Characterization of human platelet glycoprotein antigens giving rise to individual immunoprecipitates in crossed-immunoelectrophoresis

Washed human platelets were labeled with 125I by the lactoperoxidase- catalyzed method and solubilized in 1% Triton X-100. The soluble proteins were analyzed by crossed-immunoelectrophoresis in 1% agarose, employing a rabbit antibody raised against whole human platelets. Analysis of autoradiograms developed from dried agarose gels led to the establishment of a normal reference pattern that was consistent for platelets obtained from more than 50 normal individuals. Six platelet membrane glycoprotein antigens contained in four distinguishable precipitates were identified. Each identification was based on direct sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of 125I-antigens contained in individually excised precipitates. These platelet antigens include major membrane glycoproteins previously designated la, lb, lla, llb, llla, and lllb. Glycoproteins llb and llla were shown to be contained in a single immunoprecipitate, while glycoproteins la and lla were routinely detected in a single different immunoprecipitate. Analysis of soluble proteins from platelets of five patients with Glanzmann's thrombasthenia demonstrated either a complete absence or a marked reduction of only one radiolabeled precipitate, that containing membrane glycoproteins llb and llla. Platelet samples from two patients with Bernard-Soulier syndrome were devoid of a different precipitate, that containing membrane glycoprotein lb.     

Insulin glargine-based therapy improves glycemic control in patients with type 2 diabetes sub-optimally controlled on premixed insulin therapies

The AT.LANTUS trial recently demonstrated the efficacy and safety of insulin glargine initiation and maintenance using two different treatment algorithms in poorly controlled type 2 diabetes mellitus (T2DM). This sub-analysis investigated glycemic control and safety in 686 patients switching from premixed insulin (premix) with or without (+/-OADs) to once-daily glargine (+/-OADs/prandial insulin). A 24-week, multinational (n=59), multicenter (n=611), randomized study comparing two algorithms (Algorithm 1: clinic-driven titration; Algorithm 2: patient-driven titration) in four glargine+/-OADs treatment groups: alone, once- (OD), twice- (BD) or >twice- (>BD) daily prandial insulin. After switching to the glargine regimen, HbA(1c) levels significantly improved in the overall group (9.0+/-1.3 to 8.0+/-1.2%; p<0.001) and in all subgroups; fasting blood glucose levels also improved in all subgroups (overall: 167.1+/-50.0 to 106.9+/-27.2 mg/dL [9.3+/-2.8 to 5.9+/-1.5 mmol/L]; p<0.001). The incidence of severe hypoglycemia was also low in all four subgroups (< or =1.7%). Patients with T2DM switching from premix+/-OADs to glargine+/-OADs had significant reductions in glycemic control with a low incidence of severe hypoglycemia. The addition of prandial (OD, BD or >BD) insulin was associated with further improvements in glycemic control. These data provide support for the stepwise introduction of prandial insulin to a more physiologic basal-bolus regimen, which is under investigation.     

Risks and benefits of postoperative cardiac catheterization in patients with ball valve prostheses

Two studies were performed to evaluate the risks and benefits of post-operative cardiac catheterization in patients with a ball valve prosthesis. In Study 1, 106 consecutive catheterization procedures in 100 patients were retrospectively evaluated. There were no deaths, myocardial infarctions or infective endocarditis. Five patients who were in extremis were studied without complication. Major complications (occurring in 8.5 percent) included excessive bleeding (four patients), cerebral emboli (two patients), ventricular fibrillation (one patient) and other (two patients). The major complication rate for patients with transthoracic left ventricular puncture with ventriculography was 24 percent (4 of 17) and for patients with therapeutic anticoagulation 18 percent (6 of 34). Significant abnormalities were detected in 55 percent of all catheterizations. The incidence rate of significant abnormalities was 75 percent (47 of 63) when clinical-hemodynamic deterioration was an indication for catheterization and 19 percent (8 of 43) when it was not. The incidence rate of reoperation for all patients was 34 percent, 51 percent (32 of 63) in those with clinical-hemodynamic deterioration and 9 percent (4 of 43) in others. In Study 2, 45 patients with a reduced level of anticoagulation were evaluated prospectively to assess whether such reduction would eliminate most complications. One patient was in extremis at the time of study. There were no complications in these 45 patients.

We conclude the following: (1) Therapeutic anticoagulation is associated with increased risk from cardiac catheterization. In patients with a less than therapeutic level of anticoagulation, cardiac catheterization can be performed at low risk. (2) Patients with a ball valve prosthesis who manifest clinical-hemodynamic deterioration should undergo postoperative cardiac catheterization because of the high incidence rate of correctable abnormalities in these patients.     

Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation

Anti-CD20 monoclonal antibodies (mAbs) are classified into type I (rituximab-like) or type II (tositumomab-like) based on their ability to redistribute CD20 molecules in the plasma membrane and activate various effector functions. To compare type I and II mAbs directly in vivo and maximize Fc effector function, we selected and engineered mAbs with the same mouse IgG(2)a isotype and assessed their B-cell depleting activity in human CD20 transgenic mice. Despite being the same isotype, having similar affinity, opsonizing activity for phagocytosis, and in vivo half-life, the type II mAb tositumomab (B1) provided substantially longer depletion of B cells from the peripheral blood compared with the type I mAb rituximab (Rit m2a), and 1F5. This difference was also evident within the secondary lymphoid organs, in particular, the spleen. Failure to engage complement did not explain the efficacy of the type II reagents because type I mAbs mutated in the Fc domain (K322A) to prevent C1q binding still did not display equivalent efficacy. These results give support for the use of type II CD20 mAbs in human B-cell diseases.     

Van Buchem disease: Clinical,biochemical, and densitometric features of patients and disease carriers

Van Buchem disease (VBD) is a rare bone sclerosing dysplasia caused by the lack of a regulatory element of the SOST gene, which encodes for sclerostin, an osteocyte‐derived negative regulator of bone formation. We studied the demographic, clinical, biochemical, and densitometric features of 15 patients with VBD (12 adults and 3 children) and 28 related carriers of the gene mutation. The most common clinical findings in patients were facial palsy (100%) and various degrees of hearing impairment (93%); raised intracranial pressure had been documented in 20%. The clinical course of the disease appeared to stabilize in adulthood, with the majority of patients reporting no progression of symptoms or development of complications with time. Carriers of the disease had none of the clinical features or complications of the disease. Sclerostin could be detected in the serum in all but 1 VBD patients (mean 8.0 pg/mL; 95% confidence interval [CI], 4.9–11.0 pg/mL), and were lower than those of carriers (mean 28.7 pg/mL; 95% CI, 24.5–32.9 pg/mL; p < 0.001) and healthy controls (mean 40.0 pg/mL; 95% CI, 34.5–41.0 pg/mL; p < 0.). Serum procollagen type 1 amino‐terminal propeptide (P1NP) levels were also significantly higher in adult patients (mean 96.0; 95% CI, 54.6–137.4 ng/mL versus mean 47.8; 95% CI, 39.4–56.2 ng/mL, p = 0.003 in carriers and mean 37.8; 95% CI, 34.5–41.0 ng/mL, p = 0.028 in healthy controls) and declined with age. Bone mineral density (BMD) was markedly increased in all patients (mean Z‐score 8.7 ± 2.1 and 9.5 ± 1.9 at the femoral neck and spine, respectively); BMD of carriers was significantly lower than that of patients but varied widely (mean Z‐scores 0.9 ± 1.0 and 1.3 ± 1.5 at the femoral neck and spine, respectively). Serum sclerostin levels were inversely correlated with serum P1NP levels (r = –0.39, p = 0.018) and BMD values (femoral neck r = –0.69, p < 0.001; lumbar spine r = –0.78, p < 0.001). Our results show that there is a gene‐dose effect of the VBD deletion on circulating sclerostin and provide further in vivo evidence of the role of sclerostin in bone formation in humans. The small amounts of sclerostin produced by patients with VBD may explain their milder phenotype compared to that of patients with sclerosteosis, in whom serum sclerostin is undetectable. © 2013 American Society for Bone and Mineral Research.     

MR T1ρ as an imaging biomarker for monitoring liver injury progression and regression: an experimental study in rats with carbon tetrachloride intoxication

Objectives

Recently it was shown that the magnetic resonance imaging (MRI) T1ρ value increased with the severity of liver fibrosis in rats with bile duct ligation. Using a rat carbon tetrachloride (CCl₄) liver injury model, this study further investigated the merit of T1ρ relaxation for liver fibrosis evaluation.

Methods

Male Sprague-Dawley rats received intraperitoneal injection of 2?ml/kg CCl₄ twice weekly for up to 6?weeks. Then CCl₄ was withdrawn and the animals were allowed to recover. Liver T1ρ MRI and conventional T2-weighted images were acquired. Animals underwent MRI at baseline and at 2?days, 2?weeks, 4?weeks and 6?weeks post CCl₄ injection, and they were also examined at 1?week and 4?weeks post CCl₄ withdrawal. Liver histology was also sampled at these time points.

Results

Liver T1ρ values increased slightly, though significantly, on day 2, and then increased further and were highest at week 6 post CCl₄ insults. The relative liver signal intensity change on T2-weighted images followed a different time course compared with that of T1ρ. Liver T1ρ values decreased upon the withdrawal of the CCl₄ insult. Histology confirmed the animals had typical CCl₄ liver injury and fibrosis progression and regression processes.

Conclusions

MR T1ρ imaging can monitor CCl₄-induced liver injury and fibrosis.

Key Points

? MR T1ρ is a valuable imaging biomarker for liver injury/fibrosis. ? Liver T1ρ was only mildly affected by oedema and acute inflammation. ? Liver MR T1ρ decreased when liver fibrosis and injury regressed.