Scintigraphy of acute inflammatory lesions in rats with radiolabelled recombinant human neutrophil-activating peptide-2.

Radiolabelled recombinant human interleukin-8 (IL-8) with its homologue neutrophil-activating peptide-2 (NAP-2) have been compared for imaging acute sterile inflammatory lesions in rats. 125I-IL-8 and 125I-NAP-2 were prepared by reaction with chloramine-T and injected intravenously into male rats bearing subcutaneous carrageenan abscesses in their left hindlimbs. Left hindlimb and right hindlimb activities were determined from serial total-body scintigrams between 1 h and 96 h post-injection as regional per cent injected activity corrected for physical decay (%IA). Time-activity curves for 125I-IL-8 and 125I-NAP-2 in the carrageenan-containing left hindlimbs were similar in that both peaked at 1-3 h post-injection (IL-8, 4.9+/-0.5%IA; NAP-2, 4.8+/-1.9%IA) and decreased exponentially thereafter. However, while the lesioned-to-control limb activity ratio (L/C) for 125I-IL-8 only approximately doubled during the imaging period (1.7+/-0.3 at 1 h vs 3.7+/-1.0 at 24 h post-injection), L/C for 125I-NAP-2 more than tripled, rising from 1.5+/-0.4 at 1 h to 5.3+/-0.7 by 72 h post-injection. It is concluded that while both radiolabelled IL-8 and NAP-2 may prove useful for clinical imaging, radiolabelled NAP-2 may provide better discrimination of inflammatory lesions from normal tissue at later times post-injection.

Scintigraphy of acute inflammatory lesions in rats with radiolabelled recombinant human interleukin-8.

We compared 125I-labelled recombinant human interleukin-8 (125I-IL-8) with 111In-labelled human leukocytes (111In-WBC) and 67Ga-citrate for scintigraphic depiction of acute sterile inflammatory lesions in rats. Radioiodination of IL-8 was catalysed by chloramine-T, and human leukocytes were radiolabelled with 111In-oxine. Inflammatory lesions were induced in male rats by subcutaneous injection of 2% carrageenan suspension into their left hindlimbs. Twenty-four hours later, each rat received 1.8-3.7 MBq (50-100 microCi) of a single agent by intravenous injection. Sequential whole-body scintigrams were obtained between 0 and 96 h post-injection. Activities in the lesion-bearing and control hindlimbs were expressed as regional percent injected activity corrected for physical decay (%IA) by reference to concurrently imaged standards, and for 125I-IL-8 by direct tissue counting at necropsy as well. 125I-IL-8 displayed appropriate electrophoretic mobility, retained chemotactic and high-affinity receptor-binding activity in vitro, and exhibited exponentially decreasing activity in most tissues beginning shortly after intravenous injection. Scintigrams showed asymmetrically increased activity in the lesion-bearing hindlimb for all three agents. By scintigraphy, 125I-IL-8 activity in the lesion-bearing hindlimb reached a zenith 1-3 h post-injection at 4.8 +/- 0.5 %IA and decreased exponentially thereafter, with little change in lesioned-to-control limb ratios (mean L/C = 3.0 +/- 0.7) over the imaging period. By direct tissue counting, abscess-associated mean IL-8 activity per gram of tissue increased to four times that of adjacent muscle and nearly seven times that of contralateral muscle by 24 h post-injection. Lesion-bearing hindlimb 111In-WBC activity also rose rapidly, reaching 4.2 +/- 0.6 %IA by scintigraphy at 3 h and an eventual plateau (maximum of 4.5 +/- 0.4 %IA) by 24 h. 67Ga scintigraphic activity in the lesion-bearing hindlimb peaked briefly at 3-6 h post-injection (9.2 +/- 0.5 %IA) and subsequently declined to a constant level of about 7.5 %IA. However, L/C for 111In-WBC and for 67Ga-citrate each averaged only 1.5 +/- 0.3 over the imaging period, compared with a mean L/C of 1.2 +/- 0.2 for a blood pool radiotracer. We conclude that 125I-IL-8 is rapidly and selectively concentrated in regions of acute inflammation, presumably by high-affinity binding to IL-8 receptors on neutrophils within the inflammatory focus. Radioiodinated IL-8 offers an attractive alternative to 67Ga-citrate and 111In-WBC for early imaging of acute inflammatory lesions, and demonstrates significantly higher target-to-nontarget activity ratios in this model. The potential usefulness of radiolabelled IL-8 for clinical scintigraphy should be evaluated.

Development of an anti-A beta monoclonal antibody for in vivo imaging of amyloid angiopathy in Alzheimer's disease.

We evaluated the efficacy of murine monoclonal antibodies (MAbs) targeted to the A beta amyloid of Alzheimer's disease for development of procedures for the in vivo identification of amyloid angiopathy (AA). MAbs to A beta were prepared and screened for effectiveness in visualizing AA and neuritic plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with technetium-99m (99mTc) using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity toward amyloid-laden blood vessels and neuritic plaques. A highly specific murine MAb, 10H3, was identified and characterized that fulfills criteria necessary for the development of an in vivo diagnostic imaging agent. Toxicity studies in rats showed the MAb to be safe. Biodistribution studies in mice demonstrated desirable properties for use as an imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.

Development of a monoclonal antibody specific for beta/A4 amyloid in Alzheimer's disease brain for application to in vivo imaging of amyloid angiopathy.

We evaluated the efficacy of murine monoclonal antibodies (Mabs) targeted to beta/A4 amyloid for development of procedures for the in vivo identification of amyloid angiopathy (AA) in Alzheimer's disease (AD). Mabs to beta/A4 amyloid were prepared and screened for effectiveness in visualizing AA and senile plaques in postmortem AD brain sections. They were assessed again after enzymatic cleavage to produce Fab fragments and after labeling with 99mTc using a diamide dimercaptide ligand system. Modified and radiolabeled Fab fragments retained activity and specificity towards amyloid-laden blood vessels and senile plaques. A highly specific murine Mab, 10H3, was identified and characterized that fulfills criteria necessary for the development of a diagnostic imaging agent. Expansion and adaptation of these strategies may provide the methods and materials for the noninvasive analysis of AA in living patients, and permit assessment of the contribution of AA to the clinical and pathological features of AD.

Monoclonal antibodies specific for non-glycosylated porcine prolactin and for pituitary porcine prolactin.

Glycosylated and non-glycosylated forms of porcine pituitary prolactin were prepared using Concanavalin A Sepharose chromatography. Anti-prolactin monoclonal antibodies were screened for their ability to distinguish these two forms. One monoclonal antibody (17D9) exhibited high affinity binding for the non-glycosylated form of porcine prolactin, but little or no affinity for the glycosylated form. Using this antibody in conjunction with other monoclonals which equally recognize both forms, we developed immunoassays which can be used to determine the amount of the glycosylated vs. non-glycosylated prolactin in serum or other tissue samples.

Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies.

We identified two monoclonal antibodies that bind spatially distinct epitopes on insulin-like growth factor I (IGF-I). Using these two antibodies, we developed a simultaneous, two-site immunoradiometric assay (IRMA) specific for IGF-I. This IRMA has no detectable cross reactivity with insulin, proinsulin, prolactin, or somatotropin, and less than 2% crossreactivity with IGF-II. The assay response varies linearly with IGF-I concentrations of 0-800 micrograms/L in serum; the detection limit is about 10 micrograms/L. A comparison of 26 IGF-I serum values from the IRMA and from a previously reported IGF-I specific RIA gave a correlation coefficient of 0.96 with no substantial bias (slope = 1.10). IGF-I values for serum, as an aid in assessing growth abnormalities, are easily (only three pipetting steps) obtained in less than 4 h.

Antigen-induced proliferative response to murine thymus cells in vitro.

Thymus cells from 5- to 6-week-old normal (unimmunized) BALB/c mice showed an increased incorporation of [(3)H]thymidine in the presence of 2,4-dinitrophenyl-bovine serum albumin, fluorescein-bovine serum albumin, and bovine serum albumin (BSA) in tissue culture. The concentrations of antigen (BSA and haptenated proteins) required for stimulation were approximately 25- to 50-fold higher than those of the nonspecific mitogen, concanavalin A. In contrast to the stimulation by concanavalin A, which was maximal at 24 to 72 h, the stimulation by antigen was most marked earlier in the culture period (6 to 24 h). The BSA response was diminished to a statistically significant degree (especially at low BSA concentrations) in thymocytes from animals injected 72 h previously with BSA, indicating that the stimulation is immunologically specific.

D-amino acid oxidase induction in the kidneys of germ-free mice.

High levels of d-amino acid oxidase activity were found in the kidneys of conventionally reared mice, whereas the enzyme was absent from the kidneys of most germ-free mice. Injection of d-alanine or monocontamination with Bacillus cereus stimulated d-amino acid oxidase activity in the kidneys of germ-free mice.