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Severe acute respiratory syndrome (SARS) is a new disease which has spread rapidly and widely. We wished to know whether evaluation of in vitro cytokine production could contribute to improved understanding of disease pathogenesis and to better patient management. Numbers of unstimulated and mitogen-stimulated cytokine-secreting peripheral blood mononuclear cells were measured repeatedly during and after hospitalization in 13 patients with SARS using enzyme-linked immunospot technology. Numbers of interferon-gamma, interleukin (IL)-2, IL-4, IL-10 and IL-12 secreting cells induced by T cell activators were below normal in many or most patients before and during treatment with corticosteroids and ribavirin but returned essentially to normal after completion of treatment. Staphylococcus aureus Cowan 1 (SAC)-stimulated IL-10 secreting cells were increased in early SARS but fell during treatment. SAC-induced IL-12 secreting cells were deficient before, during and long after treatment. Numbers of cells induced to produce IL-6 and tumour necrosis factor-alpha by T cell or monocyte activators were higher than normal in many early SARS patients and were still increased in some during and after treatment. We conclude that prolonged dysregulated cytokine production occurs in SARS and that future studies should be directed at improving anti-inflammatory and antiviral therapies in order to limit cytokine impairment.

In order to establish normal reference ranges of serum immunoglobulin G subclasses for different age groups in Chinese, we measured the four IgG subclasses by radial immunodiffusion using polyclonal antisera in 350 normal healthy subjects (148 males and 202 females) recruited from the community. There was no significant sex difference for all the four IgG subclasses. Using Box-Cox transformation, we constructed smooth age-dependent percentile curves for the four IgG subclasses. For the 50th percentile values, the plateaus for IgG1 to IgG4 are respectively 970 mg/dl at 13 years old, 481 mg/dl at 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 IgG1 level also plateaued later and at a higher level than that in other studies.

T-suppressor function and T-helper function in healthy adults, elderly patients with non-immune diseases, and patients with rheumatoid arthritis (RA) and IgA nephropathy (IgAN) were titrated by adding graded concentrations of CD8+ cells to autologous CD8-depleted peripheral blood mononuclear cells (PBMC), or CD4+ cells to CD8- 4- PBMC, respectively. Following culture with pokeweed mitogen (PWM), numbers of CD5+ and CD5- immunoglobulin-secreting cells were determined using a combination of rosetting with anti-CD5-coated Dynabeads and reverse haemolytic plaque formation (Jones, 1990). Of 11 RA patients studied, eight had slightly reduced suppressor activity for CD5+ and CD5- IgM-secreting cells, and three with active disease and high serum levels of C-reactive protein, could not suppress IgG, IgA or IgM secretion by either B subset. Helper activity for both CD5+ and CD5- B cells was slightly but significantly increased in RA patients. One of eight patients with IgAN could not suppress IgG, IgA or IgM production by CD5+ or CD5- B cells, and all IgAN patients required strikingly fewer CD4+ cells for PWM-induced activation of CD5+ and CD5- B cells than controls. It was concluded that in two immunologically mediated diseases in which some patients have raised numbers of circulating CD5+ B cells, aberrant T-regulation affects CD5+ and conventional CD5- B cells equally.

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 patient's 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 lectins, including phytohaemagglutinin, concanavalin A and PWM, but markedly defective to OKT3.

OKT4+ (T helper/inducer) and OKT8+ (T cytotoxic/suppressor) subsets were depleted from peripheral blood lymphocytes (PBL) by complement-mediated lysis and residual cells examined for responsiveness to pokeweed mitogen (PWM) using a protein A haemolytic plaque assay for immunoglobulin secreting B cells. It was shown that: (1) three cycles of cell killing were required to totally abolish T helper function; (2) OKT4- PBL did not respond to PWM, but in a co-culture system, an equal number of unfractionated normal PBL could entirely reconstitute responsiveness of the residual B cells; (3) OKT8- PBL gave enhanced numbers of PWM-induced plaque forming cells (PFC); (4) addition of 4 micrograms/ml concanavalin A (con A) to PWM stimulated OKT8- PBL failed to suppress PFC generation, but suppression was induced by 12.5 and 25 micrograms/ml con A and (5) kinetics of PWM-induced PFC development were similar in the presence or absence of OKT8+ cells.

Pokeweed mitogen (PWM)-induced B-cell activation was entirely abrogated by pre-treating peripheral mononuclear cells (PBM) with T4 (anti-T-helper-inducer subset) or T11 (anti-sheep erythrocyte receptor, ie. anti-pan-T-cell) monoclonal antibody plus complement. Immunoglobulin secretion was restored in T11- but not T4- cultures by adding the culture supernatant from PWM-stimulated PBM (T-replacing factor, TRF). Since T8+ (T-suppressor-cytotoxic subset) cells were present in high concentrations in T4- but absent from T11- cultures, it appeared that these cells could inhibit B-cell activation by PWM plus TRF and that suppression could occur in the absence of T4+ cells. Incubation of TRF with purified, unstimulated T8+ cells prior to addition to T11- cultures abrogated subsequent PWM-induced B-cell activation, whereas T8- PBM had no effect on TRF. T8+ cells did not secrete suppressor factors during incubation with TRF. Absorption of one or more of the factors required for B-cell growth and differentiation by T8+ cells appears to be an important suppressor mechanism operating in the PWM system.

Using the protein A haemolytic plaque assay (PrA-HPA) and standardized, optimal conditions, pokeweed mitogen (PWM) stimulated peripheral blood lymphocytes (PBL) from normal donors produced 5,500-48,000 (mean 16,955) IgG plaque-forming cells (PFC) per 10(6) lymphocytes, 3,375-38,000 (mean 14,179) IgA-PFC/10(6) and 5,500-69,750 (mean 18,684) IgM-PFC/10(6). Addition of 4 micrograms/ml concanavalin A (Con A) at the start of PWM stimulation of PBL from normal donors gave 59-98% (mean 84%) suppression of IgG-PFC, 46-99% (mean 79%) suppression of IgA-PFC and 31-100% (mean 82%) suppression of IgM-PFC. When PFC/10(6) values in PWM-stimulated co-cultures of unfractionated PBL from pairs of unrelated normal donors were compared with PFC/10(6) obtained in the individual PWM-stimulated cultures, observed/expected ratios were between 0.81 and 1.24 (mean 0.99) for IgG-PFC, 0.85 and 1.23 (mean 0.98) for IgA-PFC and 0.86 and 1.25 (mean 1.03) for IgM-PFC. Cord blood lymphocytes (CBL) produced few PWM-stimulated PFC and co-culture of CBL with normal PBL gave markedly reduced observed/expected ratios, indicating hyperactivity of T-suppressor cells in cord blood. PBL from a patient with partial DiGeorge syndrome also responded poorly in the PWM-driven PrA-HPA, but co-culture of this patient's PBL with normal PBL and PWM gave rise to higher than expected values, indicating reduced T-helper cell function. These simple methods could be employed in clinical evaluation of the cellular defect in humoral immunodeficiency and autoimmune disease states.