Cloning and characterization of cDNAs coding for the heavy and light chains of a monoclonal antibody specific for Pseudomonas aeruginosa outer membrane protein I.

A set of seven monoclonal antibodies (MAb) directed against outer membrane proteins of Pseudomonas aeruginosa has been examined by Western blot analysis, indirect immunofluorescence tests and subclass typing. The hybridoma cell line secreting MAb 6A4, which reacts with outer membrane protein I, belongs to the IgG2a subclass and crossreacts with the 17 P. aeruginosa serotypes as listed in the International Antigenic Typing System, was selected as source for the preparation of poly(A)+RNA which in turn was used as template for cDNA synthesis and cloning. Full length cDNA clones of the gamma heavy chain as well as the kappa light chain were obtained and characterized by nucleotide sequence analysis. The complete cDNA sequences coding for the heavy and light chains will be the prerequisite for the construction and heterologous expression of a chimeric human-mouse monoclonal antibody which might be used in therapy of P. aeruginosa infections.

Soluble serum E-cadherin as a marker of tumour progression in colorectal cancer patients.

A pilot study was conducted to determine the concentrations of soluble serum E-cadherin in 36 patients with colorectal cancer or a high-grade dysplasia by the use of an ELISA technique. The results were compared with staging characteristics and concentrations of routine serum carcinoembryonic antigen (CEA). Sixteen patients with benign diseases and nine healthy volunteers served as internal or negative controls. Tumour specimens from seven patients were analysed by immunohistochemistry to compare concentrations of soluble serum E-cadherin with patterns of cell-bound E-cadherin or beta-catenin. Serum E-cadherin concentrations were increased in colorectal cancer patients (P = 0.009), but also in benign disease controls (P = 0.005), correlating with the T- (P < 0.05), but not N- or M-stage, and with serum CEA (P = 0.002) in case of existing liver metastases. Compared with other staining patterns, concentrations of soluble serum E-cadherin were higher in case of an exclusive membrane-bound localization of cellular beta-catenin (P = 0.071). The results suggest marker characteristics of soluble serum E-cadherin in colorectal cancer patients, but lacking specificity argues against a routine clinical use.

Enzyme replacement in Tay-Sachs disease.

Enzyme replacement therapy was attempted with two Tay-Sachs-diseased individuals--a 14-month-old child and a 7-week-old infant. Treatment consisted of repeated weekly intrathecal injections of pure hexosaminidase A. Injection of this enzyme resulted in almost complete disappearance of GM2 from the serum, but did not bring about dissolution of the GM2 membranous cytoplasmic bodies in the brain, as detected by electronmicroscopy. Both patients tolerated the treatment without apparent clinical complications, but no clear-cut improvement was noted as a result of prolonged injections of hexosaminidase A. Since this treatment was initiated in both an advanced stage and a very early stage of the disease, we conclude that enzyme replacement treatment by this route is not beneficial for patients with Tay-Sachs disease.

Prolonged reversal of diabetes in the rat by transplantation of allogeneic islets from a single donor and cyclosporine treatment.

Islets were isolated using neutral red as a dye to exclude lymphatic and acinar impurities in the transplant preparation. In a one donor-one recipient model, intraportally transplanted isogeneic islets survived indefinitely. Mean graft survival could be prolonged from 5 to 90 days, using a 3-dose rejection prophylaxis with CsA in allografts of (DA [RT1a]----Lew (RT 1l). Histological examination revealed intact islets in the grafted normoglycemic animals. Immunohistochemical staining of these islets showed B cells with insulin-rich cytoplasma. Blood glucose levels and the results of i.v. glucose tolerance tests 50 days after transplantation are presented. The different outcomes of our experiments are discussed.

Induction and rapid screening of monoclonal antibodies against cyclosporin A.

Cyclosporin C-hemisuccinate was coupled to thyroglobulin by the mixed anhydride method. Cyclosporin C was used instead of cyclosporin A (CsA) because of lack of functional groups of CsA. The resulting protein-cyclosporin C conjugate allowed us to induce high antibody titers also against cyclosporin A in rabbit and mice. Polyclonal and monoclonal antibodies were prepared following standard procedure. Since no standard methods for screening and quantification of anti-CsA-antibodies were available, two methods were adapted: (a) liquid phase radio assay (RA) and (b) solid phase enzyme-linked immunoassay (ELI-SA). For the former procedure inactivated charcoal was applied to separate the antibody-bound and the unbound CsA. CsA-coated PVC microtiter plates were used for the latter.

Prevention of type I diabetes in the non-obese diabetic (NOD) mouse with 15-deoxyspergualin (15-DS) or 15-DS + cyclosporin A (CyA).

By selectively inbreeding diabetic individuals, we have been able to establish an NOD mouse population with a genetic predisposition towards insulin-dependent diabetes mellitus (IDDM) in approximately 100% of cases. We examined the preventive effect of 15-DS or 15DS + CyA on developing IDDM in these animals. Whereas 15-DS has been proved to be effective in preventing diabetes (significant decrease of the diabetic risk ratio to 0.368 and a reduction of the incidence of the disease to 46.7%), combined treatment with CyA did not produce any additional benefit.

Identification of genes involved in human mesothelial cancer progression using a modified differential display technique.

A modified differential display method called RNA fingerprinting was used to identify mRNAs that were differentially expressed during human mesothelial cancer progression. We report the isolation of five different clones. Two clones were expressed in the metastatic mesothelioma cell line M1A and the malignant mesothelioma cell line M1, one clone was expressed uniquely in the metastatic cell line M1A and one clone was solely expressed in the normal mesothelial cells. The other clone was downregulated in the metastatic cell line M1A. The different expression patterns were confirmed by Northern blot analysis. Three clones had no homology to known genes, whereas the other two clones had a striking sequence homology to the M130 antigen and rab 12 mRNA, respectively. The clone that contained a high sequence homology to the M130 antigen mRNA was expressed in the mesothelioma cell lines M1 and M1A and not in any further investigated cancer cell line. This sequence tag may be of interest as a specific mesothelioma tumor marker.

Effect of recombinant human granulocyte colony-stimulating factor on hemodynamic and cytokine response in a porcine model of Pseudomonas sepsis.

To investigate the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on sepsis, chronically catheterized conscious pigs were challenged with Pseudomonas aeruginosa (8 x 10(7) colony-forming units kg-1 h-1) for 84 h (Group A, n = 8). Group B (n = 7) also received rhG-CSF at 5 micrograms kg-1 d-1, the first dose being given 30 min before starting bacterial infusion. Two of the animals in Group A died from pulmonary failure, whereas all those treated with rh-GCSF survived. Fever, severe pulmonary hypertension and systemic hypotension--the latter accompanied at first by a transient hypodynamic, and later a hyperdynamic response--were observed in all of the animals. In Group B, however, the rise in temperature, mean pulmonary arterial pressure (at a later stage of the observation), plasma levels of tumor necrosis factor, and endotoxin were significantly less than in Group A. In the rhG-CSF-treated pigs, an initial leukopenia completely recovered within 24 h (p < .05 vs. Group A). These data suggest that rhG-CSF might be beneficial in the treatment of sepsis.

Effect of delayed treatment with recombinant human granulocyte colony-stimulating factor on survival and plasma cytokine levels in a non-neutropenic porcine model of Pseudomonas aeruginosa sepsis.

BACKGROUND: Neutrophils are of great importance for the host's defense against invading organisms. Granulocyte colony-stimulating factor (G-CSF) has been used to augment both the neutrophil number and function, and its prophylactic administration has proved beneficial in animal models of sepsis. However, pretreatment with G-CSF is not practical under clinical conditions. We therefore investigated the effect of recombinant human (rh)G-CSF, administered only after infection, on the survival rate as well as the hemodynamic and cytokine response of the animals. METHODS: Chronically catheterized conscious pigs were challenged with Pseudomonas aeruginosa (8 x 10(7) colony-forming units kg(-1) x h(-1) for 120 h (control group, n = 10). Animals in the G-CSF group (n = 7) also received rhG-CSF (5 microg kg(-1) x day(-1)), the first dose being given 3 h after beginning bacterial infusion. RESULTS: The mortality rate was 50% (5/10) and 29% (2/7) in the control and G-CSF groups, respectively (p = NS, control vs. G-CSF group). Fever, severe pulmonary hypertension, and a hyperdynamic response were recorded in all of the animals. In spite of a prompt and significant recovery from the initial leukopenia (p < .05 vs. control group), the animals of the G-CSF group showed no significant differences in the parameters investigated from those of the controls. Compared with the survivors, the interleukin-1 receptor antagonist was markedly elevated in all nonsurvivors after 6 h of sepsis (p < .05). CONCLUSIONS: These data suggest that treatment with rhG-CSF after the onset of bacterial sepsis might not significantly improve the chances of survival for non-neutropenic patients.

Autocrine growth stimulation of SW403 colon carcinoma cell line is caused by transforming-growth-factor-alpha-mediated epidermal growth factor receptor activation.

Results of recent studies indicate that some cultured human carcinoma cell lines are capable of proliferating autonomously in serum-free medium as a result of the synthesis and secretion of transforming growth factor alpha (TGF alpha). TGF alpha interacts with epidermal growth factor receptor (EGFR) and induces its activation. In an attempt to extend these observations, we evaluated TGF alpha-mediated autonomous growth and constitutive EGFR activation in the human adenocarcinoma cell line SW403. The cell line shows synthesis of EGF receptors and TGF alpha but not EGF, and exhibits constitutive phosphorylation of the 170-kDa EGFR. Use of blocking anti-EGFR monoclonal antibodies (mAb) inhibits autonomous growth of SW403 cells and leads to a significant reduction of receptor phosphorylation. The inhibitory effect of the blocking anti-EGFR mAb is reversible upon addition of TGF alpha. In contrast, autonomous proliferation of SW403 cells is not inhibited by addition of neutralizing anti-EGF mAb. Our findings suggest that the proliferation of cells of the human SW403 adenocarcinoma cell line is regulated by an autocrine TGF alpha loop and that this regulatory pathway can be interrupted by using anti-EGFR mAb.

The Caulobacter crescentus outer membrane protein Omp58 (RsaF) is not required for paracrystalline S-layer secretion.

To identify the outer membrane protein component of the Caulobacter crescentus CB2 surface-layer export machinery we used the Serratia marcescens LipD protein to find homologs in the CB2 genome. From two homologous sequences found, one encodes a putative OMP with a predicted molecular mass of 57.5 kDa, termed Omp58 (formerly RsaF). Comparison of membrane protein profiles revealed a protein with an appropriate molecular mass present in wild-type, but not CB2 omp58::kanamycin, a mutant strain with an inactivated omp58 gene. Disruption of omp58 did not affect surface-layer production, suggesting that Omp58 is not involved in surface-layer protein secretion and, thus, may not be the outer membrane protein component of the C. crescentus surface-layer export system.

Sensitization against brain gangliosides after therapeutic swine brain implantation in a multiple sclerosis patient.

The case history is presented of a patient with multiple sclerosis who developed acute polyradiculoneuritis 11 days after subcutaneous implantation of a swine brain preparation. By means of lymphocyte transformation tests (LTT), sensitization against brain gangliosides could be demonstrated 16 days after the implantation. A second patient who underwent the same treatment showed neither clinical symptoms nor sensitization against brain gangliosides in the LTT. Patients did not show reactivity when tested with myelin basic protein. The polyradiculoneuritis was caused by the immune response to the implanted swine brain cross-reacting with human nervous system gangliosides.

A Salmonella typhimurium strain genetically engineered to secrete effectively a bioactive human interleukin (hIL)-6 via the Escherichia coli hemolysin secretion apparatus.

Human interleukin-6 (hIL-6) cDNA was genetically fused with the Escherichia coli hemolysin secretorial signal (hlyA[S]) sequence in a plasmid vector. Recombinant E. coli XL-1 Blue and attenuated Salmonella typhimurium secreted a 30 kDa hIL-6-HlyA(S) fusion protein, with an additional form of higher apparent molecular mass produced by S. typhimurium. In S. typhimurium cultures hIL-6-HlyA(S) concentrations entered a plateau at 500 to 600 ng ml(-1) culture supernatant. In contrast to E. coli XL-1 Blue, in S. typhimurium culture supernatants hIL-6-HlyA(S) was accumulated faster reaching three-fold higher maximal concentrations. The cell proliferating activity of hIL-6-HlyA(S) fusion protein(s) was equivalent to that of mature recombinant hIL-6. Furthermore. hIL-6-secreting S. typhimurium were less invasive than the attenuated control strain. Therefore, the bulky hemolysin secretorial peptide at the C-terminus of the fusion protein does not markably affect hIL-6 activity, suggesting that the hemolysin secretion apparatus provides an excellent system to study immunomodulatory effects of in situ synthesized IL-6 in Salmonella vaccine strains.

Immunogenic efficacy of differently produced recombinant vaccines candidates against Pseudomonas aeruginosa infections.

Three different variants of the recombinant hybrid outer membrane protein OprF (aa 190-342)-OprI (aa 21-83) could be obtained in high yield after expression in Escherichia coli. The hybrid protein was modified N terminally, either with a minimal histidine tag or with a homologous sequence of OprF. Both recombinant proteins were purified by nickel chelate affinity chromatography under native and denaturing conditions, and this produced three suitable candidates for a vaccination trial, protein His-F-I, which was purified in its native as well as in its refolded form; and the native purified N terminally extended protein, ex-F-I. In mice, significantly higher antibody titers and survival rates after challenge with Pseudomonas aeruginosa were observed following immunization with protein His-F-I, purified under native conditions.

Inflammatory consequences of the translocation of bacteria and endotoxin to mesenteric lymph nodes.

BACKGROUND: Translocation of intestinal bacteria to mesenteric lymph nodes (MLNs) has been documented in humans under a variety of circumstances, yet its clinical significance remains to be established. The aim of this study was to correlate detectable translocation to MLNs of bacteria and endotoxin with local and systemic signs of inflammation. METHODS: From each of 10 patients with carcinoma of the cecal region two MLNs were harvested prior to resection. The presence of bacteria and endotoxin in the lymphatic tissue and blood was determined by culture methods and DNA preparation (PCR) and by a Limulus assay, respectively. Inflammatory mediators were determined in plasma and in MLN homogenates. RESULTS: Viable bacteria were detected in MLNs of 7 patients and in 9 of 20 lymph nodes. PCR revealed traces of bacteria in 4 patients and in 6 of their MLNs. Combining both modalities, the translocation rate was 80% and 55% for patients and MLNs, respectively. There was no detectable bacteremia. Endotoxin was found in the plasma of 7 patients and in 9 MLNs from 5 patients. There was no correlation between culture findings and endotoxin concentrations. Moreover, bacteriological data did not correspond to local or systemic inflammation. The group of MLN with detectable endotoxin differed significantly from LPS-negative nodes with respect to interleukin-6, interleukin-10, and sCD14. Systemic concentrations of endotoxin and inflammatory parameters did not correspond to levels within MLNs. CONCLUSION: Translocation to MLNs occurs in patients with cecal carcinoma. This, however, seems not to be of major clinical significance if no additional physiologic insults are encountered. Irrespective of the presence of bacteria, there are variations in inflammatory reactions between lymph nodes from one and the same patient, probably reflecting fluctuating response mechanisms to low-grade translocation.

Growth of "new" coronary vascular structures by angiogenetic growth factors.

The efficacy of the human angiogenetic heparin-binding growth factor I (HBGF-I) to initiate site-directed growth of new blood vessels from the aorta into the myocardium was studied. First, manipulated Escherichia coli bacteria, which had received the human mRNA-transcript for HBGF I into their genetic material, were cultivated. The growth factor derived was purified using heparin-Sepharose affinity chromatography. The separation and characterization of biologically active alpha- and beta-chains was carried out using high pressure liquid chromatography (HPLC) of dialyzed and lyophilized samples from the heparin-Sepharose column. One microgram HBGF I (alpha-ECGF) was bound to polytetrafluoroethylene (PTFE) sponges, precoated with collagen type I, and implanted between the aorta and the myocardium of the left ventricle in experimental rats. Twelve growth factor implants in the experimental group were compared to six controls receiving uncoated PTFE sponges for 9 weeks. Digitized computed angiography showed new blood vessels between the aorta and the myocardium in 11 of the 12 experimental animals, and retrograde coronary perfusion by these "new" vascular structures could be seen. Histology showed no specific structures in the control group (without HBGF I). In the experimental group (with HBGF I) individual vessels with highly differentiated endothelial and smooth muscle cell layers were evident. Our experiments proved the feasibility of induced, site-directed angiogenesis. It is possible to initiate in vivo growth of new "coronary" vascular structures between the aorta and the myocardium.

Renal artery occlusion model in dogs for the evaluation of thrombolytic agents.

The purpose of this study was to develop a model of renal artery occlusion and to investigate the effects of various thrombolytic agents on an acute occlusion of the renal artery with respect to ischemic tolerance of renal parenchyma. In order to do this, a thrombosis model in dogs (n = 36) was established and a total of 72 dorsal renal arteries occluded using autologous clot material. For the in vitro preparing of a clot, autologous blood (20 mL) was withdrawn and 100 U thrombin immediately added. Then 1 mL of the clot material was injected into the dorsal branch of the exposed renal artery. The dogs were divided into 8 groups (2 control groups, 6 therapy groups with local and systemic thrombolytic therapy). Thrombolysis was performed using urokinase, single-chain urokinase, and recombinant tissue-plasminogen activator. In all cases the clot preparation technique allowed complete and stable occlusion of the renal arteries. Local and systemic application of the thrombolytic agents, however, resulted in complete recanalization of the clot material in all study groups. Recombinant tissue-plasminogen activator turned out to be the most effective agent in terms of recanalization time. The technique described allowed effective and reproducible artery occlusion for in vivo experimental work to study comparatively thrombolytic agents with respect to fibrin specificity, lytic efficacy, and side effects.

Cultured gingival epithelium. A possible suitable material for pre-prosthetic surgery.

In cultivating gingival epithelium from people up to the age of 66 years it is possible to gain a differentiated mucosal tissue sheet that is more than 100 times the size of the original biopsy surface. The morphological characteristics are a prickle cell layer with polygonal cells and a basal cell layer with flattened cells. Desmosomes, tonofibrils and microplicae at the superficial side of the epithelium are typical features. There is a cell biological differentiation as the distinct binding of lectins and of a cytokeratin antibody proves. The multiplication in surface area, the possibility of using cells of senior patients and the morphological and cell biological differentiation of cultured gingival epithelium are advantages in its application for the autologous bridging of intraoral defects.

The stimulation of neo-angiogenesis in the ischemic heart by the human growth factor FGF.

BACKGROUND: The present article deals with the conduct of our animal experiments with the human growth factor FGF (fibroblast growth factor) and the results obtained therefrom. METHODS: In order to establish the angiogenetic potential of FGF, this factor was first obtained from a genetically transformed strain of E. Coli, and then isolated and highly purified. Afterwards the growth factor FGF has been used in several in vitro- and in vivo experiments in order to prove its influence on neo-angiogenesis in ischemic tissue. RESULTS: In cultures of endothelial cells from the human great saphenous vein it has been possible to stimulate growth successfully with FGF obtained in this way, and a further increase in its action was brought about by the addition of heparin. In tritium-thymidine assays, the endothelial cell stimulating action of FGF was confirmed. It could also be shown angiographically that administering FGF to the ischemic myocardium of these animals initiates the development of new vessels, and we could demonstrate that a myocardial capillary network sprouting directly from the coronary vessels themselves can establish an alternative blood flow. These results were confirmed histologically by the significantly greater capillary density which appeared following the use of the growth factor. CONCLUSIONS: By using the human growth factor FGF, we have been able for the first time to understand the physiological processes of angiogenesis as they come into play during wound healing or the development of collaterals following tissue ischemia, and to use this knowledge for the production of new vessels in the ischemic hearts of rats and rabbits. Decisive for the future use of the factor in human patients -- particularly for the treatment of coronary heart disease (CHD) are the results of experimental investigations designed to exclude the possibility of the growth factor initiating or stimulating neoplasia.

A technique of chronic intravascular catheterization for monitoring haemodynamic and laboratory values in conscious swine.

In human and small animal intensive care medicine percutaneous sheath introducer (PSI) sets are commonly used for repeated insertion of an arterial or a venous catheter with only one vascular puncture. We used PSI for chronic catheterization of swine with a Swan-Ganz thermodilution catheter via a surgically exposed external jugular vein. In this way we were able to change defective catheters or correct the position of the catheter tip without renewed surgical intervention.