Intrinsic reproductive isolating mechanisms in the maintenance of a hybrid zone between ecologically divergent subspecies.

Understanding factors involved in maintaining stable hybrid zones is important for predicting the ultimate fate of the interacting taxa, but the relative importance of mechanisms such as ecological selection and intrinsic reproductive isolation remains unclear. Most studies of reproductive isolation in hybrid zones have focused either on zones with strongly bimodal patterns in genotype or phenotype frequencies, with relatively strong isolation, or unimodal zones with relatively weak isolation, whereas less is known about more intermediate classes of hybrid zone. Here, we utilize a hybrid zone of this intermediate type occurring between northern and southern subspecies of Atlantic killifish, Fundulus heteroclitus, to identify isolating mechanisms playing a role in maintaining this type of zone. The two subspecies differ in environmental tolerance, and we found some evidence of microhabitat preference between subspecies within a small tidal creek at the centre of the hybrid zone. There was also an association between sex, mitochondrial genotype and habitat within this creek. Fertilization success did not differ between consubspecific and heterosubspecific crosses, but hatching success was significantly lower for crosses involving southern males and northern females, and crosses between southern females and northern males had altered developmental rates. Southern females and northern males showed patterns consistent with positive assortative mating. Together, these results indicate a role for a combination of factors including assortative mating and/or early hybrid inviability in the maintenance of this hybrid zone and suggest that hybrid zones with intermediate levels of reproductive isolation are likely to be maintained by multiple interacting isolating mechanisms.

Genetic identification of istiophorid larvae from the Gulf of Mexico based on the analysis of mitochondrial DNA control region sequences.

Assigning relative importance of spawning and nursery habitats for threatened and endangered teleosts, such as those seen in the Gulf of Mexico (GoM), relies on the proper identification of the early life-history stages of the species of concern. Here, sequencing a portion of the mitochondrial DNA (mtDNA) control region (CR) I as barcodes is recommended to identify istiophorid (billfish) larvae in the Atlantic Ocean because of its high resolution and the intrinsic value of the levels of genetic variation that can be extracted from these data. The universality of the primers employed here demonstrates their utility for not only the positive identification of istiophorids in the GoM, but for any larval teleost occurring in areas recognized as larval hotspots worldwide.

Incidence of cutaneous squamous cell carcinoma in a New Zealand population of chronic lymphocytic leukaemia patients.

BACKGROUND: Chronic lymphocytic leukaemia (CLL) is associated with an increased incidence and aggressiveness of skin cancers, particularly cutaneous squamous cell carcinoma (cSCC), but little is known about cSCC incidence in Australasian CLL patients. AIM: In this retrospective study, we analysed the incidence of cSCC in patients seen at a tertiary hospital in New Zealand (NZ). METHODS: We retrospectively assessed the clinical history and histology data of CLL patients (n = 371) who presented to the Haematology Department, Christchurch Hospital, NZ during the period 1996-2015. Baseline characteristics, incidence of second cancers, treatment details and overall survival were analysed. RESULTS: During follow-up (median = 11.8 years), 221 second cancers were recorded in 88 patients. Of these cancers, 185 were cSCC, removed from 61 patients. In 56% of these patients, >1 cSCC was removed, and the majority of cSCC occurred following the treatment for CLL. The cumulative incidence of a first cSCC was 11% at 5 years, whereas the cumulative incidence of a subsequent cSCC was 88% at 5 years. The incidence of cSCC in male patients was threefold higher than that reported for the general NZ population. CONCLUSION: NZ CLL patients have a high incidence of cSCC relative to the levels observed in the general population, which are themselves among the highest in the world. The careful monitoring of CLL patients is warranted, particularly those who have a progressive disease or have had a first cSCC removed.

Isolation and characterization of human tonsil dendritic cells.

Human dendritic cells were isolated from tonsils by density gradient separation followed by FACS IV sorting with mAbs to remove contaminating cell populations. The resulting dendritic cell population consisted of large cells with plentiful basophilic cytoplasm, lacking in granules but containing a prominent Golgi apparatus and numerous mitochondria. The cell membrane was irregular, and marked cell protrusions were obvious when stained with anti-HLA class II reagents. Their nuclei were irregular and often indented with a visible nucleolus. These cells were not phagocytic and stimulated autologous and allogeneic lymphocytes more effectively than other tonsil cell types in MLR. Phenotypic analysis of these cells confirmed that they expressed the leucocyte common antigen and stained strongly for HLA-class II antigens. Tonsil dendritic cells also coexpressed the LFA-1 alpha and LFA-1 beta chains but did not stain with a wide variety of anti-monocyte or anti-macrophage antibodies. The cells also lacked Fc and complement receptors and failed to stain with CD1 antibodies. Extensive testing with mAbs revealed only a few positive reactions, and these were consistent with reports of these antibodies staining interdigitating cells in tissue sections. This established that tonsil dendritic cells belong to the unique haemopoietic cell lineage of dendritic cells. No cytoplasmic staining of IL-1 alpha or IL-1 beta was demonstrated, although these lymphokines were readily detected in activated monocytes.

CD38 as a prognostic marker in chronic lymphocytic leukaemia at a single New Zealand centre: patient survival in comparison to age- and sex-matched population data.

AIM: The aim of this study is to determine whether the analysis of CD38 expression by chronic lymphocytic leukaemia (CLL) cells provides useful additional prognostic information. METHODS: Clinical, laboratory, overall survival (OS) and treatment-free survival (TFS) data were collected on 130 CLL patients who had CD38 expression analysed at Canterbury Health Laboratories, New Zealand (NZ) during 1998-2008. RESULTS: The detection of any level of CD38 expression by CLL cells was associated with a significantly shorter OS and TFS. When analysis was restricted to Binet stage A patients, CD38 expression identified a subset of patients (21%) who, in common with Binet stage B/C patients, had a significantly shorter OS and TFS (P<0.0015), and a TFS at 4 years of <10%. In contrast, CD38-negative Binet stage A patients had an OS that was not significantly different from that of an age/sex-matched NZ population and a 5-year TFS of 77%. CONCLUSION: This study indicates that, when combined with clinical staging, the presence of any detectable CD38 expression can be used to further improve the identification of CLL patients with more aggressive disease (i.e. Binet stage B/C or Binet stage A and CD38 positive). This will allow better identification of those patients requiring more intensive monitoring and also allow improved patient counselling regarding prognosis.

Immune development and performance characteristics of Romney sheep selected for either resistance or resilience to gastrointestinal nematodes.

Immunological and performance characteristics were explored in Romney sheep from lines selected for either resistance or resilience to parasite infection. At a mean 78 days-of-age, twin lambs from a line selected for resistance (RT) and lambs from a line selected for resilience (RL) were infected with the intestinal nematode Trichostrongylus colubriformis for 100 days (I) while their twin remained as an uninfected control (C). Compared with RL, RT animals had lower levels of circulating CD4+ T-cells (P = 0.003) but a greater proportion of these were activated (CD4+CD25+) in response to infection (P = 0.007). Differences between the lines in humoral immune responses to nematode infection varied with higher levels of T. colubriformis specific immunoglobulin (Ig) E in RT-I than RL-I (P = 0.002) but similar levels of both IgG (P = 0.926) and IgA (P = 0.321) responses. Temporal differences in the immune response also existed between the lines with RT-I animals displaying an earlier peak and more rapid reduction in FEC and an earlier peak in T. colubriformis specific IgA. In addition, compared with their RT-C and RL-C counterparts, infection caused a 22% reduction in feed intake from day 56 (P = 0.001) with total feed intake reduced by 15% and 9% for RT-I and RL-I, respectively. Cumulative liveweight gain was greatest for RL animals (P = 0.026) and relative to RT-C and RL-C was reduced by 5.8 kg and 4.9 kg for RT-I and RL-I, respectively. Overall, the selection lines appear to have differences in immunological characteristics that are both dependent on, and independent of parasite infection. Further, the difference in growth in the uninfected animals coupled with the similar cost of infection suggests the lower liveweight gain of RT-I compared with RL-I may be due to inherent differences between the lines in their growth potential, rather than a greater cost of infection in animals selected for resistance.

Direct evidence for cooperating genetic events in the leukemic transformation of normal human hematopoietic cells.

Although genetic abnormalities associated with hematological malignancies are readily identified, the natural history of human leukemia cannot be observed because initiating and subsequent transforming events occur before clinical presentation. Furthermore, it has not been possible to study leukemogenesis in vitro as normal human cells do not spontaneously transform. Thus, the nature and sequence of genetic changes required to convert human hematopoietic cells into leukemia cells have never been directly examined. We have developed a system where the first step in the leukemogenic process is an engineered disruption of differentiation and self-renewal due to expression of the TLS-ERG oncogene, followed in some cases by overexpression of hTERT. In two of 13 experiments, transduced cells underwent step-wise transformation and immortalization through spontaneous acquisition of additional changes. The acquired karyotypic abnormalities and alterations including upregulation of Bmi-1 and telomerase all occur in acute myeloid leukemia (AML), establishing the relevance of this system. One resultant cell line studied in depth exhibits cellular properties characteristic of AML, notably a hierarchical organization initiated by leukemic stem cells that differentiate abnormally. These findings provide direct evidence for multiple cooperating events in human leukemogenesis, and provide a foundation for studying the genetic changes that occur during leukemic initiation and progression.

Transcription of the apicoplast genome.

Many members of the Apicomplexa contain a remnant chloroplast, known as an apicoplast. The apicoplast encodes numerous genes, and loss of the organelle is lethal. Here, we present a summary of what is known about apicoplast transcription. Unlike plant chloroplasts, there is a single RNA polymerase, and initial transcription is polycistronic. RNA is then cleaved into tRNA, mRNA and rRNA molecules. Significant levels of antisense transcription have been reported, together with a single case of RNA editing. Polycistronic transcription is also observed in the related algae Chromera and Vitrella, which retain a photosynthetic chloroplast. Surprisingly, a polyU tail is added to Chromera and Vitrella transcripts which encode proteins involved in photosynthesis. No such tail is added to Plasmodium transcripts. Transcription in the Apicomplexa is remarkably similar to that seen in the chloroplast of the related peridinin dinoflagellate algae, reflecting the common evolutionary origins of the organelle.

Human plasma contains a soluble form of CD86 which is present at elevated levels in some leukaemia patients.

Cell surface expression of CD86 (mCD86) provides an important co-stimulatory signal which profoundly influences immune responses. In this report, we investigated the potential presence of a circulating soluble form of CD86 (sCD86) in normal individuals and patients with acute myeloid leukaemia (AML) or B cell chronic lymphocytic leukaemia (B-CLL). Circulating sCD86 was detected in the plasma of all normal individuals (1.04 +/- 0.33 ng/ml, n = 51) and patients analysed. Plasma collected from AML patients in remission (n = 6) contained only low levels of sCD86 but significantly elevated levels (> or =2.65 ng/ml, P < 0.0001) were detected in 10/24 AML patients analysed at the time of presentation or relapse. Significantly elevated levels of sCD86 were also detected in 2/17 B-CLL patients. There was no correlation between sCD86 levels and other clinical parameters. RT-PCR analysis demonstrated that normal monocytes and dendritic cells, as well as isolated AML (n = 2) and B-CLL (n = 4) cells, expressed an alternatively spliced transcript of CD86 which encoded a soluble form absent in normal T, B and NK cells. The finding that a proportion of leukaemia patients contain elevated levels of sCD86 and that at least some leukaemic cells express sCD86 transcript suggests a potential role for sCD86 in modulating mCD86 signalling during the malignant process.

Degenerate DNA recognition by I-PpoI endonuclease.

The I-PpoI endonuclease is encoded by a group I intron found in the slime mold Physarum polycephalum. To initiate homing of its encoding intron, I-PpoI catalyzes a specific double-stranded break within a 15-bp recognition site. The high substrate specificities of I-PpoI and other homing endonucleases make these enzymes valuable tools for genomic mapping and sequencing. Here, we report on the ability of I-PpoI to cleave recognition sites that contain a wide variety of mutations generated randomly or deliberately. We find that much degeneracy is tolerated within the recognition site of I-PpoI. Few single substitutions prevent cleavage completely. In addition, many sites with multiple substitutions are cleaved efficiently. In contrast, deletions or insertions within the I-PpoI recognition site are detrimental to catalysis, indicating that proper registry between the protein and its substrate is critical. Finally, we find that the sequence of the flanking regions can influence catalysis by I-PpoI. Thus, I-PpoI has both the complex binding specificity of a transcription factor and the catalytic ability of a restriction endonuclease.

Optimisation of the conditions for generating human DC initiated antigen specific T lymphocyte lines in vitro.

Naive T lymphocytes specific for a given primary antigen occur in low frequencies and require the relevant antigen to be presented by specialist antigen presenting cells (APC), i.e., dendritic cells (DC). For these reasons, the in vitro induction of primary T lymphocyte responses remains a significant technical challenge. We have attempted to improve current strategies for generating in vitro responses by optimising (i) isolation and concomitant activation of DC from peripheral blood, (ii) uptake, processing and presentation of antigen by DC and (iii) antigen driven T lymphocyte proliferation. We established that RPMI 1640 media supplemented with 10% autologous serum resulted in the best yield of CMRF-44+, CD14-, CD19- DC after enrichment over a Nycodenz gradient. Optimal presentation of whole protein and peptide antigen was achieved by addition after the purification of the APC, i.e., at the start of the T lymphocyte proliferation assay. RPMI 1640 supplemented with 10% autologous serum or plasma supported the best antigen driven specific T lymphocyte responses. Using these optimised conditions, we compared the efficacy of PBMC and purified blood DC for priming T lymphocyte responses to the chronic myeloid leukaemia (CML) specific bcr-abl (b3a2) peptide. Peptide specific T lymphocyte responses were generated with both purified DC and whole PBMC, suggesting that T lymphocyte precursor frequency was the limiting factor in these experiments. These results will aid in the generation of human T lymphocyte lines to primary antigens, for in vitro and therapeutic applications.

Synthesis and alpha 2-adrenoceptor effects of substituted catecholimidazoline and catecholimidazole analogues in human platelets.

It is known that the steric requirements for the interactions of catecholamines and catecholimidazolines with alpha 1- and alpha 2-adrenoceptors are different. New analogues of desoxycatecholimidazoline (1), desoxycatecholimidazole (3), benzylic hydroxyl substituted imidazole (4), and the aromatic fluorine substitution analogues of 1 at the 2 (5), 5 (6), and 6 (7) positions, and a set of asymmetric 4-substituted catecholimidazolines, S-8 and R-8, were prepared and tested for interaction with alpha 2-adrenoceptors in human platelets. With the exception of 3, all compounds were selective for alpha-adrenoceptor-mediated responses in human platelets. Introduction of a double bond in imidazoline 1 to give an imidazole 3 or the introduction of a benzylic hydroxyl group to 3, as in 4, reduced the inhibition of platelet aggregation with a rank order potency of 1 greater than 3 greater than 4. Fluorine atom substitution at the 2-, 5-, or 6-positions only slightly modified the inhibitory activity of 1. Each analogue (1, 3-7) produced alpha 2-mediated inhibition of platelet adenylate cyclase and can be classified as a partial agonist. The inhibition potency of S-8 and R-8 against epinephrine-induced aggregatory responses were greatly different, and only R-8 and 4 were alpha 2-agonists on human platelet function. Our studies provide further evidence for the differential interaction of catecholamines and catecholimidazolines in alpha 1- and alpha 2-adrenoceptor systems.

Studies with a monoclonal antibody on the distribution of Thy-1 in the lymphoid and extracellular connective tissues of the dog.

BALB/c mice were immunized with canine T lymphocytes from lymph node and used in cell fusion experiments to derive monoclonal antibodies to the T lymphocyte surface. The cloned hybrid line F3-20-7 described in this paper was shown to be secreting antibodies directed at canine Thy-1 since (1) the tissue distribution of the antigen corresponded precisely to the tissue distribution expected of canine Thy-1 from previous studies and (2) the antigen purified from F3-20-7 monoclonal antibody affinity columns (a) had the same mobility on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) as pure rat Thy-1 and (b) could inhibit and assay shown previously to be directed at canine Thy-1 on the basis of cross-reactivity with rat Thy-1. Studies with the fluorescence-activated cell sorter showed that all thymocytes and lymph node T lymphocytes of the dog were Thy-1 positive, establishing that Thy-1 is a T cell marker in the dog, as in the mouse, although the presence of a small percentage of Thy-1-positive B cells could not be entirely excluded. In addition, 15% of the nucleated bone marrow cells were Thy-1 positive, which is similar to the finding in rat bone marrow. Localization studies on frozen sections of skin demonstrated large amounts of Thy-1 organized in a highly structured manner around the basal parts of the hair follicles. This covering of Thy-1 was lost as the follicles approached the surface. Epidermal cells were Thy-1 negative. In the kidney, Thy-1 was shown to be strongly associated with all of the basement membranes of the medulla. The kidney cortex, including glomeruli was essentially negative, except for the occasional staining of basement membranes, possibly of collecting ducts.

Characterization of interstitial dendritic cells in human liver.

Sensitive immunofluorescence and immunoperoxidase techniques were used to test an extensive range of monoclonal antibodies for reactivity with Kupffer cells and interstitial dendritic cells (DCs) in cryostat-cut sections of human liver. Leucocytes with a dendritic cell morphology were identified with CD45 (antileucocyte common) reagents in portal tracts, predominantly around bile ducts, and these cells stained strongly for the HLA-DP, DQ, and DR antigens. Kupffer cells stained less intensely with anti-class-II reagents, particularly anti-HLA-DQ. The interstitial DCs expressed the LFA-1 antigen but failed to stain with CD11b, CD11c, and the defined T and B cell CD antibodies; nor did they stain with antibodies to FcR1, FcR11, FcRIII, or the C3b receptor. Of the myeloid monoclonal antibodies available from the 3rd Leucocyte Differentiation Antigen Workshop, only Y2/131, Ki-M7, Ki-M8, and a minority of CD14 antibodies stained DCs, whereas Kupffer cells showed a wider reactivity with antimacrophage antibodies including those of workshop groups 11, 15, 16, and other unique antibodies. A 2nd probable DC population was identified in the liver capsule that had a similar phenotype to portal interstitial DCs. Although some minor phenotypic differences between liver portal DCs and the phenotypes of Langerhans cells and isolated tonsil DCs were noted, our results support the view that there is a unique hemopoietic lineage of DCs. The presence of DCs, which stimulate strong allogeneic T cell responses, in the portal triads is consistent with the fact that the histologic changes of graft-versus-host disease seen in bone marrow transplantation and the lymphocytic infiltrate in a rejecting liver allograft occur predominantly in the periportal region.

The effect of donor pretreatment on interstitial dendritic cell content and rat cardiac allograft survival.

Different pretreatment schedules were applied to rat heart donors and their effect on heart interstitial dendritic cell content was observed using monoclonal antibodies and immunofluorescence techniques. Interstitial dendritic cell (IDC) numbers were correlated with the histology and survival of hearts transplanted into untreated allogeneic recipients. Hearts from AS donors pretreated with cyclophosphamide and total-body irradiation showed prolonged survival in DA recipients only when more than 95% of the graft interstitial dendritic cells were depleted. Reconstitution studies established that prolonged graft survival following donor pretreatment depended on the removal of bone-marrow-derived cells and that these were IDCs. These results suggest that the IDC is the most significant "passenger leucocyte", and that very small numbers of residual IDCs were still sufficient to cause rapid rejection. DA rat heart contained three times as many IDCs as the AS strain and DA hearts were much more difficult to deplete of IDCs. Pretreated DA hearts were rejected by AS recipients, although grafts with a lower IDC content resulted in an attenuated histological rejection response and a markedly decreased recipient lymphocytotoxin response. To be effective, donor pretreatment schedules had to be initiated 5 days prior to transplantation. Pretreatment 6 hr prior to transplantation failed to deplete IDC or prolong graft survival even in he weak (ASxDA)F1 to DA model. Pretreatment protocols used clinically have probably not removed IDCs adequately and the development of methods that deplete IDCs effectively could improve graft survival at no risk to the recipient.

Adhesion molecules on human tonsil dendritic cells.

Dendritic cells are specialist antigen-presenting cells that have a unique ability to stimulate a primary T cell response. Activation of T cells by DC depends on the formation of cell clusters creating DC-T cell membrane contact that probably involves adhesion molecules. Monoclonal antibodies were used to study adhesion molecules on DC, including members of the integrin and immunoglobulin supergene families. DC expressed LFA-1, ICAM-1, LFA-3, and the Hermes antigen, but no other integrin or immunoglobulin supergene family adhesion molecules were detected using a sensitive immunoperoxidase staining technique. Monoclonal antibodies to LFA-1 alpha and LFA-1 beta inhibited DC-stimulated allogeneic T cell (MLR) responses by 75 +/- 12% and 74 +/- 8%, respectively, as did the anti-LFA-3 (56 +/- 3% inhibition) and anti-LFA-2 (60 +/- 5% inhibition) antibodies. Three different anti-ICAM-1 antibodies inhibited only to a limited degree (mean range 8-24%). The inhibitory effect of the LFA-1 and LFA-3 antibodies was maximal if added early to the MLR. The inhibitory effect of the different antibodies was associated with variable decreases in DC-T cell cluster stability. The simultaneous addition of monoclonal antibodies to MLRs and preincubation washing experiments established that DC have at least 3 independent adhesion ligand interactions (LFA-1-ICAM-1, ICAM-1-LFA-1, and LFA-3-CD2) with T cells. It seems likely that the additional ligand for LFA-1, ICAM-2, is expressed on DC and contributes significantly to DC-T cell adherence and T cell activation. The membrane mobility of these molecules may also be important in the DC-T cell activation process.

Interstitial dendritic cells.

Interstitial dendritic cells (IDC) were first identified in the interstitium of non-lymphoid organs as leucocytes which stained intensely with anti-MHC class II antibodies. These cells have been identified in several species including man, and can be distinguished from tissue macrophages by their immunological phenotype and cytochemical and functional characteristics. IDC appear to be closely related to lymphoid dendritic cells (DC), and have the capacity to bind antigen and stimulate T lymphocyte responses. It seems probable that they represent a stage of nonlymphoid dendritic cell differentiation necessary for antigen surveillance, similar to the Langerhans cell of the skin. Exposure to antigen appears to induce migration of these cells into adjacent lymphatics and subsequent localization in the interfollicular areas of lymph node, where the DC present processed antigen to activate a primary T cell response. The IDC has been identified as the passenger leucocyte within organ allografts which contributes substantially to graft immunogenicity, so that eradication of donor organ IDC improves organ graft survival.

Differential effects of G-CSF mobilisation on dendritic cell subsets in normal allogeneic donors and patients undergoing autologous transplantation.

It has been suggested that the immunological properties of cytokine primed PBSC may reflect the presence of altered levels of cellular components. In this study the changes induced in blood dendritic cell (DC) subsets following G-CSF mobilisation are analysed. Analysis of normal donors (n = 64) demonstrated considerable individual variation in the absolute numbers (x10(6)/l) of resting blood CD11c(-) DC (1.2-26.2) and CD11c(+) DC (0.9-34.7) as well as in the CD11c(-)/CD11c(+) DC ratio (0.29-4.13). G-CSF therapy increased CD11c(-) DC numbers to above the normal range in all normal donors analysed (n = 6) and the CD11c(-)/CD11c(+) ratio was also increased to >2.0 in all donors. Patients undergoing autologous PBSCT showed a heterogeneous response to mobilisation and although total DC and CD11c(-) DC numbers were increased in the majority (8/14), they remained within the normal range post mobilisation. The CD11c(-)/CD11c(+) ratio decreased in 5/15 patients and only three patients had ratios >2.0 post mobilisation. Post G-CSF the DC from all normal donors and 13/14 patients had an immature phenotype. These results demonstrate that G-CSF mobilisation induces relatively consistent changes in the number and ratio of DC subsets in normal donors, but considerable variation is seen in the response of patients undergoing mobilisation for autologous PBSCT.

Role of tumour necrosis factor-alpha in dendritic cell-mediated primary mixed leucocyte reactions.

Tumour necrosis factor (TNF alpha) is a major inflammatory cytokine with potentiating effects on specific immune responses, including graft-versus-host disease. This study examined the contribution of TNF alpha to dendritic cell (DC)-mediated primary allogeneic T lymphocyte responses. Purified blood DC were shown to produce minimal amounts of TNF alpha mRNA but no significant TNF biological activity or secreted TNF alpha as measured by ELISA. Amplification of DC mRNA by PCR using oligonucleotide primers to CD120a (TNFRI, p55) and CD120b (TNFRII, p75) and probing with specific internal oligonucleotides, suggested that DC express the CD120b but little if any CD120a. These results were confirmed using monoclonal antibodies to the TNF receptors. Polyclonal antiserum specific for TNF alpha blocked the blood DC-stimulated allogeneic mixed leucocyte reaction (MLR). The addition of TNF alpha to suboptimal MLRs (limited DC stimulators), increased the proliferation of responding T lymphocytes. Having confirmed that T lymphocytes produce TNF alpha and express CD120b after stimulation, we sought to clarify whether the contributing effect of TNF alpha to the allogeneic MLR resulted from a TNF alpha-mediated signal stimulating DC activity, or as a result of autocrine stimulation of T lymphocytes. Pre-incubation of DC with TNF alpha did not increase DC stimulatory capacity and late addition of anti-TNF serum (up to 72 h) still had a significant inhibitory effect on the MLR. We conclude that TNF alpha is probably not involved in the initial DC-T lymphocyte interaction, but acts as an autocrine growth factor for DC induced T lymphocyte proliferation.

Distribution of Thy-1 in human brain: immunofluorescence and absorption analyses with a monoclonal antibody.

A monoclonal antibody to human Thy-1 has been used to study the anatomical localization of Thy-1 in human brain and to quantitate the relative amounts of Thy-1 in different brain subregions. Quantitative absorption analyses using homogenates of carefully dissected brain subregions, together with an [125I]anti-immunoglobulin binding assay using brain homogenate as target, established that Thy-1 was present in large amounts throughout human brain, but the grey matter of cerebrum (cortical grey matter, caudate nucleus, putamen and thalamus) had 5-10 times as much Thy-1 as white matter. Grey matter of cerebellum (cerebellar cortex and dentate nucleus) also had higher amounts of Thy-1 than white matter, but the total amount of Thy-1 in cerebellum was less than in the cerebrum. Immunofluorescence studies gave interesting results and demonstrated in particular: (a) the outlining of some neuronal cell bodies and their processes (particularly the Purkinje cells of the cerebellar cortex) by spots of fluorescence; (b) staining of what appeared to be cell bodies of satellite cells in areas of grey matter; (c) granular staining in grey but not white matter; (d) staining of what appeared to be fibre tracts in the basal ganglia and thalamus, the tracts appearing duller than the surrounding grey matter of the nuclei; (e) staining of only some fibres in sciatic nerve; and (f) absence of staining of the adrenal gland.