MIF4G domain containing protein regulates cell cycle and hepatic carcinogenesis by antagonizing CDK2-dependent p27 stability.

The CDK inhibitor p27(kip1) plays crucial roles in cell cycle regulation and cancer progression. Through yeast two-hybrid screening, we identified MIF4G domain containing protein (MIF4GD) as a novel binding partner for p27. The association of MIF4GD and p27 was verified using immunoprecipitation and glutathione S-transferase (GST) pull-down assays. Interaction with MIF4GD led to the stabilization of p27 both in the nucleus and in the cytoplasm in hepatocellular carcinoma (HCC) cells as a result of suppressed phosphorylation of p27 by CDK2 at threonine187. Serum stimulation decreased the levels of MIF4GD and p27 simultaneously. In addition, MIF4GD overexpression resulted in increased p27 levels and reduced cell proliferation, while knockdown of MIF4GD promoted cell cycle progression with decreased p27 levels in cells. Furthermore, overexpression of MIF4GD reduced colony formation and inhibited xenograft tumor growth in nude mice. Finally, we found that both MIF4GD and p27 were expressed at low levels in HCC tissues compared to non-cancerous tissues, and that low expression levels of MIF4GD and p27 were associated with significantly worse prognosis in HCC patients. Our results suggest that MIF4GD is a potential regulator of p27-dependent cell proliferation in HCC. These findings provide a rational framework for the development of potential HCC therapy by targeting the MIF4GD-p27 interaction.

Activation of NF-kappa B by nontypeable Hemophilus influenzae is mediated by toll-like receptor 2-TAK1-dependent NIK-IKK alpha /beta-I kappa B alpha and MKK3/6-p38 MAP kinase signaling pathways in epithelial cells.

Nontypeable Hemophilus influenzae (NTHi) is an important human pathogen in both children and adults. In children, it causes otitis media, the most common childhood infection and the leading cause of conductive hearing loss in the United States. In adults, it causes lower respiratory tract infections in the setting of chronic obstructive pulmonary disease, the fourth leading cause of death in the United States. The molecular mechanisms underlying the pathogenesis of NTHi-induced infections remain undefined, but they may involve activation of NF-kappa B, a transcriptional activator of multiple host defense genes involved in immune and inflammatory responses. Here, we show that NTHi strongly activates NF-kappa B in human epithelial cells via two distinct signaling pathways, NF-kappa B translocation-dependent and -independent pathways. The NF-kappa B translocation-dependent pathway involves activation of NF-kappa B inducing kinase (NIK)--IKK alpha/beta complex leading to I kappa B alpha phosphorylation and degradation, whereas the NF-kappa B translocation-independent pathway involves activation of MKK3/6--p38 mitogen-activated protein (MAP) kinase pathway. Bifurcation of NTHi-induced NIK-IKK alpha/beta-I kappa B alpha and MKK3/6--p38 MAP kinase pathways may occur at transforming growth factor-beta activated kinase 1 (TAK1). Furthermore, we show that toll-like receptor 2 (TLR2) is required for NTHi-induced NF-kappa B activation. In addition, several key inflammatory mediators including IL-1 beta, IL-8, and tumor necrosis factor-alpha are up-regulated by NTHi. Finally, P6, a 16-kDa lipoprotein highly conserved in the outer membrane of all NTHi and H. influenzae type b strains, appears to also activate NF-kappa B via similar signaling pathways. Taken together, our results demonstrate that NTHi activates NF-kappa B via TLR2-TAK1-dependent NIK--IKK alpha/beta-I kappa B alpha and MKK3/6--p38 MAP kinase signaling pathways. These studies may bring new insights into molecular pathogenesis of NTHi-induced infections and open up new therapeutic targets for these diseases.

Functional characteristics of a protective monoclonal antibody against serotype A and C lipooligosaccharides from Moraxella catarrhalis.

A monoclonal antibody (MAb), designated MAb 8E7 (immunoglobulin G3), specific for Moraxella catarrhalis lipooligosaccharide (LOS) was evaluated for its functional activity in vitro and in a mouse model of colonization. Enzyme-linked immunosorbent assay (ELISA) demonstrated that the MAb 8E7 could be prepared to a high titer against LOS of the homologous strain 035E, and that it had bactericidal activity. MAb 8E7 reacted with M. catarrhalis serotype A and C LOSs but not serotype B LOS, as measured by ELISA and Western blotting. On the basis of published structures of LOSs, this suggests that the epitope recognized by MAb 8E7 is directed to a common sequence of either alpha-GlcNAc-(1-->2)-beta-Glc-(1--> at the branch substituting position 4 of the trisubstituted Glc residue or a terminal tetrasaccharide alpha-Gal-(1-->4)-beta-Gal-(1-->4)-alpha-Glc-(1-->2)-beta-Glc-(1--> at the branch substituting position 6 of the trisubstituted Glc residue. In a whole-cell ELISA, MAb 8E7 reacted with 70% of the 30 wild-type strains and clinical isolates tested. Immuno-electron microscopy demonstrated that MAb 8E7 reacted with a cell surface-exposed epitope of LOS on strain O35E. MAb 8E7 inhibited the adherence of strain O35E to Chang conjunctival epithelial cells by 90%. Passive immunization with MAb 8E7 could significantly enhance the clearance of strain O35E from mouse lungs in an aerosol challenge mouse model. This enhanced bacterial clearance was inhibited when MAb 8E7 was absorbed by M. catarrhalis serotype A LOS, indicating that the M. catarrhalis LOS-directed antibody may play a major role in the enhancement of M. catarrhalis clearance from lungs. These data suggest that MAb 8E7, which recognizes surface-exposed LOS of M. catarrhalis, is a protective antibody against M. catarrhalis.

Enhancement of clearance of bacteria from murine lungs by immunization with detoxified lipooligosaccharide from Moraxella catarrhalis conjugated to proteins.

Moraxella catarrhalis strain 25238 detoxified lipooligosaccharide (dLOS)-protein conjugates induced a significant rise of bactericidal anti-LOS antibodies in animals. This study reports the effect of active or passive immunization with the conjugates or their antiserum on pulmonary clearance of M. catarrhalis in an aerosol challenge mouse model. Mice were injected subcutaneously with dLOS-tetanus toxoid (dLOS-TT), dLOS-high-molecular-weight proteins (dLOS-HMP) from nontypeable Haemophilus influenzae (NTHi), or nonconjugated materials in Ribi adjuvant and then challenged with M. catarrhalis strain 25238 or O35E or NTHi strain 12. Immunization with dLOS-TT or dLOS-HMP generated a significant rise of serum anti-LOS immunoglobulin G and 68% and 35 to 41% reductions of bacteria in lungs compared with the control (P<0.01) following challenge with homologous strain 25238 and heterologous strain O35E, respectively. Serum anti-LOS antibody levels correlated with its bactericidal titers against M. catarrhalis and bacterial CFU in lungs. Additionally, immunization with dLOS-HMP generated a 54% reduction of NTHi strain 12 compared with the control (P<0.01). Passive immunization with a rabbit antiserum against dLOS-TT conferred a significant reduction of strain 25238 CFU in lungs in a dose- and time-dependent pattern compared with preimmune serum-treated mice. Kinetic examination of lung tissue sections demonstrated that antiserum-treated mice initiated and offset inflammatory responses more rapidly than preimmune serum-treated mice. These data indicate that LOS antibodies (whether active or passive) play a major role in the enhancement of pulmonary clearance of different test strains of M. catarrhalis in mice. In addition, dLOS-HMP is a potential candidate for a bivalent vaccine against M. catarrhalis and NTHi infections.

Biological activities of antibodies elicited by lipooligosaccharide based-conjugate vaccines of nontypeable Haemophilus influenzae in an otitis media model.

Vaccination of chinchillas with nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) conjugates protected against otitis media. Correlations between the levels of conjugate-induced LOS antibodies (Abs) in sera and middle ear fluids (MEFs) and Ab-mediated biological functions and protection were examined. Following parenteral vaccination and middle ear challenge, all vaccinated animals, but none of the controls, had high titers of anti-LOS in their sera and MEFs. There was a correlation between the levels of anti-LOS IgG + M, IgG or IgA in the sera and in the MEFs (P < 0.001). An inverse correlation was found between the level of serum IgG + M and bacterial counts and between the levels of MEF Abs and bacterial counts at the early postchallenge stage (P < 0.05). Of the 39 vaccinated animals, 44% showed complete protection against otitis media, 46% (18/39) of their sera inhibited adherence of NTHi to human epithelial cells, 49% (19/39) demonstrated bactericidal activity and 49% (19/39) showed opsonophagocytic activity. In contrast, none of the controls (19) were protected, none of their sera inhibited bacterial adherence or had bactericidal activity and only 21% showed opsonophagocytosis. Our interpretation is that vaccine-induced LOS Abs transuded into the middle ear and conferred immunity to NTHi by binding to LOS of NTHi, inhibition of NTHi adherence to epithelial cells and complement-mediated bacteriolysis (or opsonophagocytosis).

An aerosol challenge mouse model for Moraxella catarrhalis.

A simple, reproducible, and non-invasive mouse pulmonary clearance model for Moraxella catarrhalis via aerosol challenge was established. All of eight tested strains could be inoculated into mice at more than 10(5) colony-forming units (CFU)/lung with a challenge concentration of 1x10(9)-6x10(9) CFU/ml in a nebulizer. The number of bacteria retained at 6 h postchallenge was more than 10(4) CFU/lung while at 24 h postchallenge, approximate 10(3) CFU/ml or less remained in the lungs. A maximum of 100 mice could be challenged per aerosol exposure. The number of bacteria inoculated in the lungs could be adjusted by the bacterial challenge concentration, the exposure time, and the negative pressure. Lung tissue sections revealed that bacteria were evenly distributed in the lungs. Passive immunization significantly enhanced pulmonary clearance of the homologous strain in this model. These data indicate that this model will be useful for evaluating M. catarrhalis vaccine candidates and studying roles of immunity against M. catarrhalis.

The structural heterogeneity of the lipooligosaccharide (LOS) expressed by pathogenic non-typeable Haemophilus influenzae strain NTHi 9274.

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen responsible for otitis media in children and of pneumonitis in adults with depressed resistance. NTHi is acapsular and, therefore, capsular polysaccharide-based vaccines are ineffective for preventing infections by this pathogen. Recently it was found that a detoxified lipooligo-saccharide (LOS) conjugate from NTHi 9274 induced bactericidal antibodies effective against a large number of NTHi isolates, and conferred protection against NTHi otitis media in chinchillas (X.-X.Gu et al., 1996, Infect. Immun.,64, 4047-4053; X. -X.Gu et al., 1997., Infect. Immun.,65, 4488-4493). In this paper we report the chemical character-ization of the LOS from NTHi 9274 LOS. NTHi is capable of expressing a heterogenous population of LOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released from the LOS of NTHi 9274 by mild acid hydrolysis were purified using Bio-Gel P4 gel permeation chromatography. The OSs were characterized by glycosyl composition analysis, glycosyl linkage analysis, nuclear magnetic resonance spectroscopy (NMR), fast atom bombardment mass spectro-metry (FAB-MS), matrix-assisted laser desorption time of flight mass spectro-metry (MALDITOF-MS), and tandem MS/MS. At least 17 different OS molecules were observed. These contained variable glycosyl residues, phosphate (P), and phospho-ethanolamine (PEA) substituents. These molecules contained either three, four, or five hexoses, and all contained four heptosyl residues. The four heptosyl residues consisted of one D,D-Hep and three L,D-Hep. Dephosphorylation of the OSs with aqueous 48% hydrofluoric acid (HF) reduced the number of molecules to about to seven; Hex(1)-(7)Hep(4)Kdo(1). Of these seven, Hex(2)Hep(4)Kdo(1), Hex(3)Hep(4)Kdo(1), and Hex(4)Hep(4)Kdo(1)were the major constituents. Thus, this NTHi LOS preparation is very heterogeneous, and contains structures different from those previously published for Haemophilus influenzae. The tandem MS/MS analysis and glycosyl linkage data suggest that the LOS oligosaccharides have the following structures where Hex is either a Glc or Gal residue.

Immortalization of chinchilla middle ear epithelial cells by adenovirus 12-simian virus 40 hybrid virus.

In order to study the cellular and molecular mechanisms of the pathogenesis of otitis media, a chinchilla middle ear epithelial cell line (CMEE-1) with differentiated cell characteristics was established by infection of a primary culture with the adenovirus 12-simian virus 40 (Ad12-SV40) hybrid. This cell line has been in continuous culture for 42 passages, whereas the parent cells underwent senescence and died at the 8th passage. The cell line also retains epithelial morphology and expresses cytokeratin polypeptides 4, 7, and 18, characteristic markers for epithelia. In Western blots of cell proteins, bands at 94 and 53 kd were labeled after binding antibodies against SV40 large T antigen and p53, respectively. Karyotype analysis showed that the cell line is derived from chinchilla epithelial cells. These findings confirm that the cell line is a chinchilla epithelial cell immortalized by the hybrid virus.

Outer membrane proteins as a carrier for detoxified lipooligosaccharide conjugate vaccines for nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media and respiratory tract infections. Outer membrane proteins (OMP) and lipooligosaccharide (LOS) are major surface antigens of NTHi and potential vaccine candidates. De-O-acylated LOS (dLOS) or oligosaccharide (OS) was coupled to total OMP to form dLOS-OMP and OS-OMP conjugates, while a dLOS-tetanus toxoid (TT) was synthesized for comparison. These conjugates were evaluated in mice and rabbits for immunogenicity. dLOS-OMP elicited a better boostable antibody response against LOS than did dLOS-TT, while OS-OMP was not immunogenic. Formulation of the conjugates with Ribi adjuvant significantly enhanced the immunogenicity of dLOS-OMP and dLOS-TT but not that of OS-OMP. In addition, rabbit antisera elicited by dLOS-OMP but not dLOS-TT (or OMP alone) demonstrated bactericidal activity against 40% of the NTHi strains tested. These results indicate that dLOS is a better derivative of LOS than OS and that OMP is a good carrier for NTHi LOS-based conjugate vaccines.

Mutation analysis in adenylosuccinate lyase deficiency: eight novel mutations in the re-evaluated full ADSL coding sequence.

The deficiency of adenylosuccinate lyase (ADSL, also termed adenylosuccinase) is an autosomal recessive disorder characterized by the accumulation in body fluids of succinylaminoimidazole-carboxamide riboside (SAICA-riboside) and succinyladenosine (S-Ado). Most ADSL-deficient children display marked psychomotor delay, often accompanied by epilepsy or autistic features, or both, although some patients may be less profoundly retarded. Occasionally, growth retardation and muscular wasting are also present. Up to now, nine missense mutations of the ADSL gene had been reported in six apparently unrelated sibships. In the present study of 10 additional patients with ADSL deficiency, nine point mutations, among which seven unreported missense mutations, and the first splicing error reported in this disorder, have been identified. These mutations have been characterized, taking into account the finding that the cDNA of human ADSL is 75 nucleotides longer at its 5'-end, and encodes a protein of 484 rather than 459 amino acids as previously reported. Five apparently unrelated patients were found to carry a R426H mutation. With the exceptions of the latter mutation, of a R190Q mutation that had been reported previously, and of a K246E mutation that was found in two unrelated patients, all other mutations were found only in a single family.

Identification of common lipooligosaccharide types in isolates from patients with otitis media by monoclonal antibodies against nontypeable Haemophilus influenzae 9274.

Twenty-one murine monoclonal antibodies (MAbs) were induced by nontypeable Haemophilus influenzae (NTHi) 9274. Nineteen MAbs were specific for the lipooligosaccharide (LOS) as determined by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. When the MAbs were assayed with five LOS prototype strains by ELISA, all bound to strain 3198 LOS (type III), while six of the MAbs were also reactive with LOSs from strain 1479 (type I), 5657 (type IV), or 7502 (type V). Ten MAbs had complement-mediated bactericidal activity, and three MAbs were opsonophagocytic against the homologous strain. Five LOS MAbs with different specificities were used to analyze 155 NTHi clinical isolates from the United States and from Japan. These isolates were classified into nine groups by ELISA. Only four isolates (2.6%) were not recognized by any of the five MAbs. Most of the isolates (91.6%) were in four groups which bound three of the five MAbs. One of three MAbs, 6347C11, had strong activity against the homologous strain and was also bactericidal to 45 clinical isolates (29%) which belonged to the four common patterns (25 belonged to pattern 1). These data indicate that these MAbs can be used for LOS typing in which almost all NTHi strains can be typed according to the LOS antigenicity. Among NTHi, at least one conserved LOS epitope which is a target of bactericidal antibodies exists. We conclude that strain 9274 LOS, which is the target for bactericidal antibodies, is a candidate for LOS-based NTHi vaccines.

Genetic data obtained for two Chinese Han populations with a quadruplex fluorescent STR typing system (HUMVWA, HUMTH01, D21S11 and HPRT).

Abstract DNA typing of four tetrameric repeat loci (HUMVWA, HUMTH0I, D21SII and HPRT) was carried out in a Chinese Han population from Shanghai (East China) and one from Guangzhou (South-East China) using a quadruplex PCR amplification and detection of the fluorescent-labeled alleles on the ALF DNA sequencer. All loci were in accordance with Hardy-Weinberg equilibrium except for D21S11 in the Guangzhou population. A test for population differentiation showed no statistical difference in the allele frequency distribution between the two populations. Comparison of the allele frequency data with other Chinese Han populations from North and South-West China for the STR loci HUMVWA and HUMTH01 revealed heterogeneity between Northern Chinese Han and Southern Chinese Han, which is in accordance with previous studies on the basis of protein markers.

The rrs (16S)-rrl (23S) ribosomal intergenic spacer region as a target for the detection of Haemophilus ducreyi by a heminested-PCR assay.

The intergenic spacer region between the rrs and rrl ribosomal RNA genes of Haemophilus ducreyi was analysed and the DNA sequence was used for the selection of specific PCR primers. A highly sensitive and specific heminested-PCR assay for the identification of H. ducreyi was developed. The assay showed a sensitivity of 96% on genital ulcer specimens from patients with clinically diagnosed chancroid, compared with a sensitivity of 56% for culture methods. These results indicate that this PCR assay has the potential to become an accurate and easy reference method for the detection of H. ducreyi.

Synthesis and characterization of lipooligosaccharide-based conjugates as vaccine candidates for Moraxella (Branhamella) catarrhalis.

Moraxella (Branhamella) catarrhalis is an important cause of otitis media and sinusitis in children and of lower respiratory tract infections in adults. Lipooligosaccharide (LOS) is a major surface antigen of the bacterium and elicits bactericidal antibodies. Treatment of the LOS from strain ATCC 25238 with anhydrous hydrazine reduced its toxicity 20,000-fold, as assayed in the Limulus amebocyte lysate (LAL) test. The detoxified LOS (dLOS) was coupled to tetanus toxoid (TT) or high-molecular-weight proteins (HMP) from nontypeable Haemophilus influenzae through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratios of dLOS to TT and HMP conjugates were 19:1 and 31:1, respectively. The antigenicity of the two conjugates was similar to that of the LOS, as determined by double immunodiffusion. Subcutaneous or intramuscular injection of both conjugates elicited a 50- to 100-fold rise in the geometric mean of immunoglobulin G (IgG) to the homologous LOS in mice after three injections and a 350- to 700-fold rise of anti-LOS IgG in rabbits after two injections. The immunogenicity of the conjugate was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced antisera had complement-mediated bactericidal activity against the homologous strain and heterologous strains of M. catarrhalis. These results indicate that a detoxified LOS-protein conjugate is a candidate for immunization against M. catarrhalis diseases.

Detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins confers protection against otitis media in chinchillas.

Detoxified-lipooligosaccharide (dLOS)-protein conjugates from nontypeable Haemophilus influenzae (NTHi) elicited a significant rise of anti-LOS antibodies with bactericidal activity in rabbits (X.-X. Gu, C.-M. Tsai, T. Ueyama, S. J. Barenkamp, J. B. Robbins, and D. J. Lim, Infect. Immun. 64:4047-4053, 1996). In this study, we evaluated whether vaccination with the conjugates would protect against NTHi otitis media in chinchillas. Fifty-eight chinchillas received three subcutaneous or intramuscular injections of dLOS-conjugated tetanus toxoid, dLOS-conjugated high-molecular-weight proteins from NTHi, or saline (control) in Freund's adjuvant and then were challenged by intrabullar inoculation with 140 CFU of NTHi. All vaccinated animals responded with elevated serum titers of anti-LOS antibody, and 49% (19 of 39) demonstrated bactericidal activity against the homologous strain. Otitis media with culture-positive NTHi effusions developed in all 19 controls and 56% (22 of 39) of the vaccinated animals during a period of 21 days (P < 0.001). Bacterial counts of the middle ear effusions were lower in the vaccine groups than in the controls (P < 0.01). The incidences of infection in the unchallenged ear or inner ear were 26 or 28% in the vaccine groups and 53 or 58% in the controls (P < 0.05). The signs of infection observed by otoscopy were less severe in the vaccine groups than in the controls. There was no significant difference between the two vaccine groups. These data indicate that active immunization with LOS-based conjugates reduces the incidence of NTHi-induced otitis media.

Synthesis, characterization, and immunologic properties of detoxified lipooligosaccharide from nontypeable Haemophilus influenzae conjugated to proteins.

Nontypeable Haemophilus influenzae (NTHi) is an important cause of otitis media in children and of pneumonitis in adults with depressed resistance. Lipooligosaccharide (LOS) is a major surface antigen of NTHi and elicits bactericidal and opsonic antibodies. We prepared detoxified LOS (dLOS) protein conjugates from NTHi for use as experimental vaccines. LOS from NTHi 9274 was treated with anhydrous hydrazine and had its toxicity reduced to clinically acceptable levels. dLOS was bound to tetanus toxoid (TT) or high- molecular-weight proteins (HMPs) from NTHi through a linker of adipic acid dihydrazide to form dLOS-TT or dLOS-HMP. The molar ratio of the dLOS to protein carriers ranged from 26:1 to 50:1. The antigenicity of the conjugates was similar to that of the LOS alone as determined by double immunodiffusion. Subcutaneous or intramuscular injection of the conjugates elicited a 28- to 486-fold rise in the level of immunoglobulin G antibodies in mice to the homologous LOS after two or three injections and a 169- to 243-fold rise in the level of immunoglobulin G antibodies in rabbits after two injections. The immunogenicity of the conjugates in mice and rabbits was enhanced by formulation with monophosphoryl lipid A plus trehalose dimycolate. In rabbits, conjugate-induced LOS antibodies induced complement-mediated bactericidal activity against the homologous strain 9274 and prototype strain 3189. These results indicate that a detoxified LOS-protein conjugate is a candidate vaccine for otitis media and pneumonitis caused by NTHi.

Linkage analysis in three families with nonspecific X-linked mental retardation.

Nonspecific X-linked mental retardation (XLMR) is a common disorder. The number of genes involved in this condition is not known, but it is estimated to be more than 10. We present a clinical and linkage study on 3 families with XLMR. All families were analyzed using highly polymorphic markers covering the X chromosome; screening for the fragile X mutation was negative. The first family (MRX 36) consisted of 1 female and 4 male patients in 3 generations and 7 healthy individuals. Considering the female as an expressing heterozygous carrier, a maximum LOD score of 3.41 was reached in region Xp21.2-Xp22.1. Considering her phenotype to be unknown, a LODmax of 1.97 was reached in the same region. The second family consisted of 5 affected and 6 healthy males with mild to borderline mental retardation. Linkage analysis using an X-linked recessive model with full penetrance and no phenocopies excluded linkage over almost the entire X chromosome. Using alternative models, including an affecteds-only analysis, a LODmax of 1.49 was found in region Xq24-28. The third family, consisting of 4 male patients with moderate mental retardation in 1 generation yielded a LODmax of 0.9 in region Xp22.13-11.3. However, even in this small pedigree, exclusion mapping was able to exclude very large parts of the X chromosome and in this way identify a likely candidate region.

Localisation of a new gene for non-specific mental retardation to Xq22-q26 (MRX35).

Non-specific mental retardation (MR) is a condition in which MR appears to be the only consistent manifestation. The X linked form (MRX) is genetically heterogeneous. We report clinical, cytogenetic, and linkage data on a family with X linked non-specific MR. Two point and multi-point linkage analysis with 18 polymorphic markers, covering the entire chromosome, showed close linkage to DXS1001 and DXS425 with a maximal lod score of 2.41 at 0% recombination. DXS178 and the gene for hypoxanthine phosphoribosyl-transferase (HPRT), located in Xq22 and Xq26 respectively, flank the mutation. All other chromosomal regions could be excluded with odds of at least 100:1. To our knowledge there is currently no other non-specific MR gene mapped to this region. Therefore, the gene causing MR in this family can be considered to be a new, independent MRX locus (MRX35).

Quantitation and biological properties of released and cell-bound lipooligosaccharides from nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) is a major pathogen causing otitis media in children. NTHi releases lipooligosaccharide (LOS) as outer membrane fragments during its growth. The release of LOS may play an important role in the pathogenicity of otitis media caused by this organism. The amounts of LOS in bacterial cells and growth media for five NTHi strains were determined by quantitative silver staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These strains were estimated to have 1.6 x 10(6) to 4.8 x 10(6) LOS molecules per bacterium. During a 3-day growth period, these NTHi strains released variable but significant amounts of LOS into the growth medium. Cells started to release detectable amounts of LOS into the medium at 2 to 5 h and continued to do so for up to 48 or 72 h. The concentrations of LOS in the culture supernatants released by these five strains were 10 to 55 micrograms/ml at 24 h and 40 to 100 micrograms/ml at 72 h, which was 34 to 189% of the cell-bound LOS concentration. The biological properties of released and cell-bound LOSs from two representative strains were compared. Released LOS showed an approximately 10-fold increase in inducing human monocytes to produce tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6, a 13- to 28-fold increase in mouse lethal toxicity, and a 16- to 37-fold increase in the clotting of Limulus amebocyte lysate. These results suggested that released LOS or its inflammatory mediators play a more important role than the LOS in bacteria in the pathogenicity of otitis media caused by this organism.