Immunoglobulin light chain staining in paraffin-embedded tissue using a heat mediated epitope retrieval method.

We have compared light chain immunohistochemistry in reactive lymphoid tissue and a series of paraffin-embedded B-cell lymphomas using standard trypsin digestion with a heat mediated epitope retrieval method. Fifty-seven B-cell lymphomas (18 high grade, 29 low grade and 10 cases of nodular lymphocyte predominance Hodgkin's disease), two reactive lymph nodes and eight tonsils fixed for known times between 12 h and 2 years were studied. Paraffin-embedded tissue was stained with polyclonal anti-kappa and anti-lambda antibodies. For each antibody staining was performed on two sections, one treated with trypsin digestion and one with microwave heating. Sections were scored from 0 to + + + with 0 representing poor staining and + + + excellent staining. A score of ++ was considered satisfactory. Light chain restriction was recorded if present. Satisfactory staining was obtained in 34/59 cases using trypsin digestion and 56/59 cases using heat retrieval. Light chain restriction was demonstrated in 32/57 (56%) B-cell lymphomas using trypsin digestion and 52/57 (91%) using heat retrieval. Satisfactory staining was obtained in tonsils fixed for up to 48 h using trypsin digestion and up to 2 years using heat retrieval. We have shown that for light chain immunostaining a heat mediated epitope retrieval method produces more consistent and satisfactory results and is effective over a greater range of fixation times than traditional trypsin digestion.

Effect of reconstituted basement membrane components on the growth of a panel of human tumour cell lines in nude mice.

Previous reports have indicated that reconstituted basement membrane (matrigel), when co-injected with either established or primary human tumour cells, can improve the growth of subcutaneous xenografts in nude mice. The human adenocarcinoma cell lines A549, SW480, and WiDr, and the human fibrosarcoma cell line HT1080scc2 exhibit varying degrees of tumourigenicity in nude mice. All these lines showed increased tumorigenicity and/or growth rate, together with a change towards a more differentiated tissue morphology, when co-injected with matrigel into nude mice. Experiments using A549 cell line have indicated that the effect of matrigel is concentration-dependent and that increased growth rate is not maintained when xenografts grown with matrigel are passaged into further mice. These results strongly suggest that increased tumour growth results from the improved growth conditions afforded by matrigel, rather than from the selection of subpopulations of the most tumourigenic cells. Increased growth of intracaecal tumours arising from the co-injection of SW480 cells with matrigel, indicate a possible use for matrigel in the development of more relevant animal models using the orthotopic site. Purified laminin significantly increased the growth of sc tumours resultant from co-injection with either WiDr or A549 cells, whereas collagen IV or laminin with entactin showed no such effect. A role for free laminin in the stimulation of cell growth in the absence of an intact basement membrane is discussed.

Microbiochemical analysis of changes in proteoglycan and collagen in joint tissues during the development of antigen-induced arthritis in the rabbit.

Microbiochemical assays of the proteoglycan and collagen content of articular cartilage and synovial lining have been performed on tissue sections taken from rabbits with antigen-induced arthritis. This experimental arthritis is a close analogue of the natural disease-rheumatoid arthritis. Animals were killed at intervals during the first 21 days following induction of arthritis to assess changes in the composition of the extracellular matrices of the synovial lining and articular cartilage during the early development of this experimental lesion. In confirmation of earlier studies these microbiochemical assays revealed a rapid and significant loss of proteoglycan from the articular cartilage. This loss was, however, not uniform but was restricted to the intermediate zone of the cartilage. Over the period studied, there was only a slight loss of proteoglycan from the superficial zone of the cartilage facing the joint cavity. These findings demonstrate that, at least in this model, cartilage proteoglycan loss is not due to the action of proteases present in the synovial fluid. Moreover it suggests that the chondrocytes in the mid-zone of the cartilage are responsive to those signals stimulating proteoglycan breakdown. There was no significant loss of collagen from the cartilage over the time period of this study. The synovial lining from arthritic joints, in contrast, showed a progressive increase in both proteoglycan and collagen.

Adjuvanticity and ISCOM formation by structurally diverse saponins.

Adjuvant activity and immunostimulating complex (ISCOM) formation by a series of saponins and glycoalkaloids differing in the structures of their aglycones and sugar chains were examined. The only two saponins apart from Quillaia that were adjuvant-active were Gypsophila and Saponaria, which resemble Quillaia in that they contain saponins with branched sugar chains attached to positions 3 and 28 of the aglycone. Glycoalkaloids with a branched sugar chain lacked adjuvant activity. Saponaria saponins formed irregular ISCOM-like structures, and Gypsophila produced a sheet of joined pore-like structures. The alfalfa hederagenin saponin and Quinoa also formed pore-sheets, despite lacking adjuvanticity.

Pathogenesis of antigen-induced arthritis in mice deficient in neutrophil elastase and cathepsin G.

The contribution of neutrophil-derived elastase and cathepsin G to joint pathology has been examined in immune arthritis in the mouse. Neutrophils from beige mice are genetically deficient in lysosomal elastase and cathepsin G, but have normal levels of the acid hydrolases, beta-glucuronidase, and N-acetyl-beta-glucosaminidase. The development of antigen-induced arthritis in normal mice has been compared with that in beige mice. The pattern of synovitis (both leukocyte accumulation and plasma leakage) were indistinguishable in normal and beige mice. Cartilage proteoglycan depletion was quantified by measuring the decrease in safranin O staining intensity, and this, too, was unaltered in mice lacking elastase and cathepsin G. These results suggest that neutrophil elastase and cathepsin G do not contribute to these aspects of joint pathology in antigen-induced arthritis in the mouse.

Collaborative clinical pharmacokinetic services.

A program for routine pharmacokinetic interpretation of serum analyses of gentamicin, tobramycin, amikacin, phenytoin, phenobarbital, theophylline, lidocaine, digoxin, quinidine, and procainamide at the Medical University of South Carolina Hospital is described. Results of all analyses of serum for the drugs listed are evaluated by a pharmacist trained in clinical pharmacokinetics. Patient variables relevant to the determination of drug serum concentrations, drug elimination, distribution, and dosage are given appropriate consideration in each evaluation. A summary of the pharmacokinetic interpretation and any necessary modification of drug dosage regimens are then written into the progress notes of the patients' medical records. Approximately 12 patients and 20 drug concentrations are evaluated each day. The average charge for te service is +35. This service, which is reimbursed by third-party carriers, has resulted in improved use of laboratory personnel, equipment, and time and has provided a framework for education and research as well as a mechanism for direct contributions to patient care by the pharmacist.