Dark immunofluorescence: correlation with serum immunoglobulin abnormalities.

Occasional serum samples (<0.5%) tested by indirect immunofluorescence showed less fluorescence than did negative-control serum. A retrospective review of these patients' serum immunoglobulins revealed a high percentage of abnormalities (71%, versus 22% of controls). We suggest that this observation should be reported when seen and that the clinician should be alerted to an association with immunoglobulin abnormalities.

Differential expression of CD 180 and IgM by B-cell chronic lymphocytic leukaemia cells using mutated and unmutated immunoglobulin VH genes.

We have studied the surface expression of the Toll-like receptor family member CD 180 on cells from 78 patients with B-chronic lymphocytic leukaemia (B-CLL). B-CLL cells had variable levels of CD 180 expression, but this was always less than that expressed by normal blood B cells and was stable for 24 months. Significantly higher levels of CD 180 were expressed by B-CLL cells with mutated IGVH genes compared with those using unmutated IGVH genes. This was in contrast to the higher levels of expression of surface immunoglobulin M by B-CLL cells using unmutated, rather than mutated IGVH genes. CD 180 was functional on B-CLL cells from some of the patients, as shown by the increased expression of CD 86 following incubation in vitro with anti-CD 180. The differential expression of CD 180 amongst B-CLL patients is one more marker that may define more precisely the different biological properties of this heterogeneous disease.

Characterization of drug release from diltiazem-loaded polylactide microspheres prepared using sodium caseinate and whey protein as emulsifying agents.

The influence of milk protein emulsifying agents on the characteristics, particularly drug release, of polylactide microspheres was investigated. Diltiazem loaded polylactide (PL) microspheres were successfully prepared using the dairy proteins, sodium casinate (SC) and whey protein isolate (WPI) as the emulsifying agents. Microspheres were characerized in terms of microsphere yield, electron microscopy, particle size, drug loading, DSC and XRD analysis and drug release. The yields of microspheres obtained were 53-63% and were independent of the emulsifying agent used. SEM revealed that, regardless of the emulsifying agent employed, the microspheres were of good sphericity, but the surface appearance of the microspheres was not the same in all cases. The milk proteins resulted in microspheres approximately half the size of those obtained with methylcellulose (MC). Significant differences in drug loading were observed between the three emulgents, the MC systems giving the highest values. Release profiles were sigmoidal in shape and were well fitted to the equation ln (x/1 - x) = k x t - k x tmax, reflecting degradation controlled drug release. The parameter k increased with drug loading, while tmax decreased. The relationships between the release parameters [P(k and tmax)] and loading (L) could be quantified by equations of the form P = a x L(N), N being negative in the case of tmax. Apart from the effect on loading efficiency, neither SC nor WPI appeared to significantly alter drug release. The quantitative relationships observed in this study may have more general application in quantifying drug release from drug-polymer composites at low loadings where polymer degradation controls drug release.

Purification of monoclonal antibodies using protein a/g.

A major breakthrough in immunology came with the discovery that large amounts of relatively pure monoclonal antibodies (mAbs) could be prepared from the fusion of B cells (secreting the relevant mAb) with a nonsecreting myeloma cell line (1). Since then mAbs have become a central tool for researchers, enabling them to investigate previously unknown molecules. More recently mAbs have also gained an important role in medicine, e.g., in helping to prevent allograft rejection and in the treatment of digoxin poisoning.

Use of 2-[18F]fluoro-2-deoxyglucose as a potential agent in the prediction of graft rejection by positron emission tomography.

BACKGROUND: We investigated the potential of predicting allograft rejection by measuring the ability of graft-infiltrating cells to take up 2-[18F]fluoro-2-deoxyglucose ([18F]FDG). This molecule is a positron emitting glucose analogue that is taken up by metabolically active cells and can be detected using positron emission tomography. METHODS: Uptake of [18F]FDG during an alloresponse was measured both in vitro in mixed lymphocyte cultures and in vivo using allogeneic and syngeneic skin grafts. RESULTS: Uptake of [18F]FDG was seen in a mixed lymphocyte reaction. Using a mouse skin graft model, we found that mean [18F]FDG uptake was 1.5-2 times higher in allografts than in syngeneic grafts; the increase in uptake correlated with the level of T-cell infiltrate seen histologically. CONCLUSION: Assessing the metabolic activity of graft-infiltrating cells with [18F]FDG may be useful in the prediction of graft rejection episodes.

Preparation and evaluation of microspheres prepared from whey protein isolate.

Whey protein (WPI) microspheres were successfully produced containing hydrochlorothiazide, eosin, patent blue violet and sodium salicylate using a w/o emulsification method with glutaraldehyde cross linking. The release of these compounds from WPI microspheres occurred rapidly, with at least 70% of the incorporated material released for all systems within the first 20 min. Release of microsphere payload was essentially complete within 1 h. The degree of glutaraldehyde cross-linking was found to have no effect on the release profile for durations of cross-linking up to 24 h. Of a range of release equations examined, the experimental release data was best described by a biexponential equation and this agrees with the work of Tomlinson et al. (1984) for the release of drugs from albumin microspheres. Swelling of the microsphere systems was examined as this may contribute to the rapid release of drug from these systems.

Indium-111 labelled lymphocytes: isotope distribution and cell division.

Since lymphocytes continue to proliferate and divide in vivo, it is important to determine the fate of a radionulide following lymphocyte labelling. Using the mixed lymphocyte reaction (MLR), we induced indium-111 labelled lymphocytes from a specific in-bred rat strain (AS) to divide and then observed the subsequent 111In distribution between cells and supernatant. L10 and L12.4 cells, which are allospecific CD4+ T lymphocytes from the AS rat, were stimulated in the MLR by antigen-presenting cells from the August rat, a different strain. We labelled L10 or L12.4 lymphocytes on day 0, the first day of the stimulation cycle, and continued to culture the lymphocytes in vitro. The proliferation of the cells was estimated according to their increase in number. The distribution of 111In between cell and supernatant fractions and between viable and dead (but intact) cells was measured in the cell suspension each day after labelling. The metabolic activity of 111In-labelled lymphocytes was compared with control cells by measuring their uptake of fluorine-18 fluorodeoxyglucose ([18F]FDG). 111In-labelled lymphocytes showed a poor proliferative response compared with control cells 24-48 h after labelling but increased in number after this time. From 24 to 72 h, about 70% of 111In was in the supernatant but only about 5%-10% was associated with intact dead cells. These dead cells tended to retain their 111In, losing less than 30% per day, suggesting that 111In in the supernatant was the result of active elimination from viable cells. Moreover, 24 h after culture, considerably more 111In was associated with viable than with dead lymphocytes, although over the next few days this distribution reversed. 111In-labelled lymphocytes took up more [18F]FDG than control cells at 24 h but not at 0 or 72-96 h; the maximum [18F]FDG uptake coincided with the greatest reduction in cell number. Furthermore, [18F]FDG uptake correlated with the initial 111In burden in lymphocytes labelled with 111In 24 h previously. The results are consistent with active elimination of 111In by 111In-labelled lymphocytes. The energy requirements for this are diverted away from cell division, thereby increasing the probability of cell death. As lymphocytes become 111In deplete, they recover their capacity to proliferate and their risk of death decreases. These findings have important implications for 111In-labelled lymphocyte scintigraphy, suggesting that cells remaining viable immediately after labelling will either subsequently die or alternatively eliminate the label.

In vitro analysis of the release of incorporated agents from sodium caseinate microspheres.

Drug loaded microspheres were successfully prepared using the dairy protein, sodium caseinate (SC), as the carrier material and containing hydrochlorothiazide, eosin, patent blue violet and sodium salicylate. The morphology of the microspheres varied depending on the incorporated material. Release of the incorporated agents from these systems was rapid with over 90% release in each case within 1 h. Further drug release occurred at a greatly reduced rate with a certain proportion of the drug loading appearing to be indefinitely entrapped within some of the microspheres. The drug release profiles were poorly described using the square root of time and Sinclair and Peppas equations but were well described by a biexponential equation. Analysis of the parameter estimates from the biexponential equation indicated that the initial rapid release phase was essentially completed within 5 min for all but the eosin microsphere systems and this timeframe was of the same order of magnitude as that found for the initial swelling of these microsphere systems as analysed by swelling studies using optical microscopy. Considering both the swelling study and the results from the release experiments, it seems likely that addition of SC microspheres to aqueous media results in immediate hydration and swelling. This process facilitates the rapid diffusional passage of water soluble drugs through the swollen and hydrated microsphere matrix, with drug release occurring almost unhindered unless other factors favour the retention of the incorporated agent with the microsphere.

Single-chain Fv radioimmunotargeting.

The availability of engineered antibody species has catalyzed new developments in radioimmunotargeting. This chapter summarized recent studies of single-chain Fv (sFv) proteins, which are minimal antibody binding sites engineered as single polypeptide chains. The single-chain Fv can be as small as 26 kDa monomers or may be engineered as larger fusion proteins designed to self-associate into dimeric or multimeric species. They typically exhibit rapid clearance that results in high targeting specificity within a matter of hours. We have compared different modes of administration to allow further manipulation of their biodistribution and targeting properties. Results of the present study comparing intravenous (i.v.) and intraperitoneal (i.p.) administration show comparable long-term retention in circulation, but the i.v. route showed an initially high peak blood level while i.p. injection did not. As with a single sFv dose, repeated bolus injections of sFv attained high target-to-background ratios, whereas continuous sFv infusion reached a steady state level of free sFv in blood and kidney that exceeded that in tumor xenografts. We observed improved localization of radioiodinated sFv in tumor xenografts if the radioiodine label resisted dehalogenation from the protein, which was accomplished, for example, through conjugation of a para-131I-benzoyl group to Iysyl epsilon-amino groups of the protein. Modification of the sFv by genetic incorporation of a cysteinyl peptide (to form sFv') provided a chelation site for radiometals that simplified incorporation of 99mTc with the opportunity for improved diagnostic imaging in cancer and other diseases. Therapeutic applications of sFv radioimmunotargeting could rely on sFv' complexed to 186Re or 188Re. Engineering sFv of sFv' with increased antigen-binding affinity and appropriately manipulating their mode of administration should promote sustained tumor retention conducive to clinically useful therapeutic indices.

The kinetics of MHC class I and class II expression in rat renal allografts.

We have used in vivo localization of radiolabeled antibodies in a rat renal transplant model to compare the level of induction of major histocompatibility complex (MHC) class I and class II molecules in grafts undergoing rejection with grafts in which rejection was modified by cyclosporine (CsA). MHC class II expression increased in rejecting grafts, peaking on day 4, whereas a later rise in CsA-treated grafts was noted. The use of donor-specific antibodies demonstrated that this was due, in part, to a rise in class II of donor origin. No major differences in MHC class I levels were noted between the two groups until after day 4, when very little antibody localization was seen in the rejecting group. Our results suggest that therapeutic doses of CsA may not prevent the upregulation of class II that occurs during rejection, and that levels of class II are not of prognostic value in kidney transplantation.

Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide.

We describe a method to facilitate radioimaging with technetium-99m (99mTc) by genetic incorporation of a 99mTc chelation site in recombinant single-chain Fv (sFv) antibody proteins. This method relies on fusion of the sFv C terminus with a Gly4Cys peptide that specifically coordinates 99mTc. By using analogues of the 26-10 anti-digoxin sFv as our primary model, we find that addition of the chelate peptide, to form 26-10-1 sFv', does not alter the antigen-binding affinity of sFv. We have demonstrated nearly quantitative chelation of 0.5-50 mCi of 99mTc per mg of 26-10-1 sFv' (1 Ci = 37 GBq). These 99mTc-labeled sFv' complexes are highly stable to challenge with saline buffers, plasma, or diethylenetriaminepentaacetic acid. We find that the 99mTc-labeled 741F8-1 sFv', specific for the c-erbB-2 tumor-associated antigen, is effective in imaging human ovarian carcinoma in a scid mouse tumor xenograft model. This fusion chelate methodology should be applicable to diagnostic imaging with 99mTc and radioimmunotherapy with 186Re or 188Re, and its use could extend beyond the sFv' to other engineered antibodies, recombinant proteins, and synthetic peptides.

Identification of a 200-kDa glycoprotein antigen of Saccharomyces cerevisiae.

SDS-PAGE analysis of the soluble proteins of the yeast Saccharomyces cerevisiae revealed a single predominant moiety of apparent size 200 kDa which was strongly periodic acid Schiff-positive. In immunoblotting experiments, human sera containing anti-S. cerevisiae antibodies were reactive only with this glycoprotein (gp200); this was demonstrated using sera containing IgG and IgA isotype-specific anti-S. cerevisiae antibodies. The antigenicity of gp200 was substantially reduced by periodate treatment. These results show that gp200 is a major glycoprotein antigen of S. cerevisiae which is immunogenic in man.