Cell-free synthesis of functional antibodies using a coupled in vitro transcription-translation system based on CHO cell lysates.

Antibodies are indispensable tools for basic research as well as diagnostic and therapeutic applications. Consequently, the development of alternative manufacturing strategies which circumvent the hurdles connected to conventional antibody production technologies is of enormous interest. To address this issue, we demonstrate the synthesis of complex antibody formats, in particular immunoglobulin G (IgG) and single-chain variable fragment Fc fusion (scFv-Fc), in a microsome-containing cell-free system based on translationally active chinese hamster ovary (CHO) cell lysates. To mimic the environment for antibody folding and assembly present in living cells, antibody genes were fused to an endoplasmic reticulum (ER)-specific signal sequence. Signal-peptide induced translocation of antibody polypeptide chains into the lumen of ER microsomes was found to be the prerequisite for antibody chain assembly and functionality. In this context, we show the rapid synthesis of antibody molecules in different reaction formats, including batch and continuous-exchange cell-free (CECF) reactions, depending on the amount of protein needed for further analysis. In addition, we demonstrate site-specific and residue-specific labeling of antibodies with fluorescent non-canonical amino acids. In summary, our study describes a novel antibody production platform which combines the highly efficient mammalian protein folding machinery of CHO cells with the benefits of cell-free protein synthesis.

Prevention of Allograft Rejection by Use of Regulatory T Cells With an MHC-Specific Chimeric Antigen Receptor.

CD4+ CD25high FOXP3+ regulatory T cells (Tregs) are involved in graft-specific tolerance after solid organ transplantation. However, adoptive transfer of polyspecific Tregs alone is insufficient to prevent graft rejection even in rodent models, indicating that graft-specific Tregs are required. We developed a highly specific chimeric antigen receptor that recognizes the HLA molecule A*02 (referred to as A2-CAR). Transduction into natural regulatory T cells (nTregs) changes the specificity of the nTregs without alteration of their regulatory phenotype and epigenetic stability. Activation of nTregs via the A2-CAR induced proliferation and enhanced the suppressor function of modified nTregs. Compared with nTregs, A2-CAR Tregs exhibited superior control of strong allospecific immune responses in vitro and in humanized mouse models. A2-CAR Tregs completely prevented rejection of allogeneic target cells and tissues in immune reconstituted humanized mice in the absence of any immunosuppression. Therefore, these modified cells have great potential for incorporation into clinical trials of Treg-supported weaning after allogeneic transplantation.

Multimerization domains for antibody phage display and antibody production.

High-throughput generation of antibodies for proteome research has become feasible by using antibody gene libraries and in vitro selection methods like phage display. Typically monovalent antibody fragments like scFv, Fab or scFab are obtained by this technology. To mimic the IgG molecule and gain avidity, resulting in stronger binding, multimerization domains can be fused to antibody fragments. Here we systematically analyzed different multimerization domains in respect to three key parameters, crucial for the high-throughput generation of binders. (i) The compatibility to be displayed on phage (assessed for at least three different antibody formats, scFv, Fab and scFab) in combination with five different multimerization domains; (ii) production yields and (iii) oligomerization properties were analyzed for three different scFv fragments. We found that the use of a biotin acceptor domain in combination with an in vivo biotinylation system performed best concerning the key parameters and thus would be a useful tool to generate multimeric antibody complexes on demand from phage display selected antibody fragments with the least effort.

Functional knockdown of VCAM-1 at the posttranslational level with ER retained antibodies.

Vascular cell adhesion molecule 1 (VCAM-1) is involved in the recruitment of leukocytes to inflammatory sites. In this study we present the first functional knockdown of VCAM-1 using an ER retained antibody construct. We generated a knockdown construct encoding the VCAM-1 specific single chain variable fragment scFv6C7.1 fused to the C-terminal ER retention sequence KDEL. HEK-293:VCAM-YFP cells stably expressing a VCAM-YFP fusion protein were transiently transfected with the knockdown construct and showed down-regulation of surface VCAM-1. Knockdown efficiency of the system is time-dependent due to used transient transfection of the intrabody construct. Furthermore, intrabody mediated knockdown of HEK-293:VCAM-YFP cells also impaired cell-cell interaction with Jurkat cells that are endogenously expressing VLA-4, the physiological partner of VCAM-1. Posttranslational knockdown with ER retained antibodies seems to be a promising technique, as shown here for VCAM-1.

Parameters affecting the display of antibodies on phage.

Despite the fact that a multitude of antibody phage display libraries has been built, systematic comparisons of critical design parameters are rare. Here we analysed the impact of various factors on the performance of the phage display system. First, we compared several vector designs for the display of Fab fragments of antibodies. Bicistronic as well as monocistronic expression of the antibody/pIII operon and vectors using fd-pIII as well as LC-pIII fusions were tested. Further, we evaluated the influence of glucose on the promoter induction. We compared monovalent versus oligovalent display of the antibody fragments and we used antibody fragments with different folding efficiency to assess the influence of the individual antibody sequences on the performance of the system. Finally, both phage display efficiency and yield of soluble Fab fragments were analysed. The significant differences found for phage yield, display of Fabs on the phage and expression of soluble Fabs suggest to use a bicistronic vector with an fd-fragment-pIII fusion for the construction of future Fab phage display libraries.

[Intrapericardial fibrinolysis as a therapeutic option in a case of purulent pericarditis]. / Intraperikardiale Fibrinolyse als therapeutische Option bei der Perikarditis purulenta.

HISTORY AND ADMISSION FINDINGS: A 23-year-old man known to have Hodgkin's disease in remission and C-protein deficiency presented with chest pain and symptoms of respiratory infection. INVESTIGATIONS: A purulent pericardial effusion was detected by echocardiography. The chest X-ray showed a left-sided pneumonia. TREATMENT AND CLINICAL COURSE: When pericardiocentesis failed to drain the purulent but pathogen-free effusion completely, streptokinase was several times instilled intrapericardially and achieved fibrinolysis without complication. The effusion did not recur. CONCLUSIONS: Intrapericardial application of fibrinolytic agents seems to be an effective treatment of a purulent pericarditis.

[Persistent left superior vena cava with right-left shunt into the left atrium]. / Persistierende linke Vena cava superior mit Rechts-Links-Shunt zum linken Atrium.

UNLABELLED: Persistent left superior vena cava with right-left shunt into the left atrium. HISTORY AND CLINICAL FINDINGS: A 72-year-old patient was admitted to the hospital following bleeding into the basal ganglia secondary to a hypertensive crisis. INVESTIGATIONS: The patient was found to suffer from marked hypoxaemia (pO2 49 mmHg) and erythrocytosis (Hb 18,5 g/dl). Subsequent investigations raised suspicion of a right-left shunt. This was verified by a contrast echocardiogram which was performed transthoracically by injection of echo-contrast material from the left. To improve imaging of the shunt a transoesophageal contrast-echocardiogram was carried out. This showed that the persistent left superior vena cava did not, as previously expected, lead directly into the left atrium, but had a connection to the left superior pulmonary vein. This anatomical variant, which so far to our knowledge has not been reported in the literature, could be confirmed by spiral computed tomography. Apart from an atrial septal aneurysm no other cardiac anomaly could be identified. TREATMENT AND COURSE: Ligation of the left superior vena cava could have been a therapeutic option, but the patient declined operative intervention. CONCLUSION: In cases of profound hypoxemia and erythrocytosis the differential diagnosis must include a persistent left superior vena cava with anomalous connection to the left atrium. Trans-thoracic and transoesophageal contrast-echocardiography is a simple and reliable method to diagnose persistent left superior vena cava as well as concomitant cardiac anomalies.

Value of repeated multiplane transesophageal echocardiography in a patient with mitral valve ring abscess and left ventricular pseudoaneurysm.

Mitral valve ring abscess and ventricular pseudoaneurysm are rare complications of infective endocarditis. We describe the case of a 58-year-old man who was admitted to our hospital with sepsis caused by Staphylococcus aureus and in whom tricuspid and mitral valve endocarditis developed within 2 weeks. Despite widespread antibiotic therapy, the endocarditis proceeded to form a mitral valve ring abscess and a left ventricular pseudoaneurysm. The diagnosis was set by repeated multiplane transesophageal echocardiography and confirmed by heart surgery.

An immunochemical in situ approach to detect adaptation processes in the photosynthetic apparatus of diatoms of the Wadden Sea sediment surface layers.

Intertidal Wadden Sea sediment surface layers located near the North Sea shore at Dangast (Germany) were subjected to quantitative chlorophyll and protein extractions followed by SDS-PAGE and Western immunoblotting. During the study, benthic diatoms were almost exclusively the only group of microphytobenthos in this area performing oxygenic photosynthesis. Three successive extractions with 90% acetone yielded more than 98% of the extractable pigments. The absorption spectra of the extracts of sediment samples were nearly identical to those obtained from the diatom Phaeodactylum tricornutum. Ten repetitive extractions with SDS containing loading buffer used for SDS-PAGE ensured that more than 98% of the extractable protein was recovered. Subsequent Western immunoblotting with an antiserum directed against the subunits of the main light harvesting complex of the diatom Cyclotella cryptica showed that the antiserum immunodecorated selectively subunits of diatomaceous light harvesting complexes. This finding demonstrated that a taxon specific class of polypeptides could be visualized and quantified directly in sediment samples. In shading experiments, shaded sedimient areas generally revealed higher amounts of light harvesting subunits which could be immunodecorized. The improved mnethodological approach and the results are discussed in the context of the current development of direct molecular methods for the investigation of activities and adaptation processes of specific groups of microorganisms in their natural habitats.

[Neurocardiogenic syncopes in patients with implanted pacemakers]. / Neurokardiogene Synkopen bei Herzschrittmachertherapie.

BACKGROUND AND OBJECTIVE: Recently the tilting table test (tilting up to 70 degrees angle) has established itself for the diagnosis of neurocardiogenic syncope (NS). Usually patients with bradycardic arrhythmias or asystole are treated by pacemaker implantation. But if the syncope is of neurocardiogenic aetiology other therapeutic alternatives must be chosen. We here report on seven patients in whom a pacemaker had been implanted in the treatment of syncopes, in ignorance of their neurocardiogenic aetiology, yet they had recurred. PATIENTS AND METHODS: Pacemakers had been implanted in eight patients: two with sick sinus syndrome, three with sinus bradycardia, one with brady-tachyarrhythmia, one with asystole and one with Mobitz-type 2 degrees AV block. All patients continued to have syncopes, when sitting or standing, months to years after the pacemaker implantation. The tilting table test (up to 30 min at an angle of 70 degrees) was positive in all patients. There was no case of pacemaker malfunction. RESULTS: One patient declined further treatment. The remaining seven patients were symptom-free in the tilting table test on various medications: theophylline up to 2 x 350 mg, metoprolol up to 2 x 100 mg or disopyramide 3 x 100 mg. CONCLUSION: Patients with syncope while sitting or standing should undergo the tilting table test to determine whether the cause is neurocardiogenic so that pacemaker implantation can be avoided. The majority of patients with NS can be successfully treated by drugs. But in some patients pacemaker implantation may have to considered if drug treatment has failed.

Vertical migration behaviour of diatom assemblages of Wadden Sea sediments (Dangast, Germany): a study using cryo-scanning electron microscopy.

The vertical migration behaviour of diatom assemblages inhabiting Wadden Sea sediments near Dangast (Germany) was investigated using cryo-scanning electron microscopy. The diatom assemblages were dominated by small Navicula species. Intertidal sediments which were located at different distances from the high tide level or stayed submerged even throughout low tides were chosen. Samples were prepared and cryofixed in the field. Sampling was restricted to three sets: (i) before the onset of vertical migration, (ii) 3 to 5 h after the onset of vertical migration, and (iii) before the area became flooded again or just prior to dusk. The diatom assemblages inhabiting the different types of sediments did not always show the same response. When the tidal cycle exposed the sediment surfaces during the night cell densities increased in the early morning hours with the onset of light. Later on, although the photon flux density was still increasing, cell densities stayed constant or decreased before the water flooded the areas around noon. In experiments in which the water drained off around noon and the areas became exposed throughout the entire afternoon, cell densities increased even up to dusk when the photon flux density had dropped to values below 20 microM photons m-2 s-1. In an experiment in which the last sampling occurred at 10.15 pm, when the photon flux density had already declined below 10 microM photons m-2 s-1, cell densities had decreased to lower values. This was ca. 1 h before the area was flooded again. Finally, cryo-scanning electron microscopy revealed frequently occurring micro-patches of diatom assemblages which could be differentiated into typical areas of lower and higher cell densities further complicating the pattern of light or water cover induced movements.

[Puerperal sepsis caused by streptococcus group A with a severe form of progression like "toxic shock-like syndrome"]. / Puerperalsepis durch Streptokokken der Gruppe A mit schwerer Verlaufsform als "Toxic Shock-like Syndrome".

HISTORY AND CLINICAL FINDINGS: A previously healthy 32-year-old woman was admitted with shock symptoms 5 days after an uneventful home delivery. She developed a fever up to 39.5 degrees C during the days after delivery with sore throat, diarrhoea and muscle aches in all limbs. On physical examination there were swellings of the arms and lower legs as well as macular and vesicular erythrodermia, especially of the trunk. INVESTIGATIONS: Abnormal laboratory findings were thrombocytopenia (20,000/microliters), increased serum concentrations of fibrin breakdown products (102 mg/dl) and of C-reactive protein (> 200 mg/dl), increased creatine kinase (5700 U/l), transaminases (GOT 220 U/l, GPT 52 U/l), creatinine (2.0 mg/dl) and urea (114 mg/dl). Streptococcus pyogenes was grown on blood culture and from vaginal smear. Sonography, echocardiography and radiological examinations failed to demonstrate a septic focus. TREATMENT AND COURSE: Mechanical ventilation was required for 7 days because of respiratory failure and shock symptoms (toxic shock-like syndrome, TSLS). Penicillin G and tobramycin were given after the bacteriological diagnosis. Severe consumption coagulopathy was successfully treated with antithrombin III and platelet concentrates. After extubation she was found to have a flaccid tetraparesis, especially of the right and of the legs, due to soft-tissue necrosis and damage to peripheral nerves. An embolic occlusion of the right brachial artery 4 weeks after onset of the disease required upper-arm amputation. CONCLUSION: One of the decisive factors for the prognosis of TSLS is early antibiotic treatment. The prodromal symptoms in this case underline the necessity of early recognition and treatment to prevent a full-blown picture of the syndrome.

[Ball variance and fracture of a Smeloff-Cutter prosthesis 24 years after aortic valve replacement]. / Ballvarianz und Fraktur einer Smeloff-Cutter-Prothese 24 Jahre nach Aortenklappenersatz.

This report documents a case of ball variance in a Smeloff-Cutter aortic prosthesis occurring 24 years after implantation. After episodes of embolic complications the patient died in acute shock. The silicone rubber ball showed several alterations including discoloration, grooving, cracking, swelling and subtotal fracture of the poppet. Terminal valvular malfunction was caused by complete thrombosis of the prosthesis. In most patients ball variance occurred during the first years after valve replacement; thus, the observed case is a very rare late complication of a ball-valve prosthesis.

[Complete gastric wall necrosis after endoscopic sclerotherapy for a gastric ulcer with visible arterial stump]. / Vollständige Magenwandnekrose nach endoskopischer Sklerosierungstherapie eines Ulcus ventriculi mit sichtbarem Gefässstumpf.

HISTORY AND CLINICAL FINDINGS: A 43-year-old man with a gastric ulcer was admitted because of sudden onset of epigastric pain, cold sweats and dizziness. He had tachycardia (100/min); his blood pressure was 120/80 mm Hg: his epigastrium was tender to palpation. There were no tarry stools. INVESTIGATIONS: Haemoglobin concentration was 12.7 g/dl. WBC count 17,900/microliter. Gastroscopy revealed residual haematin and an ulcer with an arterial stump at the angular fold. TREATMENT AND COURSE: 3 ml epinephrine, diluted 1:20,000, and 13 ml of 1% polidocanol were injected around the arterial stump, most of the latter solution flowing back into the gastric lumen from the rather hard ulcer base. Haematemesis four days later necessitated laparotomy followed by gastrectomy with reconstruction and a Roux-Y anastomosis because of complete necrosis of the gastric wall. Histological examination of the surgical specimen showed chronic scarred gastric ulcer and ulcerating pangastritis with haemorrhagic necrosis of the wall and associated peritonitis, caused by accidental injection of polidocanol into the artery. CONCLUSION: Since the tissue-sparing injection of epinephrine, fibrin glue or salt solution is alone effective in the endoscopic treatment of bleeding gastroduodenal ulcers, polidocanol should not be injected as well.

[Löffler's parietal fibroplastic endocarditis. Echocardiographic course over 5 years]. / Endocarditis parietalis fibroplastica Löffler. Echokardiographischer Verlauf über 5 Jahre.

HISTORY AND CLINICAL FINDINGS: A 28-year-old man, who had sustained a traumatic displaced fracture of the left lateral clavicle requiring operative realignment, was admitted to hospital because of the incidental finding of pathological electrocardiographic findings (suggestive of past anterior myocardial infarction). He had dyspnoea on effort and episodes of irregular tachycardia without other cardiac symptoms, as well as regular tachycardia (120/min) without pulse deficit. Body temperature was 37.3 degrees C, blood pressure 110/70 mm Hg. INVESTIGATIONS: Transoesophageal echocardiography showed regular myocardial contractility but a 3.27 cm cap-like area of echo-poor and echo-dense layers at the left ventricular apex. There was leucocytosis (25,400/microliter) with 45% eosinophilia (11,430/microliter). IgE was raised to 134.6 kU/l. Bone marrow smear showed marked eosinophilic but little neutrophilic hyperplasia. Parasitic infection was excluded. TREATMENT AND COURSE: The clinical and echocardiographic findings suggested the diagnosis of Löffler's eosinophilic endocarditis (endomyocardial fibrosis), later confirmed histologically. After initial treatment, first only with cortisone then also hydroxyurea, the eosinophilia finally responded to combined cortisone and interferon alpha 2. The dosage schedule was governed by the clinical and echocardiographic findings. In the further course of the disease there occurred progressive heart failure with severe tricuspid and mitral regurgitation, secondary pulmonary hypertension and severe fibrosis of both ventricles, 41 months after diagnosis the patient underwent tricuspid and mitral valve reconstruction with removal of the endocardial fibrotic layers. Follow-up examinations found the cardiac condition to be adequately controlled without further cardiac infiltration since 20 months. CONCLUSIONS: Echocardiography, in conjunction with the clinical findings, provides a firm foundation for successfully treating Löffler's endocarditis. In selected cases cardiac surgery can markedly improve the course.

[Tricuspid valvulectomy in antibiotic-refractory right-heart endocarditis]. / Trikuspidalvalvulektomie bei antibiotikarefraktärer Rechtsherzendokarditis.

HISTORY AND CLINICAL FINDINGS: A 19-year-old man, known to be addicted to intravenous heroin, was admitted because of respiratory failure with a septic fever up to 40.0 degrees C. An abscess at the site of intravenously heroin injection on the right arm had been previously opened. At examination he was pale and his general condition was poor. There were numerous puncture sites over lower arm veins, some of them thrombosed. There were no signs of septic emboli to the skin and to visible mucosae. The spleen was not palpable. INVESTIGATIONS: There was leucocytosis with shift to the left, thrombocytopenia (54,000/microliter), anaemia (haemoglobin 7.9 g/dl) and markedly raised C-reactive protein (202 mg/dl). Blood culture grew Staphylococcus aureus. Transthoracic and transoesophageal echocardiography revealed a large vegetation on the tricuspid valve, confirming infectious endocarditis involving the tricuspid valve. Chest radiogram showed an infiltrate and pleural effusion of the left lung. TREATMENT AND COURSE: Infectious emboli to the lung necessitated artificial ventilation. The septic process could not be controlled by antibiotics. The whole of the tricuspid valve was therefore removed, followed by high doses of antibiotics (vancomycine, gentamicine, rifampicine). This successfully controlled the infection and a postoperative sacroileitis. Postoperative echocardiography revealed severe tricuspid regurgitation with marked enlargement of the right ventricle, paradoxical movement of the interventricular septum and a dilated inferior vena cava. 7 months postoperatively there were no clinical signs of severe right heart failure. CONCLUSION: Removal of the tricuspid valve without valve replacement can be successfully undertaken to control the infection in intravenously heroin addicts in whom the valve has been destroyed by infectious endocarditis.

[Pre-syncope and syncope at the operating table--diagnostic and therapeutic possibilities]. / Präsynkopen und Synkopen am Operationstisch--diagnostische und therapeutische Möglichkeiten.

Standing in long-lasting operations can be a trigger for neurocardiogenic syncopes for the personal in the operating theatre. We report on a 37-year-old surgeon who suffered since childhood from more than 100 syncopes and presyncopes. That was the reason for him to think of giving up his profession. The head-up tilt-test proved the mechanism of a neurocardiogenic syncope. No further syncope has occurred for 16 months during the therapy with theophyllin.

Right ventricular apical view--a new window for Doppler echocardiography of aortic valve stenosis.

We studied a new Doppler echocardiographic approach in 56 patients with valvular aortic stenosis from the right ventricular apex (AS-RV) and compared the transvalvular gradients with the results of the standard view from the left ventricular apex (AS-LV). AS-RV resulted in good or acceptable velocity curves in 59% of patients. The correlation between the two apical views for the peak/mean gradients were close (r = 0.95/0.96). Using all typical positions for Doppler investigation of aortic stenosis, highest peak gradients were best recorded in five cases by AS-RV. In one woman with a narrow left ventricular cavum and severe aortic stenosis, only AS-RV yielded a technically good spectral curve. Thus, in selected patients--probably those with a small left ventricular cavity or an enlarged right ventricle--AS-RV may be the best window or even the only possibility in Doppler investigation of aortic valve stenosis.

Transesophageal echocardiographic artifact mimicking pseudoaneurysm of the aorta.

Transesophageal echocardiography (TEE) is a sensitive and specific procedure that gives detailed images of the thoracic aortic anatomy and blood flow; however, there are few reports on pitfalls with TEE. This case describes a patient with a typical history of a possible aortic rupture; artifacts in the TEE mimicked aortic rupture and development of a pseudoaneurysm. Echocardiographers should know this possible pitfall to make the correct diagnosis.