Liposome mediated affection of monocytes.

Dichloromethylene diphosphonate (Cl2MDP) enclosed in liposomes, when injected intravenously, selectively eliminates all phagocytizing macrophages that are in direct contact with the blood circulation of the spleen and the liver. We examined whether Cl2MDP containing liposomes affect, in addition, rat monocytes and bone marrow macrophage precursors (BMMP's). In addition to Phosphatidylcholine-Cholesterol liposomes (PC liposomes), we also used mannosylated PC liposomes (PCMAN liposomes), which are reported to bind more efficiently to macrophages. Monocytes appeared to be affected 24 h after injections of 2 ml of Cl2MDP containing PC as well as PCMAN liposomes. In addition, almost no macrophages could be detected in one week cultures of blood leukocytes isolated from these animals; cultures from control animals contained +/- 50% macrophages. Bone marrow macrophage precursors did not appear to be affected.


In vivo presentation of Mls-1 antigen by T and B lymphocytes.

Previous studies of minor lymphocyte stimulatory (Mls) presenting lymphoid cells had shown that B cells rather than T cells present stimulatory Mls-1 antigen in vitro whereas B as well as T cells present Mls-1 antigen in vivo. Deletion of Mls-1 reactive T cells in the thymus of newborn mice is induced by T cells rather than by B cells. Applying a recently developed method for measuring the Mls-1 response in Mls-1- mice we assessed the Mls-1 stimulatory activity of T and B cells quantitatively. B cells are significantly more effective than T cells in this process. Both Mls-1+ T and B cells are also capable of inducing Mls-1 anergy in Mls-1- mice. Remarkably few lymphoid cells from Mls-1+ animals are needed for this effect: a few thousand B cells or 10(4) to 10(5) T cells per mouse induce substantial Mls-1 anergy in Mls-1- mice. These low cellular requirements for Mls-1 anergy production correspond well to the low T cell requirements described for the induction of Mls-1 tolerance in newborn mice. However, the high efficacy of B cells in inducing peripheral Mls-1 anergy contrasts with their failure to induce neonatal tolerance in newborn animals. We attribute this discrepancy to the previous notion that stimulatory Mls-1 antigen is not delivered to the thymus and that B cells and T cells present qualitatively different Mls-1 related signals to Mls-1 reactive T cells.


Analysis of CD16+dim and CD16+bright lymphocytes--comparison of peripheral and clonal non-MHC-restricted T cells and NK cells.

The Fc gamma RIII receptor (CD16) has been described on natural killer cells and a small subset of T lymphocytes. CD16+bright lymphocytes represent the typical population of peripheral blood CD3- NK cells. In these studies in addition to CD16+bright NK cells Fc gamma RIII expressing cytotoxic T lymphocytes in peripheral blood from one healthy individual are characterized as CD16+dim non-MHC-restricted CTLs either expressing the alpha/beta (80%) or the gamma/delta T cell receptor (20%). Both CD16+ subsets are clearly distinct in their functional capacity performing NK and ADCC activity. Freshly isolated CD16+dim T cells exert higher ADCC, CD16+bright NK cells higher NK activity. They are also differentially activated by interleukin-2 since CD16+bright NK cells reveal a bright expression of the p75 IL-2 receptor beta-chain in contrast to the very low p75 expression on CD16+dim T cells. This activation leads to a gradual increase of ADCC by NK cells. Finally the CD16 expression pattern with low and bright intensity represents a stable phenotype expressed by clones generated from these different subpopulations. On a clonal level CD16+dim non-MHC-restricted T cells can be distinguished from CD16+bright NK cells by their lower capacity in NK killing, but they are equally potent in ADCC. Finally these CD3+CD16+dim clones provide the basis for studies of Fc gamma RIII and TcR interaction.


Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli.

A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.


Resistance of different tumor cells to lysis by lymphokine activated killer cells can be mediated by distinct mechanisms.

Lymphokine activated killer (LAK) cells have been shown to exert a potent cytotoxic effect on many histologically different tumors and virally infected targets. Most normal cells but very few tumors have proven resistant to LAK lysis. The availability of two LAK resistant tumors, P815r, a murine mastocytoma, and SNUC-1, a human colon carcinoma, allowed us to study the phenomenon of LAK lysis. We examined the role of surface molecules on targets, which mediate binding to LAK cells, by cold target competition experiments and lectin dependent cellular cytotoxicity assays. The results showed that in the murine system, P815r cells do not compete for lysis of the LAK sensitive target B16 whereas other LAK sensitive murine targets compete. Alternatively, in the human system, SNUC-1 cells compete for lysis of the LAK sensitive target SNUC-4 as do other LAK sensitive human tumor cells. Furthermore, inducing binding of target and effector cells with lectin reverted the resistance of P815r but not SNUC-1 targets to lysis by LAK cells. These results imply that distinct stages of the lytic pathway might be involved in the resistance of different tumors to killing by LAK cells. The murine cell line is resistant to lysis because it cannot bind LAK cells. The human target, which does bind LAK, was insensitive to the effects of tumor necrosis factor alpha (TNF-alpha), a lymphokine released by LAK effectors and possibly involved in their lysis. Resistance to TNF-alpha was not mediated by the presence of endogenous short-lived proteins in the SNUC-1 targets. The elucidation of mechanisms of resistance may provide a tool to improve current protocols of adoptive immunotherapy as well as insights as to how tumor cells are or are not killed by LAK effectors.


Measurement of lymphocyte proliferation: critical analysis of radioactive and photometric methods.

Different methods of lymphocyte proliferation are compared to identify a non-radioactive alternative to 3H-thymidine-test. The enzymatic assays evaluating the turnover of mitochondrial dehydrogenases (MTT-test) and lysosomal hexosaminidase (NAG-test) proved not sensitive enough to substitute for 3H-thymidine incorporation. The incorporation of the nucleotide analog 5-bromodeoxyuridine (BrdU) can be exploited using an ELISA-system (enzyme linked immunosorbent assay) employing a monoclonal anti-BrdU antibody to measure cell proliferation. An optimized test protocol of the BrdU-ELISA which fulfills the requirements for a sensitive and practicable non-radioactive alternative to 3H-thymidine-test is presented.


Functional properties of heterogeneous human asialo-C4 and its isotypes C4A and C4B.

The fourth component of human complement (C4) is encoded at two separate but closely linked loci within the MHC on the short arm of chromosome 6. Thus, there are two types of C4 protein in most individual and pooled normal human sera (NHS): C4A and C4B. Incubation of individual sera, pooled NHS, or purified heterogeneous C4 (C4A/C4B) with bacterial sialidase at 37 degrees C increased C-mediated hemolysis of antibody-sensitized sheep erythrocytes 1.54- to 1.93-fold. Comparative studies of Tmax of human C2, using asialo-C4 or buffer-treated C4 on EAC1gp and extrapolation to time 0 indicated a z value 4-fold higher with asialo-C4. This indicated that more hemolytically active C42 complexes are available with sialidase-treated C4 compared to untreated C4. There was no appreciable difference in the % 125I-C4 bound to EAC1gp (sialidase- or buffer-treated). Sera from two different blood donors with C4A3 phenotype (C4BQ0), two different donors with C4B1 phenotype (C4AQ0), and serum from an individual heterozygous deficient at both C4A3 and C4B1 regions (A3, AQ0; B1, BQ0) were investigated. The C4 allotypes, purified from these sera, were treated with sialidase; the C4A3 was enhanced in hemolytic assays by sialidase-treatment (1.52- to 2.3-fold), whereas the C4B1 allotype was not enhanced. Fluorometric determinations revealed that approximately the same percentage of sialic acid was released from sialidase-treated C4A3 and C4B1. Therefore, the increase in hemolytic titer observed after treatment of NHS or purified heterogeneous C4 with sialidase is a property of C4A3 but not a property of C4B1.


Relative expression of surface IgM, IgD and the Ig-associating alpha(mb-1) and beta(B-29) polypeptide chains.

Membrane immunoglobulins are associated with a transmembrane disulphide-linked heterodimer composed of an alpha-chain (mb-1) and a beta-chain (B-29). The relative surface expression of all of the polypeptide chains comprising the Ig-alpha beta complex has been investigated using surface labelling coprecipitation analysis and two-colour flow cytometric analysis. The main conclusions are that mb-1 and B-29 are B-cell surface markers on immature and mature B cells, and that all components of the surface Ig-alpha beta complex are expressed in stoichiometrically equivalent amounts. Thus the complex was quantitatively precipitated from digitonin lysates of 125I-surface-labelled cells with anti-B-29, anti-mb-1 or anti-Ig. Secondly, by two-colour FACS analysis there was a proportionality between the relative amounts of cell surface mb-1 or B-29 and surface IgM or IgD, but not other B-cell markers (class II, B220, FcR gamma, FcR epsilon). Finally there was an insignificant number of B cells expressing membrane Ig without alpha- and beta-chains, and vice versa. Thus there appears to be a closely controlled relative synthesis and surface expression of all components of the B-cell receptor complex.


Production of rheumatoid factor in adoptively immune guinea-pigs after challenge with Treponema pallidum.

Guinea-pigs of inbred strains 2 and C4D were infused with various concentrations (1 x 10(8) to 4 x 10(8) of syngeneic nylon wool-purified Treponema pallidum-immune T lymphocytes (TPI-T) and challenged 24 hr later with virulent T. pallidum (10(8) organisms). The degree of protection depended on the number of infused T cells and was associated with an accelerated production of IgM rheumatoid factor (RF). Fully protected animals (4 x 10(8) TPI-T) did not produce treponemal antibodies or circulating immune complexes (CIC) but produced IgM RF detectable 10 days after infection. Partially protected animals (< or = 2 x 10(8) TPI-T) produced, 30 days post-infection, relatively low levels of treponemal antibodies but high levels of CIC and RF. Control animals infused with 2 x 10(8) TPI-T lymphocytes but not infected with T. pallidum, when monitored for a period of 6 weeks, did not produce treponemal antibodies, CIC, or RF, excluding the possibility that IgM RF could be generated by the donor's B cells contaminating (circa 3%) the TPI-T lymphocytes. Moreover, unprotected syngeneic control animals infused, prior to infection, with T. phagedenis biotype Reiter-immune T cells or with T. pallidum-free testicular inflammatory fluid-immune T cells responded with increasing levels of treponemal antibodies; only a few animals produced RF and CIC 5 months after infection similarly to control guinea-pigs infected only. The production of RF in partially protected animals responding to infection with treponemal antibodies and CIC was apparently associated with the presence of the CIC; but the mechanism of RF production in fully protected animals in which no antibodies or CIC were detected is currently unknown.


Kinetics of tumour necrosis factor and prostaglandin production by murine resident peritoneal macrophages as affected by dietary n-3 polyunsaturated fatty acids.

Cell-associated and secreted tumour necrosis factor (TNF), prostaglandin (PG) E2, and 6-keto PGF1 alpha were monitored at various times following in vitro stimulation of resident peritoneal macrophages with lipopolysaccharide (LPS). Macrophages were obtained from mice maintained on diets containing 1.5 wt% n-3 polyunsaturated fatty acids (PUFA)+ 1.5 wt% n-6 fatty acids; 1.5 wt% n-6 fatty acids; or 3 wt% n-6 fatty acids, for 4 weeks. Cell-associated TNF increased transiently in the resident peritoneal macrophages from mice consuming all diets and decreased after TNF secretion had reached maximum and plateaued. Macrophages from mice consuming the n-3 PUFA contained more cell-associated TNF and secreted more TNF than macrophages from mice consuming diets containing n-6 fatty acids only, at all time-points studied. Macrophages from mice consuming the n-3 PUFA showed an earlier increase in cell-associated and secreted TNF compared with macrophages from mice consuming n-6 fatty acids only. Kinetics of maximum TNF production was not affected by the diets and dietary n-3 PUFA did not cause a prolonged increase in TNF secretion. Macrophages from mice consuming the n-3 PUFA produced less PG than macrophages from mice consuming the n-6 fatty acids only. PG secretion increased following appearance of cell-associated TNF but when PG had accumulated in the medium there was no further increase in TNF production.


Adult T-cell leukaemia-derived factor/thioredoxin expression on the HTLV-I transformed T-cell lines: heterogeneous expression in ALT-2 cells.

Adult T-cell leukaemia (ATL)-derived factor (ADF), originally described as an inducer of interleukin-2 receptor-alpha (IL-2R alpha/Tac), has homology with the co-enzyme thioredoxin which is involved in many dithiol-dependent reducing processes. Using antibody against the C-terminal synthetic polypeptide of ADF and RNA probe of ADF, we examined the expression of ADF in various cell lines by immunofluorescence, immunohistochemical staining, Western blotting and in situ hybridization. ADF was intensely expressed on HTLV-I+ T-cell lines as compared with HTLV-I- T-cell lines. While the Epstein-Barr virus (EBV)+ B-lymphoblastoid cell lines were intensely positive for ADF, Burkitt-derived B cell line Jijoye with defective EBV was negative for ADF. Electron microscopic and photomicroscopic analysis of HTLV-I+ ATL-2 cells showed that ADF was localized on both the cell membrane and cytosol. In ATL-2 cells, a marked heterogeneity of ADF expression was observed. In in situ hybridization, heterogeneity of ADF messenger RNA (mRNA) expression was also demonstrated, indicating that ADF expression was regulated in the transcriptional level.


Palmitic acid conjugation of a protein antigen enhances major histocompatibility complex class II-restricted presentation to T cells.

The effect on antigenicity of covalent attachment of lipid groups to a protein antigen was investigated. Coupling of palmitic acid to ovalbumin (OVA) enhanced major histocompatibility complex (MHC) class II-restricted presentation to most OVA-specific murine T-cell clones in vitro. The enhanced antigenicity of palmitoylated antigen was localized to the level of presentation of the synthetic peptide epitope, OVA 323-339. T-cell responses to palmitoylated antigen were more difficult to block with anti-MHC class II antibodies than responses to native antigen. However, T-cell proliferation to palmitoyl (p)-OVA and native (n)-OVA were blocked equally by anti-CD4 antibodies. Taken together, the results suggest that lipid conjugation of a protein antigen leads to the formation of a lipopeptide T-cell epitope with increased affinity of binding to MHC class II and/or T-cell receptor (TcR). These results have implications for the design of synthetic peptide vaccines.


Identification of a human endothelial cell activation antigen that is co-expressed by germinal follicle centre B lymphocytes.

Endothelial cell activation antigens may play important roles in immune responses and in inflammation. This report describes the identification and characterization of a monoclonal antibody, named EAA-B, which reacts specifically with human umbilical vein endothelial (HUVE) cells pre-treated with tumour necrosis factor-alpha (TNF-alpha) but not with untreated cells. The expression of the EAA-B antigen on HUVE cells could also be induced by interleukin-1 (IL-1), bacterial lipopolysaccharide (LPS), and phorbol esters but not by interferon-gamma (IFN-gamma). By contrast, EAA-B antigen expression on neonatal foreskin and rheumatoid synovial fibroblasts, whether pre-treated with TNF-alpha or not, was not detectable. Peripheral blood leucocytes and the leukaemic cell lines U937, HL-60, Raji and Molt 4 showed no detectable expression of the EAA-B antigen. Kinetic studies demonstrated that the EAA-B antigen was rapidly expressed, peaked at 6 hr and declined to basal level by 24 hr. Western blotting revealed that monoclonal antibody EAA-B recognized a polypeptide of approximately 80,000-90,000 MW. EAA-B partially blocked the augmented adhesion of HL-60 cells to TNF-treated HUVE cells. However, it failed to inhibit the enhanced binding of peripheral blood leucocytes, U937, Raji and Molt 4 Cells to TNF-treated HUVE cells. In situ, the EAA-B antigen was detected on some vascular endothelium in tonsils, lymph nodes, psoriatic skin and rheumatoid synovium but not in normal non-lymphoid tissues. Interestingly EAA-B antigen is also expressed by B lymphocytes in germinal follicle centres (GFC) of lymphoid tissues. The co-expression of this endothelial activation antigen by GFC B lymphocytes may have significant implications for immune responses and in B-lymphocyte differentiation.


Identification and tissue distribution of rabbit leucocyte antigens recognized by monoclonal antibodies.

Three monoclonal antibodies which recognize rabbit leucocytes have been characterized by immunofluorescence staining of a variety of cell populations and also by immunochemical techniques. The evidence obtained suggests that these antibodies recognize the rabbit equivalents of the CD58/LFA-3 (VC21), CD43/leukosialin (L11/135) and CD9 (MM2/57) antigens. A fourth antibody, RPN3/57, recognizes an antigen expressed strongly on T cells, thymocytes and neutrophils and at lower levels on platelets. It has not, however been possible to characterize the antigen recognized by RPN3/57 in molecular terms. Both L11/135 and RPN3/57 are useful reagents for the detection of T cells both by flow cytometry and by immunohistochemistry.


Lymphoid precursor cell lines have capacity to migrate to multiple lymphoid sites.

Cell lines have been isolated which phenotypically resemble natural killer (NK)/lymphoid precursor cells. These cell lines were derived by in vitro culture and represent continuously replicating cell lines. Since they do not grow as tumours in vivo, it was anticipated that they may have maintained the migratory capacity of their normal cell counterpart. Capacity to migrate to various lymphoid sites, i.e. spleen, bone marrow, thymus and mesenteric lymph node, has been analysed in a short-term 3-hr homing assay following intravenous injection of cells into a syngeneic irradiated host. Frequency of cells localizing in these organs was estimated by limit dilution cloning analysis. Three out of four cell lines were found to migrate to all four organs, while the C6VL/1 T-cell lymphoma was detected in spleen and bone marrow only, and did not enter thymus and mesenteric lymph nodes. This suggests that NK/lymphoid precursor cells may exist which have capacity to recirculate widely through the lymphoid system.


Regulation of leucocyte subpopulations in the sheep endometrium by progesterone.

To determine whether progesterone causes a change in lymphocyte subpopulations in the endometrium, frozen sections of intercaruncular and caruncular endometrium from ewes receiving daily i.m. injections of 100 mg/day progesterone were evaluated by immunohistochemistry for the presence of lymphoid cells bearing CD45, major histocompatibility complex (MHC) class II, CD45R, CD4 and CD8 antigens. The pattern of lymphocyte distribution in the uterine endometrium of untreated ewes was similar to previous reports. Progesterone treatment, particularly after 60 days, caused reductions in numbers of CD45+ cells in the glandular epithelium and associated subepithelial stroma, MHC class II+ cells in all regions of the intercaruncular endometrium and CD45R+ cells in all epithelial regions of intercaruncular and caruncular endometrium. These data demonstrate a role for progesterone in regulating migration or proliferation of endometrial lymphocyte populations; this action of progesterone may represent an important mechanism by which progesterone modifies uterine immune function.


The contribution of LFA-1 (CD11a/CD18) and MAC-1 (CD11b/CD18) to the in vivo migration of polymorphonuclear leucocytes to inflammatory reactions in the rat.

Recently the critical requirement for the CD18 family of adhesion molecules on leucocytes for their adhesion and migration to inflammatory reactions has been recognized in humans and several animal models. The in vivo studies have mostly utilized antibodies to CD18, the common beta-subunit of CD11a,b,c/CD18 molecules and thus have blocked the function of all three family members, making evaluation of the role of individual subunits impossible. Furthermore, none of the reagents used were suitable for studies in rats. Here we report the effects on polymorphonuclear leucocyte (PMNL) adhesion and in vivo migration of a new monoclonal antibody (mAb) TA3, which recognizes and blocks rat CD11a/CD18 (LFA-1). These studies also evaluated mAb MRC OX42, which reacts with rat CD11b/CD18 (CR3, MAC-1). Neither antibody alone inhibited rat PMNL adhesion to interleukin-1 (IL-1)-activated rat endothelium, but the combination inhibited adhesion by 44%. OX42 treatment of rat PMNL inhibited phorbol myristate acetate (PMA) activated adhesion by 88%, while TA3 only inhibited this adhesion in combination with OX42, resulting in 99% inhibition of PMA-induced PMNL adhesion. Treatment of rats with TA3 alone partially inhibited 51Cr-labelled rat blood PMNL migration into zymosan-activated serum (C5adesArg; ZAS), but not IL-1, or endotoxin [lipopolysaccharide (LPS)] induced dermal inflammatory reactions. MAb OX42 had no such effect in vivo. However, treatment with both antibodies virtually eliminated any PMNL accumulation in all three types of inflammatory reactions. Ex vivo treatment of the 51Cr-labelled PMNL, prior to i.v. infusion showed that mAb TA3 again preferentially inhibited PMNL migration to ZAS. These results suggest that in the rat, CD11a/CD18 plays a major role in PMNL migration to C5a and that either CD11a or CD11b/CD18 can function to maintain normal PMNL migration to IL-1 or LPS dermal inflammatory reactions. More than one member of this adhesion family or their ligands may need to be targeted for effective modulation of PMNL infiltration, at least in this species.


Native and recombinant soluble CD23 fragments with IgE suppressive activity.

CD23-bearing cells are known to release 37,000 33,000 and 25,000 MW soluble CD23 (sCD23) fragments that were reported to display multiple biological activities, including the potentiation of IgE synthesis. We previously reported that tunicamycin treatment of RPMI-8866 cells switched the biological activity of the sCD23 released by these cells from IgE potentiation to IgE suppression. In this study we show that tunicamycin-treated cells release small CD23 fragments with a MW of 16,000. These fragments are formed by truncation of the N-terminal 160 amino acids and truncation of the carboxy-terminal end of CD23. Two observations indicate that the cleavage of surface CD23 into 16,000 MW fragments is not caused by tunicamycin-mediated inhibition of the N-glycosylation of CD23 but rather by the deletion of the carboxy terminal end of the molecule: (1) Chinese hamster ovary (CHO) transfectants expressing a CD23 mutant lacking the N-glycosylation site release 37,000-33,000 MW sCD23 unless they are treated with tunicamycin; (2) transfectants expressing a CD23 deletion mutant lacking the last 33 carboxy-terminal amino acids release 16,000 MW sCD23. Highly purified native and recombinant 16,000 MW sCD23 bind to IgE and down-regulate the ongoing and the interleukin-4 (IL-4)-stimulated synthesis of IgE.


Lymphocytes bearing the gamma delta T-cell receptor in acute toxoplasmosis.

Although the relative and absolute numbers of CD3+ cells (T lymphocytes) were similar in eight children with acquired Toxoplasma gondii infection and 10 uninfected age- and sex-matched healthy controls, the proportion of cells bearing the gamma delta T-cell receptor was significantly higher in the subjects with acute toxoplasmosis. The great majority of gamma delta T cells from the infected patients expressed covalently bound gamma delta chains on their surface, i.e. were BB3+ lymphocytes. Since the gamma delta T-cell subsets exert both restricted and unrestricted major histocompatibility complex cytotoxicity, further research is needed to elucidate the role of gamma delta T cells in the control of this coccidian protozoan infection.


Abnormal TNF production in prediabetic BB rats is linked to defective CD45R expression.

The genetic basis and diabetes association of aberrant tumour necrosis factor-alpha (TNF-alpha) production by activated peritoneal macrophages in diabetes-prone (dp) biobreeding (BB) rats was analysed. Southern blot analysis could not detect a restriction fragment length polymorphism for the TNF gene distinguishing dp BB rats from Wistar (Wi) rats and diabetes resistant (dr) BB rats. The contiguous genetic arrangement of lymphotoxin (LT) and TNF genes described in mouse and man was also found in the rat by cloning a chromosomal region covering both genes. In search of a polymorphic marker we amplified a (CA)n:(GT)n microsatellite in the TNF promoter region by polymerase chain reaction (PCR). We detected two alleles, (CA)26 and (CA)33, but no correlation with diabetes risk was seen. Crosses between dp BB rats and Wi or Lewis. 1A (Lew. 1A) rats, respectively, indicated that aberrant TNF-alpha production of activated macrophages is inherited dominantly with only weak penetrance. Analysis of the F2 generation and backcrosses with the two parental strains showed that aberrant TNF production co-segregates with lymphopaenia and defective CD45R expression, markers known to reflect a diabetes predisposing gene(s) outside the RT1 complex. We conclude that a single linkage group is responsible for both aberrant TNF production and defective T-cell maturation in dp BB rats.


Liposome potentiation of humoral immune response to lipopolysaccharide and O-polysaccharide antigens of Brucella abortus.

Liposomes were evaluated for their effectiveness as vaccine carriers in the potentiation of the mouse humoral response to the lipopolysaccharide (LPS) and O-polysaccharide (OPS) antigens of Brucella abortus. LPS and OPS were extracted from a pathogenic strain of B. abortus and were encapsulated within multilamellar vesicles. Groups of mice, immunized with liposome-encapsulated and free LPS or OPS, were bled weekly and the specific IgM and IgG levels in the sera were determined by an indirect fluorogenic enzyme-linked immunosorbent assay. Humoral response to these antigens were found to be dose-dependent. Mice immunized with LPS and OPS encapsulated within liposomes were found to have significantly higher IgG levels than mice immunized with free LPS and OPS. In addition, the antibody levels in mice that were immunized with liposome-encapsulated LPS and OPS were more sustained and remained at elevated levels--even after 5 weeks post immunization. As expected, OPS was found to be less immunogenic than LPS, but multiple injections of OPS encapsulated within liposomes greatly improved the immunogenicity. These results indicate that the humoral response to LPS and OPS of B. abortus can be enhanced when these antigens are encapsulated within liposomes.


Control of Langerhans' cell density by a skin tumour-derived cytokine.

Langerhans' cells (LC) are bone marrow-derived dendritic antigen-presenting cells (APC) found in the epidermis of mammals. It is not known why they accumulate in the epidermis. Human and murine skin tumours are infiltrated with large numbers of LC, however previous experiments have shown that this does not seem to be associated with immune responses against the tumours. Here we show that a squamous-derived tumour cell line (T7) produces a cytokine which increases the number of LC in normal epidermis. T7 supernatant increased the density of LC in both mice syngeneic to the T7 cells (Skh:HR-1) as well as in BALB/c mice, indicating that the cytokine is not genetically restricted. The cytokine is a protein, not a prostaglandin, with a MW of > 12,000 as its production was inhibited by cycloheximide but not indomethacin and it could not be removed by dialysis against a 12,000 MW cut-off membrane. The increased numbers of LC found in tumour supernatant-treated epidermis expressed Ia as well as the molecule defined by the J11d monoclonal antibody, which is expressed by LC but not macrophages, confirming that these cells are LC. Another squamous-derived skin tumour, T79, which is not infiltrated with large numbers of LC when inoculated into syngeneic mice, did not produce a factor capable of increasing the density of LC. Hence these studies demonstrate either the activity of a novel cytokine or a new biological activity of a previously described cytokine. It is most likely that this cytokine increased the number of LC by attracting precursors into the epidermis. As the cytokine was produced by transformed squamous cells it is also possible that this cytokine is responsible for attracting LC into normal epidermis.


Characterization of human mast cells developed in vitro from fetal liver cells cocultured with murine 3T3 fibroblasts.

Cocultures of dispersed human fetal liver cells with murine Swiss 3T3 fibroblasts resulted in the development of human mast cells after 1 to 4 weeks of culture. Mast cells were detected by immunohistochemistry using a murine monoclonal anti-tryptase antibody, before metachromasia appeared with toluidine blue. When subjected to double immunohistochemistry using murine monoclonal anti-chymase and anti-tryptase antibodies, 94% +/- 10% (SD) of the mast cells seen at day 30 of culture were of the MCT type. These results contrast with those obtained with human mast cells derived from cord blood mononuclear cells cocultured with murine 3T3 fibroblasts which are comprised of substantially greater numbers of MCTC cells, averaging 48% +/- 31% (SD) at day 30 of culture. Mast cells developed in vitro from fetal liver cells or cord blood mononuclear cells contained similar amounts (+/- SD) of histamine (0.9 +/- 0.5 pg/cell and 1.1 +/- 1 pg/cell, respectively) and tryptase (1.7 +/- 0.4 pg/cell and 1.9 +/- 1.2 pg/cell, respectively) on day 30 of culture. Fetal-liver-derived mast cells from a 30-day-old culture were identified by immunoelectron microscopy using gold-labelled antitryptase antibody. Typically, these mast cells appeared immature as they had large nuclear to cytoplasmic ratio and a small number of ill-formed cytoplasmic granules. For both fetal-liver- and cord-blood-derived mast cells, there was no evidence of conversion of the MCT type into the MCTC type provided by this study. These results suggest that commitment to develop as an MCT or MCTC type of mast cell may have occurred in mast cell precursors present in fetal liver and cord blood mononuclear cells, prior to granulation.


Down-regulation of a spontaneous arthritis in male DBA/1 mice after administration of monoclonal anti-idiotypic antibodies to a cross-reactive idiotope on anti-collagen antibodies.

We recently described a spontaneously occurring, inflammatory and erosive joint disease in male DBA/1 mice. A major question is whether specific immune reactions are involved in eliciting this disease. The possibility that collagen autoimmunity might constitute one pathogenic factor was particularly interesting as this spontaneous arthritis appears to be genetically restricted in a way similar to collagen-induced arthritis. In the present study, we demonstrate increased serum antibody levels to collagen II in a fraction of the male DBA/1 mice, but not in age-matched female controls. Administration of antibodies with an anti-idiotypic activity to anti-collagen II antibodies and with an affinity for determinants present on isolated syngeneic IgG Fc but not on intact IgG, was shown to interfere with the development of the spontaneous arthritis in a manner similar to that earlier documented for collagen arthritis. These observations suggest that mechanisms similar to those operating in collagen-induced arthritis may also be found in the spontaneously occurring arthritis in male DBA/1 mice.


Neutrophil priming by hepatocyte growth factor, a novel cytokine.

We demonstrate here that the recently defined cytokine hepatocyte growth factor (HGF) 'primes' human neutrophils. Recombinant human HGF over the concentration range 0.1-20 ng/ml increased the neutrophil response to f-met-leu-phe by up to 200%, and required only a short preincubation, 10 min producing the maximum effect. Priming was independent of changes in cytosolic-free calcium homeostasis. We conclude that HGF may be a physiologically important cytokine with 'priming' activity for neutrophils.


Development and characterization of a monoclonal antibody specific for the bovine low-affinity interleukin-2 receptor, BoCD25.

An IgM monoclonal antibody, UC-2C2 was produced using splenocytes from mice immunized with cultures of interleukin-2 (IL-2)-dependent bovine peripheral blood lymphocytes. UC-2C2 was found to recognize a cell surface antigen of apparent molecular weight 52,000-54,000 present on activated bovine peripheral blood mononuclear leucocytes (PBML) but not on resting PBML or cells of the bovine lymphoblastoid cell line BL3. The 52,000-54,000 MW antigen was expressed early following activation of PBML by mitogens or alloantigens, with the majority of cells positive by 48 h of culture. UC-2C2 was unable to block binding of phycoerythrin (PE)-conjugated human recombinant IL-2 to PHA-stimulated bovine PBML as determined by flow cytometric analysis. However, two-colour analyses indicated that the antigen recognized by UC-2C2 was present on the same cell population that expressed IL-2 receptors. All activated T lymphocytes of BoCD4, BoCD8 and gamma delta receptor positive phenotypes expressed the target antigen of UC-2C2 and IL-2 receptors. Monocytes and B lymphocytes expressed the target antigen of UC-2C2 and IL-2 receptors at a lower density. This differential expression by the various PBML subpopulations parallels that described for expression of the low-affinity IL-2 receptor (CD25) on human leucocyte subpopulations. Based upon the relative molecular weight, time-course of expression and cellular distribution of the antigen identified by UC-2C2, it is inferred that UC-2C2 recognizes an epitope on the bovine homologue of CD25 which is not involved in binding IL-2.


Interactions between interleukin-2-activated lymphocytes and vascular endothelium: binding to and migration across specialized and non-specialized endothelia.

A prerequisite for the successful immunotherapy of solid tumours with interleukin-2 (IL-2)-activated lymphocytes is their ability to home to the tumour tissue. Lymphocyte homing is a complex process which is known to involve at least two independently regulated events: adhesion to the luminal surface of vascular endothelium and the subsequent transendothelial migration of lymphocytes. In this study we have used an in vitro model of lymphocyte homing which employs specialized high endothelium to ask whether IL-2-activated lymphocytes are able to migrate across vascular endothelium in order to leave the blood vessel. Both the adhesion of IL-2-activated cells and their migration across monolayers of cultured high endothelial cells (HEC) were increased in comparison with non-activated lymphocytes. The adhesion of IL-2-activated lymphocytes was mediated by lymphocyte function-associated antigen-1 (LFA-1) and a very late activation antigen-4 (VLA-4)-related pathway. LFA-1-dependent adhesion was mediated by ligands on HEC other than the intercellular adhesion molecule-1 (ICAM-1) and the VLA-4-related pathway was mediated by ligands other than the CS1 domain of fibronectin. HEC-adherent lymphocytes were enriched in natural killer (NK) cells and CD8+ T cells which are known to be the tumour-cytotoxic cells in IL-2-activated lymphocytes. However, there was no evidence of cytotoxicity towards the endothelial layer using a syngeneic model. The interaction of IL-2-activated lymphocytes and endothelial cells was not specific for high endothelium since equal numbers of activated lymphocytes bound to and migrated across aortic endothelium. The inability of IL-2-activated lymphocytes to discriminate between high endothelium and non-specialized 'flat' endothelium could be responsible for the widespread dissemination of the cells throughout the body following their adoptive transfer and the unwanted side-effects at non-involved sites.


Inhibition of CD25 (IL-2R alpha) expression and T-cell proliferation by polyclonal anti-thymocyte globulins.

Anti-lymphocyte and anti-thymocyte globulins (ATG) are currently used as immunosuppressive agents in organ transplantation. Their administration in vivo may induce not only lymphocyte depletion but also functional effects which were investigated in the present study. In vitro ATG inhibited T-cell proliferation induced by monocyte-dependent T-cell mitogens, like CD3 antibodies, phytohaemagglutinin (PHA) and concanavalin A (Con A), by monocyte-independent mitogens, like CD2 antibodies, or by protein kinase C activators (phorbol esters) associated with a calcium ionophore. The inhibitory effect of ATG was therefore not solely accounted for by a suppression of co-stimulatory signals delivered by monocytes, but rather implied a direct action on T cells. Addition of recombinant human interleukin-2 (rIL-2) did not overcome the inhibition. Suppression of T-cell proliferation by ATG was characterized by normal RNA synthesis and IL-2 secretion contrasting with markedly reduced expression of the CD25 protein [p55, the alpha-chain of interleukin-2 receptor (IL-2R)] both in cytoplasm and on T-cell membrane, as well as a decreased secretion of interferon-gamma (IFN-gamma). Northern blot analysis revealed increased levels of CD25 and IFN-gamma mRNA, suggesting a post-transcriptional inhibition of these molecules, whereas IL-2 mRNA levels were unchanged. These data demonstrate that inhibition of T-cell proliferation by ATG can be attributed primarily to a post-transcriptional defect of CD25 expression, implying a novel mechanism different from those described with other immunosuppressive agents. Blocking of T-cell proliferation in the late G1 phase of the cell cycle may contribute to the immunosuppressive activity of ATG in prophylactic treatment of allograft rejection.


Transforming growth factor-beta enhances interleukin-6 secretion by intestinal epithelial cells.

Recent reports have suggested that transforming growth factor-beta (TGF-beta) may have an important role in IgA immune responses, e.g. induction of surface IgM+ B cells to commit to IgA. TGF-beta is also an important regulatory cytokine for the maturation of intestinal epithelial cells. Using the IEC-6 rat intestinal epithelial cell line as a model system, TGF-beta 1 was found to enhance interleukin-6 (IL-6) secretion by the IEC-6 cells. The IL-6 was produced in a dose-dependent manner and secretion could be specifically inhibited by an anti-TGF-beta 1 antibody. IL-6 production by the IEC-6 cells was confirmed by using a rabbit anti-mouse IL-6 antibody which completely neutralized the IL-6 present in the IEC-6 cell supernatant. The enhancement of IL-6 secretion was found to involve a low-level enhancement in the expression of RNA for IL-6. The induction of IL-6 secretion was also reversible when TGF-beta was removed. These results suggest that the action of TGF-beta on intestinal epithelial cells may play an important role in immune responses at the intestinal mucosa.


Histological and functional differentiation of non-lymphoid cells in the chicken spleen.

The phenotypes and functions of various populations of non-lymphoid cells in the chicken spleen were investigated with monoclonal antibodies and after in vivo administration of antigens. Monoclonal antibody CVI-ChNL-68.1 was used to detect red pulp macrophages, interdigitating cells, and monocytes, whereas CVI-ChNL-68.2 was used to detect ellipsoid-associated non-lymphoid cells (EANC). Two new monoclonal antibodies were developed: CVI-ChNL-74.2, which specifically recognized red pulp macrophages and a ring of macrophages surrounding the peri-ellipsoid lymphocyte sheath; and CVI-ChNL-74.3, which specifically recognized follicular dendritic cells (FDC) in germinal centres and small clusters of stromal cells in T-cell areas. After intravenous injection of lipopolysaccharide (LPS), the number of 68.1+ and 74.2+ macrophages decreased dramatically, whereas 68.2+ EANC and 74.3+ FDC were unaffected. After intravenous injection of heat-inactivated Brucella abortus, the numbers of both macrophages and EANC decreased. In contrast, a significant increase of 74.3+ cells was observed in the T-cell areas outside the germinal centres. As expected, intravenous injection of non-mitogenic antigens, such as keyhole limpet haemocyanin and Ficoll, and carrageenan did not affect the non-lymphoid cell populations. At least six subpopulations of non-lymphoid cells in the chicken spleen can now be discriminated with monoclonal antibodies. Our results show that mononuclear phagocytes are sensitive for mitogenic stimulators such as LPS and Brucella abortus. In contrast, stromal non-lymphoid cells are only sensitive to the particulate mitogen Brucella abortus. We conclude that the complex formed by ellipsoid cells, the peri-ellipsoid B-cell sheath, and the surrounding macrophages, is the functional equivalent of the mammalian marginal zone.


The role of antigen-presenting cells in the regulation of delayed-type hypersensitivity. I. Spleen dendritic cells.

A previously unreported mechanism for the induction of delayed-type hypersensitivity (DTH) was studied in detail. The subcutaneous injection (without adjuvant) of spleen dendritic cells (DC) pulsed with keyhole limpet haemocyanin (KLH) into syngeneic BALB/c mice caused DTH when the ear was later injected with the same antigen. When KLH-pulsed DC were transferred intravenously, DTH was not induced, although the titre of anti-KLH antibodies rose after such transfer. The intravenous transfer of KLH-pulsed DC into mice immunized subcutaneously with KLH in complete Freund's adjuvant (CFA) at the same time (in the sensitization phase), but not when the ear was challenged with KLH (in the effector phase), had a suppressive effect on DTH, in an H-2-restricted way. When radiolabelled DC were transferred intravenously, they migrated into the spleen, but when transferred by subcutaneous injection, they stayed in the skin or migrated into the lymph nodes. In splenectomized mice immunized with KLH, the intravenous transfer of KLH-pulsed DC did not cause production of anti-KLH antibodies and did not suppress DTH. These findings suggest that the anatomical sites in which antigens are presented (i.e. the spleen or lymph nodes) rather than the type of cell that first presents antigens to the immune system governs whether DTH or antibody production is induced. Antibody production was induced when antigens were presented in the spleen.


A role for tumour necrosis factor-alpha in the human mixed lymphocyte culture reaction.

The possible role and influence of tumour necrosis factor-alpha (TNF-alpha) in the human mixed lymphocyte culture (MLC) reaction was investigated. Polyclonal rabbit sera to recombinant human-TNF-alpha (rhTNF-alpha) consistently inhibited the MLC, and this could be reversed by simultaneous addition of rhTNF-alpha to the culture medium. Under optimal cell culture conditions, the exogenous addition of 1000 U/mL of rh TNF-alpha had a variable but generally small enhancing effect on the MLC reaction. Additional studies have clearly highlighted the importance of MLC assay conditions in influencing the effect of exogenous rhTNF-alpha. Enhancement of the MLC reaction was only consistently observed under suboptimal cell culture conditions. While the present study clearly supports an important role for TNF in controlling the proliferative response in the human MLC reaction it also highlights the influence of in vitro tissue culture conditions in determining the effect of exogenous TNF-alpha in this assay.


Modulation of thymocyte subsets during acute and chronic phases of experimental Trypanosoma cruzi infection.

Several observations have demonstrated the importance of T-cell-mediated mechanisms in experimental Chagas' disease. In previous studies, we have shown that mice acutely infected by Trypanosoma cruzi develop a progressive thymic atrophy with severe alterations in the lymphoid compartment. In this report we performed a kinetic analysis of the murine thymic lymphocytes comparing acute and chronic phases of infection. At the chronic phase, we observed that total thymocyte numbers returned to age-matched control values. Additionally, the decrease in the percentage of CD4+CD8+, in parallel with an increase of CD4+CD8-, CD4-CD8+, CD3high, TcR alpha beta and TcR gamma delta cells detected in the acute infection, was also restored in chronically infected mice. This thymocyte recovering is probably linked to the increase in the percentage of thymocyte precursors, such as CD4lowCD8- and CD4-CD8low cells, together with the increase in the number of IL-2R+ and cycling cells, appearing in the late stages of acute infection.


Defining the nucleic acid substrate for somatic hypermutation.

Recent reports have more precisely defined the distribution of somatic mutations around rearranged mouse V-D-J genes. The 5' boundary of mutation is most likely in the region of the transcription start site (cap) and/or the promoter (P), implying that transcription may be a prerequisite for mutations to be generated. As more than 95% of somatic mutations lie downstream of the cap site, the transcription unit itself is implicated as the target of the mutational machinery. For heavy chain genes, the 3' boundary can extend into the enhancer region (E). For kappa light chain genes, the 3' boundary extends to approximately 700 bp beyond J kappa-5 (approximately 700 bp upstream of E). In a single study on mutated derivatives of the rearranged mouse lambda 1 light chain V-J gene, it was claimed that the 3' boundary fell within the constant region (C) exon. Although more data are required, the frequency of mutations around V-D-J genes appears asymmetrical, being positively skewed with a single mode centred near the V-D-J coding region and a long tail extending into the 3' non-translated region of the J-C intron. Such a distribution may place constraints on possible molecular mechanisms. It is suggested that the apparent asymmetrical pattern of mutation is best explained by models that assume localized error-prone DNA synthesis generating variable fragment lengths of mutated DNA or cDNA retrotranscripts. The frequency distribution of these lengths of mutated DNA is positively skewed into the 3' J-C intron, with a common terminus at or near the cap site. It is then assumed that they can be integrated into the target V-D-J region via a gene conversion or homologous recombination process. The model invoking reverse transcription may be preferred as it best explains the data without too many ad hoc assumptions.


Expression of the high responder/non-responder human Fc gamma RII. Analysis by PCR and transfection into FcR-COS cells.

Distinct differences in the capacity of monocyte Fc gamma RII of different individuals to bind or not bind mouse IgG1 defines a polymorphism of Fc gamma RIIa and has previously been defined as the high responder (HR) or low responder (LR) polymorphism of Fc gamma RII. The precise definition of the molecular basis of the human HR/LR polymorphism of Fc gamma RIIa from the peripheral blood mononuclear cells of normal individuals has been determined by anti-CD3 induction of T cell proliferation, the polymerase chain reaction (PCR), nucleotide sequencing, transfection and IgG binding. Amplification of first strand cDNA from mRNA isolated from mononuclear cells was performed by PCR using primers specific for the sequences encoding the leader and cytoplasmic sequences of PCR using primers specific for the sequences encoding the leader and cytoplasmic sequences of Fc gamma RIIa, which is normally expressed in monocytes. Sequencing of the PCR products and transfection of these to Fc gamma R- cells indicated that in Fc gamma RIIa of HR or LR individuals: (i) three nucleotide substitutions (CA to TG and G to A) resulted in the change of glutamine to tryptophan at position 27 (first extracellular domain) and arginine to histidine at position 131 (second extracellular domain); (ii) expression of cDNA encoding the various combinations of these indicated that arginine at position 131 was essential for IgG1 binding whereas the amino acid changes at position 27 had no effect; and (iii) IgG1 at high concentration bound to all allomorphic forms of Fc gamma RIIa.(ABSTRACT TRUNCATED AT 250 WORDS)


Pattern of the action of a suppressor factor produced by a human macrophage-like cell line, U937.

A U937 suppressor factor (U937SF) was purified from crude supernatant by sequential chromatography using fast protein liquid chromatography. The molecular weight and isoelectric point of U937SF were 69 kDa and 4.5, respectively. The U937SF preparation inhibited the proliferative response in human PBMC stimulated with an antigen tuberculin purified protein derivative, tetanus toxoid) or a mitogen (phytohaemagglutinin concanavalin-A). U937SF depressed both interleukin-2 (IL-2) production and IL-2 receptor (CD25) expression in peripheral blood mononuclear cells (PBMC) stimulated with an antigen but not with a mitogen. Anti-CD3 monoclonal antibody-induced responses including a proliferative response, IL-2 production and CD25 expression were suppressed by U937SF. In contrast, U937SF did not affect monocyte functions such as antigen processing and IL-1 production. Neither did it modulate the expression of T cell receptor (TCR) or CD3 molecules on the surface of lymphocytes. Moreover it did not inhibit CD25 expression in PBMC stimulated with phorbol myristate acetate plus A23187. These results suggest that U937SF prevents both IL-2 production and CD25 expression in lymphocytes activated through the TCR/CD3, but not through the other receptors or molecules. In addition, U937SF does not block the early activation events following TCR-mediated stimulation, nor affect the pre-TCR activation steps.


Immunomodulatory activity of a peptide isolated from Viscum album extract (NSC 635 089).

A peptide isolated from Viscum album extract (Iscador) has been earlier reported to have cytotoxic and tumour reducing activity. Administration of the peptide (2 micrograms/ml) was found to produce increased natural killer cell activity (NK-activity) in the normal animals and tumour bearing animals. The peak activity was observed on the 3rd day after the administration of the peptide. Administration of the peptide also stimulated antibody dependent cellular cytotoxicity (ADCC) which was expressed maximally on the fourth day. There was also an increase in antibody forming cells in the spleen, and antibody titers were increased in the animals treated with the peptide. Activity of the crude plant extract coincided with the activity of the peptide, indicating that the isolated peptide is mainly responsible for the immunostimulatory activity present in Viscum album extract.


Human lymphocyte stimulation by legume lectins from the Diocleae tribe.

Lectins from eight leguminous seeds from the Diocleae tribe were compared to Concanavalin A (Con A), a well known T cell mitogen, on the stimulation of lymphocyte proliferation and Interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) from normal volunteers. Lectins from Canavalia brasiliensis and Dioclea virgata induced the highest lymphocyte proliferation, both much higher than levels obtained with Con A, whereas lectins from Dioclea guianensis var. lasiophylla and from Canavalia bonariensis induced the lowest stimulation. Lectins from Dioclea rostrata, D. grandiflora, D. violacea and Cratylia floribunda induced intermediate levels of proliferation. The highest stimulation for IFN-gamma production was obtained with the lectin from D. rostrata, followed by those of C. floribunda and C. brasiliensis; only the lectins from D. virgata and C. bonariensis induced an IFN-gamma production lower than the one obtained by Con A-stimulation. Since all these legumes belong to the same tribe of C. ensiformis (Con A), and all are supposed to exhibit very similar lectins, it is interesting the high variation in the stimulation of lymphocyte proliferation. It is also noteworthy the dissociation between this parameter and IFN-gamma production in the case of D. virgata. A detailed analysis on the structure of such lectins, and their ligand sugars on lymphocyte surface is necessary to further explore such differences.


Dysregulated proteolysis in AIDS.

Plasmin activity induced by different concentrations of added urokinase (UK) in serum from 20 patients with AIDS (P) and 10 healthy control (HC) subjects was measured using the fibrin plate assay. 125I-fibrin coated 24 well plates were exposed to UK (0.34-6.8 ng/ml) or to trypsin (T) (2.5 micrograms/ml) in the presence of serum (10%) from P or HC. Control wells were exposed to either T or tris buffer (pH 8.1) alone. Volumes were adjusted to 1 ml with buffer and after 1 hr at 37 degrees C, radioactivity (cpm) released into the medium was determined using a gamma counter. Data are expressed as % plasmin activity or as % inhibition of plasmin activity. All sera from HC totally abrogated (greater than 98%) the plasmin activating ability of UK (1.7 ng/ml). In contrast, sera from approximately 50% of P were less able to inhibit plasmin activation (to 26%). The inability of P sera to inhibit plasmin activation was specific since P and HC sera were equally capable of inhibiting T. Mixing experiments using P and HC sera demonstrated that P sera did not block the ability of HC sera to inhibit plasmin activation. The inhibitory activity of HC and active P sera eluted in the void volume of a spehadex G100 column (MW greater than 100,000 daltons) and is acid sensitive, however HC sera also contains an acid stable inhibitor(s) of plasmin activation not detected in P sera. These data suggest dysregulation of UK dependent proteolysis may be associated with AIDS.


Profile of cytokines in synovial fluid specimens from patients with arthritis. Interleukin 8 (IL-8) and IL-6 correlate with inflammatory arthritides.

The synovial fluid aspirated from patients with symptomatic arthritis was analyzed for the presence of tumor necrosis factor (TNF), interleukin 6 (IL-6) and interleukin 8 (IL-8). All three cytokines were found in both inflammatory and non-inflammatory arthritides: IL-8 levels ranged from less than 20 to 38,990 pg/ml, IL-6 from less than 10 to 72,300 pg/ml and TNF from less than 4 to 61 pg/ml. No inhibitors of cytokine activity were found. IL-8 and IL-6 were present in significantly higher levels in patients with inflammatory arthritis compared to patients with osteoarthritis, and there was significant correlation between the IL-6 and IL-8 levels. These findings document the presence of multiple cytokines in the synovial fluid specimens of patients with arthritis, and demonstrate that higher cytokine levels accompany inflammatory arthritis.


The effect of divalent metal ions on the thermostability of C3.

Thermostability studies on human complement component C3 showed that saturation of the electronegative sites in the C3 molecule with divalent cations offered some protection against heat denaturation, a finding not previously reported in the literature. To inactive C3 in about 30 minutes, the required temperature was 61 degrees C in the presence of excess Ca2+ ions, 59.5 degrees C in the presence of excess of Mg2+ ions, but only 54 degrees C in the absence of these ionized metal ions.


Negative regulation of interleukin-1 in immune reactive cells.

Interleukin-1 (IL-1) is an immunological regulator with a multitude of effects. Recently, IL-1 inhibitors from urine, monocytes, or monocytic cell lines have been described. We previously demonstrated an IL-1 inhibitor from human monocytes under immune complex or immunoglobulin stimulation. The present studies were initiated to determine the production of IL-1 inhibitor from human polymorphonuclear cells (PMN), B and T lymphocytes in response to certain stimuli using a murine thymocyte system responsive to IL-1. My results indicated that the inhibitor is constitutively present in PMN because unstimulated PMN supernatants also show inhibitory activity. B and T lymphocytes can not produce IL-1 inhibitor under zymosan, immunoglobulin, or immune complex stimulation. The presence of this PMN inhibitor may also be important in the negative regulation of IL-1.


Effectiveness of an oral enteric coated vibrio vaccine for use in salmonid fish.

An oral enteric-coated Vibrio anguillarum vaccine was developed by initially coating lyophilized bacteria onto 0.9 mm diameter dextrose sugar beads followed by an Eudragit L-30D coat to serve as enteric protection. Vaccine efficacy was determined by both an in vivo challenge with live pathogen and measurements of serum and mucus antibody levels by ELISA. Survival rates after challenge were 80.3%, 83.3% and 70.3% among the vaccine group, positive and negative controls respectively. Serum and mucus antibody levels were found to be significantly greater in the vaccine group (p less than 0.01). In cases where equivalent survival among tested groups is observed, determination of antibody titer by ELISA may be a preferable indicator of vaccine efficacy.


Augmentation of natural cell-mediated cytotoxic activity by supernatant from in vitro mitogen-stimulated, in vivo hormone-treated lymphocytes in immature male (K strain) chickens.

Newly hatched White Leghorn male chicks were used in this study. Triiodothyronine (T3; 0.1 or 1 ppm) and Thyrotropin Releasing Hormone (TRH; 1 or 5 ppm) were added to the feed for an 8-week period starting at hatch. A fifth group received the unsupplemented diet and served as controls. Peripheral blood lymphocytes from each treatment group were cultured in vitro with or without different mitogens (PHA, Con-A, or LPS), and the culture supernatants were tested for the presence of lymphokines (LK). The natural cell-mediated cytotoxicity (NCMC) assay was carried out with or without supernatant using the standard chromium (Cr51) release assay. Control untreated chicks were used as donors for effector cells and the P815 mouse mastocytoma was used as a target. Supernatant from in vivo 1 ppm T3- or 5 ppm TRH-treated lymphocytes significantly suppressed NCMC (or cells mediating NCMC, e.g., NK cells). However, supernatant from 1 ppm T3-treated, PHA-stimulated lymphocytes significantly enhanced NK cells cytotoxicity, while supernatant from 5 ppm TRH-treated lymphocytes with PHA stimulation tended to suppress cytotoxicity. These results provide evidence supporting a regulatory role of the hypothalamo-pituitary thyroid axis on lymphokine (LK) production. The results also suggest that these hormones act on different subpopulation of lymphocytes, and therefore, the mediators released by them.


An autopsy study of relationship between perinatal stomach capacity and birth weight.

Aspiration pneumonia is a common complication of overfeeding in neonates. Since overfeeding may be related to neonatal stomach capacity, it was considered worthwhile to obtain such data. To measure the capacity of stomach obtained from fresh stillbirths and liveborn infants at autopsy and correlate the same with their birth weights. Stomach capacity was measured at autopsy in 63 stillborn and 37 newborn infants with birth weights ranging from 500 g to 3500 g. Stomach capacity had a significant positive correlation with birth weight (r = 0.56, p < 0.001). A formula and a nomogram have been derived to estimate stomach capacity from birth weight. There was no significant difference in stomach capacity between liveborn and stillborn infants in any of the weight groups, except in the 1501-2000 g weight group (p < 0.001). Our results have provided normative data on stomach capacity across a wide birth weight range in the perinatal period.


Sodium and potassium content of liquid antacids.

Sodium and potassium contents of 37 commercially available liquid antacid preparations were estimated. Only nine preparations had sodium content below 60 mmol/L. The potassium content was below 1 mmol/L in 26 preparations. We conclude that caution should be exercised in selecting liquid antacid preparations in whom critically ill patients for electrolyte overload could prove harmful.


Effect of blockade of transdiaphragmatic absorption of bacteria on survival in peritonitis: an experimental study in rats.

An experimental rat model of established peritonitis was used to test the effect of intraperitoneal injection of platelet rich plasma (PRP) on blood and peritoneal fluid culture positivity and survival rates. Thirty animals divided into two groups of 15 each were studied. The first group served as control while animals in the second group received intraperitoneal injection of PRP. The use of PRP in established. Peritonitis was of no significant benefit.


Gluteal fistula--an unusual manifestation of carcinoma colon.

A case of carcinoma cecum presented as a gluteal enterocutaneous fistula. Tumor excision provided effective palliation for nearly three years.


Transmural bowel infarction: a rare complication of aortic dissection.

A 50 year old hypertensive man presented with acute epigastric pain associated with massive gastrointestinal bleed and died within 48 hours of admission. Autopsy revealed transmural infarction of the gut due to a long aortic dissection.


Benign non-traumatic inflammatory stricture of the common bile duct.

A case of isolated benign non-traumatic inflammatory stricture of the mid portion of the common bile duct presenting with recurrent jaundice and cholangitis is reported. The histological features suggested that this was not due to sclerosing cholangitis.


Primary malignant hemangioendothelioma of jejunum.

Primary malignant hemangioendothelioma is a rare tumor. We report a patient with a malignant jejunal hemangioendothelioma which had metastasized to the regional lymph nodes and the liver.


Prepancreatic preduodenal portal vein.

We report a 17 year old girl with prepancreatic and preduodenal portal vein. She presented with recurrent vomiting. Barium study revealed malrotation of the gut. Laparotomy confirmed malrotation of the gut with a prepancreatic and preduodenal portal vein. The patient is asymptomatic after gastrojejunostomy and vagotomy.


Genetics of cholera toxin.

Cholera is caused by the toxin secreted by Vibrio cholerae 01. Cholera toxin (CT) is a protein consisting of A and B subunits. The former contributes to intracellular toxicity whereas the B subunit is required for binding of CT to eukaryotic cell surface receptor. The structural genes encoding A and B subunits are designated as ctxA and ctxB respectively. These genes are located on the chromosome forming an operon in which ctxA precedes ctxB. The ctxAB have been cloned and sequenced. Classical strains contain two full copies of unlinked ctxAB. Most el tors have single copy. However, in some strains there are two copies which are arranged in tandem. The tandem duplication and amplification of ctxAB is controlled by a transposable element like DNA sequence called RS1. A number of genes have been identified which regulate the expression of ctx operon. V. cholerae seems to elaborate more than one toxin which are different from the one encoded by ctxAB genes.


Serum beta 2 microglobulin levels in HIV seropositive persons.

As beta 2 microglobulin (B2M) has been found to be elevated in immunological disorders and also in HIV infection, its levels were studied in 475 HIV seropositive, asymptomatic persons; 101 HIV seronegative persons from high risk groups for HIV and 99 healthy controls. The B2M levels in asymptomatic HIV seropositives are found to be significantly higher than healthy controls (1.0 mg/1 to 2.7 mg/1, P less than 0.001) and HIV seronegatives from high risk groups (1.1 mg/1 to 2.7 mg/1, P less than 0.001). Two hundred and thirty four (49.26%) seropositives showed increased levels of serum B2M. Thus, quantitative analysis of B2M may be useful as an early nonspecific marker of HIV infection and immune dysfunction. The prognostic value of B2M was assessed in a follow up study of 54 HIV seropositives in a 2 yr period. Within this period, B2M levels were found to be significantly increased in these subjects (1.2 mg/1 to 4.6 mg/1, P less than 0.001). Three of the subjects who showed high increase in the B2M levels, progressed to AIDS-related condition, whereas one progressed to persistent generalised lymphadenopathy. Thus, the rising levels of B2M appears to correlate well with disease progression.


HIV infection in patients of liver cirrhosis.

A total of 130 patients of liver cirrhosis (97 males, 33 females; aged 9-70 yr) of various etiologies were subjected to anti HIV antibodies testing by ELISA and supplementary Western Blot (WB) tests. Eleven patients were positive by ELISA. Of these 11 patients, 5 were WB positive, 4 were WB negative and 2 were indeterminate. Of the 5 WB positive patients none had received blood transfusions and one was a homosexual. These results indicate that HIV infection was present in 3.8 per cent patients of liver cirrhosis. Further studies are required on a large number of patients to recommend HIV testing routinely in cirrhotic patients.


Polymerase chain reaction based diagnostic assay for identification of hepatitis B virus.

A highly sensitive polymerase chain reaction (PCR)-based diagnostic assay was developed to detect the presence of hepatitis B virus (HBV) in human serum. The assay involved amplification of HBV DNA sequences using primers specific to HBV. This in vitro enzymatic amplification technique, when used in combination with molecular hybridization assay can detect HBV genome in the patient's serum, with a higher degree of sensitivity than achieved with dot blot assay. The assay can identify samples containing 3-10 virus particles.


Occurrence of multi-drug resistant Salmonella typhi in Calcutta.

Blood and faecal samples were collected from 122 hospitalised patients of Calcútta clinically suspected to have enteric fever, for isolation of S. typhi. It was isolated from 34.4, 4.9 and 4.1 per cent patients by blood culture, stool culture and by both respectively. The in vitro drug susceptibility testing showed that all the isolates were resistant to chloramphenicol, ampicillin and trimethoprim-sulphamethoxazole, but were uniformly susceptible to ciprofloxacin, norfloxacin and furazolidone. In view of the appearance of multi-drug resistant S. typhi in Calcutta, great care should be exercised in the use of newer quinolone derivatives.


Comparison of selective media for optimal recovery of Clostridium difficile from diarrhoeal stools.

Five selective media were compared for their efficacy in the recovery of C. difficile from stool specimens. Of 341 diarrhoeic stool samples, 38 (11%) yielded C. difficile. Eighty per cent of the isolates were detected on modified taurocholate cycloserine cefoxitin fructose agar (MTCCFA) and 73 per cent were detected on taurocholate cycloserine cefoxitin fructose agar (TCCFA). MTCCFA was also found superior to the other four media as it supported better growth of C. difficile colonies, by effectively suppressing the competing microflora. These results suggest that the recovery rate of C. difficile could be enhanced when routine media, incorporated with taurocholate and lower concentration of cycloserine and cefoxitin, is used for the isolation of C. difficile from diarrhoeic stool.


Mannose-resistant haemagglutination by Campylobacter jejuni--a preliminary communication.

Ten strains of C. jejuni each isolated respectively from patients with diarrhoea and from chicken intestine (10 strains from each source) were examined for presumptive colonization factor(s) by measuring their cell surface hydrophobicity and haemagglutination. None of the strains expressed cell surface hydrophobicity. However, 14 strains (7 from either source) showed variable haemagglutination pattern with human, sheep and rabbit erythrocytes in the presence of 0.5 per cent D-mannose. Thus, mannose resistant haemagglutinin(s) may be involved in the colonization of intestinal mucosal surfaces by C. jejuni.


Multiply resistant Haemophilus influenzae type b causing meningitis.

The isolation from patients of meningitis, of two multidrug resistant strains of H. influenzae is of relevance to the empirical treatment of meningitis patients. The isolates produced beta lactamase and had higher MICs as compared to the four H. influenzae strains sensitive to the drugs commonly used for the treatment of meningitis. The cephalosporins and gentamicin were found to be effective antibiotic agents. The occurrence of resistance to ampicillin, chloramphenicol, cloxacillin, cotrimoxazole, tetracycline, penicillin and erythromycin is of concern.


Kinetics of specific IgM in patients of hepatic amoebiasis.

Entamoeba histolytica (EH) specific IgM was measured in 54 patients with diagnosed amoebic liver abscess (ALA), 13 with non-suppurative hepatic amoebiasis (NSHA) and 50 controls. The mean levels of EH specific IgM, estimated by ELISA were significantly raised in patients of invasive amoebiasis (both ALA and NSHA) compared to controls (P less than 0.05). EH specific IgG was also raised in both groups of patients. Follow up of patients with ALA showed a significant decline (P less than 0.05) in the specific IgM levels three months after treatment while the specific IgG antibodies persisted in high titres (1:160). Only four patients of NSHA could be followed up and all showed a decline in specific IgM levels. Raised specific serum IgM seems to be an indicator of active (invasive) amoebiasis.


Mosquito repellent activity of oils from Vitex negundo Linn. leaves.

Oil obtained from stream distillate of V. negundo leaves was fractionated by column chromatography. Mosquito repellence activity, as evaluated against Aedes aegypti was mainly confined to the most polar fractions. The protection period against mosquito bites by polar fractions ranged between 1-3 h. However, the mean protection period values of these fractions did not show significant increase in the subsequent subfractions.


Laboratory assessment of indigenous plant extracts for anti-juvenile hormone activity in Culex quinquefasciatus.

Of 15 plants tested, five plant extracts showed anti-juvenile hormone-like activity against laboratory colonised late fourth instar larvae and adult female mosquitoes. Petroleum ether extract of Eichhornia crassipes and acetone extracts of Ageratum conyzoides, Cleome icosandra, Tagetes erectes and Tridax procumbens showed growth inhibitory (P less than 0.001) and juvenile hormone mimicing activity to the treated larvae of C. quinquefasciatus.. Larval pupal intermediates, demalanised pupae, defective egg rafts and adult with deformed flight muscles were few noticeable changes. Biting behaviour was observed to be affected only in Ageratum, Cleome and Tridax extracts (P less than 0.001). Loss of fecundity was observed in the treated mosquitoes but no sterilant effects could be seen. Adults, obtained from larvae exposed to the plant extracts produced significantly shorter egg-rafts (P less than 0.005) than in control.


Seasonal abundance, natural survival & resting behaviour of Phlebotomus papatasi (Diptera: Phlebotomidae) in Pondicherry.

A study was carried out on the seasonal abundance, natural survival and resting behaviour of Ph. papatasi in four ecologically different areas of Pondicherry. The average indoor resting density ranged from 2.25 (May 1989) to 16.70/man hour (October 1989). Ph. papatasi was observed to be predominantly endophilic in its resting behaviour. A significant positive correlation was observed between rainfall and relative density. The daily survival of adult population ranged from 0.893 to 0.949 and showed significant positive correlation with relative humidity. Examination of the abdomen of indoor resting females showed that the entire period of gonotrophic cycle was spent indoors.


Non-oliguric acute renal failure in gout.

Four patients of pure gouty nephropathy are presented. Gout was of over five years duration and asymptomatic nephropathy manifested as non-oliguric acute renal failure. Diseases commonly associated with it like uric acid stones, urinary tract infections, hypertension, diabetes mellitus, hyperlipidemid, obesity and nephrosclerosis were absent. Reduction in serum uric acid level resulted in prompt improvement in renal functions. Early detection and control of hyperuricemia may help in restoration of renal functions.


A retrospective study on scorpion sting in a pediatric age group in a hospital in Calcutta.

During 1985-1989, in Calcutta Medical College Hospitals, of 152 children of 1-6 year age group admitted with the history of scorpion sting 18 (11.8%) died. Maximum numbers of stings were inflicted in the fingers. Important clinical features recorded were circulatory failure, breathlessness, profuse sweating, vomiting, local oedema and convulsion. Incidences of scorpion stings were much more frequent in the summer and rainy seasons than in the winter season.


A new Brånemark single tooth abutment: handling and early clinical experiences.

A new prosthetic concept, today available under the name CeraOne, for single tooth replacement with the Brånemark system is described. This concept is characterized by a new design of the prefabricated components. A mechanical torque driver is used together with a gold screw and a special counter-torque device to ensure that the screw is tightened in an optimal manner to resist screw loosening and only transmit minor stress to the fixture interface. Another characteristic is the use of a prefabricated cap of sintered aluminum oxide as the basis for the ceramic crown. The crown is cemented to provide better esthetic possibilities even in situations of somewhat unfavorable fixture placement.


Management of an anterior defect with a removable partial denture supported by implants and residual teeth: a case report.

The treatment of a patient with a major defect in the anterior region of the mandible is described. A large part of the anterior dental arch and underlying mandibular bone was involved in the defect. A removable partial denture supported by implants as well as residual teeth was used to restore the defect. Compared with a traditional removable partial denture, which was used by the patient for several years as an interim prosthesis, the application of implants resulted in improved functional comfort and stability.


Titanium implants in irradiated tissue: benefits from hyperbaric oxygen.

Since the introduction of osseointegrated titanium implants for bone-anchored facial and dental prostheses, an increasing number of irradiated patients are being treated with this technique. Although the number of patients who have had titanium implants after irradiation is limited, available statistics point to a tendency of a higher implant loss frequency as compared with nonirradiated patients. This review discusses factors behind deleterious tissue effects and implant failures from irradiation and points to possibilities to improve the surgical outcome with special reference to hyperbaric oxygen therapy.


Theoretical analysis of the fatigue life of fixture screws in osseointegrated dental implants.

Metal fatigue failure of the gold screw used to retain a fixed prosthesis to Brånemark osseointegrated fixtures/abutments has been analyzed theoretically. Mechanical engineering principles show the importance of appropriate preload being applied through the gold screw to the gold cylinder and abutment. The significance of the screw design and necessity of applying the correct torque to achieve a long fatigue life for the screw are also described. The consequence of misalignment of a gold cylinder to an abutment is discussed.


Implant superstructures: a comparison of ultimate failure force.

Because of reported prosthodontic failures, alternative implant abutments and screws with suggested strength superiority have been manufactured. However, little quantitative data are available to support these claims. This study compared the force necessary to cause failure in many of the more commonly used implant and abutment combinations. An 18-mm cantilever test prosthesis was constructed for each system and loaded on an MTS machine until failure occurred (n = 5 for each sample). The mean failure forces ranged from 1.22 to 17.23 kg. The failure force and location of failure for each individual system are reported.


Failures and complications in 127 consecutively placed fixed partial prostheses supported by Brånemark implants: from prosthetic treatment to first annual checkup.

Ninety-six partially edentulous maxillae and mandibles were consecutively treated with 127 freestanding fixed prostheses supported by 354 implants. The patients were followed for 1 year and the overall success rate was 98.6% for the examined implants. None of the inserted prostheses was lost during the observation period. The most commonly reported problems during the first year of function were related to loose gold screws and esthetic complaints, complications that were easily resolved. Furthermore, the total number of complications was low and was less than has been reported for routine full-arch fixed prostheses.


Fixture placement posterior to the mental foramen with transpositioning of the inferior alveolar nerve.

The results of 10 fixture placement operations with transpositioning of the inferior alveolar nerve are presented. Nerve transpositioning increased the operating time, but with experience this time should be reduced. Neurosensory dysfunction of the inferior alveolar nerve was found in 7 of 10 operated sites 1 week after surgery. Six months postoperatively, altered sensation was still present in 2 patients. Nerve function was normal in all patients 1 year postoperatively. The stability of fixtures was satisfactory throughout the examination period and the procedure should prove useful in treatment of the resorbed mandible posterior to the mental foramina.


Patient satisfaction with overdentures supported by one-stage TPS implants.

Sixty-four edentulous patients with severe conventional denture problems who had been treated with 218 one-stage titanium plasma sprayed (TPS) screw implants and new overdentures were clinically evaluated and questioned on their experiences with treatment up to 6 years after implant insertion. The results demonstrated that only seven of the implants had failed during this period, resulting in a success rate of 97%. Most of the patients (95%) were satisfied with their new overdentures, and almost all patients (98%) found that their new dentures fit comfortably. Only 3% of the patients treated would not recommend that others undergo similar treatment.


Prosthesis fabrication using electrical discharge machining.

Fixed-removable implant prostheses provide solutions for some of the problems associated with implant dentistry, especially in the maxilla. The technique for using electrical discharge machining to create a precise passive fit between the substructure bar and the removable suprastructure is presented. The advantages, disadvantages, and complications associated with this type of prosthesis are discussed.


A morphometric and biomechanic comparison of titanium implants inserted in rabbit cortical and cancellous bone.

The removal torques for screw-shaped pure titanium implants inserted in rabbit tibia and the femoral part of the knee joint and the tissue response to these implants, as quantitated with light microscopic morphometry on ground sections, were compared after 6 weeks, 3 months, and 6 months. The bone surrounding the femoral intra-articular implants was mostly cancellous, while cortical bone was formed around the tibial implants. The torque needed to remove the intra-articular implants increased with time, but there was no such increase for the tibial implants. At 6 weeks, significantly less torque was needed to remove the intra-articular implants in spite of the fact that significantly more bone was found in the threads of these implants as compared with the tibial implants. When calculating the amount of bone in threads situated in the cortical and subchondral passage, more was found in the threads of the tibial implants, which corresponded to the higher removal torque. Additional light microscopic observations on implants unscrewed after 12 months in rabbit tibia indicated that rupture occurred between the implant surface and calcified bone. Findings indicate that the resistance to unscrewing is dependent on the amount of compact bone surrounding a titanium implant.


Osteogenesis of hydroxyapatite and tricalcium phosphate used as a bone substitute.

Hydroxyapatite (HA) and tricalcium phosphate (TCP) are useful for grafting and augmentation of bone tissue. In this study, conventional and histochemical transmission electron microscopy were used to study osteogenic events at the interface between the implanted materials and adjacent tissue from 1 to 4 weeks postoperatively. The microscopic results indicated that TCP was resorbed more rapidly than HA after implantation, with a notable breakdown of material and replacement by mesenchymal cells with ultrastructural features resembling osteoprogenitor cells and collagen up to 4 weeks postoperatively. Alkaline phosphatase and acid phosphatase reactivity in the tissues helped to identify and differentiate the histologic differences observed between HA and TCP.


A new self-tapping Brånemark implant: clinical and radiographic evaluation.

A new self-tapping Brånemark implant designed for denser bone qualities was evaluated with regard to insertion technique, complications, marginal bone remodeling, and survival rate. Thirty patients, representing 21 mandibles and 9 maxillae, participated in the study. In each patient both standard and self-tapping implants were placed, and a total of 179 implants, 88 self-tapping and 91 standard, were inserted. Thirteen of 62 mandibular self-tapping implants reached their correct positions only after using the screw tap or the cylinder wrench for manual insertion. No such problems were noted when using standard fixtures after bone pre-tapping. In the maxillae, neither of the two implant designs presented any problems. One standard and one self-tapping fixture failed to osseointegrate. Radiolucencies were seen in the bone around the apical portion of two fixtures, one of each design. The mean marginal bone resorption after 1 year of follow-up was 0.5 to 0.6 mm for the two fixture types.


Reconstructing the atrophic mandible with inferior border grafting and implants: a preliminary report.

Advanced mandibular atrophy is a serious surgical reconstruction challenge. Previous experience with an inferior border approach has been favorable. Predictably successful osseointegrated implants ultimately allow loading of the healed grafts. The technique described uses a freeze-dried, gamma-irradiated cadaver mandible packed with autogenous iliac crest bone. An extraoral approach allows augmentation without disrupting use of an intraoral prosthesis during healing. The incidence of infection and resorption can be reduced. Fewer problems with neurosensory disturbances common with some of the superior border, or sandwich osteotomy, grafting techniques were experienced. The use of implants to ultimately load the bone should result in less resorption than in those techniques using a conventional removable prosthesis.


Comparative surface microanalysis of failed Brånemark implants.

The chemical composition and topography of an implant surface determine the human immunologic system response. This study compared the surfaces of 13 Brånemark oral implants, 11 that came from retrieved specimens which failed initially or did not osseointegrate and 2 that were never implanted (controls). The period of implantation in human jaws varied between 3 and 20 months. After cleaning and sterilization, the topography, surface chemical composition, and thickness of the oxide layer were studied. The results obtained with scanning electron microscopy did not show any significant topographic differences among the specimens. X-ray spectrographic microanalysis showed very similar composition (titanium and amounts less than 0.5% of other elements) in the outermost layer of the analyzed specimens. The Auger spectroscope revealed considerable percentage differences in the amount of carbon and silicon in the last monolayers, which could be attributed to handling or to an inadequate cleaning process. This places the retrieved specimens out of the acceptable statistical limits of contamination by introducing a factor of doubt for long-term prognosis in the hypothetical situation of their re-use.


Visualization and initial characterization of the titanium boundary of the bone-implant interface of osseointegrated implants.

A simple procedure has allowed consistent visualization of the titanium boundary of the bone-implant interface of osseointegrated titanium implants at the electron microscope level. This was accomplished by embedding the intact bone-implant specimen block with low-viscosity resin prior to removal of the device in preparation for sectioning. The titanium boundary consisted of either a thin, compact amorphous electron dense layer, a broad layer of dense amorphous granules, or both. This material was removed by decalcification in formic acid (prior to embedding) and did not diffract electrons (ie, was noncrystalline). Scanning-transmission electron microscopy-EDX analysis indicated the presence of titanium, calcium, and phosphorus in the electron dense material. Field emission scanning electron microscopy-EDX dot-mapping analysis confirmed the presence of these elements and mapped them to the same locations at the implant-interface boundary.


Failures and complications in 92 consecutively inserted overdentures supported by Brånemark implants in severely resorbed edentulous maxillae: a study from prosthetic treatment to first annual check-up.

Overdentures were consecutively inserted in 92 severely resorbed maxillae and followed for 1 year. The dentures were supported by a total of 430 implants. Of these, 69 (16%) became loose and were removed during the follow-up period. The mobile implants caused 7 complete failures of the overdenture treatment. Postinsertion maintenance was more extensive for the overdentures than for fixed prostheses supported by implants. Problems arose in relation to the mucosa around the implants, and there were also fatigue fractures of the acrylic resin and the retentive clips. Fewer speech problems were reported in the overdenture group when compared to those with fixed prostheses.


Surface analysis of an original Brånemark implant and three related clones.

Selected surface characteristics of screw-type titanium dental implants from four different manufacturers were evaluated. Considerable differences in surface and near-surface contaminants were demonstrated. The fixture treated with radiofrequency glow discharge (plasma) demonstrated the thinnest titanium oxide layer and the cleanest surface.


Mandibular ridge augmentation with simultaneous onlay iliac bone graft and endosseous implants: a preliminary report.

Mandibular onlay composite grafts (autogenous iliac bone and titanium cylindrical threaded endosseous dental implants) were placed in seven patients with advanced bone resorption. All seven patients have experienced uncomplicated healing and continuous, uninterrupted prosthesis use without soft tissue or mechanical complications for 1 to 4 years. This preliminary report includes a discussion of the indications for the procedure, potential alternative management of the severely resorbed mandible, and details of the surgical procedure illustrated by two patients who received this treatment modality.


Three-dimensional force measurements on mandibular implants supporting overdentures.

The purpose of this study was to determine masticatory and functional forces in three axes on mandibular implants supporting overdentures. Five edentulous test subjects were selected, each having two mandibular implants. Three-dimensional piezoelectric force transducers were mounted on the two-part ITI Bonefit implants and rigidly connected to the denture. Forces in vertical, lateromedial, and anteroposterior directions were measured by means of electrostatic plotter records. The test modalities were light tapping, grinding, maximal occlusal force, and chewing test food. Results showed that the five subjects developed similar stress patterns but quantitatively different occlusal and chewing forces. In all but one subject, reduced maximal occlusal force was found compared to dentate subjects and edentulous subjects with fixed partial prostheses supported by implants. The recordings of chewing cycles when eating test food resulted in very regular rhythmic strokes, similar to those of dentate subjects but with slightly reduced speed. All stress patterns showed that occlusal and chewing forces were mainly directed in vertical, medial, and anterior dimensions. The dominating component was vertical.


A retrospective evaluation of endosseous titanium implants in the partially edentulous patient.

Partial edentulism in 34 patients was consecutively treated using the Brånemark osseointegration technique. A total of 102 implants was tested for mobility, signs and symptoms of infection, and radiographic bone levels. All patients had at least a 6-month follow-up of prosthesis function (mean 22.5 months). An overall success rate of 88.2% was observed. Twenty-five of 28 maxillary implants and 65 of 74 mandibular implants were successfully placed and restored. Twelve failures were observed; 7 were not integrated at the time of stage 2 surgery while 5 occurred after prosthetic reconstruction. The results of this study suggest that the Brånemark osseointegration procedure can be used to treat partially edentulous patients with a high degree of success.


Nasofacial prostheses supported by osseointegrated implants.

Surgical ablation in the nasofacial area may lead to defects that can be treated with maxillofacial prosthetic restorations retained by osseointegrated implants. When the prosthesis is large or impacted by masticatory or orbicularis muscle forces, the use of implants placed in available facial skeleton bone may be desirable. Available sites for implant placement in the facial skeleton are described, and a craniofacial site classification is suggested. Two examples of patients treated for nasofacial defects are presented.


Heat distribution in bone during preparation of implant sites: heat analysis by real-time thermography.

The purpose of this experiment was to observe and measure the distribution of heat to bones and the maximum temperature that developed when cutting bone with drills. Generation of heat that spread in the presence or absence of irrigation when drilling with IMZ, Brånemark, and ITI implant (F type) drills was observed in the pig rib via thermography. Without irrigation, the condition of heat spread in each drill and bur differed according to bur shape and drilling site. Maximum heat temperature without irrigation was higher than that with irrigation for any IMZ, ITI, and Brånemark drill.


Clinical parameters of evaluation during implant maintenance.

This paper reviews investigations concerning the clinical parameters of evaluating dental implants during the maintenance phase of therapy. Clinical parameters that are discussed include mobility, gingival alterations, tissue movement, probing and attachment level measurements, bleeding upon probing, occlusion, and microbial monitoring. The role of implant radiology is discussed and encompasses radiographic interpretation, interval, and technique.


Public awareness and acceptance of dental implants.

To investigate public awareness and acceptance of dental implants, 120 adult US citizens were asked to answer a questionnaire. Of the 109 persons who completed the questionnaire, 77% had heard about dental implants, mostly through media and lay persons. Only 17% named a dentist or physician as the source of information. Persons with many missing teeth were not more aware of implants than those with fewer missing teeth. Of 19 removable denture prosthesis wearers, 15 knew about implants but only one third learned of them from their dentist or physician. Of 84 persons with information about implants, 51 would consider implant treatment, 17 would not, and 16 were undecided. Esthetics was the most frequent motivating factor favoring implants, while high cost was the most frequent argument against them. Younger interviewees were significantly more often in favor of implants than older persons. It was concluded that public awareness and acceptance of dental implants are high. Dentists and physicians, however, play only a minor role as sources of information.


Bone regeneration around titanium dental implants in dehisced defect sites: a clinical study.

Insufficient bone volume can be a significant problem when placing dental implants. This clinical study was designed to evaluate bone regeneration potential at dehisced dental implant sites. Nineteen titanium dental implants with exposed threads were studied. To create a secluded space for bone formation, an expanded polytetrafluoroethylene (e-PTFE) membrane was placed over the exposed implant sites secured with an implant cover screw and completely covered with the flap. Three membranes perforated the overlying soft tissue during the healing time and were removed prematurely. The remaining membranes were removed after an uneventful healing period of 4.5 to 6 months. Fourteen of 19 dehisced implant sites were completely covered with newly formed bonelike tissue; 4 implants demonstrated partial bone fill at reentry and 1 implant showed partial fill with soft tissue. In five implant sites a reentry was performed between 6 and 9 weeks; nonmineralized fibrous tissue was found to fill the space under the membrane. At 16 of the 19 implant sites there were similar dehiscence-type defects that were evaluated as a group. These dehiscences varied from 2.0 to 9.0 mm. The percentage of bone fill at reentry ranged from 28.4% to 100% (mean 89.6%; SD 22.51; SE 5.63) and was highly significant (P < .0001). Six to 12 months after prosthesis connection, 12 of the 19 implants were available for radiographic interpretation and an average bone loss of 1.73 mm (SD = 0.43) was measured. This surgical application of an e-PTFE membrane suggested a viable clinical method for enhancing bone formation around dental implants.


Presurgical tomographic assessment for dental implants. 1. A modified imaging technique.

A modified technique for presurgical evaluation of intraosseous implant placement using linear tomography has been developed. The simplified technique is applicable to tomographic systems equipped with a cephalometric head positioner (cephalostat) and a fiberoptic positioning light system. It provides precise cross-sectional images for the assessment of bone morphology and associated anatomic structures. This technique is more comfortable for patients who are unable to tolerate the positioning of submentovertex projections used to estimate horizontal angulation of the head position and to determine the depth of radiographic cut. Instead, the positioning light and casts are used to make these two determinations.


Implant and prosthetic treatment of the edentulous maxilla using a bar-supported prosthesis.

Functional and anatomical considerations led to a new concept of an implant-supported bar prosthesis in the edentulous maxilla. The cantilever situation is avoided by the placement of endosseous cylindrical implants (type IMZ) in the maxillary tuberosities distal to the maxillary sinus. Full arch support is provided by the symmetrical insertion of six IMZ implants in the tuberosity, canine, and incisor regions. After 4 months of healing, a milled precision bar and removable prosthesis are fabricated and supported by the implants.


Osseointegrated Brånemark implants used to retreat a fractured mandibular bone plate staple implant: a case report.

This article describes postimplant follow-up and retreatment of a 66-year-old patient with an esthetically compromising fixed maxillary tissue-integrated prosthesis and a mobile mandibular overdenture stabilized by precision attachments with a gold clip bar cemented to the transmucosal posts of a bone plate staple implant. Following successful completion of prosthodontic treatment, the patient returned with a fracture of the left transmucosal pin in the mandibular bone plate staple implant that was retreated with Brånemark implants and a fixed prosthesis.


Determination of bone width in malnourished children under 5, and its inter relationship with mid arm muscle thickness, subcutaneous fat thickness and the mid upper arm girth.

The mid upper arm of 516 malnourished children (one to five years) were studied radiographically for changes in bone width, muscle mass and subcutaneous fat, of malnutrition at different ages. The data was studied by statistical analysis, determining the correlation coefficients of each of the factors. The findings indicate that previous assumptions about the components and the changes of the mid upper arm girth (MUG) in chronic severe malnutrition, were perhaps too simplistic.