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.

Characterisation of a lipoprotein in Mycobacterium bovis (BCG) with sequence similarity to the secreted protein MPB70.

The monoclonal antibody, mAb3C4, raised against sonicated Mycobacterium bovis (Mb) BCG (Tokyo strain 172) cells recognises a 23-kDa protein in the cell wall. The gene encoding this protein was cloned and sequenced and found to be 100% homologous to mpb83 and mpt83 and the putative protein to have a 76% sequence similarity to the secreted, Mb-specific protein, MPB70. MPB83 contains the amino acid (aa) sequence LAGC, which corresponds to the consensus sequence for bacterial lipoprotein modification and processing. MPB83 associated with the detergent phase when separated with Triton X-114 confirming that it is a lipoprotein. When the putative site of acylation, the Cys in the sequence LAGC, was substituted with Ser, the mutated MPB83 associated with the aqueous phase. The cloned gene was used to determine the distribution of mpb83 in various Mycobacterium species. The gene was present in the M. tuberculosis (Mt) complex organisms, as well as in M. kansasii. In addition, Southern blot analysis of Mb and Mt DNA indicated that the mpb83 and mpb70 genes are located close to each other on the genome. Western blot analysis of cell lysates of various Mycobacterium species indicated that only Mt H37Rv and H37Ra produced proteins which reacted with mAb3C4. Furthermore, only two out of six of the Mb field isolates produced detectable antigen, indicating that expression of the mpb83 gene is variable within the Mt complex organisms.

Immunoglobulin allotypes and genetic susceptibility to invasive Haemophilus influenzae type b and Staphylococcus aureus infections in South African children.

OBJECTIVE: The purpose of this study was to determine whether the G2m(n), G1m(f) and Km(3) immunoglobulin allotypes have any association with susceptibility to invasive Haemophilus influenzae type b (Hib) and Staphylococcus aureus (S. aureus) infections in children. METHODS: Direct enzyme-linked immunosorbent assays with commercially available monoclonal antibodies were established to quantitate G2m(n) and G1m(f) allotypes. A qualitative enzyme-linked immunosorbent assay with polyclonal rabbit anti-Km(3) antibody was established for Km(3) determination. RESULTS: The G2m(n) marker occurred in 34.4% of the mixed ancestry population and 2.9% of the Black population. There was a significantly decreased frequency of the G2m(n) allotype in mixed ancestry children with Hib meningitis (8.5%) and Hib osteomyelitis/septic arthritis and a decreased frequency of Km(3) in black and mixed ancestry children with Hib meningitis. The frequency of G2m(n), G1m(f) and Km(3) allotypes in patients with S. aureus osteomyelitis/septic arthritis were not significantly different from normal population frequency. CONCLUSIONS: This study shows a clear association between the absence of the G2m(n) allotype in mixed ancestry children and susceptibility to invasive infections caused by H. influenzae and an association between the absence of Km(3) and Hib meningitis in both black and mixed ancestry children.

IgG subclass values from normal children in Cape Town.

Sensitive and reproducible enzyme-linked immunoabsorbent assays (ELISA) have been developed to quantitate IgG subclass levels using monoclonal antibodies. Normal values for serum IgG subclass levels were determined in 300 healthy children between 6 months and 14 years of age and in 80 adults. High levels of IgG1 and delayed maturational development of IgG2 in children from Cape Town are different to results reported from developed countries. Genetic differences may account for this.

Immunological abnormalities in children with acute rheumatic carditis and acute post-streptococcal glomerulonephritis.

Immunological functions were investigated in 10 children with acute rheumatic fever and 11 children with acute nephritis to try and elucidate the cause of heart damage in acute rheumatic fever. Children with acute rheumatic fever and carditis showed an increase in serum IgG, IgA and antistreptococcal antibodies during the acute stage. Lymphocyte transformation responses to phytohaemagglutinin and streptococcal antigens were reduced but this was due to a serum suppressor effect. After recovering from acute rheumatic fever a lymphocytosis and an increased lymphocyte blastogenic response to streptococcal antigen were found. T-cells, T-helper cells and T-suppressor cells showed some changes in acute rheumatic fever but these were not statistically significant in our study. None of the changes in immunological responses that were seen in acute rheumatic fever were found in acute nephritis. These results support the hypothesis that an abnormal immune response to streptococcal products is involved in the development of carditis and the other phenomena observed in acute rheumatic fever.

HIV infection of undetermined origin during infancy.

The case histories of two children with horizontally acquired HIV infection are described. These children were diagnosed at a paediatric hospital in sub-Saharan Africa. Although the source(s) of infection was not identified, both children had had several contacts with the health service, experienced invasive procedures and ingested expressed milk from their own mothers during hospital admission. Health-care institutions, particularly those located in high HIV prevalence areas, must implement effective infection control measures to ensure that the risk of horizontal infection is minimized. Attention should be given to practices that are unique to each clinical discipline.

IgM rheumatoid factor in congenital syphilis: associations with clinical and laboratory findings.

IgM rheumatoid factor (RF), measured by means of an ELISA, was detected in 92% of infants with congenital syphilis. Elevated levels were found to correlate with liver and renal involvement as well as the extent of the disease (P less than 0.05). In addition, levels of circulating immune complexes were closely related to the RF concentration (P less than 0.001). Following treatment of the infants both RF levels and VDRL titres declined at a similar rate (P less than 0.001). These findings indicated a close relationship between the disease process and IgM RF levels. It is postulated that IgM RF may add to immune complex deposition and exacerbate tissue damage in congenital syphilis.

X-linked agammaglobulinaemia.

X-linked agammaglobulinaemia (XLA) is a rare immunodeficiency disorder affecting only male subjects. There is an absence of all serum immunoglobulins and circulating B cells. T-cell function and numbers are normal. The clinical characteristics are recurrent pyogenic infections starting in infancy, hypoplasia of lymphoid tissue and a family history in about half the cases. Ten patients with XLA have been seen over the last 14 years at Red Cross War Memorial Children's Hospital, Cape Town. Chronic infections causing significant morbidity occurred in half our patients and 2 have died. Intravenous of gammaglobulin replacement therapy is superior to the intramuscular form. The recommended dose is 200-400 mg/kg/mo., although the optimal dose and frequency of the gammaglobulin infusion should be individualised for each patient.

Deficiency in kwashiorkor serum of factors required for optimal lymphocyte transformation in vitro.

Blastogenic responses of normal human peripheral blood lymphocytes cultured in media supplemented with serum from children with kwashiorkor were, on average, 47.7% of those observed when the same cells were cultured in the presence of normal AB serum. Incorporation of radioactive uridine was also diminished in the presence of normal AB serum. Incorporation of radioactive uridine was also diminished in the presence of kwashiorkor serum indicating that lectin-induced RNA synthesis was also affected. The kwashiorkor serum effect was not due to a cytotoxic action nor could it be attributed to the presence of saccharides or other inhibitors of the inducing lectins. Mixing experiments showed that kwashiorkor serum was not inhibitory, but that it lacked factors present in normal serum that are required for optimal lymphocyte blastogenesis. The deficiency of these factors could largely be rectified by supplementing kwashiorkor serum with an ultrafiltrate of normal serum containing components with molecular weights of less than 500 Daltons. We conclude that nutritional deprivation of severity sufficient to cause kwashiorkor leads to a deficiency of low molecular weight lymphocyte growth factors. This lack may contribute to the immunodeficiency associated with the disease.

The effects of kwashiorkor serum on lymphocyte transformation in vitro.

The serum from twelve children with kwashiorkor was deficient in its ability to support lymphocyte transformation in vitro, whereas lymphocytes from these children responded to phytohaemagglutinin and al-ogeneic lymphocytes in a relatively normal manner when cultured in normal serum. This serum abnormality improved with therapy and could not be clearly correlated with the degree of malnutrition, the presence or absence of infection or other laboratory manifestations of kwashiorkor. These observations indicate that defective cellular immune reactions in kwashiorkor may be symptomatic of a lack of some humoral factor and do not necessarily reflect an intrinsic cellular defect.

Unusual features in a case of snakebite, presumably due to a Cape cobra (Naja nigricollis).

A case of snakebite, thought to be due to a Cape cobra, in a young girl is described. Unusual features were a protracted period of paralysis and severe neurological residua in the form of hemiparesis and impaired vision. The ophthalmological features started as a bilateral optic neuritis and progressed to bilateral optic atrophy. It is difficult to ascribe all these effects to the neurotoxins of the Cape cobra as the neurotoxic features usually resolve rapidly if the patient survives. The actions of the neurotoxins, the quantity of antivenom to be administered in cases of cobra bite and the possible use of anticholinesterases such as neostigmine in these cases are also discussed.

Disseminated histoplasmosis in an 'immunocompetent' child.

A rare case of severe disseminated histoplasmosis in a 7-year-old boy with apparently normal immune function is described. Current recommendations for diagnostic investigations, monitoring and the treatment of this disease with amphotericin B and itraconazole are reviewed.

Identification of cardiac autoantigens in human heart cDNA libraries using acute rheumatic fever sera.

Antigenic mimicry or cross-reactivity between Group A streptococcal antigens and cardiac autoantigens may initiate an autoimmune response resulting in cardiovascular damage in acute rheumatic fever. This study describes a molecular biological approach to the identification of such cross-reactive cardiac antigens. Two human heart cDNA libraries were constructed in the expression vector lambda gt11 and screened with patient sera, monoclonal antibodies and rabbit immune sera cross-reactive with streptococcal and cardiac antigens. Using the serum of a patient with a recurrent acute attack of rheumatic fever containing high titres of antibodies cross-reactive with both sets of antigens, we were able to identify three positive clones with insert sizes of 1.0 kb, 1.4 kb and 0.9 kb in these libraries. Acute rheumatic fever (ARF) sera reacted more strongly with these autoantigen clones than did normal sera. Autoantibodies eluted from the purified plaques of all three clones displayed different patterns of cross-reactivity against immunoblots of streptococcal M5, M6, M19 and M24 protein extracts. The cDNA inserts were sequenced and compared with known sequences in the EMBL and Genbank databases. One clone was 98% homologous with human cytokeratin 8 and showed homologies of 40 to 50% with human cardiac heavy chain myosin, tropomyosin and streptococcal M5 protein--all members of the alpha-helical coiled-coil family of proteins. Another clone was completely homologous to the G-protein alpha-subunit of adenyl cyclase, whilst the sequence of the third clone was not found in any of the data banks.

Human monoclonal antibodies: analysis of two antibodies derived from lymphocytes of a patient with acute rheumatic fever.

Human monoclonal antibodies were produced by fusion of peripheral blood lymphocytes from a patient with acute rheumatic fever, with the HGPRT-non-secreting murine (Balb-c) cell line SP2/0Ag14. Heterohybridomas were selected by screening against rheumatic fever-associated group A streptococci using an ELISA, and against paraffin wax-embedded human heart sections using an immunoperoxidase technique. Two human IgM monoclonal antibodies were selected for further analysis by Western blotting and ELISA. Both antibodies demonstrated multispecificity by immunoblotting and ELISA. One of the monoclonals bound to 48 kD and 83 kD bands common to group A streptococcal and heart antigen preparations. Both human monoclonal antibodies bound to a 43 kD constituent band common to human heart and sarcolemma membrane extract. Inhibition studies performed using a competitive solid phase immunoassay confirmed shared epitopes between group A streptococci and human heart. The significance of these monoclonal antibodies to the pathogenesis of rheumatic fever is uncertain.

Complement abnormalities during an epidemic of group B meningococcal infection in children.

During an epidemic of group B meningococcal infection mean values obtained in 96 consecutively affected children showed a reduction in classical pathway function (CH50), normal alternate pathway function (AP50), C4 and factor B levels, and raised C3 levels. CH50, C3 and Factor B were however significantly lower in those children who had a rapid onset of illness, were in shock, had signs of septicaemia, had extensive skin purpura, or who died. The presence of detectable meningococcal antigen by Counter Immuno Electrophoresis (CIE) and laboratory evidence of Disseminated Intravascular Coagulation (DIC) also correlated with lower complement levels. The significant reduction in CH50 and Factor B in the more severely affected patients suggests that activation of both classical and alternate pathways occurs in group B meningococcal infection.

Group B meningococcal infection in children during an epidemic in Cape Town, South Africa.

One hundred and thirteen children with meningococcal infection were studied during an epidemic caused by N. meningitidis group B. Fifteen per cent presented with only meningeal symptoms, the remainder showed signs of septicaemia or combined septicaemia and meningitis. Sixteen per cent of the children were in shock and 18% required admission to the Intensive Care Unit (ICU). The mortality was 4.4%. More than half the children were younger than 2 years old. There was no statistical association between the age or nutritional state of the children and any of our measures of severity. A short history of symptoms was more common in children who presented with septicaemia and severe illness, who needed admission to the ICU, or who died. Diagnosis was confirmed by routine bacteriological methods and counter-immuno-electrophoresis (CIE) in 104 children. Eighty-six per cent of the isolates were group B type 2. A history of recent antibiotic treatment was associated with fewer positive cultures, but detection of meningococcal antigen by CIE was not affected by this. CIE antigen detection was not reliable because of the high incidence of false-negative results.

Cellular immune function in marasmic and underweight infants with prolonged diarrhoea.

Lymphocytes from 10 marasmic and underweight infants with prolonged diarrhoea responded normally to phytohaemagglutinin stimulation. There was no gross deficiency in the total number of lymphocytes, serum immunoglobulins, or serum C3. Defective cellular immunity does not appear to play a major role in the pathogenesis of diarrhoea in these children.

Hyper IgM with combined immunodeficiency.

This report describes a fourteen year old girl with an unusual immunodeficiency characterized by persistent lymphadenopathy and associated with hypogammaglobulinaemia, excessive IgM production and a severe T cell defect. Total T cell and T helper cell numbers were reduced and T cell proliferative responses to mitogens were poor. Serum IgM levels showed marked fluctuations and peaks correlated with acute tender lymphadenopathy. She was treated with intravenous gammaglobulin and prophylactic antibiotics. Although defective isotype switching of B cells into IgA and IgG producing cells has been accepted as the mechanism of the hyper IgM syndrome, it is becoming increasingly evident that T cell function is not uncommonly involved and may be responsible for impaired isotype switching.

Childhood near-drowning--factors associated with poor outcome.

Anoxic cerebral damage is the limiting factor in recovery from near-drowning accidents. This study reports factors associated with poor outcome in 100 near-drowned patients admitted to the Red Cross War Memorial Children's Hospital from 1976 to 1987. The study was designed as a retrospective folder search. Children with fixed dilated pupils, flaccidity, decerebrate or decorticate posturing, or clinical signs of cerebral oedema are more likely to have a poor outcome. Patients with metabolic acidaemia and those requiring cardiopulmonary resuscitation in the emergency room or subsequent mechanical ventilation also do poorly. Those requiring more aggressive management can be identified and a more accurate working prognosis may be possible in the hours after the accident.