Forensic Engineering of Advanced Polymeric Materials-Part VII: Degradation of Biopolymer Welded Joints.

Welding technology may be considered as a promising processing method for the formation of packaging products from biopolymers. However, the welding processes used can change the properties of the polymer materials, especially in the region of the weld. In this contribution, the impact of the welding process on the structure and properties of biopolymer welds and their ability to undergo hydrolytic degradation will be discussed. Samples for the study were made from polylactide (PLA) and poly(3-hydroxyalkanoate) (PHA) biopolymers which were welded using two methods: ultrasonic and heated tool welding. Differential scanning calorimetry (DSC) analysis showed slight changes in the thermal properties of the samples resulting from the processing and welding method used. The results of hydrolytic degradation indicated that welds of selected biopolymers started to degrade faster than unwelded parts of the samples. The structure of degradation products at the molecular level was confirmed using mass spectrometry. It was found that hydrolysis of the PLA and PHA welds occurs via the random ester bond cleavage and leads to the formation of PLA and PHA oligomers terminated by hydroxyl and carboxyl end groups, similarly to as previously observed for unwelded PLA and PHA-based materials.

Prediction studies of environment-friendly biodegradable polymeric packaging based on PLA. Influence of specimens' thickness on the hydrolytic degradation profile.

Application of new biodegradable polymer packaging based on polylactide (PLA), susceptible to organic recycling, can help in the waste reduction in landfills. In this paper, the results of the study on abiotic degradation of PLA and its blend containing 15 mol% of poly[(R,S)-3-hydroxybutyrate], as a model for the first step of organic recycling were presented. The samples used for this study have different shapes and thicknesses: rigid films and cuboid-bars. Particular emphasis was placed on determining the pattern of degradation products released into the medium. Originally, the results of present study revealed that the application of electrospray ionization mass spectrometry supported by high performance liquid chromatography allowed envisaging the differences in the degradation products pattern released from the studied PLA-based samples differing in thickness. The significant differences in degradation products pattern were predominately observed in the first steps of incubation process and are caused by an autocatalytic effect, which occurs mainly during degradation of the large size PLA samples. Although, the thickness of PLA-based packaging changes the degradation product patterns, however this does not increase the total amounts of acids released to the medium. Thus, it may be concluded that thickness should not affect significantly organic recycling of the packaging.

Paraneoplastic vitelliform retinopathy associated with cutaneous or uveal melanoma and metastases.

PURPOSE: To report unusual vitelliform fundus findings in three cases of paraneoplastic retinopathy associated with metastasised cutaneous or uveal melanoma and in one case, a unique immunoreactivity response. PATIENTS AND METHODS: Observational case series. The histories of three patients with MAR-like paraneoplastic retinopathy were reviewed. Electroretinography, Goldmann perimetry, fluorescein angiography, and in one case optical coherence tomography, immunohistochemistry and Western blotting were performed. RESULTS: All patients revealed similar paraneoplastic vitelliform retinal abnormalities. Symptoms in two cases differed from the classical MAR syndrome. In one case, western blotting and immunohistochemistry demonstrated antibodies against 120-kDa, a soluble photoreceptor protein. No immunoreactivity to retinal bipolar cells was detected. CONCLUSION: The clinical, electrophysiological, and immunological findings in our patients suggest a melanoma associated paraneoplastic origin, like in MAR syndrome. However contrary to MAR syndrome, this paraneoplastic vitelliform retinopathy exhibits a peculiar fundus picture, consisting of serous macular detachment and nummular vitelliform lesions in the posterior pole. This could be an unusual presentation of MAR or a separate paraneoplastic entity.

Expression of CC chemokines and their receptors in the eye in autoimmune anterior uveitis associated with EAE.

PURPOSE: To determine the pattern of expression of CC chemokines and their receptors in the eyes of Lewis rats and to establish their role in autoimmune anterior uveitis (AU) associated with experimental autoimmune encephalomyelitis (EAE). METHODS: EAE/AU was induced in Lewis rats with myelin basic protein in complete Freund's adjuvant (CFA). The rats were scored for the development of clinical EAE and AU. The expression of CCL5/regulated on activation normal T-cell expressed and secreted (RANTES), CCL2/monocyte chemotactic protein (MCP)-1, CCL3/macrophage inflammatory protein (MIP)-1alpha, and CCL4/MIP-1beta and their receptors was examined at the preclinical stage, onset, peak, and recovery by RT-PCR and ELISA. EAE/AU rats were treated with neutralizing polyclonal antibodies against CCL3/MIP-1alpha, CCL4/MIP-1beta, CCL2/MCP-1, and CCL5/RANTES and tested for the suppression of onset of clinical AU and EAE. The control group received normal rabbit IgG at the same dose. RESULTS: The gene expression of those chemokines was upregulated concurrently with symptom onset of EAE/AU and correlated with the intensity of inflammatory changes in the eye and central nervous system (CNS). The highest expression of CCL4/RANTES, CCL2/MCP-1, and CCL3/MIP-1alpha in the eye was detected at onset of clinical uveitis, whereas CCL4/MIP-1beta was elevated at the peak of AU. The expression of chemokine receptors associated with T-helper (Th)1-type response, CCR1 and CCR5, correlated with their appropriate ligands and was the highest at the peak of AU, whereas CCR2, the receptor for CCL2/MCP-1, was present before the onset of the disease. Treatment of anti-MIP-1beta and anti-MCP-1 significantly delayed the onset and shortened the duration of AU and EAE. Anti-MIP-1alpha treatment had no effect on clinical EAE but inhibited the clinical signs of AU. Although CCL5/RANTES expression was observed during the entire course of the disease, anti-RANTES treatment had no effect on clinical disease progression. CONCLUSIONS: The data suggest that CCL2/MCP-1, CCL3/MIP-1alpha, and CCL4/MIP-beta contribute to the recruitment of inflammatory cells into the eye and CNS and to disease activity.

Synthesis of poly(2-methyl-3-hydroxyoctanoate) via anionic polymerization of alpha-methyl-beta-pentyl-beta-propiolactone.

Synthesis of an alpha,beta-alkyl branched polyester, i.e., poly(2-methyl-3-hydroxyoctanoate), has been accomplished via anionic polymerization of alpha-methyl-beta-pentyl-beta-propiolactone mediated by supramolecular complexes of potassium methoxide or potassium hydroxide, respectively. The structure of resulting polymers has been established by electrospray ionization multistage mass spectrometry (ESI-MSn), FT-IR, NMR, and GPC analyses. Previously proposed addition-elimination mechanism of the polymerization of beta-lactones containing alpha-hydrogen by alkoxide anion has been confirmed to operate also in the case of beta-lactone having alkyl substituents in both alpha and beta positions.

Water-soluble L-alanine and related oligopeptide conjugates with poly[(R,S)-3-hydroxybutanoic acid] oligomers, synthesis and structural studies by means of electrospray ionization multistage mass spectrometry.

The reactions of (R,S) beta-butyrolactone with L-alanine and related oligopeptides (Ala-Ala-Ala) were investigated. The resulting water-soluble oligomers were composed of poly[(R,S)-3-hydroxybutanoic acid] (a-PHB) covalently conjugated to L-alanine and Ala-Ala-Ala oligopeptide. The other chain end was of the carboxylic acid type. The structure of the obtained oligomers was assessed by electrospray ionization multistage mass spectrometry (ESI-MSn) and the respective structural information was completed by IR, NMR, and GPC analyses. The molecular weight and structure of the products could be controlled through reaction conditions. Using this new synthetic approach. a-PHB oligomers with well-defined end groups, as well as respective block copolymers, can be prepared via regioselective ring-opening oligomerization of (R,S) beta-butyrolactone induced by amino acids under their zwitterionic form.

A new type of thermoalkalophilic hydrolase of Paucimonas lemoignei with high specificity for amorphous polyesters of short chain-length hydroxyalkanoic acids.

A novel type of hydrolase was purified from culture fluid of Paucimonas (formerly Pseudomonas) lemoignei. Biochemical characterization revealed an unusual substrate specificity of the purified enzyme for amorphous poly((R)-3-hydroxyalkanoates) (PHA) such as native granules of natural poly((R)-3-hydroxybutyrate) (PHB) or poly((R)-3-hydroxyvalerate) (PHV), artificial cholate-coated granules of natural PHB or PHV, atactic poly((R,S)-3-hydroxybutyrate), and oligomers of (R)-3-hydroxybutyrate (3HB) with six or more 3HB units. The enzyme has the unique property to recognize the physical state of the polymeric substrate by discrimination between amorphous PHA (good substrate) and denatured, partially crystalline PHA (no substrate). The pentamers of 3HB or 3HV were identified as the main products of enzymatic hydrolysis of native PHB or PHV, respectively. No activity was found with any denatured PHA, oligomers of (R)-3HB with five or less 3HB units, poly(6-hydroxyhexanoate), substrates of lipases such as tributyrin or triolein, substrates for amidases/nitrilases, DNA, RNA, casein, N-alpha-benzoyl-l-arginine-4-nitranilide, or starch. The purified enzyme (M(r) 36,209) was remarkably stable and active at high temperature (60 degrees C), high pH (up to 12.0), low ionic strength (distilled water), and in solvents (e.g. n-propyl alcohol). The depolymerase contained no essential SH groups or essential disulfide bridges and was insensitive to high concentrations of ionic (SDS) and nonionic (Triton and Tween) detergents. Characterization of the cloned structural gene (phaZ7) and the DNA-deduced amino acid sequence revealed no homologies to any PHB depolymerase or any other sequence of data banks except for a short sequence related to the active site serine of serine hydrolases. A classification of the enzyme into a new family (family 9) of carboxyesterases (Arpigny, J. L., and Jaeger, K.-E. (1999) Biochem. J. 343, 177-183) is suggested.

Retinal anti-bipolar cell antibodies in a patient with paraneoplastic retinopathy and colon carcinoma.

PURPOSE: To describe clinical, electrophysiologic, and immunologic features of a unique paraneoplastic retinopathy with characteristics of cancer-associated and melanoma-associated retinopathy. METHODS: Serial assessment of clinical visual function, electroretinography, and assays of anti-retinal antibodies. RESULTS: A 51-year-old woman with progressive visual glare for 1 year had normal visual acuity and color vision, paracentral scotomas, and a normal-appearing retina. Electroretinography revealed no responses of the right eye and attenuated responses of the left eye, especially those recorded under scotopic conditions. Anti-bipolar antibodies were detected. Subsequent evaluation uncovered adenocarcinoma of the colon. Several months after resection of the tumor and chemotherapy, no evidence existed of cancer or anti-bipolar cell antibodies, and electroretinography responses were markedly improved. CONCLUSION: The presence of anti-bipolar cell antibodies in a patient with retinal dysfunction is not specific of melanoma-associated retinopathy. Effective treatment of cancer may result in elimination of associated anti-retinal antibodies and improved retinal function.

Importance of cryptic myelin basic protein epitopes in the pathogenicity of acute and recurrent anterior uveitis associated with EAE.

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU), which can relapse without recurring of EAE. In this study, we analyzed the repertoire of MBP epitopes that play a role in acute and recurrent AU by injection of MBP synthetic peptides. In addition to the encephalitogenic epitopes 69-89 and 87-99, several cryptic epitopes were found to be strongly uveitogenic in Lewis rats upon immunization with synthetic peptides, including 100-120, 121-140 and 142-167. However, the peptide corresponding to the MBP residues 1-20 was uniquely capable of inducing AU without EAE. Immunization with intact MBP was not essential for the induction of the recurrence of AU. The responses of T cells from lymph nodes and spleens showed a dominant response to the original disease-induced epitope with responses to secondary epitopes. In conclusion, the analysis of pathogenic determinants important for the induction of uveitis provides further evidence that MBP-specific T cells also contribute to the pathogenesis of anterior uveitis. Moreover, this also suggests that a distinct immunoregulatory mechanism exists in the eye and spinal cord because of the uniqueness of the epitope 1-20 in AU but not EAE, and the capability of MBP-specific T cells of inducing AU without EAE.

The role of T cells in autoimmune uveitis.

Autoimmune diseases result from the activation of self-reactive T cells recognizing autoantigens or foreign antigens cross-reactive with an autoantigen. T cells are thought to play a major role in autoimmune diseases in different organs, including the eye. This review focuses on the role of T cells in autoimmune uveitis in humans and in animal models of experimental autoimmune uveitis. Since rheumatoid arthritis is an autoimmune disease that has been studied far more extensively than uveitis, we have also included a review of clinical and experimental observations relevant to that disease.

Biomimetic polyesters and their role in ion transport across cell membranes.

Syntheses of biomimetic low-molecular weight poly-(R)-3-hydroxybutanoate mediated by three types of supramolecular catalysts are presented. The utility of these synthetic polyesters for preparation of artificial channels in phospholipid bilayers capable of sodium and calcium ion transport across cell membranes, is discussed. Further studies on possible applications of these bio-polymers for manufacturing drugs of prolonged activity are under way.

Epitope recognition and T cell receptors in recurrent autoimmune anterior uveitis in Lewis rats immunized with myelin basic protein.

Lewis rats immunized with myelin basic protein (MBP) develop experimental autoimmune encephalomyelitis (EAE) and associated anterior uveitis (AU). Rats recover and become resistant to further reinduction of EAE. We investigated whether the resistance to reinduction of EAE was associated with the resistance to AU in LEW rats reinjected with MBP. We demonstrated that while rats remained resistant to EAE, they become susceptible to uveitis after recovery, and suffered a second episode of disease. The susceptibility to reinduced disease was associated with the recognition of new MBP epitopes. In contrast to the initial episode of AU, TCR Vbeta8.2 predominance was not observed in the iris/ciliary body. Our results suggest that T cells specific for MBP, which are rapidly reactivated when re-exposed to antigen, are sufficient to induce clinical uveitis in LEW rats. This process may involve a shifting of T cell specificity from the major encephalitogenic peptide utilizing the Vbeta8.2 receptor to a more diverse cell repertoire.

Electrospray multistep ion trap mass spectrometry for the structural characterisation of poly

Electrospray 'soft' ionisation (ESI) and multistep mass spectrometry (MS(n)) techniques enable characterisation of a bioactive polymer, poly[(R,S)-3-hydroxybutanoic acid] (a-PHB), containing covalently bonded benzylpenicillin. The chemical structures of individual mass-selected bioactive macromolecules have been determined, and their fragmentation mechanisms have been compared with those of pure penicillin G. Copyright 2000 John Wiley & Sons, Ltd.

Electrospray ionisation tandem mass spectrometry of poly

Evaluation of polymer end-capping reactions with the aid of electrospray ionisation tandem mass spectrometry techniques (ESI-MS(n)) allows characterisation of novel poly[(R, S)-3-hydroxybutanoic acid]-(a-PHB) telechelics, containing primary hydroxyl groups at both polymer chain ends. The chemical structures of individual mass-selected macromolecules of the well-defined a-PHB telechelics have been defined in this way, and fragmentation mechanisms have been proposed. Copyright 1999 John Wiley & Sons, Ltd.

Novel synthesis of functionalized poly(3-hydroxybutanoic acid) and its copolymers.

Novel feasibility of fuctionalized poly(3-hydroxybutanoic acid), PHB, and its copolymers synthesis via ring-opening of beta-butyrolactone (ROP) mediated by activated anionic initiators or enzymes in vitro is presented. Using these new synthetic approaches, PHB with defined chemical structure of the end groups as well as block, graft and random copolymers have been obtained and characterized by IR, NMR, ESI-MS and GPC techniques. The relationship between the structure and properties of the novel polymeric materials prepared is discussed.

Intraocular transplantation of E1A-immortalized retinal precursor cells.

The purpose of this study was to examine the effect of the ocular environment on the survival, tumorigenicity, and phenotypic marker expression of immortalized retinal precursor cells transplanted into immunocompetent adult and neonatal Sprague-Dawley rats. EIA-NR.3, a rat immortalized retinal precursor cell culture, was used as an inexhaustible source of experimental graft material. These cells were prelabeled with the fluorescent marker dil (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) and transplanted intravitreally (50,000 cells per microL) into 11 adult and 31 neonatal Sprague-Dawley rat eyes. At 1 mo posttransplant, animals were sacrificed and retinal tissue sections examined histologically for the presence of grafted cells, signs of tumor formation, and retinal phenotypic marker expression. No obvious signs of tumor formation or rejection were seen in a total of 42 eyes in the immunocompetent hosts. Our results indicate that EIA-NR.3 cells survive at least 1 month in vivo, and can migrate from the vitreous into neuroretinal cell layers. Subpopulations of surviving grafted cells were seen to express photoreceptor markers rhodopsin and recoverin comparably between in vitro and in vivo conditions. However, the number of cells immunoreactive for vimentin and E1A decreased significantly under in vivo conditions. This report represents the first experimental intravitreal transplantation of E1A-immortalized retinal precursor cells into adult and neonatal rats. The intraocular location and environment appears to affect phenotypic expression of surviving grafted cells, especially with respect to vimentin and E1A expression. The fact that E1A-NR.3 cells survived intraocularly at least 1 mo without tumor formation suggests that the cells may continue to be useful for further in vivo studies of experimental retinal transplantation, and effects of histological location on retinal cell phenotype and histogenesis in immunocompetent hosts.

Antibodies to recoverin induce apoptosis of photoreceptor and bipolar cells in vivo.

Autoantibodies against recoverin are found in the sera of patients with cancer-associated retinopathy (CAR) syndrome. In these studies we examined the effect of anti-recoverin antibodies from the sera of patients with CAR and rat monoclonal antibody on the retinas of Lewis rats. Anti-recoverin autoanti-bodies penetrated into the photoreceptor and bipolar cell layers following intravitreal injection. Their presence in the retina could be detected by immunofluorescence 24 h after injection. At the same time, individual cells undergoing apoptosis were identified throughout photoreceptor and bipolar cell layers using terminal transferase-mediated dUTP nick-end labeling (TUNEL) and electron microscopy. Normal antibodies used in control experiments did not produce TUNEL labeling. At 24 h, DNA fragmentation was confirmed by DNA ladder electrophoresis. At the electron microscopic level, there was clear evidence of cells undergoing apoptotic cell death in the retinas treated with anti-recoverin antibodies. At 24 and 96 h, nuclear chromatin condensation and increased vacuolization of photoreceptor outer segments were observed. An examination of retinas from animals receiving anti-retinal antibodies revealed a loss of 1-2 rows of nuclei in the outer and inner nuclear layers whereas all controls (sham, normal IgG, phosphate buffered saline) showed an unchanged number of nuclei rows. In addition, there was an increase in spacing between the rows of nuclei of the outer nuclear layer in retinas treated with anti- recoverin antibodies, indicating additional cell loss. These studies provide clear evidence that anti-recoverin antibodies are capable of penetrating photoreceptor and bipolar cells, the normal site of recoverin expression in the retina, and that anti-recoverin antibodies produce apoptotic cell death. A similar mechanism may occur in patients with CAR, which may lead to visual loss and blindness.

EAE TCR motifs and antigen recognition in myelin basic protein-induced anterior uveitis in Lewis rats.

T cells infiltrating the iris/ciliary body of Lewis rats with anterior uveitis (AU) that had been induced by myelin basic protein (MBP) immunization were previously found to share surface markers common to the T cells that cause experimental autoimmune encephalomyelitis (EAE). To determine whether these AU-associated T cells are in fact the same as those that infiltrate the central nervous system to cause EAE, we examined TCR V gene expression in T cells infiltrating the anterior chamber in rats with AU. As with EAE, we found a biased expression of Vbeta8.2 and Valpha2 in the iris/ciliary body and, although one would expect an influx of nonspecific inflammatory T cells, these biases were still evident at the peak of AU. An analysis of the TCR Vbeta8.2 and Valpha2 sequences derived from the iris/ciliary body demonstrated the presence of the same complementarity determining region 3 motifs found in MBP-specific T cells that are pathogenic for EAE and found in T cells derived from the central nervous system of rats with EAE. Finally, T cells isolated from the iris/ciliary body of rats with AU were found to proliferate in a specific fashion to MBP Ags. Thus, it appears that MBP-specific T cells are pathogenic for AU as well as EAE in the Lewis rat. In addition, the long-term presence of this highly restricted MBP response in the iris/ciliary body indicates that distinct immunoregulatory mechanisms exist in the environment of the eye. This provides an interesting model with which to address questions pertaining to the nature of T cells infiltrating the eye and their regulation during EAE and other systemic diseases.