Transglutaminase: new insights into gelatin nanoparticle cross-linking.

Gelatin nanoparticles (GNPs) have demonstrated to be beneficial as a biodegradable and biocompatible delivery system. So far, nanoparticles prepared by the two-step desolvation technique were subsequently cross-linked by glutaraldehyde to guarantee storage stability. Although in vivo and in vitro toxicological studies have not revealed any glutaraldehyde related undesired effects, an alternative to chemical cross-linking could ease future clinical use in humans. Therefore, the recombinant enzyme microbial transglutaminase was used to examine its cross-linking abilities in nanoparticle production. Various process parameters, such as incubation time, temperature, medium, pH and the particle purification were evaluated regarding their impact on particle size and its distribution. Cross-linking reactions were best at 25°C using an ion-free solvent at a neutral pH and have been terminated after 12 h. Preliminary storage stability testing indicated adequate consistency of particle size and particle distribution making transglutaminase a potential candidate for glutaraldehyde substitution in future GNP production.

A novel technique for selective NF-kappaB inhibition in Kupffer cells: contrary effects in fulminant hepatitis and ischaemia-reperfusion.

BACKGROUND AND AIMS: The transcription factor nuclear factor kappa B (NF-kappaB) has risen as a promising target for anti-inflammatory therapeutics. In the liver, however, NF-kappaB inhibition mediates both damaging and protective effects. The outcome is deemed to depend on the liver cell type addressed. Recent gene knock-out studies focused on the role of NF-kappaB in hepatocytes, whereas the role of NF-kappaB in Kupffer cells has not yet been investigated in vivo. Here we present a novel approach, which may be suitable for clinical application, to selectively target NF-kappaB in Kupffer cells and analyse the effects in experimental models of liver injury. METHODS: NF-kappaB inhibiting decoy oligodeoxynucleotides were loaded upon gelatin nanoparticles (D-NPs) and their in vivo distribution was determined by confocal microscopy. Liver damage, NF-kappaB activity, cytokine levels and apoptotic protein expression were evaluated after lipopolysaccharide (LPS), d-galactosamine (GalN)/LPS, or concanavalin A (ConA) challenge and partial warm ischaemia and subsequent reperfusion, respectively. RESULTS: D-NPs were selectively taken up by Kupffer cells and inhibited NF-kappaB activation. Inhibition of NF-kappaB in Kupffer cells improved survival and reduced liver injury after GalN/LPS as well as after ConA challenge. While anti-apoptotic protein expression in liver tissue was not reduced, pro-apoptotic players such as cJun N-terminal kinase (JNK) were inhibited. In contrast, selective inhibition of NF-kappaB augmented reperfusion injury. CONCLUSIONS: NF-kappaB inhibiting decoy oligodeoxynucleotide-loaded gelatin nanoparticles is a novel tool to selectively inhibit NF-kappaB activation in Kupffer cells in vivo. Thus, liver injury can be reduced in experimental fulminant hepatitis, but increased at ischaemia-reperfusion.

Nanoparticulate CpG immunotherapy in RAO-affected horses: phase I and IIa study.

BACKGROUND: Recurrent airway obstruction (RAO), an asthma-like disease, is 1 of the most common allergic diseases in horses in the northern hemisphere. Hypersensitivity reactions to environmental antigens cause an allergic inflammatory response in the equine airways. Cytosine-phosphate-guanosine-oligodeoxynucleotides (CpG-ODN) are known to direct the immune system toward a Th1-pathway, and away from the pro-allergic Th2-line (Th2/Th1-shift). Gelatin nanoparticles (GNPs) are biocompatible and biodegradable immunological inert drug delivery systems that protect CpG-ODN against nuclease degeneration. Preliminary studies on the inhalation of GNP-bound CpG-ODN in RAO-affected horses have shown promising results. OBJECTIVES: The aim of this study was to evaluate the clinical and immunological effects of GNP-bound CpG-ODN in a double-blinded, placebo-controlled, prospective, randomized clinical trial and to verify a sustained effect post-treatment. ANIMALS AND METHODS: Twenty-four RAO-affected horses received 1 inhalation every 2 days for 5 consecutive administrations. Horses were examined for clinical, endoscopic, cytological, and blood biochemical variables before the inhalation regimen (I), immediately afterwards (II), and 4 weeks post-treatment (III). RESULTS: At time points I and II, administration of treatment rather than placebo corresponded to a statistically significant decrease in respiratory effort, nasal discharge, tracheal secretion, and viscosity, AaDO2 and neutrophil percentage, and an increase in arterial oxygen pressure. CONCLUSION AND CLINICAL IMPORTANCE: Administration of a GNP-bound CpG-ODN formulation caused a potent and persistent effect on allergic and inflammatory-induced clinical variables in RAO-affected horses. This treatment, therefore, provides an innovative, promising, and well-tolerated strategy beyond conventional symptomatic long-term therapy and could serve as a model for asthma treatment in humans.

Preparation of avidin-labeled protein nanoparticles as carriers for biotinylated peptide nucleic acid.

The possibility of preparing protein nanoparticles followed by covalent linkage of avidin was investigated. Free sulfhydryl groups were introduced onto the surface of protein nanoparticles either by aldehyde quenching with cysteine or reaction of free amino groups with 2-iminothiolane. The number of primary amino groups and sulfhydryl groups on the surface of the resulting particles was quantified with site-specific reagents. Avidin was attached to the surface of the thiolated nanoparticles via a bifunctional spacer which reacted in a first step with amino groups of avidin and in a second step with the sulfhydryl groups introduced onto the surface of the nanoparticles. Biotinylated peptide nucleic acid (PNA) as a model compound for biotinylated drugs was effectively coupled to the nanoparticles by complex formation with the covalently attached avidin. Since the formation of the interaction between biotin and avidin is very rapid and stable a highly effective drug carrier system for biotinylated compounds such as PNAs was achieved.

Desolvation process and surface characterisation of protein nanoparticles.

The objective of the present study was to characterise and optimise the desolvation process of human serum albumin (HSA) for the preparation of nanoparticles and to characterise the resulting colloidal system. Following the desolvation of the protein, the resulting nanoparticles were stabilised by the addition of varying amounts of glutaraldehyde or by heat denaturation. The particle size, zeta potential, and the number of available amino groups on the surface of the nanoparticles were determined. The amino groups were quantified by a spectrophotometric method using 2,4, 6-trinitrobenzenesulfonic acid (TNBS). The results indicated that the particle size depended mainly on the amount of desolvating agent added, but not on the amount of cross-linker or the kind of cross-linking procedure. Increasing amounts of glutaraldehyde reduced the number of amino groups on the surface of HSA nanoparticles and also decreased the zeta potential of the carrier system. The temperature and heat denaturation time only had an influence on the stability of the nanoparticles but not on the amount of amino groups or the particle size. It was shown that heat denatured HSA nanoparticles possessed the greatest number of amino groups on their surface. Additional experiments for the characterisation of gelatin A and B nanoparticles were performed.

Preparation of avidin-labelled gelatin nanoparticles as carriers for biotinylated peptide nucleic acid (PNA).

The possibility of preparing uniform nanoparticles consisting of proteins such as gelatin followed by covalent linkage of avidin was investigated. Gelatin nanoparticles were prepared by two step desolvation. Functional groups at the surface of the particulate system were quantified with site-specific reagents. The surface of the nanoparticles was thiolated and avidin was covalently attached to the nanoparticles via a bifunctional spacer at high levels. Biotinylated peptide nucleic acid (PNA) was effectively complexed by the avidin-conjugated nanoparticles. Avidin-conjugated protein nanoparticles should prove as potential carrier system for biotinylated drug derivatives in antisense therapy.

[Anterior high-frequency capsulotomy: advantages and problems]. / Vordere Hochfrequenz-Kapsulotomie: Chancen und Probleme.

METHODS: Since the clinical test phase, the author performed consistently more than 500 capsulotomies in his cataract surgeries by means of a radio-frequency current, using an instrument developed by Klöti et al. Some problems in the application of the instrument and their possible solution are discussed from the practical view of the user. CONCLUSIONS: The method proved as easy and safe to use especially in certain difficult cases. The main problem seems to be a reduced stability of the anterior capsular rim compared to the cases performed by capsulorhexis.

[Anterior high frequency capsulotomy. II. Clinico-surgical experiences]. / Vordere Hochfrequenz(HF)-Kapsulotomie. II. Klinisch-chirurgische Erfahrungen.

Using an instrument developed by Klöti, the author performed anterior capsulotomy by means of a radio-frequency current in 21 cases of extracapsular cataract extraction. This group is compared with 21 control cases. The advantages and limits of this technique are discussed. The procedure was found to be simple and very helpful to the surgeon. There was no evidence of any damage to structures in the patient's eye.

[Computer as replacement for the current medical record? Introduction of a pilot project of the St. Gallen ophthalmology clinic]. / Der Computer als Ersatz für die herkömmliche Krankengeschichte? Vorstellung eines Pilotprojektes an der Augenklinik St. Gallen.

Computerized data processing is more and more used in hospitals and medical offices. There are no doubts about the usefulness of a computer for administrative tasks. At our Dept. of Ophthalmology we started a pilot project to enter the medical records into the computer. The pros and cons of such an information system are discussed. Entering medical records into the computer did not prove to be definite advantage for the physician.

[Unusual cases of coronary death]. / Ungewöhnliche Koronartodesfälle.

Stenosing coronary artery sclerosis is the most common cause of sudden unexpected death. In this report we present three exceptions: 1. A mycotic coronary aneurysm ruptures and results in pericardial tamponade; 2. in a young woman Takayasu aortitis encroaches on the ostium and mainstem of the left coronary; 3. fibromuscular dysplasia of the right coronary artery causes death in a 56-year-old male.

Syphilis, hepatitis A and hepatitis B seromarkers in homosexual men.

Two hundred homosexual men were investigated for prevalence rates of syphilis, hepatitis A (HAV) and hepatitis B (HBV). A reactive Treponema pallidum haemagglutination test (TPHA) was found in 56.3% of the participants. In 81.7% HBV seromarkers were detected. Hepatitis B surface antigen (HBsAG) was present in 6.6%. Anti-HAV was found in 51.0%. Risk factors for acquisition of these infections are discussed.

Protozoal enteric infections in homosexual men.

The prevalence of enteric protozoal infections was examined in a group of 200 homosexual men. Entamoeba histolytica (E. histolyt .) was found in 22.8%, apathogen amoeba in 54.5% and Giardia lamblia (G. lamblia) in 9.1% of the participants. Antibodies against E. histolyt . were significantly higher in subjects carrying E. histolyt . than in the group with apathogen amoeba and in uninfected participants. Clinical symptoms were not associated with protozoal infection. The analysis of a number of variables showed that oral-anal sex and promiscuity were the two most important risk factors for protozoal infection. The clinical significance of protozoal enteric infections and their possible relation to the acquired immune deficiency syndrome (AIDS) is discussed.

Syphilis, hepatitis A, hepatitis B, and cytomegalovirus infection in homosexual men in Antwerp.

In a homosexual communication centre in Antwerp 196 homosexual men were screened for seromarkers of syphilis, hepatitis A (HAV), hepatitis B (HBV) and cytomegalovirus (CMV). A comparison group consisted of 118 heterosexual men attending a venereal disease clinic in Antwerp. Treponemal antibodies were found in 7.1% of homosexual men, of whom half gave no history of past or present infection. Anti HAV was present in 43.3%, HBV seromarkers in 34.4%, and CMV antibodies in 71.2% of homosexual men. Hepatitis B surface antigen (HBsAg) was detected in eight homosexual men, but not in the heterosexual control group. Prevalence rates of infections other than HAV were significantly higher in homosexual men than in heterosexual men. Answers to a questionnaire were used to evaluate risk factors for different diseases, which were: duration of active homosexuality for all infections, promiscuity (greater than or equal to 10 partners in the past six months) for syphilis and hepatitis B, and anal intercourse for hepatitis B. Visiting saunas and travelling for sexual contacts also indicated a higher risk for STD, but were an indirect expression of promiscuity.

Gelatin nanoparticles by two step desolvation--a new preparation method, surface modifications and cell uptake.

A new two-step desolvation method for manufacturing gelatin nanoparticles was developed. After the first desolvation step, the low molecular gelatin fractions present in the supernatant were removed by decanting. The high molecular fractions present in the sediment were redesolved and then desolvated again at pH 2.5 in the second step. The resulting particles can then be easily purified by centrifugation and redispersion. The different fractions obtained during the process were analysed by gel permeation chromatography (GPC). Based on these results, it can be concluded that the molecular weight of gelatin has a decisive influence on the stability of the manufactured gelatin nanoparticles. In addition, two fluorescent dyes (Texasred and fluoresceinamine) were coupled to the nanoparticles for cell uptake studies. The fluorescent nanoparticles showed a high uptake into monocytes/macrophages.

[Immunologic findings in homosexual males with generalized lymphadenopathy. Prodromal state of acquired immunodeficiency syndrome?]. / Immunologische Befunde bei homosexuellen Männern mit generalisierter Lymphadenopathie. Prodromalstadium des "Acquired Immunodeficiency Syndromes"?

Peripheral blood of 6 homosexual men with lymphadenopathy associated with fever, weight loss and night sweat, 13 homosexuals with lymphadenopathy without further clinical symptoms, 13 healthy homosexuals and 20 healthy hetero-sexual blood donors was evaluated for different lymphocyte subpopulation, mitogen induced T-cell proliferation and Gamma-interferon production. In the six patients with lymphadenopathy lymphocyte subpopulations were also analysed in lymph node-biopsies. delayed type hypersensitivity was established by skin test's using different recall antigens. Sera of all homosexual men were screened for immunoglobulin levels, complement activity, presence of immunocomplexes and autoantibodies. Most of the homosexuals with lymphadenopathy but without clinical symptoms and all patients with lymphadenopathy associated with clinical symptoms had a significantly decreased helper-(OKT 4) suppressor- (OKT 8) cell ratio. In contrast, the OKT 4/OKT 8 ratio was found to be normal in lymphocyte suspension from lymph node-biopsy from the 6 patients with lymphadenopathy and clinical symptoms. Within the group of healthy homosexuals this ratio exhibited a rather large variation, in three of the tested persons the ratio was below 1. No functional abnormalities of T-cell immunity were observed in the homosexual men as compared to the control group, with the exception of a significantly increased LAG- and PWM-induced lymphocyte proliferation in the group of healthy homosexual men. Likewise no significant differences were obtained with regard to the further analysed immunological parameters. It is concluded that the obtained immunological results do not provide the possibility to define persons at immediate risk for the development of AIDS.

Incorporation of biodegradable nanoparticles into human airway epithelium cells-in vitro study of the suitability as a vehicle for drug or gene delivery in pulmonary diseases.

PURPOSE: Nanoparticles are able to enhance drug or DNA stability for purposes of optimised deposition to targeted tissues. Surface modifications can mediate drug targeting. The suitability of nanoparticles synthesised out of porcine gelatin, human serum albumin, and polyalkylcyanoacrylate as drug and gene carriers for pulmonary application was investigated in vitro on primary airway epithelium cells and the cell line 16HBE14o-. METHODS: The uptake of nanoparticles into these cells was examined by confocal laser scan microscopy (CLSM) and flow cytometry (FACS). Further the cytotoxicity of nanoparticles was evaluated by an LDH-release-test and the inflammatory potential of the nanoparticles was assessed by measuring IL-8 release. RESULTS: CLSM and FACS experiments showed that the nanoparticles were incorporated into bronchial epithelial cells provoking little or no cytotoxicity and no inflammation as measured by IL-8 release. CONCLUSIONS: Based on their low cytotoxicity and the missing inflammatory potential in combination with an efficient uptake in human bronchial epithelial cells, protein-based nanoparticles are suitable drug and gene carriers for pulmonary application.

Lymphadenopathy and antibodies to HTLV-III in homosexual men. Clinical, laboratory and epidemiological features.

The study provides information on the epidemiology of HTLV-III infection and the lymphadenopathy syndrome (LAP) in 374 German homosexual men. Sexual contacts in the USA and rectal enemas before receptive anal intercourse are the main risk factors associated with virus transmission. HTLV-III seropositivity is significantly correlated with LAP. Prominent clinical signs are infrequent. Immunological and haematological abnormalities are prevalent, and the retrovirus infection is frequently associated with serological markers of other viruses (hepatitis B, herpes group viruses). Lymphadenopathy as a manifestation of HTLV-III infection is discussed within the context of AIDS-related disorders.