Overcoming inefficient secretion of recombinant VEGF-C in baculovirus expression vector system by simple purification of the protein from cell lysate.

The first reports about successfully expressed recombinant proteins with the use of a baculovirus vector were published over 30years ago. Despite the long time of refining this expression system, early problems with the production of baculovirus-derived secretory proteins are still not satisfactorily solved. The high expression level driven by baculoviral promoters often does not result in the desired yield of secreted recombinant proteins, which frequently accumulate inside insect cells and are only partially processed. During our attempts to produce vascular endothelial growth factor C (VEGF-C) with the use of a baculovirus vector we also faced an inefficient secretion of the recombinant protein to culture medium. We were not able to improve the outcome and obtain an acceptable concentration of VEGF-C in the medium by changing the culture conditions or utilizing different signal peptides. However, as a significant amount of native VEGF-C was detected inside the baculovirus-infected cells, we developed a simple method to purify recombinant, glycosylated VEGF-C from a lysate of the cells. The presented results indicate that the lack of a secretory protein in the insect cell culture medium after baculovirus infection does not necessarily signify failure in the production of the protein. As demonstrated by us and contrary to generally accepted views, the lysate of baculovirus-infected cells may constitute a valuable source of the biologically active, secretory protein.

Effects of the time of antihypertensive drugs administration on the stage of primary open-angle glaucoma in patients with arterial hypertension.

Many patients with glaucoma suffer from arterial hypertension (AH). It has been proved that both AH and low blood pressure (BP) at night are important vascular risk factors for primary open-angle glaucoma (POAG). The aims of this study were to assess the severity of pathological changes within the optic nerve and characteristics of blood flow in selected arteries of the eyeball and orbit in patients with POAG and controlled hypertension, in relation to the time of hypotensive drugs administration. Eighty-eight patients with POAG and treated, controlled hypertension were examined. The patients were divided into two subgroups, consisting of group A (n = 43), in whom hypotensive drugs were dosed only in the morning and group B (n = 45), in whom hypotensive drugs were also taken in the evening. In patients who were taking hypotensive drugs also in the evening (group B), there was a statistically significant lower mean perfusion pressure at night, a greater visual field loss and reduced amplitude of evoked potentials. Our analysis showed significantly worse changes in the parameters relating to the optic nerve in patients taking hypertensive medicines in the evening and also significantly lower perfusion pressures at night.

A marked fall in nocturnal blood pressure is associated with the stage of primary open-angle glaucoma in patients with arterial hypertension.

INTRODUCTION: The aim of this study was to assess blood flow in the vessels of the eyeball and changes in the optic nerve in patients with arterial hypertension and primary open-angle glaucoma. MATERIAL AND METHODS: The patients were divided into groups: 1 (night blood pressure, BP, fall, NBPF, not more than 10%; non-dippers); 2 (NBPF 10-15%, dippers) and 3 (NBPF>15%; extreme dippers). RESULTS: In the group of dippers and extreme dippers, perfusion pressure was significantly lower than that in the non-dippers group, there was reduced thickness of the nerve fibers and a greater decrease in the visual field. Significant relationships between peak systolic, end-diastolic flow in the ophthalmic and central retinal arteries and night perfusion pressure, thickness of nerve fibers, and a loss of visual field were observed. CONCLUSION: In patients with glaucoma and well-controlled hypertension, a nocturnal BP fall of more than 10% is associated with a greater visual field defect and greater degeneration of the optic nerve fibers. Low minimum diastolic pressure and the level of nocturnal BP fall, but not the absolute value of average arterial BP at night, should be included in the group of specific risk factors in patients with hypertension and open-angle glaucoma. These findings also suggest avoiding excessive lowering of BP at night in this group.

Recommendations for ultrasound examination in ophthalmology. Part II: Orbital ultrasound.

Pathological processes within the orbits are a heterogeneous group of diseases of various etiologies, clinical pictures and therapy models. Due to poor access to the orbits in a clinical examination, imaging plays a significant role in both diagnosis and treatment monitoring in patients with an orbital pathology. One of such imaging modalities is ultrasonography. It is relatively well-available, rapid and safe for the patient. This paper enumerates indications for an orbital ultrasound scan, including functional ocular disorders (vision disorders, mobility disorders), autoimmune diseases, inflammatory conditions, proliferative processes, and others. The authors present Ossoinig's standardized method which encompasses topographic, qualitative and kinetic echography, and may facilitate orbital ultrasound examinations. Moreover, the article shows management standards for ultrasound imaging of orbital pathologies with an emphasis on the relevance of equipment selection, scanning technique (transducer position, transocular technique, paraocular technique) and patient preparation for the examination, and indicates appropriate elements of an examination report. The authors discuss the ultrasound presentation of the orbital structures in physiological conditions and selected orbital pathologies, such as pseudotumor, thyroid orbitopathy, cancerous tumors of the optic nerve, and others. The ultrasonographic characteristics of the presented pathologies are shown taking into account A and B scans. Attention was paid to the evaluation of angle kappa in the A scan in echographic assessment of the orbits. Furthermore, the authors include referential values for extraocular muscle thickness and quantitative measurement of the severity of thyroid ophthalmopathy based on Ossoinig's muscle index.Pathological processes within the orbits are a heterogeneous group of diseases of various etiologies, clinical pictures and therapy models. Due to poor access to the orbits in a clinical examination, imaging plays a significant role in both diagnosis and treatment monitoring in patients with an orbital pathology. One of such imaging modalities is ultrasonography. It is relatively well-available, rapid and safe for the patient. This paper enumerates indications for an orbital ultrasound scan, including functional ocular disorders (vision disorders, mobility disorders), autoimmune diseases, inflammatory conditions, proliferative processes, and others. The authors present Ossoinig's standardized method which encompasses topographic, qualitative and kinetic echography, and may facilitate orbital ultrasound examinations. Moreover, the article shows management standards for ultrasound imaging of orbital pathologies with an emphasis on the relevance of equipment selection, scanning technique (transducer position, transocular technique, paraocular technique) and patient preparation for the examination, and indicates appropriate elements of an examination report. The authors discuss the ultrasound presentation of the orbital structures in physiological conditions and selected orbital pathologies, such as pseudotumor, thyroid orbitopathy, cancerous tumors of the optic nerve, and others. The ultrasonographic characteristics of the presented pathologies are shown taking into account A and B scans. Attention was paid to the evaluation of angle kappa in the A scan in echographic assessment of the orbits. Furthermore, the authors include referential values for extraocular muscle thickness and quantitative measurement of the severity of thyroid ophthalmopathy based on Ossoinig's muscle index.

Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies.

Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab')2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the µ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool.

A new expression vector facilitating production and functional analysis of scFv antibody fragments selected from Tomlinson I+J phagemid libraries.

Tomlinson I+J are synthetic phagemid human scFv libraries widely employed to obtain specific antibody fragments via a phage display method. The pIT2/HB2151 expression system proposed by the designers of the libraries has certain drawbacks which result in the lack of expression or low expression levels of numerous soluble scFvs. At the stage of scFv screening, this may lead to losing some excellent antibodies, which can be avoided but requires laborious and expensive work. Here we present a new, pET-30-based vector, which is compatible with Tomlinson libraries, retains all virtues of pIT2 used as a plasmid and eliminates all its flaws. We demonstrate that pET-scFv-T is frequently superior to pIT2 in terms of efficient scFv expression. Moreover, an amber suppressor bacterial strain, RosettaBlue(DE3)pLysS, transformed with the new vector, pET-scFv-T, coding for a number of scFvs, produces substantial amounts of functional, easy to purify recombinant antibody fragments, regardless of whether their coding sequences contain amber codons. Thus, pET-scFv-T/RosettaBlue(DE3)pLysS expression system seems to be a perfect tool for screening for the finest soluble scFvs selected from Tomlinson I+J, as well as from many other phagemid libraries.

Chromosome study of Lithobius forficatus (Lithobiomorpha, Chilopoda).

The diploid number 2n = 46 and the chromosome arm number NF = 74 are described in Lithobius forficatus from Olsztyn (Poland). Analyses of silver and CMA3-stained mitotic chromosomes suggest that a single chromosome pair has active NORs which correspond to G-C-rich (CMA3-positive) chromatin. Heteromorphism of the largest metacentric chromosome pair was observed. The sex chromosomes were not identified. Size polymorphism of the first chromosome pair was found.

[Surgical management of thyroid orbitopathy]. / Leczenie chirurgiczne orbitopatii tarczycowej.

Thyroid orbitopathy (TO) requires often not only pharmacological treatment but also surgical procedures. The management contains orbital decompression (wall decompression, excision of fat), correction of lid retraction and strabismus operations on extraocular muscles. Authors describe indications for surgical procedures, methods of operations, results and complications of own material of 99 patients with TO treated in the Eye Clinic in Poznan and Plastic Surgery and Eye Clinic in Kobylniki from 1999 until 2003.

[Glaucoma and ocular ischemic syndrome--case report]. / Jaskra a oczny zespól niedokrwienny--opis przypadku.

PURPOSE: Ocular ischemic syndrome (OIS) is often poorly diagnosed and treated as primary open angle glaucoma or later on, as neovascular glaucoma. We present a 54 year old male, treated topical since 23 years for glaucoma and sent to our clinic for trabeculectomy because of rapid worsening of vision on right eye with bilateral total excavation of optic disc. MATERIAL AND METHODS: Observational case report. RESULTS: Because of typical signs of IOS (iris neovascularization, mid-peripheral dot and blot hemorrhages in both eyes, narrowed arterioles in right eye, following examinations were performed: Doppler ultrasonography of carotid arteries, digital subtractional angiography of the carotid vessels and magnetic resonance angiography. The examinations showed occlusion of the right common carotid artery and with 80% stenosis of the left common carotid artery, occlusion of abdominal aorta. After phacoemulsification with implantation of intraocular lens because of rapid intumescence cataract in the right eye, and endarterectomy of left external carotid artery, the neovascularization of the iris regressed in both eyes. CONCLUSION: In case of iris neovascularization or mid-peripheral hemorrhages the Doppler sonography of carotid arteries should be performed. Quick cooperation between ophthalmologist, radiologist and vascular surgeon following endarterectomy seems to stop progressing changes of ocular ischemic syndrome.

Antibody-based antiangiogenic and antilymphangiogenic therapies to prevent tumor growth and progression.

Blood and lymphatic vessel formation is an indispensable factor for cancer progression and metastasis. Therefore, various strategies designed to block angiogenesis and lymphangiogenesis are being investigated in the hope to arrest and reverse tumor development. Monoclonal antibodies, owing to their unequalled diversity and specificity, might be applied to selectively inhibit the pathways that cancer cells utilize to build up a network of blood vessels and lymphatics. Among the possible targets of antibody-based therapies are proangiogenic and prolymphangiogenic growth factors from the VEGF family and the receptors to which they bind (VEGFRs). Here, we present molecular mechanisms of angiogenesis and lymphangiogenesis exploited by tumors to progress and metastasise, with examples of antibody-based therapeutic agents directed at interfering with these processes. The expanding knowledge of vascular biology helps to explain some of the problems encountered in such therapies, that arise due to the redundancy in signaling networks controlling the formation of blood and lymphatic vessels, and lead to tumor drug resistance. Nonetheless, combined treatments and treatments focused on newly discovered proangiogenic and prolymphangiogenic factors give hope that more prominent therapeutic effects might be achieved in the future.

Observations on degenerative changes within the optic nerve in patients with primary open glaucoma and arterial hypertension: 6-month follow-up.

The authors aimed to determine the effect of the time of hypotensive drug administration on the progress of degenerative changes within the optic nerve in patients with hypertension and glaucoma. Two groups were included in the study: group A comprised patients-dippers taking drugs in the mornings, and group B comprised patients-nondippers taking drugs both in the mornings and in the evenings. After 6 months, group B showed significant drops in nocturnal diastolic blood pressure (BP) (month 1=73.27 mm Hg vs month 6=67.50 mm Hg), nocturnal mean BP (89.34 vs 84.65 mm Hg), and minimum diastolic BP (50.74 vs 44.03 mm Hg). Group B also showed significant reductions in nocturnal ocular perfusion pressure (43.0 vs 39.73), retinal nerve fiber layer thickness (131.31 vs 113.12 µm), and flow in the eye vessels. Taking hypotensive drugs in the evening may significantly decrease blood flow in the eye arteries, cause degenerative changes within the optic nerves, and result in greater loss in the field of vision.