Improving expression of recombinant human IGF-1 using IGF-1R knockout CHO cell lines.

Chinese Hamster Ovary (CHO) cells are widely used for the large-scale production of recombinant biopharmaceuticals. However, attempts to express IGF-1 (a mutated human Insulin-like growth factor 1 Ea peptide (hIGF-1Ea mut)) in CHO cells resulted in poor cell growth and low productivity (0.1-0.2 g/L). Human IGF-1 variants negatively impacted CHO cell growth via the IGF-1 receptor (IGF-1R). Therefore knockout (KO) of the IGF-1R gene in two different CHO cell lines as well as knockdown (KD) of IGF-1R in one CHO cell line were performed. These cell line engineering approaches decreased significantly the hIGF-1 mediated cell growth inhibition and increased productivity of both KO CHO cell lines as well as of the KD CHO cell line. A productivity increase of 10-fold at pool level and sevenfold at clone level was achieved, resulting in a titer of 1.3 g/L. This data illustrate that cell line engineering approaches are powerful tools to improve the yields of recombinant proteins which are difficult to produce in CHO cells.

Skeletal muscle metastases: primary tumours, prevalence, and radiological features.

BACKGROUND: Although skeletal muscles comprise nearly 50% of the total human body mass and are well vascularised, metastases in the musculature are rare. The reported prevalence of skeletal muscle metastases from post-mortem studies of patients with cancer is inconstant and ranges from 0.03 to 17.5%. MATERIALS AND METHODS: Of 5,170 patients with metastasised cancer examined and treated at our institution during the period from January 2000 to December 2007, 61 patients with muscle metastases (80 lesions) were identified on computed tomography (CT). Genital tumours (24.6%) were the most frequent malignancies metastasising into the skeletal musculature, followed by gastrointestinal tumours (21.3%), urological tumours (16.4%), and malignant melanoma (13.1%). Other primary malignancies were rarer, including bronchial carcinoma (8.2%), thyroid gland carcinoma (4.9%), and breast carcinoma (3.3%). In 8.2%, carcinoma of unknown primary was diagnosed. RESULTS: Skeletal muscle metastases (SMM) were located in the iliopsoas muscle (27.5%), paravertebral muscles (25%), gluteal muscles (16.3%), lower extremity muscles (12.5%), abdominal wall muscles (10%), thoracic wall muscles (5%), and upper extremity muscles (3.8%). Most (76.3%) of the 80 SMM were diagnosed incidentally during routine staging CT examinations, while 23.7% were symptomatic. CONCLUSION: Radiologically, SMM presented with five different types of lesions: focal intramuscular masses (type I, 52.5% of SMM), abscess-like intramuscular lesions (type II, 32.5%), diffuse metastatic muscle infiltration (type III, 8.8%), multifocal intramuscular calcification (type IV, 3.7%) and intramuscular bleeding (type V, 2.5%).

Radial optic neurotomy for central retinal vein occlusion: long-term retinal perfusion outcome.

BACKGROUND/AIMS: To analyze the long-term changes in retinal perfusion and functional improvement induced by radial optic neurotomy (RON) in central retinal vein occlusion (CRVO). METHODS: Sixty-three eyes of 63 consecutive patients with CRVO were included. Twenty-eight (44.5%) patients underwent RON and 35 (55.5%) were followed as a control group. Time of arteriovenous transit and visual acuity were determined at baseline and after 1-year follow-up. RESULTS: After 1 year, retinal perfusion status improved in 63.1% of operated eyes and 14.3% of controls (p = 0.048). The improvement in arteriovenous retinal transit was statistically significant (p = 0.023) only in the RON group. The visual improvement in the RON group was significantly better (p = 0.043) than in the control group. Moreover, the development of chorioretinal anastomosis was significantly higher (p = 0.036) after RON than in controls and correlated with better functional results. In operated eyes there was a lower incidence of CRVO-related complications. CONCLUSION: RON improved retinal perfusion and achieved a better functional outcome. The measurement of perfusion changes as presented here may be useful for monitoring CRVO and assessing other treatment modalities.

Towards absolute quantification of therapeutic monoclonal antibody in serum by LC-MS/MS using isotope-labeled antibody standard and protein cleavage isotope dilution mass spectrometry.

Although LC-MS methods are increasingly used for the absolute quantification of proteins, the lack of appropriate internal standard (IS) hinders the development of rapid and standardized analytical methods for both in vitro and in vivo studies. Here, we have developed a novel method for the absolute quantification of a therapeutic protein, which is monoclonal antibody (mAb). The method combines liquid chromatography tandem mass spectrometry (LC-MS/MS) and protein cleavage isotope dilution mass spectrometry with the isotope-labeled mAb as IS. The latter was identical to the analyzed mAb with the exception that each threonine contains four (13)C atoms and one (15)N atom. Serum samples were spiked with IS prior to the overnight trypsin digestion and subsequent sample cleanup. Sample extracts were analyzed on a C18 ACE column (150 mm x 4.6 mm) using an LC gradient time of 11 min. Endogenous mAb concentrations were determined by calculating the peak height ratio of its signature peptide to the corresponding isotope-labeled peptide. The linear dynamic range was established between 5.00 and 1000 microg/mL mAb with accuracy and precision within +/-15% at all concentrations and below +/-20% at the LLOQ (lower limit of quantification). The overall method recovery in terms of mAb was 14%. The losses due to sample preparation (digestion and purification) were 72% from which about 32% was due to the first step of the method, the sample digestion. This huge loss during sample preparation strongly emphasizes the necessity to employ an IS right from the beginning. Our method was successfully applied to the mAb quantification in marmoset serum study samples, and the precision obtained on duplicate samples was, in most cases, below 20%. The comparison with enzyme-linked immunosorbent assay (ELISA) showed higher exposure in terms of AUC and Cmax with the LC-MS/MS method. Possible reasons for this discrepancy are discussed in this study. The results of this study indicate that our LC-MS/MS method is a simple, rapid, and precise approach for the therapeutic mAb quantification to support preclinical and clinical studies.

Modified transscleral suture for sulcus fixation of posterior chamber lenses.

We describe a modified technique to minimize the risk of hypotony during the placement of transscleral anchoring sutures. An anchor thread is used to transsclerally fixate a posterior chamber intraocular lens (IOL). The IOL is placed precisely and without trauma. The technique was used in 17 patients. Postoperatively, all IOLs were well centered. Visual acuity increased from a preoperative mean of 0.25 to a postoperative mean of 0.40. The mean postoperative refraction was -0.75 diopter (D) (range +0.50 to -4.50 D). Longer phases of hypotony do not occur, and the use of CIF needles can be omitted.

Effect of pars plana vitrectomy for proliferative diabetic vitreoretinopathy on preexisting diabetic maculopathy.

BACKGROUND: Vitrectomy is the treatment of choice for proliferative diabetic vitreoretinopathy with tractions and persistent vitreous hemorrhage. Since vitrectomy has recently been discussed as a possible therapy for diabetic maculopathy as well, we were especially interested in studying the change in diabetic maculopathy following surgery. For that purpose a grading system developed at our clinic was used. METHODS: In a retrospective study we evaluated fundus photographs and fluorescein angiograms of 33 eyes of 30 patients who had undergone vitrectomy for proliferative diabetic vitreoretinopathy at our clinic between 1990 and 1997. In all eyes diabetic maculopathy was present at the time of surgery. The grading was performed using preoperative images and images taken a median of 18 months postoperatively. RESULTS: Best corrected visual acuity increased by 3.7 lines on average. Intraretinal dot and spot hemorrhages, hard exudates, microaneurysms on fundus photos, and leakage and cysts on fluorescein angiograms decreased after surgery. The extent of the foveolar avascular zone and the extent of the perifoveolar ischemic area worsened, however. CONCLUSION: Vitrectomy seems to help diabetic eyes not only by removal of membranes, tractions, and vitreous hemorrhage; it seems to have a positive influence on the course of diabetic maculopathy as well. We suspect that the removal of the posterior vitreous membrane is one of the crucial factors in interrupting the disease process. From these results the indication for vitrectomy in diabetic patients may have to be reconsidered and extended to include diabetic maculopathy prior to the development of ischemia.

A modified technique to stain the internal limiting membrane with indocyanine green.

PURPOSE: Peeling of the internal limiting membrane (ILM) has improved the outcomes in vitreoretinal surgery of macular diseases. Indocyanine green (ICG) is used to stain and improve the visualization of the ILM. This paper aims to describe a modified approach to stain and peel the ILM avoiding potential adverse side effects. METHODS: After a core vitrectomy, 0.05 ml of ICG in a concentration of 5 mg/ml is quickly injected under water around the macular region. The tip of the syringe is positioned about 5 mm from the macular tissue, in a way that about a 3-mm diameter area around the fovea is stained by the ICG. Additional ICG is immediately removed by aspiration. Peeling of the ILM is accomplished with a forceps. RESULTS: Neither residual ICG in the vitreous cavity nor any clinical signs of phototoxicity like retinal edema were detected in any patients operated by this technique. CONCLUSIONS: This modified technique to stain the ILM limits the amount and concentration of ICG. The locally limited contact of ICG with the retinal surface seems to be a safe procedure to stain the ILM.