Leukocyte origin and profile in follicular aspirates at oocyte retrieval.

BACKGROUND: Follicular aspirates represent a snapshot in time of conditions within the follicle at oocyte retrieval in women undergoing in vitro fertilization and embryo transfer. This clinical material has been much investigated and yet its cellular composition remains unclear. In this study we investigated the origin and profile of leukocytes found within follicular aspirates. METHODS: We performed morphological and immunohistochemical analyses of follicular aspirates and peripheral blood obtained concurrently at oocyte retrieval. RESULTS: There was no correlation between erythrocyte and leukocyte numbers in follicular aspirates. The profile of leukocyte subtypes within follicular aspirates was variable and differed significantly from the peripheral circulation in a significant proportion of the analysed samples. A subset of follicular aspirates displayed a marked increase in monocytes/macrophages and an apparent concomitant reduction in polymorphonuclear leukocytes compared with peripheral blood. CONCLUSIONS: Leukocytes within follicular aspirates cannot be accounted for solely as a result of blood vessel damage during oocyte retrieval. The variation in leukocyte subtypes observed in some follicular aspirates may reflect a coordinated infiltration of these cells, characteristic of progressive inflammatory responses in other systems. The possibility that leukocyte variation is indicative of follicular maturation deserves further investigation due to its potential relevance in optimizing oocyte selection.

Alleles and haplotypes of tumor necrosis factor (TNF) alpha and beta genes in three ethnic populations of Sulawesi Indonesia.

Polymorphic variation in two cytokine genes, tumor necrosis factor (TNF) -alpha and -beta, was examined in three ethnic groups, the Bugis, the Makassans, and the Torajans, who inhabit Sulawesi, a large island in the Indonesian archipelago, and formerly a Dutch colony. TNF-alpha and -beta are key molecules in immune responses to infection, and both have been implicated in the pathogenesis and clinical manifestations of parasitic diseases. Several polymorphic variants with the potential to affect cytokine levels in autoimmune diseases and parasitic and bacterial infection have been reported. Two loci in the promoter region of TNF-alpha and two sites in the first intron of TNF-beta were scored in a maximum of 150 Bugis, 168 Makassans, and 58 Torajans. Genotypes at the two TNF-alpha loci are not in Hardy-Weinberg equilibrium because of a deficit of heterozygotes (p < 0.05). However, genotypes at the TNF-beta loci exhibit Hardy-Weinberg equilibrium. A comparison of allelic and genotypic frequencies at all TNF loci across the ethnic groups reveals that the differences are significant for TNFalpha(308) (p < 0.01) and for TNFbeta(NcoI) (p < 0.05). Overall, the distribution of the alleles differs from that seen in the few Asian populations for which data are available (p < 0.05). Construction of 4-locus haplotypes showed that, in addition to the five previously reported, four novel haplotypes were present in Sulawesi. These novel haplotypes were in low frequency, and two were seen only in Bugis (haplotypes F and J) and one (haplotype K) only in Makassans. The other, haplotype D, was present in Makassans and Torajans. Preliminary sampling of other ethnic groups suggests that three of these haplotypes (D, F, and J) may be restricted to Asian or Asian-derived populations. The frequency of the common TNF haplotypes differed between Dutch and Sulawesi populations, and these data also indicated that haplotype E, which has a relatively high frequency in the Dutch (25%), may be a useful marker of Dutch/European admixture in Indonesian populations, in which it is either rare (1%) or absent. The results suggest that unique allelic combinations with potential to influence cytokine secretion are present in Sulawesi, possibly as a consequence of parasite-driven selection, and argue for more extensive investigation of haplotype distribution in parasite-endemic areas.

Body condition and immune response in wild zebra finches: effects of capture, confinement and captive-rearing.

Behavioural ecologists attempt to predict fitness in birds from estimates of body condition and immune capacity. We investigated how the stresses associated with capture, confinement and captive-rearing of wild zebra finches (Taeniopygia guttata) affected different elements of the immune system and body condition. Wild birds had higher heterophil:lymphocyte ratios and total leucocyte counts than aviary birds, presumably an outcome of mounting specific resistance to pathogens, but this response diminished significantly within 10 days of confinement. Wild birds had lower phytohaemagglutinin-A (PHA) responses than their aviary-bred counterparts possibly because energetic costs limited a general resistance response. Wild birds were heavier and had higher haematocrits than their aviary counterparts, but had less fat, although just 10 days of captivity significantly increased fat levels. Measures of body condition were of limited use for predicting immune responsivenesss. We conclude that the different elements of the immune system and body condition respond independently, and often unpredictably, to many ecological and behavioural stressors.

Oral immunization with xenogeneic antibodies stimulates the production of systemic and mucosal anti-idiotypic antibodies.

The humoral and mucosal immune responses to oral immunization with xenogeneic antibodies were studied using an animal model in which female rabbits were fed daily doses of the MOPC-315 murine IgA antibody, and were mated during the course of the feeding programme. Serum and colostrum samples were assayed for the presence of anti-idiotypic antibodies by ELISA assay, before and after depletion of anti-IgA antibodies, by affinity chromatography using another murine IgA idiotype. It was shown that all animals responded to exposure to the MOPC-315 idiotype with the production of serum anti-murine immunoglobulin antibodies and that four of six animals produced serum anti-idiotypic antibodies. That the immune response included antibodies directed against the antigen-binding site was confirmed by competition ELISA assay. Mucosal IgG and IgA anti-immunoglobulin antibodies were present in milk from all antibody-fed rabbits tested, and IgA anti-idiotypic antibodies were detectable in the colostrum of one rabbit. The results provide some support for the hypothesis that human exposure to xenogeneic antibodies, most commonly bovine milk immunoglobulins, may provoke the production of anti-idiotypic antibodies, and that such exposure may lead to disturbances of immune regulation.

Bovine milk, including pasteurised milk, contains antibodies directed against allergens of clinical importance to man.

Pasteurised and raw bovine milk and bovine colostrum samples were assayed by enzyme-linked immunoassay for the presence of antibodies directed against a selection of allergens of importance in human atopic disease. Samples were tested for the presence of antibodies directed against or cross-reacting with ryegrass pollen, house dust mites, Aspergillus mould and wheat proteins. Antibodies of each specificity were detected in every sample tested, including all samples of commercial pasteurised milk. The results are discussed with reference to a hypothesis that dietary xenogeneic antibodies may play a role in the emergence of some human atopic disease, and the recent demonstration that oral immunisation with xenogeneic antibodies may lead to the production of anti-immunoglobulin antibodies including anti-idiotypic antibodies.

Antimitochondrial antibodies in primary biliary cirrhosis recognize both specific peptides and shared epitopes of the M2 family of antigens.

Sera from patients with primary biliary cirrhosis exhibit variable autoantibody reactivity against mitochondria, the commonest antigen (designated M2) including three structures of approximate M.W. 70, 50 and 40 kD. The nature of these antigens has only recently been established; the 70 and 50 kD are the transacetylase E2 and component X, respectively, of the pyruvate dehydrogenase complex and are distinct polypeptides. We have demonstrated, by immunoblotting, elution and rebinding of antibodies, unequivocal cross-reactivity between the major bands of the M2 antigen. In addition, cross-reactivity has been shown between antibodies binding to each of the three M2 bands of mitochondria and two major antigenic bands of both Gram-negative and Gram-positive bacteria. Conversely, antibodies eluted from these two bands of Escherichia coli were found to bind all three M2 bands of mitochondria. These results suggest that the antibodies of primary biliary cirrhosis contain both peptide-specific and cross-reacting antibodies, the latter recognizing a common "M2 epitope" that might include nonprotein components of the peptides. However, direct and competitive enzyme-linked immunosorbent assays failed to implicate the coenzyme of the pyruvate dehydrogenase complex, lipoic acid or its amide, as the common antigenic moiety.

Mitochondrial antigenic structure and enzyme activity in ageing human diploid fibroblasts.

It has been proposed that cellular ageing may be caused by loss of mitochondrial function due to the action of free radicals. To investigate this hypothesis, antigenic structures of the mitochondrial inner membrane/matrix and of the outer mitochondrial membrane of human diploid fibroblasts were monitored by immunoblotting at four stages during cellular lifespan in vitro. At the same time, specific activities of the enzymes oligomycin-sensitive ATPase (O-S ATPase), malate dehydrogenase (MDH) and glutamate dehydrogenase (GDH) were assayed to assess the functional capacity of cellular oxidative phosphorylation and of the tricarboxylic acid cycle. No changes were found with ageing in inner mitochondrial membrane-associated matrix components, or in the activities of O-S ATPase and MDH. However GDH activity increased significantly with ageing in vitro, possibly indicating greater amino acid utilization for energy production in older cells. There was loss of an outer mitochondrial membrane antigen, of approximate molecular weight 60 kilodaltons (kDa), in the oldest cells tested, which may influence outer membrane transport capacity late in the cellular lifespan. Overall, the results fail to provide support for the hypothesis that ageing primarily results from free radical-induced impairment of mitochondrial function.

Phenotype of cytotoxic effector cells infiltrating a transplanted, chemically induced rat sarcoma.

Previous work from this laboratory indicated that methylcholanthrene-induced sarcomas in the rat are infiltrated by cells with the cytotoxic properties of natural killer (NK) cells. Using a combination of velocity sedimentation and analysis and separation in the fluorescence-activated cell sorter, it has been possible to isolate and characterize these putative NK cells. The present results confirm that cytotoxicity is restricted to NK-sensitive targets (syngeneic, allogeneic and xenogeneic) and no evidence was found for cytotoxic T lymphocytes (CTL). In addition, effector cells have the morphology of large granular lymphocytes, and the pattern of expression of five monoclonal antibody-defined surface markers is identical with that of normal splenic NK cells.

Immunomodulation: NK cells activated by interferon-conjugated monoclonal antibody against human osteosarcoma.

A mouse monoclonal antibody raised against the human osteogenic sarcoma cell line 791T has been covalently coupled to purified human lymphoblastoid interferon alpha (IFN alpha). Conjugation does not interfere with antibody function, as the product binds to 791T cells and mediates complement-dependent tumour cell lysis to a degree equal to that of free antibody. The IFN activity, assessed by augmentation of natural killer (NK)-cell-mediated lysis, is reduced, but the conjugate does augment the killing of 791T and other tumour targets by peripheral blood NK cells. In admixture experiments the conjugate, when bound to unlabelled osteogenic sarcoma cells, also augments the killing of radiolabelled bystander cells. Neither free antibody nor the conjugate mediate antibody-dependent cellular cytotoxicity (ADCC), and augmented tumour cell lysis is a function of NK cell activation. This product provides for an alternative approach to cancer therapy via the activation of infiltrating hose effector cells using specifically targeted lymphokines.

Interferon-alpha conjugation to human osteogenic sarcoma monoclonal antibody 791T/36.

Human lymphoblastoid interferon-alpha (IFN-alpha) has been coupled using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to a murine monoclonal antibody (791T/36) which reacts with antigens expressed on human osteogenic sarcomas. The purified conjugates retain antibody activity as defined by their capacity to compete with binding of fluorescein isothiocyanate-labelled 791T/36 antibody to 791T cells. IFN-alpha-791T/36 antibody conjugates synthesized with 125I-trace-labelled IFN-alpha and 131I-trace-labelled antibody also bound to 791T cells, but not to bladder carcinoma T24 cells. The conjugates also retain the capacity of free IFN to activate natural killer cells in human peripheral blood lymphocytes and show specific localization in human osteogenic sarcoma xenografts developing in immunodeprived mice. These findings establish that conjugates containing IFN linked to a monoclonal antibody reacting with osteogenic sarcoma-associated antigens have potential for targeted immunotherapy and in related investigations with antibody has been shown by gamma camera imaging of patients following infusion of 131I-labelled antibody to localize in primary osteogenic sarcomas.

Quantitative studies of natural immunity to solid tumours in rats. NK activity in animals with primary or transplanted spontaneous tumours.

Natural killer (NK) cell activity was measured in a quantitative 6 h chromium release assay using sarcoma MC7 cells as targets. The total NK lytic activity present in the spleen and blood of tumour-bearing animals was compared with the corresponding values for age/sex/parity-matched animals. Rats with primary spontaneous tumours in the breast or subcutaneous sites showed normal levels of NK activity, while rats with primary spontaneous kidney tumours had elevated NK activity, the degree of augmentation being greater with increasing tumour size. A similar elevation of NK activity was generally found in animals with large, transplanted, spontaneous or chemically-induced tumours. This augmentation could only detected when total lytic activity was considered: when NK activity was measured merely on a cell-for-cell basis, it often appeared to be depressed in such animals, in agreement with previous reports. However, with one rapidly metastasizing spontaneous tumour, a real depression of both spleen and blood NK activity was found. Small inocula of cells from non-immunogenic spontaneous mammary tumours or from other non-immunogenic spontaneous tumours caused no early increase in systemic NK activity when injected into the mammary pad, a site where spontaneous tumours frequently arise. However, cells from one immunogenic spontaneous tumour and 2/3 immunogenic chemically-induced tumours did occasionally stimulate significant early increases in NK activity when placed at this site. Early changes in peritoneal exudate NK activity were also investigated using small inocula of these tumours injected intraperitoneally. Augmentation of NK activity occurred with a 3-fold greater frequency following inoculation with immunogenic tumour cells than with non-immunogenic cells in this system. It can be concluded from these studies that: (1) spontaneous tumours do not selectively arise in members of an inbred strain with subnormal NK activity; (2) most large tumours in rats stimulate rather than depress NK activity; (3) early boosting of NK activity by small inocula of tumour cells placed in the mammary pad does not occur with non-immunogenic spontaneous tumours; (4) early boosting of NK activity in the peritoneal site does occur with non-immunogenic tumours, but with a very low frequency. The latter findings suggest that developing spontaneous tumours are unlikely to stimulate the NK system, and emphasize the importance of using syngeneic, spontaneous tumours for studying tumour-host relationships in animals.

Tumour cells in metastatic deposits with altered sensitivity to natural killer cells.

If natural killer (NK) cells play a role in immunosurveillance it might by expected that, during the metastatic process, selection would occur for tumour cells with reduced NK sensitivity. This hypothesis was tested in the rat by measuring the NK sensitivity of cells freshly isolated from metastases of syngeneic transplanted spontaneous mammary carcinomas. Lysis was measured in a 6-h chromium release assay using normal syngeneic spleen cells as effectors. Our studies led to the following conclusions. (1) Metastases developing at certain tissue sites (draining lymph node and lung, but not pericardium) were frequently composed of tumour cells with markedly reduced sensitivity to NK cells. (2) This resistance could generally be detected only if freshly isolated tumour cell population were studied; after a few days in culture, resistant metastasis-derived tumour cells usually regained normal NK sensitivity. (3) Resistance to NK cells was not always due to the loss of NK target structures; it could also result from an innate resistance to the NK lytic mechanism. (4) The tissue distribution of NK-resistant metastases suggested that if NK cells exerted an immunoselective pressure they did so at the tissue site rather than in the primary tumour or in the bloodstream.

Lymph node anti-tumour effector cell mechanisms in colorectal carcinoma.

Eighty lymph nodes from 61 cases of colorectal carcinoma were studied by in vitro microcytotoxicity assay. It was found that 25 nodes (31%) from 22 of these cases (36%) contained lymphoid cells which were cytotoxic against autologous carcinoma cells in vitro. Lymph node cells (LNC) were not cytotoxic against 51Cr chicken red blood cells (CRBC). This assay was used as an indicator of natural killer cell (NK) activity. Comparably, normal control peripheral blood lymphocytes (PBL) were cytotoxic against CRBC but not against colonic target cells obtained from surgical biopsy specimens. Fc receptor-bearing cells, monitored by cytotoxicity against antibody-coated CRBC, were detected in 50 and 60% of cytotoxic and non-cytotoxic LNC populations. The varying effects of E- and EAC-rosette fractionation and iron filing treatment of effector cells indicate that regional LNC cytotoxicity in colorectal carcinoma is a complex phenomenon, which, however, is predominantly a function of E-rosetting lymphocytes. Lymphocytes obtained from all regional nodes were capable of responding to phytohaemagglutinin (PHA). The relative proportion of T lymphocytes (E-rosetting cells) was significantly higher in those lymph node suspensions which were cytotoxic against colon carcinoma cells.

Quantitative studies of natural immunity to solid tumours in rats. The nature of the killer cell depends on the type of assay.

Cell fractionation techniques were used to identify the cells in rat spleen responsible for natural killing of a syngeneic sarcoma cell in short-term (6 h and 18 h) and long-term (72 h) cytotoxicity assays. Cytotoxicity was quantified precisely using a method previously derived from consideration of natural cytotoxicity as an enzyme-substrate reaction, and by analysing results in terms of lytic units. Killing in all three assays displayed 'single-hit' kinetics implying that a single effector cell was sufficient to lyse a single target cell. The fractionation studies, using glass adherence, carbonyl iron, nylon wool, EA and EAC monolayers and congenitally athymic rats, revealed two populations of cytotoxic cells. In the 6 h assay most of the activity was due to cells with similar characteristics to the NK cells previously defined using leukaemic targets, but in the 18 h and 72 h assays macrophages played an important rôle. The activity exerted by the macrophages was cell lysis and not cytostasis. No evidence that the macrophages acted by releasing factors which stimulated NK cells could be found.

Quantitative studies of natural immunity to solid tumours in rats. Persistence of natural immunity throughout reproductive life, and absence of suppressor cells in infant rats.

Consideration of cell-mediated cytotoxicity as an enzyme-substrate reaction provided a theoretical and practical basis for the quantification of natural immunity to solid tumours. The natural cytotoxic activity of spleen cells from normal rats towards cultured target cells from solid tumours, measured in a 6 h chromium release assay, developed between 2 and 5 weeks after birth and, in contrast to previous reports of natural cytotoxicity against lymphoid tumour cells, showed no sign of decline up till at least 22 months of age. Both virgin and breeder (multiparous) females showed equally good maintenance of natural immunity, and the apparent specificity of this natural immunity on a panel of four target cells did not change between 8 weeks and 18 months of age. The low level of natural immunity in infant spleen, and also in adult lymph nodes and thymus, was not caused by suppressor cells but rather by an absence of appropriate effector cells.

Immune response to human keratoacanthoma.

Immune reactivity to human keratoacanthoma was investigated by microcytotoxicity tests and immunofluorescence. IgM and complement were consistently present in lesions; IgG and fibrin were infrequent. No evidence of in vivo bound immunoglobulin was found on the surface of keratoacanthoma cells by membrane immunofluorescence. Neither patients' sera nor peripheral blood leukocytes showed significant cytotoxicity against autochthonous tumour cells in microtitre assays. This study fails to support the view that regression of human keratoacanthoma is mediated by immunological mechanisms.

Immunoreactivity by intrinsic lymphoid cells in colorectal carcinoma.

Mononuclear leucocytes were separated by Hypaque--Ficoll from 60 unselected primary colorectal carcinomas, and then fractionated by rosetting with sheep erythrocytes, either alone (E) or coated with antibody and complement (EAC). The E-rosetting cells, putative T lymphocytes, were cytotoxic in vitro to autologous tumour cells in 18 of the 60 cases, whilst the EAC-rosetting cells were unreactive. This intrinsic T-lymphocyte anti-tumour immunoreactivity was significantly associated with the presence of "cuffs" of small dark lymphocytes at the mesocolic or pararectal edge of the primary tumours, but there was no correlation with antitumour cytotoxic lymphocytes in the patient's blood at the time of operation.