Escherichia coli K51 and K93 capsular polysaccharides are crossreactive with the group A capsular polysaccharide of Neisseria meningitidis. Immunochemical, biological, and epidemiological studies.

Eleven Escherichia coli strains, crossreactive with the capsular polysaccharide (CPS) of Neisseria meningitidis group A (GrA), were detected among 645 stool isolates from healthy families in Cairo, Egypt. 10 of these strains were of the O107:K93:H27 or O107:K93:SP serotypes and may be considered descendents of a single bacterium or as a clone. The remaining crossreactive strain was of the O7:K51:H18 serotype. None of the 11 strains produced enterotoxins and none were enteroinvasive. The purified CPS of these E. coli strains, as well as a polysaccharide (PS) from B. pumilis, strain Sh17, precipitated with equine GrA (H49) antiserum. A partial identity between the E. coli K93, K51 and Sh17 PS on the one hand and the GrA CPS on the other was observed by double immunodiffusion when reacted against the H49 antiserum. Four K93 strains and one K51 strain were found among 320 E. coli strains from patients at the Clinical Center, National Institutes of Health, and three K93 strains were found in 105 stool samples from children in Copenhagen. The data from these three surveys suggest that these crossreactive E. coli are common organisms and could serve as a stimulus for "natural" GrA CPS antibodies. Quantitative precipitation analysis showed that K51, K93, and Sh17 PS precipitated 25, 46.8, and 50% of H49 antibodies, respectively. Absorption of H49 antiserum with the GrA CPS removed its precipitating activity with the E. coli K93, K51, and Sh17 PS. Absorption of H49 antiserum with either K51 CPS or Sh17 PS removed the homologous crossreactivity only, whereas K93 CPS absorbed both K93 and K51 reactivities. Antibodies, raised by intravenous injection of formalinized E. coli K93 or K51 cells into rabbits, precipitated with GrA CPS and were bactericidal against GrA meningococci. The crossreaction between the E. coli K93 and the GrA CPS was unexpected since these two CPS are compositionally so dissimilar.

Evidence for multiple molecular weight forms of somatostatin-like material in Escherichia coli.

Extracts of Escherichia coli grown in defined medium contain somatostatin-related material (1-10 pg/g wet weight of cells). Preconditioned medium had no immunoactive somatostatin whereas, conditioned medium had 110-150 pg/l. Following purification of the extracted material on Sep-pak C18, Bio-Gel P-6 and HPLC, multiple molecular weight forms of somatostatin- (SRIF-) related material were identified. The material in one peak reacted in both the N-terminal and C-terminal SRIF immunoassay and coeluted on HPLC with SRIF-28, whereas that in a second peak eluted near SRIF-14 and was reactive only in the C-terminal SRIF assay. The two peaks are thus similar to SRIF-28 and SRIF-14 of vertebrates. These findings add support to the suggestion that vertebrate-type peptide hormones and neuropeptides have early evolutionary origins.

Purification and characterization of recombinant human thyrotropin (TSH) isoforms produced by Chinese hamster ovary cells: the role of sialylation and sulfation in TSH bioactivity.

The biological significance of glycosylation variants of pituitary glycoprotein hormones remains controversial because of the indirect methods usually employed to determine carbohydrate composition or structure as well as the use of unreliable biological/immunological ratio to determine bioactivity. We have previously characterized recombinant human TSH (rhTSH) secreted by Chinese hamster ovary cells attached to microcarrier beads in a large scale bioreactor after stable transfection of hCG alpha and hTSH beta minigenes. In the present study rhTSH has been used as a model to determine structure-function relationships of different isoforms of glycoprotein hormones. We have now produced greater than 200 mg rhTSH using a hollow fiber bioreactor. The highly purified rhTSH produced in the hollow fiber bioreactor (rhTSH-N) as well as rhTSH commercially produced in a large scale bioreactor (rhTSH-G) were quantitated by immunoassays, receptor binding assay, and amino acid analysis and further characterized by a variety of physico-biochemical methods, including chromatofocusing and carbohydrate analysis. rhTSH-G, rhTSH-N, as well as pituitary human TSH (phTSH) have been separated by chromatofocusing on a Mono P column into several isoforms with different pI values. Compositional analysis of the fractions showed higher sialic acid content in the more acidic rhTSH-G fractions. phTSH acidic isoforms showed higher total sulfate and sialic acid contents than the more basic fractions. The bioactivities of various TSH isoforms based on rigorous quantitation of mass by amino acid analysis determined in three different FRTL-5 cell bioassays showed that the more basic and less sialylated fractions of rhTSH-G were more active than the more acidic fractions. In contrast to the in vitro data, highly sialylated and acidic rhTSH-G isoforms showed longer plasma half-lives and higher in vivo bioactivity than the basic forms. These results indicate that secreted rhTSH, similar to intrapituitary phTSH, exists as a mixture of charge isoforms that are related at least in part to the degree of sialylation. The degree of sialylation, highly dependent on the bioreactor production conditions, appears to be the major factor affecting the charge heterogeneity, MCR, and bioactivity of rhTSH.

Peritransplant tolerance induction with anti-CD3-immunotoxin: a matter of proinflammatory cytokine control.

BACKGROUND: Tolerance is gaining momentum as an approach to reduce lifelong immunosuppressive therapy while improving transplant longevity. Anti-CD3 immunotoxin (IT), FN18-CRM9, has potential to induce tolerance owing to its exceptional ability to deplete sessile lymph node T cells. However, if initiated at the time of transplantation, alpha-CD3-IT alone elicits a proinflammatory cytokine response, precluding establishment of tolerance. METHODS: Four groups of rhesus monkeys received kidney allografts and immunosuppression. Three groups received alpha-CD3-IT alone or alpha-CD3-IT supplemented with 15-deoxyspergualin (DSG) and/or methylprednisolone (MP). One group received alpha-CD3-monoclonal antibody with DSG and MP. Cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: Supplementing peritransplant alpha-CD3-IT treatment with a brief course of DSG and MP promoted rejection-free kidney allograft acceptance in 75% of macaques followed for up to 550 days. Among those given alpha-CD3-IT alone or with MP, none were long-term survivors. Tolerance developed after alpha-CD3-IT, DSG, and MP treatment, but not when the unconjugated a-CD3 monoclonal antibody was substituted for IT. Systemic production of proinflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha induced after peritransplant alpha-CD3-IT was prevented only in animals given DSG. Despite high levels of interleukin (IL)-12 in the first month after transplant, tolerant recipients exhibited IL-12 resistance, as evidenced by baseline plasma levels of IFN-gamma but elevated IL-4. DSG was shown to inhibit IL-12-driven IFN-gamma production by a mechanism associated with inhibition of nuclear factor kappa-B. CONCLUSIONS: In this model, peritransplant induction of tolerance is promoted by efficient elimination of sessile lymph node T cells and control of the proinflammatory IFN-gamma response by a mechanism that appears to involve resistance to IL-12.

Evidence for an endogenous peptide ligand for the phencyclidine receptor.

Porcine brain contained an active factor that competed with [3H]-phencyclidine (PCP) for binding to rat brain membranes. On reverse phase high pressure liquid chromatography, the active material eluted between 38-42% acetonitrile. Gel filtration chromatography of the factor predicted a molecular weight of approximately 3000 daltons. The endogenous substance appeared to be selective for PCP receptors as it did not interact with either benzodiazepine, neurotensin, nor with mu, delta, or kappa opioid receptors. The active material showed a heterogenous distribution in brain, with highest concentrations found in hippocampus and cortex. It is likely to be a small peptide since various proteases eliminated or markedly reduced the potency of the compound in a [3H]-PCP binding assay. The material also possessed PCP-like activity in two bioassays. Like PCP, it induced contralateral rotational behavior after unilateral intranigral injection and depressed spontaneous cell activity after iontophoretic micropressure application in hippocampus and cerebral cortex. Thus, this small peptide is likely to be an endogenous ligand for the PCP receptor.

Large scale recovery and purification of periplasmic recombinant protein from E. coli using expanded bed adsorption chromatography followed by new ion exchange media.

Expanded bed chromatography was used for the recovery and purification of modified Pseudomonas aeruginosa exotoxin A. The exotoxin accumulates in the periplasmic space of E. coli BL21 (lambda DE3), was released from the cells by osmotic shock and captured by applying the open cell suspension directly to an anion exchanger (STREAMLINE DEAE) using an expanded bed (STREAMLINE) column. Processing of 4.5 kg of E. coli using the expanded bed process was 3 times faster and did not require clarification of the bacterial extract, in comparison with the conventional purification method. Also, the recovered protein solution was 3 times more concentrated and the yield slightly higher.

Treatment of uveitis by oral administration of retinal antigens: results of a phase I/II randomized masked trial.

PURPOSE: To evaluate the effect and safety of the oral administration of retinal antigens as a treatment of ocular inflammation. METHODS: In a phase I/II randomized masked trial, patients with endogenous uveitis who were dependent on immunosuppressive agents were randomly assigned to receive either retinal S antigen alone (10 patients), retinal S antigen and a mixture of soluble retinal antigens (10 patients), a mixture of soluble retinal antigens alone (10 patients), or placebo (15 patients). An attempt was then made to taper patients completely off their standard immunosuppressive therapy over an 8 week period. The primary study endpoint was time to ocular inflammatory attack. The secondary study endpoint was the ability to taper patients completely off their immunosuppressive or cytotoxic medication within 8 weeks. RESULTS: Time to development of the main study endpoint was not statistically significantly different for any of the four treatment groups. However, the group receiving the purified S antigen alone appeared to be tapered off their immunosuppressive medication more successfully compared with patients given placebo (P = .08), whereas all the other groups appeared to do worse than did those receiving placebo. No toxic effects attributable to any treatment were observed. CONCLUSIONS: This phase I/II study is the first to test the use of orally administered S antigen in the treatment of uveitis. Although not statistically significant, patients given S antigen were more likely to be tapered off their chronically administered systemic immunosuppressive therapy than were the other groups tested.

Production of HIV-1 gp120 in packed-bed bioreactor using the vaccinia virus/T7 expression system.

The HeLa cell-vaccinia virus system is an attractive method for producing recombinant mammalian proteins with proper post-translation modifications. This approach is especially important for the production of HIV-1 envelope glycoprotein, gp120, since more than half of its total mass is due to carbohydrates. A recombinant vaccinia virus/T7 RNA polymerase expression system was developed to express and produce large amounts of gp120 tagged with six histidine residues. In this system, the expressed T7 RNA polymerase from one virus drives the transcription of the gp120 encoded in the second virus. During the process development phase, the following parameters were studied: infection time, infection duration, multiplicity of infection, ratio of the two viruses, medium composition, and medium replacement strategy during the infection phase. The chosen production method was based on using the packed-bed bioreactor. The HeLa cells were immobilized on fibrous disks (Fibra-Cel) packed in an internal basket positioned in a vertically mixed bioreactor (Celligen Plus), and 25 g of carriers were packed in a 1.6-L (working volume) reactor. The process included a growth stage followed by a production stage. In the growth stage, the bed was perfused with a serum-containing medium, allowing the cells to grow to saturation, and in the production stage, done using serum-free medium, the cells were infected with the two recombinant viruses. The expressed protein was secreted, collected from the culture fluid, and purified. The specific production was found to be between 2 and 3 microg of protein/10(6) cells, and the volumetric production was around 10 mg/50 g carriers.

Production, purification and immunogenicity of a malaria transmission-blocking vaccine candidate: TBV25H expressed in yeast and purified using nickel-NTA agarose.

We have constructed a second generation malaria transmission-blocking vaccine candidate based on Pfs25, the predominate surface protein of Plasmodium falciparum zygotes, to overcome potential production problems with the original construct. Four modifications were made: (1) addition of the last cysteine residue of the fourth epidermal growth factor like-domain of Pfs25; (2) mutagenesis of asparagine-linked glycosylation sites with glutamine rather than alanine; (3) addition of a six histidine tag at the carboxy-terminus for highly efficient purification of recombinant protein on nickel-NTA agarose; and (4) fermentation that combines continuous glucose fed-batch methodology with pH-controlled glucose addition and a terminal ethanol feed. The resulting product, TBV25H (Transmission-Blocking Vaccine based on Pfs25 with a Histidine tag), appears to be a more potent antigen and immunogen than the original construct, and the fermentation and post-fermentation processing methodology easily lend themselves to technology transfer to the ultimate users, newly industrialized countries.