Robert A. Lindemann

Serum antibody responses to indigenous oral mucosal antigens and selected laboratory-maintained bacteria in recurrent aphthous ulceration☆

Sera from subjects with recurrent aphthous ulceration (RAU) and control subjects were tested for relative levels of IgM, IgG, and IgA antibodies against eight selected laboratory-maintained bacteria, including Streptococcus sanguis which has been implicated in the etiopathogenesis of RAU. There were no differences in relative serum antibody levels for any isotype against any bacteria between control and RAU groups. RAU subjects with active lesions were then paired with control subjects, and each serum was tested against sedimentable material derived from the oral mucosa of each pair member. The analysis of data indicated that RAU and control subjects had similar levels of serum antibodies to indigenous mucosal antigens, but RAU subjects had significantly less antigenic material than control subjects.

Generation of lymphokine-activated killer cell activity from non-NK precursor cells

It is possible to generate high levels of lymphokine-activated killer (LAK) activity in short-term culture from cells enriched for natural killer (NK) activity. To determine whether LAK activity can also be generated from non-NK cells, we have depleted peripheral blood lymphocytes (PBL) of NK cells prior to culture with IL-2. NK activity in PBL is correlated with the intensity of staining with the lysosomotropic vital dye quinacrine. Quinacrine dim PBL, which are devoid of lytic NK cells, are capable of developing LAK activity following culture with IL-2. We have also separated PBL using the NK-associated NKH-1 marker. Depleting NKH-1+ cells eliminates NK activity but the ability to develop LAK activity is retained. NKH-1-depleted cells generate less LAK activity than unseparated or NKH-1-positive cells and do not proliferate as well as unseparated cells to IL-2. When NK-depleted cells are subsequently examined for the expression of the NKH-1 antigen, this marker is absent from most cells at Day 3 of IL-1 culture, but is expressed on an increasing number of cells by Days 6-8. These results suggest that LAK derived from non-NK cells is functionally and phenotypically similar to LAK from PBL-containing NK cells, and may be the result of the activation of an NK precursor population.

Phagocyte-mediated killing of Candida tropicalis.

Human peripheral monocytes (MO), neutrophils (PMN), and lymphocytes (PBL) were tested for their ability to kill Candida tropicalis. With incubation times between 30 min and 2 h, unstimulated MO and PMN, but not PBL, were efficient killers of C. tropicalis. Both leukocyte subsets were able to kill at minimum 2.5∶ 1 effector to target ratios. Pre-incubation of MO for 24 h with interferon-gamma or tumor necrosis factor (TNF) increased their ability to kill yeast targets. TNF alone had no effect on C. tropicalis targets at concentrations up to 1000 U/ml. PBL activated for 4 d with interleukin-2 did not kill yeast targets. PMN exhibited more cytocidal efficiency per cell than MO in these assays. Direct contact of effectors and targets was required; no significant killing by PMN or MO supernatants was measured. PMN-mediated killing, but not MO killing, was inhibited by a mixture of catalase and Superoxide dismutase suggesting that oxygen-dependent killing mechanisms were partially responsible for candidacidal activity.

The effects of staphylococcal protein A on human lymphokine-activated killer cell induction

SummaryStaphylococcal protein A (Cowan strain; SpA), a biologically active molecule capable of inducing augmented natural killer (NK) cell cytotoxicity, was studied in regard to its effects on lymphokine-activated killer (LAK) cell development. SpA, when co-cultured with interleukin-2 (IL-2) for 4 days, significantly augmented both LAK activity against NK-resistant M14 (melanoma) target cells and DNA synthesis of peripheral blood mononuclear cells (PBMC). This enhancement occurred with SpA concentrations of 1–100 µg/ml in a dose-dependent fashion; concentrations above 100 µg/ml were no more effective. When SpA (10 µg/ml) was added to PBMC cultures with various IL-2 concentrations, cytotoxicity was increased over controls with IL-2 alone. The peak cytotoxic effect reached a plateau at 80 U/ml IL-2. SpA alone induced early (day 1) cytotoxicity, which rapidly declined. SpA alone did not induce PBMC proliferation but it did increase expression of CD25 (Tac), IL-2 receptor α chain, on CD56(Leu 19)-positive and -negative cells. The potentiating effect of SpA was significantly enhanced in serum-free medium. If either human AB serum or human IgG was added to cultures SpA-enhanced LAK cytotoxicity was diminished. The addition of anti-interferon γ (anti-IFNγ) antibody, but not anti-IFNα, inhibited (SpA+IL-2)-induced cytotoxicity, indicating that IFNγis partially responsible for the additive cytotoxic effect.

Oral mucosal antigen reactivity during exacerbation and remission phases of recurrent aphthous ulceration

Pooled serum from subjects with active recurrent aphthous ulceration (RAU) and from control subjects was tested against indigenous surface mucosal material from subjects with active RAU, remission RAU subjects, and normal subjects. IgM antibody reactivity in both sera was significantly higher to material from remission RAU subject mucosa than to material from control subjects or subjects with active RAU. IgG antibody activity in both sera was significantly lower to material from active RAU subject mucosa than to material from control subjects or subjects whose RAU was in remission. These results suggest that the character and amount of antigen on mucosa may differ during the pathogenesis of RAU.

Application of a new method for detecting the phenotype of target binding cells.

SummaryA new method was developed for detecting the phenotype of target binding cells (TBC) in a single-cell assay system. This methodology was evaluated during a clinical trial of recombinant interferon alfa-2a (rIFN alfa-2a) for the treatment of 10 metastatic renal cell carcinoma patients. Total TBC with K562 targets, HNK-1+ TBC, and HLA-DR+ TBC were quantitated during rIFN alfa-2a therapy. A significantly increased proportion of lymphocytes bound to target cells on day 9 of therapy bore the HNK-1 marker. This proportion subsequently declined to pretreatment levels. Total TBC paralleled the rise and fall in HNK-1+ TBC. HLA-DR+ TBC binding to targets remained constant and low throughout therapy. These findings suggest that rIFN alfa-2a early in therapy (day 9) caused the recruitment of additional HNK-1+ cells into binders. However, with continued therapy, this proportion reverts to pretreatment levels. The results of this clinical trial served to illustrate the ability of the modified single-cell assay system to detect TBC phenotype.