Genetic survey of a group of children with clefting: implications for genetic counseling.

A cleft lip, cleft palate, or both are associated with a high frequency of other anomalies. This study gives an inventory of associated anomalies in a consecutive group of children (n = 36) with clefts, referred to a local multidisciplinary cleft team in the Netherlands. In 47.2% of cleft patients associated anomalies were found, allowing diagnosis of provisionally unique syndromes or known entities. In 17 patients family history was positive for clefting; in five patients (13.9%) this influenced the occurrence risks for siblings. Both findings had an effect on genetic counseling of the parents of these children. Additional evidence is provided that all children with clefting should be carefully evaluated by a trained clinician for additional anomalies, including dysmorphic features.

Monoclonal antibody mediated targeting of enzymes. A comparative study using the mouse spleen as a model system.

The aim of this study was to target enzymes specifically to cells in the murine spleen. Monomeric and polymeric conjugates of the enzymes horseradish peroxidase or alkaline phosphatase and monoclonal antibodies against cell surface determinants were prepared. Highly specific in vivo targeting of enzymes to macrophages was obtained only when monomeric MAb-enzyme conjugates were used.

Development of antibody-forming cells in follicles of lymph nodes of mice after continuous antigenic stimulation.

It was the purpose of the present study to test a hypothesis on the direct differentiation of newly formed memory B cells into antibody-forming cells (AFC) in the follicles of lymph nodes. AFC may develop in follicles when mobile antigen is present at the moment that such memory B cells have completed their differentiation under influence of immune complexes trapped on follicular dendritic cells (FDC). In order to study whether the simultaneous presence of mobile antigen and immobilized immune complexes on FDC alters the normal distribution of AFC in lymph nodes, different immunization protocols with the thymus-dependent antigens trinitrophenylated keyhole limpet haemocyanin (TNP-KLH) and horseradish peroxidase (HRP) were used. Results confirm that, provided that mobile antigen and immobilized immune complexes are present simultaneously, AFC that are normally found in the medulla of the lymph node may also develop in the follicles.

The in situ immune response of the rat after intraperitoneal depletion of macrophages by liposome-encapsulated dichloromethylene diphosphonate.

This study concerns the contribution of peritoneal macrophages in vivo to local and systemic immune responses in the rat. Peritoneal macrophages as well as macrophages in the draining parathymic lymph nodes were selectively eliminated by intraperitoneal inoculation of dichloromethylene-diphosphonate-containing liposomes. This depletion resulted in an enhanced immune reaction to intraperitoneally administered trinitrophenyl keyhole limpet haemocyanin in the parathymic lymph nodes, as demonstrated by the higher numbers of specific anti-TNP antibody-forming cells in macrophage-depleted animals than in control animals from day 5 after immunization. The immune reaction peaked at day 7 and remained high until day 10. Specific antibody-forming cells were found occasionally in the mesenteric lymph nodes and spleen, but not in the mucosa of the gut or in the bronchus-associated lymphoid tissue. An elevated immune reaction found in parathymic lymph nodes associated with the depletion of local macrophages by liposome treatment indicates a regulatory role of peritoneal macrophages in local humoral immune response.

Fixation of cryo-sections under HIV-1 inactivating conditions: integrity of antigen binding sites and cell surface antigens.

Cryostat-sections of biopsies from HIV-infected patients or HIV/SIV-infected experimental animals pose a biohazard risk to laboratory workers. The objective of this study was to select a procedure that appropriately fixes cryo-sections and reduces the risk of HIV-1 infectivity. This inactivation procedure should preserve antigen binding capacity of host-produced antibodies and the antigenic structure of epitopes present in these tissues, while retaining sufficient morphologic detail. We tested the effect of seven different established fixation-inactivation procedures for HIV-1 on the detection of specific antibodies and membrane markers, compared to acetone fixation as a reference. Frozen sections of spleens from mice immunized with trinitrophenyl (TNP)-Ficoll were incubated with TNP-alkaline phosphatase to detect specific antibody-forming cells and follicular immune complexes containing TNP-specific antibodies. In addition, sections were stained with monoclonal antibodies directed against IgM (187-1), T-cells (anti Thy-1), and marginal metallophilic macrophages (MOMA-1). Five procedures proved useful as they gave results similar to regular acetone fixation. In contrast, two procedures with a methanol-containing fixative obscured both antigen binding sites and membrane antigens. Subsequently, these five selected procedures were tested on glass slide preparations of HIV-1 infected cell lines, expressing HIV-1 determinants defined by monoclonal antibodies. Finally, the procedures were tested on sections of an HIV-1 infected human lymph node, for detection of HIV-specific B-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Elimination of spleen and of lymph node macrophages and its difference in the effect on the immune response to particulate antigens.

To study the role of macrophages in the in situ immune response to particulate antigens in spleen and popliteal lymph nodes (PLN), mice were injected with dichloromethylene diphosphonate (Cl2MDP)-containing liposomes to eliminate macrophages, followed by immunization with trinitrophenylated sheep red blood cells (TNP-SRBC). Depletion of macrophages in the spleen caused a strong decrease in the number of antibody-forming cells (AFC), which develop after intravenous (i.v.) injection of the antigen. These results strongly suggested the involvement of splenic macrophages in the processing of TNP-SRBC. In particular, the populations of marginal zone macrophages may be involved in the inductive phase of an antibody response to particulate antigens. These macrophages are strategically positioned at the end of the white pulp capillaries in the marginal zone of the spleen and they have their cell processes between the marginal zone-B cells. Elimination of macrophages in PLN had no effect on the number of AFC, which develop after subcutaneous (s.c.) injection of the antigen in the hind footpads. This indicates that the macrophages are not essential for the induction of a local immune response to the particulate antigen TNP-SRBC. After depletion of lymph node macrophages, the number of AFC developing in the spleen after s.c. footpad injection of the antigen increased and the anti-TNP serum titers were elevated. This may well be caused by the fact that more of the antigen reaches the circulation and subsequently stimulates the spleen.

Mechanism of follicular trapping: localization of immune complexes and cell remnants after elimination and repopulation of different spleen cell populations.

The role of marginal zone macrophages, marginal metallophilic macrophages (marginal metallophils) and marginal zone lymphocytes in the follicular trapping of immune complexes was investigated in a detailed elimination and repopulation study. Intravenous injection of liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP) resulted in a complete and lasting elimination of marginal zone macrophages and marginal metallophils, while the number of marginal zone B-lymphocytes was temporarily reduced. By means of image analysis of light-microscopic images we quantified the repopulation of the above cell types and the presence of immune complexes during the repopulation process. Trapping of peroxidase-anti-peroxidase complexes was reduced up to Day 3 after administration of Cl2MDP-liposomes, but reached control values on Day 5, before reappearance of the different cell types. Therefore, marginal zone macrophages and marginal metallophils are neither directly nor indirectly involved in the transport of immune complexes to splenic follicles. It is unlikely that marginal zone B cells play a role in the transport of complexes, as a substantial reduction in B-cell number did not impair follicular trapping. At different time-points after treatment with Cl2MDP-liposome treatment, three macrophage markers (acid phosphatase, ligand for ERTR-9 and ligand for MOMA-2) were found in splenic follicles of several animals, but not in control animals. The presence of these macrophage markers in splenic follicles implies that soluble and particulate cell remnants migrate to the follicle and are retained there without the involvement of specific antibody and complement. Collectively, the data showing trapping of immune complexes despite the absence of several candidate transporter cell types and the localization of cellular remnants to splenic follicles provide evidence against a cell-mediated transport of immune complexes. The data argue in favour of diffusion as a transport mechanism of both immune and non-immune compounds to the follicle.

The in situ immune response in popliteal lymph nodes of mice after macrophage depletion. Differential effects of macrophages on thymus-dependent and thymus-independent immune responses.

Mice were subcutaneously (SC) injected in the left hind footpad with dichloromethylene diphosphonate (Cl2MDP)-containing liposomes to eliminate macrophages lining the subcapsular sinus (SCS) and those in the medulla of draining popliteal lymph nodes (PLN). In order to study the effect of depletion of these macrophages on the in situ immune response in the PLN, liposome-treated mice were SC injected in the same footpad with thymus-independent (TI) type 1 antigen trinitrophenylated lipopolysaccharide (TNP-LPS), TI-type 2 antigen TNP-Ficoll or thymus-dependent (TD) antigen TNP-keyhole limpet haemocyanin (TNP-KLH). No major differences were observed in antibody-serum titers of liposome-treated and control animals. After primary as well as secondary immunization with the TD-antigen TNP-KLH, an increase in the number of antibody-forming cells (AFC) was found and the peak of response was delayed in the PLN of liposome-treated animals. Such differences were not observed with the TI-antigens. These results indicate that macrophages lining the SCS and those in the medulla of the PLN are not essential for the induction of an immune response. The positive effect of macrophage-depletion on the number of AFC may be explained by competition for the antigen by macrophages and other antigen-presenting cells.

Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate.

Rats received a single intravenous injection with liposome-encapsulated dichloromethylene diphosphonate (C12MDP). This treatment resulted in the elimination of macrophages in spleen and liver within 2 days. Macrophages ingest the liposomes and are destroyed by the drug, which is released from the liposomes after disruption of the phospholipid bilayers under the influence of lysosomal phospholipases. Repopulation of macrophages in spleen and liver was studied at different time intervals after treatment. Macrophages in the liver (Kupffer cells) and red pulp macrophages in the spleen were the first cells to reappear, followed by marginal metallophilic macrophages and marginal-zone macrophages in the spleen. Different markers of the same cell did not reappear simultaneously. On the other hand, the same marker (recognized by the monoclonal antibody ED2) reappeared much more rapidly in the liver than in the spleen. The present results in the rat were different from those earlier obtained in the mouse. Red pulp macrophages were the first cells and marginal zone macrophages were the last cells to repopulate the spleen in both rodents after treatment with C12MDP liposomes. However, there was much more overlap in the repopulation kinetics of splenic macrophage subpopulations in the rat, when compared with the mouse.

Repopulation of macrophages in popliteal lymph nodes of mice after liposome-mediated depletion.

Macrophages lining the subcapsular sinus (SCS) and those located in the medulla of popliteal lymph nodes (PLN) of mice were eliminated after subcutaneous (s.c.) injection of dichloromethylene diphosphonate (Cl2MDP)-containing liposomes. No effect of liposome-entrapped Cl2MDP could be seen on nonphagocytic cells, e.g., interdigitating cells (IDC) and B- and T-lymphocytes. One month after injection the eliminated subsets of macrophages were still absent. After 2 mo a small number of macrophages had reappeared along the SCS and in the medulla of the PLN of a few animals. Complete repopulation of the PLN with macrophages was observed only after 5 mo. This extremely long repopulation time could be shortened drastically by local administration of Freund's complete adjuvant (FCA). A small number of macrophages reappeared along the SCS and in the medulla 1 wk after FCA and after 3 wk the repopulation of the PLN with macrophages was complete. Such rapid repopulation of macrophages was not achieved after s.c. injection of paraffin oil or paratyphoid vaccine. These results indicate that the normal rate of influx of mononuclear phagocytes into the PLN is low, but that it can be sped up after administration of FCA.

Effects of intracellular diphosphonates on cells of the mononuclear phagocyte system: in vivo effects of liposome-encapsulated diphosphonates on different macrophage subpopulations in the spleen.

The intracellular effects of different diphosphonates on splenic macrophages were compared after intravenous injection of liposomes with encapsulated dichloromethylene diphosphonate (C12MDP), 3-amino-1-hydroxypropane-1,1-diphosphonate (APD), or 1 hydroxyethane-1,1-diphosphonate (EHDP) in similar concentrations. Intracellular C12MDP was by far the most toxic compound for macrophages in the spleen. APD encapsulated in liposomes in the highest possible concentration eliminated marginal zone macrophages only and EHDP did not affect any of the macrophage subpopulations in the spleen.

Marginal zone of the murine spleen in autotransplants: functional and histological observations in the response against a thymus-independent type 2 antigen.

Splenic tissue from mice was autotransplanted; after initial necrosis, a rapid restoration of implants into a structure histologically indistinguishable from splenic tissue was observed. The development of the marginal zone in these autotransplants, as determined with monoclonal antibodies against different splenic cell types and routine histological stains, was compared with the local and systemic response against a thymus-independent (TI) type 2 antigen. Full restoration of time course and peak of anti-trinitrophenyl (TNP) serum titres against TNP-Ficoll was observed at 4 weeks after autotransplantation. Anti-TNP antibody-forming cells were observed in subnormal and normal numbers in 2- and 4-week old autotransplants, respectively. The appearance of normal numbers of antibody-forming cells, and the restoration of antibody titres at week 4 correlated with the return of newly formed B cells in a normal marginal zone. An unexpected observation was that marginal zone macrophages did not return until 10 weeks after transplantation, thereby making the necessity for these cells in the normal TI-2 response unlikely. We conclude that normal anti-TI-2 responses (onset and peak titres) can be restored by autotransplantation of splenic tissue. B cells and marginal zone organization are responsible for this response, for which marginal zone macrophages seem expendable. The partial protection against overwhelming post-splenectomy infections, given by autotransplants, can thus be explained by restorative capabilities of these implants on antigen presentation and antibody formation against TI-2 antigens, and not by an increase (compared with splenectomized individuals) of phagocytosis by marginal zone macrophages.

Macrophage subset repopulation in the spleen: differential kinetics after liposome-mediated elimination.

Different macrophage subsets can be discriminated in the well defined compartments of the mouse spleen by specialized functions and the presence of specific surface determinants. Red pulp macrophages, marginal zone macrophages, and marginal metallophilic macrophages are eliminated simultaneously within 24 hr by a single injection with liposome-entrapped dichloromethylene diphosphonate (DMDP). After such elimination, these subsets show a striking difference in their kinetics of reappearance: Red pulp macrophages are back in normal numbers after 1 week, the marginal metallophilic macrophages take 2 weeks to regain fully their position at the border of the marginal zone and periarteriolar lymphocyte sheath, but it takes over 1 month for complete reappearance of the marginal zone macrophages. Marginal zone lymphocytes, also affected by treatment with the liposome-entrapped drug, reappeared in the marginal zone within 2 weeks, indicating that marginal zone macrophages are not required for their localization and/or retention there. Approximately 2 weeks after treatment, all cells in the spleen have returned to normal numbers with the exception of marginal zone macrophages, which can be found only sporadically at that time. The results indicate that these macrophage subpopulations must have different precursor requirements. The differential reappearance of the macrophages creates the possibility of studying lineage analysis and will help to unravel the precise function of the marginal zone macrophages and marginal metallophilic macrophages in particular.

Effects of chronic injection of sphingomyelin-containing liposomes on lymphoid and non-lymphoid cells in the spleen. Transient suppression of marginal zone macrophages.

Mice were injected with sphingomyelin/cholesterol or phosphatidylcholine/cholesterol (PC/C) liposomes, from twice up to 10 times, on alternate days. Administration of sphingomyelin/cholesterol (SM/C) liposomes gave rise to hepato and splenomegaly, microgranulomatous infections and changes in macrophage numbers and activity in spleen and liver. Enzyme and immuno-cytochemical methods were used, to demonstrate the effect of liposomes on the lymphoid and non-lymphoid cell populations, on cryostat sections of the spleen. Routine histological staining, of sphingomyelin/cholesterol treated animals, showed no drastic changes in morphology or compartmentalization of the spleen, apart from a small enlargement (with some microgranulomas) of the red pulp. No significant differences were found in the presence or localization of T-helper, T-cytotoxic/suppressor, T-total-lymphocytes, B-total-lymphocytes, red pulp macrophages, marginal metallophils, or non-lymphoid dendritic cells. However, a transient suppression of cells expressing marginal zone macrophage surface marker ERTR-9, was observed between the second and eighth (intravenous) administration of sphingomyelin/cholesterol liposomes. Immunization of these animals with trinitrophenyl (TNP)-ficoll, a thymus-independent type-2 antigen which is specifically processed by marginal zone macrophages (MZM), showed that these cells were not suppressed with regard to their immunological function. We conclude that chronic administration of sphingomyelin liposomes influences macrophages, probably through a general phagocytic-system overload, but not permanent or damaging changes in splenic cell populations or immunological functions occur.

In vitro and in vivo elimination of macrophage tumor cells using liposome-encapsulated dichloromethylene diphosphonate.

It is shown in the present study that RAW 264 tumor cells can be killed by liposome-entrapped dichloromethylene diphosphonate (DMDP), both in vitro and in vivo. DMDP is ingested by phagocytic cells when entrapped in liposomes. Once phagocytized the liposomal membranes are digested and the drug is released into the cell and is ready for action. In vitro, even low doses of liposome-entrapped DMDP caused an significant reduction in cell numbers. In vivo, liposome-encapsulated DMDP markedly reduced tumor formation in the liver, when given 1 day after injection of 1 x 10(6) RAW 264 tumor cells. Liposome-encapsulated DMDP, given 4 or more days after injection of the tumor cells had no significant effect. We concluded that tumor formation by RAW 264 cells is only susceptible to in vivo treatment with liposome-entrapped DMDP during a short period of time after injection of the cells. Obviously, phagocytosis of the tumor cells is reduced after this period making the cells less susceptible to treatment with the liposome-entrapped drug.

In vivo effects of lipopolysaccharide on lymphoid and non-lymphoid cells in the mouse spleen. Suppressive and adjuvant effects of LPS on the development of specific antibody forming cells in situ.

Mice were immunized i.v. with 2,4,6-trinitrophenyl (TNP)-carrier conjugates and received lipopolysaccharide (LPS) before, simultaneously with or after these antigens, in order to study the effects of LPS on the in situ development of anti-TNP antibody forming cells in the spleen. LPS given before, simultaneously with or shortly after the thymus dependent (TD) antigen TNP-keyhole limpet haemocyanin (KLH) suppressed the development of anti-TNP antibody forming cells in the spleen, but serum IgM-anti-TNP titres were not reduced, suggesting a selective inhibiting effect of LPS on the local immune response in the spleen. Splenic responses to thymus independent (TI) antigens TNP-Ficoll and TNP-LPS were not reduced when these antigens were administered one day after LPS. LPS given 4 days after TNP-KLH and 1 day before killing of the animals had induced severe alterations in splenic histology, but we found no marked influence on numbers and distribution pattern of anti-TNP forming cells. Results indicate that the LPS induced suppression of the immune response to TD antigens in the spleen is not caused by a direct effect of LPS on antigen reactive B-lymphocytes or on the anti-TNP antibody forming cells themselves. Possible explanations for the LPS-induced immunosuppression are discussed.

Immunomodulation with liposomes: the immune response elicited by liposomes with entrapped dichloromethylene-diphosphonate and surface-associated antigen or hapten.

Macrophages in the murine spleen were eliminated by the intravenous administration of dichloromethylene-diphosphonate (DMDP) encapsulated in liposomes. The immune response against an antigen (bovine serum albumin, BSA) or hapten (dinitrophenyl, DNP) associated with the surface of the DMDP liposomes was studied. Significant effects of macrophage elimination on the primary (IgM), but not on the secondary (IgG), anti-BSA response were found. Dramatic effects on the response against liposome-associated DNP were observed in macrophage-depleted mice. The number of anti-DNP antibody-forming cells in the spleen decreased from 300 to 28 per section, and anti-DNP serum titres dropped to 12% of their normal values. Since a similar phenomenon was observed for TNP-Ficoll, a thymus-independent type-2 antigen (and not for thymus-dependent or thymus-independent type-1 antigens), we suggest that this response should be classified as thymus-independent type-2 on grounds of its in vivo behaviour. We conclude that adjuvant activity (and memory formation) of liposomes with antigen exposed on their surface occurs irrespective of the presence or absence of splenic macrophages, and that DMDP liposomes could be useful in drug targeting with antigenic liposomes.

Development of specific antibody-forming cells in various lymphoid organs of rabbit after intravenous antigen administration.

Rabbits were intravenously primed and boosted with trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) and human serum albumin (HSA); both antigens were injected simultaneously. The localization of anti-TNP-antibody-forming cells (AFCs) and anti-HSA-AFCs was determined in various lymphoid organs of the rabbit. In all lymphoid organs of primed rabbits anti-TNP-AFCs outnumbered anti-HSA-AFCs, with the exception of the thymus, in which neither of them was encountered. In the spleen the antibody-forming cells were mainly situated in the periphery of the periarteriolar lymphocyte sheaths (PALS) and in the coaxial sheaths of lymphoid tissue surrounding the terminal arterioles. In the lymphoepithelial organs AFCs were almost exclusively situated in the interfollicular areas, and in the lymph nodes largely in the medulla. An intravenous booster injection led to a secondary immune response (i.e., increase of AFCs) in the spleen. No visible change in the number of specific AFCs was observed in the lymphoepithelial organs. However, in the mesenteric and popliteal lymph node the number of anti-TNP-AFCs had increased tremendously.