditions. It was found that microencapsulation and transplantation into diabetic mice preserved a three-fold higher level of insulin content in selected RINmS cells when compared to the parental ones. Retrieval of transplanted encapsulated cells from the peritoneal cavity of diabetic mice had a significantly higher insulin content and a more intense insulin response to secretogogues in selected RINmS cells when compared to retrieved RINm cells. In conclusion, our results show that RINmS cells do not lose their improved functional characteristics after encapsulation and transplantation into diabetic mice.

Streptozotocin and alloxan-based selection improves toxin resistance of insulin-producing RINm cells.

The aim of our study was to develop a method for selection of subpopulations of insulin producing RINm cells with higher resistance to beta cell toxins. Cells, resistant to streptozotocin (RINmS) and alloxan (RINmA), were obtained by repeated exposure of parental RINm cells to these two toxins, while the defense capacity was estimated by the MTT colorimetric method, and [3H]-thymidine incorporation assay. We found that RINmS and RINmA displayed higher resistance to both streptozotocin (STZ) and alloxan (AL) when compared to the parental RINm cells. In contrast, no differences in sensitivity to hydrogen peroxide were found between toxin selected and parental cells. Partial protection from the toxic effect of STZ and AL was obtained only in the parental RINm cells after preincubation of cells with the unmetabolizable 3-O-methyl-glucose. The possibility that GLUT-2 is involved in cell sensitivity to toxins was confirmed by Western blot analysis, which showed higher expression of GLUT-2 in parental RINm compared to RINmS and RINmA cells. In addition to the higher cell defense property evidenced in the selected cells, we also found higher insulin content and insulin secretion in both RINmS and RINmA cells when compared to the parental RINm cells. In conclusion, STZ and AL treatment can be used for selection of cell sub-populations with higher cell defense properties and hormone production. The different GLUT-2 expression in parental and resistant cells suggest involvement of GLUT-2 in mechanisms of cell response to different toxins.

Enhanced glucose-dependent glucagon-like peptide-1 and insulin secretion in Crohn patients with terminal ileum disease is unrelated to disease activity or ileal resection.

BACKGROUND: Enhanced secretion of glucagon-like peptide-1 (GLP-1) has been reported in patients with Crohn disease (CD). However, the correlation between the enteropancreatic axis and the activity of CD remains unclear. METHODS: Plasma glucose, insulin, GLP-1 levels and insulin sensitivity were determined before and after oral glucose tolerance tests in 13 patients with CD of the terminal ileum, in 13 patients after resection of the terminal ileum and in 7 healthy controls. Basal and stimulated insulin sensitivities were determined using the homeostasis model assessment (HOMA) and the insulin sensitivity index (ISI) methods, respectively. RESULTS: Basal and stimulated glucose levels were comparable in patients and controls. The peak stimulated GLP-1 secretion was significantly higher in the patient group compared to controls: 12.2 +/- 1.24 pM/L and 8.1 +/- 1.72 pM/L, respectively, P=0.03. This was associated with 52% increased overall insulin secretion in the patients' group as compared to controls (P=0.007) and a higher peak insulin response: 63.5 +/- 9.69 mU/L and 41.5 +/- 6.85 mU/L for patients and controls, respectively, P=0.04. Operated patients had similar GLP-1 levels but higher peak and overall insulin secretions compared with those in non-operated patients (P=0.01). Fasting and stimulated insulin sensitivities were reduced only in patients with ileal resection as compared to controls: P=0.01 and P=0.05, respectively. No correlation was found between the CD activity index and GLP-1 or insulin secretion. CONCLUSIONS: CD of the terminal ileum is associated with enhanced glucose-dependent GLP-1 secretion, which is unrelated to disease activity or ileal resection.