Long-term follow-up of multilayer amniotic membrane transplantation (MLAMT) for non-traumatic corneal perforations or deep ulcers with descemetocele.

BACKGROUND: To evaluate the long-term efficacy of multilayer amniotic membrane transplantation for reconstruction of epithelium and stroma in non-traumatic corneal perforations (less than 2 mm) or deep ulcers with descemetocele. DESIGN: Retrospective, non-comparative, interventional case series. PATIENTS AND METHODS: Eleven consecutive patients with non-traumatic corneal perforations or deep corneal ulcers with descemetocele refractory to conventional treatments: herpetic or zoster keratitis (n = 4), Sjögren's syndrome (n = 2), rosacea (n = 1), hydrops (n = 1), mucous membrane pemphigoid (n = 1), bacterial keratitis (n = 1) and perforation after protontherapy for melanoma (n = 1). Intervention was: multilayer amniotic membrane transplantation with cryopreserved amniotic membrane. Complication rate and clinical outcome were evaluated in this long-term follow-up. RESULTS: Mean follow-up was 32 months (12 to 60). Integration of the multilayer amniotic membrane was obtained in 10 cases after one year. Corneal epithelium healed above the membrane in 10 cases within 3 weeks and remained stable after 32 months in 9 cases. Thickness of the stroma was increased and remained stable during the follow-up in 9 cases. In one case herpetic keratitis recurred with a corneal perforation. The clearing of the amniotic membrane was gradually obtained over a period of 11 months. Complications occurred in 15% of the eyes during the long-term follow-up. CONCLUSION: Multilayer amniotic membrane transplantation is a safe and efficient technique for a long restoration of the corneal integrity after non-traumatic corneal perforations or deep corneal ulcers with descemetocele. Long-term prognosis of these eyes depends of the gravity of the initial disease.

An origin of DNA replication and a transcription silencer require a common element.

A eukaryotic chromosomal origin of replication was identified in the yeast Saccharomyces cerevisiae. By several criteria, including map position, deletion analysis, and a synthetic form of saturation mutagenesis, the origin co-localized with the HMR-E silencer, which is a DNA element that represses transcription of the adjacent genes. A specific site within the silencer was required for both initiation of chromosomal replication and for repression of transcription. This analysis directly demonstrates that initiation of eukaryotic chromosomal replication is dependent on specific sequence elements and that a particular element can act in both initiation of chromosomal replication and regulation of transcription.

Silencing: the establishment and inheritance of stable, repressed transcription states.

Silencing refers to a particular type of transcriptional repression characterized by the formation of a genetically heritable, repressed transcriptional state. Examples of silencing include position-effect variegation, X-chromosome inactivation, and the repression of the silent mating-type gene loci in yeast. Recent discoveries suggest that silencing in yeast, like silencing in larger eukaryotes, results from a particular chromatin structure that defines a chromosomal domain. In addition, a chromosomal origin of DNA replication is required for silencing in yeast, suggesting that DNA replication plays a role in forming functional chromosomal domains.

Sequences far downstream from the classical tRNA promoter elements bind RNA polymerase III transcription factors.

We have examined the interaction of transcription factors TFIIIC and TFIIID with a silkworm alanine tRNA gene. Previous functional analysis showed that the promoter for this gene is unusually large compared with the classical tRNA promoter elements (the A and B boxes) and includes sequences downstream from the transcription termination site. The goal of the experiments reported here was to determine which sequences within the full promoter make stable contacts with transcription factors. We show that when TFIIIC and TFIIID are combined, a complex is formed with the tRNA(Ala)C gene. Neither factor alone can form this complex. DNase I digestion of gene-factor complexes reveals that most of the tRNA(Ala)C promoter is in contact with factors. The protected region extends from -1 to at least +136 and includes both the A and B boxes and the previously identified downstream promoter sequences. Analysis of mutant promoters shows that sequence-specific contacts throughout the protected region are required for binding. The role of 3'-flanking sequences in transcription factor binding explains the contribution of these sequences to the tRNA(Ala)C promoter. We discuss the possibility that such sequences affect promoter strength in other tRNA genes.

SAS4 and SAS5 are locus-specific regulators of silencing in Saccharomyces cerevisiae.

Sir2p, Sir3p, Sir4p, and the core histones form a repressive chromatin structure that silences transcription in the regions near telomeres and at the HML and HMR cryptic mating-type loci in Saccharomyces cerevisiae. Null alleles of SAS4 and SAS5 suppress silencing defects at HMR; therefore, SAS4 and SAS5 are negative regulators of silencing at HMR. This study revealed that SAS4 and SAS5 contribute to silencing at HML and the telomeres, indicating that SAS4 and SAS5 are positive regulators of silencing at these loci. These paradoxical locus-specific phenotypes are shared with null alleles of SAS2 and are unique among phenotypes of mutations in other known regulators of silencing. This work also determined that these SAS genes play roles that are redundant with SIR1 at HML, yet distinct from SIR1 at HMR. Furthermore, these SAS genes are not redundant with each other in silencing HML. Collectively, these data suggest that SAS2, SAS4, and SAS5 constitute a novel class of regulators of silencing and reveal fundamental differences in the regulation of silencing at HML and HMR. We provide evidence for a model that accounts for the observation that these SAS genes are both positive and negative regulators of silencing.

HMR-I is an origin of replication and a silencer in Saccharomyces cerevisiae.

There appear to be fundamental differences between the properties of the silencers at HML and HMR, with some being origins of replication and others not. Moreover, past studies have suggested that HMR-I's role in silencing may be restricted to plasmid contexts. This study established that HMR-I, like HMR-E and unlike either HML silencer, is an origin of replication. Moreover, both HMR-E and HMR-I contribute to silencing of a chromosomal HMR locus. In addition, we found that Abf1p plays no unique role in silencer function.

The role of Sas2, an acetyltransferase homologue of Saccharomyces cerevisiae, in silencing and ORC function.

Silencing at the cryptic mating-type loci HML and HMR of Saccharomyces cerevisiae requires regulatory sites called silencers. Mutations in the Rap1 and Abf1 binding sites of the HMR-E silencer (HMRa-e**) cause the silencer to be nonfunctional, and hence, cause derepression of HMR. Here, we have isolated and characterized mutations in SAS2 as second-site suppressors of the silencing defect of HMRa-e**. Silencing conferred by the removal of SAS2 (sas2 delta) depended upon the integrity of the ARS consensus sequence of the HMR-E silencer, thus arguing for an involvement of the origin recognition complex (ORC). Restoration of silencing by sas2 delta required ORC2 and ORC5, but not SIR1 or RAP1. Furthermore, sas2 delta suppressed the temperature sensitivity, but not the silencing defect of orc2-1 and orc5-1. Moreover, sas2 delta had opposing effects on silencing of HML and HMR. The putative Sas2 protein bears similarities to known protein acetyltransferases. Several models for the role of Sas2 in silencing are discussed.

Identification of SAS4 and SAS5, two genes that regulate silencing in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, chromatin-mediated silencing inactivates transcription of the genes at the HML and HMR cryptic mating-type loci and genes near telomeres. Mutations in the Rap1p and Abf1p binding sites of the HMR-E silencer (HMRa-e**) result in a loss of silencing at HMR. We characterized a collection of 15 mutations that restore the alpha-mating phenotype to MATalpha HMRa-e** strains. These mutations defined three complementation groups, two new groups and one group that corresponded to the previously identified SAS2 gene. We cloned the genes that complemented members of the new groups and identified two previously uncharacterized genes, which we named SAS4 and SAS5. Neither SAS4 nor SAS5 was required for viability. Null alleles of SAS4 and SAS5 restored SIR4-dependent silencing at HMR, establishing that each is a regulator of silencing. Null alleles of SAS4 and SAS5 bypassed the role of the Abf1p binding site of the HMR-E silencer but not the role of the ACS or Rap1p binding site. Previous analysis indicated that SAS2 is homologous to a human gene that is a site of recurring translocations involved in acute myeloid leukemia. Similarly, SAS5 is a member of a gene family that included two human genes that are the sites of recurring translocations involved in acute myeloid leukemia.

Monoclonal antibodies against carcinoembryonic antigen (CEA) used in a solid-phase enzyme immunoassay. First clinical results.

A solid-phase enzyme immunoassay using both mouse monoclonal and goat polyclonal antibodies against carcinoembryonic antigen (CEA) was developed. The assay detects 0.6 to 1.2 ng of CEA per ml of serum and has 3 incubation steps which can be performed in 1 day. Polystyrene balls coated with polyclonal goat anti-CEA antibodies are first incubated with heat-extracted serum samples. Bound CEA is then detected by addition of mouse monoclonal antibodies, followed by goat IgG anti-mouse IgG1 coupled to alkaline phosphatase. Results with this enzyme immunoassay using monoclonal antibodies (M-EIA) have been compared with those obtained by the conventional inhibition radioimmunoassay (RIA) using goat antiserum. Three hundred and eighty serum samples from 167 patients with malignant or non-malignant diseases and from 134 normal individuals with or without heavy smoking habits were analyzed by the 2 assays. Excellent correlation between the results of the 2 assays was obtained, but the M-EIA, using monoclonal antibodies from a single hybridoma, did not discriminate better than the conventional RIA between CEA produced by different types of carcinoma and between CEA associated with malignant or non-malignant diseases. Follow-up studies of several patients by sequential CEA determinations with the 2 assays showed that the M-EIA was as accurate as the RIA for the detection of tumor recurrences.

Protective effect of rabbit immune serum administered orally to rats infected by a human pathogenic strain of E. coli.

1-week-old rats were inoculated orally with a strain of E. coli (serotype 078) isolated from the blood of a newborn baby who had died of septicemia. During the 3 weeks following inoculation, approximately 50% of the animals died of septicemia and 60% of the surviving rats had pathogenic bacteria in their rectum. Some of the surviving rats were severely impaired in their development. Autopsy showed evidence of active intestinal infection localized mainly in the ileum and cecum. A rabbit anti-E. coli (strain 23) serum (agglutinating titer: 1/2,500) afforded 100% protection when as little as 0.03 mg of serum protein per gram of rat body weight was orally administered in a single dose. The immune serum had an effect both on the mortality rate and on the growth of the rats. However, it never affected the survival of pathogenic bacteria in the rectum, even when administered at a daily dose of 1.5 mg of serum protein per gram of rat body weight on 4 consecutive days. The immune rabbit serum had only a weak bactericidal effect in vitro. The hemagglutination test showed the presence in the immune serum of antibodies against the fimbriae of the pathogenic E. coli strain (titer: 1/1,000). The role of antibody in inhibiting the adherence of bacteria to epithelial cells and/or their progression across the mucous layer are discussed as possible immune mechanisms in the intestinal lumen.

Synergism between iron chelators and complement for bactericidal activity.

Iron-binding agents such as the plasma protein transferrin or the siderophore desferal from Streptomyces pilosus inhibit the growth of pathogenic bacteria, supposedly by interfering with iron uptake by those bacteria. This study shows that anti-Escherichia coli activity exerted by desferal and transferrin can be increased in a synergistic way by complement and anti-E. coli antibodies of normal serum.

Inhibitors of the adhesiveness of enteropathogenic E. coli.

The entero-pathogenic strain of E. coli 0125: K 70 was able to adhere to washed guinea-pig erythrocytes and to cause their agglutination. Electron microscopy revealed this strain to be rich in fimbriae. They were able to cause hemagglutination by themselves, generally at protein concentration of 10 to 100 mug per ml. D-mannose and alpha-methylmannoside were able to inhibit hemagglutination by the whole bacteria or their isolated fimbriae at concentrations of 0.003 to 0.012 mg/ml. L-mannose, D-lactose, D-glucose, N-acetylglucosamine, D-galactose, N-acetyl-D-galactosamine, D-fucose, did not exert any inhibiting effect at concentrations as high as 50 mg/ml. N-acetyl-neuraminic acid was also uneffective at concentration as high as 9 mg/ml.

Identification of a compound origin of replication at the HMR-E locus in Saccharomyces cerevisiae.

Eukaryotic chromosomal origins of replication are best defined in Saccharomyces cerevisiae. Previous analysis of yeast origins suggests that they are relatively simple structures comprised of three or four small DNA sequence elements contained within approximately 100-200-base pair regions (Gilbert, D. M. (1998) Curr. Opin. Genet. Dev. 8, 194-199). In contrast, the sequence elements that may comprise origins in multicellular eukaryotes are largely unknown. The yeast HMR-E region is both a chromosomal origin of replication and a silencer that represses transcription of adjacent genes through a position effect. The analysis presented here indicated that HMR-E had a novel DNA structure that was more complex than defined for other yeast origins, and thus revealed that there is variation in the structural complexity of yeast origins. In contrast to "simple" yeast origins, the origin at HMR-E consisted of at least three independent subregions that had the capacity to initiate replication. We have termed HMR-E a compound origin to reflect its structural complexity. Furthermore, only one origin within the compound origin was a silencer.

Leishmania mexicana: extracellular proton concentration is a key regulator of cysteine proteinase CPb expression.

The purpose of this work was to determine which parameters trigger expression of proteins that are potentially important for the differentiation of Leishmania mexicana from the promastigote to the amastigote stage. To this effect, a protein-free axenic incubation system was used that supported the differentiation of L. mexicana promastigotes into amastigotes at 33 degreesC and at acidic pH. The predominant modification detected in SDS-PAGE patterns of extracted soluble proteins was the appearance in parasites cultured for 4 days of a strong 28-kDa protein band that displayed the same position and intensity as seen in amastigotes extracted from a mouse lesion. These molecules exhibited in gelatin gels the typical lytic pattern of cysteine proteinases (CPs) and were shown to belong to the CPb family, as further demonstrated by N-terminal amino acid sequencing. The expression of these enzymes was quantified by their lytic activity on the fluorogenic Z-F-R-AMC CP substrate. When the parasites were incubated at 33 degreesC for 3 days at various initial pHs, CPb started to be induced when the pH dropped below 5. When comparing cultures maintained at 26 or 33 degreesC for 3 days, it was seen that a rise in extracellular proton concentration (to pH 4.2-4.6) resulted in production of CPb at both temperatures (around 20-fold over the concentration measured in promastigotes cultured at 26 degreesC, pH >6). These results demonstrate that extracellular proton concentration is a key regulator of cysteine proteinase CPb synthesis and that an increase in temperature is neither necessary nor sufficient for the expression of this enzyme.

Vaccine development against cutaneous leishmaniasis. Subcutaneous administration of radioattenuated parasites protects CBA mice against virulent Leishmania major challenge.

Experiments described in this paper were aimed at determining whether subcutaneous inoculation of live, avirulent Leishmania major would protect mice against infection by the virulent parasite. To this effect, promastigotes or amastigotes of a highly virulent strain of L. major (MRHO/IR/76), used in human trials of leishmanization, and which induces non-healing skin lesions in both CBA and BALB/c mice, were rendered non-pathogenic by gamma irradiation. A dose of 150 krad was required to abrogate the virulence of the parasite as tested on BALB/c mice. Strikingly, however, not all leishmanias were completely inactivated by this procedure since live parasites were detected in the footpads and/or the inguinal lymph nodes as long as 28 days (CBA) or 18 weeks (BALB/c) after injection. Furthermore, 150 krad-irradiated promastigotes retained the capacity to transform into amastigotes intracellularly in vitro. Subcutaneous inoculation of this irradiated 'vaccine' conferred onto CBA mice a high degree of protection against challenge by both the homologous and a heterologous (MRHO/SU/59/P) strains of L. major. Lymph node cells from protected animals acquired the capacity to activate infected macrophages in vitro to kill intracellular L. major. To allow for maximum development of immunoprotection, the irradiated promastigotes had to remain viable, perhaps reflecting a requirement for transformation into amastigotes in the vaccinated host.

Designer deletion and prototrophic strains derived from Saccharomyces cerevisiae strain W303-1a.

We report the construction of Saccharomyces cerevisiae strains isogenic to W303-1a that are designed to allow efficient genetic analysis. To facilitate the generation of null alleles of target genes by PCR-mediated gene disruption, we constructed designer deletion alleles of the ARG4, TRP1 and URA3 genes. In addition, a single pair of oligonucleotide primers were designed that can be used to amplify any of several marker genes for use in PCR-mediated gene disruption. A new version of the 'reusable' hisG-URA3-hisG cassette was constructed for use in PCR-mediated gene disruption. Finally, to facilitate the formation of isogenic diploids by selection, we constructed strains that contain combinations of wild-type alleles of ADE2, HIS3, LEU2, TRP1 and URA3.

In vivo isotype regulation of humoral responses to dextran B1355 in BALB/c mice. Double-class IgG3/IgM producers as a function of age.

This report shows that, in 8- to 10-month-old BALB/c mice immunized intraperitoneally with dextran B1355, approximately 75% of IgG3 anti-alpha (1----3) polyglucan (anti-dex) plaque-forming cells (PFC) detected in the spleen were identified as double-Ig class producers secreting simultaneously IgG3 and IgM antibodies with the same specificity for the dex epitope. Under the same conditions of immunization, however, IgA anti-dex PFC were mostly single-class secretors. IgA PFC developed in the spleen in highest numbers (equal to IgM), but in Peyer's patches IgA PFC were sevenfold more numerous than IgM. Furthermore, spleen IgG3 anti-dex PFC responses were low compared with spleen IgA and IgM anti-dex PFC responses and appeared only late in ontogeny. The possibility is discussed whether a TH dependence of the IgA anti-dex response and a TH-independent generation of the IgG3 response are responsible for the different pattern of isotype expression.

Age-dependence of the IgA anti-alpha (1 leads to 3) dextran B1355 response in vitro.

The magnitude of the Balb/c mouse IgA anti-alpha (1 leads to 3) dextran B1355 (anti-dex) response in vivo was recently found to be markedly T-cell-dependent and age-dependent. This report demonstrates that the in vitro IgA anti-dex response by mesenteric lymph nodes (MLN) is highly age-dependent and that there is an age-dependent increase of the background IgA anti-dex plaque-forming cell (PFC) response occurring in the absence of added antigen which correlates significantly with the magnitude of the antigen-stimulated response. In aging mice both background and antigen-stimulated IgA anti-dex responses appeared to be significantly higher in MLN than in spleen cultures. Moreover, it is shown that there is a striking increase with age of natural antibody in the serum of normal Balb/c to alpha (1 leads to 3) glucan determinants, particularly IgA and lesser amounts of IgM and IgG3.

The properties of a new polymerase III transcription factor reveal that transcription complexes can assemble by more than one pathway.

We have resolved a previously unidentified factor (TFIIID) that is required for in vitro transcription of polymerase III templates. Our ability to resolve factor D from each of the other components of the transcription machinery (polymerase and transcription factors IIIB and IIIC) allowed us to test the capacity of these separated components to form stable complexes with tRNA genes. We find that none of the individual components binds detectably to tRNA genes, but that certain combinations of transcription factors do bind. Our results show that TFIIID is essential for binding and that formation of a full transcription complex can proceed by either of two different pathways.

Vaccination against Leishmania major in a CBA mouse model of infection: role of adjuvants and mechanism of protection.

Gp63 is a major surface protein of Leishmania promastigotes. Its protective efficacy has been tested in several experimental models using different mouse strains, gp63 forms, adjuvants and routes of immunization, giving rise to conflicting results. This investigation was designed to determine whether these discrepancies could be ascribed to differing experimental procedures, and to compare gp63-induced protection with that achieved using live promastigotes. Preliminary experiments demonstrated that gp63 was an extremely potent immunogen compared to a standard antigen (ovalbumin). Protection against Leishmania major infection afforded by gp63 inoculation was studied in CBA mice. Injection of gp63 in saline, or of CFA, BCG, and C. parvum without antigen, induced significant protection. When gp63 and adjuvants were combined, results differed depending on the site of vaccination relative to that of the challenge infection. Vaccination with gp63 plus adjuvants in the tail (i.e. close to the site of infection) led to a stronger reduction of lesion size than the basal level of protection elicited by adjuvants alone, except in the case of CFA. Surprisingly however, when the antigen was injected at a distance from the site of infection (immunization in the hind foot pads, infection in the rump), the protective effect of gp63 was decreased by the adjuvants. Finally, vaccination at either site using live parasites (radioattenuated or virulent promastigotes) resulted in most instances in better protection than achieved by any protocol using gp63 and adjuvants. While anti-gp63 T cells proliferated in vitro in response to L. major-infected bone marrow-derived macrophages, they were unable to activate macrophages for parasite killing. This is in contrast with lymphocytes from mice immunized with live parasites, which both proliferated and stimulated significant killing of the microorganisms within 48 h.