Safety and pharmacokinetics of XOMA 3AB, a novel mixture of three monoclonal antibodies against botulinum toxin A.

Botulinum neurotoxin A is a category A bioterrorism agent. Current antitoxin therapies are scarce and produce adverse reactions. XOMA 3AB consists of 3 IgG1 monoclonal antibodies (MAbs), each with a distinct human or humanized variable region, which bind to distinct epitopes on botulinum neurotoxin serotype A. This first-in-human study evaluated the safety and pharmacokinetics (PK) of escalating doses of XOMA 3AB administered intravenously (i.v.) to healthy adults. In this double-blind placebo-controlled dose escalation study, 3 cohorts of 8 healthy subjects received a single intravenous dose of XOMA 3AB or placebo at a 3:1 ratio. Follow-up examinations included physical examinations, hematology and chemistry blood tests, electrocardiograms, and pharmacokinetics. Pharmacokinetic parameters were estimated using noncompartmental methods. There were no infusion discontinuations or hypersensitivity reactions. Two or more subjects experienced headache, hyperglycemia, or anemia; none was dose related. All adverse events (AEs) were mild to moderate except for an episode of exercise-induced elevation of a subject's creatine phosphokinase (CPK) level, unrelated to XOMA 3AB. Concentration-time plots demonstrated a peak in MAb concentrations 1 to 2 h after completion of the infusion, after which the levels declined in a biexponential decay pattern for all analytes. For each MAb, the maximum concentration of drug in serum (Cmax) and the area under the concentration-time curve from 0 to infinity (AUCinf) increased as the dose increased. Clearance of the humanized mouse MAb was more rapid than that of the two fully human MAbs, particularly at the lowest dose. None of the MAbs was immunogenic. At the doses administered, XOMA 3AB was well tolerated. These safety findings support further investigation of XOMA 3AB as a potential agent for botulism treatment and postexposure prophylaxis. (This study has been registered at ClinicalTrials.gov under registration no. NCT01357213.).

Stability of colistimethate sodium in aqueous solution.

Colistimethate sodium, increasingly used to treat multidrug-resistant Gram-negative infections, spontaneously hydrolyzes to form colistin A (polymyxin E1) and B (polymyxin E2/B) when mixed with water. High levels of these active breakdown products at the time of administration have been associated with nephrotoxicity and even death. In this study, reconstituted colistimethate sodium was shown to be stable (<1.0% colistin A/B formation) for up to 24 h when stored at 21, 0, -20, and -70°C.

Experimental gonococcal urethritis and reinfection with homologous gonococci in male volunteers.

BACKGROUND: Reinfection, a common occurrence with gonorrhea, may result from a lack of protective immune response, or from the tremendous gonococcal strain variation. GOAL: A two-phase study in human volunteers tested whether experimental infection with Neisseria gonorrhoeae MS11mkC would protect against reinfection with the same organisms. STUDY DESIGN: In phase 1, an intraurethral inoculum of 57,000 piliated, transparent (opacity protein-negative [Opa-]) MS11mkC N gonorrhoeae infected 14 of 15 (93%) volunteers. The volunteers were encouraged to delay treatment for at least 5 days. In phase 2, which began 2 weeks after treatment for the initial infection, volunteers were inoculated with 7,100 piliated, Opa- MS11mkC. RESULTS: The phase 2 challenge infected 6 of 14 (43%) previously infected volunteers and 5 of 10 (50%) naïve control subjects. Phase 1 volunteers who resisted reinfection were significantly more likely to have had a fourfold or greater increase in lipooligosaccharide immunoglobulin G during phase 1 than those who did not resist reinfection (P = 0.026). CONCLUSIONS: Although infection did not provide protection from reinfection under the conditions used, the results suggest that immunity to reinfection is more complex than anticipated by the experimental design.

CEACAM is not necessary for Neisseria gonorrhoeae to adhere to and invade female genital epithelial cells.

Neisseria gonorrhoeae has a repertoire of up to 11 opacity-associated (Opa) proteins that are adhesins. Most Opa proteins adhere to CEACAM antigens and when CEACAM molecules are present on the surface of transfected epithelial cells their binding by Opa is thought to induce invasion of these cells by gonococci. In this study, we investigated whether several malignant epithelial cell lines, normal cervical and fallopian tube epithelial cell cultures, as well as normal fallopian tube tissue express several of the CEACAM molecules, and whether gonococci use these molecules for adherence and invasion of these female genital epithelial cells. A primary cervical cell culture and metastatic cervical cell line ME180 both expressed CEACAM as shown by whole cell ELISA and flow cytometry, and increased the surface expression of total CEACAM during incubation with Opa+ gonococci. Opa+ gonococci both adhered to and invaded these cells; CEACAM-specific monoclonal antibody (MAb) partially abolished this interaction. Two primary fallopian epithelial tube cell cultures, a primary cervical cell culture and two malignant cell lines, HEC-1-B and HeLa, did not express CEACAM nor was CEACAM mRNA present. No evidence of either intracellular or secreted extracellular CEACAM was found with HEC-1-B and HeLa cells. Opa+ gonococci both adhered to and invaded CEACAM non-expressing cells; however, Opa+ gonococcal association with these non-expressing cell lines could not be inhibited with CEACAM-specific MAb. These data show that CEACAM is not always expressed on female genital epithelial cells and is not essential for gonococcal adherence and invasion. However, when CEACAM is expressed, Opa+ gonococci exploit it for the adherence to and invasion of these cells.

Pigment gallstone pathogenesis: slime production by biliary bacteria is more important than beta-glucuronidase production.

Pigment stones are thought to form as a result of deconjugation of bilirubin by bacterial beta-glucuronidase, which results in precipitation of calcium bilirubinate. Calcium bilirubinate is then aggregated into stones by an anionic glycoprotein. Slime (glycocalyx), an anionic glycoprotein produced by bacteria causing foreign body infections, has been implicated in the formation of the precipitate that blocks biliary stents. We previously showed that bacteria are present within the pigment portions of gallstones and postulated a bacterial role in pigment stone formation through beta-glucuronidase or slime production. Ninety-one biliary bacterial isolates from 61 patients and 12 control stool organisms were tested for their production of beta-glucuronidase and slime. The average slime production was 42 for biliary bacteria and 2.5 for stool bacteria (P <0.001). Overall, 73% of biliary bacteria and 8% of stool bacteria produced slime (optical density >3). In contrast, only 38% of biliary bacteria produced beta-glucuronidase. Eighty-two percent of all patients, 90% of patients with common bile duct (CBD) stones, 100% of patients with primary CBD stones, and 93% of patients with biliary tubes had one or more bacterial species in their stones that produced slime. By comparison, only 47% of all patients, 60% of patients with CBD stones, 62% of patients with primary CBD stones, and 50% of patients with biliary tubes had one or more bacteria that produced beta-glucuronidase. Most biliary bacteria produced slime, and slime production correlated better than beta-glucuronidase production did with stone formation and the presence of biliary tubes or stents. Patients with primary CBD stones and biliary tubes had the highest incidence of slime production. These findings suggest that bacterial slime is important in gallstone formation and the blockage of biliary tubes.

Functional activities and immunoglobulin variable regions of human and murine monoclonal antibodies specific for the P1.7 PorA protein loop of Neisseria meningitidis.

The meningococcal PorA protein is considered a promising vaccine candidate. Although much is understood regarding the structure of PorA proteins, little is known about the structure-function relationships of PorA antibodies. The aim of this study was to compare the functional and molecular characteristics of a human monoclonal antibody (MAb) and three murine MAbs specific for the PorA P1.7 serosubtype. Murine MAbs 207,B-4 (immunoglobulin G2a [IgG2a]) and MN14C11.6 (IgG2a) were both bactericidal and opsonophagocytic for P1.7-expressing meningococci, whereas human MAb SS269 (IgG3) and murine MAb 208,D-5 (IgA) initiated neither effector function. Epitope mapping with synthetic peptides revealed that MAbs 207,B-4 and 208,D-5 recognized the sequence ASGQ, which is the same specificity motif that a previous study had established for SS269 and MN14C11.6. Nucleotide and amino acid sequence analyses of the variable regions of the four MAbs showed that the SS269 V(H) region belonged to the VH3 family and was approximately 70% homologous to those of the murine MAbs which were all from the 7183 family, whereas the SS269 V(L) region belonged to the Vlambda1-b family and was less than 40% homologous to those of the murine MAbs which were all members of the Vkappa1 family. The Fab fragment of SS269 was cloned and expressed in Escherichia coli and was shown by enzyme-linked immunosorbent assay analyses to bind as well as intact SS269 MAb to P1.7,16 serosubtype group B strain 44/76. We conclude that distinct differences exist in the effector function activities and variable region gene sequences of human and murine P1.7-specific MAbs despite their recognition of similar epitopes.

Neisseria gonorrhoeae coordinately uses Pili and Opa to activate HEC-1-B cell microvilli, which causes engulfment of the gonococci.

This study was undertaken to examine concomitant roles of pili and colony opacity-associated proteins (Opa) in promoting Neisseria gonorrhoeae adherence to and invasion of human endometrial HEC-1-B cells. Adherence of N. gonorrhoeae to cultured HEC-1-B cells was saturable, even though organisms adhered to <50% of the cells. During 4 to 6 h of incubation, adherent mono- and diplococci formed microcolonies on the surfaces of the cells. Microvilli of the HEC-1-B cells adhered by their distal ends to individual cocci within the microcolonies. When the microcolonies grew from isogenic pilus-negative (P-) Opa-, P- Opa+, or P+ Opa- gonococci, microvilli did not elongate, and the colonies were not engulfed. In contrast, the microvilli markedly elongated during exposure to P+ Opa+ gonococci. The microvilli adhered to the organisms along their full lengths and appeared to actively participate in the engulfment of the microcolonies. Internalized microcolonies, with P+ Opa+ gonococci, contained dividing cocci and appeared to be surrounded by cell membrane but were not clearly within vacuoles. In contrast, degenerate individual organisms were within vacuoles. Low doses of chloramphenicol, which inhibits protein synthesis by both prokaryotes and eukaryotes, prevented the microvillar response to and internalization of the P+ Opa+ gonococci; higher doses caused internalization without microvillus activation. Cycloheximide and anisomycin, which inhibit only eukaryotic protein synthesis, caused dose-dependent enhancement of uptake. Cytochalasins reduced engulfment; colchicine had no effect. These results show that gonococci must express both pili and Opa to be engulfed efficiently by HEC-1-B cells.

Neisseria gonorrhoeae that infect men have lipooligosaccharides with terminal N-acetyllactosamine repeats.

Infectious Neisseria gonorrhoeae make relatively large lipooligosaccharides (LOS) that structurally resemble human glycosphingolipids. MS11mkC is an LOS variant of N. gonorrhoeae strain MS11 which was isolated from men at the onset of dysuria (Schneider, H., Griffiss, J. M., Boslego, J. W., Hitchcock, P. J., Zahos, K. M., and Apicella, M. A. (1991) J. Exp. Med. 174, 1601-1605). Delayed extraction matrix-assisted laser desorption and ionization and electrospray ionization mass spectrometry of O-deacylated MS11mkC LOS produced ions consistent with known LOS which have lacto-N-neotetraose (Galbeta1-->4GlcNAcbeta1-->3Galbeta1-->4Glc; paraglobosyl; monoclonal antibodies (mAbs) 1B2(+) and 06B4(+)) and GalNAc-->lacto-N-neotetraose (gangliosyl; mAb 1-1-M+) oligosaccharides. Ion peaks for a larger LOS which also bound mAb 1B2 indicated the addition of a hexose (+162 Da) to gangliosyl LOS or the addition of a hexose and a N-acetylhexosamine (+365 Da) to paraglobosyl LOS. Analysis of HF-treated and O-deacylated LOS revealed three major components present in a phosphoethanolamine (PEA)0 and a PEA1 series. Digestion of MS11mkC LOS by beta-N-acetylhexosaminidase and beta-galactosidase, alone and sequentially, combined with mAb binding patterns, confirmed the presence of a nonreducing terminal repeating LacNAc ((Galbeta1-->4GlcNAc)2) on the largest LOS, rather than a parallel oligosaccharide structure.

Characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae strain F62: instability of transposon Tn1545 delta3 in gonococci and evidence that multiple genetic loci are essential for lipooligosaccharide sialylation.

Neisseria gonorrhoeae strain JB1 was previously shown to be defective in the sialylation of lipoologosaccharide (LOS) by exogenous CMP-NANA. The LOS components synthesized by the mutant now have been shown by mass spectrometry to be similar to those in the parental strain, F62, and to include the 4.5 kDa widely conserved lacto-N-neotetraose component that can be sialylated. The same two LOS components could be sialylated on the surface of the mutant and parental strains. One major component was sialylatable after chemical extraction of the LOS from either strain. These data confirm that the mutant, JB1, retains the ability to synthesize the LOS target required for the conversion by sialylation of serum-sensitive gonococci to serum resistance. A single base frame-shift mutation was found in the lst gene from the mutant, resulting in the replacement of the final 61 amino acids at the C-terminus of the sialyltransferase by four residues. Seventeen independent clones of the lst gene were isolated from the parental strain, but none of them complemented the sialyltransferase defect of the mutant and no sialyltransferase activity expressed from the clones could be detected in Escherichia coli. Although the data suggest that the mutant might be defective in genes at more than one chromosomal locus and that multiple loci are essential for sialyltransferase synthesis and activity, the alternative possibility, that DNA adjacent to the lst gene encodes a product which is toxic to E. coli, cannot be excluded. The site of insertion of the transposon Tn1545-Delta3 in strain JB1 was cloned and sequenced. The transposon is located in an intergenic region adjacent to genes for a putative ATP-dependent transport protein, but encoding no recognizable function relevant to LOS sialylation. Evidence that transposon Tn1545-Delta3 is unstable in gonococci is presented.

Sialylation of Neisseria meningitidis lipooligosaccharide inhibits serum bactericidal activity by masking lacto-N-neotetraose.

Exogenous sialylation of gonococcal lipooligosaccharide causes resistance to serum bactericidal activity. The aim of this study was to determine how lipooligosaccharide sialylation affects the serum sensitivities of group C Neisseria meningitidis strains. The relationship between the degree of sialylation or expression of the lipooligosaccharide sialic acid acceptor, lacto-N-neotetraose (LNnT), of nine meningococcal strains and their sensitivities to a pool of normal human sera was assessed. All strains expressed LNnT that was variously endogenously sialylated. Susceptibility to serum bactericidal activity ranged from extremely sensitive to resistant in 50% serum. For endogenously sialylated strains, the amount of killing correlated with the amount of free LNnT above a threshold of expression; strains that expressed less than the threshold survived in 25% serum. All strains added more sialic acid when they were grown in medium that contained cytidine monophospho-N-acetylneuraminic acid. Exogenous sialylation reduced the expression of free LNnT and significantly increased serum resistance. Exogenous sialylation affected killing through both classical and alternative complement pathways. The killing of exogenously sialylated strains also correlated with the amount of free LNnT. The amounts of endogenous, exogenous, and total sialic acid bound to LNnT did not correlate with the resistance of strains to serum bactericidal activity; rather, the loss of free LNnT expression by sialylation was associated with resistance. In conclusion, the expression of free LNnT by group C meningococcal strains is directly associated with the amount of killing of organisms in pooled human sera. Both endogenous and exogenous lipooligosaccharide sialylation are associated with increased serum resistance by masking LNnT.

Immunoprophylaxis against klebsiella and pseudomonas aeruginosa infections. The Federal Hyperimmune Immunoglobulin Trial Study Group.

To determine if passive immunization could decrease the incidence or severity of Klebsiella and Pseudomonas aeruginosa infections, patients admitted to intensive care units of 16 Department of Veterans Affairs and Department of Defense hospitals were randomized to receive either 100 mg/kg intravenous hyperimmune globulin (IVIG), derived from donors immunized with a 24-valent Klebsiella capsular polysaccharide plus an 8-valent P. aeruginosa O-polysaccharide-toxin A conjugate vaccine, or an albumin placebo. The overall incidence and severity of vaccine-specific Klebsiella plus Pseudomonas infections were not significantly different between the groups receiving albumin and IVIG. There was some evidence that IVIG may decrease the incidence (2.7% albumin vs. 1.2% IVIG) and severity (1.0% vs. 0.3%) of vaccine-specific Klebsiella infections, but these reductions were not statistically significant. The trial was stopped because it was statistically unlikely that IVIG would be protective against Pseudomonas infections at the dosage being used. Patients receiving IVIG had more adverse reactions (14.4% vs. 9.2%).

Bacterial enzymes can add galactose alpha 1,3 to human erythrocytes and creates a senescence-associated epitope.

Humans have abundant circulating anti-alpha (1,3-di)-galactosyl (alpha Gal) antibodies (anti-Gal). Anti-Gal has been implicated in the clearance of senescent human erythrocytes (RBCs). The nature of the anti-Gal-binding RBC epitope has defied explanation, given that humans repress expression of the alpha 1,3 galactosyltransferase (alpha 1,3 GT) enzyme. This study explored whether alpha Gal epitopes on human RBCs might be synthesized by alpha 1,3 GTs of bacterial origin that are translocated into the circulation during commensal colonization of the gut by gram-negative bacteria. We found that an acellular Klebsiella pneumoniae sonicate could add 3H-UDP-Gal to human RBCs in the alpha configuration at 37 degrees C in the presence of 6 mM MnCl2 (pH 7.6). Gradient anion-exchange chromatography of the Klebsiella sonicate yielded four fractions that could catalyze the addition of 3H-Gal to human RBCs. Size-exclusion chromatography of these anion-exchange fractions yielded peaks of high GT activity for each, but only those derived from the first, third, and last anion-exchange fractions incorporated Gal such that the RBCs bound anti-Gal by fluorescence-activated cell sorter, suggesting that these three GTs are alpha 1,3 GTs. Thus, Klebsiella spp. make at least four GTs that can add an alpha Gal to human cell surface acceptor structures. Three of these GTs can form alpha 1,3 Gal structures on human RBCs that bind anti-Gal, thereby creating "autoimmune" senescence-associated RBC epitopes.

Anti-Gal binds to pili of Neisseria meningitidis: the immunoglobulin A isotype blocks complement-mediated killing.

alpha 1,3-Galactosyl antibodies (anti-Gal) are ubiquitous natural human serum and secretory polyclonal antibodies that bind to terminal galactose-alpha 1,3-galactose (alpha-galactosyl) residues. Serum immunoglobulin G (IgG) anti-Gal can block alternative complement pathway-mediated lysis of representative gram-negative enteric bacteria that bind it to lipopolysaccharide alpha-galactosyl structures, thereby promoting survival of such bacteria in the nonimmune host. We wanted to know whether anti-Gal also could bind to the lipooligosaccharides (LOS) of Neisseria meningitidis. To our surprise, we found that serum and secretory anti-Gal bound to pili but not to LOS of certain strains. This suggested the presence of an immunogenic pilus carbohydrate epitope. Mild periodate oxidation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated outer membrane preparations from strains that bound anti-Gal followed by labeling of the neoaldehyde groups resulted in the labeling of bands that corresponded to pilin and LOS, confirming that pilin contains carbohydrate structures. A Bandeiraea simplicifolia lectin that also binds terminal alpha 1,3-galactosyl residues also bound to pilin. Serum IgG, IgA, and IgM anti-Gal as well as colostral secretory IgA anti-Gal bound to pilin, as judged by immunoblotting, and to the pili of intact piliated organisms, as judged by immunoelectron microscopy. Total serum anti-Gal (IgG, IgA, and IgM) and purified serum IgA1 anti-Gal, but not its purified IgG isotype, blocked complement-mediated lysis of a piliated meningococcal strain that bound anti-Gal to its pili. Colostral anti-Gal secretory IgA blocked killing of the same strain. Thus, anti-Gal IgA may promote disease when it binds to the pili of N. meningitidis strains.

Anti-alpha-galactosyl immunoglobulin A (IgA), IgG, and IgM in human secretions.

Anti-alpha-galactosyl (anti-Gal) is a natural human serum antibody that binds to the carbohydrate Gal alpha 1,3Gal beta 1,4GlcNAc-R (alpha-galactosyl epitope) and is synthesized by 1% of circulating B lymphocytes in response to immune stimulation by enteric bacteria. We were able to purify secretory anti-Gal from human colostrum and bile by affinity chromatography on silica-linked Gal alpha 1,3Gal beta 1,4GlcNAc. We found similar secretory anti-Gal antibodies in human milk, saliva, and vaginal washings. Secretory anti-Gal from milk and saliva was exclusively immunoglobulin A (IgA); that from colostrum and bile also contained IgG and IgM isotypes. Serum was also found to contain anti-Gal IgM and IgA in addition to the previously reported IgG. Anti-Gal IgA purified from colostrum and bile had both IgA1 and IgA2. Secretory anti-Gal from saliva, milk, colostrum, and bile agglutinated rabbit erythrocytes (RRBC) and bound to bovine thyroglobulin, both of which have abundant alpha-galactosyl epitopes. The RRBC-hemagglutinating capacity of human saliva, milk, bile, and serum was specifically adsorbed by immobilized Gal alpha 1,3Gal beta 1,4GlcNAc but not by Gal alpha 1,4Gal beta 1,4GlcNAc, Gal beta 1,3GalNAc, Gal beta 1,4GlcNAc, Gal beta 1,4GlcNAc alpha 1,2Man, or Fuc alpha 1,2Gal beta 1,4GlcNAc. No RRBC-hemagglutinating activity could be detected in rat milk, rat bile, cow milk, or rabbit bile, suggesting a restricted species distribution for secretory anti-Gal similar to that found for serum anti-Gal. Colostral anti-GaI IgA bound strongly to a sample of gram-negative bacteria isolated from the throats and stools of well children as well as to an Escherichia coli K-1 blood isolate. Colostral anti-GaI IgA inhibited the binding of a Neisseria meningitidis strain to human buccal epithelial cells, suggesting that this antibody may play a protective role at the mucosal surface.

The structure of lipooligosaccharide produced by Neisseria gonorrhoeae, strain 15253, isolated from a patient with disseminated infection. Evidence for a new glycosylation pathway of the gonococcal lipooligosaccharide.

We studied the structure of the lipooligosaccharide (LOS) that is produced by Neisseria gonorrhoeae, strain 15253. This strain, recovered from a patient with disseminated infection, produces predominantly a single LOS component, and its oligosaccharide (OS) structure is different from those of previously studied LOSs. Definition of this OS structure provides additional information on the LOS biosynthesis. We determined that the 15253 OS has an unusual structure: 2 lactosyl residues at its nonreducing ends shown below, [formula: see text] where KDO is 2-keto-3-deoxy-mannooctulosonic acid and Hep is heptose. Comparison of this OS structure with those determined previously indicates the presence of a new glycosylation pathway for gonococcal OS biosynthesis: elongation of a GlcNAc-linked heptose, in contrast to elongation of the other heptose by sequential addition of glycoses which results in the antigenic similarity with human glycolipids. The current study provides not only additional structural information on LOS expressed during different clinical states of infection but also evidence for the diversity of gonococcal LOS biosynthesis. This evidence may be helpful in understanding the pathogenesis involving gonococcal LOS.

Meningococcal group A lipooligosaccharides (LOS): preliminary structural studies and characterization of serotype-associated and conserved LOS epitopes.

Structural studies indicate that the neisserial lipooligosaccharides (LOS) are composed of an oligosaccharide (OS) portion with a phosphorylated diheptose (Hep) core attached to the toxic lipid A moiety. A conserved meningococcal LOS epitope, defined by monoclonal antibody (MAb) D6A, is expressed on group A and many group B and C meningococci of different LOS serotypes (J. J. Kim, R. E. Mandrell, H. Zhen, M. A. Apicella, J. T. Poolman, and J. M. Griffiss, Infect. Immun. 56:2631-2638, 1988). This MAb-defined D6A epitope is immunogenic in humans (M. M. Estabrook, R. E. Mandrell, M. A. Apicella, and J. M. Griffiss, Infect. Immun. 58:2204-2213, 1990; M. M. Estabrook, C. J. Baker, and J. M. Griffiss, J. Infect. Dis. 197:966-970, 1993). In this study, we characterize this important MAb-defined LOS epitope. Serotype L10 and L11 group A meningococal LOS were chemically modified and used to investigate what portion of the LOS molecule is important for expression of the conserved (D6A) epitope and serotype-associated LOS epitopes by use of immunoblotting techniques and selected MAbs as probes. Preliminary structural characterization of the LOS was also accomplished by electrospray ionization-mass spectrometry. Our results indicate the following. (i) Antibodies that recognize the serotype-associated or conserved LOS epitopes recognize the OS portion of the LOS. (ii) The phosphorylated diheptose core region of the OS is essential for expression of the conserved D6A epitope. (iii) The lipid portion of the molecule is important for optimum expression of the LOS epitopes. (iv) The proposed compositions of the O-deacylated LOS are consistent with the presence of a phosphorylated diheptose core and are as follows: for O-deacylated L10 LOS, 3Hex (hexose), 1HexNAc (N-acetylhexosamine), 2KDO (2-keto-3-deoxy-D-manno-octulosonic acid), 2Hep (heptose), 1PEA or 2PEA (phosphoethanolamine), and O-deacylated lipid A; and for O-deacylated L11 LOS, 2Hex, 1HexNAc, 2KDO, 2Hep, 2PEA, and O-deacylated lipid A. Because the phosphorylated diheptose core region of the LOS is essential for the formation of a conserved LOS epitope (D6A) that is immunogenic in humans, care should be taken to maintain stereochemical requirements for the expression of this conserved epitope in the design of effective, nontoxic LOS vaccines.

Differences in outer membrane characteristics between gallstone-associated bacteria and normal bacterial flora.

Previous studies with scanning electron microscopy (SEM) have suggested that pigment gallstones contain bacteria. We set out to culture these bacteria and to study their membrane characteristics. We studied gallstones from 54 patients (36 men, 18 women; mean age 55.4 years) admitted consecutively to two hospitals for cholecystectomy. SEM detected bacteria in all of 14 brown pigment stones, 2 of 14 black pigment stones, and in the pigmented centres of 9 of 19 mixed cholesterol stones; no bacteria were detected in 14 pure cholesterol stones or within the cholesterol portions of mixed stones. We were able to culture bacteria from all gallstones with bacteria seen on SEM and for which sufficient material was available (n = 16). 20 bacterial species were recovered from these stones. Gallstones containing bacteria were associated with clinical sepsis and cholangitis. All bacteria obtained from gallstones agglutinated human O P1 erythrocytes, which reflects the presence of P1-specific fimbriae. 5 strains were positive for Forssman-antigen-specific fimbriae. None showed evidence of mannose-specific fimbriae. All of the organisms bound anti-Gal, a ubiquitous naturally occurring IgG specific for alpha-galactosyl residues. The presence of P1 fimbriae and alpha-galactosyl residues and the absence of mannose-specific fimbriae distinguish these organisms from gut flora. We postulate that possession of these unusual properties may enhance the ability of bacteria to colonise the biliary tree and initiate pigment gallstone formation.

HLA expression by benign and malignant prostatic epithelium: augmentation by interferon-gamma.

Chronic prostatitis is a poorly understood entity that is characterized by lymphocytic infiltration of benign prostatic epithelium. Previously, we and others have shown that prostatic epithelium involved by prostatitis is phenotypically different from uninvolved epithelium. In addition, we have shown that malignant prostatic epithelium is rarely, if ever, infiltrated by lymphocytes. We now report that benign prostatic epithelium expresses HLA-DR only in the presence of lymphocytic inflammation, and that benign epithelium without chronic prostatitis and malignant prostatic epithelium do not express HLA-DR. In order to determine whether HLA-DR expression is inducible on malignant prostatic epithelium and therefore, at least theoretically, susceptible to immune regulation, we studied the DU-145 cell line in culture under various conditions. DU-145 cells did not express HLA-DR under routine culture conditions. However, the addition of interferon-gamma (100 to 6000 U/ml.) resulted in HLA-DR expression by DU-145 cells at 24 hours with maximal expression by 72 hours. In contrast, other cytokines (tumor necrosis factor, interleukin-1, interleukin-2) had no effect on HLA-DR expression. These investigations show that interferon-gamma induces HLA-DR expression on the DU-145 prostatic adenocarcinoma cell line, raising the theoretical possibility that malignant prostatic cells may be induced in vivo to express HLA-DR and thus become susceptible to immune regulation.

Does ozone alleviate AIDS diarrhea?

Five patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) and intractable diarrhea were treated with daily colonic insufflations of medical ozone (oxygen/ozone mixture) for 21-28 days. The daily dose of ozone (O3) ranged from 2.7 to 30 mg. Three of the four patients whose diarrhea was of unknown etiology experienced complete resolution, and one patient had marked improvement. The fifth patient, whose diarrhea was due to Cryptosporidium, experienced no change. No consistent change in the absolute number of helper (CD4) or suppressor (CD8) lymphocytes was detected, and no obvious changes were seen in the PO2 or the results of routine hematologic and blood chemistry studies. Patients had mild to moderate local discomfort during ozone administration early in the course of treatment, but no adverse systemic effects were observed. The results of this series suggest that medical ozone administered by rectal insufflation is simple, safe, and effective. Should this simple treatment be used routinely to treat chronic intractable ARC/AIDS diarrhea?