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The ability of firefighting vehicles and staff to reach a fire area as quickly as possible is critical in fighting against forest fires. In this study, a Geographical Information System-based decision support system was developed to assist fire managers in determining the fastest and the safest or more reliable access routes from firefighting headquarters to fire areas. The decision support system was tested in the Kahramanmaras Forestry Regional Directoratein the Mediterranean region of Turkey. The study area consisted of forested lands which had been classified according to fire sensitivity. The fire response routing simulations considered firefighting teams located in 20 firefighting headquarter locations. The road network, the locations of the firefighting headquarters, and possible fire locations were mapped for simulation analysis. In alternative application simulations, inaccessible roads which might be closed due to fire or other reasons were indicated in the network analysis so that the optimum route was not only the fastest but also the safest and most reliable path. The selection of which firefighting headquarters to use was evaluated by considering critical response time to potential fire areas based on fire sensitivity levels. Results indicated that new firefighting headquarters should be established in the region in order to provide sufficient firefighting response to all forested lands. In addition, building new fire access roads and increasing the design speed on current roads could also increase firefighting response capabilities within the study area.

Microglia exist under physiological conditions in a resting state but become activated after neuronal injury. Recent studies have highlighted the reciprocal role of neurons in controlling both the number and activity of microglia. In this study, microglia derived from newborn rat cortices were cultured and activated by interferon-gamma (IFNgamma) treatment, then exposed to recombinant Sema3A or conditioned medium derived from stressed embryonic cortical neurons. We found that activation of microglia by IFNgamma induced differential upregulation of the semaphorin receptors Plexin-A1 and Neuropilin-1. This result was confirmed by Northern blotting, reverse transcription-PCR, and Western blotting. Furthermore, recombinant Sema3A induced apoptosis of microglia when added to the in vitro culture, and a similar result was obtained on activated microglia when Sema3A was produced by stressed neurons. Using an in vivo model of microglia activation by striatal injection of lipopolysaccharide demonstrated a corresponding upregulation of Plexin-A1 and Neuropilin-1 in activated microglia and enhanced production of Sema3A by stressed adult neurons. These results suggest a novel semaphorin-mediated mechanism of neuroprotection whereby stressed neurons can protect themselves from further damage by activated microglia.

The unique and complex nature of biotechnology-derived pharmaceuticals has meant that it is often not possible to follow the conventional safety testing programs used for chemicals, and hence they are evaluated on a case-by-case basis. Nonclinical safety testing programs must be rationally designed with a strong scientific understanding of the product, including its method of manufacture, purity, sequence, structure, species specificity, pharmacological and immunological effects, and intended clinical use. This knowledge, coupled with a firm understanding of the regulatory requirements for particular product types, will ensure that the most sensitive and regulatory-compliant test systems are used to optimize the chances of gaining regulatory approval for clinical testing or marketing authorization in the shortest possible time frame.

Insulin-like growth factor-2 (IGF-2) present in media conditioned by non-activated and interferon gamma (IFN gamma)-treated microglia reduces galactocerebroside(+) (GalC) oligodendrocyte apoptosis in cultures derived both from the CG4 cell line and primary rat cortices. Microglia-derived IGF-2 also acts in each culture system to block GalC(+) oligodendrocyte toxicity resulting from soluble microglial-derived tumour necrosis factor alpha (TNF alpha). IGF-2 inhibits TNF alpha-induced c-Jun kinase (JNK) activation of the CG4 cell line. Microglial activation results in the release of soluble factors that are potentially toxic to oligodendrocytes but this may be offset by the production of soluble factors that protect these vulnerable cells. Allowing for extrapolation of these in vitro findings to intact tissue, our observations suggest one mechanism for limiting bystander damage in the context of inflammatory brain disease.

The explosion of genuine high throughput technologies has allowed large compound libraries to be screened with ever increasing biological specificity, exacerbating the problem of lead candidate selection for subsequent drug development. To avoid creating a bottleneck, compounds identified from the high throughput screens undergo lead optimisation, a medium-throughput screen which allows ranking in terms of their basic absorption, distribution, metabolism, excretion (ADME) and toxicological properties. The historical role of the preclinical scientist in the drug discovery/development continuum has been to perform ADME and toxicology studies, simply to support the regulatory submission of lead candidates. This situation is, however, changing with the development of preclinical lead optimisation technologies (Approaches to High Throughput Toxicity Screening, London, Atterwill et al., 1999) facilitating the selection of leading candidates, thereby bridging the gap between high throughput efficacy screens and traditional safety assessment programmes.

We use spatial data representing transportation networks, elevation, stand height, and recreation use to construct and compare models of recreation use patterns and visibility in a forest. The recreation use pattern model depicts use frequencies along travel corridors. The visibility model quantifies visibility for all forest areas. We find that the models provide different but complementary types of information. Forest managers who are involved in scheduling harvest operations and want to address the visual concerns of forest visitors may benefit most from the visibility model. Managers who wish to know more about travel patterns or to reroute forest visitors affected by operations may benefit from the use pattern model. A combination of the two models has the highest potential for providing planning assistance in multiple-use forests. Both models may be able to enhance visual resource management (VRM) systems already in use by providing spatially explicit recreation use and visibility data.

We demonstrate a role for nonactivated rat microglia in the survival and maturation of oligodendrocyte precursor cells (OPCs). Media conditioned by nonactivated microglia increase the number of surviving galactocerebroside(+) (GalC(+)) oligodendrocytes in vitro at 48 h by inhibiting the apoptosis of OPCs and stimulating their maturation to GalC+ oligodendrocytes. These effects are not observed with medium conditioned by microglia activated with interferon-gamma (IFN-gamma). Conditioned medium from nonactivated microglia is associated with upregulation in OPCs of nuclear factor of kappa binding (NF-kappa B) p65 subunit. The use of antisense to the inhibitor of kappa binding (I kappa B) induces p65 subunit activation in OPCs and, in common with medium conditioned by nonactivated microglia, also inhibits OPC apoptosis and promotes cell maturation. Anti-platelet-derived growth factor (PDGF) antibody abolishes this effect even though PDGF-A chain is expressed at similar levels within both nonactivated and IFN-gamma-activated microglia and both conditioned media have similar levels of PDGF-A chain bioactivity. However, only conditioned medium from nonactivated microglia recruit phosphatidyl-3-inositol (PI-3) kinase to the PDGF-alpha receptor and synergise with endogenous PDGF-A chain to increase NF-kappa B activation. These results suggest that, dependent on their state of activation, microglia produce soluble factors that promote oligodendrocyte development through an effect on the PDGF-alpha receptor-signalling pathway.

The explosion of genuine high throughput technologies has allowed large compound libraries to be screened with ever-increasing biological specificity, exacerbating the problem of lead candidate selection for subsequent drug development. To avoid creating a bottleneck, compounds identified from the high throughput screens undergo lead optimisation by employing medium-throughput screen which permits ranking in terms of their basic absorption, distribution, metabolism, excretion (ADME) and toxicological properties. The historical role of the CRO in the drug discovery/development continuum has been to perform efficacy and toxicology studies, simply to support the regulatory submission of lead candidates. This situation is, however, changing with the development of preclinical lead optimisation technologies facilitating the selection of leading candidates, thereby bridging the gap between high throughput efficacy screens and conventional safety assessment programmes.

The elective treatment of patients with multiple sclerosis, using a humanized anti-leukocyte (CD52) monoclonal antibody (Campath-1H), has illuminated mechanisms that underlie the clinical course of the disease. Twenty-seven patients were studied clinically and by magnetic resonance imaging (MRI) before and for 18 months after a single pulse of Campath-1H. The first dose of monoclonal antibody was associated with a transient rehearsal of previous symptoms caused by the release of mediators that impede conduction at previously demyelinated sites; this effect remained despite selective blockade of tumor necrosis factor-alpha. Disease activity persisted for several weeks after treatment but thereafter radiological markers of cerebral inflammation were suppressed for at least 18 months during which there were no new symptoms or signs. However, about half the patients experienced progressive disability and increasing brain atrophy, attributable on the basis of MRI spectroscopy to axonal degeneration, which correlated with the extent of cerebral inflammation in the pretreatment phase. These data support the formulation that inflammation and demyelination are responsible for relapses of multiple sclerosis; that inflammatory mediators, but not tumor necrosis factor-alpha, cause symptomatic reactivation of previously demyelinated lesions; and that axonal degeneration, conditioned by prior inflammation but proceeding despite its suppression, contributes to the progressive phase of disability. These results provide evidence supporting the emerging view that treatment in multiple sclerosis must be given early in the course, before the consequences of inflammation are irretrievably established.

The interaction between microglia and T cells is important in the development of central nervous system inflammation. This may result in full T cell activation, a partial state of activation, anergy or apoptosis of the 'responding' T cell. Here, we demonstrate that neonatal rodent microglia not only fail to initiate a mixed lymphocyte reaction (MLR), but suppress background T cell proliferation. Even after activation with gamma-IFN or following phagocytosis, microglia remain unable to support a MLR. By contrast, gamma-IFN-activated microglia are able to activate memory T cells in a recall assay resulting in cytokine (gamma-IFN) release and modest T cell proliferation. Although the stimulation index is small, functional relevance is demonstrated. Supernatants from the recall assay stimulate gamma-IFN-dependent activation of a STAT (signal transducer and activator of transcription) factor within resting microglia. This demonstrates that memory T cells not only receive sufficient stimulation from the gamma-IFN-activated microglia to proliferate and produce cytokines, but that there is also a reciprocal stimulation of resting microglia. Importantly, this provides evidence that activated microglia have the potential to propagate immune responses in the central nervous system, but are unlikely to initiate a primary response.

A comparison of the expression and ligand specificity of the C1q (first complement component) receptor on rat microglia and peritoneal macrophages was made. This revealed that radiolabelled C1q was competed from the peritoneal macrophages with intact C1q, and additively displaced by calf-skin collagen and purified C1q globular heads, suggesting the presence of at least two receptors. This was in contrast to microglia, where radiolabelled C1q was displaced with intact C1q and to a modest degree with collagen, but not with globular heads. Taken together, this implies that under these conditions, peritoneal macrophages and microglia both express a C1q receptor which binds to the collagen-like region, and that peritoneal macrophages additionally express a molecule which binds to the globular head of C1q. Analysis of the ligand bound by these cells reflected the differences observed in the competitive binding experiments, with the novel identification of naturally-occurring peptides from the globular head of C1q bound to the peritoneal macrophages, but not the microglia.

There is no established immunological marker of multiple sclerosis activity, which reflects the poor understanding of the immunopathogenesis of multiple sclerosis. Passive measurement of the levels of soluble inflammatory markers, whose half lives are usually measured in minutes and hours, can only indicate the extent of instantaneous inflammation, which is known to fluctuate in multiple sclerosis. We favour measurement of immune responses in vitro. As healthy individuals have T cell reactivities to myelin proteins that are postulated to be pathogenic in multiple sclerosis, we prefer non-antigen specific mitogen and recall antigen assays as immunological markers. We illustrate their use in the treatment of 27 patients with multiple sclerosis using a pulse of humanised anti-lymphocyte (CD52) antibody that caused prolonged T cell depletion. The mitogen-induced proliferation, and secretion of IFN-gamma, from peripheral blood mononuclear cells in vitro was significantly reduced after treatment, suggesting that immune responses had been modulated. Such observations will only gain credence as an outcome measure if they are shown to correlate with clinical or radiological measures of multiple sclerosis activity. Perhaps more importantly, aspects of the pathogenesis of multiple sclerosis may be revealed by close immunological surveillance of patients undergoing experimental treatments.

Cultured rat oligodendrocytes are lysed by complement via antibody-independent activation of the classical pathway. This susceptibility to complement lysis has been demonstrated to be due to lack of CD59, a complement regulatory protein which inhibits assembly of the membrane attack complex. In this study the effects of homologous and heterologous complement were examined in a co-culture system of rat oligodendrocytes and peripheral neurones, where axonal ensheathment was observed as early as 4 days after the addition of glial progenitors to the neurones. Following exposure to complement, ensheathing oligodendrocytes were markedly less sensitive to antibody-independent but not antibody-dependent complement lysis than were cells grown without neurones. Immunocytochemical data revealed that co-cultured oligodendrocytes remained CD59 negative, but in contrast to oligodendrocytes cultured alone, were negative for C3b when incubated with C7-deficient serum. Taken together these data indicate that the decreased sensitivity of co-cultured oligodendrocytes to complement lysis is not attributed to the increased expression of CD59, but rather in a failure to activate complement. Incubation of oligodendrocytes with neurone-conditioned medium afforded significant protection (68%), against antibody-independent complement attack, suggesting that soluble neuronal factors can protect oligodendrocytes from complement-mediated lysis.

We describe the engineering of antibody fragments produced in bacteria for recruitment of complement effector functions. From a phage display repertoire we isolated human antibody fragments directed against complement C1q, and linked these to lysozyme-specific antibody fragments, creating bispecific antibodies (diabodies). One diabody was able to recruit C1q, resulting in efficient lysis of lysozyme-coated sheep erythrocytes, and also induced rosette-formation of erythrocytes with human monocytes and phagocytosis after phorbol ester stimulation. These diabodies may have therapeutic applications requiring the activation of complement.

beta-interferon (beta-IFN) has both pro and anti-inflammatory properties, the balance of which leads to some suppression of disease activity in multiple sclerosis patients. Here, we examine the immunomodulation of neonatal rodent microglia, the principal CNS accessory cell, by beta-IFN and consider the interaction of beta-IFN and gamma-interferon (gamma-IFN). beta-IFN and gamma-IFN inhibit microglial proliferation. beta-IFN antagonises both gamma-IFN-induced upregulation of class II expression and the ability of gamma-IFN primed cells to mount a respiratory burst. In contrast, beta-IFN upregulates microglial Fc receptor expression and augments tumour necrosis factor alpha secretion from suboptimally stimulated microglia.

The administration of the immunosuppressive humanized monoclonal antibody CAMPATH 1-H, which recognizes CD52 on lymphocytes and monocytes, is associated with a first-dose cytokine-release syndrome involving TNFalpha, IFNgamma, and IL-6 clinically. In vitro models have been used to establish the cellular source and mechanism responsible for cytokine release, demonstrating that cytokine release is isotype dependent, with the rat IgG2b and human IgG1 isotype inducing the highest levels of cytokine release, which was inhibited with antibody to CD16, the low affinity Fc-receptor for IgG (FcgammaR). Cross-linking antibody opsonized CD4 T lymphocytes failed to stimulate TNFalpha release, which together with the observation that TNFalpha release by purified natural killer (NK) cells stimulated by fixed autologous CAMPATH 1-H-opsonized targets was inhibited with anti-CD16, indicates that cytokine release results from ligation of CD16 on the NK cells, rather than Fc-receptor (FcR)-dependent cross-linking of CD52 on the targeted cell. Since the hierarchy of isotypes inducing cytokine release in these cultures matches that seen clinically, we conclude that ligation of CD16 on NK cells is also responsible for cytokine release after injection of CAMPATH 1-H in vivo.

It is traditionally held that human IgG4 MoAbs should not deplete target cells in vivo, as this isotype is inactive in a number of in vitro assays that measure effector function. We have previously challenged this dogma, and the current study was designed to investigate the in vivo biological effects in humans of a MoAb of human IgG4 isotype. Nine patients with refractory rheumatoid arthritis (RA) fulfilling ARA criteria, and one with ankylosing spondylitis (AS) received a human IgG4 Campath-1 MoAb (with specificity against the pan-lymphocyte antigen CD52) as part of a two-stage therapeutic protocol. In stage 1, patients received a single dose of this MoAb. Stage 2, starting 48 h later, comprised a 5-day course of a human IgG1 Campath-1 MoAb with identical V-region (CAMPATH-1H), as previously used in the management of RA patients. The intervening 48 h provided a window of opportunity to monitor the biological effects of the IgG4 MoAb for comparison with the IgG1. The two MoAbs were also compared for in vitro biological activity. IgG4 depleted peripheral blood lymphocytes (PBL), albeit less efficiently than IgG1. It produced a first-dose reaction of similar intensity, although associated circulating tumour necrosis factor-alpha (TNF-alpha) levels were lower. TNF-alpha release from whole blood in vitro was also greater with the IgG1 MoAb. The study design did not permit conclusions to be drawn regarding therapeutic efficacy of the IgG4 MoAb. In summary, a human IgG4 Campath-1 MoAb depletes target cells in vivo. Importantly, this study demonstrates for the first time in humans that in vitro assays may not predict the in vivo effector function of therapeutic MoAbs.

The present study analysed the susceptibility of neuronal cells to human complement. A human neuronal precursor cell line (NTera2) which can be induced to differentiate into post-mitotic neuronal cells was employed. Using immunocytochemistry, NTera2 neurones, but not their precursors (stem cells), were shown to activate complement in the absence of antibody and to lack CD59, an inhibitor of the terminal stages of the complement cascade. Following exposure to human serum, neurones but not stem cells were lysed by complement, as demonstrated by a cell viability assay and consistent rise in intracellular calcium. This response was abrogated when cells were treated with complement-depleted serum by heat inactivation or cobra venom factor. Protection from lysis was observed following incorporation of CD59 with its glycolipid anchor in the neuronal cell membrane.

Ex-vivo whole blood assays have been evaluated for their ability to accurately predict the risk of a first-dose cytokine reaction developing in vivo following therapeutic antibody infusion. Tumour necrosis factor alpha (TNF alpha) release was rapidly detected in cultures incubated with either anti-CD52 antibodies of the human IgG1 or rat IgG2b isotype, and to a lesser extent with a human IgG4 isotype. Endotoxin contamination of the antibodies was not responsive for cytokine release, since polymixin B failed to inhibit cytokine release using concentrations of this antibiotic which neutralized the enhanced cytokine release seen from LPS-spiked antibody. A rat IgG2b antibody to CD45 and a human IgG1 anti-CD3 also induced significant TNF release, however, an aglycosyl anti-CD3 mutant devoid of adverse side-effects in vivo, did not result in cytokine release in vitro. Since the pattern of cytokine release seen following the clinical use of these antibodies was in good agreement with the findings of the ex-vivo whole cultures, this demonstrates the usefulness of this assay to predict cytokine release in vivo.

Oligodendroglia (ODG) are unique among glial cell types in their capacity to activate complement in the absence of antibody, causing insertion of the potentially damaging membrane attack complex (MAC) into the plasma membrane. Using microfluorimetry of indo-1 fluorescence we have detected a complex oscillatory [Ca2+]i response in ODG following exposure to sublethal dilutions of serum-derived complement. Oscillations were transitory and preceded complete and stable return to resting [Ca2+]i levels, whereas nonoscillating ODG underwent rapid lysis. Depletion of the terminal complement component C9 from serum removed the oscillatory stimulus, which could be restored by reconstitution with purified C9. Exposure to the C9-homologous peptide melittin produced [Ca2+]i oscillations similar in pattern to those induced by whole serum. However, this type of response could not be reproduced by Ca2+ ionophores or mechanical wounding, suggesting that oscillations cannot be provoked by Ca2+ influx alone and depend on the presence of the MAC or a pore-forming lesion. Oscillations were not prevented in the continuous presence of caffeine, demonstrating independence from caffeine-releasable intracellular stores. Inhibition of the endoplasmic reticular Ca(2+)-ATPase with thapsigargin produced an abrupt elevation in [Ca2+]i but did not alter the latency between exposure to serum and the initial complement-induced transient. However, the slope of this initial transient was considerably reduced and oscillations suppressed, demonstrating dependence of the oscillatory mechanism on functional endoplasmic reticular Ca2+ stores. The coincidence of ODG recovery with oscillating [Ca2+]i suggests that the complex calcium signal that follows MAC attack may stimulate repair or protective mechanisms.

Using a human IgG-Sepharose column to which rabbit anti-human IgG was bound (rabbit anti-human/human IgG-Sepharose), human and rat C1 or C1q were isolated from serum in a single step, and the C1q further purified to homogeneity by FPLC. This procedure allowed the rapid isolation of haemolytically active C1 or C1q, with a yield equal to or greater than published methods. The availability of human and rat C1q allowed comparison of the two molecules, revealing differences in their mobility on SDS-PAGE as well as on agarose gel electrophoresis. Amino terminal sequence analysis demonstrated greater than 78% residue identity between rat C1q A, B and C chains and the published human and mouse sequences. Similar amino acid compositions suggest that the homology extends throughout the molecules. In addition to the major A:B and C:C dimer bands, rat, unlike human C1q, contained minor dimer species. These may reflect heterogeneity in glycosylation and or lysine and proline hydroxylation.

Rat oligodendrocytes, which activate the classical pathway of complement in the absence of antibody, are highly sensitive in a reactive lysis assay using human C5b6 and EDTA serum. Oligodendrocytes may be relatively deficient in glycolipid-linked complement regulatory protein(s), since digestion with phosphatidylinositol-specific phospholipase C (PI-PLC) failed to increase their sensitivity to serum, whereas complement-insensitive astrocytes, when treated with PI-PLC, became strikingly sensitive. To test the hypothesis that oligodendrocytes lack terminal complement regulatory molecule(s), human erythrocyte CD59, a recently described complement regulatory protein, was purified to homogeneity. The biological activity of the preparation was confirmed by reincorporating the protein into guinea-pig erythrocytes through its glycolipid anchor, which resulted in dose-dependent protection against human C5b6 and EDTA serum. Incorporation of 10(5) molecules of human CD59 into rat oligodendrocytes resulted in good protection against homologous human complement (76%), and significant protection against rat complement homologous to the cell (36%). Protection could be reversed using an antibody to CD59.

The interaction of human serum with oligodendroglia was investigated in vitro using purified cultured neonatal rat oligodendrocytes. Previous evidence for antibody independent classical pathway complement activation was confirmed; the results also showed a deficit in the protection of rat oligodendrocytes from complement attack suggesting a deficiency in the expression of terminal regulatory proteins of the complement cascade. Thus, rat oligodendrocytes are selectively sensitive to normal serum due both to complement activation and impaired protection from terminal complement attack.

This paper describes a T-cell targeting strategy based on the use of an antigen-monoclonal antibody heteroconjugate. A rat anti-idiotypic monoclonal antibody specific for a murine B-cell lymphoma was conjugated to the purified protein derivative (PPD) of tuberculin. This construct selectively delivered up to 4.5 x 10(4) molecules of PPD onto each tumour cell. Targeted PPD was internalized for endosomal processing and was presented in association with the I-A class II restriction element to PPD-reactive T-helper (Th) cells. Activated Th cells were demonstrated to proliferate and secrete significant levels of tumour necrosis factor (TNF). Such lymphokine secretion was observed at a PPD concentration as low as 1 ng/ml. Despite the secretion of TNF, the B-cell lymphoma was found to be resistant to autonomous Th-mediated cytotoxicity. Targeted Th cells did, however, activate tumourcidal macrophages that subsequently mediated significant tumour cytostasis. Based on this observation, it is proposed that the targeting system described may be exploited as the basis for a future immunotherapeutic strategy.

We have shown that a murine CD4+ PPD-reactive T lymphocyte clone was weakly cytotoxic towards the syngeneic tumour B16 melanoma and MC6A fibrosarcoma which had been coated with PPD using a monoclonal antibody-PPD heteroconjugate. Cell-free supernatants produced by PPD-stimulated T lymphocyte clones were however highly cytostatic for the two tumour targets when assayed over 48-72 h. In this study we have demonstrated good titres of tumour necrosis factor (TNF) and interferon-gamma (IFN-gamma) in the supernatants, which accounted for their observed cytostatic activity on the tumour targets. The high level of cytostasis seen with the B16 melanoma using the supernatants could be attributed to their sensitivity to the cytostatic activity of IFN-gamma; the lower levels of cytostasis seen with the IFN-gamma-resistant MC6A target was the result of IFN-gamma increasing the sensitivity of this target to TNF. Antibodies to IFN-gamma were able to neutralize the majority of the cytostatic activity of the supernatants on both targets, consistent with the role demonstrated for this lymphokine.

We have constructed PPD monoclonal antibody heteroconjugates specific for a tumour-associated antigen of C57BL/6 melanomas or for human complement component C3d fixed de novo to murine fibrosarcoma cells (MC6A). The ability of our heteroconjugates to target CD4+ PPD-reactive T cells against the appropriate tumour targets was then determined in vitro. Heteroconjugate-treated B16-F10 and MC6A tumour targets were both able to present PPD to the specific T cells, resulting in activation and concomitant lymphokine secretion. Secreted lymphokines were then demonstrated to cause significant tumour cytolysis and cytostasis in vitro. Preliminary experiments in vivo suggest that this targeting system may provide the basis for a future immunotherapeutic strategy.

A high non-specific background fluorescence signal was observed when cell surface antigen analysis was carried out using flow cytometry on a cell sample which contained a high proportion of dead and dying cells. To overcome this problem it was necessary to analyse the cells in three stages. First the intact cells were identified by their forward (FWD) and 90 degree scatter profile. These cells were gated-on, then analysed on the basis of their FWD scatter and propidium iodide (PI) signal, allowing the dead PI positive cells to be gated out. The PI negative cells were then displayed using their 90 degree scatter and fluorescence signals following staining with the irrelevant antibody control. This revealed a population of dead cells, which despite being PI negative, were non-specifically binding antibody molecules. Such multiparameter analysis permitted the successful analysis of cell surface antigens in preparations of low viability by gating out the high background fluorescence associated with dead PI positive and negative cells.

Cryosurgery and excision were used to treat primary tumours of HSV-2-transformed hamster tumour sublines, and post-operative survival and the extent of metastatic disease were compared in the two groups. An inferior prognosis was observed following cryosurgery although the extent of metastatic disease was similar in both groups. Using this model it would appear that cryosurgery enhances the development of micrometastases rather than affecting the number of cells shed from the primary tumour during surgery. To investigate the underlying causes of the decrease in survival following cryosurgery, in vitro assays were used to monitor host immunocompetence following surgery. The results showed that whilst natural killer cell cytotoxicity was only marginally depressed, mitogen responsiveness and lymphocyte participation in a mixed lymphocyte reaction were severely reduced 3-7 days post-cryosurgery. In parallel with immunosuppression, extensive cell proliferation in the spleen of cryosurgically treated tumour-bearing animals was observed. Histological examination of the spleen demonstrated the presence of large numbers of transformed cells which correlated with the loss of mitogen responsiveness and the ability to participate in a mixed lymphocyte reaction. Further studies (manuscript submitted for publication) have demonstrated that spleen cells from animals whose tumour is treated by cryosurgery are capable of suppressing immunocompetence in vitro, implying they have a role in the uncontrolled growth of micrometastases in vivo.

Cryosurgery of a primary HSV-2-induced hamster fibrosarcoma resulted in the generation of a population of suppressor cells. These cells were detectable in the spleen 1-10 days post-cryosurgery by their ability to suppress the proliferation of immunocompetent splenic T-lymphocytes following exposure to concanavalin A (Con A). The spleens of tumour-bearing (t.b.) animals which received cryosurgery 3 days previously displayed gross splenomegaly due to the generation of large numbers of highly proliferative erythroblasts. The erythroblast cells were unlikely to be the source of suppression since time course studies have demonstrated the presence of suppressor cells before and after their appearance in the spleen. The erythroblasts therefore probably reflected a response by the host to regenerate the erythrocytes lost during surgery and their presence was independent of the appearance of suppressor cells. Characterisation of the suppressor cell has revealed it to be non-adherent and esterase negative making it unlikely to be of macrophage (MO) lineage. This was confirmed by the ability of splenic MOs from day 3 t.b. cryosurgery-treated animals to completely restore Con A-dependent T-lymphocyte proliferation following MO depletion. As nylonwool column-eluted cells are able to suppress Con A-dependent T-lymphocyte proliferation, it seemed unlikely that B-lymphocytes play a role in cryosurgery-induced immunosuppression. These findings suggest that cryosurgery of a t.b. animal results in the generation of a population of T-lymphocytes capable of suppressing Con A-dependent T-lymphocyte proliferation, and infers that these cells contribute to the inferior prognosis following cryosurgery as compared to excision of a metastatic tumour.