Monocyte function in ageing humans.

Peripheral blood monocytes from hospitalised patients greater than 60 years of age and less than 35 years, and those from healthy normal controls less than 35 years, were tested for a range of functional and physiological properties, comprising chemotaxis under agarose, the ability to phagocytose and kill Candida albicans, adhesion to glass and spreading on glass. No significant difference was found between young and old groups, nor between hospitalized and non-hospitalized groups in respect of any parameter. There was some decline in phagocytosis and in spreading in a very old subgroup (greater than 75 years), but this was not statistically significant. This study showed that phagocytic cell function in the elderly does not decline at the same rate as the specific immune response and thus cannot directly account for the increased incidence of infection in the aged.

Human transfer factor prepared by dialysis, ultrafiltration and gel chromatography: biological activity in local transfer of skin sensitivity.

Human transfer factor (TF) was prepared by a variety of methods including dialysis using cellophane tubing, ultrafiltration through a membrane of known pore size. Sephadex G25 chromatography or combinations of some of these methods. In general the various preparations when injected locally into human skin gave greater delayed-type responses than antigen (PPD or Candida) alone. The combination of either vacuum dialysis, or ultrafiltration, with G-25 chromatography gave as good or better TF activity when compared with unchromatographed materials. Since ultrafiltration and concentration is rapid procedure and eliminates the need for freeze-drying, in contrast to vacuum dialysis against water, these results indicate that ultrafiltration and G-25 chromatography provide a convenient method for preparing large batches of relatively pure TF from leucocyte extracts.

One-way mixed lymphocyte culture: differences between Chinese and Caucasian stimulator cells.

In "standardized" one-way mixed lymphocyte cultures, a pool of blood lymphocytes from randomly chosen Chinese subjects was a significantly stronger stimulus to Caucasian responder lymphocytes than was a pool of allogeneic cells from Caucasians. In contrast, Chinese lymphocytes did not respond more strongly to Caucasian cells than to Chinese cells. This could not be explained by an inherent racial difference in the capacity of blood lymphocytes to undergo blastogenesis. The implications of these findings for organ transplantation and routine testing of cell-mediated immune function are discussed.

Lymphocyte subsets in patients with oestrogen deficiency.

We have previously shown that in patients with idiopathic premature ovarian failure there were significant changes in lymphocyte subsets. To test our hypothesis that these changes were due to oestrogen deficiency we studied lymphocyte subsets in patients with oestrogen deficiency due to other causes. Blood was taken for serum oestradiol, lymphocyte counts and lymphocyte subset counts (CD2+, CD4+, CD8+ and B cells) before oestrogen replacement in 19 patients with gonadal dysgenesis, 22 patients with hypothalamic-pituitary failure and 24 healthy female control subjects. The CD4:CD8 ratio in both groups of patients was significantly lower than that in the normal control subjects while the percentages and counts of lymphocytes and CD8+ cells were significantly higher. There was a significant positive correlation between the serum oestradiol level and the CD4:CD8 ratio. These findings support the hypothesis that the changes in lymphocyte subsets are due to oestrogen deficiency.

Mixed lymphocyte reaction in gestational trophoblastic disease: presence of a specific plasma inhibitor of the response to paternal alloantigens.

One-way wife-husband and wife-unrelated donor mixed lymphocyte reactions (MLR) were performed for 17 patients with malignant gestational trophoblastic disease (MGTD) and 17 normal pregnant subjects in the first trimester. The response of patients with MGTD to the husbands' lymphocytes did not differ significantly from the response to donors' lymphocytes when the MLR was performed in pooled human serum. Autologous plasma from patients with MGTD suppressed the MLR to husbands' lymphocytes but not that to donors' lymphocytes. Plasma from pregnant subjects in the first trimester did not suppress the wife-husband MLR. It is postulated that the specific plasma blocking activity may contribute to the failure to reject the trophoblastic tumours.

Immunological parameters in gestational trophoblastic neoplasia.

Immunological function were studied in 22 patients with hydatidiform mole and 29 patients with malignant trophoblastic disease before and after treatment; normal pregnant and post-pregnant women served as controls. The only significant abnormality in hydatidiform mole was a low granulocyte chemotaxis before evacuation. In malignant trophoblastic disease the total lymphocyte counts, T-cell counts. B-cell counts, lymphocyte responses to mitogens and serum IgA levels were significantly lower than in normal women 6 wk after pregnancy. In those who responded to chemotherapy, these indices rose to the levels of post-pregnancy controls. An 'immune profile score' based on these indices was found to be a useful prognostic index. All patients with hydatidiform mole who had a score of 7 or less developed malignant trophoblastic disease, while the two patients with malignant trophoblastic disease who died had the lowest scores of the series.

Immunoperoxidase detection of T and B cells in blood compared with conventional methods.

Peripheral blood T and B cells were enumerated in 26 normal adults by conventional immunological markers (erythrocyte rosette and surface membrane immunoglobulin) and by monoclonal markers (T11 and B1) using both membrane immunofluorescence of cells in suspension and immunoperoxidase staining of dried, fixed, cytocentrifuged cells after separation from blood by buoyant density centrifugation. The results of immunochemistry were determined independently from the results of erythrocyte rosetting and membrane immunofluorescence techniques. A high correlation was found between all three methods for enumerating T cells (erythrocyte rosetting, T11/immunoperoxidase, and T11/indirect immunofluorescence). Correlation was less good between the results of the three methods for enumerating B cells; surface membrane immunoglobulin and B1/indirect immunofluorescence techniques showed the highest correlation. The results suggested that B cells may be preferentially lost during the extensive cell washings entailed in the indirect immunofluorescence procedures. Immunochemical methods, using monoclonal antibodies, are useful for enumerating lymphocyte subsets in blood, particularly when permanent records are desired.

Distinction between antinuclear antibody and P-ANCA.

To differentiate between perinuclear immunofluorescence staining of antinuclear antibody (ANA) and the perinuclear form of anti-neutrophil cytoplasmic antibody (P-ANCA), the pattern after formaldehyde vapour fixation of normal human neutrophils was compared with that of standard ethanol fixation. Fifteen out of 17 myeloperoxidase antibody positive sera showed cytoplasmic staining on formaldehyde vapour fixed cells; 30 of the 32 ANA positive samples became negative or gave weak nuclear staining on the same substrate. Formaldehyde vapour fixation is a simple, useful technique for differentiating between ANA and P-ANCA.

ad and ay subtypes of hepatitis B antigen in a case of hypogammaglobulinaemia.

We describe the finding of d and y specificities of hepatitis B surface antigen (HBsAg) in a case of hypogammaglobulinaemia of the 'common variable' type treated with fresh frozen plasma infusions. Absorption studies show that the two specificities are on separate particles, suggesting dual infection. It raises important questions regarding the relationship between HBsAg persistence and the immune status of the carrier.

A latex slide agglutination test for rapid detection of antimyeloperoxidase antibody.

AIM: To develop and test a new latex slide agglutination test (MPO-LSAT) to detect antimyeloperoxidase (anti-MPO) antibody in serum. METHODS: Latex bead coating was adjusted to give maximum sensitivity by attending to latex size, MPO to latex ratio for coupling, ratio of diluted serum to MPO-latex, reaction time and temperature for coupling, and reaction time for agglutination. Inhibition studies were performed using MPO, proteinase 3, bactericidal/permeability increasing protein, and lactoferrin. RESULTS: There was very good correlation between this test and the conventional anti-MPO enzyme linked immunosorbent assay (ELISA): 81% of sera positive in the ELISA were positive by MPO-LSAT. MPO-LSAT results correlated better with IgM anti-MPO than with IgG anti-MPO. CONCLUSIONS: MPO-LSAT is a simple diagnostic test that is potentially useful in the clinical laboratory as a rapid screening tool for vasculitic diseases.

alpha-1 antitrypsin phenotypes by isoelectric focusing in a metropolitan southern Chinese population.

AIMS/BACKGROUND: alpha-1 antitrypsin (alpha1AT) is an abundant protease inhibitor in human plasma. Its phenotypic variability has been reported to be associated with pulmonary emphysema and chronic liver diseases. However, alpha1AT deficiency is an uncommon condition in the Chinese population. The aim of this study was to describe the phenotypic distribution of alpha1AT in a southern Chinese population. METHODS: A total of 1085 healthy blood donors underwent alpha1AT phenotyping by isoelectric focusing. RESULTS: Two thirds (66.1%) were homozygous for either M1 or M2, whereas 32.6% were heterozygous for two different M phenotypes. The frequency of allelic variants was only 0.007, and deficiency variants were absent. Compared with earlier studies on southern Chinese populations, this study found a lower frequency of M2, and a higher number of allelic variants, including E, L, N, P, and S. This phenomenon can be attributed to population migration and mixing. CONCLUSIONS: An understanding of the alpha1AT pattern is important for evaluating the predisposition of the population to selected clinical diseases.

Immunologic studies in patients with premature ovarian failure.

Tests for a range of autoantibodies, and counts of lymphocytes, B cells, T cells, and T cell subsets were performed in 45 Chinese patients with premature ovarian failure and 45 age-matched normal control subjects. Eight patients (18%) were positive for at least one autoantibody. Only one patient was positive for antiovarian antibody. Patients with autoantibodies had a significantly higher percentage of circulating B cells. The lymphocyte, T cell, CD4+, and CD8+ counts in patients with premature ovarian failure were significantly higher than those in the control group, but the CD4:CD8 ratio was significantly lower in women with premature ovarian failure. There was a significant negative correlation between plasma estradiol levels and CD8+ counts, and a significant positive correlation between plasma estradiol levels and CD4:CD8 ratios. The changes in lymphocytes and lymphocyte subpopulations in premature ovarian failure may be due to estrogen deficiency.

Autoantibody formation after allogeneic bone marrow transplantation: correlation with the reconstitution of CD5+ B cells and occurrence of graft-versus-host disease.

Manifestations of autoimmune diseases are common in patients who have received allogeneic bone marrow transplantation (BMT). Autoantibodies have been reported in these patients but the source and clinical significance of these autoantibodies are still obscure. In the present study the kinetics of autoantibody formation and the reconstitution of CD5+ B cells was followed in 21 patients who were submitted to allogeneic BMT. Anti-nuclear, anti-smooth muscle, anti-neutrophil cytoplasmic antibodies, anti-reticulin and rheumatoid factor were found at a frequency of 25%, 17%, 24%, 22% and 10% respectively after BMT. Anti-double stranded DNA levels were mildly elevated in 15% of samples. The screening for anti-extractable nuclear antigen, anti-mitochondrial, anti-gastric parietal cell, anti-proteinase III, anti-myeloperoxidase, anti-lactoferrin antibodies was negative. The percentage and absolute count of CD5+ B cells increased with time after allogeneic BMT. Those patients with anti-nuclear or anti-smooth muscle antibodies had significantly higher CD5+ B cell counts than those without these two antibodies. Correlations of CD5+ B cell counts with other autoantibodies were negative. Acute graft-versus-host disease (GVHD) occurred in eight of the patients and chronic GVHD in four patients, but the frequency of autoantibodies had no relationship with the occurrence of acute or chronic GVHD.

Measurement of anti-dsDNA: a comparative study of two ELISA and the Crithidia assay.

We compared the measurement of anti-dsDNA by a commercial ELISA test (DIASTAT), an in-house ELISA and the Crithidia luciliae assay in cross-sectional sera samples of 209 systemic lupus erythematosus (SLE) patients and 64 patients with a variety of rheumatological, autoimmune and non-autoimmune diseases in Hong Kong. The Crithidia assay was found to be the least sensitive (17%) but most specific (95%) method for detection of a positive result in SLE patients. The DIASTAT assay has a higher sensitivity (68%) but lower specificity (80%) than the in-house ELISA test (32% sensitivity and 89% specificity). The positive predictive value of the three assays are comparable at 90-92% while DIASTAT had the highest negative predictive value (44%). There was good linear correlation (r = 0.7) between the two ELISAs. ELISA can serve as a useful screening test for anti-dsDNA in SLE patients and doubtful cases can then be confirmed by another method such as radio-immunoassay.

Low predictive value of c-ANCA and anti-alpha-granule antibody for vasculitis in Hong Kong.

One thousand sera, not previously tested for ANCA, were assayed for C-ANCA and anti-alpha-granule antibody. There was low concordance (23%) between the two assays in terms of positivity. Each assay gave a positive predictive value for systemic vasculitis excluding SLE and other connective-tissue diseases) of not more than 20%.

Evaluation of phagocytic function in Mycobacterium lepraemurium infection.

Infection of mice with Mycobacterium lepraemurium caused significant functional alterations of the mononuclear phagocyte system. Accelerated clearance of sheep red blood cells was consistently demonstrated throughout the infection and the infected mice showed progressive anaemia. Infected mice showed an enhanced ability to limit growth of phagocytosed Listeria monocytogenes in spleens during the early stages of infection, whereas moribund leprous mice lost this ability. Autoradiography showed that uninfected Kupffer cells and splenic macrophages of moribund mice could still phagocytose Listeria, suggesting that MLM infection did not affect the capacity of Listeria to localize to macrophages but interfered in some way with subsequent killing of such bacteria. The possible mechanisms underlying these observations are discussed.

The effect of in vivo modulation of macrophage activities on Mycobacterium lepraemurium infection.

During the early stage of Mycobacterium lepraemurium (MLM) infection in mice, the mononuclear phagocyte system (MPS) was activated non-specifically as demonstrated by enhanced listericidal activity. Such listericidal activity could be further increased by Corynebacterium parvum treatment, indicating that MLM was not a good MPS stimulant. Corynebacterium parvum treatment conferred only marginal protection upon mice during MLM infection, as shown by the slight but significant prolongation of survival time and decreased bacillary load. In contrast, mice could not control splenic Listeria growth in the later stage of infection regardless of C. parvum treatment. Adoptive transfer of Listeria-immune spleen lymphocytes, however, did significantly suppress splenic Listeria growth. The significance of these findings is discussed.

A histopathological study of pulmonary infection of mice with Mycobacterium lepraemurium.

Intranasal instillation of Mycobacterium lepraemurium (MLM) into mice produced pulmonary infection. MLM multiplied rapidly in the lung tissue during the first few weeks without involvement of other organs. The increase in number, size and confluence of lung granulomas paralleled the multiplication of MLM which could be found both intracellularly and extracellularly. It is postulated that extracellular bacteria may find their way to the bloodstream and thus spread to other visceral organs. Extensive destruction of alveoli and occupation of airspaces by lepra-like cells invariably occurred as the disease progressed.

Antinuclear antibody detection using streptavidin-biotin-peroxidase complex on HEp-2 cell substrate.

The streptavidin-biotin-peroxidase complex (SABC) technique was compared to conventional indirect immunofluorescence (IIF) for the detection of anti-nuclear antibody (ANA) on HEp-2 cell substrate. SABC showed higher specificity and predictive value and gave more reproducible titres and clearer staining patterns than IIF in sera from a series of rheumatic disease patients. Sera from 80 patients with various types of rheumatic diseases and 20 without rheumatic disease were further tested using the SABC method. All systemic lupus erythematosus (SLE) sera were positive. The overall sensitivity was 95%, specificity 90% and predictive value 97% for rheumatic disease. The rim pattern was associated with SLE and mixed connective tissue disease. The nucleolar/homogeneous pattern was associated with scleroderma and SLE in remission. ANA titre and staining pattern have limited value in the clinical assessment of rheumatic disease; however, ANA has very high sensitivity for SLE and remains an excellent screening test.

Comparison of counter immunoelectrophoresis with immunoblotting for detection of anti-extractable nuclear antigen antibodies in systemic lupus erythematosus.

Anti-extractable nuclear antigen (ENA) antibodies were assayed by counter immunoelectrophoresis (CIE) and immunoblotting in patients with systemic lupus erythematosus (SLE). We found the two methods showed good concordance rates, the lowest being 67% for anti-SS-A. Immunoblotting was more sensitive in detecting anti-Sm, anti-SS-B and anti-PCNA (proliferating cell nuclear antigen); CIE was more sensitive for anti-nRNP and anti-SS-A. Overall, the prevalence of these anti-ENA antibodies in SLE was increased by 9-20% if immunoblotting was used in addition to CIE. Sera specific for the 52 kDa peptide of the SS-A antigen (anti-52kDa SS-A) were better detected by immunoblotting. Anti-PCNA antibody was found in 6.3% of SLE patients and was associated with active disease and hemolytic anemia. The positive rate of anti-Sm was 9% by CIE and 23.7% by immunoblotting and this antibody was a specific marker for SLE using either method. It was concluded that using immunoblotting in addition to CIE, the overall sensitivity of detection of anti-ENA antibodies in SLE was increased and clinically useful antibodies such as anti-52kDa SS-A and anti-PCNA could be detected.