Brian M. Jones

Reduced in vitro production of interferon-gamma, interleukin-4 and interleukin-12 and increased production of interleukin-6, interleukin-10 and tumour necrosis factor-alpha in systemic lupus erythematosus. Weak correlations of cytokine production with disease activity.

Production of cytokines in unstimulated and mitogen-stimulated cultures were evaluated by ELISPOT in 34 SLE patients with low to moderate disease activity and 23 healthy controls. Significantly reduced production of IFN gamma, IL4 and IL12 and significantly increased production of IL6, IL10 and TNF alpha were found in patients with SLE. Regression analysis revealed that production of all six cytokines tended to decrease with increasing disease activity, but negative correlation with SLEDAI was significant (p < 0.05) only for PHA-stimulated IL4, unstimulated and PHA-stimulated IL10 and SAC-stimulated IL6. Negative correlation of stimulated and unstimulated IL6 and TNF alpha production with anti-DNA antibody levels were also significant.

Defective neutrophil and lymphocyte function in leucocyte adhesion deficiency

We report a Chinese girl with the moderate phenotype of leucocyte adhesion deficiency (LAD), presenting with persistent omphalitis and recurrent soft tissue infections. She had subnormal adhesion‐dependent neutrophil functions, such as chemotaxis and chemiluminescence response to a particulate stimulant (opsonised zymosan). Despite her adequate humoral response to documented herpes simplex virus type 1, parainfluenza type 2 and adenovirus infection in vivo. there was marked impairment in the generation of plaque‐forming cells (PFC) driven by pokeweed mitogen (PWM) in vitro. IgM PFC were less severely affected than IgG and IgA PFC, probably because IgM production is less dependent on T cell help than IgA and IgG production. The patients B cells and accessory cells had reduced function compared with the control subsets, while helper function of her CD4+ cells was virtually absent in the PWM‐driven PFC assay. She also had marked defect in natural killer cell activity. The proliferation of her lymphocytes was normal to several plant Iectins, including phytohaemagglutinin, concanavalin A and PWM, but markedly defective to OKT3.

Percentile ranges for serum IgG subclass concentrations in healthy Chinese children

In order loestablish normal reference ranges of serum immunoglobulin G subclasses fordifferent age groups in Chinese, we measured the four IgG subclasses by radial immunodiffusion using poiyclonal antisera in 350 normal healthy subjects (148 males and 202 females) recruited Irom ihe community. There was no signifieant sex difference for all the four IgG subclasses. Using Box‐Cox transformation, we constructed smooth age‐dependent percentile curves lor the four IgG subclasses. For the 50th percentile values, the plateaus Tor IgGl lo lgG4 are respectively 970 mg/dl at 13 years old. 481 mg/dl al 18. 48 mg/dl at 17 and 80 mg/dl at 13. Methodology, reagents, environmental and genetic factors might be responsible for the observed difference among the various reports. The IgG2 level in our Chinese population seemed to be higher than that in Caucasians, which might account for the very low incidence of invasive Haemophilus influenzae type b disease among the Chinese. Our IgG l level also plateaued later and at a higher level than that in other studies.

Penicillium marneffei infection in a non-HIV infected child

We report a case of Penicillium marneffei infection in a previously healthy 3.5 years‐old Chinese boy and review the literature. The details of the case are described with emphasis on his immune function and treatment outcome. This boy had transient immunodeficiency involving phagocytic and NK cells due to P. marneffei infection which resolved after treatment with gamma interferon and amphotericin B. A prolonged course of fluconazole of 1 year was successful in preventing relapse. Gamma interferon, amphotericin B and a prolonged course of fluconazole may be useful in the treatment of life‐threatening infection by P. marneffei.

Evaluation of CD5 and other differentiation antigens on human immunoglobulin-secreting cells using a combination of immunobead rosetting and reverse haemolytic plaque formation

This paper describes a new method, with high specificity and sensitivity, for evaluating cell surface markes such as differentiation antigens and cytokine receptors on immunoglobulin-secreting cells. Mononuclear cells, freshly derived from peripheral blood or following stimulation in vitro with pokeweed mitogen or Staphylococcus aureus Cowan I, are partially depleted of T cells and monocytes using immunomagnetic beads (Dynabeads) coated with anti-CD2. The cells are incubated with Dynabeads coated with monoclonal antibody against the cell marker under investigation and then used in a protein A haemolytic plaque assay. Plaque-forming cells (PFC) with (marker-positive) and without (marker-negative) attached beads can be readily enumerated. Values are given for percentages of IgG-, IgA- and IgM-PFC bearing CD19, CD38, CD25 and CD5.

Effect of 12 neutralizing anti-cytokine antibodies on in vitro activation of B-cells. Interleukin-12 is required by B1a but not B2 cells.

Normal human peripheral blood mononuclear cells, depleted of most monocytes and virtually all CD8‐positive cells, were stimulated in vitro with pokeweed mitogen plus Staphylococcus aureus Cowan I in the presence or absence of variousneutralizing anti‐cytokine antibodies. Numbers of CD5+ and CD5− immunoglobulin‐secreting cells were determined using the protein A haemolytic plaque assay after labelling B1a cells with anti‐CD5‐coated beads. Antibodiesagainst IL‐2, IL‐5 and IL‐10 had little or no effect on plaque‐forming cell (PFC) induction; anti‐IL‐6, ‐TNFα and ‐TGFβ enhanced PFC induction; anti‐IL‐1α, ‐IL‐1β, ‐IL‐4, ‐IFNγ and ‐IL‐13 suppressed PFC induction. B1a and B2 cells were equally affected by cytokine deprivation using these 11 neutralizing antibodies. In contrast, neutralizing anti‐IL‐12 suppressed induction of CD5+ but not CD5− PFC. Furthermore, recombinant IL‐12, if added during thefirst 48 h of culture, enhanced CD5+ PFC induction while marginally suppressing (IgG‐) or not affecting (IgA‐, IgM‐) induction of CD5− PFC. IL‐12 did not preferentially increase survival in culture of B1a cells norinduce expression of CD5 on B2‐cells. Further studies are required to determine whether manipulation of B1a and B2 subsets in vivo using IL‐12 could be achieved in clinical situations where imbalances in the two populations have beenobserved.

CD5-positive and CD5-negative rheumatoid factor-secreting B cells in IgA nephropathy, rheumatoid arthritis and Graves' disease.

The relative contributions of CD5+ and CD5– B–cells in production of rheumatoid factors (RF) was evaluated in polyclonally activated B–cells from patients with IgA nephropathy (IgAN), rheumatoid arthritis (RA) and Graves’ disease (GD). In IgAN and RA, diseases in which RFs are believed to be involved in pathogenesis, there were 10– and 4–fold decreases respectively in CD5+ IgG–RF–secreting B–cells compared with controls. Furthermore, the number of CD5– IgG–RF– and IgA–RF–secreting B–cells were increased 12– and 14–fold in IgAN and 9– and 4–fold in RA. Such abnormalities were not apparent in GD, in which RFs have not been implicated in pathogenesis. These findings are compatible with the concept of CD5+ RF–secreting B–cells normally acting to prevent production of potentially pathogenic RFs by CD5– B–cells. When IgAN or RA patients’ B–cells were activated in the presence of control instead of autologous CD4+ cells, numbers of RF– secreting CD5– B–cells were reduced to the levels seen with control B–cells plus control T–helper cells. Presumably lymphokine secretion profiles of T–helper cells would be important in determining whether CD5+ or CD5– B–cells are activated to secrete RFs, and perhaps therapeutic manipulation of these profiles could restore normal activity of CD5+ B–cells in IgAN and RA.

Expression, refolding and purification of a human interleukin-17A variant.

A human interleukin-17A (IL-17A) variant was overexpressed in Escherichia coli BL21 (DE3) under the control of a T(7) promoter. The resulting insoluble inclusion bodies were isolated and solubilized by homogenization with 6 M guanidine HCl. The denatured recombinant human IL-17A variant was refolded in 20 mM Tris-HCl, pH 9.0, 500 mM arginine, 500 mM guanidine HCl, 15% glycerol, 1 mM cystamine, and 5 mM cysteine at 2-8°C for 40 h. The refolded IL-17A variant was subsequently purified using a combination of cation-exchange, reversed-phase and fluoroapatite chromatography. The final purified product was a monodisperse and crystallizable homodimer with a molecular weight of 30,348.3 Da. The protein was active in both receptor binding competition assay and IL-17A-dependent biological activity assay using human dermal fibroblasts.

Antirecipient helper and cytotoxic T-cell frequencies in bone marrow transplantation

Summary:We assayed helper T-lymphocyte precursor frequencies (HTLPf), interferon (IFN)-γ-producing cell frequencies (IFN-γPf) and CTL precursor frequencies (CTLPf) to see if they could predict the severity of acute graft-versus-host disease (aGVHD) and disease relapse after transplantation. In all, 48 bone marrow transplantation (BMT) patients and their HLA-identical sibling (n=29) or matched unrelated donors (MUD) (n=19) were recruited. HTLPf, IFN-γPf and CTLPf were measured using a limiting dilution assay (LDA). Patients were followed prospectively to assess the severity of aGVHD and the status of the primary disease after BMT. High (>5 × 10−6) HTLPf, CTLPf and IFN-γPf were significantly associated with the occurrence and severity of aGVHD in patients who received transplants from HLA-identical sibling. Among patients receiving BMT from MUD, HTLPf and CTLPf, but not IFN-γPf, were associated with aGVHD. Five patients had disease relapse post-BMT and the risk was not significantly associated with HTLPf, CTLPf or IFN-γPf. Patients with high (>5 × 10−6) HTLPf , IFN-γPf or CTLPf before BMT are at higher risk of developing aGVHD after transplantation from both matched sibling donors and MUD. Whether these parameters can predict disease relapse would have to be investigated with a larger cohort of patients.

CD5+ B-Lymphocytes are as Dependent on T-Helper Cells and as Responsive to T-Suppressor Cells in Pokeweed Mitogen-Stimulated Activation as Conventional CD5− B-Lymphocytes

Pokeweed mitogen (PWM)‐stimulated cultures were established with varying numbers of CD4+ or CD8+ cells and CD5+ immunoglobulin‐secreting cells were subsequently identified using a combination of haemolytic plaque formation and rosetting wilh immunobeads coated with anti‐Leu‐1. Il was found that CD5+ and CD5+ B‐cells required similar numbers of CD4+ cells for half‐maximum plaque‐forming cell (PFC) induction and that 50% suppression of both CD5+ and CD5− PFC occurred with similar numbers of CD8+ cells. Suppression of both CD5+ and CD5− PFC could occur when T‐helper signals were supplied by a PWM‐stimulated culture supernatant, indicating that both subsets could be directly suppressed by CD8+ cells rather than by indirect suppression of T‐helper cells. It is concluded that CD5+ B‐cells are as responsive to T‐regulatory influences as conventional CD5− B‐cells.