Treatment of post-transplant lymphomas with anti-B-cell monoclonal antibodies.

The treatment of post-transplant lymphomas still needs major improvements in order to put the patient in remission and to retain graft function. Chemotherapy is far too toxic in these patients. A more specific treatment such as anti-B-cell monoclonal antibody is very promising. The cytotoxic effect of antibody relies mainly on complement-induced and antibody-dependent cellular cytotoxicity; apoptosis may also induce tumor cell death. B-cell antigens expressed on the cell surface are the targets of antibody attack; some specificities may be chosen because of their level of expression or because of signaling induced within the cell. Anti-B-cell antibodies can be produced by genetic engineering in order to be humanized or to carry bispecific Fabs. The efficacy and safety of anti-B-cell monoclonal antibodies (mAbs) in transplant patients have been proven with different antibodies such as anti-CD21/CD24 mAb, anti-CD38 mAb and anti-CD20 mAb. In a retrospective analysis of different centers in France, rituximab (anti-CD20 mAb, Roche Products) achieved an overall 69% remission rate in 34 organ and bone-marrow transplant patients. But the conditions of use of antibody must be better defined, particularly with regard to the immunosuppressive therapy, the type of tumor and the dose of antibody. We must also improve our understanding of the in vivo mechanisms of action of antibody to develop more efficient antibody constructs.

Antibody-induced modulation of the leukocyte CD11b integrin prevents mild but not major renal ischaemic injury.

BACKGROUND: CD11/CD18 beta(2) integrins are involved in leukocyte adhesion to the activated endothelium, and therefore represent a possible therapeutic target in the prevention of ischaemic acute renal failure (ARF). METHODS: To assess the effect of an anti-CD11b monoclonal antibody (mAb) in ischaemic ARF, uninephrectomized Fischer rats were subjected to 45 or 60 min of warm renal ischaemia, then received 1 mg of anti-CD11b mAb 5 min before reperfusion. RESULTS: After 45 min of ischaemia, renal function tests at 24 and 48 h were less altered in mAb-treated than in control rats, but after 60 min of ischaemia the same level of renal insufficiency was observed in the two groups. In parallel, milder tubular necrosis and less leukocyte infiltration were observed in the treated group after 45 min of ischaemia, but no difference was seen after 60 min compared to the control group. The mAb was detected on blood neutrophils up to 48 h after infusion and a marked down-regulation of CD11b expression on neutrophil surfaces was documented by flow cytometry. CONCLUSION: These results indicate that anti-CD11b mAb administered prior to reperfusion decreases moderate ischaemic ARF but fails to prevent renal injury secondary to prolonged ischaemia in this model.

In vivo effects of monoclonal antibodies against rat beta(2) integrins on kidney ischemia-reperfusion injury.

BACKGROUND: Ischemia-reperfusion (IR) involves adhesion of leukocytes to the activated endothelium, leading to tissue damage. CD11/CD18 beta(2) integrins interact with their ligands on endothelial cells and may therefore represent a therapeutic target for the prevention of IR. We investigated the effects of three monoclonal antibodies (mAbs) that recognize epitopes of heavy or light chain of the beta(2) integrins on IR in kidneys. METHODS: Uninephrectomized Fischer rats were subjected to 45 or 60 min of renal ischemia, treated with intravenously anti-beta(2) integrin monoclonal antibodies (anti-CD11a, anti-CD11b, and anti-CD18) 5 min prior to reperfusion, and compared to a nontreated group. Serum creatinine, blood urea nitrogen (BUN), and kidney histopathological damages were assessed at 1, 2, and 7 days after ischemia. RESULTS: After 45 and 60 min of ischemia, serum creatinine and BUN were significantly higher in the control than in animals treated with anti-CD11a and anti-CD18 at 24 and 48 h. Administration of anti-CD11b had a beneficial effect on renal function after 45 min but not after 60 min of ischemia. Histologic and immunostaining studies demonstrated mild tubular necrosis and less leukocyte infiltration in the anti-CD11a- and anti-CD18-treated groups compared to the control group. CONCLUSION: These results indicate that selected antibodies to CD11a/CD18 may decrease kidney IR injury when administered prior to reperfusion.

Interleukin-10 in Epstein-Barr virus-associated post-transplant lymphomas.

We used enzyme-linked immunosorbent assays (ELISA) to investigate the presence of interleukin-10 (IL-10) in the serum of patients developing post-transplant lymphomas. Serum IL-10 was detected in 14 out of 19 cases with a lymphoma or Hodgkin's disease, with higher values being observed in patients who had developed a lymphoma within the first few months post-transplantation, and who had an aggressive form of the disease. Eleven out of the 14 patients in whom IL-10 was detected had Epstein Barr virus-positive tumors. And 11 out of 14 patients died of lymphomas. In most of the patients who had detectable IL-10 at the time of diagnosis of the lymphoma, the IL-10 had not been present previously, but it was found in the serum of 7 out of 9 dialysis patients, and in 8 out of 17 stable transplant patients. We conclude that IL-10 plays a role in the development of the more severe forms of post-transplant lymphomas, and may be secreted by tumor cells. However. data from patients with chronic renal failure or patients undergoing immunosuppressive therapy must be treated with caution.

Anti-NK antibodies injected into recipient mice enhance engraftment and chimerism after allogeneic transplantation of fetal liver stem cells.

Natural killer (NK) cells have been shown to play a role in the phenomenon of resistance to transplantation of allogeneic stem cells. To explore and prevent such resistance, we treated severe combined immunodeficiency mice (SCID) with anti-NK antibodies and analysed the improved engraftment of stem cells induced by this treatment. Two groups of nine SCID mice (H-2d) were compared: group A received two injections of anti-asialo GM1 rabbit antibodies (anti-NK) on days 1 and 4; group B received two injections of normal rabbit serum. All mice were injected intravenously with 7 x 10(6) fetal liver cells from B6 mice (H-2b) on day 2. One month after fetal liver cell transplantation, all mice from group A demonstrated engraftment and chimerism; at this time, donor cells accounted for more than 50% of peripheral blood mononuclear cells (PBMC). In contrast, in group B, only one mouse had 26% of donor cells among PBMC and all other mice had less than 10%. At two months, results were virtually identical in group A (over 72% of donor cells among PBMC from all mice) and slightly improved in group B (0-38% of donor cells). After the third month and continuously until the 12th month, the stability of chimerism was established in group A (over 55% of donor cells in 7 of the 9 mice) but had virtually disappeared in group B (0-2% of donor cells in all mice). Tissue analysis demonstrated the improved reconstitution of the thymus and the spleen in mice from group A. The proliferative responses of spleen cells to phytomitogens were significantly higher in all mice from group A than in any mouse from group B. Skin allografts from a third party (H-2k) were rejected within 10 days by group A mice but not by group B mice, one year after fetal liver cell transplantation. On the whole, anti-NK antibodies were able to improve engraftment, chimerism and stability of allogeneic stem cell transplants.

Antihypertensive effect of thymectomy in Lyon hypertensive rats. Vascular reactivity, renal histology, and sodium excretion.

The aim of this study was to search for the possible mechanisms involved in the antihypertensive effect of neonatal thymectomy that we previously observed in Lyon hypertensive (LH) rats. To that end, we studied in LH and normotensive control (LN) rats the consequences of neonatal thymectomy on vascular reactivity, renal structure, and pressure-natriuresis. The increase in pressor responses to angiotensin I and phenylephrine noted in LH rats as compared to LN animals was abolished by neonatal thymectomy. Histological study showed that kidneys from LH rats exhibited arterial wall hypertrophy, segmental hyalinization of the glomeruli, and were infiltrated by mononuclear cells. All these features of kidney injury were reduced in neonatally thymectomized LH rats. Lastly, the responses of isolated perfused kidneys from LH rats to stepwise reductions in renal perfusion pressure differed from those of LN rats by decreased renal perfusion flow and natriuresis. Neonatal thymectomy tended to improve sodium excretion in parallel with a slight decrease in renal vascular resistances. It is concluded that the normalization of vascular responsiveness to vasoconstrictor factors, the alleviation of renal lesions and, to a lesser extent, the moderate improvement of pressure natriuresis may account, at least in part, for the antihypertensive effect of neonatal thymectomy in LH rats.

Image analysis of dendritic cells in the human fetal thymus.

The distribution and evolution of thymic dendritic cells (DC) were studied during human ontogeny. Immunochemical techniques were used to detect S100 protein-positive cells on fetal thymus sections, at different post-fecundation (PF) ages; an image analysis of these positive cells was then carried out. Variations in the percentage of these cells in the medulla were determined according to age: the higher percentages were seen at 12 and 16 weeks PF. Dendritic cells were present at an early stage in the thymic rudiment (7 weeks PF), a finding consistent with an origin of these cells in the fetal liver, and with the possible existence of local attraction and/or maturation factors. The expression of the CD54 adhesion molecule revealed the existence of particular interactions between DC and lymphocytes in the medullary area, and at the cortico-medullar junction. Diffuse CD11a (LFA-1) expression on lymphocytes was consistent throughout gestation. The monocyte/macrophage markers (CD11b, CD14) and the reaction of non-specific esterases showed that the distribution pattern of these cells differed from the DC pattern. These ontogenic data are related to the significant role played by DC throughout the different stages of thymopoiesis.

IL-1 production by human thymic dendritic cells: studies on the interrelation with DC accessory function.

Thymic dendritic cells (DC) have been proposed to play a critical role in the generation of immunocompetent T lymphocytes. Since IL-1 is widely considered to be an important second signal in T cell stimulation, we have studied the ability of isolated human thymic DC to produce IL-1. Using the EL4/CTLL conversion assay standardized with recombinant IL-1 beta (rIL-1 beta), we demonstrate that upon LPS-stimulation thymic DC produce small amounts of IL-1 as compared to peripheral blood monocytes (PBM). In contrast with PBM, DC IL-1 production is not influenced by indomethacin. IL-1 activity was detected in the supernatants of DC cultures from all thymuses tested, although quantitative variability was noted among individual thymic donors. The specificity of the active factor was confirmed by neutralization assays with anti-IL-1 beta mAb. On the other hand, we demonstrate that rIL-1 beta cannot substitute for nor amplify the accessory function of thymic DC and that anti-IL-1 beta mAb fails to block the DC accessory function. Thus we conclude that IL-1 beta might not be a major factor for the efficient DC accessory function toward mature thymocytes recently demonstrated in our laboratory. Of interest, IL-1 beta was also detected in the supernatants of DC-thymocyte cocultures in the absence of mitogenic factor, suggesting that thymocyte contacts can constitute a sufficient signal to induce DC to produce IL-1. These observations indicate that human thymic DC represent an intrathymic source of IL-1 whose role in thymocyte proliferation or maturation remains to be understood.

Immunohistochemical localization of type IV collagen fibronectin and laminin in the juxtaglomerular apparatus of the rat kidney.

The juxtaglomerular apparatus (JGA) is a complex structure containing several components: the vessels, the extraglomerular mesangium and the distal tubule. These structures include cellular elements and an extracellular matrix (ECM). Collagenous (type IV collagen) and noncollagenous components of the basement membranes were studied. The localization of type IV collagen and of two extracellular glycoproteins (laminin and fibronectin) was investigated using immunofluorescent and immunoperoxidase labelled antibodies. Type IV collagen and laminin have the same localization on the JGA basement membranes. On the other hand, fibronectin is limited to the entrance of the glomerular stalk. On electron microscopy, type IV collagen is found in the basement membrane while fibronectin is restricted to certain areas of the extracellular matrix. These findings confirm data concerning the distribution of these three components in basement membranes and allow a better understanding of the histoarchitecture of the juxtaglomerular apparatus.

Cyclosporine A nephrotoxicity: evidence for mesangial foam-cells in dogs.

Nephrotoxicity of cyclosporine A was studied in dog after 3 weeks' administration of the drug at high doses (20 mg/kg/day). Cyclosporine A concentrations measured in different organs revealed a high ratio between renal tissue and plasma. Renal histopathology showed non-specific tubular lesions and glomerular modifications. There were lipids in the mesangial cells which took on a foamy aspect. Ultrastructural study and lipid staining confirmed these findings, and non-specific esterase reaction revealed no macrophagic infiltration. Using anti-CSA antibodies it was not possible to demonstrate CSA in the mesangial cells. This alteration has never been described before in CSA therapy and its meaning is not clear; however, it does suggest that there occur modifications in the lipid metabolism or in the phagocytic function of the mesangial cells when cyclosporine A is administered over long periods.

[Macrophage infiltration in human glomerulonephritis: histochemical study by the non-specific esterase method]. / Infiltration macrophagique au cours des glomérulonéphrites humaines: etude histochimique par la méthode de l'activité des estérases non spécifiques.

Detection of glomerular macrophages in glomerulonephritis (GN) was attempted in 77 renal biopsies by measuring non-specific esterase (NSE) activity. Macrophage infiltration was at its highest in two cases of cryoglobulinemia (NSE index greater than 10) both associated with a diffuse proliferative GN. In 15 cases of lupus, macrophage infiltration was low or absent (NSE index 0.44). In diffuse proliferative GN (24 cases) only GN with extracapillary proliferation had an NSE index greater than 1. In GN with predominant IgA (23 cases), apart 2 cases of extracapillary forms, there was no notable macrophage infiltration. There is a relation between monocytes and the localization and the quantity of glomerular deposits. The NSE index is higher in the case of large sub-endothelial deposits of immunoglobulins, C3 and/or fibrin.

Simultaneous staining of nuclear DNA and blood group cell surface antigens on cells from bladder irrigation fluid.

Simultaneous staining of nuclear DNA and blood group cell-surface antigens is proposed as a means of studying the prognosis of superficial bladder cancer. The quality of the results obtained on urothelial cells from bladder irrigation fluid, as shown by fluorescence microscopy, suggests that this staining technique may be suitable for flow cytometry.

Clinically occult bladder cancer diagnosis. Trial using ultraviolet cystoscopy.

Ultraviolet cystoscopy was used to demonstrate flat cancerous and precancerous bladder lesions by two techniques based on different principles: the loss of epithelium blood group antigenic expression using immunofluorescence reaction, and submucosa neoangiogenesis after fluorescein intravenous injection. The results obtained with these two techniques were disappointing, but do not preclude the use of ultraviolet cystoscopy in this type of study.

Revelation of AB cell surface antigens on bladder-washing specimens with the immunofluorescence technique. An approach to automated cytology.

Revelation of AB cell surface antigens is possible with the immunofluorescence technique on urothelial cells from bladder irrigation fluid. Normal urothelial cells show a heterogeneous expression varying with cell type and cell viability. However, the loss of any antigenic reactivity of some cells seems to be directly related to their neoplastic features. Flow cytometry appears to be the most appropriate technique to provide the necessary objectivity for this type of study.

Pathology of the pancreas after intraductal neoprene injection in dogs and diabetic patients treated by pancreatic transplantation.

The injection of neoprene into the pancreatic ducts of dogs has been used to destroy exocrine function prior to pancreatic transplantation. The subsequent histological changes and the evolution of lesions over a period of 3-36 months are described. Animals were sacrificed or biopsied at various intervals (3, 15 and 36 months) and the pancreases showed the disappearance of exocrine acini and changes of chronic pancreatitis. An immunoperoxidase procedure with insulin, glucagon, somatostatin and pancreatic polypeptide antisera was used to show the persistence of pancreatic endocrine cells. After the injections, sclerosis progressively increased and secondary lesions of the islets were seen, although functional islets persisted. This technique was then applied to pancreas transplantation in man. Eight transplants from seven diabetic patients were available for examination. In four cases, there were early technical failures, but four pancreatic transplants continued to function for 28-889 days until suppuration destroyed one of the grafts and the three other patients died. The persistence of endocrine cells in sclerotic tissue was observed in histological and immunopathological examinations.

Distant evolution of kidney arteriolar lesions and hypertension after pregnancy toxemia.

60 patients were studied 3 months to 4 years after pregnancy. All had high pressure, gross proteinuria, and edema. In many cases, typical glomerular lesions and proteinuria disappeared rapidly. When arteriolar lesions are of grade I or II, they progressively disappear or remain unchanged and blood pressure returns to normal values. When arteriolar lesions are of grade III (endotheliitis, hyperplasia and edema of the media and subendothelial deposits), blood pressure remains at a high level or increases, without changes during the time of observation of the arteriolar lesion or of renal function.