Search

A survey conducted at McGill University suggests that non-tenure track faculty are a diverse group of highly-qualified individuals whose employment status involves a high degree of uncertainty. In accordance with other Canadian and American studies, the survey also found that a disproportionate number of women occupy non-tenure track as opposed to tenured or tenure track positions. Since the 1980s, North American universities have responded to increasing student enrollments and con- tinued cuts to government funding by appointing significant numbers of faculty to full-time and part-time non-tenure track positions. Due to the precariousness of their employment status, non-tenure track faculty rep- resent an attractive buffer in times of financial restraint. Despite their increasing numbers, however, little is known about the composition and concerns of non-tenure track faculty as a group. This article describes this group at one university and puts into question the structural and power relations that have led to their increased use and abuse.

The combined 12th International Congress of Immunology (ICI) and the 4th Annual Conference of the Federation of Clinical Immunological Societies (FOCIS) was held in Montreal, Canada July 18-23, 2004 and attracted over 6000 immunologists and almost 4000 abstracts. The host society, the Canadian Society for Immunology (CSI) spent many years in preparation for this large meeting and encouraged its members to propose topics for symposia and mini-symposia and to sponsor satellite meetings. With sponsorship of CSI; the Canadian Institutes of Health Research; the University of Guelph, Guelph, ON; Queen's University, Kingston, ON; McGill University, Montreal, QU, Canada; and the American Society for Reproductive Immunology, a focused, highly successful, one day satellite meeting on human uterine immunology was held. The highlights of the presentations and discussions are reported.

Chronic ocular inflammation can be due to a disease process or to iatrogenic factors that attract inflammatory cells to the anterior chamber. This study was conducted to investigate the effect of commonly used ophthalmic preparations on leukocyte migration.A modified multi-well Boyden chamber was used to study the chemotactic and chemokinetic effects of 33 commercial ophthalmic preparations. To determine whether the chemotactic effect was a property of the commercial ophthalmic preparation or other chemicals present in the products, experiments were also done with some common preservatives and excipients.Of the drugs, 14 (42.4%) showed chemokinetic and/or chemotactic activity and 19 (57.6%) had either no effect or decreased neutrophil migration. Of the preservatives and excipients, 5 (62.5%) were found to be chemotactic. Eleven of 14 chemotactic drugs (78.6%) and 8 of 19 non-chemotactic drugs (42.1%) were positive for at least one chemotactic excipient. The correlation between chemotactic ophthalmic preparations and the presence of a chemotactic excipient in their composition was significant (p0.05).Chemotactic activity was commonly found in commercial ophthalmic preparation. Furthermore, the presence of certain chemotactic preservatives and/or excipients was a contributing factor enhancing this property. Avoiding known chemotactic compounds or adjusting the intervals of the treatment may help to eliminate this iatrogenic component of the inflammatory process especially in patients with chronic ocular inflammation.

PROBLEM: There is considerable controversy concerning the root cause and mechanisms of early embryo loss. It has been suggested that most pregnancy losses occur due to morphogenetic anomalies of the embryo. It has also been suggested that the maternal specific immune system rejects the embryo. METHODS: Existing data on the cell and molecular biology of early embryo loss in murine experimental models is reviewed. RESULTS: Using the CBA? x DBA/2? model of early embryo loss, it has been established that maternal inflammatory cells infiltrate the decidua basalis of all implantation sites within 48 hr after implantation. For most embryos, the relatively low numbers of macrophages (MΦ) and natural killer-like (NK-like) cells of maternal origin remain relatively constant after day 8, whereas 20-30% of the embryos show a significant increase in inflammatory cells in the maternal decidua, corresponding to the incidence of early embryo resorption visible at day 12. Evidence will be reviewed to suggest that decidual NK-like cells are not cytolytic but may be producing the MΦ-activating cytokine interferon gamma (IFN-γ), which activates decidual MΦ and other cells. Furthermore, embryo loss is ameliorated by in vivo treatment with anti-IFNγ or anti-NK antisera, indicating that NK-like cells and/or IFNγ are required for embryo loss, but not for embryo survival. In resorbing embryos, the inflammatory MΦ show evidence of having been primed during early pregnancy, in that in vitro incubation with lipopolysaccharide induced the production of tumor necrosis factor alpha and nitric oxide. CONCLUSION: These findings support the concept that early embryo loss is a nonspecific event mediated by the triggering of cytotoxin production by primed decidual macrophages.

Thymocytes develop in close apposition to the stromal cells of the thymus. The ontogeny of thymocytes is dependent on intimate interactions between these cells and the stromal cells. The molecular mechanisms involved in regulating thymocyte–stromal cell interactions remain to be clearly defined. In this study, we utilized a polymerase chain reaction strategy to identify members of the cadherin family of cell adhesion molecules that are expressed by CD4+CD8+thymocytes, the major cell type in the thymus. One classical cadherin (E-cadherin), three atypical cadherins (OB-cadherin, K-cadherin, and cadherin-8), and two novel cadherins (T1-cadherin and T2-cadherin) were found to be expressed by the CD4+CD8+thymocytes. The discovery that these cells display multiple cadherins opens a new area of investigation concerning the adhesive mechanisms involved in modulating thymocyte–stromal cell interactions. We speculate that cadherins will prove to play an essential role in the ontogeny of thymocytes.

The causes and precise mechanisms leading to early embryo loss in mammals remain largely unknown, especially from a molecular point of view. Using the CBA/J x DBA/2 murine model of early spontaneous embryo loss (25-30% embryo loss), we have previously demonstrated the involvement of infiltrating activated macrophages and their cytolytic products such as nitric oxide and tumor necrosis factor alpha (TNF alpha) in the etiology of early embryo loss. On the other hand, far fewer of the CBA/J x Balb/c conceptuses (5-10% embryo loss) displayed significant cellular infiltration and nitric oxide and TNF alpha. Having used probes for cellular activation markers, we now present evidence indicating that significantly increased expression of AP-1 family members, Ha-ras, Ki-ras, v-erbA, v-raf, v-abl, and c-myc was present in 24.4% of the CBA/J x DBA/2 embryonic units that also harbored significant Mac-1, F4/80, and class II major histocompatibility complex (MHC) molecule cellular infiltration. In contrast, only 7% of CBA/J x Balb/c conceptuses displayed increased proto-oncogene expression and increased cellular infiltration. Therefore, macrophage infiltration, cellular activation as identified by the increased expression of proto-oncogenes, and the production of cytotoxic macrophage products are closely linked to early embryo loss. These data add to the evidence that activated maternal macrophages may be directly responsible for spontaneous pregnancy failure.

Problem Spontaneous early embryo resorption following implantation occurs in many species, but little is known regarding the causes or the prevention of early pregnancy failure. Embryo and fetal loss have widely been assumed to be due to maternal allospecific immune rejection. Alloimmunization therapy with paternal tissues has been successfully used in human and murine pregnancies to prevent early embryo demise. The mechanisms of this treatment have been assumed to be the induction of antigen specific, fetal "graft" enhancing antibodies and suppressor cells. The purpose of this study was to investigate the validity of this assumption. Method To investigate these general assumptions, female CBA/J mice were immunized with either specific or nonspecific antigens prior to mating with DBA/2 or Balb/c males. Further, a model system for the study of bacterial lipopolysaccharide (LPS) induced abortion was used to demonstrate the nature of antigen specific immune protection against abortion. Results Whereas the administration of 1 microgram of LPS to CFW female x CFW male pregnant mice on day 7 of gestation induced complete fetal resorption, prior immunization with 20 micrograms of LPS completely prevented LPS induced abortion as long as the anti-LPS antibody titers remained above a threshold value of about 1/500. Therefore, early embryo loss could be induced by a bacterial infection and could be prevented by appropriate immunity to abortogenic factors. However, due to the short half-life of IgM antibodies, immunity to LPS was short-lived and the protective effect of LPS immunization against LPS induced abortion waned after 5 wk. Through the use of the CBA/J female x DBA/2 male model system to study spontaneous early embryo loss, previous vaccination of CBA/J female mice with Balb/c spleen cells expressing paternal MHC antigens, complete Freund's adjuvant (CFA) or LPS, all decreased the incidence of spontaneous resorption in subsequent pregnancies. Similarly, a previous mating with a Balb/c male prevented spontaneous embryo loss for a period of up to 6 wk. However, none of the immunotherapeutic vaccinations or matings had a permanent effect on CBA/J female x DBA/2 male embryo survival, which one would have expected if specific immune mediators were involved. Conclusion The results of this study indicated that the decrease in the incidence of spontaneous embryo resorption following alloimmunization was more likely to be due to nonspecific immunomodulatory effects on the immune system of the female mice, as opposed to specific antipaternal immunity. This may, in part, explain the placebo effects observed for alloimmunization therapy for human habitual pregnancy loss. The relevance of these results to the development of immunotherapy strategies for the prevention of habitual abortion is discussed.

In early embryo loss, the fetus may be considered to be an allograft and, therefore, may be rejected by maternal immunocytes. However, the cytotoxic mechanisms involved are still poorly understood. We have previously shown the involvement of natural killer (NK) cells and mononuclear cells expressing Mac-1 (CD11b) and F4/80 in resorbing compared to nonresorbing embryos. In this study, the role of nitric oxide (NO) in the mechanism of early embryo loss was studied. Pregnant CBA/J females mated with DBA/2 males (20-30% early embryo loss) and CD1 females mated with CD1 males (5-10% early embryo loss) were studied on days 8, 10, and 12 of gestation. Cells from the implantation sites of individual embryos were tested for the production of nitrite and nitrate with or without in vitro challenge with lipopolysaccharide (LPS) to determine whether decidual macrophages were primed in situ. On day 12 of gestation, when resorption was clearly visible, resorbing embryos showed more than a fivefold increase in both basal- and LPS-induced nitrite and nitrate production compared to nonresorbing embryos in both mouse strains tested, indicating that the decidual mononuclear cells were primed. Furthermore, more than 20% of CBA/J embryos showed a significant nitrate release on days 8 and 10 of gestation before any signs of embryo cytopathology. This percentage corresponded to the spontaneous resorption rate seen in CBA/J female X DBA/2 male matings. Similarly, 4% of the embryos from pregnant CD1 mice on days 8 and 12 of gestation produced a significant amount of nitrate, which again correlated with the low incidence of resorption observed in these mice. Using immunohistochemistry, the presence of inducible nitric oxide synthase (iNOS) was detected at implantation sites. Furthermore, decidual cells positive for both iNOS and the macrophage marker Mac-1 were demonstrated in implantation sites by double immunostaining. This strongly suggests that decidual macrophages could be the cellular source of NO production. Aminoguanidine, a selective inhibitor of the iNOS, inhibited the in vitro production of nitric oxide by cells isolated from individual implantation sites, and more strikingly, significantly reduced early embryo losses in CBA/J females mated by DBA/2 males when given orally or parenterally to the gravid females starting on day 6 of gestation. In addition, aminoguanidine-treated pregnant mice showed a significant increase in average litter size when the pregnancies were allowed to proceed to term.(ABSTRACT TRUNCATED AT 400 WORDS)

PROBLEM: Even though our knowledge of the phenomenon at play at the fetoplacental interface has greatly advanced during the past years, a complete understanding of the reasons why the developing embryo is not rejected by maternal immune effector cells remains largely unknown. METHODS: We have used immunohistochemistry with the macrophage-specific markers F4/80 and MHC II to study the relationship between decidual infiltration and resorption in murine models of embryo loss between days 6 and 10 of gestation. RESULTS: Analysis of day 8 CBA/J × DBA/2 pregnancies has revealed 2 distinct populations of embryos. The majority (69.4%) expressed low levels of F4/80+ cells, but a minority (30.6%) expressed much higher level of the macrophage marker. In CBA/J × BALB/c, most embryos (91.7%) expressed low numbers of F4/80+ cells. As earlier experiments established that products of activated macrophages (TNF-α and nitric oxide) were implicated in embryo loss in this model, the activation status of the F4/80+ macrophages was assessed through the cell surface expression of MHC II. Again, a similar association was established: 30.6% of the CBA/J × DBA/2 embryos were infiltrated by significantly more MHC II+ cells than the control CBA/J × BALB/c mating. Finally, when coordinate expression of F4/80, MHC II and CD11b was assessed, it was found that an embryo significantly infiltrated by cells bearing one of the 3 markers was also heavily infiltrated by cells bearing the 2 other markers. CONCLUSIONS: This study has shown that the augmented infiltration of the deciduum with maternal macrophages is an early event which precedes spontaneous abortion of the early embryo.

The anterior chamber of the eye is known to be a site of immune privilege. Particularly, the aqueous humor (AqH) appears to possess unique immunoregulatory properties. The authors have previously shown that human AqH (HAqH) may increase or decrease the proliferation of different cell types. Although no single factor has been established as solely responsible for these effects, much attention has been given to the 24-30 kD fraction of AqH, where transforming growth factor-beta (TGF-β) is found. The purpose of this study was to determine the changes occurring in the rabbit AqH (RAqH) in relation to intraocular inflammation. Heterologous lens or human serum albumin (HSA) immunization-induced uveitis models were used in two groups of New Zealand albino rabbits to study the relationship between uveitis and TGF-β. AqH and serum samples were obtained serially before, during and after the induction of ocular inflammation. Systemic humoral immunity to HSA or lens antigens was monitored using enzyme-linked immunosorbent assay (ELISA). A mink lung epithelial cell (CCL-64) bioassay for TGF-β was used to quantify the amount of this cytokine in RAqH. TGF-β levels in RAqH increased fourfold after the first immunization. A sharp decrease in RAqH TGF-β levels was found in association with the development of acute intraocular inflammation. The implications of this finding to the etiology of uveitis are discussed.

The relationship between the early cellular response to embryo implantation and subsequent embryo survival was explored. Immunohistochemistry using the anti-CD11b antibody (Mac-1) was used to localize and quantify maternal inflammatory cells present at the fetoplacental interface. CD 11b is expressed mostly on macrophages, but is also present on natural killer (NK) cells, neutrophils, and B cells. The occurrence of CD11b-positive cells at the fetoplacental interface was quantified in CBA/J females mated by DBA/2 males (20-30% embryo loss) and CBA/J females mated by BALB/c males (5-10% embryo loss) in order to investigate the relationship between infiltration by these types of cells and subsequent embryo loss. CD11b-positive cells were found to infiltrate decidua of each embryo starting at Day 6 of gestation. Their numbers sharply increased on Days 7 and 8, to a plateau on Days 8 to 10, well before any damage to the embryo is macroscopically visible on Days 10 to 12 of gestation. The resorption-prone mating of CBA/J female by DBA/2 male showed a significantly elevated number of CD11b-positive cells in 26% of the embryos on the eighth day of gestation compared to CBA/J female by BALB/c male matings which were taken as the reference mating. Moreover, experimental conditions modulating fetal survival in CBA/J mothers such as poly (I:C) treatment of DBA/2-mated females (lower survival) or mating with BALB/c males (higher survival than with the mating with DBA/2 males), were found to be associated with high or low numbers numbers of CD11b-positive cells at the fetoplacental interface. Furthermore, injection of anti-CD 11b into pregnant mice at Day 6 of gestation significantly reduced the subsequent incidence of resorption in the resorption prone CBA/J x DBA/2 mating. These results suggest that CD11b-positive cells are associated with the etiology of spontaneous abortion in this system.

The mating of CBA/j female mice (H2k) by DBA/2j male mice (H2d) typically results in an elevated incidence of spontaneous embryo loss thus providing an ideal genetically controlled laboratory model for the study of the factors causing early embryo loss during pregnancy. There is now considerable data on the cells and factors involved in fetal resorption but little is known about the events which activate this process. While the activation of the maternal response to the fetal implant could have endogenous or genetic origins, a role for exogenous factors including microbial pathogens could also be involved. In order to investigate these possibilities, the reproductive success of CBA/j female x DBA/2j male matings in a conventional animal care facility were compared with matings in a specific pathogen free (SPF) animal facility. All animals housed under these conditions were routinely screened by immunoassay and culture, for the presence of a number of viral and bacterial pathogens of mice. The incidence of spontaneous embryo loss in specific pathogen free CBA female mice mated by DBA and other male strains was found to be virtually identical to that of CBA female mice infected with multiple viral pathogens and housed under otherwise identical conditions (non-SPF). However, the numbers of implantation per pregnancy was significantly greater in an SPF facility. Therefore, exposure of mating mice to exogenous viral and bacterial pathogens did not appear to alter the overall incidence of spontaneous embryo resorption. It was concluded that the immunomodulatory effects of infection by common murine pathogens neither augmented nor reduced post-implantation embryo losses.

The use of biological assays in research often creates complex Problems of data analysis. Due to the nature of bioassays which respond to the intricate interplay between multiple factors that may either increase or decrease the readout, simple arithmetic or standard linear regression analysis often fails to provide accurate values when extrapolation from a standard curve is employed. Non-linear polynomial analysis could provide a better way for expressing such complex relationships. Using a bioassay for transforming growth factor-beta (TGF-β) employing the mink lung epithelium cell line CCL-64, the authors present a new way to analyze experimental data based on non-linear polynomial statistics to provide accurate quantitative results when other simpler techniques have failed. This technique for data analysis provided accurate consistent results with acceptably small experimental variance. Using this approach, the bioactive concentration of TGF-β in normal rabbit aqueous humor was found to be 2.72 ± 0.61 ng/ml, that is, corresponding to 2. 72 ng of PURE TGF-β2, when referring to a standard curve.

Female CBA/J mice mated with DBA/2 males exhibit an increased spontaneous resorption rate (30-35%) in their first pregnancy. Second pregnancies show a decreased resorption rate (15-20%). in contrast, resorption in CBA/J females mated with BALB/c males (identical to DBA/2 at the H-2 major histocompatibility locus) occurs with a frequency of 5-10%. Resorption is preceded by fetoplacental infiltration of natural killer (NK).Iike cells and a deficiency in a lipophilic NK-suppressive activity. The eicosanoids leukotriene B4 (LTB4) and prostaglandin E2 (POE,) are known to modulate NK activity in vitro. We measured the concentrations of LTB4 and POE2 in extracts of individual fetoplacental units at Day 8 of gestation from (1) primigravid CBA/J X DBA/2 resorption-prone matings (RES); (2) second CBA/J x DBA/2 matings (SEC); and (3) primigravid CBA/J X BALB/c control matings (CON). We detected a significant decrease in the mean concentration of LTB4 in RES fetoplacental units (176.4 ± 11.8 pg/mI; n = 42) compared with CON and SEC fetoplacental units (570.2 ± 45.5 pg/mi; n = 21 and 420.2 ± 59.5 pg/mI; n = 39, respectively). To confirm that the LTB4 deficiency is associated with decreased NK suppression in RES matings, we supplemented RES extracts, in vitro, with exogenous LTB4 (0-500 pg/mI). The effect of the addition of LTB4 to RES extracts was biphasic. Addition of LTB4 in the range of 30-125 pg/mI increased the extract’s NK suppressive capacity, whereas LTB4 alone either stimulated NK activity or was without effect. These results suggest a critical role for LTB4 in averting NK-mediated early spontaneous fetal resorption.

Lipid extraction was used to study the natural killer (NK) suppressive activity of individual feto-placental units. Normal pregnancies showed a lipophilic NK cell suppressive factor that was gestational day specific. Feto-placental units from CBA/J x DBA/2 pregnancies were deficient in the NK cell suppressive factor when compared to normal CBA/J x BALB/c pregnancies. The frequency of non-suppressive feto-placental units from CBA/J x DBA/2 pregnancies correlated with the frequency of feto-placental units infiltrated with NK cells and the frequency of spontaneous resorption. Our results implicate a deficiency of NK suppressive activity in the feto-placental unit as a contributing factor in spontaneous fetal resorption.

To compare the chemotactic activity induced by polymethyl methacrylate (PMMA), acrylic, and silicone intraocular lens (IOL) materials, regardless of the surgical and host factors.The chemotactic effect of five different IOLs was studied using a modified multi-well Boyden chamber. Two different non-coated PMMA IOLs, a heparin-coated PMMA IOL, an acrylic IOL, and a silicone IOL were assessed. Each mean migration distance of the IOLs was compared with the others using Student's t test.All IOLs induced some amount of leukocyte chemotaxis. There was no statistically significant difference between the chemotactic activity of PMMA IOLs. However, chemotactic activity was significantly higher in acrylic (p0.05) and silicone (p0.05) IOLs compared to one of the PMMA lenses.This limited study has proven that chemotaxis assay is a useful tool to assess the biocompatibility of IOLs. The IOL material itself might attract inflammatory cells to its surface in the absence of surgical and host factors. As PMMA IOLs do not cause an increase in inflammatory reaction, they may be considered safe to use. Further improvement in chemical composition and surface characteristics of IOLs should reduce the inflammatory reaction and increase the biocompatibility and safety of IOLs.

We have previously shown that both priming and triggering signals were needed for nitric oxide production by decidual macrophages and that nitric oxide was responsible for embryo wastage. In this study, we investigated the role of IFN-gamma as the primary signal for macrophage activation in early embryo loss. IFN-gamma-deficient (GKO) and heterozygous F1 control mice were injected with lipopolysaccharide (LPS) at day 7 of gestation. The results showed that the GKO mice were more resistant to LPS-induced embryo loss than the wild type. This suggested that IFN-gamma was needed for LPS-induced embryo resorption and that decidual macrophages from pregnant GKO mice were not primed and could not be activated when given LPS. Further, the results showed that IFN-gamma mRNA was simultaneously expressed in the same embryos that also expressed mRNA markers for macrophage activation (TNF-alpha and iNOS), indicating that macrophage activation could be a consequence of IFN-gamma production. Similarly, we investigated the role of IL-12 as a switch cytokine capable of eliciting TH1-associated cytokine production including IFN-gamma. The results showed that IL-12 mRNA expression was correlated with IFN-gamma expression and macrophage activation. In this in vivo study, we showed for the first time that spontaneously increased decidual IFN-gamma expression is detrimental to embryo survival.

Pregnancy outcome may be altered by both genetic and environmental factors. The mating of CBA/J female mice with DBA/2 males normally results in pregnancies characterized by a relatively high incidence of early embryo compared with most other syngeneic or allogeneic matings. This study addressed the role of normal laboratory stress in the induction of early embryo loss. The previously studied 'Bruce effect' describes the total loss of preimplantation embryos (pregnancy block) that is apparently caused by the stress induced by the presence of an alien male and mediated by neuroimmunological effects on prolactin activity. To determine whether this effect could be responsible for the high incidence of postimplantation embryo losses in the CBA/J x DBA/2 model, the original DBA/2 male was replaced on day 6 of gestation by another DBA/2 male, a CBA/J, a C57Bl/6 or a BALB/c male. The relatively high incidence of embryo loss was not affected by removing the original DBA/2 male or introducing another DBA/2 or a CBA/J male, indicating that stress induced by an alien male did not increase the postimplantation losses in this model. Furthermore, the introduction of a DBA/2 male to a CBA/J female that had been mated with a BALB/c male did not elicit early embryo loss. However, the replacement of the original DBA/2 male by a BALB/c male dramatically reduced the incidence of early embryo loss in pregnant CBA/J female mice. The introduction of a C57Bl/6 male also reduced embryo loss but to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppression of cholesterol synthesis by 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, such as lovastatin, has been shown to inhibit mitogen stimulated proliferation of natural killer (NK) cells and other lymphocytes in vitro. This effect is only partially overcome by provision of exogenous free or lipoprotein cholesterol but is reversed by mevalonate, suggesting that proliferating lymphocytes have a specific requirement for a nonsterol isoprenoid product of mevalonate. The effect of lovastatin (20 mg bid) on a range of immune function parameters was determined in a randomized, placebo-controlled, double-blind ex vivo study in 52 patients with primary hypercholesterolemia. No significant differences (P0.05) were found between lovastatin and placebo groups for basal NK or interleukin-2 (IL-2)-induced cell-mediated cytotoxicity, PHA-stimulated lymphocyte proliferation, or relative numbers of T lymphocytes (CD3+), B lymphocytes (CD19+), total NK cells (CD3-, CD16+, CD56+) and CD57+ NK cells or in immunoglobulin levels after 4 or 8 weeks of treatment. In contrast to previous in vitro data, no statistically or clinically significant changes were observed in any parameter of lymphocyte function in patients treated with lovastatin.

Certain strains of mice display an increased frequency of fetal resorption, but little is known about the effector mechanisms involved. We have examined the events associated with lipopolysaccharide (LPS)-induced fetal resorption in mice. Administration of 25 micrograms LPS on Day 12 of gestation resulted in the appearance of tumour necrosis factor-alpha (TNF-alpha) in the amniotic fluid and fetal resorption. Levels of LPS-induced TNF-alpha were reduced by 90% after pretreatment with the TNF-alpha-suppressing drug pentoxifylline (PXF). Treatment of pregnant mice during early gestation with 0.1 micrograms LPS resulted in fetoplacental resorption which was maximal when the LPS was given on Day 8. Resorption induced by 0.1 micrograms LPS on Day 8 of gestation was significantly reduced by pretreatment with PXF. Infiltration of asialo-GM1-positive cells was observed in the decidual-ectoplacental cone area of embryonic units from LPS-treated mice. In addition, treatment with anti-AGM1 antiserum prevented the LPS-induced resorption. Our results suggest that TNF-alpha and asialo-GM1-positive cells are involved in LPS-induced fetal resorption.

A simple and efficient method for the analysis of the affinity and number of functional transferrin receptors (TFR) on human tumor cells is described. The technique is designed to utilize microtitration equioment; and is suitable for easy comparison of up to 8 different cell preparations per assay. Using this technique, 5 established cell lines were evaluated for functional TFR expression. The control erythroleukemic cell line K562 possessed 3.28 × 10 5 functional TFR per cell (±3.69 × 10 4 , SEM) K d = 9.0 × 10 −9 · M −1 . Trypsin and heat-pretreated cells were compared to control erythroleukemic K562 cells from the same culture to determine both the effects of receptor removal and cell viability on the assay. Trypsin and heat pretreatment of these K562 cells severely decreased receptor function as indicated by Scatchard analysis as well as by time course and cold competition analysis respectively. Whereas the affinity of trypsin-treated receptors on cells was similar to control values, heat-killed cells displayed an altered cellular affinity for 125 I-transferrin underscoring the importance of utilizing cells of high viability in receptor assays.

The effect of serum on the K562 assay for spontaneous human lymphocyte-mediated cytotoxicity (SLMC) was examined systematically at the level of the assay system and with respect to the serum used for the long-term maintenance of the K562 target cells. It was found that the assay did not require the presence of serum, but all sera tested increased the level of cytotoxicity observed. Lymphocyte and target cell preparation in serum-free medium led to a high non-specific cell loss which could be avoided by substituting 5% human serum albumin for fetal calf serum. With this serum-free assay, the effect of long-term culture of K562 in 5% human serum was examined. High SLMC by normal donors was obtained in spite of the fact that the target cells had been cultured in human serum. This occurred even when the serum used for long-term culture was autologous to the lymphocyte donor, and as long as the experiment was continued (10 months). The relative cytotoxicity of each donor relative to the other two tested in this series of experiments was similar for each of the K562 sources tested, and every time that the experiment was performed. Persistence of susceptibility to cytotoxicity after 10 months of culture in 5% human serum, partial sensitivity to pronase and trypsin, and resistance to neuraminidase, all indicated that the antigen recognized by the effector cells in this assay is distinct from the heterologous membrane antigen described by Irie et al. (1974a, b). The ability of lymphocytes to lyse target cells cultured in serum autologous to the lymphocyte donor indicated that SLMC is not mediated by lymphocyte recognition of serum-derived antigens or serum-derived mitogenic factors. The “recovery” of the SLMC susceptibility of pronase or trypsin-treated target cells in serum-free medium also suggests that the K562-SLMC “antigen” is intrinsic to the K562 target cell and is independent of the serum used to culture these cells.

Stable, soluble labelling compounds are formed by the reaction of DNP-, TNP- and dansyl-L-lysine with cyanuric chloride at 0°C in aqueous solutions. These compounds react efficiently at room temperature with carbohydrates and with sheep erythrocyte membranes. Mice immunized with Ficoll haptenated with these reagents yield characteristic thymus-independent plaque-forming cell responses.

Immunohistochemistry was used to study a murine model which spontaneously aborts at a frequency of 25 to 30%. Our results show that natural killer (NK) cells are not only the predominant infiltrating cells in aborting feto-placental units, but that they also appear in a similar proportion of feto-placental units before abortion is detectable. The frequency of feto-placental units with significantly elevated NK infiltrates corresponds to the subsequent abortion frequency, indicating a causal relationship. Immunization of the mother with BALB/C splenocytes prevents these NK infiltrates and decreases the abortion frequency to normal levels. These results suggest for the first time that maternal NK cells may have an instrumental role in the etiology of spontaneous abortion.

The effect of various lymphocyte depletion techniques on SLMC effector cell activity against the cell line K562 could be attributed t o the effects that these procedures had on the proportions of FclC3 receptor-bearing cells in the preparations. Depletion of cytotoxic activity by removal of Fc receptor-bearing cells could be augmented by the presence of complement on the 7S-EA indicator cells used for rosette depletion. Two methods of assessing the presence of surface receptors on cytotoxic cells are presented: (1) linear regression analysis of spontaneous cytotoxicity plotted as a function of the proportions of receptor-bearing cells remaining after various depletion techniques; and (2) the use of a mathematical formula t o estimate the proportion o f cytotoxic cells with any particular receptor, based on the effects of receptor-bearing cell depletion. Linear regression analysis of cytotoxicity vs the proportions of SRBC, Fc o r C3 receptor-bearing cells demonstrated that all cytotoxic cells had the Fc receptor, but that cytotoxicity could s t i l l occur in the absence of cells with the C3 receptor. This indicated that the C3 recepor is not necessary for the demonstration of SLMC activity. The mathematical formula to predict cytotoxicity after depletion of C3 receptor-bearing lymphocytes showed, however, that a significant proportion of the Fc receptor-positive killer cells were also C3 receptor-positive. No direct evidence was found t o support a role for T cells in SLMC, although E rosette depletion resulted in lower than expected cytotoxicity i n view of the number of Fc receptor-bearing cells remaining in the preparation.

Dichlorotriazinylaminofluorescein (DTAF), the product of the reaction of aminofluorescein with cyanuric chloride, is an effective reagent for conjugating fluorescein to immunoglobulins. DTAF has absorption and emission properties nearly identical to fluorescein-isothiocyanate (FITC) and DTAF and FITC-labelled antibodies are similar in terms of preparation and specificity of immunofluorescence. However, DTAF is superior to FITC with regard to cost, purity and stability. Also, DTAF-labelled rabbit IgG conjugates can be quickly and efficiently fractionated by simple ammonium sulfate precipitation procedures to yield preparations free of both over- and under-conjugated material. In addition, over-conjugated protein can be readily removed from DTAF:IgG conjugates by appropriate adjustment of pH and temperature.

A method is described for the measurement of human natural killer cell activity using heparinized whole blood in a 51 chromium release assay. Fractionation-reconstitution experiments showed that the cytotoxic activity was abolished by removal of the Fc receptor bearing lymphocytes, but not by the elimination of monocytes and granulocytes. Autologous or pooled plasma was not found to possess inherent cytolytic activity. By analogy to an enzyme kinetic reaction, the results were expressed as kinetic lytic units (KLU) which were defined as the maximum number of target cells that could be lysed per unit time per milliliter of whole blood. The buffy coat cytotoxicity (BCC) assay is quick, easy to perform, and suitable for screening and monitoring of natural cytotoxicity. Since this methodology preserves the actual concentration of natural killer cells, it may represent a truer reflection of in vivo events.

Human lymphocytes will rosette with human red blood cells under certain conditions (H rosettes). The work presented in this paper documents the following: (1) a reproducible short-term assay for the detection of H rosettes in proportions similar to those reported recently in the literature (25-30%); (2) technical improvements on the basic assay system demonstrating that, in fact, 40-65% of human lymphocytes form H rosettes; (3) the fact that, in normal donors, auto and allo H rosettes are formed by identical lymphocyte subpopulations; (4) that H rosettes are formed independently of the ABH, Rh and Thomsen-Freidenreich (TF) red-cell antigens on the indicator red cells; and (5) that H rosette-forming cells consist almost entirely of T lymphocytes having high-affinity receptors for sheep erythrocytes. The large difference in size between sheep and human RBC made it possible to do double rosette assays in which high- and low-affinity E-rosette-forming T cells could be visualized on the same slide. As with T lymphocytes in general, a large proportion of HRFC was found to have Fc-IgM receptors while a smaller proportion has Fc-IgG receptors. Studies done on a small group of leukemic patients supported the T-cell nature of the H rosette-forming cell.

A 52-year-old Caucasian man with chronic neutropenia and recurrent infections was found to have an increased proportion of peripheral T lymphocytes having Fc receptors for IgG (T gamma ). Although levels of antibody-dependent cell-mediated cytotoxicity (ADCC) and "natural" killing (NK) by unfractionated lymphocytes were similar to those of a control donor, the frequency of KN cells was markedly increased. Removal of E rosette-forming cells eliminated both NK and ADCC by the patient's peripheral blood, in marked contrast to the enhanced cytotoxicity seen with control lymphocytes. Both normal and patient ADCC and NK functions were removed by depletion of Fc receptor-bearing cells. These depletion experiments proved that all of the patient's killer cells were E rosette-forming T gamma cells, in contrast to the heterogeneous pattern of null gamma and T gamma killer cells seen in the blood of normal donors. The homogeneity of the T gamma proliferation suggested that ADCC and NK were mediated by the same cell type, albeit acting by different mechanisms. The addition of the patient's serum and lymphocytes to chromium-labelled normal granulocytes caused a low but significant level of cytotoxicity, indicating that the patient's neutropenia may have been caused by a similar mechanism in vivo. There was no evidence of complement-dependent serum antibody-mediated neutrophil lysis, but one serum sample taken over the course of the patient's disease agglutinated granulocytes from four of five donors tested.

Natural killer cells have been postulated to play a role in the immunopathogenesis of graftversus-host disease (GVHD). This is based on reports of augmented natural killer (NK) activity, both in animal models and in humans. It has not been elucitated from previous reports, however, whether the cells mediating this activity are of donor or recipient origin. We have investigated the origin of the effector cells using highly specific antisera: Our results indicate that the augmented lytic activity seen during the course of GVHD is mediated primarily by donor lymphocytes. Depletion with anti-Class I antisera followed by immunostaining with anti-asialo GM-1 indicate that the effector cells are also asialo GM-1 positive. We report here that the augmented non-specific lytic activity seen during GVHD is mediated by asialo GM-1 positive lymphocytes predominantly of donor origin.

On analysis of in vitro assays of human natural killer (NK) cell function the inadequacy of commonly used methods of expressing lytic activity was apparent. A comparison was made of the data obtained using modifications of two equations-the simple exponential fit and the von Krogh equations. Both of these equations were found to satisfy the following essential criteria for use in these assays. First, the majority of the results obtained in the chromium-release assay could be used in data reduction; second, the resultant "dose-response" curve was reduced to linearity; and third, a single numerical expression was obtained which was directly proportional to the cytotoxic activity. Of the two methods the more conventional exponential fit was found to be the simpler to use. The closeness of fit of the experimentally derived data to the ideal curves did not support the possibility that normal lymphocyte preparations contain suppressor cells capable of inhibiting NK activity. Data have also been presented showing that NK-sensitive targets could be categorized with respect to their susceptibility by comparing the slopes of the target cell survival curves obtained using the exponential fit equation. These observations are relevant to the accurate assessment of NK activity in patient populations and to the determination of the effects of disease and its treatment on this activity.

Publisher Summary This chapter presents an assessment of natural killer (NK) cell activity in cancer patients. It has been shown that there is a relationship between the presence of malignant disease and alterations in NK activity. However, it is unclear, whether these alterations are the result of the malignant disease or whether NK cells actually influence the course of the disease. This chapter presents a study in which approximately 1650 cancer patients and 550 normal adults were investigated with respect to their NK activity. It is customary to plot NK data as per cent cell-mediated lysis versus log:LT ratio, which results in a sigmoid curve. Because the curve is not linear, the proportional difference between two donors varies markedly, depending on which LT ratio is used as the comparison point. Similarly, the use of lytic units calculated from these curves also gives different relative cytotoxicity values, depending on the percent lysis value chosen. It is absolutely essential, therefore, that cytotoxicity data be rendered linear and that comparison between two sets of values yield data that reflect the proportional difference between the two donors.

A rabbit model for the study of hypersensitivity to thimerosal was established in order to develop better techniques for screening patient sera and tears for specific antibodies to lens care re-agents. Thimerosal (sodium ethylmercury thiosalicylatelate) was successfully coupled to several protein carriers using a water soluble carbodiimide which linked the carboxyl group of thimerosal to free amino groups of the carrier proteins. Thimerosal was also shown to spontaneously react with proteins as detected by the irreversible binding of mercury to the protein carrier. Immunization of rabbits with the chemically coupled thimerosal resulted in the production of antibodies which specifically reacted with thimerosal. The rabbits also manifested delayed and immediate forms of hypersensitivity to the thimerosal conjugates. The ELISA assay for specific serum antibodies was found to be a sensitive, reliable and specific screening tool However, there was no immunological cross reactivity between the chemically coupled thimerosal and the spontaneously coupled thimerosal. Therefore, the epitopes produced by these two reaction mechanisms were probably immunochemically different even though both contained detectable thimerosal derived mercury.

The results of natural killer cell (NK) studies on 539 normal healthy donors tested from once to 213 times over a seven-year time span have been presented. NK activity did not vary with donor blood group, Rh type or (in a small sample) HLA type. There was a slight but significant increase in NK activity from birth to adulthood, and between males an females. The male/female difference was present at birth and persisted through adulthood. The relative NK activity of individual donors tested repeatedly over many years was remarkably consistent in spite of variability in the absolute cytotoxicity observed. The expression of NK data in terms of relative NK was found to be superior to other methods, and the values obtained were found to be independent of the NK-sensitive target cell used. Although age and sex differences in NK activity are slight, their existence should be considered when studies of NK activity in patients are analysed.

Previous studies to determine the nature of the specificity of natural killer (NK) cells for leukemic cells indicated that functional transferrin (Tf) receptors may be one of the determinants recognized by NK cells. To further investigate these observations, the relationship between cellular Tf receptor expression and ability to compete with a control K562 cell preparation in a standard chromium release assay was studied. K562 cells were selected at different phases of growth by removing cells from tissue culture at 1, 3, and 5 days postfeeding. Under these conditions, K562 cells, respectively, displayed relatively high, medium, and low numbers of Tf receptors and corresponding competitive activity against a control K562 cell preparation. K562 cells were modified by either trypsin, heat, or sodium butyrate (differentiation inducer) pretreatment. An NK-resistant clone was also studied. There was a good correlation between Tf receptor expression and cold competitive activity of the above K562 cell preparations (r = 0.82, P less than 0.01). The different tumor target cell lines, K562, Molt-4, Raji, HL-60, and MeWo, which would be expected to express different ranges of specificity, did not show a significant correlation between Tf receptor expression and their cold competitive activities against Cr-51-labeled K562 cells. Rabbit reticulocytes which express high numbers of Tf receptors were tested for their ability to compete with K562 cells for NK cells. These cells were able to compete with K562 cells while mature rabbit red blood cells which do not express Tf receptors did not compete well. These findings support the contention that the Tf receptor may be involved in NK cell recognition of some tumor cells.