Hans I.J. Feitsma

Binding of 99mTc-labelled polyclonal human immunoglobulin to bacteria as a mechanism for scintigraphic detection of infection.

The aim of the present study was to determine whether99mTc-labelled polyclonal human immunoglobulin (99mTc-HIG) binds to bacteria in vitro as well as in vivo. In vitro, the binding of99mTc-HIG to various gram-positive and gram-negative bacteria was determined. In vivo, mice were infected withStaphylococcus aureus Cowan I (protein A rich) orS. aureus EMS (protein A deficient) in a thigh muscle and then99mTc-HIG or99mTc-labelled human serum albumin (99mTc-HSA) was administered; scintigrams were made 1, 4, and 18 h later. In vitro binding of99mTc-HIG to bacteria was higher for gram-positive than for gram-negative forms. A positive correlation was found between the protein A content and the degree of binding toS. aureus. This was also found in vivo. The accumulation of99mTc-HIG at the site of infection was significantly (P < 0.01) higher than that of99mTc-HSA, for both strains ofS. aureus. It is concluded that vascular permeability cannot fully explain the accumulation of99mTc-HIG at the site of infection and that binding of99mTc-HIG to bacteria plays a role in this respect.

Improved detection of a staphylococcal infection by monomeric and protein A-purified polyclonal human immunoglobulin

The present study was untertaken to compare the technetium-99m labelled non-specific polyclonal human immunoglobulin (Ig) with 99mTc-labelled monomeric human immunoglobulin (m-Ig), 99mTc-labelled, protein A-purified, human immunoglobulin (A-Ig) and 99mTc-labelled monomeric, protein A-purified, human immunoglobulin (mA-Ig) as tracer agents for the detection of a thigh infection with Staphylococcus aureus. In vitro the binding of the various tracer agents to bacteria at various intervals was determined. For the in vivo evaluation, mice were infected and received one of the various labelled proteins. Scintigrams were made 0.25, 1, 4 and 24 h later. All 99mTc-labelled Igs bound to bacteria in vitro: the percentages of binding for the m-Ig (from 1 h onwards) and A-Ig and mA-Ig (from 3 h onwards) were significantly higher than that for Ig. The in vivo target-to-non-target (T/NT) ratios were significantly higher from 4 h onwards for all purified Igs than for Ig. Protein A-purifled Igs yielded higher T/NT ratios than m-Ig. Furthermore, the amount of activity in the liver was significantly lower 24 h after administration of m-Ig, A-Ig and mA-Ig than after administration of Ig. It is concluded that in this experimental infection 99mTc-labelled monomeric Ig localizes a staphylococcal thigh infection better and faster than 99mTc-labelled unpurified Ig. However, the accumulation obtained with protein A-purified Ig or protein A-purified monomeric Ig was the highest of all tracer agents tested.

Detection of experimental infections with 99mTc-labeled monoclonal antibodies against TNF-α and interleukin-8

This study was designed to assess monoclonal antibodies (MAbs) directed against tumor necrosis factor-alpha (TNF-alpha) (anti-TNF) or interleukin-8 (anti-IL-8) as radioactive agents for the detection of Staphylococcus aureus-or Klebsiella pneumoniae-infected thighs in mice. At 5 min (acute infection) or 20 h (established) post-infection, 20 micrograms of the 99mTc-labeled MAbs were injected. At various time intervals, the accumulation of the radiotracer in the infected thighs was assessed and expressed as a target-to-nontarget (T/NT) ratio. The binding of 99mTc-labeled MAbs to circulating mononuclear cells and granulocytes was quantitated 20 h after injection. The pharmacokinetics of the MAbs, in relation to the control agents 99mTc-labeled polyclonal human immunoglobulin (IgG) and a 99mTc-labeled nonspecific IgG1 MAb, were also studied. In acute infections, 99mTc-anti-TNF accumulated to a higher extent (p < 0.05) in S. aureus-infected thighs in mice until 4 h after the injection than 99mTc-IgG and was higher at 0.25 h in K. pneumoniae-infected mice (p < 0.03) compared with 99mTc-IgG. In established S. aureus and K. pneumoniae infections, 99mTc-anti-IL-8 detected the infection more intensely than 99mTc-IgG until 1 h after injection. In both S. aureus and K. pneumoniae infections, localization of sites of infection correlates (p < 0.05) with increased binding of the 99mTc-labeled MAbs to granulocytes and mononuclear cells in both acute and established infections. It was concluded that 99mTc-labeled MAbs, directed against TNF-alpha and IL-8, accumulate in bacterial infections in mice to a higher extent than does 99mTc-IgG after infection and is related to the binding of the antibodies to blood leukocytes. With these 99mTc-labeled MAbs, information might be gained about the development of an infection.

Contribution of phagocytic cells and bacteria to the accumulation of technetium-99m labelled polyclonal human immunoglobulin at sites of inflammation

The purpose of this study was to assess the contribution of phagocytic cells and bacteria to the accumulation of technetium-99m labelled polyclonal human immunoglobulin (HIG) at sites of inflammation. Mice were intraperitoneally injected withStaphylococcus aureus (SA animals), with heat-inactivated newborn calf serum (NBCS, to mimic a non-bacterial inflammation) or with physiological saline (controls); 1 h thereafter they received HIG. At various intervals after the administration of HIG the mice were killed, and the percentages of radioactivity in the peritoneal effluent and attached to the cellular and bacterial fraction thereof were established. Furthermore, the total number of cells and that of bacteria in the fluid were quantitated. The percentage of activity in the effluent in the SA animals was (P<0.02) higher than those in the NBCS-injected animals and controls from 4 h onwards. In all groups of mice this percentage was highest at 4 h and decreased (P<0.01) afterwards. The percentage of cell-bound activity and the total number of cells remained fairly constant or increased with time in the SA animals (P<0.01). The bacteria-bound activity remained rather constant throughout the experiment and ranged between 4% and 6%. In the SA-infected animals the percentage of cell-bound activity was correlated with the total number of cells (macrophages but especially neutrophils) but even more strongly with the number of cell-associated bacteria. In the NBCS-injected animals a correlation was demonstrated between the cell-bound activity and the total number of cells (only neutrophils). It is concluded that in both experimental inflammations, phagocytic cells, and especially neutrophils, contributed significantly to the accumulation of label at the site of inflammation. Their impact on this localization is augmented by the phagocytosis of bacteria.

Optimized localization of bacterial infections with technetium-99m labelled human immunoglobulin after protein charge selection

To improve the scintigraphic detection of bacterial infections a protein charge-purified fraction of polyclonal human immunoglobulin was applied as a radiopharmaceutical. This purification was achieved by attaching the immunoglobulin to an anion-exchanger column and by obtaining the column-bound fraction with buffer. The binding to bacteria in vitro and the target to non-target ratios of an experimental thigh infection with Staphylococcus aureus or Klebsiella pneumoniae in mice were evaluated to compare the purified and the unpurified immunoglobulin. The percentage of binding to all gram-positive and gramnegative bacteria used in this study was significantly (P<0.03) higher for the purified than for the unpurified immunoglobulin. For the in vivo study, mice were infected in the thigh muscle with Staph. aureus or K. pneumoniae. After 18 h 0.1 mg of technetium-99m labelled polyclonal immunoglobulin or 99mTc-labelled protein charge-purified polyclonal human immunoglobulin was administered intravenously. At all time intervals the target (infected thighs) to non-target (non-infected thighs) ratios for both infections were significantly higher (P<0.03) for protein charge-purified polyclonal immunoglobulin than for unpurified polyclonal human immunoglobulin. Already within 1 h the infected tissues could be detected by the purified immunoglobulin. It is concluded that 99mTc-labelled protein charge-purified immunoglobulin localizes both a gram-positive and a gram-negative thigh infection more intensely and faster than 99mTc-labelled unpurified immunoglobulin.