Clinical potential of mannose-binding lectin-replacement therapy.

Mannose-binding lectin (MBL; also known as mannan-binding lectin) is an important component of innate immunity. MBL levels are mainly genetically determined. Low serum MBL levels and their cognate haplotypes have been associated with a wide range of infections. However, most subjects with MBL deficiency remain healthy. MBL deficiency is also associated with non-infectious diseases including systemic lupus erythematosus, rheumatoid arthritis, cystic fibrosis and common variable immunodeficiency. MBL deficiency may affect susceptibility to (e.g. meningococcal disease), or alter the natural history of (e.g. rheumatoid arthritis, cystic fibrosis), a disease. MBL (plasma-derived or recombinant) therapy has yet to be shown to be safe and effective. Potentially it may be useful in MBL-deficient patients to reduce susceptibility to, or enhance recovery from, bacterial infection or to alter the natural history of a disease (disease-modifying drug). In practise the place of MBL therapy may be as a disease-modifying drug to reduce the severity of rheumatoid arthritis and to preserve lung and liver function in cystic fibrosis. MBL therapy may also ameliorate various immunodeficiency syndromes. A potential hazard of MBL therapy is enhanced complement-mediated host damage. The place of MBL therapy will await results of randomized controlled clinical trials.

Recurrent miscarriage and variant alleles of mannose binding lectin, tumour necrosis factor and lymphotoxin alpha genes.

Variant alleles of the mannose binding lectin (MBL) gene are associated with increased susceptibility to infection and polymorphisms of tumour necrosis factor and lymphotoxin alpha genes (TNF, LTA) are associated with increased severity of infection. Studies have associated recurrent miscarriage with low serum mannose binding lectin concentrations and premature membrane rupture and preterm delivery with elevated maternal and fetal levels of TNF and the TNF (- 308) polymorphism. In this study the frequencies of variant MBL, TNF and LTA alleles in 76 Caucasian couples with idiopathic recurrent miscarriage were compared with those in 69 Caucasian control couples with no history of miscarriage and at least one previous live birth. A new assay based on hybridization to immobilized sequence-specific oligonucleotides (SSO) was used to rapidly detect nine MBL, two TNF and two LTA sequence variants. The assay genotyped all the structural and promoter MBL variants known to influence serum MBL concentrations. This assay was more reliable than restriction digestion or nested allele-specific PCR for the structural variants at codon 54 or 52, respectively. Reliability for codon 57 alleles was not assessed because of the low frequency in this population. The MBL haplotype frequencies in antenatal controls were similar to those reported in other control populations. The frequencies of structural variant MBL genes and of low, medium and high MBL level haplotypes were similar in the recurrent miscarriage and control couples. The TNF and LTA haplotype frequencies were similar in the recurrent miscarriage and control couples. In this carefully defined population no association has been found between recurrent miscarriage and variant alleles of the MBL, TNF or LTA genes.

Virus hepatitis update.

Currently seven viruses, A, B, C, D, E, G and transfusion transmitted virus (TTV), are recognised in the hepatitis virus alphabet. Hepatitis G virus and TTV probably do not cause liver disease in humans. Hepatitis A and E usually cause a self-limiting hepatitis followed by complete recovery but occasionally cause fulminant hepatic failure. Hepatitis B and C are major public health problems worldwide due to their sequelae of chronic hepatitis, cirrhosis and primary liver cancer. Chronic hepatitis C is a particular health issue for Western Europe already, accounting for 40% of end-stage cirrhosis and 30% of liver transplants. The contribution of hepatitis C to chronic liver disease is predicted to rise in the future. Vaccines can prevent hepatitis A and B. Interferon alpha is effective treatment in 25-30% of patients with chronic hepatitis B or C. The prospects for treating chronic hepatitis B have been improved by the introduction of reverse transcriptase inhibitors. Lamivudine is the first drug of this class to be licensed. The optimal use of these new drugs is currently being studied. The success rate for treating chronic hepatitis C can be raised to about 40% with combination therapy of interferon alpha and ribavirin. A large research effort to discover new antiviral agents against hepatitis C is already giving the prospect of more effective therapies in the next few years.

Association of variants of the gene for mannose-binding lectin with susceptibility to meningococcal disease. Meningococcal Research Group.

BACKGROUND: The reasons why meningococcal disease develops in only a small proportion of individuals carrying the causative bacteria are unknown. Differences in host responses to bacterial colonisation are thought to be involved, since people with deficiencies in the terminal components of the complement pathway, or of properdin, are susceptible to meningococcal disease. We postulate that genetic variants of mannose-binding lectin (MBL), a plasma opsonin that initiates another pathway of complement activation, might similarly cause susceptibility to meningococcal disease. METHODS: The frequency of variants of the MBL gene was ascertained in children with meningococcal disease and controls from two independent studies; one hospital-based (194 patients and 272 controls [patients with non-infectious disorders]), and one community-based (72 patients and 110 controls [healthy individuals]), by means of PCR and restriction-enzyme digestion, with confirmation by DNA sequencing. FINDINGS: The proportion of people homozygous for MBL-variant alleles was higher in patients with meningococcal disease than in controls in the hospital study (15 [7.7%] vs four [1.5%]; odds ratio 6.5 [95% CI 2.0-27.2]) and in the community study (six [8.3%] vs three [2.7%]; 4.5 [0.9-29.1]). The population attributable fraction of cases attributable to MBL variants (homozygous and heterozygous) was 32%. INTERPRETATION: The MBL pathway is a critical determinant of meningococcal-disease susceptibility, and genetic variants of MBL might account for a third of all disease cases.

Mannose-binding protein in HIV-seropositive patients does not contribute to disease progression or bacterial infections.

This study set out to investigate whether plasma mannose-binding protein (MBP) deficiency caused by mutations in the MBP gene associates with pyogenic or opportunistic infections in HIV-infected patients. Plasma samples were selected randomly from 131 HIV-infected patients followed prospectively for a period not exceeding 12 months or until death. Plasma MBP concentrations were measured by an ELISA and genotyping was determined by amplification of exon 1 of the MBP gene by polymerase chain reaction (PCR) technology, followed by restriction enzyme analysis and Southern blotting using sequence-specific oligonucleotide probes. Neither MBP concentration nor genotype was found to associate with disease progression or opportunistic infection rate. There was an unexpected increased bacterial infection rate in patients with MBP levels greater than 100 ng/ml and wild type genotype. Thus, MBP does not appear to play a role in HIV infection. MBP is an acute phase reactant and this may explain the higher levels in those with more frequent pyogenic infections.

Pancreatic abnormalities and AIDS related sclerosing cholangitis.

OBJECTIVES: Biliary tract abnormalities are well recognised in AIDS, most frequently related to opportunistic infection with Cryptosporidium, Microsporidium, and cytomegalovirus. We noted a high frequency of pancreatic abnormalities associated with biliary tract disease. To define these further we reviewed the clinical and radiological features in these patients. METHODS: Notes and radiographs were available from two centres for 83 HIV positive patients who had undergone endoscopic retrograde cholangiopancreatography for the investigation of cholestatic liver function tests or abdominal pain. RESULTS: 56 patients had AIDS related sclerosing cholangitis (ARSC); 86% of these patients had epigastric or right upper quadrant pain and 52% had hepatomegaly. Of the patients with ARSC, 10 had papillary stenosis alone, 11 had intra- and extrahepatic sclerosing cholangitis alone, and 35 had a combination of the two. Ampullary biopsies performed in 24 patients confirmed an opportunistic infection in 16. In 15 patients, intraluminal polyps were noted on the cholangiogram. Pancreatograms were available in 34 of the 45 patients with papillary stenosis, in which 29 (81%) had associated pancreatic duct dilatation, often with associated features of chronic pancreatitis. In the remaining 27 patients, final diagnoses included drug induced liver disease, acalculous cholecystitis, gall bladder empyema, chronic B virus hepatitis, and alcoholic liver disease. CONCLUSION: Pancreatic abnormalities are commonly seen with ARSC and may be responsible for some of the pain not relieved by biliary sphincterotomy. The most frequent radiographic biliary abnormality is papillary stenosis combined with ductal sclerosis.

Association of mutations in mannose binding protein gene with childhood infection in consecutive hospital series.

OBJECTIVE: To determine the extent to which mutations in the mannose binding protein gene predispose to childhood infection. DESIGN: Clinical details and genotype of mannose binding protein determined in consecutive children attending a paediatric department. SETTING: Inner city hospital paediatric service in London. SUBJECTS: 617 children attending hospital between October 1993 and August 1995. MAIN OUTCOME MEASURE: Infection as the cause for attendance or admission in relation to mutations in the mannose binding protein gene. RESULTS: The prevalence of mutations in the mannose binding protein gene in children with infection (146/345) was about twice that in children without infection (64/272) (P < 0.0001). Increased susceptibility to infection was found in both heterozygotic and homozygotic children. 13 out of 17 children homozygotic for variant alleles presented with strikingly severe infections, including 6 with septicaemia. CONCLUSIONS: The findings suggest that mutations in the mannose binding protein gene are an important risk factor for infections in children. Screening for such mutations should be included in the investigation of severe or frequent infections.

The role of collectins in host defense.

Mannose-binding protein (MBP) belongs to a group of Ca(2+)-dependent lectins called collectins that play a role in first-line host defense. It recognizes specific carbohydrate residues (mannose and N-acetylglucosamine) on the surface of microorganisms and promotes the killing of microbes either by acting directly as an opsonin or by activating the lectin complement pathway. The collagenlike domain of MBP is important for the binding of MBP to the collectin receptors expressed on different phagocytes, and for activation of complement. The binding of MBP to bacteria, viruses, and parasites has been demonstrated in vitro. Three major mutations have been found in exon 1 of the MBP gene, which encodes the collagenous domain of the protein. These mutations cause low levels of serum MBP and have been linked with lifelong risk of infection. The homozygotes for these mutations are especially susceptible to severe infections.

Mutation of gene of mannose-binding protein associated with chronic hepatitis B viral infection.

BACKGROUND: Persistent with hepatitis B virus (HBV) affects 350 million people worldwide, and 20-40% of infected patients die of cirrhosis and liver cancer. Little is known about the host factors that determine the variable natural history. Studies have focused on the role of acquired rather than innate immunity. We have investigated the prevalence of mutations in the gene for mannose-binding protein (MBP), which have been associated with susceptibility to bacterial and fungal infections. METHODS: Mutations in the MBP gene were sought by sequence-specific oligonucleotide hybridisation, site-directed sequencing in Caucasian and Asian patients with HBV infection, and in HBsAg-negative controls. FINDINGS: A mutation in codon 52 of the MBP gene was present in two (11%) of 19 Caucasian patients with acute hepatitis B and nine (27%) of 33 Caucasian patients with chronic hepatitis B, compared with four (4%) of 98 Caucasian controls (p = 0.0004). By contrast the prevalence of the mutation was similar in Asian patients with chronic hepatitis B and in Asian controls (one [5%] of 20 vs two [2%] of 117). Mutations in codon 54 and codon 57 were found in similar proportions of patients and controls. INTERPRETATION: These findings show in Caucasian, but not Asian, patients an association of the codon 52 mutation of the MBP gene with persistent HBV infection. They suggest an important role for this gene, or a gene in linkage disequilibrium with MBP, in determining outcome after HBV infection in adult but not neonatal life.

Mutations in the human mannose-binding protein gene: frequencies in several population groups.

Mannose-binding protein (MBP; mannan-binding protein, mannan-binding lectin) is a member of the collectin family of proteins and is thought to be important in innate immunity. We have previously shown high frequencies of two distinct mutations in codon 54 and codon 57 of exon 1 of the MBP gene in non-African and African populations, respectively. These result in low levels of the protein and an opsonic deficiency but the frequencies also suggest some selective advantage for low MBP levels. A third mutation in codon 52 occurs at a much lower frequency. We have now extended our earlier studies to other populations. In the south-west Pacific (Papua New Guinea and Vanuatu) neither the codon 52 nor the codon 57 mutation was detected and the codon 54 mutation was significantly less common (gene frequencies of 0.07 and 0.01, respectively) than in other non-African populations (gene frequencies 0.11-0.16). This could be explained by relatively recent admixture. The ancestral Melanesian population probably diverged some 50,000-60,000 years ago and our data suggest that the codon 54 mutation may have occurred after that even but before the divergence of European-Asian groups (40,000 years ago). Two further sub-Saharan populations were also studied: a group of Xhosa from South Africa were similar to Gambians, with a high gene frequency for the codon 57 mutation (0.27) and no evidence of the codon 52 or 54 mutations. In contrast, San Bushmen from Namibia had low frequencies of both the codon 57 mutation (0.07) and the codon 54 mutation (0.03). Again the codon 52 mutation was not found. This pattern is unique amongst sub-Saharan populations studied to date and suggests that this population may have been subjected to different selective pressures.

Distinct physicochemical characteristics of human mannose binding protein expressed by individuals of differing genotype.

Mannose binding protein (MBP) is a serum collectin (collagenous lectin) believed to be of importance in innate immunity. Three point mutations, in codons 52, 54 and 57 of exon 1 of the human MBP gene, have been predicted to affect the tertiary structure of the collagenous region of the protein, and are known to be associated with low serum concentrations of MBP. However, other groups working with recombinant mutant proteins have claimed that the proteins are expressed and assembled normally. The aim of the present investigation was to characterize the effects of these mutations on the physicochemical nature of MBP that is present in the circulation in vivo, and for this we used polyacrylamide gel electrophoresis, gel filtration and sucrose density gradient centrifugation, followed by immunoblotting and enhanced chemiluminescence. The circulating wild-type MBP appeared to comprise a mixture of polymers formed from two to eight subunits (each based on three identical 32,000 MW polypeptide chains) of apparent molecular weights 200,000-700,000, with dimers and trimers constituting the predominant forms. Individuals homozygous for the codon 54 or 57 mutation had dramatically reduced concentrations of serum MBP, mainly comprising material of an apparent molecular weight of 120,000-130,000. Heterozygous individuals showed characteristics of both phenotypes. In contrast to the results obtained with artificial expression systems, our data suggest that individuals homozygous for the MBP mutations have very little circulating protein and that this comprises mainly low molecular weight material.

Mannose binding protein is involved in first-line host defence: evidence from transgenic mice.

Mannose binding protein (MBP) is a calcium-dependent C-type lectin secreted by the liver which seems to be an important component of innate or natural immunity. We have investigated the effects of Candida albicans and thioglycolate injection into transgenic mice bearing the human MBP gene. The transgenes contained a 15 kb fragment of the MBP gene which included the complete coding sequence. Northern blot hybridization showed human MBP mRNA transcripts in the liver of two transgenic lines with low and high copy number respectively. Western blot analysis showed the presence in serum of human MBP which associated into the higher multimeric forms which are capable of activating complement. Enzyme-linked immunosorbent assays (ELISA) showed that serum human MBP concentrations in the transgenes (1.90 +/- 0.16 mg/l, mean +/- SEM) were about twice as high as the levels in man. The serum concentration of MBP A, which is the mouse homologue of MBP, (13.9 +/- 0.45 mg/l) was about seven times that of human MBP. Intravenous injection of Candida albicans caused the serum human MBP level to fall by more than 50% in the first hour and then slowly recover, but it did not return the initial value by 72 hr. Candida injection caused a 25% fall in serum mouse MBP A in the first hour which then rose to supranormal levels by 72 hr. Following Candida injection mouse MBP A mRNA concentrations increased over 72 hr in contrast to human MBP mRNA which remained constant in both transgenic lines. These data indicate that the human MBP gene fragment in the transgene did not include the regulatory elements of the gene. Total haemolytic complement activity and C3 concentrations also fell immediately after Candida and thioglycolate injection while the concentrations of mannose specific immunoglobulin G (IgG) and immunoglobulin M (IgM) did not fall. The data indicate that mannose binding protein plays an important role in the initial stages of defence against infection which, in this model, is quantitatively greater than that of mannose-specific IgG and IgM antibodies. Mannose binding protein is probably most important in defense of previously unexposed and non-immune hosts.

Mannose binding protein gene mutations associated with unusual and severe infections in adults.

A defect in opsonisation can cause a common immunodeficiency. A mutation in mannose binding protein (MBP) caused by point mutations in the MBP gene will lead to such a defect. This type of syndrome can cause recurrent infections in infants between 6 and 18 months of age but is not generally believed to predispose to adult infections. We looked at 4 patients with severe and unusual infections in whom MBP gene mutations were the only identified cause of immunodeficiency and one patient with combined MBP and IgA deficiency. We analysed the MBP genotypes of all the patients in whom we suspected an immunodeficiency because of their clinical history. Infections seen were recurrent skin abscesses, chronic cryptosporidial diarrhoea, meningococcal meningitis with recurrent herpes simplex, and fatal klebsiella lobar pneumonia. Both sexes were affected and ages ranged from 15 to 56 years. Two patients were homozygous for codon 54 mutations, one patient had codon 52 and codon 54 mutations and was phenotypically homozygous, and two patients were heterozygous for codon 54 mutations. Individuals homozygous for MBP mutations are unusual in the general population (approximate frequency 0.3%). The occurrence of three homozygotes for MBP mutations among these five infected patients suggests that MBP deficiency may confer a life-long risk of infection.

Combined percutaneous transhepatic and endoscopic placement of biliary stents.

Combined percutaneous transhepatic cholangiography and endoscopic retrograde cholangiography can be used to stent biliary obstruction when attempts at endoscopic stenting have failed. Between January 1987 and August 1991 we performed 35 combined procedures in 31 patients with malignant obstruction. Post stenting serum bilirubin and serum alkaline phosphatase concentration fell after 33 and 29 procedures, respectively. In six studies there was evidence of infection prior to stenting. In spite of the use of prophylactic antibiotics, septic complications developed after eight procedures (23%). Pseudomonas was the most commonly isolated pathogen (46%). Twenty-three patients were discharged, 30-day mortality was 8 (23%) and median survival was 14 weeks (range 0-75 weeks). Seven patients required eight stent changes because of blockage (median patency time 18 weeks; range 7-75 weeks). Use of this technique allows relief of biliary obstruction. Potential infective and bleeding complications must be anticipated.

Human mannose-binding protein gene is regulated by interleukins, dexamethasone and heat shock.

Mannose-binding protein (MBP) is a plasma protein synthesized by hepatocytes. MBP, a structural analogue of the complement component C1q, can activate complement via the classical pathway and plays an important role in host defence. Expression of the human MBP gene was studied using the human hepatoma cell line HuH-7. RNA extracted from HuH-7 cells was reverse-transcribed to cDNA, amplified by the polymerase chain reaction and analysed by Southern blot hybridization. MBP mRNA expression in HuH-7 cells was increased by interleukin-6 (IL-6), dexamethasone and heat shock, decreased by interleukin 1 (IL-1), and unaffected by interferon gamma (IFN gamma), tumour necrosis factor alpha (TNF alpha) and transforming growth factor beta (TGF beta). Gel shift assays demonstrated Sp-1 binding sites in the 5' region of the gene, and formation of specific complexes between DNA and nuclear protein extracted from HuH-7 cells treated with IL-1 or IL-6. Human MBP is an acute-phase protein, and transcription of its gene is enhanced by IL-6, dexamethasone and heat shock but inhibited by IL-1. The actions of the cytokines appear to be mediated by specific transcription factors.