Recombinant variants of antibody 138H11 against human gamma-glutamyltransferase for targeting renal cell carcinoma.

The monoconal antibody 138H11 recognizes human renal gamma-glutamyltransferase (GGT), an antigen shown to allow targeting primary and metastatic renal cell carcinoma (RCC). We determined the primary structure of the antigen binding region of mAb 138H11 and generated several different recombinant antibody variants. First, monomeric single-chain Fv antibody fragments, diabodies and triabodies were obtained by constructing linker variants consisting of 18, 10, 8, 5, 3, 2, 1 and zero amino acid residues, resulting in significant differences in yield and molecular architecture. Second, two variants of disulphide bond-stabilised Fv fragments (dsFvs) were generated using two different pairs of complementary framework amino acid positions of VH and VL for disulphide stabilisation. The binding activities of diabodies to human GGT located on its tissue decreased when using shorter linker lengths. The scFv dimer containing a 3 amino acid residue linker peptide and one of the dsFv variants were not functional. Further, the work paves the way for generating new effector constructs and a systematic optimisation of the pharmacokinetics of this tumor targeting antibody by offering variants with a broad range of valencies and molecular masses.

1-alpha-calcidol modulates major human monocyte antigens and toll-like receptors TLR 2 and TLR4 in vitro.

Since vitamin D derivatives are known to interfere with the cellular immune response, we analysed the possible effect of 1-alpha-calcidol (AC) on major monocyte antigens CD14 (an endotoxin receptor), HLA-DR, and toll-like recptors 2 and 4 (TLR2, TLR4). Peripheral blood monocytes were isolated from healthy donors and cultured by standard protocol followed by incubation with various concentrations of AC in unstimulated and LPS-activated cells. After 24, 48 and 72 hours cells were harvested and analysed for the expression of antigens by flowcytometry. Compared to the controls AC increased the expression of CD14 in a dose and time dependent manner (after 72 hours culture time p < 0.01). AC was capable of further stimulating CD14 expression in LPS activated monocytes (p < 0.05). Both LPS and AC downmodulated HLA-DR dramatically after 24 (p < 0.05), 48 (p < 0.01) and 72 hours (p < 0.0001). The expression of TLR2 but not of TLR4 was inhibited by 10-7M AC. The data reveal that AC significantly modulates the expression of CD14, HLA-DR as well as of TLR2, all involved as targets and effector molecules in antigen recognition and processing, relevant to overcome infections and organ lesions.

Proinflammatory blood monocytes: main effector and target cells in systemic and renal disease; background and therapeutic implications.

Although peripheral blood monocytes are heterogeneous, they all express the CD14 molecule, a multifunctional receptor, and part of the toll-like membrane receptor complex. In healthy persons, a minor subset (8%) coexpresses CD14 and CD16, a low affinity Fc-gamma type III receptor. This subpopulation shows characteristics of tissue macrophages and expands greatly in acute and chronic infections, systemic inflammatory syndromes, hyperlipidedemia, AIDS and renal failure. CD14+/CD16+ monocytes (Mo) exhibit higher phagocytosis activity than CD14++/CD16-negative monocytes and synthesize high levels of interleukin-1, TNF-alpha and HLA-DR, -DP and -DQ antigens. Glucocorticoids (and interleukin-4), and successful therapy in patients with inflammatory and septic complications lead to a down-regulation in the expression of CD14 and deplete CD14+/CD16+-proinflammatory Mo. Recovery of low monocytic HLA-DR expression parallels clinical improvement. Serial analyses of Mo phenotypes may be useful tools for monitoring patients receiving immunosuppressive and anti-inflammatory therapy respectively.

Haemodialysis monocytopenia: differential sequestration kinetics of CD14+CD16+ and CD14++ blood monocyte subsets.

In peripheral blood the majority of circulating monocytes present a CD14highCD16- (CD14++) phenotype, while a subpopulation shows a CD14lowCD16+ (CD14+CD16+) surface expression. During haemodialysis (HD) using cellulosic membranes transient leukopenia occurs. In contrast, synthetic biocompatible membranes do not induce this effect. We compared the sequestration kinetics for the CD14+CD16+ and CD14++ monocyte subsets during haemodialysis using biocompatible dialysers. Significant monocytopenia, as measured by the leucocyte count, occurred only during the first 30 min. However, remarkable differences were observed between the different monocyte subsets. CD14++ monocyte numbers dropped to 77 +/- 13% of the predialysis level after 15 min, increasing to > or = 93% after 60 min. In contrast, the CD14+CD16+ subset decreased to 33 +/- 15% at 30 min and remained suppressed for the course of dialysis (67 +/- 11% at 240 min). Approximately 6 h after the end of HD the CD14+CD16+ cells returned to basal levels. Interestingly, the CD14+CD16+ monocytes did not show rebound monocytosis while a slight monocytosis of CD14++ monocytes was occasionally observed during HD. A decline in CD11c surface density paralleled the sequestration of CD14+CD16+ monocytes. Basal surface densities of important adhesion receptors differed significantly between the CD14+CD16+ and CD14++ subsets. In conclusion, during HD the CD14+CD16+ subset revealed different sequestration kinetics, with a more pronounced and longer disappearance from the blood circulation, compared with CD14++ monocytes. This sequestration kinetics may be due to a distinct surface expression of major adhesion receptors which facilitate leucocyte-leucocyte, as well as leucocyte-endothelial, interactions.

Targeted therapy of experimental renal cell carcinoma with a novel conjugate of monoclonal antibody 138H11 and calicheamicin thetaI1.

In search for a new therapeutic approach for metastasized renal cell carcinoma (RCC), we evaluated the cytotoxicity of a novel prodrug chemoimmunoconjugate with monoclonal antibody (mAb) 138H11 and the DNA-cleaving enediyne calicheamicin thetaI1 (Camtheta) in vitro and in vivo. Previously, mAb 138H11, produced against human renal gamma-glutamyltransferase, stained over 99% clear cell and papillary RCC on frozen sections, showing a membranous expression of the target antigen. In contrast, in normal kidneys gammaGT was restricted to the brush-border in the lumen of proximal tubules and not accessible to the circulation. Thus, human tumor-bearing kidneys perfused in an extra-corporeal system with 99mTc-138H11 revealed a high, specific uptake into the tumor. In this study, fluorescence-activated cell sorting analysis showed binding of mAb 138H11 to RCC cell lines, whereas squamous cell carcinoma lines, fibroblasts, and the murine RENCA were negative. XTT cell proliferation assays revealed efficient killing of the Caki-1 cell line by the 138H11-Camtheta conjugate using SPDP (EC50 = 5 x 10(-11) M) as a covalent linker. For in vivo testing, five groups of eight nude mice each were injected with 2.5 x 10(6) Caki-1 cells s.c. and treated with the following: (a) PBS; (b) 138H11; (c) Camtheta; (d) a mixture of 138H11 and Camtheta; and (e) 138H11-Camtheta conjugate. Treatment started on day 1 after tumor induction and was repeated three times. The data show a highly significant inhibition of tumor growth with the 138H11-Camtheta conjugate versus PBS (P = 0.004). Only mice treated with 138H11-Camtheta showed a tumor shrinkage to minimal residues. In a second experiment, lower doses of the 138H11-Camtheta conjugate were compared with an antineuroblastoma mAb (ch14.18), confirming targeted killing of RCC by the 138H11-Camtheta conjugate at tolerable toxicity in vivo. In conclusion, these combined results encourage further studies for targeted therapy of metastatic RCC with mAb 138H11 conjugates.

Induction of RANTES, HLA-DR, and intercellular adhesion molecule-1 on highly purified distal tubular cells from human kidney.

BACKGROUND: Expression of proinflammatory molecules by tubular epithelial cells plays an important role in renal allograft rejection and inflammatory kidney diseases. Different studies from patients with acute rejection point to the involvement of distal tubular segments. At present no in vitro system for the human distal tubule is established. METHODS: Human distal tubular cells were isolated immunomagnetically. Cultured cells were stimulated with cytokines (interferon-gamma, tumor necrosis factor-alpha, interleukin-1beta, or a cytokine mix). Secretion of RANTES (regulated upon activation, normal T-cell expressed and secreted) was evaluated with an enzyme-linked immunoassay. Expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 was assessed by flow cytometric analysis and immunofluorescence studies. RESULTS: Our data clearly indicate that distal tubular cells express RANTES, HLA-DR, and ICAM-1 in response to a mixture of specific cytokines. Dexamethasone inhibited the induced expression of RANTES and HLA-DR significantly, but not that of ICAM-1. CONCLUSIONS: We demonstrate an appropriate in vitro system for the human distal tubule. The present study proves the involvement of the distal tubular segment during inflammatory kidney diseases.

Effects of mycophenolic acid on human renal proximal and distal tubular cells in vitro.

BACKGROUND: Mycophenolic acid has been shown to be effective for the prevention and treatment of renal allograft rejection. Rejection episodes were found to be associated with an infiltration of lymphocytes and macrophages/monocytes into the diseased kidney. Expression of RANTES, HLA-DR and ICAM-1 may be important for the pathogenesis of this leukocyte infiltration. Therefore the aim of this study was to evaluate the effect of the antiproliferative and immunosuppressive agent mycophenolic acid (MPA) on cell growth and cytokine-induced expression of RANTES, HLA-DR and ICAM-1 of highly purified proximal (PTC) and distal tubular cells (DTC) from human kidney. METHODS: Human PTC and DTC were cultured in the presence of different concentrations of MPA (0.25-50 microM) or MPA plus guanosine (100 microM). Total cell number (DNA content) was determined after 4 days of cell culture by a non-radioactive fluorescence assay. Cells were stimulated by a combination of cytokines (IL1beta+gammaIFN+TNFalpha=cytomix) or cytomix plus MPA. Secretion of RANTES protein was evaluated with an enzyme-linked-immunosorbent assay. Cell surface expression of HLA-DR and ICAM-1 was assessed by flow cytometric analysis. RESULTS: MPA inhibited cell growth of PTC and DTC in a dose-dependent manner. This effect was totally abolished by the addition of guanosine. Cytokine-induced RANTES expression was synergistically increased in the presence of MPA, an effect that was partially prevented by the addition of guanosine. Cytokine stimulation resulted in de novo expression of HLA-DR and a marked increase of ICAM-1 expression, which was partially inhibited by dexamethasone. Addition of MPA did not influence this stimulated expression. CONCLUSIONS: We demonstrate that MPA has an effect on cell growth and chemokine release of tubular epithelial cells, and that these effects are dependent on the inhibition of cellular guanosine production. The clinical consequences of this possible pro-inflammatory effect of MPA on RANTES release may be abolished by a concomitant treatment with steroids.

Transdifferentiation of distal but not proximal tubular epithelial cells from human kidney in culture.

Human renal proximal and distal (thick ascending limb and early distal convoluted tubule) epithelial cells have been isolated according to their specific antigen expression. The cells were well characterized by flow cytometry, enzyme cytochemistry and electron microscopy and cultured for up to 3 months. Cultured tubular cells coexpressed cytokeratin and vimentin as intermediate filament proteins. While primary isolated cells, proximal as well as distal, revealed the phenotypic characteristics of their nephron origin, cultured distal cells showed the tendency to dedifferentiate/transdifferentiate. Distal cells lost their characteristic expression of Tamm-Horsfall glycoprotein and started de novo expression of the proximal marker proteins aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV. The expression of these antigens by distal cells could be shown by flow-cytometric analysis and fluorescence microscopy. Enzyme activity assays revealed the activity of aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV, but not of the proximal marker enzyme alkaline phosphatase. This antigenic shift could not be prevented in different culture media, and the original phenotype could not be restored. Cultured cells displayed characteristic hormonal stimulation patterns indicative of their proximal and distal origins, as shown by activation of adenylate cyclase by different peptide hormones. These results indicate that distal tubular cells possibly transdifferentiate to a more proximal phenotype in view of loss of the distal marker enzyme Tamm-Horsfall protein and de novo expression of proximal marker enzymes like dipeptidyl peptidase IV and aminopeptidase M.

CD14++ monocytes, CD14+/CD16+ subset and soluble CD14 as biological markers of inflammatory systemic diseases and monitoring immunosuppressive therapy.

The majority of peripheral blood monocytes strongly positive for the lipopolysaccharides (LPS)-receptor CD14 are negative for Fcgamma receptor type III (CD16). However, a subset of monocytes coexpressing CD14 and CD16 accounts for about 8% of all monocytes. This population exhibits features of tissue macrophages, and is largely expanded (> 20%) during acute and chronic inflammatory diseases including cases with pararheumatic systemic vasculitis. In addition, compared to normal controls, soluble CD14 (sCD14) is elevated (> 3 microg/ml) in serum specimens of these patients. CD14+/CD16+ monocytes show a higher phagocytosis rate than CD14+/CD16 negative cells, and express higher levels of interleukin-1 and major histocompatibility complex, such as histocompatibility antigens HLA-DR, -DP and -DQ antigens. Glucocorticoids downregulate expression of CD14 and rapidly deplete CD14+/CD16+ monocytes from peripheral blood. Patients under chronic immunosuppressive therapy exhibit low CD14/+/CD16+ rates, which may rise during infectious and non-infectious inflammatory complications, however. Thus, serial analyses for sCD14 and the proinflammatory CD14+/CD16+ subset of monocytes suggest a valuable tool monitoring patients under immunosuppressive and/or antiinflammatory therapy.

[Chronic pyelonephritis. Synopsis of laboratory values and ultrasound lead to diagnosis]. / Chronische Pyelonephritis. Synopse von Labor und Ultraschall führt zur Diagnose.

Bacteria or their antigens persisting in the kidneys may induce the classical type of chronic pyelonephritis (CP), which progresses slowly, and may finally result in end-stage renal failure requiring dialysis. Pyelonephrogenic strains enter uro-epithelial and renal epithelial cells--where they accumulate--or may invade the renal interstitium. Promoting factors are obstruction, reflux, urolithiasis, nephrolithiasis, pregnancy, diabetes mellitus, humoral and cellular immunodeficiencies, immunosuppression treatment (e.g. following transplants), autoimmune phenomena (antigenic mimicry). Therapy comprises the treatment of underlying disease, antibiotics as indicated by the resistogram, acidification of urine (L-methionine), i.v. immunoglobulins (IgM, IgG) and oral vaccination with lyophilized uropathogens.

[When lipids increase in dialysis: the role of heparin! Standard heparin increases, low-molecular-weight heparin lowers triglycerides]. / Wenn bei der Dialyse die Lipide steigen: Auf's Heparin kommt's an! Standardheparin erhöht, niedermolekulares Heparin senkt Triglyzeride.

Dyslipoproteinemia in patients on hemodialysis is characterized by a decrease in high density lipoprotein (HDL), cholesterol, hypertriglyceridemia, increased triglyceride-rich lipoproteins, such as very low density lipoprotein (VLDL) and intermediate density lipoprotein (IDL), a higher proportion of the small dense low density lipoprotein (small dense LDL) subfraction, and higher lipoprotein(a) concentration. The reason for the changes in triglyceride metabolism is an increase in the production of apolipoprotein B, and a decrease in the metabolism of VLDL as a consequence of decreased endothelial cell delipidation. The endothelial lipoprotein lipase, which plays a major role in this process, is released by heparin, which is essential for the function of the enzyme. Repeated administration of heparin for anticoagulation during hemodialysis apparently leads to an LPL depletion in the endothelium. This results in further exhaustion of lipolysis. Clinical studies in hemodialysis patients with high triglyceride and cholesterol levels indicate that a change from standard heparin to low-molecular-weight heparin improves the lipid profile by lowering triglycerides and cholesterol.

Renal failure in giant cell vasculitis.

A 59-year-old white woman with temporal arteritis developed progressive renal failure. Renal biopsy results showed focal and segmental necrotizing glomerulonephritis; furthermore, giant cells were present in the destructed vessel walls. Immunosuppressive therapy did not prevent terminal renal failure. This case shows that renal involvement may be a feature of temporal arteritis.

Expanded CD14+ CD16+ monocyte subpopulation in patients with acute and chronic infections undergoing hemodialysis.

Infections are frequent complications in end-stage renal failure patients undergoing hemodialysis (HD), and peripheral blood monocytes are important cells in host defense against infections. The majority of circulating monocytes express high levels of lipopolysaccharide receptor antigen CD14 and are negative for the immunoglobulin Fcgamma receptor type III (CD16). We studied the occurrence of a minor subpopulation coexpressing low levels of CD14 together with CD16 in HD patients. In healthy controls CD14+ CD16+ monocytes account for 8% +/- 4% of CD14+ monocytes, with an absolute number of 29 +/- 14 cells/microl. In stable HD patients the CD14+ CD16+ subpopulation was significantly elevated (14% +/- 3%, or 66 +/- 28 cells/microl), while the number of CD14(++) monocytes (monocytes strongly positive for CD14) remained constant. In HD patients suffering from chronic infections a further rise in CD14+ CD16+ monocytes was observed (128 +/- 71 cells/microl; P < 0.01) such that this subpopulation constituted 24% of all blood monocytes. In contrast, numbers of CD14++ cells did not change compared to those for stable HD patients, indicating that the CD14+ CD16+ monocyte subpopulation was selectively expanded. During acute infections the CD14+ CD16+ cell subpopulation always expanded. A whole-blood assay revealed that CD14+ CD16+ monocytes exhibited a higher phagocytosis rate for Escherichia coli bacteria than CD14++ monocytes, underlining their role during host defense. In addition, CD14+ CD16+ monocytes expressed higher levels of major histocompatibility complex (MHC) class II antigens (HLA-DR, -DP, and -DQ) and equal amounts of MHC class I antigens (HLA-ABC). Thus, CD14+ CD16+ cells constitute a potent phagocytosing and antigen-presenting monocyte subpopulation, which is expanded during acute and chronic infections commonly observed in chronic HD patients.

Nephrotoxic potential of antiinfective drugs as assessed by tissue-specific proteinuria of renal antigens.

In order to assess the nephrotoxic potential of antibiotics, various aminoglycosides and cephalosporins were tested for their potency to alter the excretion of tubular marker proteins (and brush border antigens) or to change the normal pattern of serumproteinuria as analyzed by SDS polyacrylamidgel gradient electrophoresis. After aminoglycosides, especially after gentamicin injection, a cumulative highly significant increase in the urinary output of marker proteins emerged (healthy volunteer model). In contrast, cephalosporins exhibited practically no nephrotoxic effect on proximal tubule cells. Excretion of tubular marker proteins was enhanced under combined administration of cephalosporins and aminoglycosides mainly due to the aminoglycoside component. There was no nephrotoxic synergy of both drugs. Image analysis of rat kidney sections after injection of aminoglycosides revealed that increased shedding of tubular membrane components under the toxic challenge is followed by rapid inductive repair processes (overshoot protein synthesis) of tubular cells. After a limited acute toxic damage tubular cells may recover within one week.

Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note.

After collagenase digestion and Percoll density gradient centrifugation of human renal tissue, tubular epithelial cells of the proximal and the distal segments were isolated with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT) cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly positive for aminopeptidase M (98.6%), however, cells of the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin, smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative. Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT 2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion (10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production was negligible (only 1.3x the basal level). The data of this study indicate the proximal and distal tubule origin of the cultured cells that were isolated according to their segment-specific antigens.

Immunohistochemical differentiation of metastases of renal carcinomas versus other carcinomas with anti-gamma GT monoclonal antibody 138H11.

AIMS: Adenocarcinomas account for about 60% of metastatic cancers of unknown primary (CUP) site. In such a clinical CUP situation, histopathologists are challenged to differentiate renal cell carcinomas (RCC) from other adenocarcinomas with similar immunophenotypes, especially chemotherapeutically treatable mammary and ovarian carcinomas. METHODS AND RESULTS: Recently, we produced a monoclonal antibody (mAb), designated 138H11, against human gamma-glutamyl-transferase (gamma GT), which stained over 98% primary clear cell and chromophilic RCC on frozen sections. The 138H11 epitope could not be stained using conventional techniques in most paraffin-embedded sections of the same origin, due to destruction by formalin fixation below the detection level. Here, we demonstrate that mAb 138H11 can specifically stain gamma GT in paraffin-embedded primary and metastatic RCC after enhancement with an ultrasensitive immunohistochemical method. We analysed a selected subgroup of adenocarcinomas with immunophenotypes which would not allow a differentiation from RCC in a CUP situation. We found a predominantly membranous expression of the 138H11 target antigen in 26/51 primary RCC and 15/ 34 metastatic RCC. In contrast, all 43/43 primary ovarian and bronchial carcinomas as well as 54/54 metastases of ovarian, mammary, bronchial and gastric carcinomas were negative for mAb 138H11. CONCLUSIONS: The data suggest that mAb 138H11 is useful for the immunohistochemical differentiation of RCC from other metastatic adenocarcinomas if the primary site of the tumour is not known.

Expression and release of the monocyte lipopolysaccharide receptor antigen CD14 are suppressed by glucocorticoids in vivo and in vitro.

The effect of glucocorticoid (GC) treatment on expression and release of the monocyte cell surface LPS receptor Ag CD14 was studied in vivo and in vitro. In patients with acute inflammatory diseases receiving GC pulse therapy serum concentrations of soluble CD14 and CD14 expression by peripheral blood monocytes decreased significantly. The LPS-binding capacity correlated positively with the amount of cell surface CD14 by human blood monocytes. In vitro, a time- and dose-dependent effect of GC preparations on monocyte membrane and soluble CD14 by cultured peripheral blood monocytes was found. Incubation with 2 x 10(-8) M prednisolone down-regulated cell surface CD14 after 72 h, and 2 x 10(-7) M suppressed CD14 expression even after 24 h. Prednisolone also decreased release of the soluble CD14 Ag, where a 10-fold higher GC concentration was required for a significant suppression compared with membrane CD14 during culture. Expression of other monocyte membrane Ags were either unchanged (CD33, CD35), diminished (CD13, CD89), or increased (CD32) by GC, indicating no general down-modulation of cell surface Ag expression. Preincubation with glucocorticoids for 24 h significantly down-regulated CD14 expression during subsequent steroid-free culture for at least 7 days. In cultured monocytes, the LPS-induced increase of membrane and soluble CD14 was markedly but not completely inhibited by prednisolone. Therefore, GC treatment suppresses the up-regulation of the LPS receptor during endotoxin challenge, and likewise, the IL-1 secretion after LPS stimulus was significantly diminished. Taken together, the suppression of the monocytic cell surface and soluble endotoxin receptor CD14 by GC may contribute to the increased risk of infections in patients undergoing steroid therapy.